assessing the use of conjugated polymers and electric

Assessing the use of conjugated polymers and electric

fields for cell culture

Ana Filipa Soares Pires

Thesis to obtain the Master of Science Degree in

Bioengineering and Nanosystems

Examination Committee

Chairperson: Professor Doctor Luís Joaquim Pina da Fonseca Supervisors: Professor Doctor Jorge Manuel Ferreira Morgado

Doctor Frederico Castelo Alves Ferreira Members of the Committee: Professor Doctor José Paulo Sequeira Farinha

December 2013

III

“It always seems impossible until it is done”

Nelson Mandela

V

Acknowledgments

Firstly, I would like to express my gratitude to Professor Joaquim Manuel Sampaio Cabral for giving

me the opportunity to develop my master´s thesis at the SCBL-RM Research Group.

In second place, I would like to thank my advisor, Professor Jorge Morgado, who introduced me to the

field of conducting polymers, offered his vision and guidance in planning experiments, and led me to a

proper understanding of the experimental results. I also like to thank my co-supervisor Doctor

Frederico Ferreira for its guidance, support, ideas and availability to answer my doubts, at any time of

day.

I am very grateful to Doctor Carlos Rodrigues by the contribution of his expertise in cell culture,

patience, weekends spent in the laboratory and especially by the fun share in the work environment. I

want to thank Miriam Sousa who was the first person who greeted me in SCBL and who taught me

and gave me support in my first experiments.

I want to express my sincerest gratitude to all colleagues of the Organic Electronic group that always

have been available to help me. Particularly, I want to thank Dr. Ana Luisa Mendonça for all her

support, ideas, advice and care. Thanks for everything.

I also want to thank Tânia Braz for helping me with the electronic equipment in the lab and for the time

spent explaining things to me whenever I had doubts.

Maria Coromoto, Tiago Felício, Braúlio Vieira, Frederico Martins, Diana Santos, Daniela Gomes, Ana

Rodrigues and Tiago Santos for their friendship, support, many laughs and for being such good

friends for so many years.

To my mum, dad, brother and family, for all their love, encouragement, support and for always

believing in me. This is dedicated to you.

Finally, I would like to thank Nelson for all the happiness, patience and boundless love.

VII

Abstract

Neural stem cell-based tissue engineering therapies still remain one of the most attractive therapeutic

strategy for treating neurodegenerative disorders like Alzheimer’s disease. In vivo, neural stem cells

(NSCs) are adherent to extracellular matrix, which provides chemical, mechanical and topographical

cues, controlling in this way cell behavior and functionality.

In this study, different substrates were designed and tested to control fetal NSCs alignment and

elongation through specific control of the topography and electric stimulation setup. RenCell VM NSCs

was used to evaluate the efficacy of poly (3,4-ethylenedioxythiophene) doped with polystyrene

sulfonate (PEDOT:PSS) substrates to promote cell adhesion, proliferation and differentiation.

Our findings suggest that PEDOT:PSS exhibits adequate physicochemical properties and a good

biocompatibility that promote cell attachment and proliferation. Our results suggest that, the

application of the AC electric field (EF) of 1 V/cm elongate and stretch more the cells as compared to

control surfaces that not were exposed to EF. The differentiation potential of electrically

PEDOT:PSS:glass substrate was evaluated by immunostaining key neuronal and glial markers that

are expressed when NSCs differentiate. The conductive PEDOT:PSS:glass substrates promoted

differentiation into neuronal and glial lineage and the NSCs cultured in their surface increased β-III

tubulin expression and the neurite length as compared with PEDOT:PSS:glass substrates that not

were exposed to EF.

Findings from this study suggest that combining EF and PEDOT:PSS provides a promising strategy to

modulate NSCs elongation and differentiation, having a great potential to be used in regenerative

medicine and disease therapies.

Keywords: PEDOT:PSS, Neural Stem Cells, Electric field, Differentiation

IX

Resumo

A terapia de engenharia de tecidos baseada em células estaminais neurais é uma das mais atractivas

estratégias terapeuticas para tratar doenças neurodegenerativas como a doença de Alzheimer´s. In

vivo, as células estaminais neurais estão aderentes à matrix extracelular, a qual fornece pistas

químicas, mecânicas e topográficas, controlando desta forma o comportamento e a funcionalidade

celular.

Neste estudo, diferentes substrates foram desenhados e testados para controlar o alinhamento e

elongamento de células estaminais neurais fetais, através do controlo específico da topografia e do

setup eléctrico. As RenCell VM foram as células usadas para avaliar a eficácia dos substratos

revestidos com poli(3,4-etilenodioxitiofeno) dopado com sulfonato polistireno (PEDOT:PSS) para

promover a adesão, proliferação e diferenciação celular.

Os nossos resultados sugerem que o PEDOT:PSS exibe e propriedades físico-químicas adequados e

uma boa biocompatibilidade que, promove a adesão e proliferação celular. Os nossos resultados

também sugerem que, a aplicação de um campo eléctrico alternado de 1V/cm, alonga e estica mais

as células comparando com as células do controlo que não foram expostas a nenhum campo

eléctrico. O potencial de diferenciação dos substratos PEDOT:PSS:vidro estimulados electricamente

foi avaliado marcando imunohistoquimicamente marcadores neuronais e gliais que as células

expressam quando se diferenciam. Os substratos que foram expostos a um campo eléctrico

promoveram a diferenciação celular numa linhagem neuronal e glial e as células cultivadas na sua

superfície aumentaram a expressão do marcador β-III tubulin e o comprimento das neurites

comparativamente às células nos substratos que não foram expostos ao campo eléctrico.

Os resultados deste estudo sugerem que combinando o campo eléctrico e os substratos revestidos

com PEDOT:PSS fornece uma estratégia promissora para modular a elongação e diferenciação das

células estaminais neurais, tendo um grande potencial para serem usados na medicina regenerativa e

na terapia de doenças.

Palavras-chave: PEDOT:PSS, células estaminais neurais, campo eléctrico, diferenciação

XI

Table of Contents

Acknowledgments ................................................................................................................................... V

Abstract.................................................................................................................................................. VII

Keywords: .............................................................................................................................................. VII

Resumo .................................................................................................................................................. IX

Palavras-chave: ...................................................................................................................................... IX

Table of Contents ................................................................................................................................... XI

List of Abbreviations ..............................................................................................................................XV

List of Figures .......................................................................................................................................XIX

List of Tables ...................................................................................................................................... XXV

Aim of Studies ......................................................................................................................................... 1

I. Introduction ........................................................................................................................................... 3

I.1. Tissue engineering and stem cells ................................................................................................ 3

I.2. Neural stem cells ........................................................................................................................... 3

I.2.1. Isolation of NSCs..................................................................................................................... 4

I.2.2. NSCs expansion...................................................................................................................... 5

I.3. Stem niche ..................................................................................................................................... 7

I.3.1. NSC niche ............................................................................................................................... 7

I.3.2. Signaling in the NSC microenvironment: molecular aspects .................................................. 8

I.4. Biomaterials for NSC culture ......................................................................................................... 9

I.4.1. Conjugated polymers ............................................................................................................ 10

I.4.2. Polypyrrole ............................................................................................................................ 11

I.4.3. Poly(3,4-ethylenedioxythiophene) ......................................................................................... 12

I.4.4. Others conjugated polymers ................................................................................................. 13

I.5. Surface strategies to control cell response .................................................................................. 15

I.5.1. Topography ........................................................................................................................... 15

I.5.2. Stiffness ................................................................................................................................. 16

I.5.3. Electrical stimulus.................................................................................................................. 17

I.5.4. Surface functionalization ....................................................................................................... 18

I.6. NSCs applications ........................................................................................................................ 18

XII

II. Materials and Methods ...................................................................................................................... 21

II.1. Conducting polymers .................................................................................................................. 21

II.2. Conductivity measurement of PEDOT: PSS films ...................................................................... 21

II.3. Current transient technique ........................................................................................................ 22

II.4. Cytotoxicity assays ..................................................................................................................... 22

II.5. Spin coating ................................................................................................................................ 23

II.6. Adhesion test .............................................................................................................................. 24

II.7. Constructs ................................................................................................................................... 24

II.7.1. Conductive substrates ............................................................................................................. 24

II.7.1.1. Perpendicular electrical field setup ................................................................................... 24

II.7.1.2. Longitudinal electrical field setup ...................................................................................... 25

II.7.2. Anisotropic and conductive substrates .................................................................................... 25

II.7.2.1. Polyacrylamide gels .......................................................................................................... 25

II.7.2.2. Replica molding ................................................................................................................. 27

II.7.2.3. Transparent vinyl disc ....................................................................................................... 28

II.8. SEM analysis .............................................................................................................................. 28

II.9. Cell culture .................................................................................................................................. 29

II.9.1. Cell line ................................................................................................................................. 29

II.9.2. NSCs thawing....................................................................................................................... 29

II.9.3. NSCs expansion................................................................................................................... 29

II.9.4. NSCs cryopreservation ........................................................................................................ 29

II.9.5. NSCs differentiation ............................................................................................................. 30

II.9.6. NSCs morphology analysis .................................................................................................. 30

II.9.6. Immunocytochemistry .......................................................................................................... 30

III. Results and Discussion .................................................................................................................... 33

III.1. Research strategy ...................................................................................................................... 33

III.2. Electrical characterization of polymers ...................................................................................... 34

II.2.1. Dichloroacetic acid treatment ............................................................................................... 36

III.3. Transient ionic current measurements ...................................................................................... 36

III.4. Cytotoxic tests with fibroblasts ................................................................................................... 38

III.5. Adhesion test with fibroblasts .................................................................................................... 41

XIII

III.6. Constructs & NSCs .................................................................................................................... 45

III.6.1. Electric field effect in NSCs ................................................................................................. 45

III.6.2. Combined anisotropy and electric field effect in NSCs ....................................................... 54

IV. Conclusions ...................................................................................................................................... 63

V. Future Trends .................................................................................................................................... 65

VI. References ....................................................................................................................................... 67

VII. Annexes .......................................................................................................................................... 73

VII.1. Fluorescence AlamarBlue Calibration Curves ......................................................................... 73

XV

List of Abbreviations

1t-hESNSCs – one time human embryonic stem cell derived neural stem cells

AHPC- adult rat hippocampal progenitor cells

ALS – amyotrophic lateral sclerosis

APC – adenomatous polyposis coli

APES – aminopropyltriethoxysilane

APS – ammonium persulfate

BPs – basal progenitors

CAMs – cell adhesion molecules

Cl – chloride

ClO4 – perchlorate

CNS – central nervous system

CP – conjugated polymers

CSF – cerebrospinal fluid

CSPG – chondroitin sulfate proteoglycans

DAPI – 4´,6-diamino-2-phenylindole

DBS – dodecylbenzenesulfonate

DBSA – dodecylbenzenesulfonic acid

DC – direct current

DCA – dichloroacetic acid

DIV – days in vitro

DMEM – Dulbecco´s modified Eagle´s medium

DMSO – dimethylsulfoxide

DRG – dorsal root ganglia

EBs – embryonic bodies

ECM – extracellular matrix

EDC – 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EF – electrical fields

EG – ethylene glycol

EGF – epidermal grow factor

ESC-NP – embryonic stem cells derived neural progenitor

ESCs – embryonic stem cells

F8T2– poly(9,9-dioctylfluorene-alt -bithiophene)

FBS – fetal bovine serum

FDA – food and drug administration

FEG-SEM – field emission gun scanning electron microscope

FGF-2 – basic fibroblast growth factor

GFAP- glial fribrillary acidic protein

GOPS – 3-glycidoxypropyltrimethoxysilane

XVI

hESCs – human embryonic stem cells

hNPCs – human neural progenitor cells

IKVAV – isoleucine-lysine-valine-alanine-valine

IMDM – Iscove´s modified Dulbecco´s medium

iPSCs – induced pluripotent stem cells

ITO – indium-tin oxide

LEDs – light-emitting diodes

LN – laminin

MAP2 – microtube-associated protein 2

MAPK – mitogen-activated protein kinases

MEH-PPV – poly(2-methoxy-5(2´-ethyl)hexoxy-phenylenevinylene)

MSCs – mesenchymal stem cells

NaHCO3 - sodium hydrogen carbonate

ND – neurodegenerative disorders

NEPs- neuroepithelial progenitors

NGF – nerve growth factor

NGS – normal goat serum

NS – neural stem cell line

NSCs – neural stem cells

OTFT – organic thin film transistors

P19 EC – P19 pluripotent embryonal cell

P3HT – poly(3-hexylthiophene)

PAni – polyaniline

PBS – phosphate buffered saline

PBT – poly(2,20-bithiophene)

PD – polydopamine

PDMS – polydimethylsiloxane

PEDF – pigment epithelium derived factor

PEDOT – poly (3,4-ethylene dioxythiophene)

PEG – poly(ethylene glycol)

PEI – polyethyleneimine

PFA – paraformaldehyde

PLA – poly(D,L-lactide)

PLL – poly-L-lisine

PolyA – polyacrylamide

PPy – polypyrrole

PSS – polystyrenesulfonate

PTh – polythiophene

Rc – contact resistance

RG – radial glia

XVII

RGD – arginine-glycine-aspartic acid

rNSCs –rat neural stem cells

rr-P3HT:PCBM – region-regular poly(3-hexylthiophene-2,5-diyl) blended with phenyl-C61-butyric-acid-

methyl ester

Rsp – spreading resistance

RT – room temperature

SEBS – styrene ethylene butylene styrene

SGZ – subgranular zone

SOX1 – sex determining region Y-box 1

SOX-2 – sex determining region Y-box 2

Sulfo-NHS – N-hydroxysuccinimide

SVZ – subventricular zone

TEMED – tetramethylenediamine

TIC – Transient ionic current

TnC – tenascin C

TsO – tosylate

Tuj1 – β-tubulin

VEGF – vascular endothelial growth factor

WST-1 – water soluble tetrazolium salt 1

YIGSR – tyrosine-isoleucine-glycine-serine-arginine

XIX

List of Figures

Figure I.1. The different NSC populations that can be obtained in vitro correspond to stage-

specific neural progenitors present at defined in vivo developmental stages (taken and adapted

from 14

). ................................................................................................................................................... 5

Figure I.2. Sources and culture system to NSCs (taken from 14

). NSCs derived from ESCs or iPS

cells and from germinative areas of the fetal and human brain can grow in monolayer or in

neurospheres. Culture as a monolayer results in a higher neurogenic potential comparing to

neurospheres since, in monolayer culture systems, there are a homogenous composition of NSCs. ... 6

Figure I.3. Inputs that regulate stem-cell function in the niche. Several factors are important to

regulate stem cell fate within the niche: interactions between cell and neighboring differentiated cells,

interactions between cells and growth factors and physicochemical nature of environment 25

.............. 7

Figure I.4. Differences between the cytoarchitectural organization SVZ in (a) adult rodent and

(b) adult human neural stem cells niches (taken from 10,23

). In adult human SVZ the neurons (A),

the astrocytes (B), the microglia (C), the oligodendrocytes (D), the ependymal cells (E) and the blood

vessels (F) interact with each other. The human SVZ have a hypocellular gap that separates astrocyte

ribbon from the layer of ependymal cells and has a lower number of proliferating cells and neurons

compared to the rodent SVZ (not shown). The most structural difference between the SVZ and SGZ is

the absence of ependymal cells on SGZ. ................................................................................................ 8

Figure I.5. Neural repair and regeneration strategy based on immobilization of growth factors

on biomaterial scaffold (taken and adapted from 34

). The NSC are seeded on scaffold and

transplanted to injury tissue to promote neurogenesis. ........................................................................... 9

Figure I.6. Structures of conducting polymers commonly studied for biomedical applications

(taken from 102

). .................................................................................................................................... 11

Figure I.7. Molecular structure of PEDOT:PSS (taken from 45

)......................................................... 12

Figure I.8. Scanning electron microscope images of (a) micropatterned polystyrene substrate with

groove dimensions 16/13/4μm (groove width/ groove spacing/groove depth). (b) On micropatterned

substrate AHPCs were aligned in the direction of the grooves in contrast to the non-patterned

substrates (c) where AHPCs were oriented randomly (taken and adapted from 69

). ............................ 15

Figure I.9. Tissues exhibit a range of stiffness, as measured by the elastic modulus, E. Native

brain is soft ( E~500 Pa) compared with precalcified bone (E ~Pa)(taken and adapted from 75

). .... 16

XX

Figure I.10. NSCs transplantation-based therapy. NSCs can be isolated from different tissues and

when they are transplanted they proliferate, migrate and differentiate leading to functional recovery.

