j. cell sci. 4, 25-37 (1969 2) 5 printed in great...

J. Cell Sci. 4, 25-37 (1969) 25Printed 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 oxygenpartial pressures (pOt). At low pOt (i-6 mmHg) the growth and respiration rates and thefinal cell population were all limited. At high pOt (320 mmHg), cell division was inhibitedafter an initial doubling of the cell number. At intermediate values of pOt, the growth ratewas constant but the final cell population varied. Within the^>O2 range of 40-100 mmHg, thefinal cell population was constant and maximal at 1-2 x 10s viable cells/ml. Except at 320 mmHgpOt, about 90 % of the glucose consumed served as an energy source and could be accountedfor as lactate and CO,. In the culture at 320 mmHg, only 60 % of the glucose consumed couldbe accounted for in this way. During growth the production of lactate and pyruvate washighest at low pOt. A sharp increase in lactate production was observed as logarithmic growthceased in each culture, except at high pO% (160 mmHg). These observations indicate thatpOt 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 yetfully understood. It has been found that anaerobic conditions generally halt orseverely depress cell growth (Clark, 1964; Dales, i960; Danes, Broadfoot & Paul,1963). At the other extreme, high oxygen partial pressure (pO2) also inhibits replicationbut does not necessarily interfere with the synthesis of macromolecules (Brosemer &Rutter, 1961; Rueckert & Mueller, i960). Between these limits an optimumpO2 rangefor cell growth must exist. Although several workers have demonstrated a beneficialeffect 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 oncellular growth and metabolism except at the extreme pO2 values.

Specific studies on the effect of dissolved oxygen partial pressure on animal cellgrowth or metabolism have been made both in static and suspension cultures. Theresults relating to static monolayer cultures are difficult to interpret, because thestatic 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 permitsthe pO2 environment to be specified much more exactly, although one cannot assumethat the gaseous and liquid phases of these cultures are in equilibrium and hence thepO2 of the medium must be measured (Kilburn & Webb, 1966). The presence inthe literature of a number of conflicting reports on the effects of dissolved oxygenmay have resulted from the assumption that the measured oxygen partial pressurein the gas phase can be equated with the liquid pO2.

Our previous work on the measurement of pO2 in suspension cultures of animalcells led to the development of a culture apparatus in which cultures could be grownat controlled pO2 values. The cultivation of cells at a number of discrete pO2 valuesbetween the inhibiting levels at high and low pO2 has permitted the influence ofdissolved oxygen on cellular metabolism to be studied in much more detail than haspreviously been possible. It has been found that maximal populations of mouse LSfibroblasts are produced when the pO2 in the culture is controlled at values fromabout 40 to 100 mmHg (Kilburn & Webb, 1968). The present paper presents furtherinformation on the effect of pO2 on growth rate, maximum cell concentration andcarbohydrate metabolism.

METHODS

Organism

The mouse LS cell used in this work was kindly supplied by Dr J. Paul. Thiscell grows spontaneously in suspension, showing little tendency to attach to glassand has been maintained in our laboratory for 3 years.

Cell culture technique

All experiments were with 3 1. suspension cultures in Eagle's minimal essentialmedium (Eagle, 1955) supplemented with 2% (v/v) horse serum, 2-5 g/1. lactalbuminhydrolysate and 1 g/1. carboxymethyl cellulose. The glucose concentration in themedium was increased to 2 g/1. The temperature of the cultures was controlled at35 +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 dissolvedoxygen partial pressure have been described elsewhere (Kilburn & Webb, 1968).

Viable cell counts were determined directly by counting a 1:1 dilution of cellsuspension with stain (0-05 % trypan blue in o-8 % saline) on a haemocytometer(Fuchs-Rosenthal ruling, Cristalite, Gallenkamp, London). Cells which did nottake 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 analysedfor 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 measuringNAD+ reduction during the oxidation of lactate to pyruvate in the presence oflactate dehydrogenase (EC1.1.1.27) (Olson, 1962). Pyruvate was determined bymeasuring the oxidation of NADH in the reverse reaction (Segal, Blair & Wyn-gaarden, 1956).

