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Cryoinjury in endothelial cell monolayers.
Stacey L Ebertz1, Locksley E McGann
1Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alta., Canada T6G 2R8. stacey.ebertz@ualberta.net
Cryobiology
|July 22, 2004
Summary
Cryopreservation of human corneal endothelial cell (HCEC) monolayers is challenging. Optimal cryopreservation involves specific cryoprotectants and a storage temperature of -80°C, not -196°C, to maintain cell viability and reduce detachment.
Area of Science:
- Ophthalmology
- Cell Biology
- Cryobiology
Background:
- Corneal cryopreservation is hindered by endothelial cell viability loss and detachment.
- Human corneal endothelial cell (HCEC) cryoinjury mechanisms require further elucidation.
Purpose of the Study:
- To understand cryoinjury in HCEC monolayers.
- To evaluate cryoprotectant effects and temperature impacts on HCEC cryopreservation.
Main Methods:
- HCEC monolayers on collagen-coated glass were exposed to cryoprotectants (DMSO, propylene glycol) at 1-2M.
- Cooling rates of 1°C/min were used, with various temperatures (-5 to -40°C) and thawing protocols.
- Cell membrane integrity and detachment were assessed using SYTO/ethidium bromide staining.
Main Results:
- Cryoprotectants significantly improved membrane integrity and reduced detachment when thawed directly from -40°C.
- Storage at -80°C resulted in better HCEC recovery and lower detachment than storage at -196°C.
- Higher cryoprotectant concentrations did not enhance monolayer recovery.
Conclusions:
- Cryoprotectants are essential for preserving HCEC integrity during cryopreservation.
- -80°C storage is superior to -196°C for HCEC cryopreservation, suggesting a need to re-evaluate current corneal storage temperatures.