What does this research mean for the field?

Single mammalian cells can be successfully cultured into pure, genetically uniform clonal colonies using specific plating techniques, enabling quantitative study of their genetics, growth, and morphological responses to different environments. Novelty: ClaimNovelty.METHODOLOGICAL. Consensus alignment: ConsensusAlignment.ESTABLISHES_NEW_DIRECTION.

What question did this study set out to answer?

To describe and compare two methods for plating single HeLa cells for clonal growth and isolated mutant development.

February 1, 1956The Journal of Experimental Medicine1,141 citationsOpen Access

Clonal Growth of Mammalian Cells in Vitro

TPTheodore T. PuckAmerican Cancer Society PMPhilip I. MarcusThe Wistar Institute SCSteven J. CieciuraSabin Vaccine Institute

Key Points

To describe and compare two methods for plating single HeLa cells for clonal growth and isolated mutant development.
Describes two plating methods for single HeLa cells: one using irradiated feeder cells and another simpler method similar to bacterial plating.
Cells were cultured in different serum types to observe changes in morphology and behavior.
Growth curves of colonies were analyzed for reproducibility and growth characteristics.
Highly reproducible growth curves observed from cloned colonies, displaying a generation time of 18 to 20 hours.
Distinct cell morphologies noted: ameboid in human serum and columnar in non-human sera, despite identical volumes and nuclear cross-sections.
Improved cell growth and isolation achieved using gentler plating methods as opposed to standard trypsinization techniques.

Abstract

Two methods for simple and rapid plating of single HeLa cells, human, carcinomatous cells, are described. These result in growth and formation of colonies from each single cell. One of these procedures uses irradiated, non-multiplying "feeder" cells to condition the medium. The second requires more gentle handling of the cells, but otherwise is virtually the same as that used in plating bacteria on semisolid, nutrient media. By extension of these methods, it is possible to isolate single mutant colonies and grow pure clonal stocks of animal cells. These genetically uniform strains are much more homogeneous in their behavior than the parental HeLa cell population. Growth curves obtained from developing colonies are highly reproducible. The most active mutant stocks so far isolated display a generation time of 18 to 20 hours. In pooled human serum HeLa cells assume a highly stretched, ameboid form, with marked motility; whereas growth of the same cells in a variety of non-human sera results in tightly packed, columnar, epithelial-like morphology. The two cell types possess volumes, nuclear cross-sections, plating efficiencies, and generation times which are identical within experimental error, but display widely different cross-sectional areas, suggesting that the basic change occurs in the cell surface. It is conceivable that this change may be related to that which enables the cells of a compact tumor to become invasive. Animal cells subjected to the standard trypsinization procedures which involve mechanical trauma and repeated washings in incomplete media leak large amounts of P and suffer impaired ability to reproduce as isolated cells. Application of the methods described in this paper as a tool for quantitative study of normal mammalian cell growth, physiology, genetics, and biochemistry, and the response of cells to drugs, viruses, high energy radiation, and other agents have been indicated.

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