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y. Cell Sci. s, 135-142 (1969) 135 Printed in Great Britain

THE CHOLINE AND SERUM PROTEIN REQUIREMENTS OF MOUSE FIBROBLAST CELLS (STRAIN LS) IN CULTURE

J. R. BIRCH AND S. J. PIRT Department of Microbiology, Queen Elizabeth College (University of London), Campden Hill, London, W. 8, England

SUMMARY The maximum cell population density of mouse fibroblast (strain LS) cells growing in static suspension culture was found to be directly proportional to the dialysed calf serum con- centration. This was due to choline limitation and the fact that serum protein was a major source of choline. The growth yield (Y) was 3'2 x io5 cells//tg . Studies on the role of serum in the presence of excess choline showed the following. When protein was omitted from the medium, cell death occurred. Whole serum protein could be replaced by either (1) bovine serum albumin fraction V, or (2) crystalline bovine serum albumin + sodium pyruvate and a-ketoglutarate, or (3) polyvinylpyrrolidone + methylcellulose + pyruvate and a-ketoglutarate. The population doubling time was 24 h in the presence of whole serum protein and in- creased considerably with the substitutes (1—3). The increase in maximum cell population density (without medium changes) exceeded 2-9 x io" cells/ml with either whole serum protein or substitutes (1) and (2). With serum substitute (3) the maximum increase in population density was reduced to i-6 x ior' cells/ml.

INTRODUCTION Many studies on cell culture are concerned with obtaining maximum population densities of cells. In mouse L cell cultures an increase in the population density of 1 to 2 x io(! cells/ml is considered the normal maximum without change of medium. Certain basic media such as that of Eagle (1959), unsupplemented, will give a far lower maximum population. The nature of the growth-limiting factors remained obscure until recently when, from a quantitative study of the amino acid require- ments, Griffiths & Pirt (1967) showed that for the mouse L cells in commonly employed media supplemented with serum (10%, v/v) certain amino acids would be limiting. With excess of amino acids and in the presence of serum (10%, v/v) the maximum yield of cells was raised to 2-3 x io°/ml without changing the medium. In the present study the work of Griffiths & Pirt (1967) was extended to investigate the nature of growth-limiting factors in serum. In general, the growth-promoting properties of serum have been attributed to serum protein and, more specifically, to the a-globulins (Puck, Waldren & Jones 1968; Tozer & Pirt, 1964; Healy & Parker, 1966; Michl, 1961; Katsuta, Takaoka, Hattori, Kawada, Kuwabara & Kuwabara, 1959). In some instances low molecular weight, but unidentified, substances which stimulate growth have been separated 136 J.R. Birch and S. J. Pirt from serum protein by prolonged (Metzgar & Moskowitz, i960) or by enzymic digestion (Eagle, i960). The importance of nutrients bound to serum proteins has been indicated by several workers and it has been shown that serum proteins are a source of vitamins (Dupree, Sanford, Westfall & Covalesky, 1962), amino acids (Dupree et al. 1962; Eagle, Oyama & Piez, i960) and lipids (Bailey, 1967). On the other hand, a-globulins which promote growth at very low concentrations have been isolated (Puck et al. 1968; Holmes, 1967). A preliminary report of this work was given at the British Society for Cell Biology meeting, at Oxford, in September 1968.

MATERIALS AND METHODS The materials and methods used were based on those of Griffiths & Pirt (1967). The composition of the culture medium is given in Table 1.

Table 1. Defined culture medium Phosphate-buffered saline containing serum or other proteins is added to medium in the proportion 1 to 10 ml medium.

Inorganic salts mg/1. Amino acids mg/1. KC1 400 L-Arginine HC1 4SO CaCl2 1 L-Isoleucine 1 So MgCl.,. 6H..O 200 L-Leucine 180 NaH2PO4.2H2O 500 L-Histidine HC1 99 NaCl 5900 L-Lysine HC1 175 NaHCO3 2500 L-Methionine 30 MnCl2.4H.2O 1 L-Phenylalanine 70 ZnSO4.7H.,O 1 L-Threonine 100 FeSO4.7H.,O 1 L-Valine 15° CuSO4.5H2O o-5 L-Alanine 90 Glycine IS Vitamins L-Serine 20 /-Inositol 2-O --Tryptophan 20 rf-Biotin o-i ^-Cystine 75 Choline chloride •2 (20) --Tyrosine 70 Folic acid 0 ^-Glutamine IOO Nicotinamide •2 ^-Glutamic acid 1200 Calcium-rf-pantothenate •2 Pyridoxine hydrochloride •0 Antibiotics Thiamine hydrochloride 2-0 Penicillin 200000 U/l. Riboflavin O-2 200 mg/1. Hvpoxanthine IOO B12 IO Sugar Glucose 2000 mg/1. * Figure in parentheses represents level found by experiment tobe an excess.