Materials

'Fermcozyme' glucose oxidase preparation and peroxidase (60 units/mg) weresupplied by Hughes and Hughes Ltd., London, W. 1.

Nicotinamide adenine dinucleotide (NAD+) and dihydronicotinamide adeninedinucleotide (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 conditionsof pH and^>02 was determined from the rate of CO2 evolution measured using tubingprobes as described previously (Kilburn & Webb, 1968). An independent estimateof respiration rate in the presence of excess oxygen was obtained by following thedepression of pO2 in a cell suspension taken from the fermentor and sealed in thecuvette of a Rank Oxygen electrode (Rank Bros., Bottisham, Cambridge). Samplestaken from cultures at low pO2 were aerated before the cuvette was closed so thata 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 inrespiration rate effected by the uncoupling agent. In most cases the maximumuncoupling 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 ofLS cells reached in batch cultures grown at controlled dissolved oxygen partialpressures (j>02) was optimal in thepO2 range from about 40 to 100 mmHg. Figure 1shows the batch growth curves for four cultures at different controlled values ofdissolvedpO2, and illustrates three separate effects: (1) at low^O2 (i-6 mmHg) boththe 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 growthrates are constant (initially the cultures at 48 and 140 mmHg follow the same growthcurve) but the maximum cell population varies.

Cultures grown within the range of pO2 from 12 to 160 mmHg exhibited thesame 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 duringthe 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 consumptionmeasured 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 oxygenpartial 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

Controlled pOt (mmHg)Respiration rate

(mmole O,/h io8 cells)

12324096

160320

0-1300-1660 1 7 50 1 8 00 1 80 1 80-228

dry weight of the cells. At 12 mmHg respiration may be slightly depressed by thelow/>O2, while at i-6 mmHg the cells are definitely oxygen-limited. This is confirmedby the increase in respiration rate from 0-13 mmoles/h io6 cells at^>02 = i-6 mmHg


to 0-21 mmoles/h io6 cells when respiration was measured in the Rank electrodewith oxygen in excess.

The effect of low pO2

In the batch culture at i-6 mmHg pO2 the growth rate and maximum cell countwere both less than in cultures at higher pO2.

It seems unlikely that the low final cell count in this culture can be attributedentirely to an increased requirement for some limiting nutrient; particularly sincethe cell viability, measured by trypan blue exclusion, was always lower than in culturesat higher pO2. Possibly some cells were unable to adapt to growth at low pO2. Thepresence of dead cells could then create toxic conditions, e.g. by releasing proteolyticenzymes, leading to low viability.

The exponential growth rate at i-6 mmHg was about half that observed for cellsgrown in the intermediate pO2 range. Presumably this decreased growth rate reflectsthe lower rate of energy production under oxygen-limited conditions.

The effect of high pO2

The results from the culture at 320 mmHg/>O2 can be summarized as follows:(a) Growth rate was sharply inhibited after about 18 h. There was a slight increase

in the total cell count over the next 48 h, but the viable count remained constant(during this period the percentage viability dropped about 10 %).

(b) The dry weight/cell was about 30 % higher than normal.(c) The respiration rate/cell was about 25 % higher than normal.(d) Very little lactate was formed.

Table 2. The effect of DNP on the respiration rate of cells grown at high pO2

Respiration ratef

Measured Multiplication* No DNP DNPJ UncouplingSample pOt (mmHg) in 2 days , * , factor

1 145 3-3 0-122 0-277 2-3

2 290 2-i 0-191 0348 i-8

3 270 24 0186 0-372 20

, ,. . 2-day viable count• Multiplication = . . . .—r-r~, .

initial viable countf Respiration rate = mmoles Os/h io8 cells. % DNP concentration = 5 x io~6 M.

respiration rate with DNP§ Uncoupling factor =respiration rate without DNP

The reason why cell division is blocked while other cell processes proceed unchanged(initially at least) is still obscure. The continued operation of glycolytic enzymes,containing functional sulphydryl groups, makes it unlikely that the oxidation of suchgroups was extensive.