Nevertheless, some transplanted NSCs will die due to hostile environment in the injured brain. ........ 19

Figure II.1. The four-point probe setup. A high impedance current source supplies a current through

the outer two probes and a voltmeter measures the voltage across the inner two probes in order to

determine sample resistivity. ................................................................................................................. 21

Figure II.2. Spin coating process. In this process an excess of solution is placed onto the center of

substrate, which is spinner at high speed in order to spread the fluid. ................................................. 23

Figure II.3. Schematic illustration of the electric field setup used in the present study to

stimulate cells. A layer of 50 nm of gold (yellow) was evaporated on the glass substrate and, after

that, PEDOT:PSS (blue) was spin coated on top. This substrate works as an electrode and was kept

parallel to a gold plate positioned above this, that was used as another electrode. Using an external

AC power supply was possible to generate a perpendicular electric field between the electrodes…..25

Figure II.4. Setup to prepare flat hydrogels. A) The gel-glass composite includes the amino-

silanated coverslip, polymerizing solution and glass slide. B) The polymerizing solution was added to

the glass slide and the amino-silanated coverslip with the treated side down was placed on top of it

105…………………………………………………………………………………………………………………26

Figure II.5. A scheme of PDMS double casting method. A) A mixture of PDMS was spread on a

piece of transparent vinyl disc. B) Curing reaction was carried out at 65° for 2h. C) Peeling. D e E)

Another mixture of PDMS was spread on the stamp obtained. F) Peeling of new stamp. G) Stamp with

a replica of the vinyl disc pattern……………………………………………………………………………...27

Figure III.1. Scheme of the research strategy followed in this project. ......................................... 33

Figure III.2. Conformational change in PEDOT after treatment with EG and DBSA.This

conformational change results in an increase of the intrachain and interchain charge-carrier mobility,

so that the conductivity is enhanced. Taken from 107

. ........................................................................... 34

Figure III.3. Transient current technique apparatus. An external fixed dc potential ‘V’ is applied

across the sample sandwiched between two electrodes (platinum disc on top of surface sample and

platinum wire in edge of sample) and the current is monitored as a function of time............................36

Figure III.4. Current transients in PEDOT:PSS sample in A,B) PBS, C,D) IMDM medium, E,F)

IMDM+10%FBS medium and G,H) IMDM+20% FBS medium after an external dc voltage be applied

across sample. ...................................................................................................................................... 37

XXI

Figure III.5. Images obtained by optical microscopy of L929 fibroblasts incubated with the

liquid extracts of each polymer. ........................................................................................................ 38

Figure III.6. Cytotoxicity assays for P3HT, F8T2, F8T2:PCBM, MEH-PPV, Neutralized Pedot Aldrich,

Pedot Aldrich and Pedot Clevios, following the ISO standards for biomaterials with tissue culture plate

(polystyrene) as negative control and a piece of latex glove as positive control. Triplicates were

performed for each condition. ................................................................................................................ 39

Figure III.7. Conjugated polymers spin-coated coverslips. A) MEH-PPV, B) P3HT, C)F8T2, D)

F8T2:PCBM, E) Pedot Aldrich, F) Neutralized Pedot Aldrich, G) Pedot Clevios and H) Glass coverslip.

............................................................................................................................................................... 39

Figure III.8. Cytotoxicity assay for the direct contact assay. The positive control is a piece of latex

glove and the negative control is the culture plate of the polystyrene. Conjugated polymer-coverslips

(A) were put in contact with cells cultured on polystyrene plate (B). ..................................................... 40

Figure III.9. Fibroblasts morphology at day 2. .................................................................................. 42

Figure III.10. Fibroblasts growth range in gelatin-coated polymers and in polymers without any

coating. All substrates allowed the adhesion and cell growth, being PEDOT:PSS the substrate that

allowed higher cell viability over the days. The coating of gelatin improves cell adhesion. The *

indicates technical problems in detaching of cells from the substrates due to their great adhesion to

the surface polymer. .............................................................................................................................. 43

Figure III.11. Images obtained by fluorescence microscopy of fibroblasts cultured in gelatin-

coated polymers and in polymers without any biological coating (Magnification 100X)…………44

Figure III.12. Images obtained by fluorescence microscopy of fibroblasts cultured in gelatin-

coated polymers and in polymers without any biological coating (Magnification 200X)…………45

Figure III.13. Electric field setup developed to apply vertical alternating electric field during in

vitro cell culture. A layer of 50 nm of gold was evaporated to the glass substrate and, after that,

PEDOT:PSS was spin coated on them (A). This substrate works as an electrode and was kept

parallel to a gold plate (B) positioned above this that was used as another electrode (C). Using an

external AC power supply (D) was possible generate a perpendicular and gradual electric field

between the electrodes………………………………………………………………………………………...46

Figure III.14. Replating of NSCs after 7 DIV under the influence of vertical electric field………..46

XXII

Figure III.15. A) Schematic illustration of the electric field setup used in this experiment to

stimulate cells. Ten layers of PEDOT:PSS were deposited on glass slide. A hollow cylinder in center

of substrate was made using a biocompatible glue in order to stanch the culture system. In order to

enhance the electrical contact, strips of gold with 40 nm of thickness were deposited on two ends of

the central ring which occur the cell culture. Platinum wires were placed in these strips and an external

AC power supply, connected to the electrodes, was used to apply an electric field vector that runs

horizontally. B) Electric field setup. C) Control where no electric field was applied……………………47

Figure III.16. Experimental scheme with the electrical stimulation during the proliferation…….47

Figure III.17. NSCs morphological changes after AC electrical field application over several

days…………………………………………………………………………………………………………….. 48

Figure III.18. SEM images of NSCs on control (no electric field applied) and on

PEDOT:PSS:glass substrates where AC electric field was applied …………………………………49

Figure III.19. Experimental scheme with the electrical stimulation during the NSCs proliferation

and differentiation……………………………………………………………………………………...……..50

Figure III.20. The bright-field images during neural differentiation under electric field and

control condition (no electric field applied)……………………………………………………………..51

Figure III.21. Electrically conductive PEDOT:PSS substrates supported long-term maintenance

and differentiation of fetal NSCs. Cells were stained with antibodies directed against βIII-tubulin

(Tuj1) in red, GFAP in green and counterstained with DAPI in blue. Fetal NSCs differentiated into

astrocytes (GFAP positive) and neurons (marked with Tuj1) under electric and non-electric conditions.

Images were taken with magnification of 100x……………………………………………………………..52

Figure III.22. Electrically conductive PEDOT:PSS coated glass enable NSCs differentiation and

neuronal growth. NSCs differentiation on PEDOT:PSS coated glass without electric stimulation are

shown for comparison. A) Immunofluorescence images of anti-Tuj1 (red) and anti-GFAP (red) followed

by DAPI (blue) staining for the nuclei of the fetal NSCs on PEDOT:PSS coated glass. Magnification is

200X. B) ImageJ software quantification of cells showing positive immunostaining for Tuj1 in NSCs at

day 7 of differentiation…………………………………………………………………………………………53

Figure III.23. Qualitative assessment of the AC electric field on the neurite length measured

from differentiated fetal NSCs……………………………………………………………………………... 54

XXIII

Figure III.24. Influence of substrate stiffness on the spreading of NSCs. NSCs adhere fully on flat

and grooved polyacrylamide (PolyA) substrates with stiffness equals to 10 kPa. Cells seem to align on

grooved PolyA gels in contrast to the cells cultured on flat hydrogels that seems randomly oriented..55

Figure III.25. Influence of different substrate stiffness on the spreading of NSCs……………….57

Figure III.26. Constructs based on replica molding technique. A) PDMS stamp with patterns

similar with to those of vinyl disc. B) PEDOT:PSS spin-coated PDMS. C) Au layer deposited in PDMS

in order to improve conductivity………………………………………………………………………………58

Figure III.27. A) SEM image (top) showing the micropattern on vinyl surface. B) PEDOT:PSS spin-

coated vinyl disc. The PDMS cylinder was created to stanch the culture system and two platinum

wires were used as electrodes………………………………………………………………………………. 58

Figure III.28. NSCs spreading on PEDOT:PSS spin-coated-vinyl disc…………………….………..59

Figure III.29. A) Schematic illustration of vinyl disc pattern and its dimensions. B) Electric field setup.

C) Experimental scheme with the electrical stimulation during the NSCs proliferation and

differentiation. D) Pulsed electric field during in vitro cell

culture………………………………………………………………………………………….…………….…..60

Figure III.30. Effect of electric field stimulation on cell number in proliferating cultures……….60

Figure III.31. PEDOT:PSS vinyl substrates supported NSCs adhesion. NSCs nuclei

counterstained with DAPI and SEM images of NSCs on control (no electric field applied) and on

PEDOT:PSS:glass substrates where AC electric field was applied……………………………………....61

Figure VII.1. AlamarBlue fluorescence calibration curve for high numbers of NSC (microplate

reader parameter: gain 89)………………………………………………………………………………..…73

XXV

List of Tables

Table 1. Molecular structure of MEH-PPV, P3HT, PCBM and F8T2. ................................................... 14

Table 2. Expected modulus of elasticity after polymerization of various acrylamide and bis-acrylamide

concentrations 105

. ................................................................................................................................. 26

Table 3. Primary antibodies used in immunocytochemistry. ................................................................. 30

Table 4. Secondary antibodies used in immunocytochemistry. ............................................................ 31

Table 5. Conductivity values obtained for different PEDOT:PSS films. ................................................ 35

Table 6. Electrical conductivities - PEDOT:PSS films before and after DCA treatment and baked at

different temperatures………………………………………………………………………………………….36

Table 7. NSCs elongation under AC electric field and in control condition (no electric field applied).48

Table 8. Elongation of NSCS and NSCs nuclei cultured on flat and grooved PolyA hydrogels. .......... 56

1

Aim of Studies

Over the last 20 years, there have been several breakthroughs in the development of stem cell to be

used in investigation and treatment of neurodegenerative diseases. In the traditional cell culture plate

the cells are forced to make dramatic changes to their morphology. Gene expression mediated

changes to the cytoskeleton result in a flattened cell morphology, and these changes can impair

cellular functions. However, advances in materials science, bioengineering, and microfabrication

technologies are allowing the development of in vitro microenvironments that better model the

complex spatial, mechanical, and chemical conditions of in vivo environments. In order to circumvent

these limitations, researchers have tested several natural and synthetic polymers and have modeled

topography, surface chemistry, electricity and stiffness of the material in order to get a substrate with

similar properties of the extracellular matrix, the natural in vivo niche for stem cells. Organic

conjugated polymers are a new class of polymers that have gained attention in the sense that they

respond to light and electrical stimuli and their molecular structure can be tuned to minimize toxic

effects. The ability of the PEDOT:PSS to sustain the growth and differentiation of ReNcell VM

progenitor cells was assessed during this work. The ReNcell VM cells is a human cell line which

undergo differentiation into neurons and glial cells.

One disadvantage of the photolithography, the most popular microfabrication technique used to mimic

the topography of the in vivo cell niche, is its cost. In this study, we chose a vinyl disc to be used as

substrate to cell culture since it is a cheap material and has an adequate grooved- pattern to align

cells.

The first task of this work was to assess the toxicity of all selected conjugated polymers using L929

cell line for material ISO cytotoxicity tests. The second task was to design conjugated polymer-based

structures to provide electrical and patterned stimuli to cell culture. Replica molding and microcontact

printing were the methods used for fabricating topographically patterned substrates for use as

engineered microenvironments for cells. The patterns were obtained using a vinyl disc as mold.

Therefore, cells were grown on substrates with topographic patterns with micron-scale features.

The final task was to test the effect of material constructs on ReNcell VM cells behavior such as cell

adhesion, expansion and differentiation, confirming the effect of topography in cell morphology and

organization.

The improvement of the platforms for cell culture is essential not only for a better understanding of the

cell behavior in vivo, but also to contribute to the design of scaffolds for tissue replacements.

3

Chapter 1

I. Introduction

I.1. Tissue engineering and stem cells

Stem cells, scaffolds and growth-stimulating signals are the key components of the tissue engineering,

whose principal aim is to develop biological substitutes to restore, replace or regenerate defective

tissues 1. Stem cells are distinguished from the other cells because they have the ability to divide

producing copies of themselves (self-renewal) and/or to differentiate into mature cells that have

characteristic shapes and specialized functions, such as nerve cells 2. Embryonic and adult tissues are

sources of stem cells. Stem cells present in adult tissues, which undergo very limited cellular

regeneration or turnover, form only a limited number of cell types contrary to the stem cells of the

mammalian embryo that have the potential to form any cell type 3. Due to these differences, embryonic

stem cells are considered pluripotent cells and adult stem cells are multipotent cells.

Various types of stem cells, such as induced pluripotent stem cells (iPSCs), embryonic stem cells

(ESCs), and neural stem cells (NSCs) have been established and isolated for regenerating and

repairing defects in various organs and tissues 4. In the current work the human adult neural stem cells

(NSCs) will be studied. These are interesting cell sources for cell therapies, providing models for drug

screening and offering hope for the treatment of several diseases, namely in the case of a stroke,

Alzheimer´s disease, amyotrophic lateral sclerosis and other neurodegenerative diseases 5.

I.2. Neural stem cells NSCs are multipotent cells present in the central nervous system (CNS), that are able to self-renew

and generate neurons, astrocytes, and oligodendrocytes 6. Since the 1960s adult neurogenesis, a

lifetime process by which neurons and glial cells are generated, has been known 7 and this genesis

has been documented in the subventricular zone (SVZ) of the lateral ventricles and the subgranular

zone (SGZ) of the dentate gyrus of the hippocampus 8. The occurrence of this process and the

activation of endogenous stem cells 9 and exogenous NSCs are very important once these offer

possible sources for neural repair and NSC-based treatment of neurodegenerative disorders 10

. The

endogenous NSCs can be induced to proliferate, migrate and differentiate into specific neuronal

populations that can be integrated into host tissue and repair damaged tissue by enhancing

neurogenesis and host axonal growth 11

. Nevertheless, at injury sites in the brain, the transplanted

NSCs lack cell-matrix interactions 12

and may undergo anoikis, that is a form of programmed cell death

which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix

(ECM), and/or apoptosis which can coincide with unfavorable cellular processes such as inflammation

and ischemia 11

. Oxidative stress, hypoxia and inflammatory responses are hostile factors that

transplanted or endogenous NSCs are exposed to and that reduce therapeutic efficacy of these cells

in the brain lesions 11

.

http://en.wikipedia.org/wiki/Programmed_cell_death

http://en.wikipedia.org/wiki/Extracellular_matrix

4

I.2.1. Isolation of NSCs The NSCs populations can be isolated from in vivo sources, the fetal and adult nervous systems, or in

vitro derived from ESCs (- derived from blastocyst stage) or from iPSCs (- derived from reprogrammed

cells)10,13,14

. This type of cells has been tested in experimental models of neurodegenerative diseases

and neurological disorders. The limited supply of human fetal tissue and ethical concerns about

deriving neural progenitors from aborted fetuses, have limited the use of fetal neurons in clinical

applications thus making ESCs and iPSCs attractive alternatives. The ESCs have the potential to

divide indefinitely in culture, producing large quantities of cells for research or therapy although some

defects such as adaptive chromosomal changes frequently appear in these cells15

. In an iPSCs-based

therapy the cells are obtained from patient's own tissues eliminating the risk of immunological

complications and they can be subjected to genetic modifications to correct genetic mutations in order

to allow these cells to generate specific neuronal types for treating neurodegenerative conditions16

.

However, these cells and the ESCs may form tumors, and safety cannot yet be guaranteed. A novel

possibility exists to generate neurons from other adult cells such as fibroblasts via direct

reprogramming without the intermediate iPSCs state which eliminates the necessity of having neural

progenitors, opening very novel avenues for restorative medicine 16

.

