mammalian cells in chemically defined media in suspension cultures

MAMMALIAN CELLS IN CHEMICALLY DEFINED MEDIA IN SUSPENSION CULTURES*

Jay C. Bryant Tissue Culture Section, Laboratory of Biology, National Cancer Institute,

National Institutes of Health, Berhesda, M d .

INTRODUCTION

This paper is concerned with the application and development of batch-type suspension culture methods to the cultivation of mammalian cells. It deals primarily with research in our tissue culture laboratory. Major emphasis is on certain biophysical or biochemical parameters, especially chemically defined media, with less emphasis on the instrumentation involved in controlling the in vitro environment.

For the defenseless mammalian cell no longer in its normal milieu, the strictly axenic culture is the only kind ordinarily possible, in marked contrast to cultures of some free-living microorganisms, which can tolerate at least limited competi- tion. When the mammalian cell culture is no longer axenic the cells are either dead or no longer present. An exception to this generalization is the troublesome PPLO (Mycoplasma) , which may coexist in the same culture with viable mammalian cells.

Early Suspension Culture Experiments with Serum-Containing Media

Such cultures of clone 929-L cells in our laboratory were first grown success- fully in 1953.l This was shortly after Owens, Gey, and Gey2 first showed that mammalian cells would grow in “tumbling tube” cultures using a strain of mouse lymphoblast cells. We used modified roller tubes rotated in different drums at various speeds up to 40 rpm. Cultures at the higher rotation rates gave higher growth rates, validating our speculation that animal cells from fixed tissue would proliferate in suspension in rapidly rotated roller tubes.

We extended these studies to fluid suspension cultures in stoppered flasks on a rotary shaker enclosed in an incubator. In 1955 we went further toward controlling the environment: (1) A special two-necked shaker flask modified from a 1.5-liter Florence flask provided for simultaneous entrance and exit of sterile gas over the fluid suspension; (2) A multiple peristaltic pumping mechanism metered a continuous supply of humidified gas, e.g. 5 per cent COz in air, to each culture, usually at a rate of about 140 ml./hr. The fluid was changed completely every two to four days; the entire suspension was pipetted into centrifuge tubes and centrifuged at about 250g, the supernatant was pipetted off, and the cells were resuspended in fresh fluid for replacement in shaker flask.

With this system we carried out an extensive series of ~ t u d i e s ~ - ~ with serum- containing media and characterized four established mammalian cell strains with respect to growth curves, generation times during the logarithmic phase, and glucose utilization. Maximum cell populations of 1 billion to 2.3 billion cells in one 400-ml. culture in a 1.5 liter flask (2.5 to 5.75 million cells per ml.) were reached in many cultures of each strain.

* The major part of this research constituted a thesis submitted by the author to the Faculty of the Graduate School of Georgetown Univkrsity, Washington, D. C., in partial fulfillment of the requirements for the degree of Doctor of Philosophy, February, 1963.

143

144 Annals New York Academy of Sciences

During the years following this pioneer work,6 many other laboratories applied and extended the agitated fluid suspension culture method to mammalian cells. Comprehensive reviews of developments up to 1962 are a ~ a i l a b l e . ~ . ~

+ 300 z 2 E: I00 Yi\iV\\K\\\\\ d SO',

1 3 I E 0 * ' I ' ' I l ' , '

Potentials of Shaker Cultures with Serum-Containing Media

Kuchler and M e r ~ h a n t , ~ Siminovitch et al.,1° and Takaoka," as well as ourselves,12 found that with agitated fluid suspension cultures of mammalian cells in serum-containing media, the logarithmic phase of the cells in the culture could be prolonged indefinitely. Since that time other investigators have confirmed this finding. This procedure can provide large numbers of cells for biochemical analysis at frequent intervals. Some pertinent ex- amples may be cited of our experiments in 1956 and 1957 with large shaker cultures of mouse cells, all in our medium NCTC 109, supplemented with 20 per cent horse serum. In one case13 we carried cells of our mouse fibroblast clone 929-L in two pairs of replicate 1.5-liter flasks through nine harvesting cycles. For one pair, the glucose concentration was 1.0 g. / l . , and for the other pair, shown in FIGURE 1 , it was 2.6 g . / l . The cultures were gassed continuously with

Yields from Frequent Harvests.

TISSUE CULTURE GENERATION c C l ~ 2 - - . C - - 3 ~ 4 4 5 - + - 6 + 7 + 8 - b - 9 4

TOTAL POPULATION PER CULTURE

z 0 i 200

- 100 W _1 AVERAGE POPULATION PER M L FLUID

L t W m 1 0

2 06

04 VOLUME OF FLUID PER CULTURE

g500 I , I I w 400 c_

k 300 - - - =I 200 100 ---

5 0 / / I ' I N I " ' ' , I I

Bryant: Mammalian Cells 145

10 per cent COr in air. FIGURE 1 shows cell growth, fluid volumes, and glucose levels during 38 days in each of the cultures. The logarithmic phase was maintained by harvesting part of the cells and subculturing the rest before that phase ended. Average population increase per cycle was from 240 X lo6 to 550 X lo6 per culture, with an average generation time of 83 hours. Harvest yield averaged 56 per cent. Slightly lower population values were obtained from the pair of strain 929-L cultures receiving glucose at 1.0 gJI. in the fresh fluid. Average rates of glucose utilization by the cells in the two cultures receiving the lower amount of glucose were only about half the rates of glucose utilization in the cultures receiving the higher amount of glucose. This clearly showed the de- pendence of glucose use on the concentration of substrate.

