Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media - Vitrification versus Slow Freezing Methods

PloS One
Achim von BomhardNicole Rotter

Abstract

Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco's modified Eagle medium Ham's F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1...Continue Reading

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Citations

Aug 8, 2018·Organogenesis·Gesine Pless-PetigUrsula Rauen
Nov 7, 2019·Tissue Engineering. Part C, Methods·Yuzhao SunSophie X Deng
Apr 17, 2020·Frontiers in Bioengineering and Biotechnology·Þóra SigmarsdóttirÓlafur E Sigurjónsson
Feb 1, 2022·Chemical Reviews·Thomas BiedenbänderBjörn Corzilius

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Methods Mentioned

BETA
Assay
fluorescence-activated cell sorting

Software Mentioned

TiProtec
GraphPad
GraphPad Prism

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