marc at nanomat_md_june_06

Form and FunctionForm and FunctionEngineering approaches on a Engineering approaches on a

cellular levelcellular level

Marc R. DusseillerMarc R. Dusseiller

Groupmeeting @ nanomat, 19 June 2006Groupmeeting @ nanomat, 19 June 2006

Groupmeeting June 2006 2

OutlineOutline

• IntroductionIntroduction• Biomaterials - BiointerfaceBiomaterials - Biointerface• Cells in Culture Cells in Culture • Form an FunctionForm an Function• Cell Patterning – Cell shapeCell Patterning – Cell shape• DimensionalityDimensionality

• My ProjectMy Project• Micro 3-D Cell Culture ConceptMicro 3-D Cell Culture Concept• MicrofabricationMicrofabrication• Surface modificationSurface modification• Cell experimentsCell experiments• Conclusions and outlookConclusions and outlook

• AcknowledmentsAcknowledments


AcknowledgmentsAcknowledgments

Prof. Marcus TextorProf. Marcus Textor DoktorvaterDoktorvaterMirabai Koch, B. NiederbergerMirabai Koch, B. Niederberger Diploma studentsDiploma students BioInterfaceGroupBioInterfaceGroupD Falconnet, J Lussi, J VörösD Falconnet, J Lussi, J Vörös DiscussionsDiscussionsProf. Nic SpencerProf. Nic Spencer Head ofHead of LSSTLSST

Dr. Stefan BlunierDr. Stefan Blunier MicrofabricationMicrofabrication IMES, D-MAVTIMES, D-MAVTWulf GlatzWulf Glatz SU-8 processingSU-8 processing Micro, D-MAVTMicro, D-MAVT

Matthias Gössi, Jerome LefèvreMatthias Gössi, Jerome Lefèvre Polymer processingPolymer processing Polymer TechnologyPolymer TechnologyBrandon BürglerBrandon Bürgler SEM, mental supportSEM, mental support CeramicsCeramics

Dr. GDr. Gáábor Csbor Csúúcscs CLSMCLSM Light Microscopy CenterLight Microscopy Center

Dr. Ruth KroschewskiDr. Ruth KroschewskiDominik Schläpfer, Aldo Ferrari Dominik Schläpfer, Aldo Ferrari MDCK experimentsMDCK experiments BiochemistryBiochemistryJudith Zaugg, Luzia ReutimannJudith Zaugg, Luzia Reutimann

Dr. Michael Smith, Sheila LunaDr. Michael Smith, Sheila Luna FN, Soft FN, Soft μμ-wells-wells nanomatnanomatProf. Viola Vogel Prof. Viola Vogel Co-refereeCo-referee

Matthias LütolfMatthias Lütolf stem cells, PEG gelsstem cells, PEG gels Stanford, School of MedicineStanford, School of Medicine

Prof. Jeff Hubbell Prof. Jeff Hubbell Co-refereeCo-referee LMRP, EPFLLMRP, EPFL

IntroductionIntroduction


BiologyMaterial Science

&Engineering

From atom, proteins and cells to human beings and society


Biomaterials - BiointerfacesBiomaterials - Biointerfaces

Implants Cell culture modelsTissue engineering scaffolds


Cell – Surface InteractionsCell – Surface Interactions

• Different size scale - atoms, molecules, proteins and cellsDifferent size scale - atoms, molecules, proteins and cells• Different time scalesDifferent time scales• Dynamic complex biological systemDynamic complex biological system


History of cells in cultureHistory of cells in culture

Harrison, R.G., The cultivation of tissues in extraneous media as a method of morphogenetic study. The Anatomical Record, 1912. 6(1): p. 181-193.



Harrison, R.G., The reaction of embryonic cells to solid structures. Journal Of Experimental Zoology, 1914. 17(4): p. 521-544.

....

