JOURNAL OF CELLULAR PHYSIOLOGY 150:99-103 (19921

Effects of pH-Variations on the Kinetics of Growth and Energy Metabolism in Cultured

T-Lymphoma Cells: A Microcalorimetric Study PER BACKMAN, TAKAYOSHI KIMURA, ARNE S C H O N AND INCEMAR W A D S O * Division of Thermochemistry, Chemical Center, University of Lund, Lund, Sweden

The progression of T-lymphoma cells (CCRF-CEM) growing in suspension has been monitored during long term ( 1 2-28 h ) batch experiments using microcalo- rimetry. In parallel with the calorimetric measurements, changes in cell concen- tration, pH, p ( 0 2 ) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, P& and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1 . 2 J per ml (corresponding to an incubation time of 20 h of a batch having a n initial cell concentration of 1 x 1 O6 cells per ml). The lowering of p ( 0 , ) to a level of 0.02 mmolil or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmolil, respectively, did not influence P,,,, or the growth pattern. No "crowding effect" was observed for the cells in the investigated concentration range (0.6-1.3) x 10' cells per ml.

Many investigations have described how drugs and experimental parameters such as pH and p(O,), influ- ence specific metabolic pathways and cellular growth. However, with the exception of calorimetry, no meth- ods used in this type of study have the feature of being nonspecific in that any complex or unexpected changes in biological activity will be observed. By measuring the heat production rate (P = dQidt), calorimetry can be used to follow the rates of overall metabolism and growth in a variety of biological systems. Hence, direct calorimetric measurements have proven to be useful both in basic physiological investigations and in work oriented towards applied areas such as clinical analy- ses and biotechnology (see, e.g., James, 1987). Method- ological investigations from microcalorimetric mea- surements on T-lymphoma cells (CCRF-CEM) have been reported for both non-growing and growing con- ditions (Schon and Wadso, 1986; Backman, 1990). These investigations were limited to short-term mea- surements (=2 h) where changes in experimental con- ditions of the batch of cells are negligible. It was thus possible to study how a change in, e.g., medium pH affected the overall metabolism without interference from other changes in the cellular environment. Short- term calorimetric measurements have also been used in combination with biochemical analyses in order to assign the origin of heat production to metabolic reac- tions. During non-growing conditions, 75% of the heat production from T-lymphoma cells of the same line as studied here was correlated to the production of lactate and carbon dioxide (Schon and Wadso, 1986). Recent investigations indicate that under growing conditions, nearly all of the heat production can be related to reactions in central catabolic pathways (Backman, in publication). Attempts have also been made to estimate

the effects of various antineoplastic drugs during long- term (= 18 h) calorimetric measurements on the same T-lymphoma cell line (Schon and Wadso, 1988; Kimura et al., 1990). In the latter studies problems occurred, however, of how to distinguish effects of the added drugs from effects due to changes in the status of the medium caused by the metabolic activity. Changes in experimental conditions are ubiquitous and the effects on proliferation and metabolism might influence re- sults obtained by methods other than calorimetry. The aim of the present investigation is to show how exper- imental factors influence the overall metabolism and growth during long-term batch culture of T-lymphoma cells.

MATERIALS AND METHODS Cell culture

T-lymphoma cells, CCRF-GEM (Foley et al., 19651, kindly provided by Dr C. Borrebaeck, Division of Immunotechnology, Chemical Center, University of Lund, were cultured under non-stirred conditions in tissue culture flasks (Costar, Cambridge, MA) a t 37"C, using RPMI 1640 medium supplemented with 10% fetal calf serum. The medium contains 10.1 mmolil a,P-D-glucose and 1.8 mmol/l L-glutamine. Medium and supplements were obtained from Flow Laborato- ries, Inc. (Rockville, MD). It should be noted that the cells used here have been made free from mycoplasma

Received November 27, 1990; accepted July 30, 1991 *To whom reprint requestsicorrespondence should be addressed. Takayoshi Kimura's present address is Department of Chemistry, Faculty of Science and Technology, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577, Japan.

0 1992 WILEY-LISS, INC.

