Cryopreservation and culture of human corneal keratocytes

Abstract

Purpose: To assess the effects of two different concentrations of albumin in a cryoprotective solution and two freezing methods on human corneal keratocyte ctyopreservation.

Methods: Isolated keratocytes were used for cryopreservation. Solutions of 10% dimethylsulfoxide with either 2% or 10% human albumin were used as cryoprotective agents. Cells either were transferred directly into a -80 degrees C freezer (freezing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 degrees C/min until -40 degrees C and then 10 degrees C/min), which resulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently were cultured. Keratocytes were studied by means of trypan blue staining, growth assay, apoptosis assays, transmission electron microscopy, and immunochemistry.

Results: The percentage of cells that were alive after thawing ranged from 80% to 99% by trypan blue staining and from 45% to 60% by flow cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryopreservation was significantly (P=0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, whereas 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 noncryopreserved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dramatically 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 cryopreservation-induced cell apoptosis, was revealed during primary culture after thawing. Direct transfer into the -80 degrees C freezer resulted in better postcryopreservation growth in the culture than controlled-rate freezing. A change in albumin concentration from 2% to 10% did not affect the results.