**Cryopreservation of Adherent Smooth Muscle and Endothelial Cells with Disaccharides**

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

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

#### **1. Introduction**

16 Current Frontiers in Cryopreservation

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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).

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

Cryopreservation of Adherent Smooth Muscle and Endothelial Cells with Disaccharides 19

presented in the discussion. Cells were plated at 10,000-20,000 cells/well the night before in 96 well microtiter plates. The next day, the cells were washed with DMEM containing 1 mM EDTA for 2 minutes and then again with DMEM to remove the EDTA. 0.2M trehalose was added and incubated for 20 minutes at 37oC followed by the appropriate concentration of H5 for the respective cell type. Cells were porated and loaded with trehalose for 1 hour at 37oC before addition of DMEM with 25 µM ZnSO4 or 10% serum to close the pores. Trehalose in DMEM was then added to the wells followed by cryopreservation using a controlled rate freezer (Planar) at ~-1.0oC/min from 4ºC to -80ºC with a programmed nucleation step at -5.0oC. Cryopreserved cells were stored overnight at <-135oC. The next day, the cells were placed at -20oC for 30 minutes followed by rapid thawing at 37oC (Campbell et al., 2003; Taylor et al., 2001). The cell cultures were washed twice and then placed at 37oC for 1 hour to recover under normothermic cell culture conditions before

Cells were plated at 10,000-20,000 cells/well and placed in culture. The next day, the culture medium was replaced with EMEM or DMEM containing trehalose (0-0.6M) and cultured at 37oC for varying periods of time. After culture, the solution was replaced with fresh medium containing trehalose (0-0.6M) and the cells were cryopreserved using a controlled

Cells were plated at 10,000-20,000 cells/well and placed in culture. The next day, the cells were washed with poration buffer (phosphate-buffered saline [PBS] with 1X essential amino acids, 1X Vitastock, 5.5 mM glucose) designed to optimize binding of ATP4- to the receptor and facilitate formation of the pore. The cells were then placed in 50 µl poration buffer, pH of 7.45, with 0.2M trehalose. A stock solution of 100 mM ATP4-, pH of 7.45, was made fresh and added to each well to achieve a final concentration of 5 mM. After addition of the ATP4-, the cells were left at 37oC for 1 hour to allow sugar uptake. Following incubation, 200 µl of DMEM plus 1 mM MgCl2 was then added to the cells at 37oC to close the pores. After 1 hour

Cell viability was determined using the non-invasive metabolic indicator alamarBlue™ (Trek Diagnostics). A volume of 20 µl was added to cells in 200 µl of DMEM (10%FCS) and the plate was incubated at 37oC for 3 hours. Plates were read using a fluorescent microplate reader (Molecular Dynamics) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Viability was measured before and after sugar loading, immediately

All experiments were repeated at least four times with four replicates in each experiment. Statistical differences were assessed by two way analysis of variance. P-values 0.05 were

of recovery from the loading procedure cryopreservation was initiated.

after thawing and at several later time points post-thaw.

assessment of cell viability.

**2.4 Cell poration with ATP** 

**2.5 Assessment of cell viability** 

**2.6 Statistical methods** 

regarded as significant.

**2.3 Pretreatment (Incubation) with trehalose** 

rate freezer as described for H5 above.

sucrose and trehalose has been known for years, conventional cryopreservation protocols have generally not employed them even though early work with them demonstrated their ability to protect proteins and membrane vesicles during freezing (Rudolf & Crowe, 1985; Crowe et al., 1990). Trehalose has both major advantages and disadvantages for potential preservation of mammalian cells. On the negative side mammalian cells do not have an active trehalose transport system for uptake of trehalose from the extracellular environment, while on the plus side if you can get it in mammalian cells it is not metabolized giving the opportunity for trehalose to be accumulated to potentially effective preservation concentrations. The purpose of the studies presented here were; 1) to assess or review alternative strategies for delivery of trehalose into mammalian cells, and; 2) to determine whether the benefits were specific to trehalose by investigating alternative sugars employing the same loading strategies.