At high pO2 cell division could be blocked for lack of energy, if the generation ofATP was uncoupled from NADH oxidation. To test this proposition, a series of


cultures at high pO2 was set up in 500-ml bottles rotated at 40 rev/min on a set ofrollers. Previous measurements showed that this type of culture gave good equi-libration between the gaseous and liquid phases.

After 2 days the respiration rate of the cells in these bottles was measured with, andwithout, added 2,4-dinitrophenol (DNP). Table 2 shows the results of these experi-ments.

In a similar experiment, using static bottle cultures, the uncoupling factor was2-0 for cells grown at 150 mmHg pO2 and 2-2 for cells grown at 290 mmHg pO2.Thus the uncoupling factors appear to be independent of pO2, which suggests thatthe degree of coupling at different pO2 is the same. If cells at high/>02 were appreciablyuncoupled, one would not expect a large increase in respiration rate upon addingDNP.

Variation of carbohydrate metabolism with pO2

Fate of glucose. If it is assumed that the C02 and lactate produced during a cultureare derived mainly from glucose and not from other components of the medium, it ispossible to estimate the amount of glucose oxidized for energy production and theamount incorporated into the cells.

Except for the culture at 320 mmHg pO2, about 90 % of the glucose consumedduring the growth phase of a culture (i.e. to the peak of the growth curve) could beaccounted for as lactate and C02. This is consistent with the findings of Papacon-stantinou & Colowick (1961). During the growth phase the assumption that onlyglucose is oxidized appears justified, because it was found that during this periodthe alkali consumed for pH control was equivalent to the increases in C02 and lactate.If significant amino acid oxidation had occurred this balance would have been upset.After the peak in the growth curve the amount of C02 and lactate produced some-times exceeded the amount of glucose consumed, indicating that other constituentsof the medium were being oxidized.

In the culture at 320 mmHg pO2 almost 40 % of the glucose consumed was in-corporated into the cells. In this culture cell division was inhibited after the initialpopulation had doubled, although the cell dry weight continued to increase to 1-3times its normal value. This is in accord with the observations of Rueckert & Mueller(i960) and Brosemer & Rutter (1961), who found that the synthesis of macromoleculescontinued after cell division was halted. In the present work the soluble proteincontent per cell was the same as in cultures at lower pO2, so it is possible that thecell composition changed. This might relate to the finding of King, Edward &Bensch (1959), that under conditions in which protein synthesis and cell divisionwere inhibited, L-cells accumulated considerable quantities of lipid. Alternatively,the cells may have begun to store glycogen.

Lactate production. Figure 2 shows the lactate produced by cultures at differentpO2 values. The rate of production is clearly a function of pO2; being very high atlow pO2, but dropping as pO2 is increased. A sharp increase in rate occurs in thefinal stages of the cultures (except those at high pO2). This probably results fromthe breakdown of the citric acid cycle, and hence its inability to accept pyruvate.


Failure of the citric acid cycle could be caused by an increased demand for its inter-mediates, perhaps for amino acid synthesis when amino acids in the medium approachexhaustion. Support for this hypothesis comes from an experiment in which glutaminewas added to a culture after growth had stopped, shown in Fig. 3. Glutamine halted

Fig. 2. Variation of extracellular lactate concentration in cultures grown at controlledpO2 values: O, i-6 mmHg; • , 12 mmHg; A, 96 mmHg; • , 160 mmHg. Thecorresponding growth curves are shown in the lower part of the figure.

lactate production and restored respiration, presumably by entering the citric acidcycle via glutamic acid and a-ketoglutarate, or alternatively by reducing the demandfor TCA intermediates. There was no increase in the lactate dehydrogenase activityat the inflexion point, except in the culture at i-6 mmHg/>02. In the other cultures,lactate dehydrogenase must have been in large excess.

