J. Cell Sci. 8, 693-700 (1971) Printed in Great Britain THE QUANTITATIVE GLUCOSE AND MINERAL NUTRIENT REQUIREMENTS OF MOUSE LS (SUSPENSION) CELLS IN CHEMICALLY DEFINED MEDIUM J. R. BIRCH* AND S. J. PIRT Department of Microbiology, Queen Elisabeth College (University of London), Campden Hill, London, W. 8, England SUMMARY The influences of glucose, phosphate, magnesium and potassium concentration on growth rate and maximum population density were studied. From the data obtained the growth yields for glucose and phosphorus were found to be 2-4x10* cells (i.e. 0-76 mg dry weight)/mg glucose and 2-2 x io 6 (i.e. 69 /ig dry weight)//ig P. The growth response to magnesium and potassium was complex since there were threshold concentrations of these metals below which no growth occurred. The growth yield for potassium (2-4 x 10' cells (i.e. 75 fig dry weight) /figk) was obtained by measuring potassium uptake during growth. Omission of calcium ions from the medium resulted in poor reproducibility of growth. INTRODUCTION Until recently, studies on culture media for animal cells have mostly been limited to the determination of qualitative nutrient requirements. These studies have resulted in the development of several chemically defined media, which support the growth of a few cell lines. In general, however, growth rates and maximum cell populations in defined media are depressed compared with media containing natural supplements such as serum. Previous studies in this Department have shown that the determination of quantitative requirements is essential to an understanding of factors limiting growth rate and maximum populations of cultured cells (Griffiths & Pirt, 1967; Birch & Pirt, 1969, 1970). The data obtained were used to design a chemically defined medium in which the LS cell grows to maximum populations in excess of 5 x io 6 /ml (Birch & Pirt, 1970). Recently, Higuchi (1970) has reported similar improvements in the productivity of a chemically defined medium for L cells, also based on the determination of quantitative nutrient requirements. In the present study we have investigated the quantitative requirements of the LS cell for glucose and the mineral nutrients, particularly potassium, magnesium and phosphate ions. Present address: Tate and Lyle Ltd., Research Centre, 'Ravensbourne', Westerham Road, Keston, Kent, England.
694 J- R - Birch and s - J- Pirt MATERIALS AND METHODS The general methods of culture and the cell strain have been described by Griffiths & Pirt (1967). Medium The defined medium of Birch & Pirt (1970) was used. The non-essential supplements (methylcellulose, keto acids, polyvinylpyrrolidone and antibiotics) were included for the present study. Preparation of inocula Cells for inocula were taken from cultures in the exponential phase of growth with population densities in the range 1-3 x io 6 cells/ml. Cells were harvested by centrifugation in sealed McCartney bottles at 170 g for 3 min. They were washed and resuspended in either phosphatefree basal salt solution (Paul, 1958) (for phosphate experiments) or 0-85 % sodium chloride solution (for magnesium, potassium and glucose experiments). Methylcellulose was included in both solutions at a final concentration of 0-5 %. Static suspension cultures Cells were grown as static suspension cultures in 8-oz (200-ml) medical flat bottles. The culture volume was 10 ml and the inoculum i-o-2-o x io 6 cells/ml. Cultures were incubated at 37 C and growth was measured by counting cells on a Fuchs-Rosenthal slide using trypan blue to distinguish viable and non-viable cells. Adherence of cells to the glass surface of culture vessels was a problem in this study, particularly in cultures which had just been inoculated, and under some nutritional conditions (low potassium concentrations). To prevent this, culture vessels were coated with silicone by rinsing with 2 % silicone fluid (MS 1107, Hopkins and Williams Ltd.) in methyl ethyl ketone, followed by drying at 160 C for about 4 h. Cell dry weight estimations The cells used for this estimation were taken from cultures just entering the stationary phase of growth, and the viability was 99'5%. Cells were suspended in phosphate-buffered saline (Pirt & Thackeray, 1964) containing methylcellulose (0-5 %). They were then dried at 105 C for 18 h in tared centrifuge tubes and weighed. Allowance was made for the salts and methylcellulose present. The dry weight of io 6 cells was 320 ± 9-6 fig (mean of 6 determinations) and this figure was used for growth-yield calculations. Estimation of potassium Potassium was determined using an EEL flame photometer. Determinations were carried out in quadruplicate on samples pooled from triplicate cultures and were always within 2 % of the mean. RESULTS Glucose requirement Cells were grown in denned medium with a range of glucose concentrations from o to 2 mg/ml. Glucose concentration had no effect on the length of the lag phase or maximum growth rate (population doubling time = 38 h). Below about 1 mg glucose/ml, maximum cell population density (inoculum deducted) was proportional to the glucose concentration (Fig. 1). There was no significant growth in the absence of glucose.
