Studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels.
Studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels.
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DOI：
10.3791/2089
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发表时间：
2010-08-10
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影响因子：
--
通讯作者：
Assoian, Richard
中科院分区：
文献类型：
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作者：
Cretu, Alexandra;Castagnino, Paola;Assoian, Richard
组织硬度是细胞功能的重要决定因素，组织硬度的变化通常与纤维化、癌症和心血管疾病有关。研究细胞功能的传统细胞生物学方法包括将细胞培养在刚性衬底(塑料碟子或玻璃盖片)上，这无法解释弹性细胞外基质的影响或组织间细胞外基质硬度的变化。为了在体外模拟体内组织顺应性条件，我们和其他人使用了ECM涂层水凝胶。在我们的实验室中，这种水凝胶是以聚丙烯酰胺为基础的，它可以模拟生物学上看到的组织顺应性的范围。通过与氢氧化钠孵育，然后添加3-APTMS，可产生“反应性”盖片。用戊二醛对3-APTMS和聚丙烯酰胺凝胶进行了交联。丙烯酰胺(AC)、双丙烯酰胺(Bis-AC)和过硫酸铵的溶液用于水凝胶的聚合。将N-羟基琥珀酰亚胺(NHS)引入AC溶液中，使ECM蛋白与水凝胶发生交联反应。水凝胶聚合后，凝胶表面覆盖有一种选择的ECM蛋白，如纤维连接蛋白、玻璃连接蛋白、胶原等。水凝胶的硬度可以通过流变学或原子力显微镜(AFM)来确定，并通过改变溶液中AC和/或双AC的百分比来调整。通过这种方式，基质硬度可以与生物组织的硬度相匹配，生物组织的硬度也可以用流变学或原子力显微镜进行量化。然后，细胞可以种植在这些水凝胶上，并根据所需的实验条件进行培养。细胞的成像和它们的回收用于分子分析是简单的。在本文中，我们将弹性模数为3000帕斯卡的软基片和具有20,000帕斯卡的硬质基片/组织定义为软基片/组织。
Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc. The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) < 3000 Pascal and stiff substrata/tissues as those with E > 20,000 Pascal.