Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers

MingHan Yu; Leah A Marquez-Curtis; Janet A W Elliott

doi:10.1016/j.cryobiol.2024.104857

Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers

Cryobiology. 2024 Feb 11:115:104857. doi: 10.1016/j.cryobiol.2024.104857. Online ahead of print.

Authors

MingHan Yu¹, Leah A Marquez-Curtis¹, Janet A W Elliott²

Affiliations

¹ Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, T6G 1H9, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, T6G 1C9, Canada.
² Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, T6G 1H9, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, T6G 1C9, Canada. Electronic address: janet.elliott@ualberta.ca.

PMID: 38350589
DOI: 10.1016/j.cryobiol.2024.104857

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.

Keywords: Cryopreservation; Delayed cryo-injury; Fluorescence microscopy; Human umbilical vein endothelial cells; Interrupted slow cooling; Membrane integrity; Monolayer; Porcine corneal endothelial cells.