An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats

Abstract

Purpose: 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 coming from albino versus pigmented rats were also investigated.

Methods: A total number 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.

Results: Immediately after isolation (day 1), 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 (day 1: 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. (day 1: 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).

Conclusion: 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 [3].