Adult NSCs were first found in the rodent striatum 17

and subsequently in the SVZ and in the

embryonic mammalian forebrain during neonatal development 18

. During and after gastrulation,

mammalian neurogenesis starts with the process of neural induction that allows the formation of

neuroectoderm 13

. Neuroectoderm forms the neural plate and, after neurulation, occurs the formation

of the neural tube 14

. These structures have a heterogeneous and complex population of cells called

neuroepithelial progenitors (NEPs) 13

. NEPs, radial glia (RG), basal progenitors (BPs) and adult

progenitors are the main types of cells that can be distinguished in the brain. NEPs undergo

symmetric division that gives rise to new neuroepithelial cells and, at a later stage of brain

development, they give rise to RG and BP by undergoing asymmetric division 14

. RG serve as

scaffolds for migrating newborn neurons and undergo symmetrical proliferative and asymmetric

neurogenic divisions. In addition, RG cells are able to generate glial, neuronal and oligodendroglial

lineages 14

. BPs are considered neurogenic transit-amplifying progenitors once they increase the

production of neurons 14

. Adult progenitors are cells present in neurogenic region (SVZ, SGZ) and in

non-neurogenic regions (spinal cord), which maintain the neurogenesis and gliogenesis throughout

adult life 10

.

Protocols based in in vivo neurogenesis have been performed in vitro to differentiate ESC into NSC,

enabling the generation of a range of distinct neural precursor populations that are similar to the

populations from stage-specific transitions (blastocyst, neural plate, neural tube, fetal and adult brain)

13. The developmental stages of NSC populations isolated or generated in vitro and in vivo are

illustrated on Figure I.1. In order to allow stable in vitro maintenance, cells need to be immortalized, a

procedure that blocks the progression of developmental programs by pushing the cells to remain in

enduring proliferation13

.

5

In the present work, the human neural progenitor cell line ReNcell VM, derived from 10-week-old fetal

ventral mesencephalon and immortalized by retroviral transduction with the v-myc oncogene was

used.

I.2.2. NSCs expansion NSCs and their progeny are often isolated and expanded using the neurosphere culture system.

Reynolds and Weiss demonstrated that NSCs cultured as floating spherical aggregates

(neurospheres) 5 can be stimulated to proliferate when they are exposed to growth factors, like

epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2), remaining multipotent 17

.

Neurosphere culture systems have some limitations like the heterogeneity that occurs within and

between neurospheres, waste accumulation in the cluster center and nutrient and oxygen diffusion

limitations. In addition, the cells expanded as neurospheres differentiate much more readily into

astrocytes than in neurons in vivo 5. Neurospheres contain NSCs and progenitors mixed with

differentiated cells embedded in a complex ECM 19

, so their application as a quantitative in vitro assay

for measuring NSC frequency is limited. As an alternative, to circumvent most of these limitations,

adherent neural stem (NS) cell lines can be derived from ESCs or from the fetal forebrain 20

. The

derivation and expansion of NS cells occurs in adherent monolayer culture where ESCs are

differentiated into neural progenitors 5. The expansion of a progenitors subset occurs in a defined

basal media with EGF plus FGF-2 20

and, over several passages, the culture acquires a homogenous

morphology, maintains the capacity to generate neurons, astrocytes and oligodendrocytes 19

, shows

self-renewal capacity and express nestin, sex determining region Y-box 2 (SOX2) and sex determining

region Y-box 1 (SOX1), characteristics similar to radial glia 5. In order to evaluate the differentiation of

cells into different neural phenotypes, the cells are immunostained for specific markers: nestin,

microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) for neurons, glial fribrillary acidic protein

Figure I.1. The different NSC populations that can be obtained in vitro correspond to stage-

specific neural progenitors present at defined in vivo developmental stages (taken and

adapted from 14

).

6

(GFAP) for astrocytes and NG2 and adenomatous polyposis coli (APC) for oligodendrocytes 21

. The

neurospheres and monolayer NSCs culture procedures are illustrated in Figure I.2.

In this experimental work, ReNcell VM progenitor cells are expanded on laminin coated T25 cm2 tissue

culture flasks in Dulbecco’s modified Eagle’s medium DMEM:F12 in conjugation with N2 supplement,

B27 neural cell supplement mix, glucose, penicillin, streptomycin and insulin, in the presence of FGF-

2 and EGF as described by Donato et al. (2007) 22

. The ReNcell VM line is an immortalized hNSCs

line derived from the ventral mesencephalon of ten-week gestation fetal neural tissue. The

immortalization using the myc transcription factor serves to extend the normal life span of fetal-derived

hNSCs since their major limitation is the high rate of mortality when they grow in culture.

ReNcell VM line has a normal karyotype and proliferates indefinitely, showing a positive signal for the

NSCs marker nestin. When the growth factors are omitted from the medium, these cells undergo

differentiation in neuronal and glial direction, showing a strong labeling for the neuronal marker Tuj1

and for the dopaminergic marker tyrosine hydrolase (TH). The ReNcell VM neurons are functional

neurons since they have the ability to generate action potentials. However, this cell line is purely used

for research and cannot be used in human therapy. This line can be used for the understanding of

stem cells and might ultimately lead to the design and selection of future cell lines for the treatment of

neurodegenerative diseases 22

.

Figure I.2. Sources and culture system to NSCs (taken from 14

). NSCs derived from ESCs

or iPS cells and from germinative areas of the fetal and human brain can grow in monolayer or

in neurospheres. Culture as a monolayer results in a higher neurogenic potential comparing to

neurospheres since, in monolayer culture systems, there are a homogenous composition of

NSCs.

7

I.3. Stem niche Schofield (1978) proposed the term “niche” to describe the particular cellular microenvironment in

which stem cells are kept after embryonic development and that provides the appropriate milieu to

support the survival as well as (in the adult CNS) the production of actively dividing precursors leading

to the generation of post-mitotic progeny 13,23,24

. Figure I.3., illustrates the factors that are important to

regulate stem cell fate within the niche. The cell function and fate (migration, differentiation, mitosis)

are regulated by the constraints of the architectural space, by the signaling interactions at the interface

of stem cells, by the paracrine and endocrine signals from local or distance sources, by the neural

input and by the physical engagement of the cell membrane with tethering molecules on neighboring

cells14,25

.

Figure I.3. Inputs that regulate stem-cell function in the niche. Several factors are important to regulate stem

cell fate within the niche: interactions between cell and neighboring differentiated cells, interactions between cells and growth factors and physicochemical nature of environment

25.

I.3.1. NSC niche After embryonic development, stem cells are kept in a niche to produce new cells. The niche provides

microenvironmental cues essential to the balance between stem cell quiescence and proliferation and

to directs neurogenesis versus gliogenesis lineage decisions. 23

. The maintenance of the germinal

characteristics of the niche and the keeping of the stem cells within this is due to the connection

between stem cells and their somatic neighbors 23

.

In the adult brain, the SVZ and SGZ are well-characterized germinal regions that support

neurogenesis and the place where NSCs reside. The adult rodent SVZ is located in the lateral walls of

the lateral ventricles 10

and contains four basic cell types: ependymal cells, SVZ astrocytes, transitory

amplifying progenitor cells and neuroblasts10

. The ependymal cells mobilize the cerebrospinal fluid

(CSF) and modulate the SVZ proliferation 23

. SVZ astrocytes that express GFAP, after asymmetric

division, give rise to transit-amplifying progenitor cells that proliferate rapidly generating neuroblasts

that migrate to the olfactory bulb and differentiate into neurons 26

.The human SVZ is quite different

from that of rodents because it has a hypocellular gap that separates astrocyte ribbon from the layer of

8

ependymal cells 10

and has a lower number of proliferating cells and neurons compared to the rodent

SVZ23

. SGZ is restricted to the thin subgranular zone near the dentate gyrus hilus and within this zone,

astrocytes divide to generate intermediate precursors (D cells) which divide in order to produce

immature granule neurons 27

. The most structural difference between these two neurogenic niches,

illustrated in Figure I.4., is the absence of ependymal cells on SGZ being a zone with neuronal

progenitors with restricted self-renewing capacity 28,29

.

In addition to the restraints imposed by niche cytoarchitecture, cells within the neurogenic niche rely

on growth factor signaling, cell to cell contact and cell to ECM interactions for homeostatic cell

turnover and increased cell production in response to stimulation.

I.3.2. Signaling in the NSC microenvironment: molecular aspects The NSC and neural progenitors behavior is regulated by the interactions of cells with the ECM

components. The ECM gives structural support for cells to attach, grow, migrate and respond to

signals, contributes to the mechanical properties (elasticity and rigidity) of tissues, provides bioactive

(a) Adult rodent NSCs niche (b) Adult human NSC niche

Figure I.4. Differences between the cytoarchitectural organization SVZ in (a) adult rodent and

(b) adult human neural stem cells niches (taken from 10,

23

). In adult human SVZ the neurons (A),

the astrocytes (B), the microglia (C), the oligodendrocytes (D), the ependymal cells (E) and the blood

vessels (F) interact with each other. The human SVZ have a hypocellular gap that separates astrocyte

ribbon from the layer of ependymal cells and has a lower number of proliferating cells and neurons

compared to the rodent SVZ (not shown). The most structural difference between the SVZ and SGZ is

the absence of ependymal cells on SGZ.

9

cues to regulate the activities of residing cells, is a reservoir of growth factors and provides a flexible

physical environment to allow neovascularization and remodeling in response to tissue dynamic

processes such as homeostasis and morphogenesis1. NSCs have cell adhesion molecules (CAMs)

located on their surface through which they interact with other cells and the ECM components

surrounding them 30

. Integrins, immunoglobulins, selectins and cadherins are families of CAMS that

interact with the glycoprotein tenascin C (TnC), chondroitin sulfate proteoglycans (CSPG), heparin

sulfate proteoglycans, collagen IV, fibronectin, laminin and thrombospondin (ECM components) 31,32

and that play an key role in maintenance of the architecture and shape of NSC niche and that are

involved on signaling transduction that regulate cell survival, proliferation, differentiation and

migration30

. The blood vessels, the CSF and cells within niche can originate growth factors, such as

vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF), EGF and FGF-

2, which play an essential role in regulation of NSC proliferation and self-renewal 10,23,31

. The release

of neurotransmitters, like serotonin and dopamine, by axonal inputs of local and distant origins that

innervate neurogenic region also play an essential role in the regulation of precursors at different

stages of stem cell lineage 27

.

I.4. Biomaterials for NSC culture Biomaterial scaffolds combined with bioactive factors and multipotent cells have been used in tissue

engineering strategies to, after transplantation into damaged tissue, promote tissue development and

function improvement. This strategy for neural repair and regeneration is illustrated in Figure I.5. A

biomaterial is defined as material which is used in body and that by contacting with biological

components of proteins, cells and due to their physicochemical properties, such as wettability, electric

charge, roughness and surface modification with chemical immobilization of CAMs, is able to affect

the adhesion, proliferation and differentiation of cells33,34

.

Figure I.5. Neural repair and regeneration strategy based on

immobilization of growth factors on a biomaterial scaffold (taken and

adapted from 34

). The NSC are seeded on the scaffold and transplanted to

injury tissue to promote neurogenesis.

10

Materials of natural and synthetic origin have been shown to promote adhesion, proliferation and

neuronal differentiation of neural cells in vitro and in vivo. Natural biomaterials such as collagen,

chitosan, fibrin, cellulose, hyaluronic acid and gelatin possess desired cell recognition sites to control

cell behavior, provide cues for stem cells and tend to be biocompatible, biodegradable and

inflammation resistant 35

. Limitations of these proteins and polysaccharides include the immunogenic

response, since the body recognizes the foreign material and attempts to destroy it and the possible

loss of biological activity during their processing. Furthermore, they have insufficient mechanical

properties and also have problems with purity and the availability of large-scale sources 32,33,36,37

. Lu

et al. (2012) developed a chitin-alginate microfibrous culture system that controls the neuronal

differentiation and maturation of human pluripotent stem cells due to the manipulation of medium

components38

. Synthetic polymer-based biomaterials have a defined chemical composition and have

the advantage of controlling the mechanical properties, shape and degradation rate allowing the

release or display of neurotrophic factors33,36,39

. However, the byproducts of these materials, that are

chemically modified to sustain cell adhesion, are inflammation-active and change the environmental

pH to a lower level than normal which is harmful to seeded cells and surrounding tissues33,36,37

.

Synthetic substrates such as polydimethylsiloxane (PDMS), polystyrene, polyacrylamide (PolyA),

poly(D,L-lactide) (PLA), polydopamine (PD), poly(ethylene glycol) (PEG), hydrogels and self-

assembled protein constructs have been used to culture and differentiate of NSC11,12,40,41

.

The ideal neural scaffold should be biocompatible, biodegradable, resistant to structural collapse

during implantation and should have a porous connectivity to allow tissue vascularization and cell

migration, and also a three dimensional shape with mechanical, electrical, biochemical and

topographical properties to mimic the ECM 35

.

I.4.1. Conjugated polymers Several tissues are responsive to electrical fields and stimuli which has made electrically conductive

materials an attractive approach to control cell state and fate via regulation of conductivity, mechanical

properties, volume and oxidation states42

.

Conjugated polymers (CP) are molecular materials whose backbone is made of alternating double-

and single- bonds. These result from the bonding between sp2

hybridized atoms (mostly carbon), so

that the overlap of pure p-orbitals of neighboring atoms leads to the formation of π molecular

orbitals43

. It is this special conjugation that enables electron delocalization along the polymer chains

and is at the origin of their conductive properties44

in contrary to the traditional polymers that are made

up of essentially σ-bonds and hence a charge once created on any given atom on the polymer chain is

not mobile. When in the pure state, CP behave very much like inorganic semiconductors as there is an

energy gap separating the bonding π orbitals from the anti-bonding π* molecular orbitals. Only upon

doping, that corresponds to an oxidation or a reduction of the polymer chains, their conductivity can be

changed from insulating to conducting, with the conductivity increasing as the doping level increases.

In some special cases, conductivity values that approach those of the metals can be obtained. CPs

are easy to synthesize, by chemical or electrochemical polymerization reactions, and to process and

are inexpensive. Various methods such as electropolymerization, spin coating and inkjet printing, have

11

been used to deposit and pattern this type of polymers. In this study the spin coating method was

selected to deposit polymers on substrates. These polymers have attractive properties for biological

applications. Namely, they are able to transfer charge from a biochemical reaction, they are usually

biocompatible, allow the control over the level and duration of electrical stimulation for tissue

engineering applications and possess the ability to entrap and controllably release biological

molecules43

. The ability to modify their chemical structure, namely by adding functional groups to the

CPs backbone, and/or their ability to be combined with various organic compounds, opens the

possibility to easily control and modulate their electrical, physical, chemical and other properties in

order to better suit the nature of the specific applications 43

.

Figure I.6. illustrates the most commonly investigated conjugated polymers, namely polypyrrole

(PPy), polyaniline (PAni), polythiophene (PTh) and its derivatives, such as the widely common poly

(3,4-ethylene dioxythiophene) or (PEDOT). These polymers can be doped with small salt ions,

peptides, proteins and neurotrophins 45

.

I.4.2. Polypyrrole The first CP studied in the biomedical field was PPy due to its high electrical conductivity, ease of

synthesis, biocompatibility and ability to release drugs or growth factors.

PPy has show the ability to promote growth of primary neurons, mesenchymal stem cells (MSCs) and

NSCs when doped with immobilization factors such as extracellular matrix cell adhesion proteins or

peptides42

. Schmidt et al. (1997) showed that neuron-like pheochromocytoma PC12 cells attach and

extend neurites on PPy films due to increased protein adsorption from serum containing media after a

steady potential of 100mV for 2h has been applied through the medium 46

. One limitation is that this

effect only extends over the first 24h of growth and stimulations performed beyond this time frame

showed no significant difference to PPy films without electrical stimulation and polystyrene, used as

control 47

.Zhang et al. (2010) cultured human embryonic stem cells (hESCs) and adult rat neural stem

cells (rNSCs) on PPy substrates doped with polystyrenesulfonate (PSS) or with laminin fragments p20

and p3148

. These authors concluded that PPy/PSS promotes a poor attachment of hESCs unlike

Figure I.6. Structures of conducting polymers commonly studied

for biomedical applications (taken from 102

).

12

PPy/p31 that promote cell adhesion and PPy/p20 neuronal differentiation. In case of rNSCs the

PPy/p20 enhanced neuronal differentiation and neurite outgrowth48

. Lundin et al. (2011) investigated

the biocompatibility of NSCs of fetal and embryonic origin with PPy substrates containing four anionic

dopants of varying chemical character and molecular weight: chloride (Cl-), tosylate (TsO

-), perchlorate

(ClO4-) and dodecylbenzenesulfonate (DBS

-)

49. The best suited system for long term maintenance as

well for NSC differentiation along neural lineages was found to the PPy(DBS), in contrast to the others

counter ions that were not compatible for NSC culture 49

.

