Human mesenchymal stem cells (hMSC) are currently in use in over 400 clinical trials and are critical components of tomorrow’s cell-based products and devices (1, 2, 3). Secretion of biomolecules by hMSC influences many biological processes and is thought to be central to the mechanism of action. Since widespread clinical use of hMSC and cell-based therapies with positive economic outcomes will be facilitated by frozen storage, cryopreserved hMSC must maintain high levels of biological function upon thaw. Additionally, while hMSC have an excellent clinical track record in terms of safety, efficacy data has been difficult to come by, suggesting that more standardized cell formats are needed. This too could be addressed by effective means of cryopreservation, allowing off-the-shelf hMSC products to be widely used in Regenerative Medicine, Tissue Engineering and for 3D BioPrinting of cells and tissues.
To date, there have been conflicting results on the impact of cryopreservation on hMSC function. The Galipeau lab showed that cryopreserved MSC have impaired immunosuppressive function in response to the pro-inflammatory cytokine, IFN-γ (lower IDO response, and decreased T-cell suppression) relative to proliferating cells (5, 6). The LeBlanc group similarly found that cryopreserved hMSC have reduced responsiveness to IFN-γ, decreased production of anti-inflammatory mediators, and impaired blood regulatory properties (7). In contrast, other studies support the use of cryopreserved hMSC. The Mueller lab showed that cryopreservation of hMSC did not change the cells’ immunomodulatory activity, viability, or differentiation (8). The Weiss group also performed in vivo tests of thawed hMSC and found that “in an immunocompetent mouse model of allergic airways inflammation … thawed MSCs are as effective as fresh MSCs.” (9) The difference in results is likely due to differences in the cryopreservation formulations, controlled rate freezing protocols, and how the cells are thawed and handled prior to implantation.
To address the critical issue of cryopreservation in our hMSC systems, we compared the biological activity of RoosterBio hMSCs from 2 donors either (a) with cells straight out of cryopreservation (THAW) or (b) with cells that had been in culture for at least 5 days (FRESH), while controlling for PDL. Based on the literature, we established a conservative hypothesis for this study that cryopreserved hBM-MSC would exhibit diminished immunosuppression and altered angiogenic cytokine secretion compared to proliferating hBM-MSC in response to challenge by inflammatory cytokines. We tested this hypothesis with RoosterBio’s hBM-MSC, produced with GMP-compatible and scalable manufacturing processes, by comparing the immunomodulatory activity and angiogenic cytokine secretion of proliferating (FRESH) to cryopreserved and thawed (THAW) hBM-MSC. By presenting the results of this study, we hope to provide additional data points for the industry on the use of cryopreserved, off-the-shelf hMSCs for Regenerative Medicine, Tissue Engineering and 3D BioPrinting.
METHODS AND EXPERIMENTAL DESIGN: