Summary

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Published: August 10, 2010
doi:

Summary

可以仿照下层刚度对细胞功能的影响<em>在体外</em>使用不同的符合标准的聚丙烯酰胺凝胶。

Abstract

组织刚度,是一个重要的细胞功能因素,并组织刚度的变化通常与纤维化,癌症和心血管疾病的1-11的关联。传统的细胞研究细胞功能的生物方法涉及一个刚性基质上培养细胞(塑料餐具或盖玻片),它可以不考虑弹性ECM或流脑组织之间的刚度变化的影响。 在体内组织遵守的条件,在体外模型,我们和其他人使用ECM涂水凝胶。在我们的实验室,是基于水凝胶聚丙烯酰胺可以模仿生物12见过的组织符合。除了3 APTMS孵化用NaOH产生“反应”的盖玻片。戊二醛是用于交叉连接的3 APTMS和聚丙烯酰胺凝胶。用于水凝胶的聚合丙烯酰胺(AC),双丙烯酰胺(BIS – AC),过硫酸铵溶液。 N -羟基琥珀酰亚胺(NHS)是纳入交联的ECM蛋白凝胶交流解决方案。聚合凝胶,凝胶表面涂有流脑的首选,如纤维连接蛋白,vitronectin,胶原蛋白等蛋白质

水凝胶的硬度,可以由流变或原子力显微镜(AFM)和由不同的解决方案的12的交流和/或双AC百分比调整。在这种方式下,下层刚度可以匹配生物组织的刚度,也可使用流变或AFM量化。细胞可以接种这些水凝胶和培养根据所需的实验条件。成像的细胞和分子生物学分析他们的恢复很简单。在这篇文章中,我们定义为那些有<3000帕斯卡与E和僵硬的组织下层/> 20,000帕斯卡的弹性模量(E)软下层。

Protocol

制备盖玻片应高压灭菌。 应使用无菌蒸馏水或去离子水,准备解决方案和洗涤盖玻片。 AC(40%W / V)和双AC(1%W / V)的解决方案是0.2微米的过滤消毒。不久之前使用无菌过滤,准备10%过硫酸铵(APS;100μg/ml水)。每月更换的APS解决方案。 不能进行高压灭菌,如3 APTMS,氯仿,戊二醛,NHS的,并SurfaSil化学试剂均保存在分配瓶仅用于水凝胶的制备。 凝胶为了达?…

Discussion

凝胶聚合过程的一个关键因素是,以避免气泡的形成,这将使细胞绑定到ECM涂层凝胶本身,而不是玻璃盖玻片。这是可以预防的仔细吹打后震荡和可视化,确保无气泡已成为被困在凝胶的聚合解决方案。我们始终建议准备额外的“反应”盖玻片和凝胶,以确保有足够的实验。

凝胶用PBS冲洗时,应特别注意支付。吸痰过程中必须避免与水凝胶本身,因为它可能被抓住,撕裂的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

工作是我们的实验室是由美国国立卫生研究院的赞助支持。

Materials

Material Name Type Company Catalogue Number Comment
Glutaraldehyde, 70%   Sigma-Aldrich G7776 Store at -20°C
3-APTMS (3-Aminopropyltrimethosysilane 97%)   Sigma-Aldrich 281778 Store at room temperature
SurfaSil Siliconizing Fluid   Thermo Scientific 42800 Store at room temperature
NHS (N-hydroxysucinimide Ester)   Sigma-Aldrich A-8060 Store at 4°C Replace monthly
Albumin, bovine serum, essentially fatty acid free   Sigma-Aldrich A6003-100G Store at 4°C
Coverslips (25mm)   Fisher Scientific 12-545-86 25 Cir 1D  
Coverslips (18mm)   Fisher Scientific 12-545-84 18 Cir 1D  

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Cite This Article
Cretu, A., Castagnino, P., Assoian, R. Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels. J. Vis. Exp. (42), e2089, doi:10.3791/2089 (2010).

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