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Thermo-Responsive Polymers for Cell-based Therapeutic
Applications.
A thesis submitted to the
Graduate School
of the University of Cincinnati
in partial fulfillment of the
requirements for the degree of
MASTER OF SCIENCE
in the Biomedical Engineering Program
in the School Biomedical, Chemical, and Environmental Engineering
of the College of Engineering and Applied Science
November, 2014
by
Hodari-Sadiki James
B.A., Berea College, Berea, KY, 2012
Committee Chair: Daria A. Narmoneva, Ph.D.
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Abstract
Poly (N-isopropylacrylamide) (PNIPAAm) is a well-known thermo-responsive polymer that has be
shown to be biocompatible, with surfaces coated with PNIPAAm supporting the culture of cells. These
surfaces support the adhesion and proliferation of multiple cell phenotypes at 37 °C, when surface is
hydrophobic, as the polymer chains are collapse and lose their affinity for water. Reducing the
temperature below the polymers lower critical solution temperature (LCST) elicits hydration and
swelling of the polymer chains and leads to cell detachment. In vitro culture on thermo-responsive
surfaces can be used to produce cell sheets for the use of different therapeutic treatments. PNIPAAm
coated membranes were used to culture human keratinocyte cells to confluence, with cell release
possible after exposing the membranes to room temperature (~25 °C) for 10 minutes. Cell sheet
transfer was possible from the coated membrane to cell culture dishes using a protocol that we
developed. There was also a trend towards similar cell apoptosis on both PNIPAAm coated and
uncoated surfaces.
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Acknowledgements
I would like to acknowledge the University Of Cincinnati Department Of Biomedical
Engineering for Graduate Scholarship and Assistantship support which allowed me to fund my studies
at the University of Cincinnati. The research performed in this thesis was supported by funds from the
University of Cincinnati Department of Biomedical, Chemical and Environmental Engineering SEED
Grant.
I would like to express my gratitude to my advisor Dr. Daria Narmoneva for welcoming me into
her lab and giving me an opportunity to work on multiple biomedical research projects. Without her
unending support, guidance and encouragement, this process would have certainly been more difficult.
I am also indebted to Dr. Dale Schaefer, and Dr. T Douglas Mast, for taking the time to serve as
members of my thesis committee.
I would also like to thank Chukwuemeka Chikelu, Naiping Hu, Toloo Taghian, Andrea Lalley,
Brandon Brown, and Bo Yang for being valued colleagues and friends. I am also thankful to staff and
faculty members in Biomedical Engineering, particularly Dr. Jing-Huei Lee and Barbara Carter, for all
the help and support during my difficult times. Finally, I want to thank my mother, brother, and other
close family and friends for their love, and support and prayer throughout this entire process.
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Table of Contents
Abstract..............................................................................................................................2
Acknowledgements ............................................................................................................ 4
Table of Contents ................................................................................................................ 5
List of Tables and Figures .................................................................................................. 6
Chapter 1: Emerging Technologies for Cell Sheet Engineering using Thermo-
Responsive Polymers. ........................................................................................................ 8
Chapter 2 :Experimental Studies with Thermo-Responsive Membranes ........................ 21
2. 1 Background and Introduction: ................................................................................... 21
2.2 Cell Detachment and Transfer Study ......................................................................... 23
2.3 Comparison of Keratinocyte Apoptosis on Different Culture Surfaces ..................... 28
Chapter 3: Conclusions and Future Directions ................................................................ 32
Bibliography ..................................................................................................................... 34
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List of Tables and Figures
Chapter 1:
Figure 1: Schematic illustration of cell sheet on PNIPAAm modified surface. ................ 9
Table 1: Showing the Responses of Multiple Cell Phenotypes Cultured on Thermo-
Responsive Surfaces........................................................................................................ 10
Figure 1.2: An illustration of cell sheet detachment by different types of water supply . 13
Chapter 2 :
Figure 2.1: Wound healing schematic and the proposed treatment strategy .................... 22
Figure 2.2 Image of the plastic insert which contains the porous PVDF membrane that
was spin coated with PNIPAAm: ..................................................................................... 23
Figure 2.3 : Schematic showing the different surfaces that were explored in preliminary
studies.. ............................................................................................................................. 24
Figure 2.4 : Schematic of the cell detachment and transfer approach using collagen ..... 25
Figure 2.5 : 4x fluorescent image of KC cells after being transferred from PNIPAAm
coated membrane to sterile uncoated dishes………………………...………….………26
Figure 2.6 : 20x image of keratinocyte cells held in collagen after being transferred from
coated membranes to a culture dish. ................................................................................ 27
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Figure 2.7 : Schematic showing an overview of KC cell apoptosis study.. ..................... 28
Figure 2.8 (A): 20x image of KC nuclei co-stained with fluorescent DAPI (blue) and
caspase3 (red) ................................................................................................................... 30
Figure 2.8 (B) : 4x image KC nuclei co-stained with fluorescent DAPI (blue) and
caspase3 (red). .................................................................................................................. 30
Figure 2.9: Graph showing the percentage KC cell death………………………………… 31
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Chapter 1: Emerging Technologies for Cell Sheet Engineering using Thermo-
Responsive Polymers.
