A method of isolating cells incapable of multiplication in supension culture

Experimental Cell Research 66 (1971) 203-208

A METHOD

OF ISOLATING CELLS INCAPABLE OF

MULTIPLICATION

IN SUSPENSION CULTURE J. WYKE 1

Department of Tumour Virology, Imperial Cancer Research Fund, London, WC2, UK

SUMMARY Hamster cells transformed by polyoma virus acquire the ability to grow in semi-solid media. All viable transformed cells show this property, whilst untransformed cells remain viable in suspension without multiplying. This difference is used to select cells lacking the transformed cell characteristic of multiplication in suspension. Cells synthesising D N A in suspension incorporate the thymidine analogue 5-bromodeoxyuridine (BUdR) and are then killed by exposure to blue light. Most cells surviving this treatment do not grow in suspension. The applications of this selective technique are discussed.

The study of cell transformation by polyoma virus has revealed many differences between transformed and normal cells. These differences are manifested by changes in in vivo and in vitro growth and by alterations in metabolism and antigenicity of transformed cells. To elucidate the mechanism of transformation one must determine the relationship of these altered transformed cell characters, both to one another and to the activity of viral genes. Two approaches to this problem seem profitable at present: (1) the use of conditional lethal virus mutants defective in lytic or transforming capacities [1-4]; (2) the study of the loss of characters from transformed cells [5-7]. The second approach is used in the present work and since reversion of transformed cells is a rare event a method of obtaining revertants by selecting against a unit character 1 Present address: Department of Microbiology, School of Medicine University of Washington, Seattle, Wash. 98105, USA.

of transformation is required. When the hamster cell line BHK21 is transformed by polyoma the ceils acquire the ability to multiply in agar or methyl cellulose suspension cultures [8, 9]. This paper describes a method of selecting against the property of growth in suspension culture, thus isolating cells which do not grow in suspension. The accompanying paper [18] describes the properties of variants of polyoma-transformed BHK21 which are obtained by such a selection. A preliminary report of this work has appeared [10]. MATERIALS A N D M E T H O D S Cell cultures All cell lines used were derived from the Syrian hamster fibroblast clone BHK21/13 (C13) [11]. PyY [12] and Py6 [13] are polyoma-transformed derivatives of C13, and B1Py is a polyoma-transformed derivative of a 5-bromodeoxyuridine-resistant subclone of C13. The latter line, which was kindly provided by D r G. Marin, lacks the enzyme thymidine kinase [14]. The ceils were cultured in Dulbecco's modification of Eagle's medium supplemented, unless otherwise stated, with 5 % or 10 % foetal calf serum

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Z Wyke

(Flow Laboratories Ltd, Rockville, Md). All cultures were maintained at 37~ in 10 % CO~ in air.

Table 1. Growth in agar of transformed cells

(Py Y) in the presence of Cl3 feeder cells

Chemicals

Calf serum concentration

Thymidine, hypoxanthine and 5-bromodeoxyuridine (BUdR) were obtained from Sigma Chemical Co. "Methotrexate" (4-Amino-Nl~ pteroylglutamic acid, sodium) was provided by Lederle Laboratories. Solutions of chemicals were prepared in distilled water at 100 times the concentration normally used in the medium, and sterilised by filtration.

Efficiency of plating (e.o.p.) on a surface This was determined by plating appropriate numbers of trypsin-dispersed cells in 50 mm "Nunc" plastic Petri dishes or 4 oz. glass bottles. After 8 days incubation colonies were fixed, stained with Giemsa and counted.

Number of cells plateda

10 %

5%

2%

1%

104 PyY 250 PyY 250 PyY + I04C13~ 250 PyY+ 105C13~

46 38 42 62

36 34 40 65

12 0.0 0.5 42

1.4 0.0 0.0 1.3

Figures quoted are % e.o.p, of PyY, means of 3 determinations. a per 1.5 ml overlay in 50 mm dish. 104 or 105C13 plated alone always failed to form colonies.

Efficiency of plating in suspension culture Trypsin-dispersed cells were mixed in medium containing 0.33 % agar, and 1.5 ml aliquots of the mixture were added to 50 mm plastic Petri dishes containing a preset base of 0.5 % agar medium [8]. Cell density and serum concentration were varied as detailed in Results. Colonies of more than 0.4 mm diameter were counted after 14 days incubation.

