A08 Session 2: Biopreservation of biologics: Translation from stem cells to cancer : Differential activation of stress pathways in human mesenchymal stem cells following biopreservation.

Research on human mesenchymal stem cells (hMSC) has expanded considerably in recent years as continued investigations have demonstrated their potential for reestablishment of in vivo functions. Further, as hMSC studies have increased our understanding of their utility, this has in turn driven the development of hMSC-based technologies. These technologies range from the exploitation of innate in vivo functions to target and repair damaged tissue, to removal and purification of hMSC for use in numerous other settings such as tissue engineering, cell therapies, etc. Essential to these developments is the ability to process and maintain these unique biologics without the loss of viability and functionality. As such, bioprocessing has become a limiting factor in the downstream use of hMSC as non-uniform samples and variation in function impairs efficacy, outcome and application of these advancements. In an effort to improve hMSC bioprocessing protocols, this study focused on the analysis and modulation of hMSC stress response following biopreservation. Two biopreservation stress regimes (hypothermic storage and hypoxic normothermic storage) were utilized to examine cell stress pathway activation. Specifically the unfolded protein response (UPR) was examined as a mediator of post-storage stress response given its recent link to preservation failure in other cell systems. Additionally, the incorporation of two chemical agents (i.e. salubrinal and resveratrol) during storage was conducted to examine the effect of targeted molecular modulation. The results of these studies demonstrated a differential response of hMSC to targeted modulation dependent upon storage temperature and carrier medium. The addition of resveratrol to MSCGM (complete growth media), HBSS (balanced salt solution) or ViaSpan (UW solution) yielded marked improvement to immediate post-storage viability (increases of 53%, 50%, and 10%, respectively) and regrowth of hMSC following hypothermic storage. Interestingly, salubrinal addition yielded modest improvement to MSCGM and HBSS stored hMSC survival (16% and 8% increases) while its addition to ViaSpan resulted in decreased viability and impaired ability to repopulate. In contrast, use of these modulators during hypoxic normothermic storage in either MSCGM or HBSS yielded negative impacts as growth inhibition or cell loss was observed in the samples. Further in-depth analyses revealed changes to both apoptotic and necrotic populations post-storage through the use of these modulators. Western blot analysis confirmed changes in the apoptotic signaling pathways as well as implicated the involvement of the UPR pathway signaling following storage. These findings illustrate the potential for storage specific cell stress pathway modulation to improve hMSC biopreservation. Further, the differential response of pathway modulation demonstrates that a more in-depth understanding of hMSC response is needed to improve each step throughout the biopreservation process (pre-storage processing, preservation and post-preservation recovery) to enable the delivery of optimal hMSC samples.