A multiwell platform for studying stiffness-dependent cell biology.
A multiwell platform for studying stiffness-dependent cell biology.
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DOI：
10.1371/journal.pone.0019929
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Tschumperlin DJ
中科院分区：
文献类型：
--
作者：
Mih JD;Sharif AS;Liu F;Marinkovic A;Symer MM;Tschumperlin DJ
贴壁细胞通常在刚性基底上培养，所述刚性基底比其来源组织硬几个数量级。在这里，我们描述了一种快速制造96孔和384孔平台的方法，用于在组织相关刚度背景下进行细胞的常规筛选。简言之，将聚丙烯酰胺（PA）水凝胶浇铸在玻璃底板中，用胶原蛋白官能化，并灭菌用于细胞培养。每种基质的杨氏模量可以指定为0.3至55 kPa，胶原蛋白表面密度在刚度范围内保持恒定。使用自动荧光显微镜，我们捕获了在384孔板内在生理硬度范围内培养的7种细胞类型的形态变化。我们在96个威尔斯孔中进行了细胞数量、增殖和凋亡的测定，并解析了原代细胞系和永生化细胞系中作为刚度的函数的细胞生长的不同概况。我们发现正常人肺成纤维细胞的硬度依赖性生长在很大程度上与胶原蛋白密度无关，并且其积累的差异通过增加血清浓度而放大。此外，我们对18种生物活性小分子进行了筛选，并鉴定了对软基质和硬基质具有增强或减弱作用的化合物，包括blebbistattin，它在1 kPa下消除了对肺成纤维细胞生长的抑制。在多孔板中部署PA凝胶以用于在组织相关环境中对细胞进行高通量分析的能力为发现在特定刚度制度下操作的细胞反应开辟了新的机会。
Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.
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影响因子：
3.4
作者：
Saha, Krishanu;Kim, Jungwook;Hayward, Ryan C.
通讯作者：
Hayward, Ryan C.
影响因子：
3.4
作者：
Engler, A;Bacakova, L;Discher, D
通讯作者：
Discher, D
DOI：
10.1083/jcb.200405004
发表时间：
2004-09-13
期刊：
The Journal of cell biology
影响因子：
--
作者：
Engler AJ;Griffin MA;Sen S;Bönnemann CG;Sweeney HL;Discher DE
通讯作者：
Discher DE
DOI：
10.1126/science.1191035
发表时间：
2010-08-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gilbert PM;Havenstrite KL;Magnusson KE;Sacco A;Leonardi NA;Kraft P;Nguyen NK;Thrun S;Lutolf MP;Blau HM
通讯作者：
Blau HM
影响因子：
3.4
作者：
Byfield, Fitzroy J.;Wen, Qi;Janmey, Paul A.
通讯作者：
Janmey, Paul A.