Very cold but still too warm; functional characterization of transient warming events in stem cell grafts.

Abstract

Transient warming events (TWEs) are warming and cooling cycles that can exacerbate the loss of function in frozen cells or tissues during storage. We previously showed that cord blood (CB) hematopoietic stem and progenitor cell (HSPC) grafts can be exposed to TWE during banking operations and that HSPCs are sensitive to TWE, stressing the importance of further investigations. Herein, we first established a model to study TWE that replicated observed reductions in cell function. Notably, TWE near to and past the reported intracellular glass transition temperature (Tg, ∼ -50°C) led to a far greater loss in cell viability and graft potency than TWE at cooler temperature (≤ -80°C). Also, HSPCs' natural resistance to delayed onset cell death was abolished by TWE ≥ -80°C. Next, we compared the cryoprotective properties of three different commercial solutions; a dimethyl sulfoxide (DMSO)/dextran-40 solution (CryoSolve) and two DMSO-free freezing solutions. All solutions afforded similar protection under normal cryostorage but provided different outcomes after TWE. We also investigated the impact of cryopreserving CB grafts with a lower DMSO concentration (5%, v/v). Interestingly, lowering DMSO did not accentuate the loss of function due to TWE. Rather, lower DMSO concentration in both control and TWE samples was associated with slight increases in the recovery of CB CD34+ cells. Finally, we sought to clarify the contribution of ice recrystallization to the loss of cell function during TWE. We observed an inverse relationship between ice-crystal size and cell function in the different cryosolutions. Moreover, supplementation with an ice recrystallization inhibitor protected HSPCs from TWE. In conclusion, this study presents new insights into TWE and freezing solutions for stem cell grafts. It also provides guidance on the appropriate storage of frozen stem cell grafts under unexpected circumstances. Lastly, prevention of ice recrystallization during warming and cooling cycles can prevent the ensuing loss of function.