Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Nat Protoc. 2011 Feb;6(2):187-213. doi: 10.1038/nprot.2010.189. Epub 2011 Jan 27.
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.
我们描述了一种微制造的细胞培养基底的使用，该基底由一个均匀排列的、紧密间隔的垂直弹性微柱阵列组成，用于研究基底刚性对细胞功能的影响。弹性微柱基底是通过硅质主模具微成型的，主模具由不同高度的微柱组成，从而产生不同刚性的基底。弹性微柱的尖端通过微接触印刷功能化细胞外基质，以促进细胞黏附。因此，这些基底在向黏附细胞呈现相同的形貌线索的同时，仅通过微柱高度的操纵来改变基底刚性。本方案描述了如何制造硅质微柱阵列模具（~2 周完成）和弹性基底（3 天），以及如何在这些基底上进行细胞培养实验（1-14 天）、免疫荧光成像（2 天）、牵引力分析（2 天）和干细胞分化测定（1 天），以研究基底刚性对干细胞形态、牵引力产生、焦点黏附组织和分化的影响。