The mouse LS cells were grown as static suspension cultures in 8-oz (about 200-ml) medical flat bottles. The culture volume per bottle was 11 ml. Cultures were inoculated with 1-5-3-0 x io5 cells/ml and incubated at 37 °C. Routine tests were Mammalian cell nutrition 137 made for mycoplasma infection, both by comparing growth with and without kanamy- cin sulphate (200 /tg/ml) and by attempting to isolate mycoplasma using the methods of Hayflick (1965). There was never any evidence of mycoplasma. Dialysed calf serum was prepared in the following way: 50-ml amounts of calf serum (Wellcome Research Laboratories), which had been heated at 56 °C for 30 min, were dialysed aseptically against 2 1. of phosphate-buffered saline, with 3 changes of buffer, over 48 h, at 4 °C.

RESULTS In our medium supplemented with dialysed calf serum the maximum cell population density was directly proportional to serum concentration; this, we found, was due to choline limitation and the fact that serum was a major source of choline. By increasing

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1 2 3 4 5 6 7 8 //g choline chloride/ml defined medium Fig. 1. The effect of choline concentration on growth of LS cells. The points on the graph are the means of triplicate determinations. The vertical lines indicate the ranges of the results. the level of choline chloride in our medium to 20 /'g/ml it was possible to replace the serum by bovine serum albumin fraction V (2 mg/ml) (Armour Pharmaceutical Co.) as the only protein supplement. In such an albumin-supplemented medium, maxi- mum cell growth was linearly proportional to choline concentration (Fig. 1). The growth yield (Y), which is given by the slope of the graph in Fig. 1, is 3-2 x io5 cells (approximately 250 /tg dry weight)//y,g choline chloride. With excess choline chloride (20/tg/ml) maximum cell yields of 3-5 x io6/ml were realized. Previous work in this department (Griffiths & Pirt, 1967) shows that at the latter population density certain amino acids become growth limiting. 138 J.R. Birch and S. J. Pitt In an experiment to determine the choline sparing effect of serum it was found that 0-5 ml dialysed and non-dialysed serum/11 ml culture contributed the equivalent of 2-1 and 6-4 fig choline chloride/ml respectively. Therefore dialysis removed about two-thirds of the serum choline available to the cells.

001 002 003 004 005 0-50 Dialysed serum concn. (ml/11 ml culture)

Fig. 2. The effect of dialysed calf serum in defined medium with excess choline and supplemented with crystalline serum albumin (2 mg/ml).

In a medium supplemented with albumin fraction V alone, omission of the protein led to rapid cell death. Crystalline bovine serum albumin (Armour) would not sub- stitute for the fraction V unless a small amount of dialysed serum, above a threshold level, was included in the medium (Fig. 2). It seemed probable therefore that some essential nutrients bound to the fraction V albumin were not present in the crystalline albumin. Subsequently it was found that the small amount of serum could be re- placed by sodium pyruvate (220 /tg/ml) and a-ketoglutaric acid (80 //,g/ml). There is a threshold level of pyruvate (between no and 220 /tg/ml) below which little growth occurs. a-Ketoglutarate is ineffective alone, but has a marked stimulatory effect on growth rate and maximum cell yield in the presence of the optimum level of pyruvate. Linoleic acid, which Ham (1963) found could replace albumin, was ineffective over a wide range of concentrations. In the presence of a small level of serum just above the threshold level shown in Fig. 2 the stimulatory effect of albumin was supplied by polyvinylpyrrolidone (Kollidon Mammalian cell nutrition

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50 40 80 120 160 200 240 Incubation time (h)

Fig. 3. The effects of crystalline serum albumin and polyvinylpyrrolidone (Kollidon 25) on cell growth in presence of 0-5 ml dialysed calf serum/11 ml culture, with excess choline. O, serum alone; #, serum + Kollidon (img/ml); A, serum + crystalline serum albumin (2 mg/ml).

Table 2. Effects of serum substitutes on growth rates and maximum cell population densities

Minimum Maximum cell Supplement to defined medium population population/ with excess choline doubling time (h) ml x io~5*

