Serum supplements for the propagation of HeLa-229 and McCoy cells

Clinical Microbiology Newsletter m

January 15 1983

Vol. 5, No. 2

Serum Supplements for the Propagation of HeLa-229 and McCoy Cells Allan L. Truant, Ph.D. and Richard E. Hepler, B.S. Clinical Microbiology Division Clinical Laboratories Department of Pathology The University of Texas Medical Branch Galveston, Texas 77550 Due to the limited availability and rising expense of fetal bovine serum (FBS) as a supplement to tissue culture minimal essential medium (MEM), several investigators have examined the potential of alternate sera or synthetic serum substitutes (1, 2). To date a clear alternative supplement for growth and maintenance of cells in vitro has not been delineated. This report describes comparative growth efficiencies of HeLa-229 and McCoy cells grown in medium supplemented with either fetal bovine serum (FBS), donor bovine serum (DBS), or newborn bovine serum (NBS). HeLa-229 and McCoy cell cultures were chosen in an attempt to more clearly define serum substitutes that would be acceptable in the isolation and identification of Chlamydia trachomatis from clinical specimens. HeLa-229 and McCoy cells (both courtesy of Dr. Paul Price, Centers for Disease Control, Atlanta, Georgia) were grown in minimal essential medium with Earle's salts (Autopow, Flow Laboratories, McLean, Virginia) supplemented with either 5% FBS, as growth medium, or 2% FBS, as maintenance medium. Both HeLa and

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McCoy cells were found to be free of mycoplasma contamination as tested by 4'-6-Diamidino-2-phenylindole (Bioassay Systems Corp., Cambridge, Massachusetts). Cells grown in FBSsupplemented media were subcultured with 0.25% trypsin, centrifuged, and rinsed with phosphate-buffered saline. Cell suspensions of each cell type were divided into three equal volumes, centrifuged, and resuspended into minimal essential medium (Autopow) supplemented with 5% of either FBS (Lot No. 29106024), NBS (Lot No. 29121023), or DBS (Lot No. 4027001) and seeded into separate flasks. All sera (Flow Laboratories, McLean, Virginia) were heat inactivated at 56°C for 30 min. Growth assays were performed with HeLa-229 cells and McCoy cells grown in medium supplemented with each of the three sera types. Cells were planted in plastic tissue culture dishes (Lux Scientific Corp., Newberry Park, California) and quantified at 24-hr intervals, replenishing the medium with MEM plus 5.% serum every 24 hr. Aliquots of duplicate plates were counted in triplicate, and the counts were averaged. The first cell passage of both HeLa-229 ceils and McCoy cells grew equally well when minimal essential medium was supplemented with either FBS, NBS, or DBS. Initial granularity appearing in NBS- and DBS-supplemented media disappeared after

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several cell passages. No significant differences appeared in the microscopic anatomy of the cell monolayer when cells were grown in media supplemented with either FBS, NBS, or DBS. Assay results of comparative growth rates can be seen in Table 1. The results indicate that numerically FBS is the optimal supplement for HeLa-229 cells and NBS is the optimal supplement for McCoy cell cultures. The optimal alternative serum for these cultures was determined to be NBS. However, DBS also appeared to be a satisfactory serum substitute for HeLa-229 [post 48 hr] or McCoy cell cultures. Studies requiring the use of

In This Issue Serum Supplements for Preparation of HeLa-229 and McCoy Cells . . . . . . . . . . . . . .

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Comparison of fetal, newborn, and bovine donor sera Fee-for-Service and the Public Health Laboratory . . . . . .

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What might it cost? Guidelines for Processing AIDS Patient Specimens . . . . . . . .

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Precautions for laboratory workers Letters to the Editor . . . . . . . . . . . . .

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Workshop, and Meetings . . . . . . . . .

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Table 1 C o m p a r a t i v e G r o w t h Rates of HeLa-229 a n d M c C o y Cells G r o w n in Either Fetal, N e w b o r n , or Donor Bovine S e r u m

HeLa-229 Time (hr)

0 24

48

72

96

FBS

2.0

x

I0 '~"

NBS

2.0

DBS

10s

x

McCoy

2.0

X

FBS

IOs

3.06 x 10s

2.43 x l 0 s

0.61 b

0.28

0

39"

86

0

4.91 x 105 0.68 35

4.15 x lOs 0.77 31

1.77 x lOs 0 0

8.39 x 10a 0.77 31

5.87 x 10~ 0.50 48

2.06 x lOs 0.22 llO

1.13 x tO6 0.43 56

8.25 x tO~ 0.49 49

5.1 x 10~ 1.31 81

NBS

DBS

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UQ 9 1 3 5 8 1.28 19

1.32 18

0.81 30

a 2.0 x 10~ viable cells (by Trypan blue exclusion) planted per dish at initiation of study n Number of generations for 24-hr period " Generation time (hr)

HeLa-229 cells for periods up to 48 hr, however, should avoid the use of DBS because both NBS and FBS clearly are superior supplements. The rationale for the sole use of FBS as an additive to minimal essential medium in early studies has been based on several factors. The early developmental stage of the bovine fetuses immune system dictated minimal immune responsiveness. The low level of circulating antibodies appeared to be a distinct advantage in investigations using infectious agents. In addition, the restricted exposure time of the donor animal to exogenous microorganisms present in utero further implied lower levels of circulating antibodies and other factors, which ma~, have complicated experimental designs. Alternative sera, however, may be equally effective as supplements to minimal essential medium while maintaining the needs formerly attributed primarily to FBS. Newborn bovine serum (used in this stud~ was obtained from blood collected from calves approxiuately

