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Marc at nanomat_md_june_06 Marc at nanomat_md_june_06 Presentation Transcript

  • Form and FunctionForm and FunctionEngineering approaches on aEngineering approaches on acellular levelcellular levelMarc R. DusseillerMarc R. DusseillerGroupmeeting @ nanomat, 19 June 2006Groupmeeting @ nanomat, 19 June 2006
  • Groupmeeting June 2006 2OutlineOutline• IntroductionIntroduction• Biomaterials - BiointerfaceBiomaterials - Biointerface• Cells in CultureCells 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
  • Groupmeeting June 2006 3AcknowledgmentsAcknowledgmentsProf. 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 LSSTLSSTDr. Stefan BlunierDr. Stefan Blunier MicrofabricationMicrofabrication IMES, D-MAVTIMES, D-MAVTWulf GlatzWulf Glatz SU-8 processingSU-8 processing Micro, D-MAVTMicro, D-MAVTMatthias Gössi, Jerome LefèvreMatthias Gössi, Jerome Lefèvre Polymer processingPolymer processing Polymer TechnologyPolymer TechnologyBrandon BürglerBrandon Bürgler SEM, mental supportSEM, mental support CeramicsCeramicsDr. GDr. Gáábor Csbor Csúúcscs CLSMCLSM Light Microscopy CenterLight Microscopy CenterDr. Ruth KroschewskiDr. Ruth KroschewskiDominik Schläpfer, Aldo FerrariDominik Schläpfer, Aldo Ferrari MDCK experimentsMDCK experiments BiochemistryBiochemistryJudith Zaugg, Luzia ReutimannJudith Zaugg, Luzia ReutimannDr. Michael Smith, Sheila LunaDr. Michael Smith, Sheila Luna FN, SoftFN, Soft μμ-wells-wells nanomatnanomatProf. Viola VogelProf. Viola Vogel Co-refereeCo-refereeMatthias LütolfMatthias Lütolf stem cells, PEG gelsstem cells, PEG gels Stanford, School of MedicineStanford, School of MedicineProf. Jeff HubbellProf. Jeff Hubbell Co-refereeCo-referee LMRP, EPFLLMRP, EPFL View slide
  • IntroductionIntroduction View slide
  • Groupmeeting June 2006 5BiologyMaterial Science&EngineeringFrom atom, proteins and cells to human beings and society
  • Groupmeeting June 2006 6Biomaterials - BiointerfacesBiomaterials - BiointerfacesImplants Cell culture modelsTissue engineering scaffolds
  • Groupmeeting June 2006 7Cell – 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
  • Groupmeeting June 2006 8History of cells in cultureHistory of cells in cultureHarrison, R.G., The cultivation of tissues in extraneous media as a method ofmorphogenetic study. The Anatomical Record, 1912. 6(1): p. 181-193.
  • Groupmeeting June 2006 9History of cells in cultureHistory of cells in cultureHarrison, 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 withThe behavior of the cells withreference to the surface of thereference to the surface of thecover slip and spider web showscover slip and spider web showsnot only that the surface of a solidnot only that the surface of a solidis a necessary condition but alsois a necessary condition but alsothat when the latter has a specificthat when the latter has a specificlinear arrangement, as in the spiderlinear arrangement, as in the spiderweb, it has an action in influencingweb, it has an action in influencingthe direction of the movement, asthe direction of the movement, aswell as upon the form andwell as upon the form andarrangement of the cells.arrangement of the cells.
  • EngineeringEngineeringapproaches on aapproaches on acellular levelcellular level
  • Groupmeeting June 2006 11Soft Lithography – Microcontact printingSoft Lithography – Microcontact printingSinghvi, R., et al., Engineering Cell-Shape and Function.Science, 1994. 264(5159): p. 696-698.
  • Groupmeeting June 2006 12Soft Lithography – Microcontact printingSoft Lithography – Microcontact printingSinghvi, R., et al., Engineering Cell-Shape and Function.Science, 1994. 264(5159): p. 696-698.
  • Groupmeeting June 2006 13Cell shape influences cell behaviorCell shape influences cell behavior• Surface chemistry and patterningSurface chemistry and patterningenable 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 adhesiveislandislandChen, C.S., et al., Geometric control of cell life and death.Science, 1997. 276(5317): p. 1425-1428.
  • Groupmeeting June 2006 14Shape/spreading controls stem cell differentiationShape/spreading controls stem cell differentiationMcBeath, R., et al., Cell shape, cytoskeletal tension, and RhoA regulatestem 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 lineageCell shape controls stem cell lineagecommitment into adipocytes orcommitment into adipocytes orosteoblastsosteoblasts• Increased spreading leads to anIncreased spreading leads to anincrease in Rho dependant contractilityincrease in Rho dependant contractility• These studies demonstrate thatThese studies demonstrate thatmechanical cues experienced inmechanical cues experienced indevelopmental and adult contexts,developmental and adult contexts,embodied by cell shape, cytoskeletalembodied by cell shape, cytoskeletaltension, and RhoA signaling, aretension, and RhoA signaling, areintegral to the commitment of stem cellintegral to the commitment of stem cellfate.fate.
