Dissociated Retinal Cell Culture

Abstract

The retina is a relatively simple and accessible part of the central nervous system, making it a powerful model to study cell fate specification mechanisms. Multipotent retinal progenitor cells (RPCs) give rise to seven major classes of retinal cell types. Mechanisms regulating cell fate choice in the retina depend on both cell intrinsic and environmental factors, but their relative contribution to specific cell fate decisions remains unclear. Dissociated retinal cell cultures provide a great assay to study this problem. RPCs are cultured in serum-free and extract-free medium, providing the investigator with a control over the environment to address questions related to the effects of a particular molecule on the development of retinal neurons. In addition, dissociated cell cultures can be used to study the importance of cell intrinsic mechanisms by isolating RPCs from their normal environment (Cayouette et al., 2003; Jensen and Raff, 1997). The method described below is suitable for the clonal-density culture of RPCs. In such cultures, RPCs are isolated from each other and from the postmitotic neurons. They divide and differentiate into different retinal cell types to form small colonies, or “clones”. In a recent study, we found that these clones are indistinguishable from the clones that develop in situ in the retina, both in terms of cell number and cell type composition, suggesting that intracellular mechanisms play a key role in retinal development (Cayouette et al., 2003)., 视网膜是中枢神经系统相对简单易用的部分，是研究细胞命运规范机制的有力模型。多能视网膜祖细胞（RPC）产生七大类视网膜细胞类型。调节视网膜细胞命运选择的机制取决于细胞内在和环境因素，但它们对特定细胞命运决定的相对贡献仍不清楚。分离的视网膜细胞培养物提供了一个很好的分析来研究这个问题。 RPC在无血清和无提取物培养基中培养，为研究者提供对环境的控制，以解决与特定分子对视网膜神经元发育的影响有关的问题。此外，分离的细胞培养物可用于通过从其正常环境中分离RPC来研究细胞内在机制的重要性（Cayouette等人，2003; Jensen和Raff，1997）。下述方法适用于RPC的克隆密度培养。在这样的培养物中，RPC彼此分离并且从后神经元分离。它们分化和分化成不同的视网膜细胞类型以形成小菌落或“克隆”。在最近的一项研究中，我们发现这些克隆与细胞数量和细胞类型组成方面在视网膜原位发生的克隆是无法区分的，这表明细胞内机制在视网膜发育中起关键作用（Cayouette et al。 ，2003）。