Purchase this article with an account.
Y. Miura, H. Hasselbach, K. Lange, B. Nölle, J. Roider; Cell Biological Characteristics of RPE cells in Retina-RPE-Choroid Perfusion Organ Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2540.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Perfusion tissue culture systems provide the possibility to cultivate highly differentiated tissues in vitro. We evaluated the morphological and functional integrity of procine RPE-cells after incubation in a perfusion tissue culture system of porcine retina-RPE-choroid complex.
A retina-RPE-choroid complex was prepared from freshly enucleated pig eyes and transferred to a commercially available perfusion tissue chamber (Bad Abbach, Germany) as previously described. RPE-cell viability was detected with trypan-blue exclusion test. To assess proliferation ability, RPE cells after 5 days were isolated from the choroid and seeded with the concentration of 6x105 into 60mm culture dish . After 5 days, all cells were collected and counted using a hemacytometer. For adhesion ability, the cells were collected as in proliferation assay and seeded on a 60mm culture dish. After 6 hours, the numbers of RPE cells adherent to the bottom of the culture dish were counted. The morphology of the RPE cells was analyzed by electron and phase contrast microscopy. As a control were used: fresh porcine RPE cells, porcine RPE cell cultures in second passage and porcine RPE-complex cultivated in a conventional culture dishes.
In contrary to the RPE-complex cultivated in a conventional culture dish the morphology of the porcine RPE-choroid complex was well preserved when a perfusion tissue culture system was used. The retina was detached in the majority of cases and various degrees of degeneration were noted. In some experiments "bubble formation" of the medium in the perfusion tissue chamber occurred and in these cases the number of living RPE cells was significantly reduced (p<0.001). Cell viability after 5 days cultivation in a perfusion tissue system was about 85%(p<0.001). Compared to fresh and second passage cultured RPE cells, the proliferation an adhesion abilities of RPE cells from day 5 perfusion tissue culture were 83% and 67% respectively.
The morphological and functional integrity of the RPE-choroid complex can be well maintained in culture by using a perfusion tissue culture system. Formation of bubbles in the perfusion medium significantly reduces the morphological outcome. Further improvements to avoid this bubble formation and to achieve a better preservation of the retina are necessary. Overall, we think the perfusion tissue culture of porcine RPE-choroid can be a suitable model for in-vitro investigations of the RPE.
This PDF is available to Subscribers Only