I.4.3. Poly(3,4-ethylenedioxythiophene) PEDOT is, probably, the most promising candidate for long-term implantation in the central nervous

system because, when compared to other conductive polymers such as PPy, possesses several

advantageous properties: it combines a moderate energy gap located at the transition between the

visible and near-IR regions of the spectrum and a low oxidation potential with good stability in the

oxidized state 45

and has superior thermal and electrochemical stability 50

. Nevertheless, PEDOT is

insoluble in many solvents and unstable in its neutral state, as it oxidizes rapidly in air. So, in order to

circumvent the problems of solubility of PEDOT, PSS is used as counter ion during polymerization of

PEDOT (illustrated in Figure I.7.), giving rise to a water-soluble polyelectrolyte, PEDOT:PSS with a

good film forming properties, high conductivity, stability and high visible light transmissivity45

.

Herland et al. (2011) used heparin as counter ion of PEDOT resulting in a biocompatible and stable

polymer 51

. FGF2 is a growth factor that can bind to heparin and this binding regulates intracellular

signaling pathways that maintain NSCs in proliferative and undifferentiated state. This study showed

that when FGF2 is anchored on neutral PEDOT through heparin, NSCs proliferate and remain in an

undifferentiated state 51

. In contrast, when PEDOT is oxidized, there is an increase of NSCs

differentiation and a decrease of proliferation due to the decrease of the bioavailability of the bound

FGF 2

51. Strategies such as adsorption, covalent tethering and entrapment allow the immobilization

and incorporation of biomolecules on conductive polymers improving cell attachment. The long-term

neuronal survival and growth was possible due to electro-adsorption of polylisine (PLL) on

Figure I.7. Molecular structure of PEDOT:PSS (taken from 45

).

13

PEDOT:PSS surface50

. Neural cell development was strongly inhibited by an additional layer of PSS

or heparin, which could be coated with spermine to restore the cell growth 50

.

Ostrakhovitch et al. (2012) proved that substrate conductivity is a key factor to induce differentiation of

NSCs. These authors tested the ability of PEDOT blended with polyethyleneglicol (PEDOT:PEG),

PEDOT doped with polystyrene sulfonate (PEDOT:PSS), electrodeposited PEDOT, poly(3-

hexylthiophene) (P3HT), poly(2,20-bithiophene) (PBT) and indium-tin oxide (ITO) to sustain mouse

NSCs and P19 pluripotent embryonal (P19 EC) carcinoma cells differentiation. They concluded that

only substrates with conductance higher than S/cm2, such as electrodeposited PEDOT,

PEDOT:PEG and ITO, sustain the NSCs and P19 cells differentiation without requirement of external

stimulation such as retinoic acid and poly-L-ornithine 42

. During this work, strategies such as the

addition of compounds with two or more polar groups, like ethylene glycol into an aqueous solution of

PEDOT:PSS and the exposure of PEDOT:PSS films to dichloroacetic acid were used to enhance

the conductivity of PEDOT:PSS films.

Richardson-Burns et al. (2007) used 0.5-1 μA/mm2 galvanostatic current to electrochemically deposit

PEDOT around neurons cultured on electrodes concluding that neurons partially embedded within the

polymer matrix are viable for at least 120h following polymerization but PEDOT-surrounded neurons

began to die by apoptosis 52

. Neuron-templated PEDOT coatings were generated by removal of the

cells from the PEDOT matrix after polymerization around the cells. Such coatings could encourage

cells in the host tissue to re-populate the cell-shaped PEDOT once the intimate contact between cells

and the conductive polymer is established allowing a possible continuous electrical contact between

the electrode and the tissue 52

.

Srivastava et al. (2013) tested the differentiation of mouse embryonic stem cells derived neural

progenitors (ESC-NP) into neurons on stretched and electrified PEDOT:PSS coated styrene-ethylene-

buthylene-styrene (SEBS) substrates concluding that the increase of strain on conducting polymers

promotes a decrease in ESC-NP differentiation into neurons and affects the cell distribution

comparatively to unstrained PEDOT:PSS coated substrates 53

. The cells aggregated on PEDOT:PSS

coated SEBS substrates differentiated less in neuronal cells once they have a actin cytoskeleton

disrupted in contrast to the differentiated neurons on non-conducting SEBS substrates that have actin

fibers uniformly arranged 53

.

PEDOT has been used as coating of neural electrodes and nano-fiber electrodes due to its high

charge delivery capacity leading to very low impedance and highly effective charge transfer, which

results in higher signal-to-noise ratios for improved electrophysiological recordings, a more effective

interface for constant current stimulation 54

and an excellent substrate to regulate adhesion,

proliferation and signaling of neuronal cells 55

. Microelectrode arrays coated with PEDOT:PSS were

successfully used to obtain electrical recordings during rat NSC development, contributing to

understand the NSC differentiation conditions 56

.

I.4.4. Other conjugated polymers Poly(2-methoxy-5(2’-ethyl)hexoxy-phenylenevinylene) (MEH-PPV) is a conducting polymer that has a

low conductivity due to its low hole and electron mobility and that allows the immobilization of

14

biomolecules without use of any additional reagent due to high density of hole-traps 57

. This polymer

has been used in electronic applications, such as light-emitting diodes (LEDs) and photovoltaic

devices and in biosensors 57,58

.

Poly(3-hexylthiophene-2,5-diyl) (P3HT) is a hole-conducting polymer which has been used in organic

solar cells, field effect transistors and in LEDs 59

. Ostrakhovitch et al. (2012) concluded that P3HT

substrates do not support mouse NSCs differentiation due to it lower conductivity (~

S/cm2) showing that conductivity is a key factor to regulate cell fate

42. A research group in Italy grew

primary hippocampal neurons onto spin-coated regio-regular P3HT blended with phenyl-C61-butyric-

acid-methyl ester (rr-P3HT:PCBM) onto a glass pre-coated with ITO, observing no differences in

viability of cells up to 28 days in vitro (DIV) with respect to control glass substrates covered with ITO

and poly-L-lisine (PLL) only 60,61

. This group also showed that P3HT:PCBM coated on ITO, after

photo-stimulation, is able to raise electrical activity in primary neurons grown on top of it 61

. In another

study, Ghezzi el al. (2013), seeded blind retinas from albino rats on P3HT-coated ITO concluding that

a 10 ms light pulse (4 mW/mm2) is able to stimulate the ganglion cell layer at the same levels as those

of control retinas of normal rats 62

. These studies suggest that these organic devices can play an

important role in subretinal prosthetic implants.

Poly (9,9-dioctylfluorene-alt-bithiophene) (F8T2) is a fluorene-based air stable aromatic polymer that is

a promising material for organic thin film transistors (OTFT), biosensors and photovoltaic cells,

showing typical p-type semiconducting behavior 63,64 ,65.

In this study, P3HT, F8T2 and MEH-PPV, illustrated in Table 1, are tested as possible substrates for

cell culture.

Table 1. Molecular structure of MEH-PPV, P3HT, PCBM and F8T2.

15

I.5. Surface strategies to control cell response The classic cell culture conditions provide a homogenous adhesion substrate which is flat and rigid

having little in common with the in situ microenvironment in which cells reside 66

. Researchers have

modeled topography, surface chemistry, electricity and stiffness of the material in order to get a

substrate with properties similar to those of ECM.

I.5.1. Topography In vivo cells are adherent to ECM, which provides chemical, mechanical and topographical cues, at

micro- and nanoscale, controlling in this way the cell behavior and functionality. The developments in

nanotechnology allowed the production of precise patterns on material surfaces for in vitro cell culture.

Photolithography, microcontact printing, photoresist lift-off and microfluidic techniques have been used

to produce patterned structures 67

since the discovery that topography influences the adhesion,

proliferation and differentiation of the cells. The phenomenon that mediates the interaction and

response of cells with these topographies and, consequently, affects cellular behavior such as

adhesion, migration and differentiation is known as contact guidance 68

. Recknor et al. (2006)

fabricated microgrooves on a polystyrene substrate (illustrated on Figure I.8.) with 16×13×4 μm

(groove width, mesa width, groove depth) and coated them with laminin and with astrocyte monolayer

in order to test the synergistic effects of these cues on adult rat hippocampal progenitor cells (AHPC)

growth and differentiation. These authors concluded that the micropatterned substrates direct the

AHPC alignment in groove direction and facilitate its differentiation into cells that acquire neuronal

morphology, comparatively to the cells that were not exposed to physical and biological guidance cues

69. In order to evaluate the differentiation of human neural progenitor cells (hNPCs) isolated from fetus

cortex, Blong et al. (2010) coated micropatterned polystyrene microgrooves, which had the same

dimensions as those prepared by Recknor et al. (2006), with entactin, collagen and laminin (ECL).

The patterns allowed the growing, alignment and migration of hNPCs. However no differences in

proliferation or differentiation potential was observed in comparison with hNPCs culture on the

nonpatterned substrates 70

.

(a) (b) (c)

Figure I.8. Scanning electron microscope images of (a) micropatterned polystyrene substrate with groove

dimensions 16/13/4μm (groove width/ groove spacing/groove depth). (b) On micropatterned substrate AHPCs were aligned in the direction of the grooves in contrast to the non-patterned substrates (c) where AHPCs were oriented randomly (taken and adapted from

69).

16

Béduer et al. (2011), made PDMS grooves with dimensions widths comparable to cell dimension (~ 20

μm) and functionalized them with PLL or with double wall carbon nanotubes getting an optimum cell

density, differentiation rate and neurite alignment 71

. In another study, Béduer et al. (2012) engineered

fibronectin and laminin-functionalized - PDMS substrates with different terraces (t) and grooves (g)

widths in order to investigate whether size of these microchannels influence the cell adherence,

differentiation and neurite outgrowth on the polymer surface. These authors demonstrated that the

smallest structures (t = 5 μm and g = 5 μm) induce a perfect alignment of neurites in a specific

direction but reduces the differentiation rate, as opposed to wider micropatterns (t = 20 μm - g = 20 μm

and t = 10 μm – g = 60 μm) that allows a intercommunication between neurons and a high rate of cells

differentiation into astrocytes or neurons although the neurite alignment is altered and neurites tend to

escape out of their “guiding” microchannel 72

.

Nevertheless, the mechanism by which microtopography cues influence cell proliferation and

differentiation it not well known but seems to involve changes in the protein adsorption on substrates,

cellular cytoskeletal organization and structure in response to the geometry and size of the underlying

features of the ECM. The clustering of integrin and other cell adhesion molecules is influenced by

feature size of the substrate that, subsequently, affect the number and distribution of focal adhesions

responsible for cell-ECM attachment and adhesion-mediated signaling 73

.

I.5.2. Stiffness Stem cell fate or lineage commitment can depend on the mechanical properties of a surrounding or

underlying environment. The relationship between mechanical stiffness of ECM and the contractile

forces resulting of the cells adherence to substrates, have an influence on migration, apoptosis and

proliferation of cells 74

. Figure I.9., illustrates the different stiffness of each tissue in the body, where

native brain is soft (E’~ 1kPa) compared with muscle (E~ Pa) and bone (E~ Pa) 75

.

Engler et al. (2006), grew mesenchymal stem cells (MSCs) on collagen I-coated- PolyA gels and

concluded that alteration of the stiffness of substrates defines the differentiation lineage of the MSC.

On soft substrates that mimic brain elasticity MSC were found to be neurogenic, on substrates of

intermediate stiffness that mimic muscle were myogenic and on stiff substrates with bone-like

properties found to be osteogenic 76

.

Saha et al. (2008), grew adult NSC on synthetic hydrogel culture system with ranging moduli between

10 and 10.000 Pa. These authors concluded that soft substrates (~10 Pa) inhibit cell spreading, self-

renewal and differentiation, whereas substrates with moduli of 500 Pa promote cell proliferation and

Figure I.9. Tissues exhibit a range of stiffness, as measured by the elastic modulus, E. Native brain is soft (

E~500 Pa) compared with precalcified bone (E ~Pa)(taken and adapted from 75

).

17

exhibit peak levels of neuronal markers such as β III tubulin 77

. The neuronal differentiation is

promoted by softer substrates (E~ 100-500 Pa), whereas glial differentiation is promoted by stiffer

substrates (E~1-10 kPa 77

).

I.5.3. Electrical stimulus All cells of human body respond to electrical fields (EF) but nerve cells are specifically designed to

transmit electrical impulses from one site of the body to another and to receive and process

information implying that an ideal neural scaffold should possess electrical conductivity to promote

neurite outgrowth and enhance nerve regeneration in culture. The ion influx across membrane, the

membrane potential and the intracellular signal transduction pathways are affected by the unequal

distribution of ions between the intra- and extracellular compartments which, subsequently, affects cell

behavior. The standing voltage gradients of the extracellular milieu are crucial to cell adhesion,

migration and differentiation 78

. Modalities such as micro electrode arrays, conductive polymers, static

and continuous direct current (DC) EF and electromagnetic fields have been used to electrically

stimulate cells 79

. Several studies have reported that extracellularly applied EF, on the order of 1 V/cm,

promotes neurite outgrowth cathodically 78,80 ,81 ,82 ,83, 84,85

. Wood et al. (2009, showed that different

surface coatings, cell culture mediums, growth supplements and EF magnitude and application time,

influence the growth of chick embryonic dorsal root ganglia (DRG) neurites 86

. Yamada et al. (2007),

reported that colonies of embryonic bodies (EBs) that received 10 V stimulation assume specifically a

neuronal fate compared to controls (no electrical stimulation received) that have a low differentiation

efficiency. The resulting neuronal cells have potential to differentiate into various types of neurons in

vivo and, when injected in blastocysts of adult mice, they contribute to the injured spinal cord as

neuronal cells 87

. The influence of pulse EF and alternating current fields on neural stem cells behavior

has been studied. Kimura et al. (1998) planted PC12 cells on ITO electrodes and subjected them to a

rectangular peak-to-peak pulse wave potential of 100 mV, with frequency of 100 Hz, for different

times. These authors showed that a potential application for more than 60 min is harmful to cells but a

good rate of differentiation was obtained when an electric potential was applied for 30 min every 24h,

repeated 3 times 88

. Park et al. (2009) cultured PC12 cells on gold nanoparticles deposited on

polyethyleneimine (PEI) - precoated surfaces and evaluated its response to a pulsed and constant

electrical stimulation of 250 mV for 1 h once every 3 days, concluding that alternating current

stimulation promotes a better viability of cells (~ 90% viable cells) comparatively to the constant

current stimulation (~70% viable cells) 89

. Chang et al. (2011) used a biphasic electrical current

stimulator chip to stimulate fetal NSCs at 100 Hz with a magnitude of 4,8,16 or 32 μA/cm2 and duration

of 50 or 200 μs for 4 days 90

. The rate of proliferation increased when a current density of 4 or 8

μA/cm2 was applied for 200 μs, at 100 Hz, not being affected by an electrical stimulation with relatively

higher amplitudes or lower pulse durations. The current density of 4 μA/cm2 for 200 μs increases

neural differentiation of fetal NSCs 90

.

New therapeutic strategies based on nanotechnology and functional biomaterials having the ability to

induce EF must be developed in order to control proliferation, differentiation and guidance of

regenerating neurons and implanted neural stem cells.

18

I.5.4. Surface functionalization Several strategies such as adsorption, entrapment and covalent attachment of desired biomolecules

have been used by the researchers in order to improve the biological properties of a CP to enhance

the adhesion and growth of cells. One of the key adhesion ligands for NSC growth and survival is the

laminin (LN). LN is a protein that is part of the ECM and that has “arms” that, upon association with

other LN molecules, form sheets and bind cells. The LN protein is constituted by specific amino acid

sequences such as arginine-glycine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine

(IKVAV) and tyrosine-isoleucine-glycine-serine-arginine (YIGSR) that are responsible to guide cell

adhesion and neurite outgrowth 32, 91,92

. Integrins are transmembrane αβ heterodimers that, after LN

bind on their receptors, suffer a change of conformation which in turn, triggers intracellular signals that

synergistically act with growth factors in order to regulate self-renewal and maintenance of NSCs

through mitogen- activated protein kinases (MAPK) signaling pathway 93, 94,95

. The growth factors

added to culture medium or secreted by NSCs are chemical messengers that mediate intracellular

communication that, as referred before, affect cell fate 75

. The EGF and FGF2 are examples of these

growth factors that act on regulation of the several genes required to sustain self-renewal and

differentiate potential of NSCs in vitro 14

. However their action on cell fate is dependent on the cell

origin, the stage of differentiation and on the concentration, time and duration of exposure used 96,97

.