Pyruvate production. Figure 4 shows the extracellular pyruvate concentration forthree batch cultures at controlled dissolved pO2. With the exception of the experimentat i-6 mmHg pO2, the pyruvate in the medium was approximately constant. Thebuild-up of pyruvate in the culture medium at i-6 mmHg suggests again that the


Fig. 3. Batch culture at 96 mmHg pO2) showing the effect of glutamine addition(shown by arrow) on lactate production: O, viable cells per mix io~6; •» lactate inmedium, mmoles/1.

Days

Fig. 4. Variation of extracellular pyruvate concentration in cultures grown at controlledpOt values: O, 1-6 mmHg; A, 96 mmHg; • , 320 mmHg. The correspondinggrowth curves are shown in the lower part of the figure.


lactate dehydrogenase was operating at near maximum rate (the Km of lactate de-hydrogenase for pyruvate is about o-i rruvi).

Lactate to pyruvate (L/P) ratio. Assuming that lactate and pyruvate equilibratewith their environment by diffusion, then their concentrations in the external environ-ment should relate to the intracellular values, and the ratio L/P should be proportionalto the NADH/NAD+ ratio in the cytoplasm of the cell.

Fig. 5. Variation of extracellular lactate/pyruvate molar ratio in cultures grown atcontrolled pO2: O, i-6 mmHg; • , 12 mmHg; A, 96 mmHg. The correspondinggrowth curves are shown in the lower part of the figure.

Figure 5 shows the variations of L/P ratio for three batch experiments at differentpO2. The high L/P ratio for the culture at i-6 mmHg pO2 indicates that the cellswere relatively more reduced than in the cultures at higher pO2. The sharp increasein L/P ratio at the end of the logarithmic phase of growth reflects the rise in lactateproduction discussed previously. If this ratio is a true representation of the redox

3 Cell. Sci. 4

34 D. G. KiUmrn and others

potential in the cytoplasm, one must conclude that highly reducing conditions occurat the end of the growth cycle.

Figure 6 shows the variation of the L/P ratio with the pO2 at which the culture isgrown. An average L/P ratio has been taken from the early part of the cultures (i.e.the flat part of the curves in Fig. 5). It can be seen that the L/P ratio is high only atvery low pO2 and drops to a more or less constant value from 32-320 mmHg pO2.The values in the range from 50-100 mmHg pO2 may be slightly lower, i.e. moreoxidized. It is of interest that the same cytoplasmic redox potential can be maintainedat 320 mmHg pO2 as at 32 mmHg pO2. In contrast to the other cultures, there wasno appreciable rise in the L/P ratio at the end of the culture grown at 320 mmHg^>02.

3 0 *

10

100 200(mmHg)

300

Fig. 6. Variation of the average extracellular lactate/pyruvate molar ratioduring exponential growth of LS mouse cells at controlled pO2 values.

DISCUSSION

The ability to cultivate mammalian cells at specific pO2 levels affords an excellentopportunity to study cellular metabolism and its regulation. From the present resultsit can be seen that carbohydrate metabolism is markedly influenced by^>02, particularlyat the lower levels. In the intermediate range of pO2 values (32-96 mmHg) theexponential growth rate is almost constant and this is reflected in the respirationrate and pattern of carbohydrate metabolism. The interpretation of the observationson the variation of maximum cell populations is complicated by the lack of exactknowledge of the growth-limiting factors in the medium. On the basis of the studiesof Griffiths & Pirt (1967) it seems likely that at least for the cultures in the inter-mediate range of pO2 values the primary factor limiting growth was glutamine,although other amino acids also may have become limiting. The variation in maximum


cell population with pO2 (except at i-6 and 320 mmHg) probably reflects a changein the pattern of amino acid utilization in response to pO2. This might be associatedwith the production of specific proteins or mucopolysaccharides. An effect of thiskind might be expected if, as has been suggested, the specialized functions of somecells are oxygen-dependent (Brosemer & Rutter, 1961; Paul, 1965). We have someevidence that extracellular protein production was initiated in continuous culturewhen the pO2 was less than 80 mmHg. This phenomenon was not detected in batchcultures, suggesting that the function is also dependent on the growth rate.