Our second example of the efficacy of frequent harvests with complete fluid renewal (experiment 2631) was with cells of mouse liver clone strain 1469;12 experimental conditions were identical with those for the first example. Popula- tion levels were higher, but otherwise the results were similar to those with strain 929-L cells except for a prolonged initial lag phase of about ten days. After day 1 1 , the cultures were carried for 38 more days through ten cycles, most of which

TOTAL POPULATION 4oM) , I t I , , , , , , ( , , , , , , ,

2000 2769

3000

- 800 600 :EEv 200 ' /

1 I L L L L i I l i l l l l

A V E R A G E POPULATION P E R ML FLUID l!!z/ !FA GLUCOSE - i l i l i l L CONCENTRATION ""i I N FLUID

2

I

V O L U M E OF FLUID

I00

look - i

5: 2 5 6 8 10 12 14 16 18 20

TIME I N DAYS

FIGURE 2. Experiment 2769: Mouse fibroblast clone strain 929-L cells in one culture in a 1.5-liter flask with 2.6 g./l. glucose in medium NCTC 109 and 20 per cent horse serum The objective was maximum population by means of a complete fluid change every 24 hours after day 5 . Total population of each culture and average population per ml. are plotted on the log 10 scale, while volume of Auid and glucose concentration during each fluid change interval are plotted on a linear scale.


were three or four days in length. Average population increase was from 228 X lo6 to 745 X lo6 per culture, with an average harvest yield of 71 per cent per cycle; average generation time was 53 hours. Again, glucose utilization seemed to depend directly on glucose concentration in the fresh medium.

Our third example was also with mouse liver cells of strain 1469.13 This experiment was longer, but otherwise it was carried out under identical conditions. We carried several replicate 400 ml. cultures through 39 cycles during 200 days. Cell concentrations were higher than in previous experiments; the average increase was from 480 X lo6 to 1.8 X lo9 cells per culture during each cycle. The harvest yield from each cycle averaged 73 per cent. The maximum population in one culture was 3.84 X lo9 cells, or 9.6 X lo6 cells per ml.

We concluded that frequent subculturing and harvesting with complete replacement of fluid at three to four day intervals was a feasible means of produc- ing large numbers of cells over extended periods of time.

Maximum Population Levels. We also examined the potentialities of our 400 TOTAL POPULATION

20,000 [ - T ! I I , 1 1 1 1 1 1 l l l i l l l 1 l l l i l ' T f l l ~

W

3 AVERAGE POPULATION PER M L F L U I D = 100

.^ a" tr

; I O O L

L E , / I (

0

400 - . * ? 1

T I M E I N DAYS

FIGURE 3. Experiment 2757: Mouse liver clone strain 1469 cells in one culture in a 1.5-liter flask with 2.6 g./l. to 3.8 g./L glucose in medium NCTC 109 and 20 per cent horse serum. The objective was maximum population by means of a complete fluid change every 24 hours after day 7. Total population of each culture and average population per ml. are plotted on the log 10 scale, while volume of fluid and glucose concentration during each fluid change interval are plotted on a linear scale. This chart was presented originally in 1958 by Earle.I2


ml. shaker cultures for maximum growth without harvesting. Our concept was that more frequent fluid changes, e. g., one every 24 hours instead of every 48, 72, or 96 hours, if started soon after the culture had begun logarithmic growth, should remove metabolic by-products from the cell milieu before possible accumulation to inhibitory levels, and should maintain concentrations of glucose and other critical compounds above limiting levels. In the same medium, NCTC 109 supplemented' with 20 per cent horse serum and containing glucose at 2.6 gm./liter, one culture of clone 929-L cells (experiment 2769, FIGURE 213) increased steadily from an inoculum of 1.3 million per ml. in 200 ml. to 7.5 million per ml. in 400 ml. in 18 days; that is, from 260 X 106 to 3 X 109 cells with an estimated wet weight of 12 gm. Fluid changes were made daily after day five.

In another culture with mouse liver cells (experiment 2757, FIGURE 3l2), total population increased 50-fold during 25 days from an inoculum of 230 X lo6 in 180 ml. to 11.6 X lo9 cells in 400 ml., i.e. cell concentrations increased from 1.5 X lo6 to 29 X lo6 per ml. Glucose concentrations did not fall to exhaustion levels until day 22, near population maximum.

We concluded that the 24-hour fluid change was highly effective, and the population levels were much higher than had been reached in any laboratory. Also, according to the recent summary of maximum animal cell populations in suspension cultures by Perlman and Giuffre,B this concentration of 29 million per ml. has not been approached since that time. While the procedure as it stands is probably not feasible for extrapolation to larger scale production of cells because of its time and labor requirements, these experiments clearly indicate the possibility of growing much greater concentrations of mammalian cells in suspension cultures than are usually grown.