The withdrawal of EGF and FGF2 or the addition of differentiation factors to the medium induces the

NSCs to spontaneously acquire a neuronal or glial phenotype 75,96,97,98

. Fibronectin, heparin, retinoic

acid, hyaluronic acid and nerve growth factor (NGF) are examples of others proteins used to

functionalize polymeric substrates. However, their adsorption is influenced by chemical structure,

surface energy, surface topography, roughness and hydrophobicity of polymer surface.

I.6. NSCs applications

In neurodegenerative disorders (ND) such as Alzheimer´s disease, Huntington´s disease, amyotrophic

lateral sclerosis (ALS) and Parkinson’s disease, there is a massive loss of one or several types of

neurons that promotes chronic or progressive decline in cognitive function affecting the memory,

behavior, language, learning and emotion 99,100

. According to the DEMENTIA - A Public Health Priority

report, annual, is estimated that the number of new cases of people with dementia is about 7.7 million,

that is, a new case every 4 seconds. Current therapies for ND alleviate only poorly the symptoms once

they are inefficient to rescue or regenerate cellular function or even halt the neuronal death process.

Stem-cell transplantation provides a novel and attractive strategy for these diseases that remain

without effective therapy. The intravenous injection or the local transplantation of NSCs isolated from

different sources have been used as strategies to treat severe injuries in CNS once, in these

situations, the endogenous repair is not enough for functional recovery. The administration of growth

factors, drugs and antioxidants is another strategy used to modulate the proliferation, migration and

differentiation of endogenous NSCs to increase their efficacy in neurogenic areas of the brain.

19

Figure I.10. NSCs transplantation-based therapy. NSCs can be isolated from different tissues and when they

are transplanted they proliferate, migrate and differentiate leading to functional recovery. Nevertheless, some transplanted NSCs will die due to hostile environment in the injured brain.

The transplanted NSCs can cooperate with other cells in the brain to activate endogenous NSCs of

niche and also secrete trophic factors and modulate the immune system providing neuroprotection 101

.

NSC have been used in pre-clinical and clinical trials in order to promote the neural regeneration. In

2009, the first pre-clinical trial was authorized in rats that used spinal cord grafts of oligodendrocyte

precursors derived from hESCs to restore function102

.

The NSCs can be used as in vitro models to screen possible drug candidates to treat some diseases,

to study disease mechanisms and test drug toxicity in way to avoid toxic and side effects due to high

dosages103

.

The challenges for regenerative medicine future are the determination of ways to promote survival and

integration of neural precursor transplants in the adult brain and the study of the mechanisms to

reverse behavioral and cognitive deficits in ND104

.

21

Chapter 2

II. Materials and Methods

II.1. Conducting polymers Different conjugated polymer solutions were prepared and used to make thin films by spin coating. Sample 1: Cross-linked PEDOT:PSS solution

The ethylene glycol (Sigma-Aldrich) was added in a volume ratio of 1:4 to PEDOT:PSS (Heraeus,

CLEVIOS P AI 4083, solids content 1.3-1.7%). Dodecylbenzenesulfonic acid (DBSA) (0.5μL/mL) and

3-glycidoxypropyltrimethoxysilane (GOPS) (10 mg/mL) were added to the solution to improve film

formation and stability.

Sample 2: Neutralized PEDOT:PSS solution

The ethylene glycol was added in a volume ratio of 1:4 to PEDOT:PSS. Then, the crosslinker GOPS

(20mg/mL) was added to improve film stability. Sodium hydrogen carbonate (NaHCO3) was added

until neutral pH.

Sample 3, 4, 5: P3HT, MEH-PPV, F8T2 The poly (3-hexylthiophene) (P3HT) and poly-(2-methoxy-,-5-(2’-ethyl-hexyloxy)-p-phenylenevinylene)

(MEH-PPV) were dissolved in chloroform and the poly(9,9-dioctylfluorene-alt-bithiophene) (F8T2) was

dissolved in xylene to be used in the preparation of thin films.

II.2. Conductivity measurement of PEDOT: PSS films Before the conductivity measuring, PEDOT:PSS was spin coated on square glass substrates and four

strips of gold with 50 nm of thickness were evaporated ( Edwards Coating System E 306A) on top, as

illustrated in Figure II.1.

Figure II.1. The four-point probe setup. A high impedance current source supplies

a current through the outer two probes and a voltmeter measures the voltage across the inner two probes in order to determine sample resistivity.

22

The thicknesses of the films were measured using a Dektak 3.21 profilometer. The four-point probe

method and the Labview program were used to find the films resistance and resistivity.

II.3. Current transient technique In the current transient technique, the electrode potential is abruptly changed from 0 to 1V and the

resulting current variation is recorded as a function of time. In a second experiment the electrode

potential is changed from 1 to 0 V and the current variation is recorded. The PEDOT: PSS dispersion

was spin coated at 1800 rpm (60s) onto square glass and this substrate was immersed in different

culture media such as phosphate buffered saline (PBS), Iscove's Modified Dulbecco's Medium

(IMDM), IMDM supplemented with 10% of fetal bovine serum (FBS) and IMDM supplemented with

20% FBS in order to evaluate the current variation in each condition.

II.4. Cytotoxicity assays Cytotoxicity assays in vitro were performed according to the ISO 10993-5:2009(E) guidelines in order

to assess the biocompatibility of materials. Materials were exposed directly and indirectly in contact to

the cell culture system (L929 mouse fibroblast cell line), allowing the production of reproducible

results. For indirect contact assays the PEDOT: PSS substrates were sterilized with UV during 1h30

min and they were immersed in 3mL of pen strep (antibiotic solution) during 2h. The others materials

were sterilized with ethanol overnight. Triplicates for each material were placed on 6-well plates

containing 2 mL of IMDM 10% with 10% (v/v) of FBS and kept in an incubator (37ºC, 5% CO2, fully

humidified) for 72 hours. The L929 fibroblasts were seeding in 24-well plate, at an initial density of

8×104 cells/cm

2, and were incubated with liquid extracts of materials for 24h. The cell proliferation was

analyzed using the cell proliferation reagent WST-1 (Roche) that is a colorimetric assay that is based

on the cleavage of a tetrazolium salt, MTS, by mitochondrial dehydrogenases to form formazan in

viable cells. The greater the number of viable, metabolically active cells, the greater the amount of

formazan product produced following the addition of WST-1. The detection of the formazan level in the

cells allows the quantification of the cell number. The results are normalized to the negative control

(cells that were cultured with IMDM 10% medium that was not in contact with any material) and

compared with positive control (IMDM 10% medium with latex pieces).

In order to evaluate the effect of the direct interaction between materials and L929 fibroblasts, the

materials were placed in contact over the cells in 12-well plates (11,7×104 cells per well) with IMDM

10% (v/v) of FBS and kept in an incubator (37 ºC, 5% CO2, fully humidified) for 48h. After the

incubation period, cells were observed under an inverted fluorescence microscope (Leica DMI 3000B,

Germany) in order to qualitatively evaluate if they are confluent or formed a halo of inhibition.

23

II.5. Spin coating Spin coating is a procedure used to prepare uniform thin films on flat substrates. In this process, as

illustrated on Figure II.2., an excess of a solution is placed on the substrate, which is then rotated at

high speed in order to spread the fluid by centrifugal force. The film thickness is controlled by

adjusting the polymer solution concentration and the spinning speed. In general, higher spinning

speeds lead to thinner films. PEDOT: PSS, P3HT, F8T2 and M3H-PPV are deposited by spin coating.

Before the deposition by spin coating, the 2 cm2 lamellas

are cleaned with isopropanol, dried with

nitrogen and, particularly prior to the deposition of the PEDOT:PSS, surfaces are exposed to oxygen

plasma (PlasmaPrep2, GaLa Instrument) to remove organic compounds from the sample and to

increase the hydrophilicity of the surface. The aqueous solutions of PEDOT:PSS was spin coated

(Spin-Coater KW-4A, Chemat Technology) on lamellas at 1800 rpm (60s) and post annealing ( 150°, 2

min) films with ~ 90μm thickness was obtained. The others polymers were spin coated on lamellas at

1500 rpm suffering the same annealing process. The annealing process allow the clearance of

residual traces of organic solvents (isopropanol, acetone that are toxic for biological systems) in order

to prepare films to cell culture and also enhances the morphology of polymeric film.

These lamellas were used on cytotoxicity assays and on adhesion tests in order to assess the toxicity

and cell growth on polymers, respectively.

Conjugated polymer

solution

Substrate

Rotating

plate

Vacuum

Figure II.2. Spin coating process. In this process an excess of solution is placed

onto the center of substrate, which is spinner at high speed in order to spread the fluid.

http://en.wikipedia.org/wiki/Thin_film

http://en.wikipedia.org/wiki/Substrate_(printing)

http://en.wikipedia.org/wiki/Solution

http://en.wikipedia.org/wiki/Centrifugal_force

24

II.6. Adhesion test The coverslips were clean with isopropanol and dried with a nitrogen stream. The solution of F8T2

was spin coated at 1500 rpm (60s) on surface of the coverslips. The others coverslips had to be

exposed to oxygen plasma prior the PEDOT:PSS spin coating at 1800 rpm (60s) in order to enhance

the hydrophilicity of their surface. The coverslips with PEDOT: PSS and the coverslips with F8T2 were

sterilized with UV for 1h30min and with ethanol overnight, respectively. The coverslips were glued on

24-wells ultra-low attachment plate and a solution of pen-strep (1:100) in PBS was added in order to

enhance the sterility of materials. Two conditions were tested: polymers spin coated on coverslips and

coverslips with polymers and 0,1% gelatin adsorbed. In every experiment there were duplicates of

each condition. A positive control was done, plating cells in glass coverslip with and without gelatin.

The coverslips with polymers were incubated (37ºC, 5% CO2, in a fully humidified environment) with

IMDM containing 10% (v/v) FBS for one hour. After that, cells were plated at 15000 cells/cm2 in IMDM

+ 10% FBS.

II.7. Constructs Given that combining biomaterial scaffolds with some anisotropy and electrical conductivity coupled

with NSCs may provide a novel strategy for the treatment of neurodegenerative diseases, some new

setups constructs were designed in order to test their ability to sustain the growth and differentiation of

this type of cells using cheap techniques such as replica molding and microcontact printing.

II.7.1. Conductive substrates

II.7.1.1. Perpendicular electrical field setup The scheme of the electric field setup developed to apply a perpendicular pulsed electric field during in

vitro cell culture is shown in Figure II.3. The PEDOT:PSS coated glass substrate that supports the

cells was used as an electrode and the gold plate positioned above the substrate was used as another

electrode. Both electrodes were kept parallel to each other and a perpendicular electric field was

generated between the electrodes. An external AC power supply (Digital Sweep Function Generator

8150, Topward), connected to the electrodes, was used to apply the electric field. Previously to the

seeding of cells, the substrate was sterilized with UV for 1h30min and with pen-strep (1:100) in PBS

during 2h.The substrates were incubated with poly-L-Ornithine for 30 minutes, at 37°C and 5% CO2

humidified environment, washed once with PBS and incubated for 4 hours with laminin (20μg/mL,

Sigma-Aldrich®)) diluted in PBS. Cells were plated at an initial cell density of 230 000 cells/cm2

in

DMEM medium supplemented with EGF (20ng/mL), FGF (20ng/mL) and B27 (20μg/mL).

25

II.7.1.2. Longitudinal electrical field setup Square glass slides were cleaned with isopropanol, dried with nitrogen and exposed to oxygen plasma

(3min) previously to the spin coating of PEDOT:PSS at 1300 rpm (60s). Ten layers of PEDOT:PSS

were deposited making an annealing at 130º during 15 min after each deposition. A hollow cylinder

was glued on the substrate with a biocompatible glue FDA approved (Silastic® medical adhesive

silicone, type A) in order to stanch the culture system (see Figure III.15). In order to enhance the

electrical contact, strips of gold with 40 nm of thickness were evaporated on each end of the central

ring which contains the cell culture. Platinum wires (Sigma-Aldrich®) were placed in these strips and

an external AC power supply, connected to the electrodes, was used to apply an electric field vector

that runs horizontally. Previously to the seeding of cells, the substrate was sterilized with UV for 1h

and with a solution of Antibiotic-Antimitotic overnight. The substrates were incubated with poly-L-

Ornithine for 30 minutes, at 37°C and 5% CO2 humidified environment, washed once with PBS and

incubated for 3 hours with laminin (20μg/mL, Sigma-Aldrich®)) diluted in PBS. Cells were plated at an

initial cell density of 140 000 cells/cm2

in DMEM medium supplemented with EGF (20ng/mL), FGF

(20ng/mL) and B27 (20μg/mL).

II.7.2. Anisotropic and conductive substrates

II.7.2.1. Polyacrylamide gels

II.7.2.1.1. Preparation of amino-silanated coverslips

A solution of 0.1M NaOH (70μl) was added to 2cm

2 coverslips and these were heated (80°) until the

solvent evaporates. Then, 40 μl of aminopropyltriethoxysilane (APES; Sigma-Aldrich®) was added for

5 min and the coverslips were immersed in distilled H2O (~10mL) to completely rinse off the unreacted

APES. The coverslips were immersed in a solution of 0,5% glutaraldehyde (Sigma-Aldrich®) in PBS

for 30 min and then they were left drying naturally in air.

II.7.2.1.2. Preparation of flat hydrogels

In order to test the effect of substrate stiffness on cell behavior, different elastic moduli substrates

were prepared varying the relative concentration of acrylamide and bis-acrylamide The acrylamide

Figure II.3. Schematic illustration of the electric field setup used in the present study to stimulate cells. A layer of 50 nm of gold (yellow) was

evaporated on the glass substrate and, after that, PEDOT:PSS (blue) was spin coated on top. This substrate works as an electrode and was kept parallel to a gold plate positioned above this, that was used as another electrode. Using an external AC power supply was possible to generate a perpendicular electric field between the electrodes.

26

(Sigma-Aldrich®) (40%) and bys-acrylamide (Sigma-Aldrich®) (2%) were mixed in the desired

concentrations in distilled H2O according with Table 2 and the solution was degassed in an excicator

for 15 min. A volume of 1/100 (v/v) of ammonium persulfate (APS, Sigma-Aldrich®) and 1/1000 (v/v)

of tetramethylethylenediamine (TEMED, Sigma-Aldrich®) was added to the solution. The solution was

stirred in the vortex and 50μl of the final solution was added to the glass slide and the amino-silanated

coverslip, with the treated side down, was placed on top of it (procedure illustrated on Figure II.4.).

The gel was polymerized inside the excicator and an unused solution was used to monitor the

polymerization degree. At the end of the polymerization, the glass slide with the gel and the amino-

silanated coverslip was immersed in water to discard the glass slide and the hydrogels were stored at

-4°C in a Petri´s dish with distilled H2O 105

.

Table 2. Expected modulus of elasticity after polymerization of various acrylamide and bis-

acrylamide concentrations 105

.

Acrylamide (ml)

Bis-acrylamide

(ml)

Water (ml)

E ± St. Dev. (kPa)

0.75 0.3 8.95 0.48 ± 0.16

1.25 0.75 8 4.47 ± 1.19

2.5 0.5 7 10.61

II.7.2.1.3. Microcontact printing: hydrogels with grooves In microcontact printing the mold is "inked" with the material to be transferred to the substrate and

contact is made between the substrate and the protruding features of the mold. Thus, the material is

transferred from the mold to the substrate and the substrate is patterned as desired. This technique

was used to create grooves in PolyA gels using a vinyl disc as model. The acrylamide (Sigma-

Aldrich®) (40%) and bys-acrylamide (Sigma-Aldrich®) (2%) were mixed to the desired concentrations

in distilled H2O, the solution was degassed and a 1/100 (v/v) of APS and 1/1000 (v/v) of TEMED were

added to the solution. The solution was stirred in the vortex and 50μl of the final solution was added to

the piece of vinyl disc and the amino-silanated coverslip with the treated side down was placed on top

of it. After the polymerization, the piece of vinyl disc with the gel and the amino-silanated coverslip

was immersed in water to release the piece of vinyl disc and the hydrogels were stored at -4°C in a

Petri´s dish with distilled H2O 105

.