In the culture grown at i-6 mmHgpO2 the respiration rate measured by the Rankelectrode was higher than that of cells grown in the intermediate pO2 range, suggestingthat the cytochrome content of cells grown at i-6 mmHg was higher than normal.Increases in the cytochrome contents of bacteria and yeast in response to low pO2

have been reported by Herbert (1965) and Moss (1956). The pO2 value at whichrespiration rate is half maximal has been compared to the K,n of the Michaelis-Mentenequation for enzyme kinetics (Longmuir, 1957; Froese, 1962). Reported values of'Km' for oxygen for animal cells range from o-1 to 2-0 mmHg pO2', approximatelythe same as the range of the Km for oxygen with cytochrome oxidase (Hayaishi, 1962).Johnson (1967) has shown that the half-maximal respiration rate of bacteria isa function of the growth conditions and is often not related to an enzyme Km. Thesearguments apply equally well to the present animal-cell cultures at low pO2, henceone cannot define the saturation level of the enzyme system coupled to oxygen. It canonly be concluded that at i-6 mmHg pO2 (and possibly 12 mmHg pO2) the cellularrespiration rate was oxygen limited.

Although the culture at 1-6 mmHg pO2 grew at a reduced rate, replication wasstill exponential, whereas without pO2 control, cultures grew linearly after reachinga very low pO2. This demonstrates an important distinction between oxygen-limitedcultures.

In the first case the controlled pO2 is constant but at such a low level thatgrowth rate is limited. Nevertheless, each succeeding generation of cells encountersthe same pO2 conditions and hence growth is exponential. The amount of oxygenconsumed per cell remains constant, while the amount of oxygen consumed by theculture increases as the cell number increases.

In the second case the cells depress the pO2 to a very low growth-limiting level,after which the amount of oxygen transferred to the medium remains constant. As thecell number increases, the amount of oxygen available per cell decreases, and thegrowth rate drops progressively, with the result that the cell number increaseslinearly.

The results of the experiments at high pO2 are in general agreement with previousreports in the literature but do little to clarify the mechanism by which oxygen blockscell division. Rueckert & Mueller (i960) found that the replication of Hela cellsgrown with 95 % O2 in the gas phase was inhibited after 24 h; DNA, RNA and proteinsynthesis continued for a further 12 h, after which the viability dropped sharply.In contrast to the present results, they found that glucose was converted entirelyto lactate. Perhaps in their experiments the citric acid cycle was inoperative due to

3-2


inactivation of lipoic acid by oxygen, as suggested by Thomas, Neptune & Sudduth(1963). This clearly did not occur at the lower pO2 used in the present work, becausethe respiration rate (and presumably the citric acid cycle activity) was higher thannormal. The increase was in proportion to the increase in cell size so that, on a weightbasis, respiration was unchanged. The rapid decrease in viability after 36 h reportedby Rueckert & Mueller was not observed, but this again can probably be attributedto the different pO2 used. Brosemer & Rutter (1961) found no change in the patternof glucose utilization when AH cells were incubated with 45 % oxygen in the gasphase. Cell division was blocked but protein synthesis continued.

It can be seen that there is a need for more research on the effect of moderatelyhigh pO2. The mechanisms might be much more subtle than those acting at highpO2, e.g. complete oxidation of sulphydryl groups appears to be ruled out at moderatepO2. It would be of interest to determine the virus-producing capacity of cells grownat high pO2, and also to study the effect of moderately high pO2 on virus replication.This might show whether functions such as nucleic acid synthesis are abnormal.Brosemer & Rutter showed that 32P incorporation into nucleic acid was unchangedbut they did not show that the nucleic acid was functional. If pO2 operates onlyon the cell division mechanism one might expect virus replication to be unhamperedby hyperbaric pO2.

The authors wish to thank the Wellcome Foundation for their support of this project.

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(Received 6 February 1968)

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