1.1 Introduction:
During a typical cell culture process, cell adhesion to the culture surface is important in order to
foster the healthy growth, differentiation, migration, and survival of cells [1], [2]. Adhesion, however,
presents a problem when cells grown in vitro need to be transported to an in vivo target area. The
conventional procedure involves proteolytic degradation of extracellular matrix (ECM) that holds the
cells together via enzymatic action, for example trypsinization. This procedure usually produces a
suspension of cells that can then be transported to another culture vessel. However, destruction of the
cell sheet's ECM is not very efficient from a tissue-engineering viewpoint, as the cell-cell interactions
present are usually integral to their in vivo function [3]. In addition nonspecific proteases can damage
crucial cell surface proteins [3], [4]. These major drawbacks have fueled research into alternative cell
harvesting methods.
Poly (N-isopropylacrylamide) (PNIPAAm) is a well-known thermo-responsive polymer which
has been studied since the late 1960's [5]. Its use as a culture surface for the in vitro growth of intact
cell sheets, however, was not studied until the early 1990's [4], [6]-[8]. In recent years, cell culture on
PNIPAAm coated surfaces or within PNIPAAm hydrogels, has been explored as a possible solution to
the problems presented by conventional cell harvesting methods [9]. A surface grafted with the polymer
has the unique property of switching from a hydrophobic to a hydrophilic state when the temperature is
decreased below its lower critical solution temperature (LCST) (~32 °C) [10]. A confirmation change
in the polymer chains of the grafted PNIPAAm layer, caused by reduced temperature, can elicit
detachment of confluent cell sheets as shown by the schematic in Figure 1 [4], [7], [11]–[16].
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This chapter will; 1) Review recent cell sheet engineering studies using PNIPAAm, or
PNIPAAm copolymers, as a cell culture surface, and 2) Come up with a unique approach of producing
keratinocyte cell sheets to be used in the treatment of chronic wounds. Focus will be placed on cell to
surface interactions such as, adhesion, proliferation, and detachment. Since cellular responses to
PNIPAAm grafted surfaces have been found to be both cell-type specific, and dependent on PNIPAAm
grafting thickness in previous studies [20],[25]. Promising applications of cell sheet engineering
research using PNIPAAm coated culture surfaces will also be highlighted at the end of this chapter.
Figure 1.1: Schematic illustration of cell sheet on PNIPAm modified surface. A: At 37 °C, cells can adhere on
dehydrated PNIPAm layer. B: At 20 °C, cells can detach from hydrated PNIPAm layer with ECM, resulting in
an intact and contiguous cell sheet [14].
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Surface Description
Cell Phenotype Cell Adhesion &
Proliferation
Cell Detachment Refs.
Flat (F) and Micro-patterned (MP)
PNIPAAm films on TCPS
Rat bone marrow
mesenchymal stem
cells (BMSC)
Static: ↑proliferation rate at 2 weeks
on both F and MP
surfaces.
Dynamic: ↑ Total cell proliferation at
21 days on MP surface.
N/A [17]
PIPAAm Graft on
Polystyrene Dish (PSD) &
Polystyrene Nano-fibrous Mat (PNM)
Human fibroblasts
↑ Adhesion and cell spreading on
PNM surface.
↑ Detachment on PNM compared
to PSD.
[13]
PNIPAAm on
Chitosan membranes
Human fetal lung
fibroblast cell line
(MRC-5)
Best proliferation rate on P-IPrOH75-
grafted surface
Complete cell sheet detachment on
P-IPrOH75 surface.
[18]
(PNIPAAm)-grafted on
Flat PDMS substrate &
Micro-patterned PDMS substrates
Bovine aortic vascular
smooth muscle
cell (VSMCs)
Good adhesion on flat surface &
micro-patterned surfaces with cell
alignment along channels.
Complete cell detachment on both
surfaces.
[19]
(PNIPAAm) on
Glass substrates
NIH/3T3 fibroblast
cells.
Best attachment and spreading on
graft thickness ≤ 14nm
Best detachment on thicknesses
between 5 and 14 nm
[20]
(P(NIPAAm-r-MPDSAH)) on
Glass substrates
NIH3T3 fibroblast
cells.
Very Good Attachment and
spreading on surfaces with ≤ 75:1
PNIPAAm:MPDSAH concentration
Complete detachment only on
surfaces with a with a 75:1
PNIPAAm:MPDSAH mixture.
[20]
PNIAM-co-AM on
TCPS
Human Chondrocyte
Cells
Very good attachment and spreading w/o PVDF 69% detachment [21]
PNIAM-co-AM on
TCPS
PNIPAAmSt microgel films
Spin-coated (SC) on glass &
Drop-coated (DC) on TCPS.
Human Chondrocyte
Cells
NIH/3T3 fibroblast
cells
Very good attachment and spreading
Attachment and spreading similar on
both uncoated and DC TCPS
with PVDF100% detachment [21] [22]
90% cell detachment in 2-3 mins
from DC TCPS with pipetting.
PNIPAAmSt microgel films
Spin-coated (SC) on glass &
Drop-coated (DC) on TCPS.
Gelatin/PNIPAAm-PHB-PNIPAAm
Copolymer on Glass cover-slips
(GCS)
NIH/3T3 fibroblast
cells
Mouse embryonic stem
(ES) cells
Attachment and spreading similar on
both uncoated and SC glass
substrates
↑ detachment on SC 1.0 wt %
microgel films compared to SC 0.5
and 0.75 wt % films
[22] [23]
↓ proliferation rate compared to
gelatin coated GCS
↑ cell detachment compared to
other surfaces
NGMA coated tissue culture plates
Corneal endothelial
cells (CEC)
grew to confluence
within seven days
4 °C the cell sheet
detached
[24]
Flat (F) and Micro-patterned (MP)
PNIPAAm films with elastin-like
protein (ELP) on TCPS
Rat bone marrow
mesenchymal stem
cells (BMSC)
Static: Proliferation similar on F and
MP to noELP surface.
