# Electrophysiological analysis of mesenchymal stem cells post-cryopreservation highlights the need for a recovery period: implications for cell-based therapies

## TL;DR

This study shows that mesenchymal stem cells need time to recover after freezing, which is important for their use in therapies.

## Contribution

The study introduces electrophysiological methods to assess MSC recovery post-cryopreservation, revealing incomplete membrane recovery and DMSO's potential role.

## Key findings

- MSC health significantly deviates from pre-freezing state immediately after thawing.
- Membrane capacitance does not fully recover even after three days post-thaw.
- DMSO as a cryopreservant may be linked to prolonged recovery periods in MSCs.

## Abstract

Human mesenchymal stem cells (MSCs) are characterized by their ability to differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes, making them promising candidates for cell-based therapies. Whilst the optimum method of clinical use is to use MSCs immediately after harvesting and expansion, there is often a need to cryostore MSCs before transplantation; this negatively impacts MSCs, affecting phenotypic marker expression, viability, differentiation potential, and other properties. There is consequently a requirement for methods to determine the biophysical state of MSCs post-thaw, in order to determine an optimum time for implantation after cells have recovered to a “normal” state. Typically, the primary method of assessing this is by measurement of cell viability; the cellular membrane is one of the key indicators of cell health and cell-cell interactions. Membrane-integrity dyes such as trypan blue are commonly used for binary viability checks, and ion tracking dyes offer insight into channel activation. However, these are typically expensive and time-consuming to use, limiting their efficacy in relatively high-throughput manufacturing scenarios. We used novel electrophysiological methods to assess MSC-health following freezing and thawing. Our results indicate that MSC health deviates significantly from its original phenotype immediately after thawing and only begins to resemble the pre-freezing state after three days. Notably, cell membrane capacitance does not fully recover to pre-freezing levels, even after this period. Results also suggest that the use of DMSO as a cryopreservant may be associated with the prolonged recovery period.