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Appl Environ Microbiol. 1970 August; 20(2): 171-175
Copyright © 1970 American Society for Microbiology. All Rights Reserved.

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks ¹

Department of the Army, Fort Detrick, Frederick, Maryland 21701

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 pH, 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 x 10⁷ to 4 x 10⁷ 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 x 10⁶ to 3 x 10⁶ viable cells/ml (2 x 10⁸ to 3 x 10⁸ 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.

² Present address: U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Md. 21701.

³ Present address: National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014.

Presented in part at the 19th Annual Meeting of the Tissue Culture Association, San Juan, Puerto Rico, 10 June 1968.