BACKMAN ET AL. 100

(BM-Cyklin, Boehringer-Mannheim Scandinavia AB, Bromma, Sweden). Thus, they are not strictly identical with the originally derived CCRF-CEM cells, which are described as mycoplasma infected by ATCC. The cells were tested for the presence of mycoplasma using a detection system from Gen-Probe (San Diego, CA). Prior to the measurements, the cell suspensions were prepared according to the standard method described earlier (Backman, 1990). By following this standard- ized method, which includes a preincubation period of 3.5 h , the distribution of cells in the growth cycle could be assumed to be equal at the beginning of each experiment.

Calorimetry A 4-channel microcalorimeter of the heat conduction

type (“Thermal Activity Monitor,” LKB/Thermometric AB, Jarfalla, Sweden), equipped with four stirred ves- sels, was used in all calorimetric measurements (Suurkuusk and Wadso, 1982; Gorman et al., 1984).

A series of experiments was performed where changes in cell concentration, pH and cellular heat production rate (Pcell) were followed during measure- ments which lasted 12-28 h. Samples of 2.7 ml were transferred to the vessels, which were inserted into the calorimeter. The suspension was stirred by means of a “turbine stirrer” at a rate of 90 rpm. A gas phase of 0.7 ml was present above the cell suspension. The initial cell concentration was varied between 0.5 and 1.3 x lo6 cells per ml. All experiments were performed at 37”C, with the initial pH at 7.17 -+ 0.03. Instrument baselines were established between each measurement with 2.7 ml water in the vessel. For details concerning the calorimetric technique, see Schon and Wadso (1986).

At regular intervals, (2-6 h, depending on the initial cell concentration), one vessel was removed from the calorimeter and values of pH and cell concentration were determined. The viable cell count was estimated by trypan blue exclusion in a Burker chamber. Mea- surements of pH were made using a Radiometer G297162 capillary electrode (Radiometer, Copenhagen, Denmark).

In some calorimetric experiments, oxygen concentra- tion in the medium was continuously monitored by use of a miniaturized oxygen electrode, positioned in the calorimetric vessel (Backman and Wadso, 1991).

Determination of metabolite levels In some experiments, samples were taken from the

stirred vessels upon removal from the calorimeter and immediately frozen in dry ice. After thawing and deproteinization with percloric acid, the samples were centrifuged and the supernatant analyzed enzymati- cally for glucose (Bergmeyer et al., 1974) and glu- tamine (Beutler and Michal, 1974). All chemicals used in the enzymatic assays were obtained from Boeh- ringer-Mannheim Scandinavia AB (Bromma, Sweden).

Determination of the growth rate as a function of pH

In a series of experiments, changes in cell concentra- tion with time were measured a t various pH-values. During a period of 9 h, pH and cell concentration were

. . .

A I I

B I

3.2

r-? 0 %

0.1 7 3

P Time(h)

Fig. 1. A. Typical P-t curves from six parallel measurements (-) terminated at different times, are shown together with an ideal curve (- -1. The cell suspension was prepared at zero time and the vessels were loaded after approximately 3.5 h. Power values were obtained 1-2 h later when the calorimetric system had reached equilibrium. B. Changes in cell concentration (-+I during the experiment, together with an ideal curve (- -). C. Changes in pH (-1 and oxygen concentration (-1 as measured during the experiment.

measured in samples taken with 90 min intervals from a cell suspension incubated in a shaking water bath at 37°C. The mean pH-value during the incubation was taken as the pH during the experiment.

RESULTS In a typical long-term experiment, the total heat

production rates (Ptot) were recorded from six vessels run in parallel by use of two 4-channel calorimeters (Fig. 1A). The recorded curves (P-t curves) can be described by two equations. Equation (1) expresses the increase in cell number (n,) with time ( t ) ,

(1) n, = ni x 2t’tg

as a function of the initial number of cells (ai), and the generation time (tJ: Equation (2), relates Ptot to n,, and to the heat production rate per cell (Peen).

(2) The ideal P-t curve indicated in Figure 1A was

pto t = Pcell x nt

MICROCALORIMETRIC STUDY OF T-LYMPHOMA CELLS 101

14 c I

b 10

I I

0 10 20 30 Time (h)

Fig. 2. The heat production rate per cell (Pcell) as measured during six independent experiments. The initial cell concentrations (in units of lo6 cells per ml) were as follows: 0.561 (0 ) ; 0.592 (0); 0.830 (W; 0.977 (0); 1.250 (A); 1.265 ( A ) .

calculated by use of Equations (1) and (2) with the initial values of tg and Peel, as constants. This curve describes the “chemostat” case where no changes in environmental constraints occur during the experi- ment.