  • Groupmeeting June 2006 15ECM geometry guides orientation of mitosisECM geometry guides orientation of mitosisThery, M., et al., The extracellular matrix guides the orientation ofthe cell division axis. Nat Cell Biol, 2005. 7(10): p. 947-53.• Geometry of islands of adhesive ECM components (FN) control cell shapeGeometry of islands of adhesive ECM components (FN) control cell shapeand orientation of the mitotic spindle during cell divisionand orientation of the mitotic spindle during cell division
  • Groupmeeting June 2006 16Multicellular Patterning – Global InfluenceMulticellular Patterning – Global Influence• Large adhesive patterns to study theLarge adhesive patterns to study theeffect of forces in multicellular systemseffect of forces in multicellular systems• Contractile forces generated by theContractile forces generated by thecells lead to an increased proliferationcells lead to an increased proliferationin those areas with the highest forcesin those areas with the highest forcesNelson, C.M., et al., Emergent patterns of growth controlled by multicellularform and mechanics. PNAS, 2005. 102(33): p. 11594-11599.
  • Groupmeeting June 2006 17Cell 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 newapproach to micro- and nanochemical patterning of surfaces forbiological applications. Langmuir, 2002. 18(8): p. 3281-3287.Falconnet, D., et al., A combined photolithographic and molecular-assemblyapproach to produce functional micropatterns for applications in thebiosciences. Advanced Functional Materials, 2004. 14(8): p. 749-756.
  • Groupmeeting June 2006 18The Third DimensionThe Third Dimension““3-D culture will allow a lot of basic questions to be3-D culture will allow a lot of basic questions to beanswered before having to turn to whole animalanswered before having to turn to whole animalresearch”, Friedlresearch”, Friedl““Our preliminary analysis shows that the expressionOur preliminary analysis shows that the expressionprofile in 3-D is much closer toprofile in 3-D is much closer to in vivoin vivo expressionexpressionprofiles 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
  • Groupmeeting June 2006 20Microenvironment of Cells in CultureMicroenvironment of Cells in Culture• Soluble cuesSoluble cuesGrowth factorsGrowth factorspHpHIonsIonsMedia supplementsMedia supplementsetc.etc.• Insoluble cuesInsoluble cuesCell-cell contactsCell-cell contactsCell-matrix contactsCell-matrix contactsForces by other cellsForces by other cellsMechanical properties of ECMMechanical properties of ECMTransmission of forcesTransmission of forces• Spatial organizationSpatial organizationof all cuesof all cues
  • Groupmeeting June 2006 21Micro 3D cell culture systemMicro 3D cell culture systemDusseiller, M.R., et al., Microfabricated three-dimensional environmentsfor single cell studies. Biointerphases, 2006. 1(1): p. P1.
  • Groupmeeting June 2006 22• SiliconSiliconvariety of techniques from microelectronicsvariety of techniques from microelectronicsand MEMS fieldand MEMS field• PhotolithographyPhotolithography• deep Reactive Ion Etching (dRIE, Boschdeep Reactive Ion Etching (dRIE, Boschprocess, ICP etching)process, ICP etching)Microfabrication - Silicon etchingMicrofabrication - Silicon etching
  • Groupmeeting June 2006 23ReplicationReplicationPDMS PEG-hydrogelPolystyrenePS (polystyrene)PS (polystyrene)Young’s Modulus ~ 10Young’s Modulus ~ 1099PaPacheap, transparentcheap, transparentwidely used as cell culture materialwidely used as cell culture materialhot embossinghot embossingPDMS (polydimethylsiloxane)PDMS (polydimethylsiloxane)Young’s Modulus ~ 10Young’s Modulus ~ 1044Pa –10Pa –1066PaPaelastomeric, transparentelastomeric, transparentreplica moldingreplica moldingPEG-hydrogelsPEG-hydrogelsYoung’s Modulus ~ 100 Pa – 10Young’s Modulus ~ 100 Pa – 1044PaPaTissue like stiffnessTissue like stiffnessDiffusion of nutrients etc.Diffusion of nutrients etc.Incorporation of functional groupsIncorporation of functional groups
  • Groupmeeting June 2006 24PDMS 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
  • Groupmeeting June 2006 25Mechanical 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
