Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants

Abstract

The storage and transport of cells is a fundamental technology which underpins cell biology, biomaterials research, and emerging cell-based therapies. Inspired by antifreeze and ice-binding proteins in extremophiles, macromolecular (polymer) cryoprotectants are emerging as exciting biomaterials to enable the reduction and/or replacement of conventional cryoprotective agents such as DMSO. Here, we critically study post-thaw cellular outcomes upon addition of macromolecular cryoprotectants to provide unambiguous evidence that post-thaw culturing time and a mixture of assays are essential to claim a positive outcome. In particular, we observe that only measuring the viability of recovered cells gives false positives, even with non-cryoprotective polymers. Several systems gave apparently high viability but very low total cell recovery, which could be reported as a success but in practical applications would not be useful. Post-thaw culture time is also shown to be crucial to enable apoptosis to set in. Using this approach we demonstrate that polyampholytes (a rapidly emerging class of cryoprotectants) improve post-thaw outcomes across both measures, compared to poly(ethylene glycol), which can give false positives when only viability and short post-thaw time scales are considered. This work will help guide the discovery of new macromolecular cryoprotectants and ensure materials which only give positive results under limited outcomes can be quickly identified and removed.

Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes

“…This is important as shorter post-thaw times lead to false positives if cells are evaluated before apoptotic processes have had a chance to complete. 13,54 Therefore the number presented is the number of cells after 24 hours, relative to those prior to freezing, and hence includes some time for potential cell growth, but remains a robust measure to avoid overestimation if shorter times to assay are used.…”

Low DMSO Cryopreservation of Stem Cells Enabled by Macromolecular Cryoprotectants

“… 24 These studies highlight that immediate post-thaw viability is not a good predictor of cell health. 28 …”

Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation

“…In order to gain a more detailed account of the cell population, both floating cells and attached cells were counted at 24 h post-thawing (Figure 2). However, even this will not capture the fate of all cells, since some cryopreserved cells will be mechanically destroyed during the freezing and thawing process due to irreversible intracellular ice crystal formation (Murray and Gibson, 2020). These destroyed cells will be missing from the live/dead and attached/floating cell counts.…”