Protein-free solution containing trehalose and dextran 40 for cryopreservation of human adipose tissue-derived mesenchymal stromal cells

Abstract

We tested the efficacy of lactated Ringer's solution with 3% trehalose and 5% dextran 40 (LR-3T-5D) as a vehicle solution for cryopreservation using human adipose-derived mesenchymal stromal cells (hADSCs) with dimethyl sulfoxide (Me2SO). We also tested the effect of the Me2SO concentration in the cryopreservation solution, and the effect of washing with lactated Ringer's solution with 3% trehalose (LR-3T) and replacement with LR-3T or LR-3T-5D. LR-3T-5D was more effective for cell viability, viable cell recovery ratio, annexin V-positive ratio, and colony-forming capacity as a vehicle solution for cryopreservation with 10% Me2SO than LR. The additive effects as cryoprotectants of trehalose and dextran 40 were confirmed to be dose dependent. The cell viability, cell proliferation ability, cell differentiation ability, and the ratio of cell surface positive/negative markers of hADSCs were well maintained after cryopreservation with LR-3T-5D containing 10% Me2SO in liquid nitrogen or in a −80 °C freezer. The cell viability and the proliferation curve in LR-3T-5D with 5% Me2SO were comparable to those with 10% Me2SO. LR-3T-5D was superior to LR-3T as a replacement solution in terms of viability and annexin V positivity. Our data showed that LR-3T-5D is effective as a vehicle solution for cryopreservation. Reducing Me2SO concentration to 5%, and washing and replacement with fresh LR-3T and LR-3T-5D after thawing, are feasible approaches to maintain cryopreservation efficacy.