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Mutation Research, 35 (1976) 173--178 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

Short communication

AN ALTERNATIVE TO PETRI DISHES IN QUANTITATIVE MUTATION EXPERIMENTS WITH CULTURED MAMMALIAN CELLS

ROGER COX, W.K. MASSON and D.A. BANCE

M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon 0 X l l ORD (Great Britain)

(Received September 15th, 1975) (Accepted November 28th, 1975)

Quantitative mutation experiments with cultured mammalian cells have, in the past, used petri dish cloning techniques for the detection of spontaneous and induced mutants and, for small scale mutation experiments, these are both suitable and convenient. However, improvements in experimental techniques have made it worthwhile to increase the scale of mutation estimation experi- ments to increase the numbers of mutant clones scored and hence improve the statistical resolution of experiments. The problem of providing a large growth area for mutant selection is particularly acute in the case of cultured human di- ploid cells where the relatively low mutation frequencies observed, the con- stmints of cell plating density and the dependence of induced mutant frequen- cy on expression time [6] mean that the number of dishes required for a quan- titative mutation experiment with a single mutagen at 3 or 4 doses may be ~103.

The problem of experimental scale has been largely overcome by cloning mu- tants on the growth surface of disposable, commercially produced bulk cell cul- ture vessels instead of in conventional petri dishes. The Sterilin bulk cell culture vessel (Sterilin Ltd., Teddington, Middlesex, England. Cat. No. 337) developed by House et al. [4] incorporates a spiral of polystyrene film as an internal growth surface (Fig. 1). The total available growth surface is approx. 8500 cm 2, equivalent in area to approx. 140 petri dishes of 9 cm diam. The principle of selecting mutant cells using the cell culture vessel is the same as that em- ployed using dishes, i.e. cells are evenly distributed over the growth surface and incubated in a selective growth medium to allow clonal growth of mutants.

Optimum conditions for cell distribution and clonal growth of normal cells in the Sterilin vessel were examined by adding cell suspensions containing 103 viable plus 7" 106 radiation inactivated (3 krad X-ray) diploid human fibro- blasts to vessels containing 1.6 1 normal growth medium (Eagles M.E.M. plus 10% foetal calf serum; Gibco Biocult, Paisley, Renfrewshire, Scotland) pre- warmed to 37 ° C and equilibrated with 95% air plus 5% CO2. After sealing, the vessels were inverted twice to distribute the cells throughout the medium and

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Fig. 1. Diagrammat ic represen ta t ion of bu lk cell cul ture vessel.

then rolled on their longitudinal axes at speeds between 1 and 60 rev/h for 18 h at 37 ° C. After this period, which was sufficient for complete cell attachment, the vessels were incubated in the vertical position for 14 days at 37°C and sub- sequently stained for clonal growth with 0.25% azur A stain for 1 h. The stain was siphoned from the vessel and excess stain removed by gently washing for 1 h with cold tap water. The base of the vessel was then removed and the whole vessel dried overnight at 37°C. The dried spiral was extracted from the body of the vessel and the stained clones counted by eye against a white background. Under these conditions, optimal clonal distribution and cloning efficiency was observed when the rolling speed for cell attachment was 5 rev/h. Cloning effi- ciencies for diploid human fibroblasts were usually between 50 and 70% and comparable to those obtained on petri dishes [2].

In unpublished experiments using petri dish cloning techniques {Cox and Masson), spontaneous and radiation-induced mutants of human diploid fibro- blasts capable of clonal growth in normal growth medium containing 2 #g/ml 6- thioguanine (6TG) were found to be phenotypically stable with less than 2% of wild type activity of the purine salvage enzyme, hypoxanthine-guanine phos- phoribosyl transferase (HGPRT) and sensitive to the glutamine analogue, aza- serine [3]. It was also found that 6TG resistant mutants induced by 150 tad X- rays required a 7 day post-irradiation growth period {"expression time") in non- selective medium before the induced phenotype was fully expressed in the cell population.