A B

Figure II.4. Setup to prepare flat hydrogels. A) The gel-glass composite

includes the amino-silanated coverslip, polymerizing solution and glass slide. B) The polymerizing solution was added to the glass slide and the amino-silanated coverslip with the treated side down was placed on top of it 105

.

27

II.7.2.1.4. Functionalization of polyacrylamide hydrogels A two-step zero-length crosslinking procedure was used to functionalize the PolyA hydrogels

106.

Laminin (Sigma-Aldrich®)(20μg/ml in PBS) was incubated, at room temperature, for 15 minutes with 2

mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Sigma-Aldrich®) in the presence of 5 mM

N-hydroxysuccinimide (sulfo-NHS; Sigma-Aldrich®). The reaction is stopped by the addition of β-

mercaptoethanol and 1mM NaOH was added to raise the pH. The PolyA hydrogels were incubated

with this final solution during 2 hours at room temperature106

.

II.7.2.1.5. Sterilization of polyacrylamide hydrogels Hydrogels were put in 24 wells ultra-low attachment cell culture plates (Dow Corning®) and were

sterilized with a solution of Antibiotic-Antimitotic (Gibco®) for 2 hours.

II.7.2.1.6. Seeding cells on hydrogels The hydrogels were washed with PBS twice to remove any traces of the antibiotic-antimitotic solution

and the cells were plated at initial density of 140 000 cell/cm2 in DMEM medium supplemented with

EGF(20ng/mL), FGF (20ng/mL) and B27 (20μg/mL). In every experiment there were triplicates of each

condition.

II.7.2.2. Replica molding The replica molding is a process that transfers a pattern from a rigid or elastomeric mold into another

material by way of a liquid solidifying when in contact with the mold. A 10:1 ratio of elastomer

monomer: curing agent (Sylgard 184 silicone elastomer, Dow Corning®) was thoroughly mixed and

degassed in vacuum (Heraeus Vacutherm, Thermo Scientific) for 30 min. The mixture was spread on

a piece of transparent vinyl disc and the curing reaction was then carried out at 65° for 2h. The piece

of transparent vinyl disc was previously cleaned with isopropanol and dried with a nitrogen stream.

Next, the obtained stamp was placed in an oven for thermal aging (100º) during 48h. Then, this stamp

is used as a secondary master and another stamp is made from it. The Figure II.5. illustrates the

scheme of replica molding process used during this strategy. The second stamp was exposed to a

oxygen plasma (3 min) and an aqueous solution of PEDOT:PSS was spin coated on top at 1800 rpm

(60s).

A B C D

E F G

Figure II.5. A scheme of PDMS double casting method. A) A mixture of PDMS was

spread on a piece of transparent vinyl disc. B) Curing reaction was carried out at 65° for 2h. C) Peeling. D e E) Another mixture of PDMS was spread on the stamp obtained. F) Peeling of new stamp. G) Stamp with a replica of the vinyl disc pattern.

28

II.7.2.3. Transparent vinyl disc In the first experience, the transparent vinyl disc was cut into circles with 9 cm

2 and these were

washed with isopropanol and dried with nitrogen stream. The circles of vinyl were exposed to an

oxygen plasma (3min) and a aqueous dispersion of PEDOT:PSS was spin coated at 1800 rpm (60s)

followed by an annealing (150°, 2min). In order to ensure that the cells would grow just on the surface

of vinyl disc, a PDMS cylinder was created to confine the culture system. The PDMS cylinder was

glued to the surface of vinyl circles using Araldite®. The circles of vinyl disc were put on 6-wells cell

culture plate (Dow Corning®) and sterilized with UV during 1h30 min and with a solution of pen-strep

(1:100) in PBS during 2h. The vinyl circles were incubated with poly-L-Ornithine for 30 minutes, at

37°C and 5% CO2 humidified environment, washed once with PBS and incubated for 4 hours with

laminin (20μg/mL, Sigma-Aldrich®)) diluted in PBS. Cells were plated at an initial cell density of 140

000 cells/cm2

in DMEM medium supplemented with EGF (20ng/mL), FGF (20ng/mL) and B27

(20μg/mL).

In the second experience, the transparent vinyl disc was cut into circles with 9cm2 and these were

wash with isopropanol and dried with nitrogen stream. The circles of vinyl were exposed to a oxygen

plasma (3min) and ten layers of PEDOT:PSS were deposited making an annealing of the 130º during

15 min between each deposition. A hollow cylinder was made using biocompatible glue in order to

stanch the culture system and strips of gold with 40 nm of thickness were evaporated on each end of

the central ring which contains the cell culture. Platinum wires were placed in these strips and an

external AC power supply, connected to the platinum wires, was used to apply an electric field vector

that runs parallel to the grooves.These substrates were immerse in isopropanol during three days and

then in ethanol for 1 day. The circles of vinyl disc were put on 6-wells cell culture plate and sterilized

with UV during 1h30 min and with a solution of Antibiotic-Antimitotic during 2h. The substrates were

incubated with poly-L-Ornithine for 30 minutes, at 37°C and 5% CO2 humidified environment, washed

once with PBS and incubated for 3 hours with laminin (20μg/mL,) diluted in PBS. Cells were plated at

an initial cell density of 140 000 cells/cm2

in DMEM medium supplemented with EGF (20ng/mL), FGF

(20ng/mL) and B27 (20μg/mL).

II.8. SEM analysis To characterize the cells subjected to the longitudinal electric field, the cells were fixed with

glutaraldehyde 1,5% (v/v) in PBS at 37° for 1h. The samples were washed three times with PBS and,

after that, the samples were immersed in ethanol solutions at different concentrations, 25%, 50%, 75%

and 99% for 30 min each at 37°. Before the observation of the substrates they were coated with a

Au/Pd layer of 30 nm using a Polaron model E5100 coater (Quorum Technologies). Images were

obtained using a Field Emission Gun Scanning Electron Microscope (FEG-SEM) (JEOL, JSM-7001F

model).

29

II.9. Cell culture

II.9.1. Cell line ReNcell VM (Millipore®) is an immortalized human neural progenitor cell line that was derived from the

ventral mesencephalon region of a human fetal brain tissue and that was immortalized by retroviral

transduction with the v-myc oncogene. This cell line has the ability to differentiate into neurons and

glial cells 22

.

II.9.2. NSCs thawing The cells were thawed from liquid nitrogen and the frozen vials were submerged in a 37ºC water bath,

and resuspended in DMEM/F12 (Life Tecnologies). The mixture was centrifuged for 3 min at 1000

rpm, and the supernatant was discarded from the mixture. The cells were resuspended in the

expansion medium: DMEM/F12 supplemented with 20 ng/mL EGF (Prepotech), 20 ng/mL FGF-2, 1%

N2 supplement (Life Tecnologies), 20 μl/mL B27 supplement (Life Tecnologies), 20 μg/mL additional

insulin (Sigma), 1,-6 g/L additional glucose (Sigma) and 1% penicillin/streptomycin. The Trypan Blue

(Gibco®) exclusion method is used to determine cells number and viability. This process was carried

out fast to avoid losing cell viability.

II.9.3. NSCs expansion In order to expand cells, RenCell VM were platted on T-flasks (Falcon®, BD Biosciences) that had

been previously incubated with poly-L-Ornithine (Sigma) for 30 minutes, at 37°C and 5% CO2

humidified environment, washed once with PBS and incubated for 4 hours with laminin (20μg/mL,

Sigma) diluted in PBS. The T-flasks with cells were incubated at 37ºC and 5% CO2 in a humidified

atmosphere using DMEM/F12 medium. The medium was changed every 2 days until cells reached an

80-90% confluence. They were then harvested using Accutase (Gibco) by incubation at 37ºC and 5%

CO2-humidified atmosphere for 3-5 min. Then, the cell suspension was diluted with DMEM/F12 and

centrifuged at 1000 rpm for 3 min. The pellet was resuspended in an expansion medium and cells

number and viability was determined through Trypan Blue exclusion method using a hemocytometer

under an optical microscope (Olympus).

II.9.4. NSCs cryopreservation After expansion, the remaining cells were dissociated using accutase, centrifuged for 3 minutes at

1300 rpm and resuspended in culture medium with 10% (v/v) dimethyl sulfoxide (DMSO). Then, they

were transferred to cryogenic vials for storage at - 80ºC. The freezing in liquid nitrogen at -196 ºC is

used for cryopreserve cells for long time.

30

II.9.5. NSCs differentiation The withdrawal of growth factors EGF and FGF2 and the culture medium change to a 1:1 mixture of

Neurobasal media (Invitrogen, Life Technologies) supplemented with B27 (20μl/mL) and DMEM/F12

supplemented with 1% penicillin streptomycin (Invitrogen), 1% N2 Supplement (Invitrogen), 20μg/mL

insulin (Sigma), 1,.6 g/L glucose (Sigma) induces differentiation. The differentiation process was

carried out for 7 days, changing the medium every 3 days.

II.9.6. NSCs morphology analysis The NSCs nuclei and cytoskeleton were labeled with 4’,6-diamino-2-phenylindole (DAPI, Sigma) and

rhodamine phalloidin probe (Sigma), respectively, in order to analyze the adhesion, morphology and

spreading of these cells on the polymeric substrates. NSCs were fixed with 4% paraformaldehyde

(PFA, Sigma) for 15 min, washed with PBS, and permeabilized with staining solution (0,1% Triton-X-

100 (Sigma) in PBS with 5% Normal Goat Serum (NGS, Sigma)) for 15 min. Secondly, cells were

stained with 300 μL of rhodamine phalloidin probe (1 μL.mL-1 in PBS) for 45 min at room temperature

(RT). After washing once in PBS, cells were incubated in 300 μL of DAPI (1.5 μg/mL in PBS) for 5 min

at 37ºC under a 5% CO2 humidified atmosphere. Finally, cells were twice washed in PBS, kept in PBS,

and protected from light. Blue-stained nuclei and red-stained cytoskeleton were visualized under a

fluorescence optical microscope (DMI 3000B, Leica) and digital images were taken with a digital

camera (DXM 1200F, Nikon).

II.9.6. Immunocytochemistry

Cells were fixed with PFA 4% (2mL per well of a six well-plate) for 30 minutes at room temperature,

and then washed twice with PBS. Cells were incubated for 30 minutes at room temperature with

blocking solution (90%PBS, 10% NGS and 0,1% Triton X) and, after this, the primary antibodies

diluted in staining solution were incubated at 4°C overnight. After the incubation with the primary

antibody, cells were washed once with PBS and incubated with the appropriate secondary antibody for

1 hour at room temperature in a dark container. Finally, cells were washed once with PBS and

incubated with DAPI nucleic acid stain for 2 minutes at room temperature and washed twice with PBS.

The stained cells were visualized under a fluorescence microscope (Leica DMI 3000B), using the

software Nikon-AcT1.

Table 3. Primary antibodies used in immunocytochemistry.

Primary Antibodies Type Source Dilution

Anti-TUJ1 mouse IgG Covance 1:4000

GFAP mouse IgG Millipore 1:100

31

Table 4. Secondary antibodies used in immunocytochemistry.

Secondary

Antibodies Type Source Dilution

Anti-Alexa 546 goat anti mouse IgG Life Technologies 1:500

Anti-Alexa 488 goat anti rabbit IgG Life Technologies 1:500

33

Chapter 3

III. Results and Discussion

III.1. Research strategy

Cell behavior is influenced by a high variety of extracellular factors including chemistry, mechanics,

and geometry. Engineered microenvironments can be used to assess a wide range of such influences

on cell behavior and identify parameters for future implementation into tissue engineered scaffolds.

The main goal of this study consists on the fabrication of conjugated polymers-based constructs that

mimic in vivo cell niche topography and that allow the application of electric fields in order to promote

NSC adhesion, alignment, expansion and differentiation rendering a high number of homogeneous

NSC populations.

Replica molding and microcontact printing were the soft lithography techniques used to make grooves

on substrates using a piece of transparent vinyl disc as mold. The conjugated polymers are highly

versatile semiconducting materials in the sense that they respond to light and electrical stimuli and

their molecular structure can be tuned to minimize toxic effects, making them ideal platforms to test a

variety of stimuli on cells behaviour. This project aims to prepare conjugated polymers-based

structures adequate to provide cues for neuronal stem cell growth and differentiation.

In order to reach this aim, it was followed the research strategy schematized in Figure III.1.

Figure III.1. Scheme of the research strategy followed in this project.

34

III.2. Electrical characterization of polymers

In this study, the electrical properties, namely the conductivity are obtained using the two- and four-

probe techniques.

The four-point probe method relies on the application of a constant current (I) across two outer

electrodes at the surface material and on measurement of the potential between the inner pair of

electrodes. Applying the following equations:

Equation (1)

Equation (2)

where ρ is the surface resistivity, R is the surface resistance, L is sample length, w is the sample

thickness and l the length between electrodes measuring voltage and the conductivity (σ) of the

material.

In the two-probe method a voltage sweep is applied to the PEDOT:PSS films and the current is

recorded with a PC.

The conductivity of PEDOT:PSS films (Aldrich and Clevios) prepared from the as-received are

compared with films prepared with PEDOT:PSS:GOPS and PEDOT:PSS:GOPS:DBSA. Table 5

shows the results obtained for each type of film. The addition of the cross-linker GOPS to the aqueous

dispersion yields an insoluble and more stable PEDOT:PSS film 45

although it leads to a reduction of

the electrical conductivity case of PEDOT Clevios. The addition of DBSA and organic solvent such as

EG to the PEDOT:PSS aqueous solution increases the interchain interaction and change the

structure of PEDOT chain from a benzoid to a quinoid structure, promoting an increase on conductivity

(Figure III.2.)107,108

. The interaction between the dipole of one polar group of the organic compound

and the dipoles or the positive charges on the PEDOT chains can be the driving force for the

conformational change of the PEDOT chains.

Figure III.2. Conformational change in PEDOT after treatment with EG and DBSA.This conformational change results in an increase of

the intrachain and interchain charge-carrier mobility, so that the conductivity is enhanced. Taken from

107.

35

Table 5. Conductivity values obtained for different PEDOT:PSS films.

The PEDOT Aldrich seems to be more conductor comparatively to PEDOT Clevios. The weight ratio

of PEDOT:PSS purchased from Sigma and Clevios was 1:1,6 and 1:6 , respectively. PEDOT

Clevios has a highly excess of PSS in solution which lowers the conductivity of aqueous dispersion.

Although the two-point probe method is capable of calculating the resistivity, the four-point probe

method is superior due to the use of two additional probes that measure the voltage potential of the

material. These probes do not carry any current, thus eliminating the contact resistance (Rc) and the

resistance caused by current flowing into the sample surface (spreading resistance, Rsp) measured in

the two-point probe method.

However, some considerations such as the spacing of the probes, the temperature effects and the use

of correction factors (sample thickness, edge effects, location of the probe on the sample, quality four-

point head) have to be taken in account in order to perform accurate four-point measurements and to

obtain repeatable and reliable resistivity values.

Samples

Pedot

Aldrich

Pedot

Aldrich+

GOPS

Pedot

Aldrich+

GOPS+

DBSA

Pedot

Clevios

Pedot

Clevios+

GOPS

Pedot

Clevios

+GOPS+

DBSA

Cross-section (m2)

Resistance

(kΩ)

2

probe

method

4

probe

method

Resistivity,

ρ

(Ω*m)

2

probe

method

4

probe

method

Conductivity,

σ

(Ω-1

m-1

)

2

probe

method

4

probe

method

36

II.2.1. Dichloroacetic acid treatment Based on a process of secondary doping

109, the PEDOT:PSS films were exposed to DCA in order to

improve their conductivity. The PEDOT:PSS:GOPS:DBSA dispersion was spin coated on glass

substrate at 1000 rpm and baked at 100° for 3 min. Then a DCA solution (99%, Sigma) was heated at

100° for 3min and the polymeric films were agitated in this solution. The treated films were then baked

at elevated temperatures (100° and 160°) for 30 min.

