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. 1970 Aug;20(2):171–175. doi: 10.1128/am.20.2.171-175.1970

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

Francis J Weirether 1, Jerry S Walker 1,2, Richard C Carter 1,3, Ralph E Lincoln 1
PMCID: PMC376894  PMID: 5529630

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 × 107 to 4 × 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 × 106 to 3 × 106 viable cells/ml (2 × 108 to 3 × 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brown B. L., Nagle S. C., Jr Preservation of mammalian cells in a chemically defined medium and dimethylsulfoxide. Science. 1965 Sep 10;149(3689):1266–1267. doi: 10.1126/science.149.3689.1266. [DOI] [PubMed] [Google Scholar]
  2. HSU T. C., MERCHANT D. J. Mammalian chromosomes in vitro. XIV. Genotypic replacement in cell populations. J Natl Cancer Inst. 1961 May;26:1075–1083. [PubMed] [Google Scholar]
  3. Mazur P., Farrant J., Leibo S. P., Chu E. H. Survival of hamster tissue culture cells after freezing and thawing. Interactions between protective solutes and cooling and warming rates. Cryobiology. 1969 Jul-Aug;6(1):1–9. doi: 10.1016/s0011-2240(69)80002-7. [DOI] [PubMed] [Google Scholar]
  4. NAGINGTON J., GREAVES R. I. Preservation of tissue culture cells with liquid nitrogen. Nature. 1962 Jun 9;194:993–994. doi: 10.1038/194993b0. [DOI] [PubMed] [Google Scholar]
  5. PHILLIPS H. J., TERRYBERRY J. E. Counting actively metabolizing tissue cultured cells. Exp Cell Res. 1957 Oct;13(2):341–347. doi: 10.1016/0014-4827(57)90013-7. [DOI] [PubMed] [Google Scholar]
  6. SCHERER W. F., HOOGASIAN A. F. Preservation at subzero temperatures of mouse fibroblasts (strain L) and human epithelial cells (strain HeLa). Proc Soc Exp Biol Med. 1954 Nov;87(2):480–487. doi: 10.3181/00379727-87-21419. [DOI] [PubMed] [Google Scholar]
  7. SWIM H. E., HAFF R. F., PARKER R. F. Some practical aspects of storing mammalian cells in the dry-ice chest. Cancer Res. 1958 Jul;18(6):711–717. [PubMed] [Google Scholar]
  8. Weirether F. J., Walker J. S., Lincoln R. E. A precise method for replicating suspension cultures of mammalian cells. Appl Microbiol. 1968 Jun;16(6):841–844. doi: 10.1128/am.16.6.841-844.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]

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