Institute of Applied Ophthalmobiology, University of Valladolid, Valladolid, Spain.
J Immunol Methods. 2013 Mar 29;389(1-2):61-8. doi: 10.1016/j.jim.2013.01.003. Epub 2013 Jan 11.
Culturing of human retinal pigment epithelial cells (hRPE) is the initial step in cell therapy of some retinal diseases. To transfer these cells into clinical use, it is necessary to guarantee that they are well differentiated and contamination free. Fluorescence microscopy is the easiest method to do this, but it is associated with operator subjectivity, and the results are highly variable. The aim of this study was to demonstrate the practicality of implementing flow cytometry (FC) analysis to determine the purity of human RPE primary cell cultures. An ARPE19 cell line, human skin fibroblasts, hRPE, and human corneal epithelial cells were analysed by FC to determine the percentage of the hRPE population expressing RPE65 and epithelial and fibroblast proteins. The cell viability and DNA content also were determined. FC analysis showed that the hRPE cells were healthy, stable, and expressed RPE65 protein in the study working conditions. The density of RPE65 protein expression decreased during passages 2 to 10, which was confirmed using a Western blot technique. However, the hRPE cells did not express the 112-kDa epithelial and fibroblast proteins in the current working conditions. These findings suggested that FC facilitates a detailed analysis of human RPE primary cell cultures, a necessary step in developing new cell therapies for retinal diseases.
培养人视网膜色素上皮细胞（hRPE）是某些视网膜疾病细胞治疗的初始步骤。为了将这些细胞应用于临床，有必要保证它们分化良好且无污染。荧光显微镜是最容易的方法，但它与操作者的主观性有关，结果高度可变。本研究旨在证明实施流式细胞术（FC）分析以确定人 RPE 原代细胞培养物纯度的实用性。通过 FC 分析 ARPE19 细胞系、人皮肤成纤维细胞、hRPE 和人角膜上皮细胞，确定表达 RPE65 和上皮及成纤维蛋白的 hRPE 群体的百分比。还确定了细胞活力和 DNA 含量。FC 分析表明，在研究工作条件下，hRPE 细胞健康、稳定且表达 RPE65 蛋白。在第 2 至第 10 代时，RPE65 蛋白的表达密度下降，这一点通过 Western blot 技术得到了证实。然而，在当前工作条件下，hRPE 细胞不表达 112kDa 的上皮和成纤维蛋白。这些发现表明，FC 促进了对人 RPE 原代细胞培养物的详细分析，这是开发新的视网膜疾病细胞治疗方法的必要步骤。