The behavior of the cells with The behavior of the cells with reference to the surface of the reference to the surface of the cover slip and spider web shows cover slip and spider web shows not only that the surface of a solid not only that the surface of a solid is a necessary condition but also is a necessary condition but also that when the latter has a specific that when the latter has a specific linear arrangement, as in the spider linear arrangement, as in the spider web, it has an action in influencing web, it has an action in influencing the direction of the movement, as the direction of the movement, as well as upon the form and well as upon the form and arrangement of the cells.arrangement of the cells.

Engineering Engineering approaches on a approaches on a

cellular levelcellular level


Soft Lithography – Microcontact printingSoft Lithography – Microcontact printing

Singhvi, R., et al., Engineering Cell-Shape and Function. Science, 1994. 264(5159): p. 696-698.


Cell shape influences cell behaviorCell shape influences cell behavior

• Surface chemistry and patterning Surface chemistry and patterning enable new experimentsenable new experiments

• Endothelial cells on adhesive islandsEndothelial cells on adhesive islands

• Cell shape controls cell behaviorCell shape controls cell behavior

• Cell shape is defined by the amountCell shape is defined by the amountof spreading allowed by the adhesiveof spreading allowed by the adhesiveislandisland

Chen, C.S., et al., Geometric control of cell life and death. Science, 1997. 276(5317): p. 1425-1428.


Shape/spreading controls stem cell differentiationShape/spreading controls stem cell differentiation

McBeath, R., et al., Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 2004. 6(4): p. 483-95.

• Human mesenchymal cells (hMSCs) Human mesenchymal cells (hMSCs) on adhesive islands of different sizeson adhesive islands of different sizes

• Cell shape controls stem cell lineage Cell shape controls stem cell lineage commitment into adipocytes or commitment into adipocytes or osteoblastsosteoblasts

• Increased spreading leads to an Increased spreading leads to an increase in Rho dependant contractilityincrease in Rho dependant contractility

• These studies demonstrate that These studies demonstrate that mechanical cues experienced in mechanical cues experienced in developmental and adult contexts, developmental and adult contexts, embodied by cell shape, cytoskeletal embodied by cell shape, cytoskeletal tension, and RhoA signaling, are tension, and RhoA signaling, are integral to the commitment of stem cell integral to the commitment of stem cell fate.fate.


ECM geometry guides orientation of mitosisECM geometry guides orientation of mitosis

Thery, M., et al., The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol, 2005. 7(10): p. 947-53.

• Geometry of islands of adhesive ECM components (FN) control cell shape Geometry of islands of adhesive ECM components (FN) control cell shape and orientation of the mitotic spindle during cell divisionand orientation of the mitotic spindle during cell division


Multicellular Patterning – Global InfluenceMulticellular Patterning – Global Influence

• Large adhesive patterns to study the Large adhesive patterns to study the effect of forces in multicellular systemseffect of forces in multicellular systems

• Contractile forces generated by the Contractile forces generated by the cells lead to an increased proliferation cells lead to an increased proliferation in those areas with the highest forcesin those areas with the highest forces

Nelson, C.M., et al., Emergent patterns of growth controlled by multicellular form and mechanics. PNAS, 2005. 102(33): p. 11594-11599.


Cell Patterning @ BioInterface GroupCell Patterning @ BioInterface Group

• Model surfacesModel surfaces• Controlled surface chemistryControlled surface chemistry• Controlled mechanical propertiesControlled mechanical properties

• Surface patterningSurface patterning• Control of cell shapeControl of cell shape

• Cell shapeCell shape• cell behaviorcell behavior• Adhesion sitesAdhesion sites• cytoskeleton organizationcytoskeleton organization• cell mechanicscell mechanics• stem cell differentiationstem cell differentiation

• 2-D !2-D !

Michel, R., et al., Selective molecular assembly patterning: A new approach to micro- and nanochemical patterning of surfaces for biological applications. Langmuir, 2002. 18(8): p. 3281-3287.

Falconnet, D., et al., A combined photolithographic and molecular-assembly approach to produce functional micropatterns for applications in the biosciences. Advanced Functional Materials, 2004. 14(8): p. 749-756.