Dynamic: ↑ Total proliferation on
both F & MP surfaces.
N/A [17]
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Surface Description
Cell Phenotype Cell Adhesion &
Proliferation
Cell Detachment Refs.
CD-90 immobilized-
P(IPAAm-co-CIPAAm) on TCPS
I) Thymic carcinoma
cell line (Ty-82).
II) Human dermal
fibroblasts (NHDFs).
III) Primary Mouse
Adipose tissue-derived
(ADP) cells
Best adhesion and spreading of Ty-82
cells and NHDFs on avidin-biotin
binding surfaces.
Ty-82 & ADP cell detachment
required application of shear stress
& low temperature treatment.
NHDFs detached from all coated
surface with low temperature
treatment.
[25]
bFGF-bound heparin-fictionalized
P(IPAAm-co-CIPAAm) on TCPS
NIH/3T3 fibroblast
cells.
Cells reached confluence in 3 days on
surfaces with bFGF
densities ≥ 20ng cm-2 compared to 5
days on TCPS dishes.
BFGF-8 and bFGF-20 showed best
detachment rate.
[26]
(PCL-g-P(NIPAAm)) films NIH/3T3 fibroblast
cells
↑ proliferation with increased
collagen content.
↓ Cell detachment with increase in
collagen.
[27]
Table 1: Responses of Multiple Cell Phenotypes Cultured on Thermo-responsive Surfaces.
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1.2 Cell interactions with PNIPAAm Coated Surfaces
Multiple studies have shown that cell adhesion and detachment are dependent upon the thickness
of the PNIPAAm layer that is grafted onto the underlying base surface [11], [12], [20], [28]. Grafted
layers thinner than 14 nm showed good cell adhesion in a study carried out by Kong et al. However, in
Kong's study, low-temperature detachment of cells from the PNIPAAm coated glass substrates was
only optimal on surfaces with 11 nm and 13 nm thicknesses respectively, cells on detached poorly from
layers less than 5nm thick [20]. Different concentrations of the polymer Poly ((3-
(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)- ammonium hydroxide) (PMPDSAH) were
added to PNIPAAm, creating new polymer composites to improve cell detachment [20]. They observed
complete detachment from surfaces grafted with 4 nm thickness of (P(NIPAAm-r-MPDSAH)) which
had a 75:1 PNIPAAm to MPDSAH composition [20].
The improvement of cell detachment has been approached in multiple ways. Thai researchers
observed improved cell release from a temperature-responsive poly (N-isopropylacrylamide)-co-
acrylamide (PNIAM-co-AM) surface with the help of a hydrophilically modified
poly(vinylidenefluoride) (PVDF) membrane [21]. The group cultured human chondracyte cells on
their co-polymer coated surface, but only observed 69 ± 11.45% cell release when dishes were
incubated at 10 °C for 30 minutes [21]. The use of the PVDF membranes along with low temperature
treatment, not only improved the cell yield to 100%, but also facilitated cell sheet transfer and
decreased cell sheet shrinkage [21].
So far we have only looked at culture on traditional coated base surfaces, such as TCPS and glass
substrates, which are known to support in vitro cell culture. There are two studies in Table 1 that
explore the use of non-traditional base surfaces coated with PNIPAAm to improve adhesion, growth
and detachment of specific cell phenotypes. In 2008 Da Silva et al. used PNIPAAm coated chitosan
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membranes as a thermo-responsive culture surface. The group correlated chitosan's biocompatibility,
non-toxicity and membrane porosity with improved mass transfer of nutrients and metabolic wastes
[18], [29]. Results from the study showed favorable adhesion and spreading on coated surfaces, with
ungrafted chitosan membranes showing very poor cell compatibility [18]. Plasma treatment of coated
surfaces increases not only the surface's cell proliferation, but also enhances the detachment of
confluent cell sheets after 16 °C incubation [18].
More recently, a Korean study, led by Hwan Hee Oh, fabricated thermo-responsive polystyrene
(PS) nano-fibrous mats, by the electro-spinning of PS solution. These mats were then grafted with
PIPAAm to produce thermo-responsive PS nano-fiberous mats for use in in vitro cell culture [13]. They
observed improved adhesion and growth of human fibroblasts that were cultured on coated PS mats as
opposed to coated PS dishes [13]. The investigators proposed that the three-dimensional structure of
electrospun mats provided better conditions for cell metabolism, which resulted in improved
proliferation. There was no cell detachment on uncoated surfaces. However cells detached from both
polymer coated PS mats and dishes, with a higher percentage of cell detachment seen on coated PS
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nano-fibrous mats [13]. The porous structure of the mats was identified as the reason for rapid
hydration and improved detachment compared to solid surfaces [13]. The schematic in Figure 1.2
illustrates how different hydration methods can cause variations in cell detachment.
The culturing of cell sheets with specific architectures is desirable since most normal tissue has
some structure that is difficult to replicate in vitro. The effect of surface chemistry, mechanical stress
and topography on bone marrow mesenchymal stem cells (BMCS) cultured on PNIPAAm -coated
surfaces was investigated by Ozturk et al. Micro-patterned PNIPAAm films encouraged cell nuclei
alignment and elongation parallel to the direction of trenches fabricated on the surface [17].
Unpatterned surfaces exhibited random growth [17]. Detachment studies were not performed because
the goal was to impose mechanical stress on the cells via temperature variation [17].
