LB33 - Transient warming events occurring after freezing impairs umbilical cord-derived mesenchymal stromal cell functionality

Abstract

Mesenchymal stromal cells (MSC) have shown promising results for the treatment of refractory acute graft-versus-host-disease and are considered for many other therapeutic applications. While safety of MSC infusion has been demonstrated, the use of cryopreserved MSC in clinical trials has raised concerns about the retention of their functional activity. This has led to the recommendation by several experts in the field to use freshly harvested MSC, even though this approach is much less practical from a logistic point of view. In the present study, we revisited the impact of cryopreservation on MSC and addressed the possibility that warming events on frozen cells rather than cryopreservation per se could impair MSC functionality and affect cell integrity. Following controlled-rate freezing to −130°C, umbilical cord-derived MSC were left at room temperature for 2 to 10 minutes (group 1) or on dry ice for 10 minutes (group 2), before being transferred into liquid nitrogen. MSC of each experimental group were subsequently thawed and tested together with their freshly harvested counterpart. We first determined viability by DAPI staining and found a slight (<10%) albeit significant decrease in post-thaw viability of cells from groups 1 and 2 compared to freshly harvested cells. We next determined the ability of MSC to inhibit the proliferation of CD3/CD28 activated T cells as a measure of their immunosuppressive effect. MSC from group 1 had a dramatically reduced immunosuppressive effect (>75%) while MSC from group 2 were as efficient as their freshly harvested counterpart. IFN-γ-induced IDO expression was also impaired in group 1 MSC. Post-thaw cell integrity was evaluated by adhesion to fibronectin-coated dishes, lactate dehydrogenase release and actin filament staining. Group 1 MSC had a reduced ability to bind to fibronectin and exhibited a higher level of lactate dehydrogenase release compared to MSC from group 2. Defects in actin cytoskeleton were observed in thawed MSC from group 1 but not group 2. We thus conclude that warming events after freezing and not cryopreservation per se significantly impair MSC functionality and induce cellular damage upon thawing indicating that properly cryopreserved MSC can be an advantageous alternative to freshly harvested cells for therapeutic purposes.