Cryobanked Mscs Have A Markedly Increased Susceptibility to T-Cell Mediated Contact Cytolysis in Vitro Immediately Post Thaw and Shortened in Vivo Persistence Relative to Physiologically Fit Culture-Rescued Mscs

The near totality of clinical trials examining the therapeutic use of MSC-like cells utilizes cryobanked MSCs that are administered to patients within a few hours following thawing. We have previously demonstrated that the process of thawing leads to a reversible molecular genetic heat-shock response and altered functionalities. Here, we further analyzed the impact of thawing on MSC's fitness. We have shown that thawing induces significant disruption of MSC's actin cytoskeleton polymerization. Although pharmacological disruption of actin cytoskeleton is associated with a reduction of MSC's lung trapping in mice, in vitro immunosuppressive properties were not significantly affected. Thus thawing induced a defect in actin polymerization, which predominantly shortens MSC's in vivo persistence, yet in itself does not provide a full explanation for loss of suppressive effects in vitro. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Microscopic and flow cytometry analysis demonstrated that unlike actively growing fresh MSCs, thawed MSCs were lysed upon coculture with activated autologous PBMCs and the lysing effect was further enhanced with allogeneic PBMCs (MSC Survival Fresh:Cryo allogeneic (7383 ± 865:1018 ± 334), autologous (17696 ± 961:9647 ± 2081). Thawed MSC's cytolysis correlated with higher levels of caspase and granzymeB activities in MSCs (Fresh 11 ± 0.2%, Cryo 69 ± 10) upon coculture with allogeneic activated T cells. We tested the hypothesis that IFNγ pre-licensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by IDO blocker. In addition, both IFNγ prelicensed thawed and fresh MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do that. However, ± /-IFNγ prelicensed thawed MSCs do not lung engraft in vivo as well as fresh MSCs (Bioluminescence Total Flux: Control: 5110 ± 50.3, Live:20933 ± 8203, Cryo:10878 ± 5027, IFNgCryo:13705 ± 5786) suggesting that IFNγ prelicensing does not rescue thawing induced defect on MSC's lung trapping properties. Our study identified reversible and irreversible cryoinjury mechanisms arising from susceptibility to host T-cell cytolysis that affect MSC's immunosuppressive and lung trapping properties.