Cryopreserved mesenchymal stem cells regain functional potency following a 24-h acclimation period

Abstract

Background Mesenchymal stem cells (MSCs) are attractive cell-therapy candidates. Despite their popularity and promise, there is no uniform method of preparation of MSCs. Typically, cells are cryopreserved in liquid nitrogen, thawed, and subsequently administered to a patient with little to no information on their function post-thaw. We hypothesized that a short acclimation period post-thaw will facilitate the recovery of MSC’s functional potency. Methods Human bone-marrow-derived MSCs were divided into 3 groups: FC (fresh cells; from existing culture); TT (thawed + time; acclimated for 24 h post-thaw); and FT (freshly thawed; thawed and immediately used). The 3 groups were analyzed for their cellular and functional potency. Results Phenotypic analysis demonstrated a decrease in CD44 and CD105 surface markers in FT MSCs, with no change in the other two groups. All MSCs were able to differentiate down the osteogenic and chondrogenic lineages. In FT cells, metabolic activity and apoptosis was significantly increased with concomitant decrease in cell proliferation; clonogenic capacity; and key regenerative genes. Following 24-h acclimation, apoptosis was significantly reduced in TT cells with a concomitant upregulation in angiogenic and anti-inflammatory genes. While all MSCs significantly arrested T-cell proliferation, the TT MSCs were significantly more potent. Similarly, although all MSCs maintained their anti-inflammatory properties, IFN-γ secretion was significantly diminished in FT cells. Conclusions These data demonstrate that FT MSCs maintain their multipotent differentiation capacity, immunomodulatory function, and anti-inflammatory properties; yet, various aspects of cell characteristics and function are deleteriously affected by cryopreservation. Importantly, a 24-h acclimation period ‘reactivates’ thawed cells to recover their diminished stem-cell function.

Low DMSO Cryopreservation of Stem Cells Enabled by Macromolecular Cryoprotectants

“…Antebi et al reported the 24 h post-thaw viability of MSC frozen with 10% DMSO to be ∼80%, highlighting that supplementation with our polyampholyte can achieve the same results but using just a quarter of the [DMSO]. 23 In a clinical context, this is a highly desirable achievement, as lowering the DMSO content of transfused stem cells has been shown to reduce the incidence of adverse clinical side effects 5-fold. 7 Critically, we have reported 24 h post-thaw results as MSCs require 24 h post-thaw to regain function and enable true evaluation of recovery.…”

“… 7 Critically, we have reported 24 h post-thaw results as MSCs require 24 h post-thaw to regain function and enable true evaluation of recovery. 23 This point is crucial to show the impact of this work; Zhao et al reported post-thaw viabilities of ∼90% when freezing 3T3 cells with polyampholytes. However, the majority of cells died after 24 h in culture.…”

The Effect of Blood-Derived Products on the Chondrogenic and Osteogenic Differentiation Potential of Adipose-Derived Mesenchymal Stem Cells Originated from Three Different Locations

“…Cryopreservation has been shown to influence MSCs' therapeutic value with regard to impaired immunosuppressive activities, increased proapoptotic features, and impaired regenerative potential [52][53][54]. More recent studies show that MSCs do maintain their differentiation capacity and also immunomodulatory features after cryopreservation, but that a "reactivation" period of at least 24 h after thawing is beneficial [55]. Our P2 cells after thawing had more than 24 h to "reactivate" before experiments were started.…”

Regeneration of a full-thickness defect of rotator cuff tendon with freshly thawed umbilical cord-derived mesenchymal stem cells in a rat model

“…Conversely, T-UC MSCs, in this study, showed above 97% viability of cells with comparable morphology and proliferation compared with the C-UC MSCs even after fresh thawing procedure. Cell viability depends on the thawing method, duration of cold storage, and reagents used [36]. Relatively short storage periods [37] and optimal concentration of DMSO (10%) are crucial factors contributing to high cell viability in cryopreserved conditions [35,[37][38][39].…”