Williams et al. attempted induced cell alignment by culturing vascular smooth muscle cells
(VSMS) on a thermo-responsive surface micro-patterned with the cell adhesive protein fibronectin. The
group maintained alignment within 30° of the fibronectin lanes [30] with successful detachment and
transfer of the aligned cell sheets [30]. This group also observed good adhesion and growth of VSMCs
on PNIPAAm-grafted micro-textured PDMS surfaces. Hoechst staining revealed elongated nuclei
aligned with lanes of the textured surface [19]. Complete cell sheets detached after low temperature
exposure, with reduced shrinkage compared to sheets grown on flat PNIPAAm-coated PDMS surfaces
[19].
The majority of the studies employed the photo polymerization method. This method allows for
chemical bonding between the grafted polymer layer and the underlying base surface, and is believed
foster better cell response and detachment [31], [32]. Thermo-responsive p(NIPAm-co-St) was used to
spin-coat glass substrates and drop-coat TCPS dishes in Xia et al's study. Styrene was included in the
co-polymer to optimize the amphiphilicity of the microgels in an attempt to improve cell detachment
[22]. NIH/3T3 cells showed similar cell adhesion and spreading properties on both spin-coated and
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uncoated glass substrates. However there was no cell detachment observed upon low temperature
treatment (20 °C) with cells only becoming more rounded, and requiring the application of shear stress
to be removed from the surface [22]. Drop coated surfaces only supported good adhesion and spreading
with a 0.5 wt % polymer composite coatings [22]. The investigators again reported the need for shear
stress along with low temperature to detach cells [22].
Loh et al. also looked at drop coating method for creating a thermo-responsive cell culture
surfaces. Loh's team used the tri-block copolymer, poly(N-isopropylacrylamide)- block-poly[(R)-3-
hydroxybutyrate]-block-poly(N-isopropylacrylamide) (PNIPAAm-PHB-PNI- PAAm), to drop coat
glass coverslips, and then added a layer of gelatin onto the polymer surface [23]. Mouse embryonic
stem (ES) cells were observed to proliferate better on the gelatin-triblock polymer coated surface, than
on surfaces coated with gelatin/PNIPAAm [23]. Detachment was tested by incubating substrates at 4 °C
for 20 minutes, with 91.3% ± 17.6% detaching from gelatin/triblock polymer, compared to 59.2% ±
10.4% from gelatin/PNIPAAm coated surface [23].
Most cell culture protocols employ growth factors, which are soluble stimulants used to
accelerate proliferation [33]. Because growth factors induce the degradation of other signaling
molecules surface immobilization of growth factors is seen as a promising method to reduce
degradation [33], [34]. A Japanese group explored thermo-responsive culture surfaces using poly(N-
isopropylacrylamide-co-2-carboxyisopropylacrylamide) P(IPAAm-co-CIPAAm) [25], [26]. They
covalently tethered heparin onto the CIPPAAm chains, and then added heparin binding proteins, such
as CD-90 and basic fibroblast growth factor (bFGF) to the surface [25], [26].
In the first study, CD-90 antibody-immobilized P(IPAAm-co-CIPAAm) TCPS surfaces were used
to culture two cell lines, thymic carcinoma cell line (Ty-82), and neonatal normal human dermal
fibroblasts (NHDFs), both of which are known to express CD-90 on their cell surface [25]. TY-82 cells
adhered better than NHDFs to the antibody-immobilized surfaces. However both cell types formed a
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confluent cell sheet after 4 days. There was only partial detachment of TY-82 cells after low
temperature treatment (20°C) on all grafted surfaces, with the application of shear stress required to get
complete detachment [25]. NHDFs spontaneously detached in response to the same low temperature
treatment, indicating that the detachment process is “cell specific” [25].
The same group looked at cell responses to surface immobilization of the growth factor bFGF on
the thermo-responsive surface [26]. NIH/3T3 fibroblast cells were cultured on bFGF-bound heparin-
fictionalized P(IPAAm-co-CIPAAm) surfaces with varying densities of bFGF. Cells reached confluence
in 3 days on surfaces with bFGF densities of 20 ng cm-2 and above, two days faster than cells cultured
on non-immobilized surfaces [26]. Density of bFGF also affected cell detachment with cells taking 45
minutes to detach from surfaces with densities of 8 and 20 ng cm-2 , compared with 120 minutes for
densities above 100 ng cm-2 , with the fastest detachment, 30 minutes, seen on non-immobilized
P(IPAAm-co-CIPAAm) surface [26]. The benefit of better adhesion and faster culture time on certain
bFGF-bound heparin-fictionalized P(IPAAm-co-CIPAAm) surfaces is helpful despite the drawback of
decreased detachment rate [26].
Table 1's final entry is a study of thermo-responsive poly (N-isopropylacrylamide)-grafted
polycaprolactone (PCL-g-P(NIPAAm)) films that were immobilized with cell-adhesive collagen to
improve cell sheet formation [27]. The higher the content of the immobilized collagen, the higher the
density of the attached cells [27]. PCL-g-P(NIPAAm)-collagen with thinner collagen coatings had
100% detachment. The percentage detachment decreased with increased collagen thickness [27].
1.3 Applications of Cell Sheet Engineering
Given the promising results on growth and release of different phenotypes on PNIPAAm
substrates or hydrogels it is logical that different research groups have looked into the medical
applications of cell sheet engineering.