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seven days old. Donor bovine serum (used in this study) was processed from whole blood taken from a controlled herd of calves up to eight months of age, weighing 3 5 0 - 6 5 0 lb. This serum differs from other bovine sera because the animal is not slaughtered at the time of bleeding. Animals (for DBS) were maintained on a predetermined diet, kept isolated from other farm animals, and removed from the bleeding program when they weighed in excess of 650 lb. All three sera types appear to be adequate potential supplements because their exposure to extrinsic factors is either totally or partially controlled. There may be less risk of containing significant levels of circulating antibodies with the use of the fairly rigidly (exposure) controlled FBS. However, the limited exposure of newborn and donor calves makes NBS and DBS very attractive substitutes. Due to the current minimal cost differences between D B s and NBS there would appear to be no advantage to choosing either on a purely

fiscal basis. However, individual decisions for particular studies or clinical tests need to be based on analysis of potential neutralization or other effector mechanisms that have not been ruled out in NBS or DBS. In addition to fiscal restraints, recent investigations have detected suppression of Chlamydia trachomatis inclusion formation by selected lots of FBS in cycloheximide-treated McCoy cells (3). Recent evaluations of newborn calf serum, however, have indicated that fetal calf serum may be superior for the isolation of a number o f serotypes of C. trachomatis (4). Also, clinical identification of viruses and C. trachomatis and basic investigations of viruses and Chlamydia rely on tissue culture cells grown in media supplemented with bovine sera (5). Therefore, it is imperative that optimal serum supplements be investigated further. In summary, newborn bovine serum and donor bovine serum have been shown to be potentially adequate serum substitutes for fetal bovine

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Additional recommendations also are made for individuals providing care to AIDS patients and for individuals performing studies with experimental animals that use specimens obtained from AIDS patients. The

exact cause of AIDS remains unknown, and these precautions have been recommended because of the distinct possibility that AIDS may be caused by an infectious agent.

Reference 1. U.S. Department of Ilealth and lluman Services. 1982. Acquired immune deficiency syndrome (AIDS): precautions for clinical and laboratory staffs. Morbidity and Mortality Weekly Report 31:577-580.

sor Reber's letter in volume 4(20) concerning the statistical expression of the distribution of results, I wish to indicate that the information did not show a normal population distribution. Publication of the actual graphic displays would have required too much space. Perhaps we should have reported only the mean, and/or median, and range of values. If

anyone would like a copy of the actual graphic display of the distributions, they may be obtained by writing directly to me.

been missed initially because of concomitant spots due to Malassezia infection. He had absconded from the hostel for immigrants before examination by a local specialist and lived with relatives. Test results in all the members of the family proved negative. It is important to keep in mind that given an incubation period of several years leprosy may continue to appear in immigrants whose health has been

cleared after thorough examination at the time of arrival. The table in the paper would have been even more helpful if the total number of persons at risk in each group was specified.

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Letters to the Editor

Microbiology L a b o r a t o r y Management To the Editors: With regard to the "Results of the Survey of Microbiology Laboratory Management: Summary and Comments" in volume 4(14) of the Newsletter, specifically in response to Profes-

L e p r o s y in

Refugee

To the Editors: I read Dr. Gruninger's feature on infectious disease in Indo-Chinese refugees (October 1, 1982) with great interest because we have just diagnosed our first case of leprosy in a Kampuchean engineer, who had arrived in this country three months ago. His borderline extensive skin lesions might have

Raymond C. Bartlett, M.D. Director, Division of Microbiology Department of Pathology Hartford Hospital Hartford, Connecticut 06115

H. K. Ghosh, M.B.B.S., PhD, F.R.C.P.A. Clinical Microbiology Department The Royal Newcastle Hospital Newcastle, N.S.IV., Australia 2300

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Workshops and Meetings

I n f e c t i o n C o n t r o l for Extended C a r e Facilities. Columbia, Missouri. February 9 - 1 1 . Contact: Continuing Education Program, UMC School of Nursing, Columbia, MO 65212.

A S M A n n u a l M e e t i n g (83rd). New Orleans, Louisiana. March 6 - 1 1 . Contact: Meetings Dept., American Society for Microbiology, 1913 I St. N.W., Washington, DC 20006.

and Molecular Biology, Catholic University of America, Washington, D.C. 20064.

I n f e c t i o n C o n t r o l : Basic Concepts. Columbia, Missouri. February 2 1 - 2 5 . Contact: Continuing Education Program,-UMC School of Nursing, Columbia, MO 65212.

Laboratory Safety: Management and Responsibility. Washington,

New York City. March 1 7 - 1 8 . Contact: Charlene Landis, Conference Planner, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

C o n t r o v e r s i e s in M a n a g e m e n t o f Infectious Complications

of Neoplastic Diseases. D.C. March 1 7 - 1 8 . Contact: Dr. Roland M. Nardone, The Center for Advanced Training in Cell