Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity

Abstract

We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different heights to yield substrates of different rigidities. The tips of the elastomeric microposts are functionalized with extracellular matrix through microcontact printing to promote cell adhesion. These substrates, therefore, present the same topographical cues to adherent cells while varying substrate rigidity only through manipulation of micropost height. This protocol describes how to fabricate the silicon micropost array masters (2 weeks to complete) and elastomeric substrates (3 d), as well as how to perform cell culture experiments (1–14 d), immunofluorescence imaging (2 d), traction force analysis (2 d) and stem cell differentiation assays (1 d) on these substrates in order to examine the effect of substrate rigidity on stem cell morphology, traction force generation, focal adhesion organization and differentiation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Flow diagram of the different sections of the protocol.
Figure 2: Characterization of micropost array masters and substrates.
Figure 3: Replica molding of a micropost array master.
Figure 4: Microcontact printing on micropost array substrates.
Figure 5: Basic imaging of cells on micropost array substrates.
Figure 6: General algorithm for analyzing traction forces from the micropost array substrate.
Figure 7: Representative images of cells on microposts.
Figure 8: Analysis of stem cell differentiation on micropost array substrates.

References

  1. 1

    Fuchs, E., Tumbar, T. & Guasch, G. Socializing with the neighbors: stem cells and their niche. Cell 116, 769–778 (2004).

    CAS  PubMed  Google Scholar 

  2. 2

    Scadden, D.T. The stem-cell niche as an entity of action. Nature 441, 1075–1079 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Jones, D.L. & Wagers, A.J. No place like home: anatomy and function of the stem cell niche. Nat. Rev. Mol. Cell Biol. 9, 11–21 (2008).

    CAS  PubMed  Google Scholar 

  4. 4

    Morrison, S.J. & Spradling, A.C. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Guilak, F. et al. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5, 17–26 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    CAS  Google Scholar 

  7. 7

    Discher, D.E., Mooney, D.J. & Zandstra, P.W. Growth factors, matrices, and forces combine and control stem cells. Science 324, 1673–1677 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Mammoto, T. & Ingber, D.E. Mechanical control of tissue and organ development. Development 137, 1407–1420 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Emerman, J.T., Burwen, S.J. & Pitelka, D.R. Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture. Tissue Cell 11, 109–119 (1979).

    CAS  PubMed  Google Scholar 

  10. 10

    Deroanne, C.F., Lapiere, C.M. & Nusgens, B.V. In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. Cardiovasc. Res. 49, 647–658 (2001).

    CAS  PubMed  Google Scholar 

  11. 11

    Engler, A.J. et al. Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. J. Cell Biol. 166, 877–887 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Google Scholar 

  13. 13

    Saha, K. et al. Substrate modulus directs neural stem cell behavior. Biophys. J. 95, 4426–4438 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Khatiwala, C.B., Kim, P.D., Peyton, S.R. & Putnam, A.J. ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK. J. Bone Miner. Res. 24, 886–898 (2009).

    CAS  PubMed  Google Scholar 

  15. 15

    Gilbert, P.M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9, 518–526 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Winer, J.P., Janmey, P.A., McCormick, M.E. & Funaki, M. Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. Tissue. Eng. Part A 15, 147–154 (2009).

    CAS  PubMed  Google Scholar 

  18. 18

    Geiger, B., Bershadsky, A., Pankov, R. & Yamada, K.M. Transmembrane crosstalk between the extracellular matrix—cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2, 793–805 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Discher, D.E., Janmey, P. & Wang, Y.L. Tissue cells feel and respond to the stiffness of their substrate. Science 310, 1139–1143 (2005).

    CAS  Google Scholar 

  20. 20

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

    CAS  Google Scholar 

  21. 21

    Orr, A.W., Helmke, B.P., Blackman, B.R. & Schwartz, M.A. Mechanisms of mechanotransduction. Dev. Cell 10, 11–20 (2006).

    CAS  PubMed  Google Scholar 

  22. 22

    Clark, K., Langeslag, M., Figdor, C.G. & van Leeuwen, F.N. Myosin II and mechanotransduction: a balancing act. Trends Cell Biol. 17, 178–186 (2007).