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One such application is the construction of mesenchymal stem cell (MSCs) sheets for use in
tissue regeneration without the differentiation of the stem cells. MSCs have self-renewal ability and
multi-differentiation potential, making them ideal for treatment of certain debilitating injuries and
diseases [35]. In Yang et al's study, primary rat bone marrow-MSC (BM-MSCs) and human adipose
tissue-MSC (AT-MSCs) were cultured on PNIPAAm copolymer films in an attempt to reduce the loss
of its cell adhesion and differentiation caused enzymatic treatment [36]–[38]. Although the mechanism
by which MSCs lose their primitive nature is not fully understood, the deficiency in their plasticity may
arises from multiple exposure to proteolytic enzymes that degrade extracellular matrix (ECM) proteins
[39], [40]. Using temperature release instead of trypsinization should prolong the primitive nature
during in vitro culture [35].
Yang's group cultured intact cell sheets on PNIPAAm copolymer surfaces [35]. They observed
greater cell viability, more colonies and stronger differentiation of MSCs harvested via temperature
drop in comparison with the trypsinization [35]. This result was linked to the preservation of ECM
proteins caused by whole sheet retrieval using the thermo-responsive surface [35]. In Ozturk et al's
study demonstrated that rat BM MSCs retain their primitive nature after in vitro culture. Here MSCs
cultured dynamically on a PNIPAAm surface showed reduced MSC differentiation when compared to
static culture [17].
In contrast to Yang’s work, Peroglio et al sought to foster the differentiation of MSC cells in vitro
for treatment of intervertebral disc (IVD) pathologies. Injection of human MSC (hMSCs) into the
intervertebral disc (IVD) space is being explored to combat IVD degeneration [41]–[43]. A drawback
to this treatment is the harsh conditions present within the disc tissue. Low glucose, oxygen levels, pH
adversely impact undifferentiated MSCs [44]. The quality of repair hinges on the ability of the MSCs to
differentiate to the disc cell phenotype after injection, and also on the cell carrier that is used to initially
support the injected cells. Pre-culture of hMSCs to encourage differentiation toward the IVD cell
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phenotype before injection is a possible path to improved outcome [46]. Peroglio's ex vivo study, used
the thermo-reversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-
PNIPAAm) for the pre-culture of hMSCs, and also as a carrier for cell delivery. The hydrogel's
reversible gelation caused by its PNIPAAm content, made it more suited for pre-culture of hMSCs
before delivery to the target area [47]. The HA-PNIPAAm hydrogel induced hMSC differentiation
toward the disc phenotype more effectively than alginate gel, with neither pre-differentiation nor
growth factor supplementation required for hMSC disc-like differentiation [47].
Schwann cell transplantation has emerged as an attractive candidate in treating demyelinating
diseases resulting from peripheral and central nervous system injuries [48]. Most of the existing
methods involve the in vitro culture of schwann cells, which are then harvested and transported to the
target site via direct injection or via scaffold seeding [48]. These conventional harvesting and transfer
methods often involve proteolytic treatment, which leads to the degradation of cell-surface proteins and
also breakdown of important cell-cell interactions [49]. Pesirikan et al explored the construction of
schwann cell sheets on polystyrene tissue plates coated by PNIPAAm, which preserves cell-surface
proteins and cell-cell interactions through temperature initiated release. The investigators were able to
construct sheets of viable schwann cells that maintained cell–cell interactions as indicated by high
expression of cadherins as well as the level of neurotrophins [48].
Cell sheet engineering has also been used clinically in the treatment of injured corneas caused by
trauma or disease [50]. Japanese scientists constructed epithelial cell sheets by culturing autologous
oral mucosal epithelial cells on a thermo-responsive surface, which included the PNIPAAm polymer
[50]. Although the sample size of this study was small (4), the group reported a 100% success with
complete reepithalization of the previously damaged corneal surface in less than a week after the cell
sheets were introduced [50].
In a more recent study, human undifferentiated immature dental pulp stem cells (hIDPSC) were
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used to construct cell sheets for implantation on the cornea of rabbits with limbal stem cell deficiency
(LSCD). LSCD can be caused by multiple conditions, including genetic disorders, contact lens–
induced keratopathy, and iatrogenic multiple ocular surgeries [51]. The pathology causes a persistent
corneal epithelial defect or abnormal re-epithelialization by conjunctival epithelial cells, and can be
treated by a transplant of tissue from the patients’ non-affected eye [52]–[54]. Gomes et al looked at
culturing hIDPSCs cell sheets in vitro on a thermo-responsive surface, and transplanting the sheets onto
the cornea of impaired rabbits. Results showed continued improvement in corneal transparency in
comparison to the control group that had total opacification [51].
The final application considered is repair and regeneration of skin defects in mice. Cerqueira et
al. engineered a construct made by stacking multiple layers of human keratinocytes (hKC) cell sheets,
dermal fibroblasts (hDFb) cell sheets, and dermal microvascular endothelial (hDMEC) cell sheets,
which were cultured in vitro on thermo-responsive culture dishes [55]. Recognizing the complexity of
skin regeneration, the investigators choose hKCs for their wound re-epithelialization, hDFbs for ECM
remodeling, and hDMECs for dermal angiogenic signaling [56]–[58]. The rate of wound closure
improved in wounds treated with the cell-sheet constructs [55]. The outcome was dependent on the
arrangement of the different cell types in the construct, with some stacking configurations showing
only minimal wound healing improvement when compared to the control [55].
1.4 Proposal for a Novel Thermo-Responsive Cell Culture Surface
The information gathered from this review of cell interactions with these thermo-responsive
surfaces allowed us to construct our own approach for the culture, retrieval, and transfer of KC cell
sheets. Spin coating was chosen as our method of grafting PNIPAAm to the base surface due to its
shorter processing time and ability to support cell growth based on previous studies [22]. To counter act
the issues that were observed with cell detachment from spin coated surfaces however we proposed the
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use of a porous PVDF membrane as our base surface.
