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

Graefe's Archive for Clinical and Experimental Ophthalmology volume 253, pages 1493–1502 (2015)Cite this article

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 from albino versus pigmented rats were also investigated.

Methods

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.

Results

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).

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.

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Acknowledgments

The authors thank Monika Rittgarn, Sabine Hofmeister, and Sigrid Schultheiss for their excellent technical assistance and Judith Birch for her great editorial support. (Section of Experimental Vitreoretinal Surgery, Centre for Ophthalmology, Tuebingen, Germany).

Conflict of interest

The authors have no conflict of interest concerning this work. Ulrich Schraermeyer has funding for his research by Novartis Pharma; however, this is not related to the present project.

Ethical approval

All authors hereby declare that "Principles of laboratory animal care" (NIH publication No. 85–23, revised 1985) were followed, as well as specific national laws, where applicable. All experiments have been examined and approved by the appropriate ethics committee.

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Affiliations

  1. Section of Experimental Vitreoretinal Surgery, Centre for Ophthalmology, Schleichstrasse 12/1, 72076, Tuebingen, Germany

    Analena Langenfeld, Sylvie Julien & Ulrich Schraermeyer

Authors
  1. Analena Langenfeld
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  2. Sylvie Julien
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  3. Ulrich Schraermeyer
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Corresponding author

Correspondence to Ulrich Schraermeyer.

Additional information

Analena Langenfeld and Sylvie Julien contributed equally to this work.

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Langenfeld, A., Julien, S. & Schraermeyer, U. An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats. Graefes Arch Clin Exp Ophthalmol 253, 1493–1502 (2015). https://doi.org/10.1007/s00417-015-3011-5

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  • DOI: https://doi.org/10.1007/s00417-015-3011-5

Keywords

  • RPE isolation
  • Retinal adhesion
  • Rat
  • Novel technique
  • Immunocytochemistry