The Third DimensionThe Third Dimension

““3-D culture will allow a lot of basic questions to be 3-D culture will allow a lot of basic questions to be answered before having to turn to whole animal answered before having to turn to whole animal research”, Friedlresearch”, Friedl

““Our preliminary analysis shows that the expression Our preliminary analysis shows that the expression profile in 3-D is much closer to profile in 3-D is much closer to in vivoin vivo expression expression profiles than the profile we’ve seen in 2-D”, Griffithprofiles than the profile we’ve seen in 2-D”, Griffith

• adhesion fundamentally differentadhesion fundamentally different• less prominent actin stress fibersless prominent actin stress fibers• a lot of unanswered questionsa lot of unanswered questions

• 3-D models difficult3-D models difficult• limited control of interactionslimited control of interactions• no control of cell shapeno control of cell shape

My ProjectMy Project


Microenvironment of Cells in CultureMicroenvironment of Cells in Culture

• Soluble cuesSoluble cues Growth factors Growth factors

pHpH

IonsIons

Media supplementsMedia supplements

etc.etc.

• Insoluble cuesInsoluble cuesCell-cell contactsCell-cell contacts

Cell-matrix contactsCell-matrix contacts

Forces by other cellsForces by other cells

Mechanical properties of ECMMechanical properties of ECM

Transmission of forces Transmission of forces

• Spatial organization Spatial organization of all cuesof all cues


Micro 3D cell culture systemMicro 3D cell culture system

Dusseiller, M.R., et al., Microfabricated three-dimensional environments for single cell studies. Biointerphases, 2006. 1(1): p. P1.


• SiliconSiliconvariety of techniques from microelectronics variety of techniques from microelectronics

and MEMS fieldand MEMS field• PhotolithographyPhotolithography• deep Reactive Ion Etching (dRIE, Bosch deep Reactive Ion Etching (dRIE, Bosch

process, ICP etching)process, ICP etching)

Microfabrication - Silicon etchingMicrofabrication - Silicon etching


ReplicationReplication

PDMS PEG-hydrogelPolystyrene

PS (polystyrene)PS (polystyrene) Young’s Modulus ~ 10Young’s Modulus ~ 1099 Pa Pacheap, transparentcheap, transparentwidely used as cell culture material widely used as cell culture material hot embossinghot embossing

PDMS (polydimethylsiloxane)PDMS (polydimethylsiloxane) Young’s Modulus ~ 10Young’s Modulus ~ 1044 Pa –10 Pa –1066 Pa Paelastomeric, transparentelastomeric, transparentreplica moldingreplica molding

PEG-hydrogelsPEG-hydrogelsYoung’s Modulus ~ 100 Pa – 10Young’s Modulus ~ 100 Pa – 1044 Pa PaTissue like stiffnessTissue like stiffnessDiffusion of nutrients etc.Diffusion of nutrients etc.Incorporation of functional groupsIncorporation of functional groups


PDMS thin film devicePDMS thin film device

• Array of microwells in PDMSArray of microwells in PDMSDifferent shapes and dimensions (10 to 30 micron diameter)Different shapes and dimensions (10 to 30 micron diameter)

Different mechanical properties by changing the cross linker densityDifferent mechanical properties by changing the cross linker density

• Assembled into a glass bottom petri-dishAssembled into a glass bottom petri-dish

• Selective passivation of plateauSelective passivation of plateau


Mechanical Properties of PDMSMechanical Properties of PDMS

• Standard tensile testStandard tensile test• 12 mm dogbones punched out from 1 mm thick cast pdms films12 mm dogbones punched out from 1 mm thick cast pdms films• Different crosslinker densitiesDifferent crosslinker densities


Surface ModificationSurface Modification

OO22 plasma treatment of PS or PDMS plasma treatment of PS or PDMS• PS: surface oxidation – negative charges PS: surface oxidation – negative charges

