Clonal Growth of Mammalian Cells in Vitro

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.

Molecular Growth Requirements of Single Mammalian Cells

“…of the above trypsin solution. In the present serum-free experiments the enzyme action could not be stopped by serum, as in our earlier procedure (4). Instead, the cell suspension was rapidly diluted serially in N15 (40 per cent) + saline G (60 per cent) to a concentration such that an inoculum of 0.10 ml.…”

Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter

“…The HepG2.1 cell line is a dedifferentiated hepatoma cell line which originated from the differentiated hepatoma cell line, HepG2, as described previously (Raney et al, 1990). The human cervical carcinoma cell line HeLa $3 (Puck et al, 1956) was grown in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 mg/ml glucose and 10% calf serum at 37 °C in 5% COJair. Transfections were performed as previously described (Graham & Van der Eb, 1973;Sorge et al, 1984).…”

Chemical transformation of Chinese hamster cells. I. A comparison of some properties of transformed cells

“…Cells were grown in a mixed-serum medium which Yerganian and Lavappa (1971) (Puck, Marcus and Cieciura, 1956 (iii) Transformation assay.-9-cm plastic dishes (Sterilin) were seeded with 5 x 103 or 5 x 104 cells (depending on survival after treatment) and were treated as above (10 dishes per treatment). The carcinogen-containing medium was removed, the cultures were fed weekly for 4-5 weeks, and observed regularly for morphological changes.…”

“…At the same time, marked colonies of different morphological types from unstained treated and untreated cultures were isolated by ring cloning (Puck et al, 1956). If the isolated subclones were seen to be heterogeneous then recloning was carried out by the same procedure, and the resultant subclones, when cultured to sufficient numbers (5-6 passages), were subjected to various tests.…”