However, a full description of experimental P-t curves has to account for variations in t, and Pcell caused by changes in experimental conditions during batch experiments. Not surprisingly, the observed ex- perimental P-t curves deviate considerably from the ideal curve, Figure 1A. The deviations are clearly caused by a reduction in Pcell with time since the variation in cell concentration during these experi- ments closely follow the ideal curve, Figure 1B. Both medium oxygen concentration and pH decreased sig- nificantly during these experiments, Figure 1C.

The changes in Pcell were examined in a series of six experiments, each performed as the previous set of experiments. In all cases, a decrease in Pcell with time was observed (Fig. 2). The rate of decrease varied between experiments carried out with different initial cell concentrations. However, when the Poll-values from Figure 2 were plotted against the total heat produced per ml of cell suspension (Q) , Figure 3A, the variation disappeared (Q was obtained by integrating the P-t curve and dividing by the sample volume, 2.7 ml). From the regression curve calculated for the values in Figure 3A, the initial value of Pcell was estimated to be 13.5 pW per cell. During these experiments, pH decreased with increasing Q (Fig. 3B) without any variation with respect to initial cell concentration. The values in Figure 3B could be fitted to a linear equation:

pH = pHi + k x Q (3) where the initial pH-value (pH, = 7.17) and slope (k = -0.30 J-l) were calculated by minimum square regression (r = 0.97).

The regression curve in Figure 3A was corrected for pH-changes using results reported by Backman (1990) on the variation of Pcell with medium pH in short-term measurements. The corrected curve shows no signifi- cant deviation from the ideal behavior until Q exceeds 1.2 Jiml. With an initial cell concentration of 1 x lo6 cells per ml this corresponds to approximately 20 h of measurement.

25 t 6 5 B

I

0 0 5 10 15

Q(J/cm3)

Fig. 3. A. P,,,,-values from Figure 2 as a function of the thermal energy ( Q ) produced per ml cell suspension after preparation time. The regression curve calculated (-), is compared with a curve corrected for pH-changes during the measurements ( - - - - I . The horizontal line represents the initial value for P,,,, (- -1. B. Decrease in pH as a function of Q during the experiments shown in Figures 2 and 3A. Symbols are the same as in Figure 2.

From the in situ oxygen measurements (Fig. lC), the medium oxygen concentration at Q = 1.2 Jim1 was found to be 0.02 mmolil. Concentrations of glucose and glutamine decreased during this period from initial concentrations of 10.1 and 1.8 mmolil to 7.7 and 1.3 mmolil respectively.

Even though the cell concentration curve in Figure 1A shows only minor variations from the ideal curve, the growth rate (UtJ is clearly affected by pH-changes, as can be observed in Figure 4. A second- order polynomial was fitted to the values in Figure 4 using a minimum square regression method (Equa- tion 4):

(4) where A = -4.007 h-’; B = 1.150 h-*; C = -0.08167 h-l. The maximum growth rate was estimated to be 0.042 h-’ a t pH 7.0. This corresponds to a t, equal to 24 h. At the reference pH 7.2, litg was equal to 0.040 h-.l which corresponds to a t, of 25 h.

Based on Equations (14) and the pH-dependence of Pcell (Backman, 1990) P-t curves were simulated. The simulation is based on an iterative algorithm, using pH, and ni as input data. In Figure 5, three typical experimental P-t curves are compared with curves predicted from the experimental values of pHi and n,.

DISCUSSION As can be seen from Figure 1, changes in medium

status due to cellular processes induces a significant decrease in overall metabolic rate (as reflected in P,,,,) and a minor decrease in growth rate during the exper- iments. Thus, the shape ofP-t curves varies with initial cell concentration. This is also the case for the variation of Pcell with time (Fig 2.). Assuming that the medium status would be better reflected by Q than by the time,

lit, = A + B x pH + C x (pH)’

BACKMAN ET AL.

I I

I I

I I

6.5 7.0 PH

0 10 20 30 Time ( h )

Fig. 5 . Experimental P-t curves starting with different initial cell Concentration (-1, as compared to the curves predicted by computer simulation (- -1.

