Philipps University Marburg, Institute of Neuropathology, Marburg, Germany.
Philipps University Marburg, Core Facility for Mouse Pathology and Electron Microscopy, Marburg, Germany.
Curr Protoc. 2022 Aug;2(8):e524. doi: 10.1002/cpz1.524.
The composition of the cell culture environment profoundly affects cultured cells. Standard cell culture equipment such as plastic and glass provide extremely stiff surfaces compared to physiological cell environments (i.e., tissue). A growing body of evidence documents the artificial behavior and morphology of cells cultured on supraphysiologically stiff surfaces, such as glass (elastic modulus ca. 70,000 MPA) or plastic (e.g., polystyrol ca. 3300 MPA). Therefore, polymer-based hydrogels are increasingly employed as more physiologically appropriate (<100 kPA) supports for 2D or 3D culture. Since multiple properties that influence the cultured cells may be easily adjusted, hydrogels have become versatile tools for studying cells in a more native in vitro environment. Polyacrylamide-based hydrogels can be used as culture substrates for a broad variety of adherent cells and are easy to handle in most downstream biological assays, such as immunohistochemistry or molecular biology methods. We faced, however, serious difficulties with processing high stiffness polyacrylamide-based hydrogels for electron microscopy. To overcome this problem, we developed a simple protocol for embedding and processing cells grown on high stiffness polyacrylamide hydrogels that do not require modifications of routine embedding protocols. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Embedding of polyacrylamide-based hydrogels for transmission electron microscopy Alternate Protocol 1: Procedure for detached hydrogels Alternate Protocol 2: Procedure for attached hydrogels.
细胞培养环境的组成会深刻影响培养细胞。与生理细胞环境（即组织）相比，标准的细胞培养设备（如塑料和玻璃）提供了极其坚硬的表面。越来越多的证据表明，在超生理硬度的表面（如玻璃（弹性模量约 70,000 MPA）或塑料（例如聚苯乙烯约 3300 MPA）上培养的细胞具有人工行为和形态。因此，聚合物基水凝胶越来越多地被用作更适合生理条件的（<100 kPA）二维或三维培养的支撑物。由于影响培养细胞的多种特性可以轻松调节，水凝胶已成为在更自然的体外环境中研究细胞的多功能工具。基于聚丙烯酰胺的水凝胶可用作多种贴壁细胞的培养基质，并且在大多数下游生物学测定（如免疫组织化学或分子生物学方法）中易于处理。然而，我们在处理高刚性基于聚丙烯酰胺的水凝胶进行电子显微镜检查时遇到了严重的困难。为了解决这个问题，我们开发了一种简单的方案，用于嵌入和处理在高刚性聚丙烯酰胺水凝胶上生长的细胞，该方案不需要对常规嵌入方案进行修改。©2022 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案：用于透射电子显微镜的聚丙烯酰胺基水凝胶的嵌入替代方案 1：用于分离水凝胶的程序替代方案 2：用于附着水凝胶的程序。
