Cryopreservation of heart cells from the eastern oyster

Conditions were developed to cryopreserve cells from pronase-dissociated at ria and ventricles of eastern oysters (Crassostrea virginica). The effect o f three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfox ide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 7 5 degrees C), and three cooling rates (slow. medium, fast) were compared. C ells were frozen at -80 degrees C and plunged in liquid nitrogen. Thawed ce lls were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4 ,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-te trazolium, and by comparing the relative spreading of cells between treatme nts. The best conditions for freezing and thawing of cells for each cryopro tectant were selected and a final study was performed to compare cryoprotec tants. For this final study, ive measured the number of cells and their via bility 3 d after thawing, in addition to determining cell metabolic activit y and cell spreading. Primary cultures of cells frozen without cryoprotecta nt and of nonfrozen cells were used as controls in all studies. Atrial cell s were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45 degrees C. After thawing, atrial cells sho wed 53 +/- 5% of the metabolic activity, 84 +/- 5% of the number, and 92 +/ - 2% of the viability of nonfrozen cells. For ventricular cells, 10% glycer ol, a medium cooling rate, and thawing at 25 degrees C yielded the best res ults. The thawed ventricular cells showed 83 +/- 5% of the metabolic activi ty, 91 +/- 5% of the number, and 96 +/- 2% of the viability of nonfrozen ce lls.