**3.1 H5 poration**

One of the first strategies for utilizing disaccharide sugars as cryoprotectants involved the use of a modified pore forming complex. In our initial studies we evaluated the H5 mutant αhemolysin (Bayley, 1994) using two adherent cell lines, A10 and CPAE. The earlier studies had been done with cells in suspension (Eroglu et al., 2000). We also evaluated sucrose, another disaccharide sugar that is commonly found in nature, for its potential usefulness as a cryoprotectant. Using the protocol of Eroglu et al as a starting point, a protocol was established for adherent cells. Several parameters were evaluated and included the H5 concentration, time of poration, concentration of trehalose loaded, and time for loading trehalose. Conditions that worked best with adherent cells included 20 minutes for poration followed by 60 minutes for trehalose loading. The highest concentration of trehalose that caused the least drop in cell viability was 0.2M. The optimum H5 concentration varied according to cell type. The A10 smooth muscle cells were porated with 12.5 µg/mL of H5 while the endothelial CPAE cells were porated with 50 µg/mL. In contrast, the fibroblasts and keratinocytes in the literature were porated with 25 µg/mL (Eroglu et al., 2000). Other changes to the protocol were made that benefited viability for adherent cells specifically and included addition of trehalose prior to the addition of H5, the base solution used for poration, and the amount of EDTA (1 mM versus 10 mM) for the removal of Zn+ prior to poration. After cryopreservation, however, poor viability was obtained with both cell types. A10 cells demonstrated a viability of 5.57±0.17%. The endothelial cells demonstrated similar viabilities. These values were not as good as those observed when suspended cells were cryopreserved with sugars in the literature. However, it is our experience that adherent cells are generally more difficult to cryopreserve regardless of the cryoprotectant used.
