Mutation Research, 264 (1991) 183-186 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-7992/91/$03.50 ADONIS 016579929100103U

MUTLET 0550

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Growth of Chinese hamster ovary (CHO) cells in the presence of MMC in low-serum medium

Kamlesh Sharma, Dennis Milanowski and Gyula Ficsor

College of Arts and Sciences, Western Michigan University, Kalamazoo, All 49008 (U.S.A.)

(Received 2 July 1991) (Accepted 25 July 1991)

Keywords: CHO cells; MMC; Low-serum cultures

Summary

Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10070 fetal bovine serum (FBS) were adapted to 0.5070 FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10- 7 M and 10- s M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5070 serum proliferate, though at a slower rate than cells in 10070 serum. Treatment with MMC revealed that at 10-7 M, MMC is cytotoxic to cells in both the media; at 10-8 M, MMC is non-cytotoxic to cells in both media.

In vitro short term tests with mammalian cells are used to determine the genotoxicity of drugs and environmental chemicals. These tests are quick, easy and inexpensive. Mammalian cells proliferate in growth media only if they are supplemented with 5-20°70 serum. Serum is an undefined component. It is made up of some 500 biomolecules, many of which are not needed for cell growth while others are cytotoxic. Sera exhibit lot-to-lot variation, e.g., in 112 samples tested by Hyclone Laboratories, Inc., the progesterone range was 0.2-238 ng/dl

Correspondence: Dr. Kamlesh Sharma, Department of General Studies, College of Arts and Sciences, Western Michigan University, Kalamazoo, MI 49008 (U.S.A.), Tel. (616) 387-5390.

showing more than a 793-fold variation; the en- dotoxin range was 0.003-0.9 ng/dl, showing more than a 300-fold variation (Hyclone Laboratories, Inc., 1986). It is also found by Hyclone that there is a 20-fold variation in the 12 hormones that con- trol the growth and function of cultured cells. Kato and Sandberg (1977) showed that increasing the concentration of untreated serum in CHO cells in- creased the incidence of sister-chromatid exchanges (SCEs). Wiencke et al. (I 984) showed that different types of sera, e.g. fetal bovine serum, new-born calf serum, and autologous plasma affect the in- duced levels o f SCEs in human lymphocytes. It ap- pears that serum components interact with test chemicals to mask their true ability to induce genetic damage. Thus, reducing or eliminating

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serum should improve the definition of culture media and the consistency, sensitivity, and reliability of short term tests.

Commercial preparations of serum-free media are available (Chandler, 1990; Gorfein and Weiss, 1990). These serum-free media are supplemented with growth factors, attachment factors, hor- mones, and minerals to replace components essen- tial for cell growth (Barnes and Sato, 1980).

In the present study our objective was to lower the serum level significantly in one of the commer- cially available serum-free media and to test the ability of these cells to withstand the stress of test chemicals. We used CHO-I Complete Media System from Ventrex. CHO cells grown in McCoy's 5a medium with 10070 serum, were adapted to 0.507o serum in CHO-1 complete Media System from Ventrex. As serum level was lowered, cells began to exhibit difficulty in attaching firmly to the substratum. They began to form grape-like clumps. After careful adaptation, we were able to bring this serum level down to 0.507o.

These cells were cryopreserved and revived suc- cessfully. We observed that at lower than 0.507o (at 0.25% and 007o), continuous sub-culturing pro- duced cells with clumps. Therefore, we compared growth efficacy of cells in 10070 serum with cells in 0.5070 serum. We also exposed these cells to MMC, a known mutagen. The results show that cells even at 0.5070 serum can withstand the stress of the genotoxic agent MMC. We found that in both types of media, MMC is cytotoxic to cells at 10- 7 M concentration, while at 10-8 M genotoxic con- centration MMC does not affect the growth of cells. Thus cells adapted to very low level serum do go through cell replication and can be used for in vitro short-term tests to determine the genotoxicity of test agents.

Materials and methods

Culture media and conditions Chinese hamster ovary ceils (CHO-WBLT) were

obtained from the Environmental Health Research and Testing (EHRT) laboratories in Lexington, Kentucky. The cells were cultured in T-75 Corning

flasks in 10 ml of McCoy's 5a Medium (Gibco), supplemented with 10070 fetal bovine serum (Hyclone), 2 mM z-glutamine (Gibco), and an- tibiotics (penicillin and streptomycin). The ceils were subcultured 2 × a week. The flasks were kept in 5070 CO2 incubator at 37°C.

The cells were then weaned gradually from 1007o FBS in CHO-1 Complete Media System (obtained from Ventrex Laboratories, Inc.). The CHO-1 Complete Media System was supplemented with 2 mM of L-glutamine, and antibiotics. The serum level was gradually reduced, from 10070 to 5070, 2.5070, 2070, 1070 and finally 0.5070. Cells in 0.5070 were cryopreserved and revived successfully.

Cell harvesting Just before confluency, the medium was discard-

ed and cells washed with 2 ml Hanks' balanced salt solution (without Ca or Mg). The cells were dislodged with 0.5 ml trypsin (0.25°7o for serum- supplemented and 0.02507o for the low-serum cells) in HBS without Ca or Mg. Cells were suspended in 4.5 ml of the appropriate medium and counted on a Coulter Counter. An inoculum of 2-5 × 105 for the 10070 serum and 7-10x105 for the 0.5070 medium was seeded in T-75 flasks and placed in 5070 CO2 incubator at 37°C.

