IN-VITRO METHODS TO DECREASE MHC CLASS II-POSITIVE CELLS IN RETINAL-PIGMENT EPITHELIUM CELL GRAFTS

Rejection of RPE transplants may be caused by expression of MHC class II molecules on the graft. We wanted to determine if passage level, hy peroxia, low temperature, or magnetic separation had an influence on t his expression or on several other physiological parameters of bovine RPE cells. For this, bovine RPE cells were cultured under normal condi tions for several passages (I-5) or were incubated with a high level o f oxygen (75%) for 24 h or at low temperature (24 degrees C) for seven days. Magnetic beads coated with monoclonal antibody against MHC clas s II antigens were used to separate the positive cells from the total cell population. The number of MHC class II-positive RPE cells was not affected by increased passage, oxygen concentration, or low temperatu re. Using magnetic beads, approximately 7.5% of the cells were separat ed from the total RPE population as MHC class II-positive cells. Hyper oxia initially increased the number of RPE cells to 178% that of the u ntreated controls, but the subsequent MTT test showed a decrease in pr oliferation to 95% of control levels. Similarly, low temperature incre ased cell number to 110% that of controls, but prolonged proliferation decreased to 76% of the controls. None of the experimental conditions had a significant influence on the viability of the cells. Culture co nditions can be modified to increase the yield of RPE cells, and MHC c lass II-positive RPE cells can be detected and selectively separated f rom the total cell population, both of which may prove to be useful fo r RPE cell transplantation.