Langenfeld Analena, Julien Sylvie, Schraermeyer Ulrich
Section of Experimental Vitreoretinal Surgery, Centre for Ophthalmology, Schleichstrasse 12/1, 72076, Tuebingen, Germany.
Graefes Arch Clin Exp Ophthalmol. 2015 Sep;253(9):1493-502. doi: 10.1007/s00417-015-3011-5. Epub 2015 Apr 26.
Since adult rats are used in pre-clinical studies, and due to the necessity of investigating the side-effects of drugs on RPE cells in vitro, there is a great need for primary RPE cells from these animals. The aim of this study was to develop a reproducible and quantifiable method of isolation, culture, and maintenance of adult rat RPE cells. Moreover, potential differences between RPE cells from albino versus pigmented rats were also investigated.
A total of 180 pigmented rats and 340 albino rats aged 6-14 weeks were used. RPE cells were isolated and cultured for several weeks by using three different methods: 1) growing directly on flat mounts, 2) after enzymatic isolation, and 3) after they spontaneously detached from the flat mounts and continued to grow on the plastic. Yield, cell survival, and morphological characteristics were investigated using light and electron microscopy as well as immunohistochemistry.
After 0 weeks, the yield of the first method was 30,000 cells/eye; after 2 weeks 18,000 cells/eye; and after 4 weeks 11,000 cells/eye. The yield of RPE cells was very low after enzymatic isolation in method 2 (0 weeks, 13.000 cells/eye; 2 weeks, 30,000 cells/eye; 4 weeks 38,000 cells/eye), whereas it was higher when the RPE cells spontaneously detached from the flat mounts and then continued to grow on the plastic in method three. (0 weeks, 30,000 cells/eye; 2 weeks, 314,000 cells/eye; 4 weeks, 659,000 cells/eye). The second method often showed contamination with fibroblasts, whereas the two other methods showed pure RPE cultures. The RPE cells were able to proliferate when using the second and the third method, but not when they were cultivated directly on the flat mounts (first method).
The qualitative and quantitative best method for isolating adult rat RPE cells is the culture of RPE cells which spontaneously detach from flat mounts. No differences were observed between albino and pigmented RPE cells.
由于成年大鼠用于临床前研究，且有必要在体外研究药物对视网膜色素上皮（RPE）细胞的副作用，因此非常需要从这些动物中获取原代RPE细胞。本研究的目的是开发一种可重复且可量化的成年大鼠RPE细胞分离、培养和维持方法。此外，还研究了白化大鼠与有色大鼠的RPE细胞之间的潜在差异。
总共使用了180只6 - 14周龄的有色大鼠和340只白化大鼠。通过三种不同方法分离并培养RPE细胞数周：1）直接在扁平载玻片上生长；2）酶解分离后；3）自发从扁平载玻片上脱离并在塑料上继续生长后。使用光学显微镜、电子显微镜以及免疫组织化学研究产量、细胞存活率和形态特征。
0周后，第一种方法的产量为30,000个细胞/眼；2周后为18,000个细胞/眼；4周后为11,000个细胞/眼。方法2酶解分离后RPE细胞产量非常低（0周，13,000个细胞/眼；2周，30,000个细胞/眼；4周，38,000个细胞/眼），而方法3中RPE细胞自发从扁平载玻片上脱离然后在塑料上继续生长时产量更高（0周，30,000个细胞/眼；2周，314,000个细胞/眼；4周，659,000个细胞/眼）。第二种方法经常显示有成纤维细胞污染，而其他两种方法显示为纯RPE培养物。使用第二种和第三种方法时RPE细胞能够增殖，但直接在扁平载玻片上培养（第一种方法）时则不能。
分离成年大鼠RPE细胞的定性和定量最佳方法是培养自发从扁平载玻片上脱离的RPE细胞。白化和有色RPE细胞之间未观察到差异。