Methyl cellulose suspension cultures Eagle's medium containing 1.6 % (w/v) methylcellulose ("Methocel", Standard Grade Type MC, viscosity 4 000 centipoises, Dow Chemical Co.) was prepared by the method of Stoker et al. [9]. Four volumes of this Methocel medium were mixed with 1 vol of cells suspended in Eagle's medium to yield 1.28 % Methocel medium containing cells at the required density and serum at the required concentration as detailed in Results. The mixture was added in 5 ml amounts to 50 mm "Nunc" bacteriological grade plastic Petri dishes containing a preset base of 0.9 % agar medium, and incubated as described below.

accurately the e.o.p. A t t e m p t s were m a d e to investigate e.o.p, at higher cell densities by i n c l u d i n g n o n - g r o w i n g cells in the m e d i u m . The growth of P y Y at various serum concentrations in the presence of 104 or 10~ C13 feeder cells is shown in table 1. The presence of feeder cells can c o m p e n s a t e in part for the depression of e.o.p, at low serum c o n c e n t r a tions. Since t r a n s f o r m e d cells may exert a qualitatively or quantitatively different feeder effect to C13, the experiment was repeated using n o n - g r o w i n g t r a n s f o r m e d cells as feeders. This was achieved by plating the cells in agar m e d i u m c o n t a i n i n g Methotrexate (10 .6 M), h y p o x a n t h i n e (4 • 10 .5 M), thymidine (4 • 10 -5 M) a n d glycine (10 -5 M), a

RESULTS

m o d i f i c a t i o n of the selective m e d i u m described by Littlefield [15]. The folic acid

Cell multiplication and survival in suspension culture

i n h i b i t o r Methotrexate prevented e n d o g e n o u s thymidylate synthesis, b u t P y Y a n d Py6 cells were able to use exogenous thymidine a n d grew normally. B1Py cells, which lacked t h y m i d i n e kinase, were u n a b l e to utilise t h y m i d i n e a n d hence failed to grow. Fig. 1 summarises the effect of plating 250 P y Y or Py6 at various serum c o n c e n t r a t i o n s in the presence of 104 or 105 B1Py feeder cells. T h e e.o.p, of Py6 is more m a r k e d l y depressed t h a n that of PyY on decreasing cell density

Low passage stocks of C13 consistently failed to f o r m colonies in soft agar w h e n plated at u p to 105 cells per overlay. P o l y o m a t r a n s f o r m e d cells u n d e r similar c o n d i t i o n s f o r m e d m a n y macroscopic colonies. The e.o.p, in agar of t r a n s f o r m e d lines increased as b o t h cell density a n d serum c o n c e n t r a t i o n increased, b u t at cell densities a b o v e 104 per overlay it became impossible to score

Exptl Cell Res 66

Cell growth in suspension 205 100

80

60

40

20

0

1

2

1

2

5

Fig. 1. Abscissa: % serum concentration; ordinate: % e.o.p. Comparison of the e.o.p, of Py6 ( I ) and PyY ( n ) in agar suspension culture containing B1Py feeder cells. (a) 250 ceils alone; (b) 250 cells+ 104 B1Py; (c) 250 cells+ 105 B1Py.

or serum concentration, and only at the highest cell densities and serum concentrations is the e.o.p, of the two lines comparable. However, with both cell lines it is clear that under suboptimal conditions cells which are potentially capable of forming colonies fail to do so. Whether or not such non-growing cells survive is crucial to the success of a selection against transformed cells based on their capacity to multiply in suspension culture. Cell survival after suspension culture was investigated by culturing cells in Methocel medium for 90 h, the period used for the selective killing experiments described below. The cells were recovered from Methocel by harvesting the suspension into four times its volume of cold Eagle's medium and pelleting the cells by centrifugation at 300 g [9]. The cell pellet was resuspended, either gently or by vigorous pipetting to disperse incipient cell colonies. The cell suspension was counted, scoring cell clumps as the equivalent of individual cells, and clumps and cells were plated at known numbers to determine their e.o.p, on a surface. Table 2 shows the different results of gentle and vigorous resuspension when applied to Py6 which had been cultured at 105, 2 x 104 or 5 • 103 cells per ml Methocel in 2 or 0.5 % serum. When colonies

were not dispersed the cell recovery was generally less than the input cell number, as expected. However, the decrease in recovery does not account for the marked decrease in cell viability in non-dispersed cultures, when compared with dispersed cultures, after suspension at low cell densities and low serum concentrations. An explanation consistent with this result is that only cells which grow in suspension remain viable, and thus pipetting to break up colonies markedly increases the proportion of viable cells, particuTable 2. Survival of transformed cells (Py6)

in Methocel medium Serum concentration in Methocel Cell density per ml Methocel 10a 2 • 10* 5 • 103