Chemically Defined Media and Suspension Cultures In our laboratory we have devoted much time and effort to the develapment

and study of chemically defined media. Our most generally useful protein-free medium, formerly designated NCTC 109 and more recently (without cysteine) known as NCTC 135, contains a total of 68 chemical corn pound^.^*-^^ These include 26 amino acids, 18 vitamins, six coenzymes, five nucleic acid derivatives, six inorganic salts, d-glucose, and six other compounds. We have made available to the cells the maximum array of compounds which the cells may either need of find useful, rather than the minimum. In stationary (monolayer) cultures we have grown nearly all of our established cell strains in media NCTC 109 or 135; the first of these strains, 2071-L, has grown steadily since January 1955. We have also grown many strains in several less complex NCTC media."

Our success with cells in chemically defined media in stationary cultures soon led us to try the same strains of cells in shaker cultures. We found that whenever serum was not included with medium 109, the cells in shaker cultures disinte- grated entirely within two or three days, with no exceptions. It appeared that disintegration occurred because of physicochemical factors rather than nutritional deficiencies. During this time Merchant and Kahnls reported that they had succeeded in growing mammalian cells in shaker cultures after elimination. of serum from otherwise chemically defined media. However, their medium, Mor- gan's 199P,19 contained a supplement of 0.5 per cent peptone. In a later study Kuchler, Marlowe and Merchantz0 included Methocel 15 cps. with medium 199P at 0.12 per cent as well as peptone at 0.5 per cent. We deliberately omitted their peptone from our cultures because it would be no less undefined and chemically heterogeneous than is serum.


When we began to include Methocel with our chemically defined medium for shaker cultures, our concept was that the protective effect of the large protein molecules of serum in shaker culture medium is physical more than nutritional. It would follow that the large molecules of some chemically characterized, non- toxic, nutritionally inert polymer such as methylcellulose might also exert a primarily physical protective effect on the suspended cells.

MATERIALS AND METHODS

Culture flasks. The modified 500 ml. and 250 ml. flat bottom boiling flasks used were smaller than those ( 1.5 liters) that had been used for our experiments with serum containing media described above and had one neck instead of two. FIGURE 4, from an earlier paper of our laboratory,21 shows a 500 ml. flask and a 1.5-liter flask with stoppers and cotton traps in place. Direct centrifugation of the culture flask and contents in a modified swinging basket centrifuge head (IEC #925A in an IEC #2 centrifuge) thus became possible. The cells could then be left in the culture flask during each fluid change at a considerable saving of time and labor.

Culture methods. For experiments with only two or three cultures, equal volumes of pooled suspension from stationary cultures were dispensed directly into each flask by careful pipetting. For replicated experiments with 12 to 24 cultures, the pooled cell suspension was dispensed into each flask in aliquots to

FIGURE 4. Two kinds of shaker flasks for suspension cultures. The 500 ml. modified short necked flat-bottom boiling flask on the left was used for most of our experiments with Methocel-supplemented medium. The two-hole stopper with two cotton traps for simultaneous inflow and outflow of gas is in place: a solid stopper replaces it at each fluid change when the flask is centrifuged. The 1.5-liter modified two-necked boiling flask on the right with separate one-hole stoppers and cotton traps, had been used for previous experiments with serum- supplemented medium. This flask is too large for direct centrifugation.


FIGURE 5. Solenoid valve gas metering device for delivering gas to each culture at controlled rates over a wide range of values. Gas from the tank and the usual reducing valves passes through the final pressure reducer E (usually set at 1.5 psi) into the large manifold D (1 inch 0. D.). Ten solenoid valves control the gas flow from manifold D through separate tubes leading to outlets controlled secondarily by hand valves F. The solenoid valves are activated by a spring switch B that rides the edge of the two-layered adjustable notched wheel C , that is rotated by a motor through reducing gears at 1 rpm. The switch opens the solenoid valves only when it rides in the notch; the length of the notch can be adjusted, by changing the relative positions of the two component wheels on the shaft, to cause gas to flow during intervals of 0.6 to 30 seconds of each minute, i.e., from 1 to 50 per cent of the time. A rubber tube on each outlet carried gas through a coarse porcelain filter immersed in dilute acid solution (0.5 per cent H2S04) in a tall humidifying tube (not shown here), thence through a rubber tube into the culture flask and then out through the cotton traps (FIGURE 4) .

insure uniform inocula by the method of Evans et dZ2 Complete fluid changes were carried out twice weekly. Antibiotics were not used routinely in our media. Complete reliance was placed upon careful and consistent use of sterile glass- ware and culture media with unfailingly sterile techniques. Procedures were always carried out in sterile rooms, air-conditioned through absolute filters. Re- peated checks have shown that all of our cell strains are free from the insidious mycoplasmas (PPLO) .

Methocel as a Supplement. Methocel (Dow Chemical Co., Midland, Michi- gan) is a methycellulose polymer consisting of repeating glucose units joined in beta-1,4 glucosidic linkage. An aqueous Methocel solution is almost wholly non- ionic and is compatible with saline solutions of physiological concentration, It is too viscous to be filtered, so a 1OX concentrated aqueous Methocel solution is autoclaved, then diluted sterilely with appropriate volumes of both single and double salines and 2.5 X concentrated medium NCTC 109.17,23

Silicone-coated Culture Flasks. Dow-Corning liquid silicone (DC-200) was prepared as a 5 or 10 per cent solution in reagent benzene; the flask was flushed with this solution. The flask was then drained, dried, baked, rinsed and re-dried


in a certain time sequence.23 This hydrophobic coat appeared to reduce cell ad- hesion. Only 500 ml., 250 ml., or 125 ml. flasks were siliconized.

pH Control. Buffering action in our shaker cultures depends on the dominant bicarbonate system rather than phosphate buffers. Stabilization of the pH within physiological limits requires a high level of COz in the air over the culture fluid. Phenol red was the indicator at 0.002 per cent concentration. A constant metered flow over the cell suspension of either air or an O2-Nz mixture containing either 5 or 10 per cent COz was provided to stabilize pH and to lessen the possibility that oxygen might be a limiting factor.

