J. Cell Sci. 4, 25-37 (1969) 25 Printed in Great Britain THE EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE GROWTH AND CARBOHYDRATE METABOLISM OF MOUSE LS CELLS D. G. KILBURN,' M. D. LILLY, DAPHNE A. SELF AND F. C. WEBB Biochemical Engineering Section, Department of Chemical Engineering, University College London, England SUMMARY Batch cultures of mouse LS cells were grown in suspension at controlled dissolved oxygen partial pressures (pOt). At low pOt (i-6 mmHg) the growth and respiration rates and the final cell population were all limited. At high pOt (320 mmHg), cell division was inhibited after an initial doubling of the cell number. At intermediate values of pOt, the growth rate was constant but the final cell population varied. Within the^>O2 range of 40-100 mmHg, the final cell population was constant and maximal at 1-2 x 10s viable cells/ml. Except at 320 mmHg pOt, about 90 % of the glucose consumed served as an energy source and could be accounted for as lactate and CO,. In the culture at 320 mmHg, only 60 % of the glucose consumed could be accounted for in this way. During growth the production of lactate and pyruvate was highest at low pOt. A sharp increase in lactate production was observed as logarithmic growth ceased in each culture, except at high pO% (160 mmHg). These observations indicate that pOt markedly influences cell growth and carbohydrate metabolism in these cells. INTRODUCTION The role of oxygen in the regulation of the metabolism of animal cells is not yet fully understood. It has been found that anaerobic conditions generally halt or severely depress cell growth (Clark, 1964; Dales, i960; Danes, Broadfoot & Paul, 1963). At the other extreme, high oxygen partial pressure (pO2) also inhibits replication but does not necessarily interfere with the synthesis of macromolecules (Brosemer & Rutter, 1961; Rueckert & Mueller, i960). Between these limits an optimumpO2 range for cell growth must exist. Although several workers have demonstrated a beneficial effect of moderately low^O2 (Zwartouw & Westwood, 1958; Cooper, Burt & Wilson, 1958; Cooper, Wilson & Burt, 1959; Pace, Thompson & Van Camp, 1962; Jones & Bonting, 1956), there is little quantitative information on the influence of pO2 on cellular growth and metabolism except at the extreme pO2 values. Specific studies on the effect of dissolved oxygen partial pressure on animal cell growth or metabolism have been made both in static and suspension cultures. The results relating to static monolayer cultures are difficult to interpret, because the static cell can establish its own micro-environment, which may vary considerably • Present address: Vaccine Department, Rijks Instituut voor de Volksgezondheid, Bilthoven, The Netherlands. 26 D. G. KiUnirn and others from conditions in the bulk of the medium. Culture of cells in suspension permits the pO2 environment to be specified much more exactly, although one cannot assume that the gaseous and liquid phases of these cultures are in equilibrium and hence the pO2 of the medium must be measured (Kilburn & Webb, 1966). The presence in the literature of a number of conflicting reports on the effects of dissolved oxygen may have resulted from the assumption that the measured oxygen partial pressure in the gas phase can be equated with the liquid pO2. Our previous work on the measurement of pO2 in suspension cultures of animal cells led to the development of a culture apparatus in which cultures could be grown at controlled pO2 values. The cultivation of cells at a number of discrete pO2 values between the inhibiting levels at high and low pO2 has permitted the influence of dissolved oxygen on cellular metabolism to be studied in much more detail than has previously been possible. It has been found that maximal populations of mouse LS fibroblasts are produced when the pO2 in the culture is controlled at values from about 40 to 100 mmHg (Kilburn & Webb, 1968). The present paper presents further information on the effect of pO2 on growth rate, maximum cell concentration and carbohydrate metabolism. METHODS Organism The mouse LS cell used in this work was kindly supplied by Dr J. Paul. This cell grows spontaneously in suspension, showing little tendency to attach to glass and has been maintained in our laboratory for 3 years. Cell culture technique All experiments were with 3 1. suspension cultures in Eagle's minimal essential medium (Eagle, 1955) supplemented with 2% (v/v) horse serum, 2-5 g/1. lactalbumin hydrolysate and 1 g/1. carboxymethyl cellulose. The glucose concentration in the medium was increased to 2 g/1. The temperature of the cultures was controlled at 35 +0-2° C and the pH was controlled at 7-4 + 0-1 by the addition of 0-5 N NaOH. Details of the culture vessel and the methods used to control pH and dissolved oxygen partial pressure have been described elsewhere (Kilburn & Webb, 