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**2**

*USA* 

**Cryopreservation of Adherent Smooth Muscle** 

*2Georgia Tech / Emory Center for the Engineering of Living Tissues, The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 3Medical University of South Carolina, Department of Regenerative Medicine and Cell* 

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

One strategy for finding interesting new cryoprotectants and cryopreservation strategies is by evaluating what happens in nature (Brockbank et al., 2011). No examples of organisms synthesizing DMSO to survive freezing conditions have been found to date, however several creatures have been found that employ glycerol (Brockbank et al., 2011) the first effective cryoprotectant to be discovered (Polge, 1949). Nature has developed a wide variety of organisms and animals that tolerate low temperatures and dehydration stress by accumulation of large amounts of disaccharides, particularly trehalose, including plant seeds, bacteria, insects, yeast, brine shrimp, fungi and their spores, cysts of certain crustaceans, and some soil-dwelling animals. While the cryoprotective capabilities of

of collagen mRNAs in a dose-dependent manner (Zeng et al., 2010).

**1. Introduction** 

**and Endothelial Cells with Disaccharides** 

Lia H. Campbell1 and Kelvin G.M. Brockbank1,2,3

*1Cell & Tissue Systems, Inc., North Charleston, SC* 

*Biology, Charleston, SC* 