    CAS  PubMed  Google Scholar 

  23. 23

    Peyton, S.R., Ghajar, C.M., Khatiwala, C.B. & Putnam, A.J. The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function. Cell Biochem. Biophys. 47, 300–320 (2007).

    CAS  PubMed  Google Scholar 

  24. 24

    Chen, C.S. Mechanotransduction—a field pulling together? J. Cell Sci. 121, 3285–3292 (2008).

    CAS  PubMed  Google Scholar 

  25. 25

    Schwartz, M.A. & DeSimone, D.W. Cell adhesion receptors in mechanotransduction. Curr. Opin. Cell Biol. 20, 551–556 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Geiger, B., Spatz, J.P. & Bershadsky, A.D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

    CAS  PubMed  Google Scholar 

  27. 27

    Choquet, D., Felsenfeld, D.P. & Sheetz, M.P. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell 88, 39–48 (1997).

    CAS  PubMed  Google Scholar 

  28. 28

    Yeung, T. et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskeleton 60, 24–34 (2005).

    PubMed  Google Scholar 

  29. 29

    Solon, J., Levental, I., Sengupta, K., Georges, P.C. & Janmey, P.A. Fibroblast adaptation and stiffness matching to soft elastic substrates. Biophys. J. 93, 4453–4461 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Wang, N., Butler, J.P. & Ingber, D.E. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 (1993).

    CAS  Google Scholar 

  31. 31

    Wang, N. et al. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells. Am. J. Physiol. Cell Physiol. 282, C606–C616 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Janmey, P.A. & McCulloch, C.A. Cell mechanics: integrating cell responses to mechanical stimuli. Annu. Rev. Biomed. Eng. 9, 1–34 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Burridge, K. & Chrzanowska-Wodnicka, M. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12, 463–518 (1996).

    CAS  PubMed  Google Scholar 

  34. 34

    Sastry, S.K. & Burridge, K. Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics. Exp. Cell Res. 261, 25–36 (2000).

    CAS  PubMed  Google Scholar 

  35. 35

    Bershadsky, A.D., Balaban, N.Q. & Geiger, B. Adhesion-dependent cell mechanosensitivity. Annu. Rev. Cell Dev. Biol. 19, 677–695 (2003).

    CAS  Google Scholar 

  36. 36

    Wozniak, M.A., Modzelewska, K., Kwong, L. & Keely, P.J. Focal adhesion regulation of cell behavior. Biochim. Biophys. Acta. 1692, 103–119 (2004).

    CAS  PubMed  Google Scholar 

  37. 37

    Giancotti, F.G. & Ruoslahti, E. Integrin signaling. Science 285, 1028–1032 (1999).

    CAS  PubMed  Google Scholar 

  38. 38

    Hynes, R.O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).

    CAS  Google Scholar 

  39. 39

    Schwartz, M.A. & Ginsberg, M.H. Networks and crosstalk: integrin signalling spreads. Nat. Cell Biol. 4, E65–E68 (2002).

    CAS  PubMed  Google Scholar 

  40. 40

    Katsumi, A., Orr, A.W., Tzima, E. & Schwartz, M.A. Integrins in mechanotransduction. J. Biol. Chem. 279, 12001–12004 (2004).

    CAS  PubMed  Google Scholar 

  41. 41

    Berrier, A.L. & Yamada, K.M. Cell-matrix adhesion. J. Cell Physiol. 213, 565–573 (2007).

    CAS  PubMed  Google Scholar 

  42. 42

    Hynes, R.O. The extracellular matrix: not just pretty fibrils. Science 326, 1216–1219 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Galbraith, C.G., Yamada, K.M. & Sheetz, M.P. The relationship between force and focal complex development. J. Cell Biol. 159, 695–705 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Shemesh, T., Geiger, B., Bershadsky, A.D. & Kozlov, M.M. Focal adhesions as mechanosensors: a physical mechanism. Proc. Natl. Acad. Sci. USA 102, 12383–12388 (2005).

    CAS  PubMed  Google Scholar 

  46. 46

    Sawada, Y. et al. Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Cell 127, 1015–1026 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Harris, A.K., Wild, P. & Stopak, D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208, 177–179 (1980).