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Chapter 2: Experimental Studies with Thermo-Responsive Membranes
2. 1 Background and Introduction:
Tissue engineering is a promising field of medicine which aims to develop biological substitutes
that restore tissue functions [59]. Wound closure is a key process during natural wound healing, as the
presence of barrier between the affected tissue and outside elements not only reduces the risk of
infection, but also creates a stable micro-environment for the regeneration of the extracellular matrix
(ECM) by cells [60]. The natural wound closure process is hampered in diabetic patients due to a
dysfunctional ECM and deficient blood vessel formation process [61], [62]. A common treatment of
diabetic wounds is wound management, which involves the regular cleaning and coverage of the
affected area to assist the natural healing process [63].
In the past our group has looked at the novel use of a nanofiber hydrogel as an ECM scaffold to
improve healing in the diabetic wound micro-environment [61], [62]. More recently, we have focused
on providing protection for unstable region while the healing process is taking place. To do so we have
proposed the in vitro growth of a keratinocyte cell sheet to be used in wound coverage, and is
illustrated in Fig 2.1 shown below. As explained in Chapter 1, cell sheet recovery using traditional cell
culture methods (eg. enzymatic action) is nearly impossible, as it requires the disassociation of the
cultured cell layer into a solution during the harvesting process [3]. The growth of KCs on a thermo-
responsive surface is a novel approach to produce a transferable cell sheets for use in diabetic wound
treatment.
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In this study we focused on the development of a method to culture and harvest human KCs on
thermo-responsive PNIPAAm coated PVDF membranes. In the first section of the study, we
investigated different cell detachment and transfer techniques, using information gained from extensive
literature review, as described in Chapter 1. The second section of our study focused on demonstrating
that PNIPAAm coating does not negatively affect cell viability, by comparing the levels of KC
apoptosis (programmed cell death) between different PNIPAAm substrates and plastic control.
Figure 2.1: Wound healing schematic (left) and the proposed treatment strategy (right). The normal wound
shows the presence of growth factors (GFs) and extracellular matrix (ECM) laid down by cells as well as
formation of blood vessels to the wound site. In contrast, in diabetes, there is increased inflammation and cell
death, degradation of GFs and ECM, leading to non-healing diabetic ulcers. The proposed treatment (right)
includes filling the wound with the stable nano-fiber matrix for cell infiltration, covered by keratinocyte sheet
layer delivered by smart release technology.
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2.2 Cell Detachment and Transfer Study
2.2a Overview
In this first section, porous PVDF membranes housed in plastic inserts, shown in figure 2.2, were
spin coated with PNIPAAm before they were used to culture KCs. The ability of these coated
membranes to support cell adhesion and proliferation were observed qualitatively. The objectives of
this study were to; 1) Conduct a preliminary study to determine optimal PNIPAAm coating parameters
for cell attachment and growth; 2) Demonstrate cell sheet detachment from the PNIPAAm coated
membrane, and 3) develop a procedure for the transfer cultured sheets to a secondary in vitro site.
2.2b Methods
Cell Culture
Frozen human primary Keratinocytes (KCs) obtained from healthy skin cell donors at the University of
Cincinnati Hospital were thawed and cultured in 75cm2 (T-75) cell culture flasks (Fisher brand) in
Medium 154 (Life Technologies, Grand Island, NY), supplemented with 0.1% CaCl2 0.2mM (Life
Technologies, Grand Island, NY), 1% antibiotic/antimycotic (Atlanta Biologicals, Lawrenceville, GA)
Figure 2.2 Image of the plastic insert which contains the porous PVDF membrane that was spin coated
with PNIPAAm. The thin fabric-like membrane is surrounded by a circular plastic barrier, which forms
an inner well around the membrane.
24
and Human Keratinocyte Growth Supplement (Life Technologies, Grand Island, NY). Cell cultures
were maintained at 37°C in 100% humidified air containing 6% CO2. Cells of passage 1-3 were used in
all experiments, in order to maintain the phenotype of the KCs.
Determination of Coating Parameters that Supported Cell Attachment and Growth.
To determine the best PNIPAAm casting parameters for PVDF membranes to support KC cell growth,
membranes were spun and drop coated with three different concentrations (0.5 % wt, 1.0 % wt, and
2.0 % wt) of PNIPAAm. The schematic in figure 2.3 shows an overview of this preliminary study.
Cell Culture on Thermo-Responsive Membranes
From culturing of KCs on the different PNIPAAm coated membranes described above, we observed
that cells attached and proliferated best on surfaces spin-coated with 2%wt PNIPAAm. Therefore, 2-
wt % PNIPAAm was used to spin-coat the membranes held within the inserts (30mm diameter, 0.4µm
pore size, 4.2 cm2 effective membrane; Millipore, Billerica, MA), followed by pretreatment with
formaldehyde. All coating procedures were carried out by our collaborator Dr Naiping Hu in Prof. Dale
Schaefer’s laboratory. Keratinocytes (KC) harvested from the T-75 culture flasks, via trypsinization,
and seeded on PNIPAAm coated membranes (n=2) that were sterilized with ethanol. The cells were
seeded at a density of 4.5×104 cells/cm2.
Figure 2.3: Schematic showing the
different surfaces that were explored in
preliminary studies to determine the best
surface to support KC cell growth.
25
Cell Sheet Detachment and Transfer
After 4 days, cells were stained with Orange Cell Tracker Dyes, CMTMR (Invitrogen, Carlsbad, CA).
