Cryopreservation of pancreatic islet cells

Our study describes a useful procedure for cryopreservation of pancreatic islet cells. The pancreatic islets of adult hamsters were collected by collagenase digestion succeeded by gradient centrifugation and were dispersed by EDTA-Dispase treatment. The dispersed cells were suspended in medium consisting of 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum, supplemented with 10% dimethyl sulfoxide or glycerol. One milliliter each of the cell suspensions containing 10(6) cells was distributed into 2 ml polypropylene tubes, processed for freezing under six cooling conditions, and stored in liquid nitrogen (-196 degrees C). Of the various conditions tested, the cells suspended in dimethyl sulfoxide-containing medium and cooled in a program freezer at a rate of 0.5 degrees C/min down to -40 degrees C, succeeded by a rate of 5 degrees C/min down to -70 degrees C, resulted in the highest recovery rate of cells, 61.2% +/- 3.1%. This rate was comparable to those of several tissue culture cell lines frozen under similar conditions. Recovered cells preserved their morphologic characteristics under light and phase-contrast microscopy and formed cell sheets in culture. Response of insulin secretion to 3 mg/ml glucose appeared 6 hours after thawing, and the response to both 3 mg/ml glucose and 10 mmol/L theophylline was recovery to the same level as nonfrozen islet cells after 12 hours. The applicability of cryopreserved cells for the detection of islet cell surface antibody was demonstrated by the indirect method of immunofluorescence using islet cell surface antibody-positive human sera.