    CAS  PubMed  Google Scholar 

  48. 48

    Lee, J., Leonard, M., Oliver, T., Ishihara, A. & Jacobson, K. Traction forces generated by locomoting keratocytes. J. Cell Biol. 127, 1957–1964 (1994).

    CAS  PubMed  Google Scholar 

  49. 49

    Pelham, R.J. Jr & Wang, Y. High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate. Mol. Biol. Cell 10, 935–945 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Dembo, M. & Wang, Y.L. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys. J. 76, 2307–2316 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Galbraith, C.G. & Sheetz, M.P. A micromachined device provides a new bend on fibroblast traction forces. Proc. Natl. Acad. Sci. USA 94, 9114–9118 (1997).

    CAS  PubMed  Google Scholar 

  52. 52

    Balaban, N.Q. et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3, 466–472 (2001).

    CAS  PubMed  Google Scholar 

  53. 53

    Tan, J.L. et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc. Natl. Acad. Sci. USA 100, 1484–1489 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Butler, J.P., Tolic-Norrelykke, I.M., Fabry, B. & Fredberg, J.J. Traction fields, moments, and strain energy that cells exert on their surroundings. Am. J. Physiol. Cell Physiol. 282, C595–C605 (2002).

    CAS  PubMed  Google Scholar 

  55. 55

    Schwarz, U.S. et al. Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization. Biophys. J. 83, 1380–1394 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Willert, C.E. & Gharib, M. Digital particle image velocimetry. Exp. Fluids 10, 181–193 (1991).

    Google Scholar 

  57. 57

    Sabass, B., Gardel, M.L., Waterman, C.M. & Schwarz, U.S. High resolution traction force microscopy based on experimental and computational advances. Biophys. J. 94, 207–220 (2008).

    CAS  PubMed  Google Scholar 

  58. 58

    du Roure, O. et al. Force mapping in epithelial cell migration. Proc. Natl. Acad. Sci. USA 102, 2390–2395 (2005).

    CAS  PubMed  Google Scholar 

  59. 59

    Yang, M.T., Sniadecki, N.J. & Chen, C.S. Geometric considerations of micro- to nanoscale elastomeric post arrays to study cellular traction forces. Adv. Mater. 19, 3119–3123 (2007).

    CAS  Google Scholar 

  60. 60

    Fu, J.P. et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 7, 733–736 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Georges, P.C. & Janmey, P.A. Cell type-specific response to growth on soft materials. J. Appl. Physiol. 98, 1547–1553 (2005).

    PubMed  Google Scholar 

  62. 62

    Taipale, J. & KeskiOja, J. Growth factors in the extracellular matrix. FASEB J. 11, 51–59 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Storm, C., Pastore, J.J., MacKintosh, F.C., Lubensky, T.C. & Janmey, P.A. Nonlinear elasticity in biological gels. Nature 435, 191–194 (2005).

    CAS  Google Scholar 

  64. 64

    Wen, Q., Basu, A., Winer, J.P., Yodh, A. & Janmey, P.A. Local and global deformations in a strain-stiffening fibrin gel. New J. Phys. 9, 1–9 (2007).

    Google Scholar 

  65. 65

    Houseman, B.T. & Mrksich, M. The microenvironment of immobilized Arg-Gly-Asp peptides is an important determinant of cell adhesion. Biomaterials 22, 943–955 (2001).

    CAS  PubMed  Google Scholar 

  66. 66

    Keselowsky, B.G., Collard, D.M. & Garcia, A.J. Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation. Proc. Natl. Acad. Sci. USA 102, 5953–5957 (2005).

    CAS  PubMed  Google Scholar 

  67. 67

    Mei, Y. et al. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat. Mater. 9, 768–778 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Saez, A., Buguin, A., Silberzan, P. & Ladoux, B. Is the mechanical activity of epithelial cells controlled by deformations or forces? Biophys. J. 89, L52–L54 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Ghibaudo, M. et al. Traction forces and rigidity sensing regulate cell functions. Soft Matter 4, 1836–1843 (2008).

    CAS  Google Scholar 

  70. 70

    Pelham, R.J., Jr. & Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13661–13665 (1997).

