Algorithm Optimization of non-DMSO Cryopreservation Protocols to Improve Mesenchymal Stem Cell Post-Thaw Function

Abstract

Mesenchymal stem cells (MSCs) are a common transfusion cell therapy that have been used in over 300 clinical trials to treat over 2000 patients with diseases ranging from Crohn’s disease to heart failure. These cells are frequently cryopreserved to better coordinate the timing of cell administration with patient care regimes and to accommodate transport of samples between different sites of collection, processing, and administration. However, cryopreservation with DMSO (the current gold standard) can result in poor cell function post-thaw and adverse reactions upon infusion. We hypothesize that non-DMSO cryopreservative molecules, including sugars, sugar alcohols, amino acids, and other small molecule additives, can be used in combination to protect cell viability and function post-thaw. This research demonstrates that some combinations of non-DMSO cryopreservatives preserve cell functionality better than others, and these effects are dependent not on osmotic or physical changes in solution, but on biological changes that affect the cell during the freezing process. We observe that there is likely a sweet spot concentration combination that produces maximum recovery for each combination of molecules, and demonstrate that an evolutionary algorithm can be used to identify optimized combinations of molecules that yield high cell recovery post-thaw. Additionally, we demonstrate that these novel solutions maintain MSC functionality when evaluated using surface markers, attachment, proliferation, actin alignment, RNA expression, and DNA hydroxymethylation. These advances in cryopreservation can improve cell therapy, and ultimately patient care.