Fig. 4. Growth rate (llt,) as a function of the medium pH. The uncertainty limits correspond to twice the SEM. The horizontal line represents the reference value of lit, (at pH 7.2).

values of Pcell was plotted against Q (Fig. 3A). This reduced the variation between experiments signifi- cantly and at Q s 1.2 Jiml, the reduction in Pcell could be entirely accounted for by the decrease in pH. The applied pH-corrections where obtained from short-term experiments where pH could be varied independently of other experimental factors (Backman, 1990). This ex- cludes the possibility that another (covarying) experi- mental factor (e.g., lactate concentration) would cause the reduction in Pcell. These observations were further supported by the successful predictions of P-t curves on the basis of independent investigations of pH-effects on growth and Pcell (Fig. 5).

A critical oxygen concentration of 0.02 mmolil has been observed for the present cell line (Backman and Wadso, 1991). This is in agreement with critical oxygen concentrations observed for, e.g., hepatocytes (0.01- 0.02 mmolil), (Jones and Mason, 1978) and sheep lymphocytes (0.0002-0.02 mmolil), (Cheung and Mor- ris, 1984). Apparent Michaelis-Menten constants for intact cells (e.g., Jones and Mason, 1978) are typically 10 times higher than for isolated mitochondria (Jones and Mason, 1978; Cole et al., 1982; Sugano et al., 1974) which is explained by intracellular diffusion gradients, presumably due to clustering of mitochondria (Jones, 1986). As the oxygen concentration here is 30.02 mmolil when Q ~ 1 . 2 J/ml, the observed decrease in oxygen activity should have no effect on the rate of metabolism. However, oxygen limitations do provide a possible explanation for the non-pH induced inhibition of Pcell observed at Q > 1.2 J/ml.

Variations in oxygen uptake with cell concentration (“crowding effect”) have been observed for human lymphocytes (Hedeskov and Esmann, 1966) and for cell lines derived from lymphocytes and lymphoma cells (Sand et al., 1977). The presence of a “crowding effect” would be reflected as a decrease in Pceil with increased cell concentration. No such variation was found here (in agreement with earlier observations of ours [Backman, 199011, which is consistent with the conclusions of

Hedeskov and Esmann (1966) that the “crowding ef- fect” can be ascribed to oxygen limitations at high cell concentrations.

Glucose and glutamine are the main available car- bon sources in the medium. For the present cell line, glucose is mainly converted into lactate whereas glu- tamine enters the citric acid cycle a t a-ketoglutarate where it is partly oxidized to carbon dioxide. These processes account for a large part of the measured heat production (Backman, in publication). At Q < 1.2 Jiml, less than 23% of the glucose and 25% of the glutamine has been consumed. It is therefore not likely that these changes will influence the metabolic rate.

It is well established that pH has large effects on various parts of cellular metabolism (see, e.g., Mi- nakami and Yoshikawa, 1966; Chambard and Pouys- segur, 1986). The pH effects on lit, determined here are in agreement with pH-effects on the rate of DNA synthesis in cultured fibroblasts (Ober and Pardee, 1987). Specific conclusions regarding the pH-sensitiv- ity of cellular processes should, however, refer to intra- cellular pH-measurements since ApH over the cell membrane can be large and differs between cell lines (Ober and Pardee, 1987).

Although pH-effects are ubiquitous phenomena in cell biology, it is an interesting and surprising obser- vation that a reduction in medium pH is the only change in medium status which significantly influ- ences proliferation and overall metabolism during batch experiments extending over an entire growth cycle. From a methodological point of view this obser- vation could allow effects from addition of various agents (Borrebaeck and Schon, 1987; Schon and Wadso, 19881, to be separated from effects of medium “deteri- oration” over extended periods. Furthermore, the present observations suggest that results from experi- ments with, e.g., different initial cell concentrations should be compared on the basis of medium status (here reflected by Q and/or pH) and not on a time basis (Figs. 2 and 3A).

P-t curves similar to those obtained here have re- cently been observed for HeLa cells and human adeno- carcinoma gastric cells (SGc 7901; Dong et al., 19891, and Molt 4 leukemia cells (Nittinger et al., 1990) cultured under aerobic conditions. This indicates that

MICROCALORIMETRIC STUDY OF T-LYMPHOMA CELLS 103

the general conclusions reached in the present investi- gation can also be applied to other studies on animal cell lines in batch culture.

ACKNOWLEDGMENTS This work was supported by grants from The Swedish

Natural Science Research Council and The Research Council of the Swedish Board for Technical Develop- ment.

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