Table 6 shows the electrical conductivities of DCA treated-PEDOT:PSS films baked at different

temperatures. Exposing PEDOT:PSS films to a simple postdeposition solvent annealing treatment

does not improve the electrical conductivity.

Table 6. Electrical conductivities - PEDOT:PSS films before and after DCA treatment and baked at different

temperatures.

Cross-section

(m2)

Resistance

(kΩ)

Resistivity

(Ω*m)

Conductivity

(Ω-1

m-1

)

PEDOT:PSS

Clevios

PEDOT:PSS:DCA

(T=100°)

PEDOT:PSS:DCA

(T=160°)

III.3. Transient ionic current measurements

The transient Current (TC) technique was used to assess the relevance of the overall ionic

conductivity of different biological fluids such as PBS and IMDM medium (Figure III.3.).

In this method, an external fixed dc potential ‘V’ is applied to the liquid “sandwiched” between two

electrodes (platinum disc on top of surface sample and PEDOT:PSS bottom contact) and the current

is monitored as a function of time. Before the application of a potential to the liquid, the charge carriers

are homogenously distributed. The applied ‘V’ polarizes and the electric field forces positive and

negative ions to migrate to the electrodes obtaining a peak current, which decreases with time until it

reaches a steady state. In all conditions tested, the peak current decreases approaching to zero

means that the level of any electronic contribution to the total electrical conductivity must be negligible

(Figure III.4.).

Figure III.3. Transient current technique apparatus. An external fixed dc potential ‘V’ is applied

across the sample sandwiched between two electrodes (platinum disc on top of surface sample and platinum wire in edge of sample) and the current is monitored as a function of time.

37

E)

C) D)

G)

H)

F) E)

B) A)

Figure III.4. Current transients in PEDOT:PSS sample in A,B) PBS, C,D) IMDM medium, E,F) IMDM+10%FBS

medium and G,H) IMDM+20% FBS medium after an external dc voltage be applied across sample. The blue line is the external fixed dc potential ‘V’ applied across the sample sandwiched between two electrodes and the black line is the system response.

38

III.4. Cytotoxic tests with fibroblasts

Indirect and direct cytotoxic tests were performed following the ISO 10993-5 guidelines for medical

devices in order to assess the cytotoxicity of each polymer.

To carry out indirect tests the L929 fibroblasts cells were seeding in 24-wells plate and incubated with

the liquid extracts of the materials that before were been incubated with IMDM supplemented with

10% (v/v) FBS for 48 h. The morphology of cells was observed by optical microscopy (Figure III.5.)

and the cell proliferation was analyzed using the cell proliferation reagent WST-1 (Figure III.6.).

Coverslips (negative control)

Latex glove (positive control)

MEH-PPV

F8T2

F8T2-PCBM P3HT

Pedot (Sigma)

Neutralized

Pedot (Sigma)

Pedot

(Clevios)

Figure III.5. Images obtained by optical microscopy of L929 fibroblasts incubated with the liquid extracts of each polymer.

39

The values of cell metabolic activity obtained in cytotoxicity assays and the morphology of the cells

(Figure III.5.) showed that none of the polymers has cytotoxic effects when compared to commonly

used tissue culture polystyrene (negative control).

When direct assays (Figure III.7.) where performed to evaluate polymers cytotoxicity no inhibition halo

was observed (Figure III.8.) Therefore, these polymers are suitable to promote stem cell adhesion and

proliferation.

A) B) C) D)

E) F) G) H)

Figure III.7. Conjugated polymers spin-coated coverslips. A) MEH-PPV,

B) P3HT, C)F8T2, D) F8T2:PCBM, E) Pedot Aldrich, F) Neutralized Pedot Aldrich, G) Pedot Clevios and H) Glass coverslip.

Figure III.6. Cytotoxicity assays for P3HT, F8T2, F8T2:PCBM, MEH-PPV, Neutralized Pedot Aldrich, Pedot Aldrich and Pedot Clevios, following the

ISO standards for biomaterials with tissue culture plate (polystyrene) as negative control and a piece of latex glove (toxic) as positive control. Triplicates were performed for each condition.

40

Coverslips

(negative control)

Latex

(positive control)

MEH-PPV

F8T2

F8T2-PCBM

P3HT

Pedot (Sigma) Neutralized Pedot (Sigma) Pedot (Clevios)

Figure III.8. Cytotoxicity assay for the direct contact assay. The positive control is a piece of latex glove and

the negative control is the culture plate of the polystyrene. Conjugated polymer-coverslips (A) were put in contact with cells cultured on polystyrene plate (B).

A

A

A

B

A

B

A

B

A

B

A

B

B

B

B

B

41

III.5. Adhesion test with fibroblasts

The ability of CP to stimulate NSCs is limited by the minimal interaction of these materials with

biological tissue. To test the ability of CP to sustain growth of cells, two conditions have been tested:

CP spin-coated onto coverslips and bioactive CP spin coated onto coverslips trough adsorption of

biological protein: gelatin. The two CP chosen to be tested were the PEDOT:PSS Clevios, once it

presented the major cell viability percentage when cytotoxic tests were performed, and F8T2 that was

planned to be used in a planned experiment to test the light effect on behavior of NSCs. The cell line

used was the L929 fibroblasts. In order to have positive and negative controls, the cells were plated

for 4 days in a glass coverslip with and without gelatin and in a non-adherent plate with and without

gelatin, respectively.

The morphology (shape and appearance) of cells inspected by a microscope at day 2 (Figure III.9.),

confirms the healthy status of cells without any signs of contamination or detachment of the cells from

the substrate. Analyzing the images, the gelatin coating seems to increase cell adhesion to

substrates.

Without gelatin With gelatin

F8T2

Pedot Clevios

42

Glass coverslips

Non-adherent plate

Figure III.9. Fibroblasts morphology at day 2.

Analyzing the shape and appearance of cells is possible to confirm the healthy status of cells. The

adsorption of gelatin onto polymer surface seems to increase cell adhesion. As expected the cells do

not adhere to the non-adherent plate showing a spherical morphology.

The trypan blue dye exclusion test was used to count viable and non-viable cells, in each day, in order

to confirm if gelatin improves cell adhesion and proliferation relatively to the polymers without any

coating (Figure III.10.)

Cells adhered and proliferated in all polymers that have been coated or not with gelatin. Results,

which are displayed in Figure III.10, evidence that after 24 h of culture the relative number of cells on

the PEDOT:PSS surface is similar to the number of cells on F8T2 and, furthermore, significantly larger

than the number of cells on the control plate. This indicates that Pedot spin-coated on the coverslips is

an appropriate substrate to support cellular adhesion, its behavior being similar to that of the

extracellular matrix. After 48 and 72h of culture, the cellular activity was re-evaluated, in terms of

proliferation, on the materials using the same procedure. As can be observed in Figure III.10, there

was an increment for the three substrates. Furthermore, the number of cells on the PEDOT:PSS

surface was significantly larger than that on the control culture plate. These results clearly show that

PEDOT:PSS is not cytotoxic and does not have an anti-proliferative effect after a prolonged exposure.

As expected, the gelatin, that is a heterogeneous mixture of water-soluble proteins derived from the

hydrolysis of collagen, improves the attachment and growth of cells in polymers due to their

hydrophilic nature and due to large number of glycine, proline, and hydroxyproline residues that it

contains. The different ligands provided by the gelatin, bound to the surface cell receptors, which

increased the adhesion110

. The cells adhered to the substrates, in such a way that it was difficult to

release them and, consequently, it was not possible to obtain meaningful results for day 4.

43

In order to observe morphological changes of attached cells, these were immunostained at day 4 with

DAPI and Phalloidin that stain nucleus and cytoskeleton, respectively (Figure III.11. and Figure III.12.).


F8T2

*

*

* *

Figure III.10. Fibroblasts growth range in gelatin-coated polymers and in polymers without any

coating. All substrates allowed the adhesion and cell growth, being PEDOT:PSS the substrate that allowed

higher cell viability over the days. The coating of gelatin improves cell adhesion. The * indicates technical

problems in detaching of cells from the substrates due to their great adhesion to the surface polymer.

44

Pedot Clevios

Glass coverslips

Figure III.11. Images obtained by fluorescence microscopy of fibroblasts cultured in gelatin-coated polymers and in polymers without any biological coating (Magnification 100X).


F8T2

Pedot Clevios

45

Glass coverslips

Figure III.12. Images obtained by fluorescence microscopy of fibroblasts cultured in gelatin-coated polymers and in polymers without any biological coating (Magnification 200X).

There are large populations of cells on the substrates containing gelatin, when comparing with

substrates without any coating. The gelatin can regulate the adhesion of cells through signal

transduction pathways between intracellular and extracellular environments 110

.

As a conclusion, these conducting polymers present an excellent behaviour as substrate to support

cellular adhesion and proliferation suggesting its potential role as scaffold for tissue engineering.

III.6. Constructs & NSCs III.6.1. Electric field effect in NSCs III.6.1.1.Perpendicular electrical field setup

Besides chemotactic signals and topography, electric fields can be a source of directional cues in vitro

to control cellular processes including microfilament reorganization, proliferation, differentiation and

migration of NSCs towards specific targets.

When cells are exposed to electric fields there is a redistribution of charged cell-surface receptors and

a charge movement is generated on the outer surface of the cells. Cations such as Na+ will drag along

water, generating an electro-osmotic flow, which asymmetrically redistribute membrane proteins to the

cathode-facing side of cells. The ability of electric fields to control cell shape, the release of signaling

molecules and cell migration, could have potential clinical applications. However, the response to

electric fields is diverse depending on cell type, developmental stage, and species. Electric fields of 1-

2V/cm occur during morphogenesis and wound healing and have been shown to orient the

movements of a wide variety of cells in vitro111

. Taking this into account and also in order to avoid the

electrolysis of water (occurs at 1,2V) that would change pH of medium due to the ions produced or

consumed, the 1V/cm was the magnitude chosen to be applied.

The purpose of this study was to determine whether 1 V/cm vertical alternating current electric field,

applied at 100 Hz using the substrate illustrated in Figure III.13, influences survival and proliferation of

NSCs.

46

The proliferation assays and the NSCs culture were compromised due to some characteristics of

substrates such as the leakage of cells because they were not stanched and also due to its opacity,

making impossible to observe the cells under the optical microscope. After 7 days, cells were re-plate

in a 24-well-plate in order to analyze their viability (Figure III.14).

Analyzing Figure III.14 , it was possible to observe that cells did not adhere, meaning that they were

not viable. Why did this happen? The surface topography and the vertical electric field may have

negatively influenced the laminin adsorption and the interactions that retain the proteins at the surface

of substrates. The absence of strong interactions between proteins and the surface of substrate

causes desorption of biomolecules and, consequently, the failure of cell adhesion.

A B

C D

Figure III.13. . Electric field setup developed to apply vertical alternating electric field during in vitro cell culture. A layer of 50 nm of gold was evaporated to the glass

substrate and, after that, PEDOT:PSS was spin coated on them (A). This substrate works as an electrode and was kept parallel to a gold plate (B) positioned above this that was used as another electrode (C). Using an external AC power supply (D) was possible generate a perpendicular and gradual electric field between the electrodes.

Figure III.14. Replating of NSCs after 7 days in vitro under the influence of vertical electric field.

47

III.6.1.2. Longitudinal electrical field and in vitro culture system

The physiological function of in vitro cultured cells could be altered in response to physical stimuli as

electric field, mechanical force and topography. In this study, we engineered a substrate (Figure

III.15.) that modulates electrical fields on living cells to investigate the effects of square AC electric

field on fetal NSCs proliferation and differentiation processes.

We exposed the cells to a 100 Hz electrical stimulation with a magnitude of 1V/cm with 10 ms pulse

duration in a continuous manner according to the culture schedule shown in Figure III.16.

NSCs adhered and proliferated under applied AC electric field and in control experiment with no

applied electric field (Figure III.17.).

A B C

Figure III.15. A) Schematic illustration of the electric field setup used in this experiment to stimulate cells.

Ten layers of PEDOT:PSS were deposited on glass slide. A hollow cylinder in center of substrate was made using a biocompatible glue in order to stanch the culture system. In order to enhance the electrical contact, strips of gold with 40 nm of thickness were deposited on two ends of the central ring which occur the cell culture. Platinum wires were placed in these strips and an external AC power supply, connected to the electrodes, was used to apply an electric field vector that runs horizontally. B) Electric field setup. C) Control where no electric field was applied.

Figure III.16. Experimental scheme with the electrical stimulation during the proliferation.

Electrical stimulation

Days

Seeding

0 1 2 3 4

Media change

SEM

48

PEDOT:PSS substrates

Condition Without electric field With electric field

Day 1

Day 2

Day 4

Figure III.17. NSCs morphological changes after AC electrical field application over several days.

The elongation is one of the adaptation methods that cells undergo in order to perform certain

functions and through which they connect to adaptor proteins and push their membrane in the

direction of movement. Taking this into account, ImageJ software was used to measure the influence

of the electric field in cell elongation from pictures obtained by SEM (Figure III.18.), since it is an

important parameter to analyze cell migration. Elongation of cells was assessed by measuring the

length of the long axis and short axis, using built-in functions of ImageJ software. At least 30 cells

were evaluated.

49

Without electric field With electric field

Figure III.18. SEM images of NSCs on control (no electric field applied) and on PEDOT:PSS:glass substrates where AC electric field was applied.

50

When cells were placed in a low conducting medium under a non-uniform electric field they suffered

changes in their shape. The AC electric field introduced a mechanical stress in cell, elongating and

stretching more the cells comparatively to control (Table 7).

Table 7. NSCs elongation under AC electric field and in control condition (no electric field applied).

Cells exposed to AC stimulation appear larger and have more lamellipodia, as opposed to their

nonpolarized morphology under normal culture conditions. The implications of AC electric field in

cellular morphology change need further investigations. The charge surface of this conductive

substrate can have affected adjacent ECM or can have attracted or repelled ions or proteins in the

media influencing cell behavior.

III.6.1.3. Differentiation of NSCs on electrically conductive substrate

Native nerve tissue is composed of neurons and glial cells, that provide nutrition and operational

support for the neurons. Thus, the generation of functional nerve tissue from stem cells requires

differentiation down both neuronal and glial lines.

NSC differentiation was studied under applied AC electric fields and compared to control experiments

with no applied electric field. We exposed the cells to a 100 Hz electrical stimulation with a magnitude

of 1V/cm with 10 ms pulse durations in a continuous manner during expansion time and over 12h per

day during differentiation time, according to the culture schedule shown in Figure III.19.

The bright-field images during neural differentiation under electric field and control condition are

illustrated on Figure III.20.

Cell Elongation

Electric field

No electric field

Differentiation

Electrical stimulation

(12h/day)

Days 0

Seeding

1 4 5 8 12

Expansion

Electrical

stimulation

Figure III.19. Experimental scheme with the electrical stimulation during the NSCs proliferation and differentiation.

51

Without electric field With electric field

Day 6

Day 8

Day 10

Day 12

Figure III.20. The bright-field images during neural differentiation under electric field and control condition (no electric field applied).

52

Cells were fixed with 4% PFA after 4 expansion days and 7 days of exposure to AC electric field for

12h/day in the absence of growth factors. Immunofluorescence study was performed using specific

antibodies against Tuj1, which labels neurons in early development as well as mature neurons and

GFAP to identify the differentiated level of astrocytic populations. The fetal NSCs on

PEDOT:PSS:glass substrates under and without electric stimulation, differentiated into neurons and

into astrocytes confirmed by Tuj1 and GFAP immunoreactivity, respectively (Figure III.21).

DAPI Tuj1 Tuj1 GFAP Tuj1 GFAP DAPI

No

electric

field

Electric

field

Adherent

culture

plate

Figure III.21. Electrically conductive PEDOT:PSS substrates supported long-term maintenance and differentiation of fetal NSCs. Cells were stained with antibodies directed against βIII-tubulin (Tuj1) in red, GFAP

in green and counterstained with DAPI in blue. Fetal NSCs differentiated into astrocytes (GFAP positive) and neurons (marked with Tuj1) under electric and non-electric conditions. Images were taken with magnification of 100x.

