Weirether F J, Walker J S, Carter R C, Lincoln R E
Appl Microbiol. 1970 Aug;20(2):171-5. doi: 10.1128/am.20.2.171-175.1970.
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 10(7) to 4 x 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 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 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.
已开发并评估了用于保存悬浮培养中生长的组织细胞以及直接从储存在 -175°C 的接种物重建悬浮培养物的方法。所研究的因素包括处理时的pH值、处理温度、冷冻培养基以及从冷冻储备（-175°C）接种起始悬浮培养物的方法。使用细胞活力、细胞大小和冷冻后悬浮培养中的生长潜力这三个参数来评估各种因素。结果表明，在4°C下处理的细胞，在含有5%二甲基亚砜加10%牛血清、细胞浓度为2×10⁷至4×10⁷个细胞/毫升的培养基中，以每分钟1°C的速度冷冻至 -50°C，并储存在 -175°C，将直接从冷冻种子重建悬浮培养物。将1毫升冷冻储备接种到99毫升培养基中，通常在4至5天内在悬浮培养中产生2×10⁶至3×10⁶个活细胞/毫升（2×10⁸至3×10⁸个总细胞）。通过此程序保存的接种物在无血清或含血清的生长培养基中生长同样良好。
