Cryopreservation Using Proline and Biocompatible Polymers: PVA, PVP, and HA

Abstract

Intracellular and extracellular ice crystals also damage cells through osmotic stress during freezing and thawing. Although DMSO is commonly used as a cryoprotectant, its cytotoxicity limits its use. Fetal bovine serum (FBS) also presents immunogenicity challenges, restricting the safety of standard cryopreservation. This study aimed to find freezing conditions for MCF-7 cells that remain effective while reducing or avoiding DMSO and eliminating FBS. To achieve this, various combinations of FBS, DMSO, proline, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), hyaluronic acid (HA), and bovine serum albumin (BSA) were used. FBS alone did not provide a significant cryoprotective effect. However, DMSO treatment increased survival rates compared to the DMSO-free control group, so 2% DMSO was selected as the baseline. Proline showed no cryoprotective effect: it had no benefit alone and did not provide any additional protection when added to other formulations. PVA also showed no beneficial effect on its own and was even harmful when combined with DMSO. The non-permeable polymer PVP provided significant cryoprotective effects; even 5% PVP alone maintained a meaningful survival rate post-thaw, and the highest survival rate was achieved when 5% PVP and 2% DMSO were combined without FBS. HA was ineffective alone and provided only limited benefits under FBS-containing conditions. Additionally, replacing FBS with BSA at equivalent albumin content also maintained high survival rates, showing that 1.15% BSA and 2% DMSO reproduced the protective effects of the conventional 10% DMSO protocol. In summary, this study identified two strategies for safer cryopreservation of MCF-7 cells. First, 5% PVP can be used without DMSO or FBS. Second, 2% DMSO combined with 1.15% serum albumin can substitute for conventional protocols that use higher DMSO and FBS.