Ga5 Metering Systern. The multiple peristaltic pumping mechanism3 that had metered gas satisfactorily for all of our earlier 1.5-liter shaker flasks had two shortcomings: (1) the section of rubber tubing that was alternately com- pressed and released by the circumferential wheels tended to wear and fail unpredictably, and (2) since the mechanism was fixed to deliver 140 ml. gas per hour through each line, rates of delivery were limited to that value or to multiples of it. In 1960 we replaced that mechanism with the solenoid valve gas-metering device shown in FIGURE 5; it controlled gas flow over a wide range of rates during one to 50 per cent of the elapsed time.

Cell Viability. Trypan blue was used at certain times in a dye-exclusion methodz4 to estimate the proportion of viable cells in a culture.

Cell Enumeration. Cell numbers were estimated at each fluid change by the nuclei count procedure25 modified by Bryant et aL4 for cells in suspension cultures, using either 0.5 or 1.0 ml. aliquot samples.

Relative Viscosity. Ostwald-type viscometers26 were used at 37.5 "C to determine the relative viscosity of various culture media. Relative viscosity is the ratio between seconds of outflow time of the test solution and seconds of outflow time of water.

Glucose. Glucose was estimated at each fluid change in sterile samples of the cell-free supernatant from centrifugation4 in most cases by the Nelson-Somogyi copper reduction method.27 All shaker experiments were monitored for glucose.

EXPERIMENTAL

Cells in Shaker Cultures with Medium NCTC 109

Mouse Fibroblast Cells. In preliminary experimentsz3 strain 207 1-L mouse fibroblast c e l W grew well in shaker cultures, as stated above. These results were confirmed in many experiments with replicated shaker culture flasks of different sizes and with different low-viscosity types of Methocel 10, 15, 25, and SO centi- poises (cps.) at either of two low concentrations of Methocel, 0.06 and 0.12 per cent. The cells proliferated at steady rates in the logarithmic phase, average generation times ranging from 2.7 to 9.5 days and with maximum populations of 1.1 X lo6 cells per ml.

Human Skin Epithelial Cells. A human strain, also already growing in medium 109 in stationary c u l t ~ r e s ~ ~ ~ ~ ~ was then tried out in Methocel-supplemented medium 109 in shaker cultures. Cells proliferated slowly but steadily through a four-week logarithmic phase and then leveled off into a long stationary phase. These skin cells grew at a slower rate and with considerably more clumping than did strain 2071-L cells.

Monkey Kidney Cells. We then applied our method to Rhesus monkey kidney cell strain LLC-MK2 originated by Hull, Cherry and Johnson.28 This strain NCTC 3526 had been growing in medium 109 since August 1959 in stationary

TASLE 1 SHAKER FLASK EXPERIMENTS WIm VARIOUS METHOCELS

Fluid volume Number of cells (x 106) mask Duration Extent of

Experiment Methocel

Per size Start Final (days) Inoculum growth‘ Type cent (ml.) (ml.) (d.) (x) Maximum Minimum End No. code (CPS.)

Lab

1 3556

2 3601b

3 3617

4 3626

5 3635c

6 3663

7 3696

25 1.65 4000 0.45

50 0.87 1500 0.39

25 1.65 50 0.87

1500 0.39 4000 0.45

25 0.825 50 0.435

1500 0.195 4000 0.225

25 0.825 50 0.435

25 0.825 50 0.413

10 1.02 50 0.435

1500 1500

1500 1500

500 500 500 500

500 500 500 500

500 500

500 500

500 500

95 95

100 100

70 70 70 70

70 70 70 70

70 70

70 70

60 60

75 75

55 55

70 70 70 70

70 70 70 70

130 180

110 110

180 180

81 27.8 84 24.2

41 25.8 41 20.9

24 31.1 24 39.8 24 34.6 21 36.2

29 49.0 29 29.5 10 33.4 22 35.5

24 67.3 35 69.4

23 22.1 23 29.3

24 40.1 24 47.7

79.6 16.7 75.0 3.0~ 65.7 17.1 38.7 2.7

40.4 18.2 35.2 >X 43.2 20.9 34.4 >x 34.8 18.9 26.6 <X 45.4 24.8 32.3 <x 57.7 34.6 51.7 1.7 42.0 35.6 35.9 =X

67.3 32.8 67.3 1.4 69.4 27.2 69.4 2.4 48.4 27.5 48.4 1.4 61.8 35.5 58.6 1.7

89.9 44.9 89.9 1.3 146.4 54.0 56.2 21

60.0 22.1 39.2 2.7 60.7 29.3 48.2 2.0

179.3 40.1 179.3 4.5 179.3 47.7 179.3 3.7

a. Growth: = x, no change from inoculum size; > X, slight increase; < x , slight decrease;

3.0 x , increase to 3 times inoculum size. b. 3601: cultures transferred on day 13 to 250 ml. flasks. c. 3635: each culture was continued from the respective 25 cps. and 50 cps. cultures of experiment 3617.


