Carter Debra A, Balasubramaniam Balini, Dick Andrew D
Bristol Eye Hospital, School of Clinical Sciences, University of Bristol, Bristol, UK.
Methods Mol Biol. 2013;935:271-83. doi: 10.1007/978-1-62703-080-9_19.
The identification of stem/progenitor cells within the retinal neural environment has opened up the possibility of therapy via cellular replacement and/or reprogramming of resident cell populations. Within the neuro-retinal niche, following injury or in disease states (including inflammation and degeneration), cellular responses affect tissue homeostasis, reduce cell density, disrupt tissue architecture, and produce scar formation. Microglia (resident retinal immune cell tissue macrophage) are key to the maintenance of retinal homeostasis and are implicated in responses that may influence the control and behavior of retinal progenitors. Factors to consider in the generation of a transplantable cell resource with good migratory and integrative capacity include their yield, purity, and functional viability. Utilizing human postmortem retina, we have created a research platform to isolate, culture, and characterize adult retinal microglia as well as analyze their effect on retinal progenitors. Here, we describe techniques using magnetic labeled bead cell separation to isolate pure populations of retinal CD133(+) precursor cells and CD11b(+) microglia from primary adult retinal cell suspensions (RCSs), enabling flow cytometric cell phenotypic and qPCR genotypic analysis, as well as functional analysis by real-time ratiometric calcium imaging.
视网膜神经环境中干细胞/祖细胞的鉴定为通过细胞替代和/或重编程驻留细胞群进行治疗开辟了可能性。在神经视网膜微环境中，受伤后或处于疾病状态（包括炎症和退化）时，细胞反应会影响组织稳态，降低细胞密度，破坏组织结构并形成瘢痕。小胶质细胞（视网膜常驻免疫细胞组织巨噬细胞）是维持视网膜稳态的关键，并且参与可能影响视网膜祖细胞控制和行为的反应。在生成具有良好迁移和整合能力的可移植细胞资源时需要考虑的因素包括其产量、纯度和功能活力。利用人类死后视网膜，我们创建了一个研究平台，用于分离、培养和表征成人视网膜小胶质细胞，并分析它们对视网膜祖细胞的影响。在此，我们描述了使用磁性标记珠细胞分离技术从原代成人视网膜细胞悬液（RCS）中分离出纯的视网膜CD133(+)前体细胞和CD11b(+)小胶质细胞群体的技术，从而能够进行流式细胞术细胞表型分析和qPCR基因分型分析，以及通过实时比率钙成像进行功能分析。
