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


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.

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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.


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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.

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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.

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Correspondence to Christopher S Chen.

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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).

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