~u1tures.l~ Unexpected difficulties arose, but the potential importance of this MK strain impelled us to carry out an extensive series of experiments to resolve these problems.

In single shaker cultures the MK cells remained essentially non-proliferative but apparently healthy for periods up to four months. In one group of experiments involving several transfers to fresh flasks during 124 days, the cells generally failed to proliferate but would grow rapidly when they were returned to stationary T-60 flasks in medium 109 without Methocel.

Methocel Concentration and Viscosity Grade

TABLE 1 summarizes experimental conditions and MK cell population in seven experiments designed to study the effect of concentration and viscosity of Metho- cel. In the first, the cells in two cultures in 1.5-liter shaker flasks proliferated irreg- ularly and slowly for more than 80 days, with maxima at 3 X inoculum size. In experiments 2 and 3, cell populations remained essentially stationary from three to six weeks. The Methocel concentrations for each viscosity grade had been selected from Methocel viscosity-concentration graphs so as to impart similar relative viscosity to all the media in these experiments, i. e. the higher viscosity grades were used at the lower concentrations. The resultant fluid media in experiments 1 through 3 were too viscous for accurate pipetting, so in experi-

"_ VOCUME OF FLUID IN EACH CULTURE

c. - __r_

$+..->\>, 1 k./.\\.\ 1 0 3 7 10 14 17 El 23 C 3 7 10 14 I7 21 24

TIME IN DAYS

FIGURE 6. Experiments 3663 and 3696 from TABLE 1: Population and glucose curves for two shaker cultures in each experiment, with monkey kidney cells (NCTC 3526) in Methocel- supplemented medium NCTC 109. The earlier experiment 3663 showed only a short-lived growth period, while the later experiment 3696 showed continuous slow logarithmic growth.


ments 4 to 7, all the Methocel concentrations were reduced by exactly one-half to intermediate concentrations.

The experimental pattern for experiment 4 was otherwise identical to that for experiment 3; during four weeks or less, cell numbers increased very slowly but consistently to levels 1.4 X to 2.4 X inoculum. Further study with intermediate concentrations of Methocel was indicated. In experiments 5 and 6, conditioned (used) fluid was mixed with fresh medium with glucose concentrations set at 1.5 gm./liter. Again very limited proliferation occurred; no stimulatory effect of conditioned medium was evident.

In experiment 7 two cultures in 500 ml. flasks yielded, during 24 days, the highest proliferation we had obtained to that time from shaker cultures of monkey kidney cells. The 10 cps. Methocel culture from the lower inoculum yielded 4.5X inoculum level and the 50 cps. Methocel culture yielded 3.7X inoculum level. The graph in FIGURE 6 for experiments 6 and 7 (3663 and 3696) presents total cell numbers, cell numbers per ml., fluid volumes, and glucose concentration for each fluid change interval. The short-lived proliferative period in experiment 6 on the left contrasts with the consistently sustained growth in experiment 7 on the right, where each culture reached populations of 180 million in 180 ml. fluid. While the population of these MK cultures was similar to strain 2071-L populations in some of our earlier shaker cultures,23 and while the cells proliferated steadily, the growth rate of 11 days mean generation time was very low. The lowest viscosity grade of Methocel, 10 cps. at the rather high concentration of 1.02 per cent, protected these cells even more effectively than any heavier grade listed in this table. The relative viscosity of that medium was found to be 3.17, but nonetheless the solution could be pipetted without much difficulty. We concluded that for further experiments with MK cells in medium 109, we would use 10 cps. Methocel at low concentration, e.g., 0.12 per cent used in earlier studies with strain 2071-L cells. Glucose in both fresh and used fluids was monitored in these experiments; these results will be presented el~ewhere.2~

Replicated Cultures in Small Shaker Flasks

We next explored the possibility that slower rates of circumferential flow of fluid in the shaker flasks might lessen the stresses on the suspended cells. We did this by using a smaller flask (125 ml.) rather than by decreasing the oscilla- tion rate of the platform ( 145 rpm.) . Smaller flasks would allow the replication necessary for adequate statistical evaluation, i.e., three or four cultures for each variable. Two 24-culture experiments (3744 and 3808) were carried out with MK cells in silicone-coated 125 ml. flasks. Each culture was maintained at 30 ml. volume throughout each experiment with a complete fluid change twice weekly. The cultures were planted with uniform inocula from a stirring funnel for exact replication of cultures,22 in contrast to our previous shaker culture experiments in which the culture was planted by direct pipetting from a pooled suspension.