Mammalian cell nutrition 695 The growth yield for glucose, given by the slope of the curve, is 2-4 x io 6 cells (i.e. 0-76 mg dry weight)/mg glucose. Our normal medium, therefore, contained sufficient glucose to support a population of 4-7 x io 8 cells/ml. In an earlier experiment the growth yield for glucose in medium containing 5 % (v/v) dialysed calf serum was found to be 3-3 x io 6 cells/mg glucose. This higher value may be due to the glucose-sparing action of serum components such as lipids and to the faster growth rate in medium containing serum (population doubling time = 24 h). Phosphate requirement Our medium normally contained 3-2 mm phosphate (500 mg NaH 2 PO 4.2H 2 O per 1.). Reducing this to o-6 mm had no effect on growth. Below this concentration maximum cell populations were directly proportional to phosphate concentration (Fig. 2). Phosphate concentration had no significant effect on the length of the lag 32 28 5 2-4 20 16 12 8 4 200-400 600 800 1000 Glucose concentration in medium (/ig/ml) 2000 Fig. 1. The effect of glucose concentration on growth. Each point is the mean of at least 3 determinations. Variation is shown by the vertical lines. phase or on growth rate (population doubling time = 40 h). There was a small amount of growth in the complete absence of added phosphate. The growth yield for phosphorus calculated from the slope of the curve is 2-2 x io 6 cells (i.e. 69 fig dry weight)//tg P. From this result it can be seen that the normal phosphate concentration (3-2 mm) is sufficient to support a population of 2-1 x io 7 cells/ml. We had previously shown (Birch & Pirt, 1969) that serum in tissue culture medium is a major source of choline, presumably in the form of bound phospholipid. An experiment was therefore set up to determine the phosphorus-sparing action of serum in
696 J. R. Birch and S. J. Pirt medium containing a growth-limiting concentration of phosphate; 5 % (v/v) dialysed calf serum had no detectable sparing effect and was not therefore a major source of phosphorus in cells grown in medium containing serum. 40 r a 35 E 30 o ~ 25 * 20 o I 15 =5 10 u 3 E c 20 40 60 80 100 Phosphate concentration In medium (,i/g NaH 2PO 4.2H 2O/ml) Fig. 2. The effect of phosphate concentration on growth. Each point is the mean of 3 determinations. Variation is shown by the vertical lines. Magnesium requirement The normal concentration of MgCl 2. 6H 2 O in our medium was 0-98 mm (200 mg/1.). There was little effect on growth down to approximately 0-25 mm (50 mg/1.). Below this concentration growth rate was markedly reduced (Fig. 3). Maximum cell populations were reduced at concentrations below 0-15 mm (30 mg/1.). Below 0-06 mm (13 mg/1.) little growth occurred, but survival was prolonged. In the complete absence of magnesium, death was rapid (18% survivors after 42 h). A threshold concentration of magnesium between 0-05 and 0-07 mm had to be exceeded for growth to occur. Because of the pronounced effect of magnesium concentration on growth rate and the known relationship between growth rate and maximum cell population (Griffiths & Pirt, 1967), a meaningful growth yield cannot be derived from the maximum cell population values. It is apparent, however, that in terms of the requirement for maximum growth rate the normal medium contained approximately a 4-fold excess of magnesium.
