John E. Shannon

Enzyme polymorphisms as genetic signatures in human cell cultures

The electrophoretic resolution of seven relatively polymorphic human gene-enzyme systems expressed in tissue culture cells can be used as a sensitive genetic monitor for intraspecific cell contamination. An identical genotype at each of the same allozyme loci provides a 95% (or greater) confindence estimate of the identity of two cultured lines, on the basis of the allelic frequencies of the seven enzyme loci in natural populations and in populations of independently derived cultured cells. Of 27 commonly used human cell lines examined, only one of 351 pairwise comparisons proved genetically indistinguishable.

A molecular approach to the identification and individualization of human and animal cells in culture: Isozyme and allozyme genetic signatures

SummaryThe electrophoretic resolution of a group of geneticallymonomorphic gene-enzyme systems that are developmentally and biologically ubiquitous has been used to provide a species-specific and type-specific biochemical characterization of various cultured cells. The relative mobilities of gene-enzyme systems representing nine distinct gene products from cell cultures of 25 species fromDrosophils to man are presented. These isoenzymes effectively discriminate interspecies cell-to-cell contamination and almost invariably serve to identify the contaminating species. The resolution of eightpolymorphic gene-enzyme systems in human cell cultures provides a virtually unique allozyme genetic signature as a monitor of intraspecies cellular contamination. The genetic signatures of 47 commonly used human cells are presented. Included in the test were seven putative HeLa (human cervical carcinoma) contaminants each of which expressed a signature identical with that of HeLa. The probability that an unrelated human cell line will have a signature identical to a typed cell is computed for each line from the genotypic frequencies at each locus in a population of cultured human cells. The gene frequencies of this cell population are comparable to the same frequencies in natural human populations. The most common human signature has a frequency (and therefore a probability) of 0.02. The majority of the 17,010 possible signatures are far less probable. A calculation of the theoretical incidence of chance matching of signatures within test groups of two or more individuals is presented. The probability of a chance match between any two randomly selected individuals is 0.004 and among five randomly selected individuals is 0.034. The allozyme genetic signature represents a definitive monitor of cell identity and is presented as a standard of cell and tissue identification for a variety of biological studies.

Isoenzyme Characterization of Animal Cell Cultures

Summary We have found the isoenzyme analysis of cells to be a significant aid in the characterization and identification of animal cell cultures from a variety of species. The G6PD and LDH patterns of 86 animal cell lines were examined using starch gel electrophoresis. From the data accumulated it was possible to construct a “fingerprint” identification chart for ready identification of animal cells from 20 out of 22 different taxonomic groups. Although there are limitations to the technique the systematic study of additional polymorphic enzymes should provide valuable information for the more precise characterization of animal cells.

Freezing, Storage, and Recovery of Cell Stocks

Publisher Summary This chapter focuses on freezing, storage, and recovery of cell stocks. The preservation of animal cells in liquid nitrogen is now a routine practice. Although some animal cells can be stored for years in a dry ice chest, the percentage of viable cells that can be recovered is very low. Although glycerol and dimethyl sulfoxide (DMSO) appear to be almost equally effective in preserving many cell lines, glycerol is best for certain lines and DMSO for others. If it is necessary to label the ampoules by hand, it may be done by using an ordinary stick pen and most laboratory inks, provided the ink is subsequently annealed. The refrigerators should be kept in well-ventilated areas because of the constant release of the nitrogen gas. Stock cultures are maintained as monolayers in T-60 flasks, plastic T-75 flasks, or other vessels, such as roller bottles. In most instances, fresh trypsin or a trypsin-Versene solution is used in dislodging the cells from surfaces of the culture vessels. After centrifugation, the cells are resuspended in the freshly prepared freeze medium by gentle aspiration with a pipette and are then introduced into a dispensing apparatus for distribution into ampoules.

Biochemical Identification of Cells in Culture: B. Enzymatic “Fingerprinting”

Publisher Summary This chapter discusses starch–gel method for the study of glucose-6-phosphate dehydrogenase (G6PD) and lactic dehydrogenase (LDH) electrophoretic patterns. The extracts are prepared by either treatment with n -octyl alcohol, or by freezing the cells in liquid nitrogen and thawing them at room temperature. Cell lines to be assayed are selected from active log phase monolayer cultures or from freshly thawed ampoules. After electrophoresis is completed (overnight), the gel is sliced in half horizontally, inverted with the cut surface facing up, and covered with a thinner gel consisting of 3.5 g of starch in 30 ml of 0.25 M Tris–HCl buffer, pH 7.5, to which the incubation mixture is added. Isoenzyme analysis provides a means of complementing or supplanting immunological techniques for species identification. Isoenzyme analysis has been useful in determining the species of presumably transformed cells that have been submitted for identification. Cultures containing cells from two different species clearly exhibit the isoenzyme patterns characteristic of both species but the lower limits of sensitivity of the isoenzyme tests has not been determined. For detection of low-level contaminations with cells of another species, more refined techniques would be necessary.

Reference Animal Cell Lines

Much of our knowledge about human chromosomes has been made possible through the study of cells cultured in vitro (Hsu, 1952; Tjio and Levan, 1956; Ford and Hamerton, 1956). The ready adaptability of cells in tissue culture to study by phase-contrast microscopy, electron microscopy, and biochemical methods has laid the foundation for modern cytogenetics and recent advances in the study of genetic diseases. Cell cultures have also been of tremendous importance in the isolation, propagation, and study of viruses, and in the production of vaccines (such as polio and measles vaccines, etc.). In fact, today there are a myriad of ways that cell cultures are used productively in cancer research, human cytogenetics, immunological research, toxicological studies, and the biochemistry of diseases (for reference to uses of specific cell cultures in research, see Tables 3–6).