Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Abstract

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at −175 C. The factors investigated were processing p H, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (−175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to −50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 × 10 7 to 4 × 10 7 cells/ml, and stored at −175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 × 10 6 to 3 × 10 6 viable cells/ml (2 × 10 8 to 3 × 10 8 total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.