Experiment 3744. This experiment was designed to answer three questions: ( 1 ) Must Methocel be included with medium 109 when the cells in suspension are under lower stress? (2) Is continuous aeration necessary? (3) What is the influence of inoculum size? We chose the lightest viscosity grade of Methocel, 10 cps. at concentration 0.12 per cent, giving to the resulting medium a relative viscosity of about 1.2. To test the effect of Methocel, half of the cultures received medium 109 with Methocel and half received medium 109 only. To test the

154

E. AERATED-HIGH INOC G NOT AERATED-HIGH INOC. 50 - -

J-? -.+*

- -

2 -

I ’

-

F AERATED-LOW INOC H NOT AERATED-LOW INOC. - 50 -

FIGURE 7. Experiment 3744: Growth of monkey kidney cells during 28 days in Methocel- supplemented medium NCTC 109 in 12 replicated 125 ml. shaker flasks from two inoculum levels and in the presence or the absence of metered aeration. Each curve averages the cell population of three replicate cultures in one group.

effect of aeration, half of the cultures were aerated continuously, as usual, with 5 per cent COz in air, while half were gassed with the same mixture for 20 seconds at each fluid change and then stoppered tightly. To test the effect of inoculum size, half of the cultures were planted with 200,000 cells per ml. and the other half with 67,000.

Cells in the 12 cultures receiving medium 109 without Methocel disinte- grated within a few days, confirming earlier experiments. The graph in FIGURE 7 therefore represents only the 12 cultures in groups E, F, G, and H receiving medium 109 with Methocel. The population curve in each of the four boxes is the average of the three replicate cultures in each group. The cells proliferated well in all cultures, from inoculum levels of either 2 or 6 X 106 to maxima of 37 to 53 X lo6 on day 24 or 28; the increases over inoculum ranged from 7 X to 20 X . The lag phase was most sharply defined in group H, not aerated and low inoculum. In each box a straight dashed line connects the populations on day 0 with those on day 14, when the better defined part of the logarithmic phase ended, although all cultures continued to increase considerably after day 14.

An analysis of variance based on individual culture populations showed that residual errors, i.e. variations between cultures within groups, were not significant. In contrast, the effect of aeration was significant: Continuously aerated cultures of Groups E and F increased to higher Ievels on day 14 than did the non-aerated cultures of Groups G and H. Cultures in Group E reached the maximum of 53 X 106.

Differences due to inoculum size were highly significant. Growth rates were much greater when the inoculum was low. Mean generation times for low inoculum Groups F and H from days 0 to 14 (broken line) were 3.8 days, and from days 8 to 14, 1.7 days; both contrasted sharply with 6.4 days mean generation time from days 0 to 14 with high inoculum groups E and G . Actual propula-


tions during the entire 28 days, however, were much higher from the high inoculum groups than from the low inoculum.

We concluded that when only a limited number of shaker cultures could be maintained, both the higher inoculum and continuous aeration would be advan- tageous, i.e., the conditions of Group E. Day 14 would seem to have been the optimum day for harvesting, although this was not done until day 28.

Experiment 3808. Two variables were examined in this second replicated experiment with 24 cultures of MK cells: (1) the effect of aeration, as in experiment 3744, and (2) the effect of three different methods of removing cells from the flask walls into the suspension, mostly the cells that tended to collect and multiply in a horizontal circumferential ring at wavecrest height. The average growth curves for each group of four replicate cultures are shown in FIGURE 8. The first method, “no removal” (Groups A and B ) , we already were using routinely in shaker flask experiments; we shook the flask vigorously by hand just before each fluid change but we made no attempt between fluid changes to shake the cells loose. The second method, “hand shaking daily” (Groups C and D ) , included shaking each flask by hand, not only at fluid change but also once each day between fluid changes. The third method, “scraping at each fluid change,” refers to the sterile removal with a glass rod having an appropriately bent tip and a small piece of perforated cellophane; this method had appeared to be more efficient than the others.

Substantial growth characterized all groups of cultures in the logarithmic phase, which lasted at least to day 10 in all cultures and to day 14 in Groups A and D. From uniform inocula of 3.2 X lo6 cells per culture, the maximum populations ranged from 13 to 20 X lo6 per 30 ml. culture, i.e., 4.4X to 6.6X inoculum size. Analysis of variance of population data in this experiment showed again that variations between cultures within groups were not significant. Continuous aeration enhanced growth consistently and, at most points, signifi-

- -

1 I I I 1 I I I 1 I I I I I 1 I / L

3808 AVERAGE POPULATION PER CULTURE I00

60 A. AERATED-NO REMOVAL C. AERATED-HAND SHAKING DAILY E AERATED-SCRAPED AT FC

20

6

2

I

FIGURE 8. Experiment 3808: Growth of monkey kidney cells during 21 days in Methocel- supplemented medium NCTC 109 in replicated 125 ml. shaker flasks from one inoculum level in the presence or the absence of metered aeration, with three different methods for removing adherent cells from the walls of the flasks. Each curve averages the cell populations of four replicate cultures in one group.


cantly, i.e. the average generation time of aerated cultures in Groups A, C and E was 4.6 days while that for non-aerated Groups B, D and F was 5.2 days.

Different methods of removal of cells from the flask walls had no consistent effect.