(COO(COO--))• PDMS: generation of glass like surface (SiOPDMS: generation of glass like surface (SiO22))

PLL-PLL-gg-PEG -PEG Poly(L-Lysine) Poly(L-Lysine) graftedgrafted Poly(Ethylene Glycol) Poly(Ethylene Glycol)

• self assembles on negatively charged self assembles on negatively charged surfacessurfaces

metal oxides, TCPSmetal oxides, TCPS• resistant to protein adsorptionresistant to protein adsorption• functional groupsfunctional groups

peptide sequences (cont. RGD - integrin peptide sequences (cont. RGD - integrin binding)binding)

specific binding sites (PLL-specific binding sites (PLL-gg-PEG/biotin, -PEG/biotin, PPB)PPB)

• specific interactions with cellsspecific interactions with cells


PLL-g-PEG PrintingPLL-g-PEG Printing

• Variety of techniques investigatedVariety of techniques investigated

• Wet printing of PLL-g-PEG using Wet printing of PLL-g-PEG using a hydrogela hydrogel

• Passivation of plateauPassivation of plateau

• Backfilling with proteinsBackfilling with proteins

• ReproducibilityReproducibility• HomogeneityHomogeneity


Cell Experiments using the micro-3-D deviceCell Experiments using the micro-3-D device

• feasibility to put cells in wells using MDCK cells feasibility to put cells in wells using MDCK cells (Madin-Darby Canine Kidney = cocker spaniel = dog)(Madin-Darby Canine Kidney = cocker spaniel = dog)

• Single Cell Polarization of MDCK cellsSingle Cell Polarization of MDCK cells• epithelial morphology epithelial morphology - polarized cells in confluent layer- polarized cells in confluent layer

- columnar shape- columnar shape

• 3-D adhesion and fibronectin matrix assembly 3-D adhesion and fibronectin matrix assembly

• CLSM - 3d reconstructionCLSM - 3d reconstruction


Confocal Laser Scanning MicroscopyConfocal Laser Scanning Microscopy


Control of Cell Shape - MDCKsControl of Cell Shape - MDCKs

yfp-plasma membrane gp58 (green) lateral markerCLIP170 (red) growing microtubules


Control of Cell Shape in 3DControl of Cell Shape in 3D


Single Cell Polarization of MDCKsSingle Cell Polarization of MDCKs

gp58 (green) lateral markerCLIP170 (red) growing microtubules


FN Matrix assemblyFN Matrix assembly

actin (green)FN (red)


Control of Cell Shape - cytoskeletonControl of Cell Shape - cytoskeleton


Conclusions & OutlookConclusions & Outlook

• New tool for model studies on single cellsNew tool for model studies on single cells

• simple replication methodssimple replication methods• different mechanical properties different mechanical properties • passivation of plateau surface (further optimization needed)passivation of plateau surface (further optimization needed)• functionalization of microwell surface with functionalization of microwell surface with

proteins and PLL-g-PEG/func (other chemistries inside the wells)proteins and PLL-g-PEG/func (other chemistries inside the wells)• practical devicepractical device

• Applied to different biological questionsApplied to different biological questions

• optimized culture and microscopy conditionsoptimized culture and microscopy conditions• controlled 3-D shape of single cells (depends on volume of microwell)controlled 3-D shape of single cells (depends on volume of microwell)• first interesting result first interesting result • further investigation will lead to a deeper understanding of how the 3-D further investigation will lead to a deeper understanding of how the 3-D

shape controls cell functionshape controls cell function


Future ApplicationsFuture Applications

Deeper understanding of cells in cultureDeeper understanding of cells in culture

Advanced cell culture modelsAdvanced cell culture models

Integration & MultiplexingIntegration & Multiplexing

• Fundamental Cell BiologyFundamental Cell Biology• mechanostransductionmechanostransduction• stem cell differentiationstem cell differentiation

• Pharmaceutical IndustryPharmaceutical Industry• high through put drug testinghigh through put drug testing• reduce animal testsreduce animal tests

