CRYOPRESERVATION AND CULTURE OF HUMAN CORNEAL KERATOCYTES

PURPOSE. To assess the effects(1) of two different concentrations of a lbumin in a cryoprotective solution and two freezing methods on human corneal keratocyte cryopreservation. METHODS. Isolated keratocytes wer e used for cryopreservation. Solutions of 10% dimethylsulfoxide with e ither 2% or 10% human albumin were used as cryoprotective agents. Cell s either were transferred directly into a -80 degrees C freezer (freez ing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 d egrees C/min until -40 degrees C and then 10 degrees C/min), which res ulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently w ere cultured. Keratocytes were studied by means of trypan blue stainin g, growth assay, apoptosis assays, transmission electron microscopy, a nd immunochemistry. RESULTS. The percentage of cells that were alive a fter thawing ranged from 80% to 99% by trypan blue staining and from 4 5% to 60% by now cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryo preservation was significantly (P = 0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, w hereas the albumin concentration had no significant influence on this ratio (P = 0.45). The percentage of apoptotic cells was significantly higher after cryopreservation than in the control group of noncryopres erved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dr amatically decreased the serum-induced cell expression of alpha smooth muscle actin, whereas cryopreservation had no influence on this cell expression. CONCLUSIONS. A freeze-thaw trauma, which was related to cr yopreservation-induced cell apoptosis, was revealed during primary cul ture after thawing. Direct transfer into the -80 degrees C freezer res ulted in better postcryopreservation growth in the culture than contro lled-rate freezing. A change in albumin concentration from 2% to 10% d id not affect the results.