Drug treatment Cultures were set up in duplicates in T-25 flasks

to be counted every 24 h for l0 days. After the first 24 h of incubation, ceils were treated with 10- 7 M and 10- g M MMC (initial dilutions were made in HBS but the final dilutions were made in the ap- propriate media to be added in 0.5-ml portions to each flask). After 24 h of treatment with MMC, the medium was discarded, and the cells washed 2 × with 2-ml aliquots of HBS. 10 ml of the ap- propriate medium was added to each flask and the cultures were replaced in the incubator. Two flasks were removed every 24 h, and the number of cells in each flask counted on a Coulter Counter.

Results

For each type of medium, four variables were

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tested: (i) control, (ii) solvent control, (iii) treated with 10 -7 M MMC, and (iv) treated with 10 -8 M MMC (see Figs. 1 and 2). The control group (i) shows that cells in both types of medium plateaued around the 5th day after culture initiation. Cells in 10070 serum, however, went through almost 5 doublings whereas cells in 0.507o serum went through almost 3 doublings. Ceils in the control group (ii) showed that simply adding the ap- propriate medium and then washing cells slows down cell growth slightly - - but it does not inhibit cell growth. The treatment with 10 - 7 M MMC group (iii) showed that cells in 1007o serum maintain the same number (no proliferation) for about 2 days but then start to decrease in number. In 0.5% serum, cells were observed to be affected im- mediately as they start to decline within 24 h. Cells treated with 10-8 M MMC group (iv) showed no effect of MMC on cell growth. These cells behaved quite similarly to the solvent controls (ii). The

8O

60

40

~ 2O

I) . , - , • , 0 2 4 6 8 10 12

Days

Fig. 2. Growth of CHO cells in CHO-I Complete Media System

from Ventre×, supplemented with 0.50/0 serum. Legend and ex-

perimental conditions as in Fig. 1.

200

2~ 1oo

E

z

, . i

2 4 6 8 10 12

Days

Fig. 1. Growth of CHO cells in McCey's 5a medium sup-

plemented with 10% serum (O) controls; ( ' ) solvent controls; (A) treated with l0 -7 M MMC; (Lx) treated with 10 - s M

MMC. Duplicate cultures were set up and incubated for 24 h. Cells were then treated with the solvent and MMC for 24 h.

Total number of cells in each flask were counted every day for

10 days. There is no difference in cell growth of controls and sol-

vent controls. At 10 -7 M MMC, all growth is completely in- hibited. At 10-8 M MMC cell growth is almost normal.

results indicate that MMC is non-cytotoxic at 10- s M to cells in media with 10070 serum and 0.5°7o serum. This shows that cells in 0.5070 serum con- tinue to proliferate, a necessary condition to deter- mine genotoxic damage.

Discussion

Reducing or eliminating serum should make in vitro short-term tests more sensitive, reproducible, and reliable. Chinese hamster ovary cells do pro- liferate in serum-free media. We wanted to know whether treating them with test agents affected their normal growth. We found that low-serum cultures went through cell division even in the presence of test chemicals. It is hoped that this would lead to an improved in vitro test system for determining chromosomal damage. As serum-free media continue to improve, it may become possible to eliminate serum completely.

Acknowledgement

This work was supported by a Fellowship and

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Grant from the Faculty Research and Creative Ac- tivities Support Fund, Western Michigan Univer- sity.

References

Barnes, David, and Gordon Sato (1980) Methods of growth of cultured cells in serum-free medium, Anal. Biochem., 102, 255-270.

Chandler, Joseph P. (1990) Cultivation of mammalian cells in serum-free medium, Am. Biotechnol. Lab., 18-28.

Gorfien, Stephen F., and Stefan A. Weiss (1990) A new serum- free medium for growth of Chinese hamster ovary (CHO) cells in suspension, Focus, 12, No. 3, 75-76.

Hyclone Laboratories, Inc. 0986) Fetal bovine serum: varia- tions in components that influence cell growth and function, Art to Science in Tissue Culture, 5, 3-4.

Jayme, D.W., D.A. Epstein and D.R. Conrad (1988) Fetal bovine serum alternatives, Nature (London), 344, 547-548.

Kato, H., and A.A. Sandberg (1977) The effects of sera on sister chromatid exchanges in vitro, Exp. Cell Res., 109, 445-448.

Maurer, H.R. (1986) Towards chemically-defined, serum-free media for mammalian cell culture, in: R.I. Freshney (Ed.), Animal Cell Culture: a Practical Approach, IRL Press, Washington.

Nardone, Roland M. 0987) Cell culture methodology from donor to cell line, Biotechniques, 5, 122-127.

Wiencke, J.K., K. Kelsey, R. Krieger and V.F. Garry (1984) Serum- and plasma-dependent variations of benzo[a]pyrene- induced sister chromatid exchange in human lymphocytes, in: R.R. Tice and A. Hollaender (Eds.), Basic Life Sciences, Plenum, New York.

Communicated by J.M. Gentile