Later Replicated Experiments with Monkey Kidney Cells

Subsequently we carried out two 12-culture experiments to test the effect on cell proliferation and glucose utilization of two new variables, COz concentration ( 5 and 10 per cent) and flask size (125, 250 and 500 ml.). The uniform cell inoculum concentration was 70,000 per ml. The population curves for the first experiment, 4014, are shown in FIGURE 9. The major result was unexpected,

4014 AVERAGE POPULATION PER CULTURE A. 5% CO,-125 YL FLASK C. 5% C0,-250 YL FLASK L. 5% C0,-500 YL FLASK 50

1 I I t t 1 +

C0,-125 YL FLASK l O . l O % C0,-250 YL FLASK F 10% C0,-500 WL FLASK t t i

3 7 10 14 17 21 3 7 10 14 17 21 3 7 10 14 17 21 TIME IN DAYS

FIGURE 9. Experiment 4014: Populations of monkey kidney cells during 21 days in Methocel-supplemented medium NCTC 109 in 12 replicated shaker flasks. Experimental variables were: (1) size of flasks-125, 250, and 500 ml.-and volumes of fluid-30, 60 and 110 ml., respectively; and (2) percentages of CO, in air used as the aerating gas. Each curve is the average of the cell populations of two replicate cultures in one group.

complete absence of significant proliferation during 2 1 days, regardless of treat- ment. The second definite result was the unusually high average rate of glucose utilization and lactic acid production, for which data will be presented in a later paper. Despite the absence of significant proliferation in this experiment, three consistent relationships appeared: ( I ) between concentration of COz in the aerating gas and level of cell populations in the larger flasks; ( 2 ) between concentration of COz in the aerating gas and pH of the culture; and (3) between size of flask and cell populations. The population curves of the larger cultures showed significantly higher cell numbers in cultures receiving 5 per cent COe than in cultures receiving 10 per cent COz. In 500 ml. flasks on days 10, 14, 17, and 21 the culture populations ( X lo6) on 5 per cent COz (Group E in FIGURE 9) and those on 10 per cent C o n (Group F) averaged 8.3 and 6.2, 7.8 and 4.0, 6.0 and 4.2, 7.8 and 5.0, respectively. Similar differences in favor of 5 per cent COz during the same time characterized the cultures in 250 ml. flasks (C versus D).


Colorimetric estimations of pH in experiment 401 4 immediately preceding each fluid change showed that the cultures on 5 per cent COP averaged approxi- mately 7.45, and those on 10 per cent COz averaged about pH 7.25.

The effect of flask size in experiment 4014 could be seen in terms of cell concentration per ml. fluid rather than total cell numbers. The overall averages of cell concentrations during the entire experiment for the four cultures in each of the flask size groups, 125 ml., 250 ml., and 500 ml., respectively were 63.6, 61.2 and 54.8 ( X lo3) cells per ml.; the difference in favor of either of the two smaller sizes was consistent throughout the experiment.

A second replicated experiment, 4040. identical in pattern but with a higher uniform inoculum of about 116 X lo3 per ml., yielded the same results; no proliferation, high glycolysis, and the same relationships among COz concentration, pH, cell population and flask size.

At the end of each of these experiments the cells from all cultures were pooled into a shaker flask. During several weeks the cells proliferated very slowly to 2.5 X inoculum level. When part of these cells were subcultured into T-60 flasks they proliferated at a moderate rate, with about 65 per cent viability, showing that growth potential had been retained both during the replicated experiment and for many weeks thereafter.

Relative Viscosity of Mediuni I09 with Various Supplements

To determine if the higher viscosity of media that included Methocel might be a factor in reducing stresses on cells in suspension cultures and in cushioning the cells against breakdown, we determined the relative viscosity (RV) of samples of medium 109 in various combinations with Methocel and with serum. RV's ranged from 1.03 for medium 109 to 9.71 for medium 109 supplemented with 25 cps. Methocel at high concentration, 1.65 per cent. When medium 109 was supplemented with 10 cps. Methocel at several different concentrations, the RV's were directly correlated with Methocel concentration; at the 0.12 per cent concentration used in our later experiments, the RV of 1.2 was comparable with the 1.14 value of medium 109 supplemented with 20 per cent serum. Also, the RV of Methocel-supplemented medium 109 decreased consistently and significantly during typical fluid change intervals. This suggests loss of some methycellulose from the fluid medium, either onto the walls of the cells or onto the walls of the flask. Metabolic degradation appears to be because reducing sugars found in Methocel-containing medium 109 have been no higher than those found in medium 109 alone. Further studies of the viscosity and the protective effect of Methocel-supplemental media are being carried out in our laboratory.31

DISCUSSION AND SUMMARY

An attempt was made in this study to identify some of the forces impinging on suspended cells in shaker cultures. Relative viscosity was directly related to concentration and grade of Methocel supplement, which in turn, however, were not related to cell proliferation. Size of flask was inversely related to cell proliferation, for both monkey kidney and strain 2071-L cells. Speed of rotation of shaker platform was constant for the monkey kidney cell study. The forces involved are probably considerable. Bungay and Wiggert,32 in a theoretical study of the motion of suspended microbial cells relative to turbulently agitated fluid culture medium, state that centrifugal forces, shear planes, and Bernoulli forces operating in such velocity fields appear to be much greater than in laminar flow.


The mechanism by which methylcellulose protects cells from these forces in chemically defined media is not understood. As a working hypothesis we have suggested that methylcellulose molecules tend to attach to the cell wall, with these results: (1 ) the cell wall might be strengthened physically against the breakdown which occurs in the absence of methylcellulose; (2) methylcellulose might also reduce the permeability of the cell wall and thus inhibit or prevent the leak- age of some diffusible essential materials from the cell. Both results would probably be of major significance.

