PHYSIOLOGICAL FEATURES OF PRIMARY AND SUBCULTURES OF HUMAN RETINAL PIGMENTED EPITHELIAL CELLS PRIOR AND FOLLOWING CRYOPRESERVATION FOR CELL TRANSPLANTATION
K. Engelmann1, M. Valtink1, R. Krüger1, O. Strauß2, G. Richard1
Purpose: Propagation of retinal pigmented epithelial (RPE) cells in vitro was performed to study cell biology, but also as a base for RPE cell transplantation mainly performed in animals. One of the most striking disadvantages of primary cell strains is the decline in typical epithelial cell behaviour and morphology during multiplication. These changes might relate to loss of cell function and consequently to the failure of cell transplantation success. The expression of typical characteristics and behaviour was determined during primary and subculture and after thawing of cryopreserved cells.
Methods: For isolation of RPE the uvea of donor eyes was mechanically removed from the surrounding tissue and incubated in a collagenase/dispase solution. The isolated cells were seeded onto coated cell culture dishes and treated with a specially designed growth medium including uvea-conditioned medium and other defined supplements. HLA class-I and -II typing was performed prior to cryoconservation and after thawing. Moreover, membrane currents in primary, 1st passage and cryopreserved RPE cells were studied using the patch-clamp technique.
Results: After cryopreservation no loss of any HLA-antigen was detectable in 12 cell strains which were studied. The range of cryopreservation was 9 to 66 month. The patch-clamp experiments demonstrated, that potassium and calcium channels could be detected in 1st passages and cryopreserved RPE cells only if our previously described improved culture conditions were used in contrast to "conventionally" cultured cells. The characteristics of the delayed and the inward rectifier potassium channels showed only little changes in subcultured compared to primary cultured cells.
Discussion: The results demonstrated that the culture environment has a strong influence on the preservation of characteristics typical for epithelial cells after cell multiplication and storage.
1Universitäts-Augenklinik, Martinistrasse 52, D-20246 Hamburg
2Inst. f. klin. Physiol. der FU Berlin, Hindenburgdamm 30, D-12200 Berlin,