2%

A B A B A B

0.5 %

C

D

C

D

300 20 150 18 144 15

110 13 60 0.5 80 0.0

290 16 150 11 72 9.6

75 7.3 36 0.0 56 0.0

A, % recovery of cells from Methocel suspension after 90 h at 37~ B, Viability of cells from Methocel suspension (expressed as % e.o.p.). C, Cell clumps dispersed after harvesting Methocel. D, Cell clumps not dispersed. Exptl Cell Res 66

206

Z Wyke

larly in conditions when only a small fraction of the input cells are growing. A similar conclusion was suggested by experiments in which transformed cells were mixed with C13 in known ratios before culturing in Methocel for 90 h and surviving colonies were scored as "normal" or "transformed" on a morphological basis. As in table 2, the viability of PyY or Py6 was greatly reduced after suspension culture at lower cell densities or serum concentrations. However, the recovery of C13, after correcting for its low input, showed less depression than that of transformed cells under the same cultural conditions. Stoker et al. [9] have also observed that C13 cells retain a high viability after culture in Methocel suspension. These results suggest that PyY and Py6 differ from C13 in that only the transformed cells which multiply in suspension remain viable. The successful killing of transformed cells in the reconstruction experiments described below confirms this interpretation.

Selective killing of cells which multiply in suspension culture Mammalian cells which synthesise D N A can incorporate the thymidine analogue B U d R and are killed by subsequent exposure to blue light [16]. The experiments described above suggest that polyoma-transformed cells either multiply or are non-viable in suspension culture. Thus, by applying the B U d R + b l u e light selection to suspension cultures of transformed cells, the only survivors should be those which have lost the ability to multiply in suspension whilst retaining their viability. The efficiency of selection was tested in reconstruction experiments in which transformed cells (PyY or Py6) were mixed with C13 in the ratio 100:1 and suspended at a density of 105/ml in Methocel medium containing 2 % calf serum. Cultures were incuExptl Cell Res 66

1.0

0.1 I-

0.01

0 S

\

10

30

9

60

Fig. 2. Abscissa: time of exposure to blue light (rain); ordinate: % of input cells surviving. Effect of B U d R and blue light on a mixed cell population, Py6/C13 in the ratio 100:1. o, total cells from cultures containing no BUdR; A, Py6

from cultures containing 5 • 10-8 M BUdR; m, C13 from cultures containing 5 x 10-6 M BUdR.

bated for 45 h to allow completion of D N A synthesis in non-dividing cells [9], and BUdR was then added in 0.5 ml Eagle's medium. Incubation was continued for a further 45 h, a period sufficient for two cycles of D N A replication [9] which would thus result in partly single-stranded and partly doublestranded incorporation of the analogue into cell D N A [17]. After gently harvesting the cells as described above they were seeded in 4 oz. glass bottles and incubated for 4-6 h to allow complete cell attachment to the glass. The bottles were then inverted and the cells irradiated through the glass 10 cm from a Philips 40 W "Actinic Blue" fluorescent lamp. Irradiation was continued for up to

Cell growth in suspension Table 3. Effect of 5 x lO-6M BUdR and blue light on a mixed population Colony counts Cell number plated in 4 oz. bottles

Period of irradiation (min)

A

B

Py6

C13 Py6

C13

104 105 3•

0 30 30 60

130 81 113 24

0 3 7 10

0 0 2 2

120 96 254 27

Py6/CI3 in the ratios A, 104 : 1, and B, 105 : 1.