1968). Viable cell counts were determined directly by counting a 1:1 dilution of cell suspension with stain (0-05 % trypan blue in o-8 % saline) on a haemocytometer (Fuchs-Rosenthal ruling, Cristalite, Gallenkamp, London). Cells which did not take up trypan blue were termed 'viable'. Analyses of culture medium Culture samples taken aseptically from the fermentor were centrifuged at 350^ for 10 min. The supernatant was deproteinized with perchloric acid and analysed for glucose, lactate and pyruvate. Glucose was analysed by the method of Marks (1959), using D glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) with o-toluidine as the chromogenic Effect of oxygen on growth of mouse LS cells 27 oxygen acceptor. Lactate was determined spectrophotometrically by measuring NAD+ reduction during the oxidation of lactate to pyruvate in the presence of lactate dehydrogenase (EC1.1.1.27) (Olson, 1962). Pyruvate was determined by measuring the oxidation of NADH in the reverse reaction (Segal, Blair & Wyn- gaarden, 1956). Materials 'Fermcozyme' glucose oxidase preparation and peroxidase (60 units/mg) were supplied by Hughes and Hughes Ltd., London, W. 1. Nicotinamide adenine dinucleotide (NAD+) and dihydronicotinamide adenine dinucleotide (NADH) were obtained from Seravac Laboratories, Maidenhead, Berks., and lactate dehydrogenase from Koch-Light Laboratories Ltd., Colnbrook, Bucks. Determination of respiration rate The rate of cellular respiration in the culture vessel under the controlled conditions of pH and^>02 was determined from the rate of CO2 evolution measured using tubing probes as described previously (Kilburn & Webb, 1968). An independent estimate of respiration rate in the presence of excess oxygen was obtained by following the depression of pO2 in a cell suspension taken from the fermentor and sealed in the cuvette of a Rank Oxygen electrode (Rank Bros., Bottisham, Cambridge). Samples taken from cultures at low pO2 were aerated before the cuvette was closed so that a significant pO2 drop with time could be measured. In several experiments, 2,4- dinitrophenol (DNP) was added to the Rank electrode to determine the increase in respiration rate effected by the uncoupling agent. In most cases the maximum uncoupling occurred with 5 x io~6 M DNP. RESULTS The effect of pO2 on cell growth As reported previously (Kilburn & Webb, 1966), the maximum concentration of LS cells reached in batch cultures grown at controlled dissolved oxygen partial pressures (j>02) was optimal in thepO2 range from about 40 to 100 mmHg. Figure 1 shows the batch growth curves for four cultures at different controlled values of dissolvedpO2, and illustrates three separate effects: (1) at low^O2 (i-6 mmHg) both the growth rate and the maximum population level are limited; (2) at high pO2 (320 mmHg) cell division is inhibited; (3) at intermediate values of pO2 the growth rates are constant (initially the cultures at 48 and 140 mmHg follow the same growth curve) but the maximum cell population varies. Cultures grown within the range of pO2 from 12 to 160 mmHg exhibited the same exponential growth rate equivalent to a mean generation time of 27-5 + 2-5 h. The effect of pO2 on respiration rate The average respiration rates, calculated from the rate of CO2 production during the rising portion of the growth curve, for cells growing at controlled pO2 levels are 28 D. G. Kilburn and others shown in Table i. These results agree well with the rates of oxygen consumption measured with the Rank oxygen electrode, except for the culture at i-6 mmHg. Within the range of pO2 from 32 to 160 mmHg the respiration rate is constant. At 320 mmHg the rate is higher, but this is probably associated with the increased Days Fig. 1. Batch growth curves for LS cells cultured at controlled dissolved oxygen partial pressures (pO2): A, i-6 mmHg; O, 48 mmHg; •, 140 mmHg; A, 320 mmHg. Table 1. Average respiration rates for cultures of mouse LS cells at controlled pO2 Respiration rate 8 Controlled pOt (mmHg) (mmole O,/h io cells) 0-130 12 0-166 32 0175 40 0180 96 018 160 018 320 0-228 dry weight of the cells. At 12 mmHg respiration may be slightly depressed by the low/>O2, while at i-6 mmHg the cells are definitely oxygen-limited. This is confirmed 6 by the increase in respiration rate from 0-13 mmoles/h io cells at^>02 = i-6 mmHg Effect of oxygen on growth of mouse LS cells 29 to 0-21 mmoles/h io6 cells when respiration was measured in the Rank electrode with oxygen in excess. The effect of low pO2 In the batch culture at i-6 mmHg pO2 the growth rate and maximum cell count were both less than in cultures at higher pO2. It seems unlikely that the low final cell count in this culture can be attributed entirely to an increased requirement for some limiting nutrient; particularly since the cell viability, measured by trypan blue exclusion, was always lower than in cultures at higher pO2.