Ultrastructure of cultured and cryopreserved human corneal keratocytes

Purpose. To describe the ultrastructural features of cultured and cryopreserved 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 cooled at 2 degrees C/min, then thawed at 37 degrees C, and subsequently recultured. They were studied by means of transmission electron microscopy (TEM). Results. TEM of cultured keratocytes before cryopreservation showed a network of intact connecting cells. The average cell thickness was 2.4 mu m in cross sections and 5.8 mu m in frontal sections. The average nuclear thickness was 1.6 mu m in cross sections and 3.7 mu m in frontal sections. Nuclei appeared regular and oval in cross sections and indented in frontal sections. 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 intact 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 cell-membrane disruption. By the end of reculture after cryopreservation, cultured keratocytes displayed the same ultrastructural features as before cryopreservation. Conclusion. Cultured human keratocytes display many ultrastructural features of in situ keratocytes. These features are still present after reculture after cryopreservation. Cryopreservation induces necrosis in a small percentage of cells, which seems to be related to a relative lack of cell-membrane protection by the cryoprotectants used.