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Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers.
- Source :
-
Cryobiology [Cryobiology] 2024 Jun; Vol. 115, pp. 104857. Date of Electronic Publication: 2024 Feb 11. - Publication Year :
- 2024
-
Abstract
- The cryopreservation of endothelial cell monolayers is an important step that bridges the cryopreservation of cells in suspension to that of tissues. Previous studies have identified clear distinctions in freezing mechanisms between cells in suspension and in monolayers, as well as developed novel protocols for monolayer cryopreservation. Recently, our group has shown that human umbilical vein endothelial cell (HUVEC) and porcine corneal endothelial cell (PCEC) monolayers grown on Rinzl plastic substrate can be cryopreserved in 5% dimethyl sulfoxide, 6% hydroxyethyl starch, and 2% chondroitin sulfate, following a slow-cooling protocol (-1 °C/min) with rapid plunge into liquid nitrogen from -40 °C. However, membrane integrity assessments were done immediately post thaw, which may result in an overestimation of cell viability due to possible delayed injury responses. Here, we show that for the optimal protocol condition of plunge at the -40 °C interrupt temperature, HUVEC and PCEC monolayers exhibited no significant immediate post-thaw injuries nor delayed injury responses during the 24-h post-thaw overnight culture period. HUVEC monolayers experienced no significant impact to their natural growth rate during the post-thaw culture, while PCEC monolayers experienced significantly higher growth than the unfrozen controls. The difference in the low-temperature responses between HUVEC and PCEC monolayers was further shown under high temperature plunge conditions. At these suboptimal plunge temperatures, HUVEC monolayers exhibited moderate immediate membrane injury but a pronounced delayed injury response during the 24-h post-thaw culture, while PCEC monolayers showed significant immediate membrane injury but no additional delayed injury response during the same period. Therefore, we provide further validation of our group's previously designed endothelial monolayer cryopreservation protocol for HUVEC and PCEC monolayers, and we identify several cell-type-specific responses to the freezing process.<br />Competing Interests: Declaration of competing interest J.A.W. Elliott and L.A. Marquez-Curtis are co-inventors on a filed patent: N. Eskandari, J.A.W. Elliott, L.E. McGann, J.A. Nychka, L.A. Marquez-Curtis, “Cryopreservation of cell monolayers”.<br /> (Copyright © 2024. Published by Elsevier Inc.)
- Subjects :
- Humans
Animals
Swine
Chondroitin Sulfates pharmacology
Endothelial Cells cytology
Hydroxyethyl Starch Derivatives pharmacology
Cells, Cultured
Endothelium, Corneal cytology
Endothelium, Corneal injuries
Cryopreservation methods
Human Umbilical Vein Endothelial Cells
Cell Survival drug effects
Cryoprotective Agents pharmacology
Dimethyl Sulfoxide pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1090-2392
- Volume :
- 115
- Database :
- MEDLINE
- Journal :
- Cryobiology
- Publication Type :
- Academic Journal
- Accession number :
- 38350589
- Full Text :
- https://doi.org/10.1016/j.cryobiol.2024.104857