Mammalian cell nutrition 697 100 1S0 200 Incubation time (h) 250 300 Fig. 3. The effect of magnesium concentration on growth. The points represent the means of triplicate cultures. Concentrations of MgCl 2.6H,O (jig/ml medium): # 9, 200; O 0,40; A A, 35; A A, 3 ;, 25; D, 20; T T, 15; V V, 10. Growth was identical to the control (200 fig MgCl 2.6H,O/ml) with 100 and 50 fig Potassium requirement Growth experiments. The normal potassium chloride concentration in our medium was 5-36 mm (400/ig/ml). Reducing this concentration to 0-7 mm (50/ig/ml) had no effect on growth. However, below this concentration both growth rate and maximum cell population were reduced (Fig. 4). It can be seen that, as with magnesium, there was a threshold concentration of potassium chloride (0-40 mm, 30 /tg/ml) below which no growth occurred. Potassium uptake during growth. Because of the complex response to limiting K+ concentrations, it was impossible to derive a meaningful growth yield for potassium from the data presented in Fig. 4. For this reason potassium uptake during growth was followed by flame photometry. To demonstrate a significant uptake, the potassium chloride concentration of the medium was reduced to o-8o mm. At this concentration, growth was identical with that in control cultures with the normal level of potassium.
698 J. R. Birch and S. J. Pin 40 80 120 Incubation time (h) Fig. 4. The effect of potassium concentration on growth. Points represent the means of triplicate cultures. Concentrations of KCl Oig/ml medium): #, 400; O O, 40; A A, 35; A A, 30; M, 25; D, 20. Growth was identical to the control (400 /ig KCl/ml) with 200, 100 and 50 fig KCl/ml medium. 20 40 60 80 100 120 140 160 Incubation time (h) Fig. 5. Potassium uptake during growth. O O, Viable cell count; 9, K + concentration.
Mammalian cell nutrition 699 Disappearance of potassium from the medium during growth is shown in Fig. 5. When decrease in potassium concentration is plotted against corresponding increase in cell number a straight-line relationship is found. The growth yield for potassium obtained from the slope of this graph is 2-4 x io B cells (i.e. 75 //.g dry weight)//<g K. Since this figure is based on uptake of potassium rather than on maximum growth with limiting potassium concentrations, it represents a minimum value for the growth yield. It follows that the normal potassium concentration in our medium (i.e. 5-36 m\i) is sufficient to support a population of at least 5 x io 7 cells/ml. DISCUSSION There appear to have been no previous reports of the growth yield (Y = yield of cells/unit weight of nutrient) of animal cells from glucose and mineral nutrients, with the exception of the results of Griffiths (1970) who determined the growth yield of WI-38 cells from glucose. Likewise no quantitative data are available on the effect of these nutrients on cell growth rate. Such information is essential in the design of media to produce given populations of cells and in the elucidation of the role of different nutrients. One can estimate the growth yield of L cells from glucose, from earlier data on disappearance of glucose from media during growth. Sinclair (1966), for instance, reported a consumption of 5 /IM glucose/10 6 cells in a semi-continuous culture system using a chemically defined medium. This corresponds to a growth yield of I-I x io 6 cells/mg glucose. The higher yield obtained in the present study (2-4 x io 6 cells/mg glucose) may be explained by the fact that our calculation of Y is based on maximum population reached at limiting glucose concentrations. It is therefore a maximum value of Y and is likely to differ from values based on glucose utilization when glucose is in excess. A much lower growth yield for glucose (0-05-0-10 mg dry weight/mg glucose) has been reported for WI-38 cells (Griffiths, 1970). Such variations from one cell system to another presumably depend on the relative proportions of glucose converted to products such as lactic acid and on factors such as cell growth rate. Previous studies on the phosphate requirement for the growth of L cells (Waymouth, 1954; Eagle, 1956) did not establish the quantitative effect of this nutrient on growth rate or maximum cell populations. Waymouth (1954) claimed that phosphate concentrations up to 2 rain stimulated 'survival, maintenance and proliferation of L cells'. She concluded that a high phosphate concentration was probably essential in maintaining the appropriate conditions for phosphorylation and the production of highenergy phosphate. However, in the present study phosphate concentration had no influence on growth above o-6 mm and below this concentration only affected maximum cell population density. At cell populations approaching io 7 /ml, which can be achieved in defined medium (Birch & Pirt, 1970), the phosphate concentration in some commonly used salt solutions would be limiting. The concentrations in Earle's and in Hanks's solutions, for instance, would limit the maximum population of LS cells to 5-6 x io 6 cells/ml. A growth yield similar to that which we obtained can be calculated from the data recently reported by Higuchi (1970).