Two approaches for cell detachment were attempted. The first involved the transfer of the membrane to
cell culture plates (Fisher Scientific, Pittsburg, PA) with cells inverted onto the surface of the dish.
These sections were then left at room temperature with a drop of media for 10 minutes to allow for cell
detachment, after which the membrane was peeled off the surface using sterile tweezers. In the second
approach, illustrated in Figure 2.4, cells were covered with 2.2 mg/ml collagen solution (Inamed
Biomaterials, Fremont, CA) mixture containing 5% 0.1M DPBS, 5% 0.2M HEPES and 55% PBS, and
incubated for 1 hour. The membrane from this insert was then transferred at room temperature to cell
culture plates with the collagen-cell layer inverted onto the surface of the well. After 10 minutes at
room temperature the membrane was peeled off using sterile tweezers. The detached cells in both
approaches were fixed with 2% formaldehyde, rinsed with PBS, and aqueous mounting medium
(Vector Laboratories, Burlingame, CA) was added afterwards. Images of detached cells were then taken
using an inverted fluorescent microscope (Olympus IX81; Olympus America, Center Valley, PA).
Figure 2.4: Schematic of the cell detachment and transfer approach using collage. Cells cultured on
PNIPAAm coated surface for 4 days before a layer of collagen is used to cover the cells. The
membrane, cell, and collagen structure is then inverted onto a cell culture dish at room temperature,
before the membrane is peeled off.
26
2.2c Results
The KC cells adhered and proliferated well on the 2% wt PNIPAAm coated membranes. After 4
days in culture a uniform layer of KCs were present, with cells bearing the same cell morphology as
those cultured in cell culture flasks. Fig 2.5 shows 4X images of KCs after being detached from the
PNIPAAm coated membranes using both transfer methods. The left image shows transfer cells from the
coated membranes directly, where cells were observed to detach in clumps. In contrast, cells transferred
after the addition of collagen layer showed more uniform cell-sheet detachment from the coated
membrane, shown in the right image.
Cell detachment from the coated membranes was observed after the membranes were left at room
temperature for very short time periods, between 5 and 10 minutes. The cell sheet could be viewed
floating in media without the use of a microscope, after detaching from underlying membrane surface.
This made it difficult to transfer cell sheets without the addition of collagen, as cells would be detached
from the membrane before inversion onto the cell culture dish. However with collagen added to the
layer of cells prior to membrane transfer, we observed detachment of cells on the well surface after the
Figure 2.5. (Right) Addition of collagen layer over cultured human keratinocytes on PNIPAAm substrate resulted in a better and more uniform sheet release, as compared to the more clump-like cell release without the collagen support (Left). Bar-150um.
27
membrane was peeled off. The difference in cell detachment using these two transfer methods is shown
in the 4x images in figure 2.5. Figure 2.6 shows a 20x image of cells after transfer using collagen, with
the cells maintaining a 3d structure in the collagen after transfer.
Figure 2.6: 20x image of keratinocyte cells (stained red) held in collagen (stained green) after being
transferred from coated membranes. The image shows that the cell/collagen construct was able to
maintain a 3D structure after transfer. Bar 5 𝜇m.
28
2.3 Comparison of Keratinocyte Apoptosis on Different Culture Surfaces
2.3a Overview and Objective
The objective of the study was to determine if the PNIPAAm coated surface does not negatively
affect KC cell viability. Based on our observations from the carrying out the cell detachment and
transfer study (Section 2.2) we hypothesized that there would be no detectable difference in KC
apoptosis between PNIPAAm coated surfaces (membranes and dishes) and uncoated surfaces. This
hypothesis was tested by measuring the percentage of caspase-3 positive KC present on PNIPAAm
coated membranes, PNIPAAm coated dishes and uncoated dishes after 24 and 72 hours of culture.
Figure 2.7 shows an overview of this study.
2.3b Methods
Cell Culture
Same as in Chapter 2.2b
Cell Culture on Thermo-Responsive Membranes
Same as in Chapter 2.2b except the cell seeding density for each membrane was changed to 105
cells/cm2 with one measurement (n = 1 ) for each time point (24 hours and 72 hours). One
measurement was viewed as sufficient since the area of a membrane was more than three times that of
Figure 2.7: Schematic showing an overview of KC cell apoptosis study. KC are seeded on three different
surfaces, PNIPAAm coated membranes, PNIPAAm coated dishes and uncoated dishes. Cells were then
cultured for 24 and 72 hours before staining with DAPI to identify all cell nuclei, and Caspase-3 to identify
apoptotic nuclei (red shaded cells).
29
the individual coated and uncoated wells.
Cell Apoptosis Detection
Three experimental groups were established: Cells seeded on (1) PNIPAAm-coated PVDF membranes
(n = 2), (2) a PNIPAAm-coated 24-well plate (n = 4 per time point) (UpCellTM Thermo Fisher
Scientific, Roskilde, Denmark), and (3) uncoated 24-well plate (n = 4 per time point) serving as the
control experiment, at a seeding density of (5 × 103 cells/cm2) for both 24 h and 3 days of culturing (n
= 4 per experimental group). At the end of the respective culture periods, cells cultured on all three
surfaces were fixed with 2% formaldehyde and immunostained afterwards with active caspase-3
monoclonal antibody (Life Technologies, Grand Island, NY) followed by goat anti-rabbit Alexa Fluor
488 antibody (Invitrogen, Eugene, OR) and DAPI.
