Ultrastructure of cultured and cryopreserved human corneal keratocytes

Purpose. To describe the ultrastructural features of cultured and cryoprese rved keratocytes. Methods. Isolated human keratocytes were cultured with 10 % fetal calf serum and 10 ng/ml acidic fibroblast growth factor. The 10% Me ,SO and 10% human albumin were used as cryoprotective agents. Cells were co oled at 2 degrees C/min, then thawed at 37 degrees C, and subsequently recu ltured. They were studied by means of transmission electron microscopy (TEM ). Results. TEM of cultured keratocytes before cryopreservation showed a ne twork of intact connecting cells. The average cell thickness was 2.4 mu m i n cross sections and 5.8 mu m in frontal sections. The average nuclear thic kness was 1.6 mu m in cross sections and 3.7 mu m in frontal sections. Nucl ei appeared regular and oval in cross sections and indented in frontal sect ions. Organelles were found in greater amounts in frontal sections than in cross sections. Gap junctions, fenestrations along the cell surface, omega- shaped structures, fibrils, and filamentous networks also were found. Most of the just-thawed, suspended cells were elongated and condensed but had in tact plasma membranes. These cells were surrounded by a granular material, corresponding to the albumin-containing thawing medium. Scattered isolated round cells displayed nuclear damage, cell edema, loss of organelles, and c ell-membrane disruption. By the end of reculture after cryopreservation, cu ltured keratocytes displayed the same ultrastructural features as before cr yopreservation. Conclusion. Cultured human keratocytes display many ultrast ructural features of in situ keratocytes. These features are still present after reculture after cryopreservation. Cryopreservation induces necrosis i n a small percentage of cells, which seems to be related to a relative lack of cell-membrane protection by the cryoprotectants used.