  • Groupmeeting June 2006 26Surface ModificationSurface ModificationOO22 plasma treatment of PS or PDMSplasma treatment of PS or PDMS• PS: surface oxidation – negative chargesPS: surface oxidation – negative charges(COO(COO--))• PDMS: generation of glass like surface (SiOPDMS: generation of glass like surface (SiO22))PLL-PLL-gg-PEG-PEGPoly(L-Lysine)Poly(L-Lysine) graftedgrafted Poly(Ethylene Glycol)Poly(Ethylene Glycol)• self assembles on negatively chargedself assembles on negatively chargedsurfacessurfacesmetal oxides, TCPSmetal oxides, TCPS• resistant to protein adsorptionresistant to protein adsorption• functional groupsfunctional groupspeptide sequences (cont. RGD - integrinpeptide sequences (cont. RGD - integrinbinding)binding)specific binding sites (PLL-specific binding sites (PLL-gg-PEG/biotin,-PEG/biotin,PPB)PPB)• specific interactions with cellsspecific interactions with cells
  • Groupmeeting June 2006 27PLL-g-PEG PrintingPLL-g-PEG Printing• Variety of techniques investigatedVariety of techniques investigated• Wet printing of PLL-g-PEG usingWet printing of PLL-g-PEG usinga hydrogela hydrogel• Passivation of plateauPassivation of plateau• Backfilling with proteinsBackfilling with proteins• ReproducibilityReproducibility• HomogeneityHomogeneity
  • Groupmeeting June 2006 28Cell Experiments using the micro-3-D deviceCell Experiments using the micro-3-D device• feasibility to put cells in wells using MDCK cellsfeasibility 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 morphologyepithelial morphology - polarized cells in confluent layer- polarized cells in confluent layer- columnar shape- columnar shape• 3-D adhesion and fibronectin matrix assembly3-D adhesion and fibronectin matrix assembly• CLSM - 3d reconstructionCLSM - 3d reconstruction
  • Groupmeeting June 2006 29Confocal Laser Scanning MicroscopyConfocal Laser Scanning Microscopy
  • Groupmeeting June 2006 30Control of Cell Shape - MDCKsControl of Cell Shape - MDCKsyfp-plasma membrane gp58 (green) lateral markerCLIP170 (red) growing microtubules
  • Groupmeeting June 2006 31Control of Cell Shape in 3DControl of Cell Shape in 3D
  • Groupmeeting June 2006 32Single Cell Polarization of MDCKsSingle Cell Polarization of MDCKsgp58 (green) lateral markerCLIP170 (red) growing microtubules
  • Groupmeeting June 2006 33FN Matrix assemblyFN Matrix assemblyactin (green)FN (red)
  • Groupmeeting June 2006 34Control of Cell Shape - cytoskeletonControl of Cell Shape - cytoskeleton
  • Groupmeeting June 2006 35Conclusions & OutlookConclusions & Outlook• New tool for model studies on single cellsNew tool for model studies on single cells• simple replication methodssimple replication methods• different mechanical propertiesdifferent mechanical properties• passivation of plateau surface (further optimization needed)passivation of plateau surface (further optimization needed)• functionalization of microwell surface withfunctionalization of microwell surface withproteins 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 resultfirst interesting result• further investigation will lead to a deeper understanding of how the 3-Dfurther investigation will lead to a deeper understanding of how the 3-Dshape controls cell functionshape controls cell function
  • Groupmeeting June 2006 36Future ApplicationsFuture ApplicationsDeeper understanding of cells in cultureDeeper understanding of cells in cultureAdvanced cell culture modelsAdvanced cell culture modelsIntegration & 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
  • Groupmeeting June 2006 38The EndThe End
  • Groupmeeting June 2006 39AcknowledgmentsAcknowledgmentsProf. 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 LSSTLSSTDr. Stefan BlunierDr. Stefan Blunier MicrofabricationMicrofabrication IMES, D-MAVTIMES, D-MAVTWulf GlatzWulf Glatz SU-8 processingSU-8 processing Micro, D-MAVTMicro, D-MAVTMatthias Gössi, Jerome LefèvreMatthias Gössi, Jerome Lefèvre Polymer processingPolymer processing Polymer TechnologyPolymer TechnologyBrandon BürglerBrandon Bürgler SEM, mental supportSEM, mental support CeramicsCeramicsDr. GDr. Gáábor Csbor Csúúcscs CLSMCLSM Light Microscopy CenterLight Microscopy CenterDr. Ruth KroschewskiDr. Ruth KroschewskiDominik Schläpfer, Aldo FerrariDominik Schläpfer, Aldo Ferrari MDCK experimentsMDCK experiments BiochemistryBiochemistryJudith Zaugg, Luzia ReutimannJudith Zaugg, Luzia ReutimannDr. Michael Smith, Sheila LunaDr. Michael Smith, Sheila Luna FN, SoftFN, Soft μμ-wells-wells nanomatnanomatProf. Viola VogelProf. Viola Vogel Co-refereeCo-refereeMatthias LütolfMatthias Lütolf stem cells, PEG gelsstem cells, PEG gels Stanford, School of MedicineStanford, School of MedicineProf. Jeff HubbellProf. Jeff Hubbell Co-refereeCo-referee LMRP, EPFLLMRP, EPFL
  • Form and FunctionForm and Function
