title: 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.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Yang
authors.forename: Jianping
authors.initials: J
authors.lastname: Fu
authors.forename: Yang-Kao
authors.initials: YK
authors.lastname: Wang
authors.forename: Ravi A
authors.initials: RA
authors.lastname: Desai
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Chen
mesh_terms: D001696:Biomechanical Phenomena; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D020724:Elastomers; D005109:Extracellular Matrix / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D055616:Microtechnology / Q000379:methods*; D011108:Polymers / Q000737:chemistry*; D012825:Silicon; D013499:Surface Properties
chemical_list: D020724:Elastomers; D011108:Polymers; C036925:elastomeric polymer; D012825:Silicon