• Cell based sensorsCell based sensors• Environmental monitoringEnvironmental monitoring• Antigen detectionAntigen detection

• Hybrid devicesHybrid devices• Bio Fuel CellsBio Fuel Cells


The EndThe End


AcknowledgmentsAcknowledgments

Prof. Marcus TextorProf. Marcus Textor DoktorvaterDoktorvaterMirabai Koch, B. NiederbergerMirabai Koch, B. Niederberger Diploma studentsDiploma students BioInterfaceGroupBioInterfaceGroupD Falconnet, J Lussi, J VörösD Falconnet, J Lussi, J Vörös DiscussionsDiscussionsProf. Nic SpencerProf. Nic Spencer Head ofHead of LSSTLSST

Dr. Stefan BlunierDr. Stefan Blunier MicrofabricationMicrofabrication IMES, D-MAVTIMES, D-MAVTWulf GlatzWulf Glatz SU-8 processingSU-8 processing Micro, D-MAVTMicro, D-MAVT

Matthias Gössi, Jerome LefèvreMatthias Gössi, Jerome Lefèvre Polymer processingPolymer processing Polymer TechnologyPolymer TechnologyBrandon BürglerBrandon Bürgler SEM, mental supportSEM, mental support CeramicsCeramics

Dr. GDr. Gáábor Csbor Csúúcscs CLSMCLSM Light Microscopy CenterLight Microscopy Center

Dr. Ruth KroschewskiDr. Ruth KroschewskiDominik Schläpfer, Aldo Ferrari Dominik Schläpfer, Aldo Ferrari MDCK experimentsMDCK experiments BiochemistryBiochemistryJudith Zaugg, Luzia ReutimannJudith Zaugg, Luzia Reutimann

Dr. Michael Smith, Sheila LunaDr. Michael Smith, Sheila Luna FN, Soft FN, Soft μμ-wells-wells nanomatnanomatProf. Viola Vogel Prof. Viola Vogel Co-refereeCo-referee

Matthias LütolfMatthias Lütolf stem cells, PEG gelsstem cells, PEG gels Stanford, School of MedicineStanford, School of Medicine

Prof. Jeff Hubbell Prof. Jeff Hubbell Co-refereeCo-referee LMRP, EPFLLMRP, EPFL

Form and FunctionForm and Function


Aristotle – Form and FunctionAristotle – Form and Function

• The material causeThe material cause

• The efficient cause The efficient cause • The formal causeThe formal cause• The final causeThe final cause

From „On the Parts of Animals“From „On the Parts of Animals“

Moreover, when any one of the parts or structures, be it which it Moreover, when any one of the parts or structures, be it which it may, is under discussion, it must not be supposed that it is its may, is under discussion, it must not be supposed that it is its material composition to which attention is being directed or material composition to which attention is being directed or which is the object of the discussion, but the relation of such which is the object of the discussion, but the relation of such part to the total form. Similarly, the true object of architecture part to the total form. Similarly, the true object of architecture is not bricks, mortar, or timber, but the house;…is not bricks, mortar, or timber, but the house;…


Lloyd Wright - Form and FunctionLloyd Wright - Form and Function

American architect (1867–1959) American architect (1867–1959)

““Form follows function- that has been Form follows function- that has been misunderstood. Form and function should be misunderstood. Form and function should be one, joined in a spiritual union."one, joined in a spiritual union."


TensegrityTensegrity

Ingber, D.E., Cellular mechanotransduction: putting all the pieces together again. FASEB J., 2006. 20(7): p. 811-827.


Single Cell Polarization of MDCKsSingle Cell Polarization of MDCKs

gp58 (green) lateral markerCLIP170 (red) growing microtubules


Goal of this Work - Goal of this Work - Micro-3-D Cell CulturingMicro-3-D Cell Culturing

• 2.5 dimensional cell array2.5 dimensional cell array

• single cells (or small clusters)single cells (or small clusters)

• control of 3-D shapecontrol of 3-D shape

• closer to closer to in vivo ?in vivo ?