700 J. R. Birch and S. J. Pirt A semi-quantitative study was made by Eagle (1956) and Wyatt (1961) of the potassium and magnesium requirements of L and HeLa cells. These studies were complicated by the presence of serum in the media and no values were given for the growth yields from these cations. Furthermore, since monolayer cultures were used, it is difficult to distinguish between the role of the cations as nutrients and their role in promoting attachment of cells to glass. It is interesting that in contrast to the phosphate requirement there are threshold concentrations of potassium and magnesium which have to be exceeded for growth to occur. Wyatt (1961) also found that there was little growth of HeLa cells in monolayer culture at low potassium and magnesium concentrations, although precise threshold levels were not established. Eagle (1956) and Wyatt (1961) found that o-i mm magnesium gave optimal growth of L and HeLa cells respectively. We found that this concentration limited growth rate to well below the maximum. This may in part be due to the use of serum in the earlier studies which would be a source of magnesium. Likewise the potassium concentration reported by Wyatt (1961) to be optimal for the growth of HeLa cells (0-5 mm) limited growth rate to below the maximum in the present study. It should be added, however, that we were working with much higher cell population densities than Wyatt. The other mineral nutrients in our medium, apart from sodium chloride, are sodium bicarbonate and a number of trace-metal salts. Bicarbonate is incorporated at a relatively high concentration to maintain adequate buffering. We were unable to show a definite requirement for trace metals other than iron, which is essential (Birch & Pirt, 1970). However, omission of calcium resulted in poor reproducibility of growth. REFERENCES BIRCH, J. R. & PIRT, S. J. (1969). The choline and serum protein requirements of mouse fibroblast cells (strain LS) in culture. J. Cell Sci. 5, 135-142. BIRCH, J. R. & PIRT, S. J. (1970). Improvements in a chemically defined medium for the growth of mouse cells (strain LS) in suspension. J. Cell Sci. 7, 661-670. EAGLE, H. (1956). The salt requirements of mammalian cells in tissue culture. Archs Biochem. Biophys. 61, 356-366. GRIFFITHS, J. B. (1970). The quantitative utilization of amino acids and glucose and contact inhibition of growth in cultures of the human diploid cell, WI-38. J. Cell Sci. 6, 739-749. GRIFFITHS, J. B. & PIRT, S. J. (1967). The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate. Proc. R. Soc. B 168, 421-438. HIGUCHI, K. (1970). An improved chemically defined culture medium for strain L mouse cells based on growth responses to graded levels of nutrients including iron and zinc ions. J. cell. Physiol. 75, 65-72. PAUL, J. (1958). Determination of the major constituents of small amounts of tissue. Analyst, Lond. 83, 37-42. PIRT, S. J. & THACKERAY, E. J. (1964). Environmental influences of the growth of ERK mammalian cells in monolayer culture. Expl Cell Res. 33, 396 405. SINCLAIR, R. (1966). Steady state suspension culture and metabolism of strain L mouse cells in simple defined medium. Expl Cell Res. 41, 20 33. WAYMOUTH, C. (1954). Some effects of inorganic phosphate and bicarbonate on cell survival and proliferation in chemically defined nutrient media. Biochem. J. 56, iv. WYATT, H. V. (1961). Cation requirements of HeLa cells in tissue culture. Expl Cell Res. 23, 97-107. (Received 24 September 1970)