  • Groupmeeting June 2006 41Aristotle – Form and FunctionAristotle – Form and Function• The material causeThe material cause• The efficient causeThe efficient cause• The formal causeThe formal cause• The final causeThe final causeFrom „On the Parts of Animals“From „On the Parts of Animals“Moreover, when any one of the parts or structures, be it which itMoreover, when any one of the parts or structures, be it which itmay, is under discussion, it must not be supposed that it is itsmay, is under discussion, it must not be supposed that it is itsmaterial composition to which attention is being directed ormaterial composition to which attention is being directed orwhich is the object of the discussion, but the relation of suchwhich is the object of the discussion, but the relation of suchpart to the total form. Similarly, the true object of architecturepart to the total form. Similarly, the true object of architectureis not bricks, mortar, or timber, but the house;…is not bricks, mortar, or timber, but the house;…
  • Groupmeeting June 2006 42Lloyd Wright - Form and FunctionLloyd Wright - Form and FunctionAmerican architect (1867–1959)American architect (1867–1959)““Form follows function- that has beenForm follows function- that has beenmisunderstood. Form and function should bemisunderstood. Form and function should beone, joined in a spiritual union."one, joined in a spiritual union."
  • Groupmeeting June 2006 43TensegrityTensegrityIngber, D.E., Cellular mechanotransduction: putting all the pieces togetheragain. FASEB J., 2006. 20(7): p. 811-827.
  • Groupmeeting June 2006 44Single Cell Polarization of MDCKsSingle Cell Polarization of MDCKsgp58 (green) lateral markerCLIP170 (red) growing microtubules
  • Groupmeeting June 2006 45Goal 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 tocloser to in vivo ?in vivo ?• Material engineering aspectsMaterial engineering aspects• microfabrication techniquesmicrofabrication techniques• optical propertiesoptical properties• mechanical propertiesmechanical properties• area selective surface chemistryarea selective surface chemistry• device developmentdevice development
  • Groupmeeting June 2006 46FN Matrix assemblyFN Matrix assemblyactin (green)FN (red)
  • Groupmeeting June 2006 47History of cells in cultureHistory of cells in cultureHarrison, R.G., The reaction of embryonic cells to solid structures.Journal Of Experimental Zoology, 1914. 17(4): p. 521-544.
  • Groupmeeting June 2006 48Cell Culture ModelsCell Culture Models• Petri dish -Petri dish - German bacteriologist Julius Richard PetriGerman bacteriologist Julius Richard Petri(1852-1921) who invented it in 1877(1852-1921) who invented it in 1877
  • Groupmeeting June 2006 49Cell PatterningCell Patterning• Cell shape is randomCell shape is random• The complex ECM protein mixtureThe complex ECM protein mixturetriggers various intracellular signalstriggers various intracellular signalsTissue culture polystyreneTissue culture polystyreneSerum proteinsSerum proteins100 µmBiologically designed model surfaceBiologically designed model surface100 µm• Cell shape is controlledCell shape is controlled• Defined ligand chemistry onDefined ligand chemistry onadhesive spotsadhesive spots• Controlled density of ligandsControlled 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.
  • Groupmeeting June 2006 50Cell 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 cytoskeletonregulation of cell behaviorregulation of cell behaviorspreadingspreadingactin stress fibersactin stress fibers
  • Groupmeeting June 2006 51Cell BiologyCell Biology• Polarized epithelial cellPolarized epithelial celltypical columnar morphologytypical columnar morphologyapical / basal part (outside / inside)apical / basal part (outside / inside)• Cell partsCell partsmembranemembranenucleusnucleusorganellesorganellescell - cell contacts, cell - ECM contactscell - cell contacts, cell - ECM contacts• CytoskeletonCytoskeletonregulation of cell behaviorregulation of cell behaviortransport and communicationtransport and communicationactin filamentsactin filamentsmechanical structuremechanical structure
  • Groupmeeting June 2006 523-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 detectedconfocal planes - pinholeconfocal planes - pinhole3 dimensional, confocal slices3 dimensional, confocal slicesz 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 confocalslices
  • Groupmeeting June 2006 53XPS of embossed PSXPS of embossed PS ↔↔ TCPSTCPS• After plasma treatmentAfter plasma treatmentembossed PS very similarembossed PS very similarsurface chemistry to TCPSsurface chemistry to TCPS• ContaminationsContaminationsPDMS (Si)PDMS (Si)FFZn (NW / B-stock)Zn (NW / B-stock)
  • Groupmeeting June 2006 54MechanotransductionMechanotransduction