Dialysed calf serum (0-5 ml/11 ml culture) 24 31 Bovine serum albumin fraction V (2 mg/ml) 42 35 Crystalline serum albumin (2 mg/ml) 53 29 + Na pyruvate (220 /tg/ml) + a-ketoglutaric acid (80 /tg/ml) Methylcellulose (1 mg/ml) 78 16 + polyvinylpyrrolidone (1 mg/ml) + Na pyruvate (220 /tg/ml) + a-ketoglutaric acid (80 fig/ml) * Inoculum deducted. 140 J. R. Birch and S. J. Pirt 25, Fluka AG) (Fig. 3). It was reported by Katsuta, Takaoka, Hosaka, Hibino, Otsuki, Hattori, Suzuki & Mitamura (1959) that polyvinylpyrrolidone could replace 99-5% of the serum used in the culture of rat ascites hepatoma cells. Attempts to maintain growth of the cells without any serum protein were unsuccessful until methylcellulose (1 mg/ml) (Methocel 15 cps Dow Chemical Co.) and polyvinylpyrrol- idone were included together. Omission of either , in the absence of serum protein, resulted in inconsistent growth. Carboxymethylcellulose (1 mg/ml) would not substitute for methylcellulose. Table 2 gives a comparison of the population growth rates and maximum population densities with whole serum proteins and various substitutes. Since the fastest growth rate was obtained only with whole serum protein, it follows that the serum protein must contain other unknown factors which, though not essential, considerably stimu- late the cell growth rate. The maximum population density showed a significant decrease with the artificial in place of serum protein. This decrease can be accounted for by the decreased yield from amino acids when the growth rate is reduced (Griffiths & Pirt, 1967).

DISCUSSION Until recently studies on the nutrition and growth of cultured animal cells have been largely qualitative and, in particular, almost no regard has been paid to two quantitative parameters of great significance: (i) growth yield, that is, quantity of cells formed from a given amount of nutrient; and (ii) specific cell growth rate or population doubling time. It seems probable that lack of knowledge about these fundamental parameters has caused a general lack of awareness about growth-limiting conditions in cell cultures. In a systematic study of the growth yields of animal cells, Griffiths & Pirt (1967) showed that first glutamine and then leucine and isoleucine would be the growth- limiting factors for mouse L cells in several standard tissue culture media. The possibility that choline (which is presumably required for phospholipid synthesis) would be limiting was suggested to us by discussions with Dr K. Higuchi, who found that choline could be growth-limiting in his culture system, in which the medium was changed at regular intervals. The present study gives the growth yield of mouse L cells for choline. It follows that many widely used media are choline deficient and that a major role of serum in such media is to provide choline. For instance, in the medium of Eagle (1959) the choline concentration would limit cell populations to 3-2 x ro5/ml in the absence of serum. Similar amounts of choline are included in other commonly used media, an exception being Waymouth's 752/1 medium, which, for LS cells, contains a large excess (250/yg/ml). It is concluded from the present study that the growth-promoting ability of serum and serum protein fractions, reported by Tozer & Pirt (1964), was due to the presence of choline in the fractions. This may also be true of other studies where maximum population density has been used as the parameter of growth response. In a recent study of factors influencing maximum cell populations of 3T3 cells, Holley & Kiernan (1968) have used the term 'cessation of growth caused by exhaustion Mammalian cell nutrition 141 of a nutrient' synonymously with 'contact inhibition'. The term 'contact inhibition' was originally applied to cell movement (Abercrombie & Heaysman, 1954), and subsequently it appeared in growth studies, with the implication that growth inhibition is caused by contact between cells. There is increasing evidence, however, that in cell cultures cessation of growth is not due to 'contact inhibition' but to exhaustion of one or more nutrients in the medium. Even the classical diploid cell line, WI j8, grows indefinitely, layer upon layer, if a continuous nutrient supply is maintained (Kruse & Miedema, 1965). Such broadening of the meaning of 'contact inhibition' makes it misleading and completely at variance with the original definition. Holley & Kiernan (1968) found that maximum cell growth was proportional to the serum con- centration in the medium. They isolated a growth-promoting fraction from serum, and also from urine. Their growth factor could be choline, since it is present, together with protein, in urine, as well as in serum (Long, 1961). Perhaps the role of keto acids (and of serum in the absence of keto acids) is to prime metabolism. This seems plausible, as the L cell is reported to be 'leaky' to keto acids (Danes, Broadfoot & Paul, 1963); such an effect would account for the threshold concentration of the nutrient required for growth. The third role of serum or of serum albumin which we have looked at is the 'protective' or detoxifying effect. This role would be consistent with the well known capacity of albumin for binding a large variety of small molecular species. The fact that both methylcellulose and poly- vinylpyrrolidone were essential for consistent growth in the absence of serum protein could mean that the protein has two non-specific functions. Finally, we find, in common with other workers, that in the absence of serum growth rate is reduced. The work of Bailey (1967) has shown that all the cell lipids of the L cell come from serum when this is provided, but that in the absence of serum the cells are able to synthesize lipids. This finding suggests that the growth rate decrease, in the absence of serum, might result from lipid synthesis in the cell becoming the growth- limiting factor.

We gratefully acknowledge a grant to one of us (J.R.B.) from the Medical Research Council.

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(Received 28 November 1968)