Cryopreservation of Adherent Smooth Muscle and Endothelial Cells with Disaccharides

There is a need for mammalian cell cryopreservation methods that either avoid or improve upon outcomes employing dimethyl sulfoxide (DMSO) as a cryoprotectant. DMSO was the second effective cryoprotectant to be discovered (Lovelock, 1959). Cell cryopreservation usually involves slow rate freezing with DMSO in culture medium and storage below -135°C for later use. Typically as long as there are enough cells surviving to start an expanding proliferating culture the yield of viable cells after thawing is not an important consideration. However, there are instances where cell yield and viability can be very important. Examples include minimization of expensive delays when starting cultures for bioreactor protein manufacturing runs and cellular therapies that involve administering cells into patients for treatment of various diseases, such as cancer. While some cells, for example fibroblasts, are easily cryopreserved other cell types like keratinocytes, hepatocytes, and cardiac myocytes do not freeze well and cell yields are often <50%. Furthermore, current opinion is that DMSO should be removed before cells are infused into patients (Caselli et al., 2009; Junior et al., 2008; Mueller et al., 2007; Otrock et al., 2008; Schlegel et al., 2009). The mechanism for DMSO cytotoxicity has not been determined, however, it is thought to modify membrane fluidity, induce cell differentiation, cause cytoplasmic microtubule changes and metal complexes (Barnett 1978; Katsuda et al., 1984, 1987; Miranda et al., 1978). DMSO also decreases expression of collagen mRNAs in a dose-dependent manner (Zeng et al., 2010).