• Material engineering aspectsMaterial engineering aspects

• microfabrication techniques microfabrication techniques • optical properties optical properties • mechanical propertiesmechanical properties

• area selective surface chemistryarea selective surface chemistry • device developmentdevice development


FN Matrix assemblyFN Matrix assembly

actin (green)FN (red)



Harrison, R.G., The reaction of embryonic cells to solid structures. Journal Of Experimental Zoology, 1914. 17(4): p. 521-544.


Cell Culture ModelsCell Culture Models

• Petri dish - Petri dish - German bacteriologist Julius Richard Petri German bacteriologist Julius Richard Petri (1852-1921) who invented it in 1877(1852-1921) who invented it in 1877


Cell PatterningCell Patterning

• Cell shape is randomCell shape is random

• The complex ECM protein mixture The complex ECM protein mixture triggers various intracellular signals triggers various intracellular signals

Tissue culture polystyreneTissue culture polystyrene

Serum proteinsSerum proteins

100 µm

Biologically designed model surfaceBiologically designed model surface

100 µm

• Cell shape is controlledCell shape is controlled

• Defined ligand chemistry on Defined ligand chemistry on adhesive spotsadhesive spots

• Controlled density of ligands Controlled density of ligands (peptides or proteins)(peptides or proteins)

Falconnet, D.; Koenig, A.; Assi, T.; Textor, M. Falconnet, D.; Koenig, A.; Assi, T.; Textor, M. Advanced Functional MaterialsAdvanced Functional Materials 2004, 2004, 1414, 749-756., 749-756.


Cell BiologyCell Biology

• Kidney tubuleKidney tubuleSingle epithelial layerSingle epithelial layer

• Cell - ECM contactCell - ECM contactECM proteins (fibronectin, RGD sequence)ECM proteins (fibronectin, RGD sequence)

αβαβ Integrin binding (transmembrane) Integrin binding (transmembrane)

connection to actin cytoskeletonconnection to actin cytoskeleton

regulation of cell behaviorregulation of cell behavior

spreadingspreading

actin stress fibersactin stress fibers


Cell BiologyCell Biology

• Polarized epithelial cellPolarized epithelial celltypical columnar morphologytypical columnar morphology

apical / basal part (outside / inside)apical / basal part (outside / inside)

• Cell partsCell partsmembranemembrane

nucleusnucleus

organellesorganelles

cell - cell contacts, cell - ECM contactscell - cell contacts, cell - ECM contacts

• CytoskeletonCytoskeletonregulation of cell behaviorregulation of cell behavior

transport and communicationtransport and communication

actin filamentsactin filaments

mechanical structuremechanical structure


3-D Microscopy3-D Microscopy

• Confocal Laser Scanning Microscopy (CLSM)Confocal Laser Scanning Microscopy (CLSM)local probe scanning microscopy (STM, AFM, SEM)local probe scanning microscopy (STM, AFM, SEM)

local fluorescence is detectedlocal fluorescence is detected

confocal planes - pinholeconfocal planes - pinhole

3 dimensional, confocal slices3 dimensional, confocal slices

z resolution (600nm) lower than xy (200 nm)z resolution (600nm) lower than xy (200 nm)

crosssection through single wellleft unstamped; right 2x stampedbelow projection of all confocal slices


XPS of embossed PS XPS of embossed PS ↔ ↔ TCPSTCPS

• After plasma treatmentAfter plasma treatmentembossed PS very similar embossed PS very similar

surface chemistry to TCPSsurface chemistry to TCPS

• ContaminationsContaminationsPDMS (Si)PDMS (Si)FFZn (NW / B-stock)Zn (NW / B-stock)


MechanotransductionMechanotransduction

marc at nanomat_md_june_06

Technology

engineering cellshape

culturehistory of cells

biointerface cells

cells withreference

d cell culture conceptmicro

cells withthe behavior

peg gelsstem cells

reaction of embryonic