Quantitative assessment of the impact of cryopreservation on human bone marrow-derived mesenchymal stem cells: up to 24 h post-thaw and beyond

Abstract

Background The effects of cryopreservation on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are still ill-defined. In this study, a quantitative approach was adopted to measure several post-thaw cell attributes in order to provide an accurate reflection of the freezing and thawing impact. Methods Fresh and cryopreserved passage-matched cells from three different donors were discretely analysed and compared for their viability, apoptosis level, phenotypic marker expression, metabolic activity, adhesion potential, proliferation rate, colony-forming unit ability (CFUF) and differentiation potentials. Results The results of this study show that cryopreservation reduces cell viability, increases apoptosis level and impairs hBM-MSC metabolic activity and adhesion potential in the first 4 h after thawing. At 24 h post-thaw, cell viability recovered, and apoptosis level dropped but metabolic activity and adhesion potential remained lower than fresh cells. This suggests that a 24-h period is not enough for a full recovery. Beyond 24 h post-thaw, the observed effects are variable for the three cell lines. While no difference is observed in the pre- and post-cryopreservation proliferation rate, cryopreservation reduced the CFUF ability of two of the cell lines and variably affected the adipogenic and osteogenic differentiation potentials of the three cell lines. Conclusion The data collected in this study clearly show that fresh and cryopreserved hBM-MSCs are different, and these differences will inevitably introduce variabilities to the product and process development and subsequently imply financial losses. In order to avoid product divergence pre- and post-cryopreservation, effective strategies to mitigate freezing effects must be developed and implemented.

Comparative Bioactivity Analysis for Off-the-Shelf and Culture–Rescued Umbilical Cord-Derived Mesenchymal Stem/Stromal Cells in a Xeno- and Serum-Free Culture System

“…In contrast, a study comparing BM-MSCs in fresh, freshly thawed, and 24-hour-culture-rescue post-thaw conditions indicated that compared to the other two groups, the freshly thawed group showed decreased CD44 and CD105 expression, decreased proliferation and clonogenic capacity associated with increased apoptosis 7 . Supporting these results, Bahsoun et al recently reported that cryopreservation altered the cellular and functional potency of BM-MSCs and that a 24 h rescue did not fully recover the properties of the cells 14 . The diverse observations might be due to the different tissue sources and culture conditions used in these studies.…”

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations

“…Previously, functional differences between pre-freeze (fresh) and post-freeze MSCs were also reported by others [ 43 , 44 ]. Cryopreservation has been shown to affect viability, apoptosis and metabolic activity for up to 24 h post plating and this can be tissue source or donor-related [ 45 , 46 ]. These findings will need further investigation for validation.…”

Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics

“…In turn, Galleu et al found that triggering of T-cell mediated apoptosis may be a hallmark of MSC-immunomodulation in vivo [ 31 ]. Intriguingly, Bashoun et al found that highest apoptosis and lowest viability peaked at 4 and 24 h post-thawing, respectively, but that there was no difference between fresh and cryo MSCs in functional outcome [ 76 ]. Nonetheless, these studies demonstrate that increased T- and NK-cell recognition of thawed vs. fresh MSCs in the clinical setting should be anticipated (Fig.…”

“…There is great clinical and commercial potential for employing cryopreserved MSC products with direct thawing and “bed-side” infusion of MSC products to patients [ 39 , 70 ], but there are still questions considering optimal manufacturing and standardization of this process. As Bahsoun et al pointed out [ 76 ], cryopreservation of MSCs may be considered an essential part of the process-chain for effective and economically viable distribution to healthcare facilities / patients, but differences in viability and functionality of cryo MSCs versus fresh MSCs cannot be overlooked.…”