Experiments reported here compare spontaneous and radiation induced mu- tation frequencies using petri dishes with those obtained using the cell culture vessel technique. After expression time growth, cells were distributed at ~103 cells/cm 2 on the growth surface of petri dishes and bulk culture vessels contain- ing 6TG selective medium and subsequently incubated at 37°C for 21 days. Af- ter this time 6TG resistant clones were stained, washed, dried and counted using the method described above. Mutant frequencies in control and irradiated cell populations were expressed as the ratio between the number of 6TG resis-

1 7 5

tant clones and the total number of viable cells present in the populat ion at the time of mutan t selection. Mutant frequencies obtained using the two assay methods were indistinguishable (Table I). The only problem encountered with the cell culture vessel mutan t assays was that small mutant clones (<2 mm dian~) were often difficult to distinguish from aggregates of cell debris on the surface of the polystyrene film and these were examined with a low power microscope to ensure that the maximum number of mutan t clones were scored. Clonal morphology and size distribution was found to be the same using the two methods (Fig. 2).

With 6TG at 2 pg/ml as the selective agent, no increase in overall yield of mutants was observed if medium changes were made during the mutan t selec- tion incubation period and in experiments reported here no medium changes were made during mutant selection. With 8-azaguanine as the selective agent for HGPRT deficient human diploid fibroblasts, f requent medium changes may be necessary for maximum recovery of mutant clones [1,5] . In this instance the cell culture vessel offers the advantage of a single medium change operation compared with 140 operations for the equivalent number of petri dishes, thus saving time and reducing the risk of contamination. The one serious disadvan- tage of the cell culture vessel technique was that the sterile isolation of mutant clones from the growth surface was complicated by the size of the polystyrene film (approx. 23 cm × 200 cm) and the difficulty in manipulating it in the wet

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177

state. However, these problems are not insurmountable and with careful sterile handling of the film the technique should facilitate the isolation of other mammalian somatic cell variants arising in cell populations at frequencies less than 10 -6 .

In terms of cost, the cell culture vessel is cheaper, by about a factor of 3, than the equivalent number of plastic tissue culture grade petri dishes. At a conservative estimate the cell culture vessel reduced handling time by a factor of 10; in addition there is a space saving factor of 10. The technique also ob- viates the need for large numbers of expensive CO2 gassing incubators. In our experience, mutant yields for cell culture vessels are more reproducible than those from petri dishes, possibly because the growth conditions are more uni- form in the large volume (1.6 l) of the vessel than in large numbers of individ- ual petri dishes containing 10 ml of growth medium.

The technique described appears to have wide applications in studies with cultured mammalian cells that use the clone forming ability of single cells and especially in quantitive studies where it is advantageous to score large num- bers of such clones.

References

1 Albertini , R.J. and R. Demara, Somatic cell muta t ion : Detect ion and quant i f ica t ion of X-ray-induced muta t ion in cul tured, diploid human fibroblasts, Mutat ion Res., 18 (1973) 199--244.

2 Cox, R. and W.K. Masson, Changes in radiosensit ivi ty during the in vi tro growth of diploid human fl- broblasts, Int . J. Radiat . Biol., 26 (1974) 193--196.

3 Fuj imoto , W.Y., J.H. Subak-Sharpe and J.E. Seegmiller, Hypoxanthlne-guaulne phosphozibosyl trans- ferase deficiency: Chemical agents selective for mu tan t or normal cul tured fibroblasts in mixed and heterozygote cultures, Proc. Natl. Aead. Sei. (US), 68 (1971) 1516--1519.

4 House, W., M. Shearer and N.G. Maroudas, Method for bu lk culture of animal cells on plastic film, Expt. Cell. Res., 71 (1972) 293--296.

5 Maher, V.M. and J.E. Wessel, Mutat ion to azaguaulne resistance induced in cul tured diploid human fibroblasts by the carcinogen, N-acetoxy-2-aeety]~mlr~o-fluorene, Mutat ion Res., 28 (1975) 277--284.

6 Simons, J.W.I.M., Dose-response relat ionships for mutan ts in mammal ian somatic cells in vi tro: Muta- t ion Res., 25 (1974) 219--227.

7 Zeeland, A.A. van, M.C.E. van Diggelen and J.W.I.M. Simons, The role of metabol ic co-operat ion in se- lect ion of hypoxanthine-guanine-phosphoribosyl- t ransferase (HGPRT)-deficient mutan ts from diploid mammal ian cell strains, Mutat ion Res., 14 (1972) 355--363.