60 min, turning the bottles at intervals to wash medium over the cells. Control cultures were treated similarly but shielded from the lamp. Colonies developing in the bottles were stained and counted after 8 days incubation, scoring them as "transformed" or "normal" on morphological criteria. Preliminary experiments showed that the most effective selective killing of transformed cells was obtained using 5 • 10-~M BUdR. The survival of Py6 and C13 after suspension culture in BUdR of this concentration followed by exposure to blue light is shown in fig. 2. Cultures that were not treated with B U d R or not exposed to blue light showed an e.o.p. of about 10%. Sixty minutes irradiation reduced the e.o.p, of B U d R treated Py6 to about 0.02 %, whilst the e.o.p, of C13 was unaffected. The resolution of the selective technique was tested by repeating the reconstruction experiments with Py6 and C13 in the ratios 104:1 and 105:1 (table 3). The killing of Py6 and survival of C13 was comparable to that shown in fig. 2 and a single cycle of B U d R + b l u e light selection thus permitted the detection of C13 when masked by a 105-fold excess of transformed cells. Similar results were obtained using PyY in place of Py6.

207

DISCUSSION Cells which synthesise D N A in suspension culture incorporate BUdR and are effectively killed by exposure to blue light. This permits a powerful selection to be exerted in favour of cells lacking the ability to multiply in suspension. Thus this technique can be used to detect variants of polyoma-transformed cells incapable of multiplication in suspension [10, 18], and it could also be useful in searching for mutants of cells or viruses incapable of expressing or inducing this function or any other altered growth property which results from virus transformation. Partial "revertants" of p o l y o m a - o r SV40-transformed cells have been obtained by selecting either for cells showing a high degree of growth control in dense cell cultures [6] or for cells able to grow on glutaraldehyde-fixed layers of normal cells [7]. The use of multiplication in suspension as the criterion of transformation against which selection is applied has advantages over these other techniques. Since cell to cell contact is not necessary the selection can be applied to any number of cells, and suspension cultures permit very large cell numbers to be handled with ease. Moreover, colony formation in suspension is a parameter that is very simply quantitated, and relevance is added to its study by the findings of Kakunaga & Kamahora [19] that it is the feature of in vitro cell growth most commonly associated with in vivo tumorigenicity. The selective procedure described here is valid only if all viable transformed cells multiply in suspension. In some C13 lines which have been newly transformed by polyoma 1% of the cells remain viable in suspension without multiplying [20]. However, repeated cycles of B U d R + b l u e light selection applied to such cells does not increase this proportion. Thus this feature Exptl Cell Res 66

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a p p e a r s to be physiologically rather t h a n genetically determined, a n d it does n o t invalidate the application of the selection procedure to newly t r a n s f o r m e d cells. I thank Dr I. A. Macpherson for his advice and encouragement. This work was carried out in part fulfilment of the requirements for the Ph.D degree of the University of London, and was supported by a Veterinary Research Training Scholarship of the Horserace Betting Levy Board. REFERENCES 1. Fried, M, Virology 25 (1965) 669. 2. Eckhart, W, Virology 38 (1969) 120. 3. di Mayorca, G, Callender, J, Marin, G & Giordano, R, Virology 38 (1969) 126. 4. Benjamin, T L, Proc natl acad sci US 67 (1970) 394. 5. Marin, G & Littlefield, J W, J virol 2 (1968) 69. 6. Pollack, R E, Green, H & Todaro, G J, Proe natl acad sci US 60 (1968) 126.

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7. Rabinowitz, Z & Sachs, L, Nature 220 (1968) 1203. 8. Macpherson, I & Montagnier, L, Virology 23 (1964) 291. 9. Stoker, M, O'Neill, C, Berryman, S & Waxman, V, Intern j cancer 3 (1968) 683. 10. Wyke, J, Intern symp of the centre national de la recherche scientifique 183 (1970) 151. 11. Macpherson, I & Stoker, M, Virology 16 (1962) 147. 12. Stoker, M, Virology 18 (1962) 649. 13. Macpherson, I, J natl cancer inst 30 (1963) 795. 14. Littlefield, J W & Basilico, C, Nature 211 (1966 250. 15. Littlefield, J W, Science 145 (1964) 709. 16. Puck, T T & Kao, F T, Proc natl acad sci US 58 (1967) 1227. 17. Djordjevie, B & Szybalski, W, J exptl reed 112 (1960) 509. 18. Wyke, J, Exptl cell res 66 (1971) 209. 19. Kakunaga, T & Kamahora, J, Biken's j 11 (1968) 313. 20. Wyke' J, Ph.D Thesis University of London (1970). Received December 21, 1970