The results from ImageJ software and from immunocytochemistry seem to indicate that there is a

higher number of Tuj1-positive cells in the electrically conductive PEDOT:PSS:glass substrates

compared to controls (Figure III.22.). Several studies suggest that conductivity of substrate can be an

essential factor for differentiation and determination of cell fate42

. The electrical stimulation influences

the neuronal differentiation, so, when the electrically PEDOT:PSS:glass substrate was placed into an

electrolyte solution, it was able to sustain local electrochemical currents between the substrate and

cell monolayer. These redox reactions between the conductive surface and cell monolayer may have

changed the intracellular distribution of redox couples and, consequently, the intracellular redox

potential, regulating in this way the cell differentiation. Changes in the effective redox potential may

+ - + - + - + -

53

alter the conformation of proteins or may stimulate signaling molecules that make cells more oxidized,

which is a prerequisite for neural differentiation. However, deeper investigation is needed to

understand the effects of electrically conducting polymers on cell differentiation.

Tuj1 Tuj1 GFAP DAPI

Withoutelectric

field

With electric

field

Figure III.22. Electrically conductive PEDOT:PSS coated glass enable NSCs differentiation and neuronal growth. NSCs differentiation on PEDOT:PSS coated glass without electric stimulation are shown for comparison.

A) Immunofluorescence images of anti-Tuj1 (red) and anti-GFAP (green) followed by DAPI (blue) staining for the nuclei of the fetal NSCs on PEDOT:PSS coated glass. Magnification is 200X. B) ImageJ software quantification of cells showing positive immunostaining for Tuj1 in NSCs at day 7 of differentiation.

+ - + -

A

B

54

III.6.2.4. Electrical stimulation enhances neurite outgrowth

The neurite outgrowth is a complex and fundamental process during neuronal migration and

differentiation. This process begins with the neurite formation, being followed through elongation of

neurites over long distances and guidance through tissues in order to recognize proper targets where

the synapse formation and functional maturation of the newly formed connections occurs.

Taken into account the immunofluorescence images, it seems that there was no directional growth of

neurites at 1V/cm (Figure III.22.) as the neurites emerged from many directions. A qualitative

assessment of the AC electric field on the neurite length measured from differentiated fetal NSCs was

performed (Figue III.23.). The neurite length was ~ 64 μm under control culture condition (no electric

field applied), but a longer neurite length of ~ 96 μm was measured at field strength of 1 V/cm.

.

Several studies suggest that electrical stimulation regulate neurite outgrowth through regulating

intracellular signaling pathways and gene expression and interacting with cation-dependent

cytoskeletal components that modulate the morphology and motility of the growth cone85

.

III.6.2. Combined anisotropy and electric field effect in NSCs

III.6.2.1. Alignment of NSCs on grooved- PolyA gels

In addition to the chemical factors and the biological cues, the mechanical stimuli provided by ECM

play an important role in deciding stem cell fate and in regulating cell migration, proliferation and

differentiation.

In this study, we tested the hypothesis that NSCs accept mechanical cues for growth and spread from

the substrate by culturing them on flat and grooved PolyA gels of varying stiffness. PolyA was the

polymer chosen due to facility in varying its modulus of elasticity by changing the concentrations of

Figure III.23. Qualitative assessment of the AC electric field on the neurite length measured from differentiated fetal NSCs.

55

acrylamide and bis-acrylamide and due to their porous structure that prevents cells entering to the

substrate.

A series of PolyA substrates with bulk stiffness in the 0,5 kPa-10kPa range was prepared by varying

the monomer and the crosslinker to monomer ratio.

After PolyA surface covalent functionalization with laminin using Sulfo-NHS, it was found that NSCs

could spread fully on all flat and grooved substrates tested (Figure III.24.).

The substrates used in this study had grooves with the following dimensions: around 60 µm width, 70

µm spacing and 0,3 µm depth. The average area of substrates for cell culturing was approximately 2

cm2.

Polyacrylamide gels

(Stiffness = 10kPa)

Flat Grooves

Bri

gh

t fi

eld

Dap

i

& p

halo

idin

Figure III.24. Influence of substrate stiffness on the spreading of NSCs. NSCs adhere fully on flat and

grooved polyacrylamide (PolyA) substrates with stiffness equals to 10 kPa. Cells seem to align on grooved PolyA gels in contrast to the cells cultured on flat hydrogels that seems randomly oriented.

Mechanical topography

(Grooves)

56

NSCs could respond to topography patterns, especially micro- and nano-grooves simultaneously

aligning and elongating the cell bodies in the direction of the groove axis (Table 8).

NSCs adhere to all substrate surfaces on the first day after seeding. To verify the influence of stiffness

and topography on morphology of cells, the elongation and spreading of cells were characterized

(Table 8).

Elongation of cells was assessed by measuring the length of the long axis and short axis. At least 30

cells were evaluated. The factor E equals the long axis divided by the short axis, shown on Equation

(3).

Equation (3)

Table 8. Elongation of NSCS and NSCs nuclei cultured on flat and grooved PolyA hydrogels.

Analyzing Figure III.24. and Table 8 it appears that nuclei on grooved PolyA hydrogels are more

elongated that on flat PolyA hydrogels. The cell bodies and extending branches of NSCs exhibited

random distribution and showed no preference of direction on the flat PolyA hydrogel, while cell

nucleus elongated along the axis of the grooves and cell extended branches that were guided by

topological directionality on grooved substrates.

The production of PolyA substrates with stiffness equal to 0,5 and 5 kPa has been more difficult

because they are so soft that it becomes almost impossible to obtain uniform substrates. Hereupon,

large percentage of cell adhesion was conditioned. However, the small portion of cells that adhere to

these substrates exhibited random distribution on flat hydrogels and aligned and elongated along the

grooves (Figure III.25.).

Elongation ( E )

Flat hydrogels Grooved hydrogels

NSCs

NSCs nuclei

57

III.6.2.2. Replica molding: PDMS substrates

The interaction of NSCs with substrates that are able to replicate the structure and length scale of

native topography modulates diverse cellular responses, including survival, adhesion, spreading,

migration, proliferation, and expression of differentiated phenotypes.

The use of advanced micro- and nanofabrication techniques, such as photolithography and soft

lithography, have enabled the fabrication of tissue engineered substrates with precise topology and

biochemical composition. Soft lithography has a lower cost than traditional photolithography and is

based on the replica molding of microstructures in elastomeric materials such as PDMS, which is a

biocompatible elastomer, inexpensive, transparent and amenable to surface modification.

Taking this into account, the purpose of this study was to create a biocompatible substrate in order to

evaluate the influence of the topography and the electric field in cell behavior. To do this, PDMS was

spread on a piece of transparent vinyl disc and the stamp obtained was placed in an oven for thermal

aging (100ºC) during 48h. Then, this stamp was used as a secondary master and another stamp was

made from it. A dispersion of PEDOT:PSS was spin coated at 1800 rpm (60s) on the surface of the

second stamp obtained (Figure III.26.)

.

Polyacrylamide gels

Flat Grooves

Bright

Field

(Day1)

Dapi

&

Phalloidin

(Day 4)

Bright

Field

(Day 1)

Dapi

&

Phalloidin

(Day 4)

0,5

kPa

5

kPa

Figure III.25. Influence of different substrate stiffness on the spreading of NSCs.

58

These substrates had a major limitation: they were no conducting surfaces. This may be due to

surface activation by oxygen plasma not render a PDMS sufficiently hydrophilic to the PEDOT:PSS

adhesion. In order to circumvent this problem a gold (Au) layer with 40nm of thickness was

evaporated to the PDMS substrate before the spin coating of PEDOTPSS. However, the substrate

remained nonconductive. The network polymer structure of PDMS that makes it highly permeable and

their porous nature enabled the diffusion of PEDOT:PSS and also Au into the bulk polymer, which

resulted in non conducting surface.

III.6.2.3. NSCs adhesion on transparent vinyl disc substrate A new substrate was designed using a transparent vinyl disc, in order to study the influence of

topography on cell fate. The vinyl disc was cut into circles and a aqueous dispersion of PEDOT:PSS

was spin coated on its surface. In order to ensure that the cells would grow just on the surface of vinyl

disc, a PDMS cylinder was created to stanch the culture system (Figure III.27).

Results, displayed in Figure III.28, evidence that after 24h of culture the cells adhered and elongated

along grooves of PEDOT:PSS vinyl disc.

B C

A B C

Figure III.26. Constructs based on replica molding technique. A) PDMS stamp with patterns similar

with to those of vinyl disc. B) PEDOT:PSS spin-coated PDMS. C) Au layer deposited in PDMS in order to improve conductivity.

Figure III.27. A) SEM image (top) showing the micropattern on vinyl surface.

B) PEDOT:PSS spin-coated vinyl disc. The PDMS cylinder was created to stanch the culture system and two platinum wires were used as electrodes.

A B

59

PEDOT:PSS spin-coated- vinyl disc

Day 1

Nevertheless, the healthy cells died within 48 hours. This fact could be due to possible lixiviates

released by epoxy glue or Araldite glue that were used to glue platinum electrodes and PDMS

cylinder, respectively. These lixiviates could alter the pH that will promote a protein denaturation and,

subsequently, disruption of cell activity and cell death.

Vinyl discs incorporate chlorine atoms and this material may contribute to toxicity of system once this

is degraded by heat, promoting a combination between the chloride and the water vapor inside of the

humidified ambient of incubator forming hydrochloric acid.

This phenomenon permits a greater amount of hydrogen ions and maybe a subsequent acidification of

the medium that was in contact with cells promoting a premature death of cells.

III.6.2.4. NSCs proliferation on electrically PEDOT:PSS: vinyl disc substrate

Our previous studies showed that, after 24h of culture, the cells adhere and elongate along the

grooves of PEDOT:PSS vinyl disc, although they die within 48 hours.

We related this cell death with a possible weak sterility of the vinyl disc and with a possible toxicity of

the glue used. To solve this problem the new substrates were immersed in organic solvents such as

ethanol and isopropanol to release some lixiviates and we used a biocompatible glue to make a hollow

cylinder that was necessary to confine the culture system (Figure III.29.).

We exposed the cells to a 100 Hz electrical stimulation with a magnitude of 1V/cm in a continuous

manner. The AlamarBlue indirect method (the calibration curve is depicted in the annex VII.1.1) was

used to quantitatively measure the cell proliferation in substrate that was exposed to AC electric fields

and in control substrate that was not exposed to electric field.

Figure III.28. NSCs spreading on PEDOT:PSS spin-coated-vinyl disc.

60

A) Vinyl disc pattern dimension B) Electric field setup

C) Experimental scheme D) Voltage pulses in oscilloscope

Figure III.29. A) Schematic illustration of vinyl disc pattern and its dimensions. B) Electric field setup. C)

Experimental scheme with the electrical stimulation during the NSCs proliferation and differentiation. D) Pulsed electric field during in vitro cell culture.

The initial NSCs seeding on constructs was about 280.000 cells. The results for the 2nd

and 4th

day of

culture, depicted in Figure III.30., show that the cell number in electrically treated culture are

significantly higher than those in the controls. This data suggest that rapidly proliferating cells are

sensitive to electric field stimulation.

Figure III.30. Effect of electric field stimulation on cell number in proliferating cultures.

61

Cells were fixed with 4% PFA after 4 expansion days and 7 days of exposure to AC electric field for

12h/day in the absence of growth factors. Immunofluorescence study was performed using specific

antibodies against Tuj1, which labels neurons in early development as well as mature neurons and

GFAP to identify the differentiated level of astrocytic populations. However, this study was affected by

PEDOT:PSS and vinyl disc. PEDOT: PSS and vinyl disc removed some fluorescence which

conditioned the acquisition of fluorescence images for specific differentiation markers. Only images

with immunostained cells with DAPI was possible to acquire (Figure III.31.). Immunostained images

suggested that cells were confined to the grooves. To confirm this supposition, we visualized these

cells using SEM. Figure III.31., shows that a high number of cells adhered to the vinyl although it is

unclear if they were differentiated or not.

Without electrical field With electric field

Figure III.31. PEDOT:PSS vinyl substrates supported NSCs adhesion. NSCs nuclei counterstained

with DAPI and SEM images of NSCs on control (no electric field applied) and on PEDOT:PSS:glass substrates where AC electric field was applied.

63

Chapter 4

IV. Conclusions

The development of new scaffolds, focusing on the use of NSCs and conjugated polymers able to

sustain proliferation and differentiation of NSCs and neurite outgrowth, represents a promising

strategy, for instance to test drugs targeted against neurodegenerative disorders such as Alzheimer´s

and Parkinson´s diseases.

In their in vivo microenvironment, cells grow in a complex 3D environment and are exposed to various

endogenous electric fields, different stiffnesses and biomolecules that modulate their behavior.

In this study, we explore the strategy of combining conjugated polymers and soft lithography

techniques in order to design a different and innovative scaffold for NSCs adhesion, proliferation and

differentiation.

When NSCs were cultured on grooved-PolyA hydrogels they aligned and elongated their cell bodies

along the direction of the groove axis, while NSCs exhibited random distribution and showed no

preference of direction on flat PolyA hydrogels. In this study, microcontact printing, an easy and cheap

technique, was used to obtain the grooves and the results indicated that the micropatterned PolyA

substrates provided a 3D support for spatial guidance and cell growth.

After performing the cytotoxicity tests, we found that all conjugated polymers tested are not toxic,

offering new possible coatings for scaffolds for tissue engineering. As the PEDOT:PSS was the more

conductive and transparent polymer and offered a higher cell viability and cell growth, its potential to

sustain NSCs growth and differentiation was tested with and without AC electrical stimulation. The AC

electric field introduces a mechanical stress in cells, elongating and stretching more the cells

comparatively to control. Cells cultured on the PEDOT:PSS:glass substrates with and without electric

stimulation, remained capable of acquiring a neuronal or glial phenotype. AC electric fields might

benefit cell survival and appear to increase the number of Tuj1-positive cells and the neurite length in

the electrically conductive PEDOT:PSS:glass substrates compared to controls. These studies

demonstrated that laminin-fuctionalized PEDOT:PSS substrates may be used to control complicated

cellular functions of fetal NSCs such as cell morphology, proliferation and differentiation.

65

Chapter 5

V. Future Trends

Recent advances in conjugated polymers and in nanotechnology have been found to cover a broad

range of applications in regenerative medicine and offer new possibilities to repair neural defects.

The work developed in this thesis may serve as a basis for the design of constructs capable of

delivering AC electric fields to cell culture in order to guide NSCs adhesion, proliferation, migration and

differentiation.

Several challenges have been identified and should be the starting focus point for the progress of

future research. The loss of conductivity of the PEDOT:PSS on PDMS must be overcomed in order to

obtain cell alignment in groove’s direction using an inexpensive technique (replica molding). In order to

overcome these challenges, protocols to enhance the hydrophilicity of PDMS can be tested or

fluorosurfactants can be added to PEDOT:PSS, rendering a wettable solution on hydrophobic

surfaces. Modifications of the substrate and of the polymer coating should be investigated with the aim

to create a substrate that should the analysis of the role of vertical electric field application on NSCs

proliferation and differentiation. One task could be to modify the dimensions and the electric field setup

in order to stanch cell culture to promote cell confinement only to the substrate, as well the

transparency to allow the visualization of cells morphology under optical microscopy.

Several studies suggest the relevant physiological role of the electrical stimulation for control and

adjustment of the cellular and tissue homeostasis. The application of electrical stimulation to cells is

complicated because cells are highly conductive, allowing for large current flow, which generates heat

and changes in pH, significantly increasing the detrimental effects in stimulated cell culture. A study to

optimize parameters such as intensity, frequency, direction and duration of the applied electric field

must be performed. In addition, quantitative studies, such as Alamar blue assay, PCR, flow cytometry

and LDH assays, must be done to correlate the alterations on signaling pathways and gene

expression with electrical stimulation.

A future study could examine the combined influence of substrate conductivity and other adhesion

ligands (fibronectin, RGD) on cell behavior with topographically patterned substrates.

Finally, a wide range of other possible micropatterns may be examined with the use of other soft

lithography techniques to look for the influence of grooves size on cell behavior.

This study paved a road, however further investigations are needed to know whether we are going the

right direction.

67

Chapter 6

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Chapter 7

VII. Annexes

VII.1. Fluorescence AlamarBlue Calibration Curves

Figure VII.1. AlamarBlue fluorescence calibration curve for high numbers of NSC (microplate reader parameter: gain 89).

y = 0,0402x + 2204,9 R² = 0,9957

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0 200000 400000 600000 800000 1000000 1200000

Flu

ore

sce

nce

Cell number

assessing the use of conjugated polymers and electric

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