    CAS  PubMed  Google Scholar 

  71. 71

    Wang, N. et al. Mechanical behavior in living cells consistent with the tensegrity model. Proc. Natl. Acad. Sci. USA 98, 7765–7770 (2001).

    CAS  PubMed  Google Scholar 

  72. 72

    Nelson, C.M. et al. Emergent patterns of growth controlled by multicellular form and mechanics. Proc. Natl. Acad. Sci. USA 102, 11594–11599 (2005).

    CAS  PubMed  Google Scholar 

  73. 73

    Ruiz, S.A. & Chen, C.S. Emergence of patterned stem cell differentiation within multicellular structures. Stem Cells 26, 2921–2927 (2008).

    PubMed  PubMed Central  Google Scholar 

  74. 74

    Liu, Z.J. et al. Mechanical tugging force regulates the size of cell-cell junctions. Proc. Natl. Acad. Sci. USA 107, 9944–9949 (2010).

    CAS  PubMed  Google Scholar 

  75. 75

    Sniadecki, N.J. et al. Magnetic microposts as an approach to apply forces to living cells. Proc. Natl. Acad. Sci. USA 104, 14553–14558 (2007).

    CAS  PubMed  Google Scholar 

  76. 76

    Saez, A., Ghibaudo, M., Buguin, A., Silberzan, P. & Ladoux, B. Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates. Proc. Natl. Acad. Sci. USA 104, 8281–8286 (2007).

    CAS  PubMed  Google Scholar 

  77. 77

    Rabodzey, A., Alcaide, P., Luscinskas, F.W. & Ladoux, B. Mechanical forces induced by the transendothelial migration of human neutrophils. Biophys. J. 95, 1428–1438 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78

    Liu, Z.J., Sniadecki, N.J. & Chen, C.S. Mechanical forces in endothelial cells during firm adhesion and early transmigration of human monocytes. Cell. Mol. Bioeng. 3, 50–59 (2010).

    PubMed  PubMed Central  Google Scholar 

  79. 79

    Ganz, A. et al. Traction forces exerted through N-cadherin contacts. Biol. Cell 98, 721–730 (2006).

    CAS  PubMed  Google Scholar 

  80. 80

    Liang, X.M., Han, S.J., Reems, J.-A., Gao, D. & Sniadecki, N.J. Platelet retraction force measurements using flexible post force sensors. Lab Chip 10, 991–998 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81

    Saez, A. et al. Traction forces exerted by epithelial cell sheets. J. Phys. Condens. Matter 22, 1–9 (2010).

    Google Scholar 

  82. 82

    Lemmon, C.A. et al. Shear force at the cell-matrix interface: enhanced analysis for microfabricated post array detectors. Mech. Chem. Biosyst. 2, 1–16 (2005).

    PubMed  PubMed Central  Google Scholar 

  83. 83

    Holst, J. et al. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nat. Biotechnol. 28, 1123–1128 (2010).

    CAS  PubMed  Google Scholar 

  84. 84

    Adamo, L. et al. Biomechanical forces promote embryonic haematopoiesis. Nature 459, 1131–1135 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85

    Chowdhury, F. et al. Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells. Nat. Mater. 9, 82–88 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86

    Evans, N.D. et al. Substrate stiffness affects early differentiation events in embryonic stem cells. Eur. Cell Mater. 18, 1–13; discussion 13–14 (2009).

    CAS  PubMed  Google Scholar 

  87. 87

    Reinhart-King, C.A., Dembo, M. & Hammer, D.A. Cell-cell mechanical communication through compliant substrates. Biophys. J. 95, 6044–6051 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 88

    Krishnan, R. et al. Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness. PLoS One 4, e5486 (2009).

    PubMed  PubMed Central  Google Scholar 

  89. 89

    Laermer, F. & Schilp, A. Method of anisotropically etching silicon. U.S. Patent No. 5,501,893 (1996).

  90. 90

    Sniadecki, N.J. & Chen, C.S. Microfabricated silicone elastomeric post arrays for measuring traction forces of adherent cells. Methods Cell Biol. 83, 313–328 (2007).