Fluorescent Microscopy and Cell Counting
An inverted fluorescent microscope (Olympus IX81; Olympus America, Center Valley, PA) was used to
take images of the fixed cells on all three surfaces at 4X magnification. With DAPI staining
fluorescing at a different wavelength than Caspase-3 and allowing quantification of percentage cell
death. Figure 2.8A shows the result of the staining process at high magnification (20x), with the
nucleus of a dead cell colored both blue and red. Images were randomly taken at five different areas
around the well or membrane. The cells were then counted using Adobe Photoshop™ image software.
Figure 2.8B shows a 4X image randomly taken of a section of an uncoated dish, with all cell nuclei
stained blue and apoptotic cell nuclei also stained red.
Statistical Analysis
A univariate ANOVA with two factors – time (2 levels) and substrate (3 levels) was carried out on the
data set after the counting procedure was finished.
30
2.3c Results
During cell culture it was observed that the KCs attached and proliferated better in the uncoated
dishes than on the PNIPAAm coated dishes. Good cell growth was also observed in the coated inserts,
mimicking what was seen in the first section of the study, an indication that successful culturing of KC
on this surface is a repeatable process.
DAPI and Caspase-3 staining showed that percentage cell death after both 24 and 72 hours of
culture was greatest on PNIPAAm coated dishes. With Fig 2.6 illustrating 4.5 ± 0.4% and 5.5 ± 0.3%
cell apoptosis after day 1 and day 3 respectively on coated dishes. The figure also shows similar
percentages for apoptosis on uncoated dishes and coated membranes after one day of culture. After day
3 apoptosis was less on the coated membrane than both the uncoated and coated dish, with only 1.7 %
cell death. It is possible that here again, the porous nature of the membranes allowed for improved
nutrition and waste management between the KCs and the culture media, or better attachment of
PNIPAAm polymer to the surface.
Statistical analysis carried out on our data set showed that these results were not statistically
(B)
Figure 2.8: (A) 20x image of KC nuclei co-stained with fluorescent DAPI (blue) and caspase3 (red).
Nuclei that are stained with both blue and red indicate dead cells. Bar 5 um. (B) 4X image of a random
section of an uncoated dish, all cell nuclei present (Blue) from DAPI staining and all caspase-3 positive
cells (red) were counted to calculate the percentage cell apoptosis on each surface.
(A)
31
significant (two-way ANOVA testing for the effects of substrate and time in culture). Post-hoc
comparison with Bonferroni correction demonstrated a trend (p-value of 0.14) for the increased
apoptosis levels for PNIPAAm coated dishes (UpCellTM) compared to PNIPAAm coated membranes
and uncoated dishes, as also evidenced by the standard deviation bars in Figure 2.9
Figure 2.9: Culture of human keratinocytes (epithelial cells) on PNIPAAm-coated porous membrane
did not increase cell apoptosis (Caspase 3 marker), as compared to standard plastic dish control. In
contrast, there was a trend (p=0.14) for the increased apoptosis for keratinocytes cultures using
commercial UpCellTM PNIPAAm-coated dishes.
32
Chapter 3: Conclusions and Future Directions
From Chapter 1 we can clearly see that PNIPAAm grafted surfaces and hydrogels can be used for
the successful culture of multiple cell phenotypes. With specific cell responses such as adhesion,
growth, alignment, and differentiation being controlled via the composition, thickness, density and
topography of the PNIPAAm surface. Promising applications of cell sheet engineering in medical and
tissue engineering fields are also being explored given the biocompatibility of thermo-responsive
PNIPAAm surfaces and their ability produce viable cell sheets.
Our group’s exploratory studies into using KCs cultured on a thermo-responsive membrane as a
part of a treatment method for diabetic wounds are promising. As qualitative observations showed good
cell adhesion and proliferation of KCs on the PNIPAAm coated membranes in both sections of the
study. The rapid cell detachment, which was seen in Chapter 2.2c, being attributed to the membranes
porous nature. Transfer of the cell sheets was the main challenge of our study. The plastic barrier
around the insert made the removal of the membrane a demanding process. For future studies it would
be advantageous to remove the inserts circular border after spin coating with PNIPAAm. The use of a
slightly flexible “transport surface” to transfer the KCs from the coated membrane to a desired final
location, would also be an improvement on our current experimental design. Maneuvering the
membrane when it is moist is currently very challenging, so the use of this intermediary would improve
the efficiency of the process. This solution would be analogous to Pasuwat et al's hydrophilically
modified (PVDF) transfer membrane, which allowed the in vitro transfer of chondrocyte cell sheets
[21].
The second section of the study also produced promising results, as the trend that was observed
via caspase-3 staining did not show a difference between KCs cultured on PNIPAAm coated and
uncoated surface. Here we would like to extend the study to quantitatively look at KC proliferation on
33
all three surfaces (uncoated dishes, coated dishes, coated membranes). Qualitative analysis from the
experiments conducted thus far show superior cell growth on our PNIPAAm coated membranes.
After the completion of the above modifications, experiments incorporating an animal model will
be more manageable. Our group has considerable experience studying the diabetic wound healing
process in the rat animal model [61], [62]. Fusing our current KC cell sheet work with the previous in
vivo wound healing studies, would prove a huge step forward towards the creation of a therapeutic
smart band aid for the treatment of diabetic wounds, as illustrated earlier in Figure 2.1. Hopefully, the
PNIPAAm coated membranes cell compatibility is not limited to KC, as we can name a multiple other
therapeutic applications from Chapter 1 alone where improved cell viability and increased cell release
would be greatly advantageous.
34
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