Other mammalian cell strains from fixed tissue, in addition to the mouse, monkey and human strains reported here, have been grown more recently in Methocel-supplemented chemically defined media in suspension cultures. Other strains have been grown under these conditions in our laboratory and in other laboratories.

Merchant and Hellman in 1962 reported growth of their mouse strain L-M during six- or seven-day experiments in a modified Eagle’s medium with double concentrations of amino acids and vitamins, without serum, hydrolysate or peptone and with a Methocel supplement, 15 cps., at 0.12 per cent concentration.33 Their growth rates were similar to those we found for 207 1-L cells in a replicated experiment to be reported later.29 Kuchler (personal communication, 1965) is also growing strain L-M cells continuously in modified Eagle’s medium with no supplements except Methocel. Merchant and Eidam recently reported a study on the plateau phase of growth of cells of the L-M strain in a Methocel-supplemented “protein-free” medium in shaker cultures.34 However, since their culture medium, a modified Morgan’s medium 199,19 was supplemented with 0.5 per cent Bacto-peptone, it was not strictly a chemically defined medium.

Our study has been confirmed by the recent work of Nagle et al.35 and Brown and Nagle.36’They found that strains of fixed tissue cells from mouse, human, and cat sources would grow satisfactorily in nonaerated suspension cultures in 2-liter shaker flasks in their chemically defined medium when it was supplemented only with low viscosity Methocel at 0.1 per cent concentration. Their medium contained fewer chemicals than NCTC 109, but the amino acids and the vitamins were at much higher concentrations. Insulin had been included in their earlier medium for three additional strains35 but in later experimenW it was omitted. Their six cell strains include a monkey kidney strain, established in their laboratories, that grew in insulin-containing media and appears to be less fragile than our strain.

The anomalous behavior of our monkey kidney cells reported here leaves us with this conclusion: These relatively fragile cells in suspension cultures of Methocel-supplemented medium 109 could remain in a nonproliferative but viable state for many weeks or months, and then upon transfer to a stationary flask, could resume satisfactory growth. It appeared that during the long-term existence of this strain in chemically defined medium in stationary cultures,17 the cells, when transferred into shaker flasks at certain times, could proliferate and at other times could not. Slight differences in the physical stresses in shaker cultures appeared to dominate this situation. Viscosity of the fluid did not appear to be significant. Small flask size usually favored cell proliferation of both this strain and strain 2071-L. To our knowledge this cell strain has not been grown in other laboratories under the conditions of this study. In Hull’s laboratory, where the parent LLC-MK2 strain was originated,28 stirrer suspension cultures are carried with one per cent serum included with chemically defined medium (personal communication). Our strain derived from LLC-MK2 appears


to find the experimental conditions of the present study an extremely critical tension zone in which maintenance is possible but growth occurs unpredictably.

Our concept that the factors limiting growth of our monkey kidney cells in shaker cultures are primarily physical rather than nutritional has met with two different pieces of evidence in our laboratory, one conflicting and the other supporting: (1) Possible importance of a nutritional factor is favored by the fact that when Methocel-supplemented medium 109 still included cysteine, MK cells proIiferated well in replicated experiments 3744 and 3808, while when Methocel-supplemented medium 109 no longer contained cysteine (now designated as NCTC 135)17 MK cells failed to proliferate in replicated experiments 4014 and 4040. The reason for the omission of cysteine has been discussed by Sanford et ul.37 and by Bryant.I3 It appeared that possibly the absence of exog- enous cysteine in the culture medium might have been responsible for failure of the cells to proliferate. In no other experiments under any conditions in our laboratory has any indication been found that medium NCTC 135 is not fully as adequate for cells as medium NCTC 109 had been. (2) Probable unimpor- tance of a nutritional factor when compared with physical factors is supported by the following fact.I7 In stationary cultures in our laboratory this monkey kidney strain grows well in several simplified NCTC media 117, 126, 131, and 132, of which I17 no longer contains cysteine and the other three never did contain it. All coenzymes and three nucleic acid derivatives present in NCTC 109-135 have been omitted from all four media, sodium acetate from 117 and several vitamins from the other three media. Growth is always better in medium 135.

In shaker cultures we have used only medium 109 and not any of the simpler NCTC media except cysteine-less NCTC 109. Continuous aeration was always provided in shaker cultures, except in certain replicated experiments; in those, aerated cultures gave consistently better proliferation than nonaerated ones. It would appear, on balance, that the nutritional needs of our monkey kidney strain have been met in our experimental situations,

ACKNOWLEDGMENTS Grateful acknowledgment is made to Mr. Edward L. Schilling, Tissue Culture

Section, retired 1964, for his skill and experience in handling cells in vitro; he was my close colleague and always dependab!e coworker in carrying out the experimental work. For the early phases of this series of studies acknowledgment is made of the highly original concepts of Dr. Wilton R. Earle, formerly Chief of the Tissue Culture Section, deceased 1964, in our extension of the culture of mammalian cells from the stationary culture to the agitated fluid suspension culture.

The appropriate analysis of variance was selected and applied to the loga- rithms of the cell numbers estimated in each culture at planting and each fluid change thereafter, by Miss Joan Gurian, formerly a member of the staff of the Biometrics Branch, National Cancer Institute, NIH, and presently Mathematical Statistician, Biometrics Research Branch, National Heart Institute, NIH. This careful work is gratefully acknowledged.

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