    CAS  PubMed  Google Scholar 

  91. 91

    Madou, M.J. Fundamentals of Microfabrication (CRC Press, 2002).

  92. 92

    Zhao, Y., Lim, C.C., Sawyer, D.B., Liao, R.L. & Zhang, X. Cellular force measurements using single-spaced polymeric microstructures: isolating cells from base substrate. J. Micromech. Microeng. 15, 1649–1656 (2005).

    Google Scholar 

  93. 93

    Schoen, I., Hu, W., Klotzsch, E. & Vogel, V. Probing cellular traction forces by micropillar arrays: contribution of substrate warping to pillar deflection. Nano Lett. 10, 1823–1830 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    Addae-Mensah, K.A. et al. A flexible, quantum dot-labeled cantilever post array for studying cellular microforces. Sensors Actuators A Phys. 136, 385–397 (2007).

    CAS  Google Scholar 

  95. 95

    Fuard, D., Tzvetkova-Chevolleau, T., Decossas, S., Tracqui, P. & Schiavone, P. Optimization of poly-di-methyl-siloxane (PDMS) substrates for studying cellular adhesion and motility. Microelectronic Eng. 85, 1289–1293 (2008).

    CAS  Google Scholar 

  96. 96

    Qin, D., Xia, Y.N. & Whitesides, G.M. Soft lithography for micro- and nanoscale patterning. Nat. Protoc. 5, 491–502 (2010).

    CAS  Google Scholar 

  97. 97

    Delamarche, E., Schmid, H., Michel, B. & Biebuyck, H. Stability of molded polydimethylsiloxane microstructures. Adv. Mater. 9, 741–746 (1997).

    CAS  Google Scholar 

  98. 98

    Tan, J.L., Tien, J. & Chen, C.S. Microcontact printing of proteins on mixed self-assembled monolayers. Langmuir 18, 519–523 (2002).

    CAS  Google Scholar 

  99. 99

    Tan, J.L., Liu, W., Nelson, C.M., Raghavan, S. & Chen, C.S. Simple approach to micropattern cells on common culture substrates by tuning substrate wettability. Tissue Eng. 10, 865–872 (2004).

    CAS  PubMed  Google Scholar 

  100. 100

    Ye, H.K., Gu, Z.Y. & Gracias, D.H. Kinetics of ultraviolet and plasma surface modification of poly(dimethylsiloxane) probed by sum frequency vibrational spectroscopy. Langmuir 22, 1863–1868 (2006).

    CAS  PubMed  Google Scholar 

  101. 101

    Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique (Wiley-Liss, 2005).

  102. 102

    Zamir, E. et al. Molecular diversity of cell-matrix adhesions. J. Cell Sci. 112, 1655–1669 (1999).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge financial support from the National Institutes of Health (EB00262, HL73305 and GM74048); the Army Research Office Multidisciplinary University Research Initiative; the Material Research Science and Engineering Center, the Nano/Bio Interface Center, the Institute for Regenerative Medicine, and the Center for Musculoskeletal Disorders of the University of Pennsylvania; and the New Jersey Center for Biomaterials (RESBIO Resource Center). M.T.Y. was partially supported by the National Science Foundation Integrative Graduate Education and Research Traineeship program (DGE-0221664). J.F. and Y.-K.W. were both partially supported by the American Heart Association Postdoctoral Fellowship. R.A.D. was partially supported by a National Science Foundation Graduate Research Fellowship. We acknowledge the Massachusetts Institute of Technology Microsystems Technology Laboratories for support in microfabrication.

Author information

Affiliations

Authors

Contributions

M.T.Y. and J.F. designed and fabricated the micropost array masters. M.T.Y. wrote the traction force analysis software. J.F., Y.-K.W. and C.S.C. conceived and designed stem cell experiments with micropost array substrates. J.F., Y.-K.W., M.T.Y. and R.A.D. performed experiments and analyzed data. M.T.Y., J.F., R.A.D. and Y.-K.W. wrote the manuscript.

Corresponding author

Correspondence to Christopher S Chen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yang, M., Fu, J., Wang, YK. et al. Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity. Nat Protoc 6, 187–213 (2011). https://doi.org/10.1038/nprot.2010.189

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing