Survival of Animal Tissue Cells in Primary Culture in the Absence of Serum - Applied and Environmental ...
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APPLiED MIcRoBIoLOGy, Jan. 1973, P. 49-54 Vol. 25, No. 1
Copyright 0 1973 American Society for Microbiology Printed in U.S.A.
Survival of Animal Tissue Cells in Primary
Culture in the Absence of Serum
HENRY C. ORR, JAMES BAKER, AND JUDY 0. CHEESMAN
Cell Biology Section, Laboratory of Virology and Rickettsiology, Division of Biologics Standards, National
Institutes of Health, Bethesda, Maryland 20014
Received for publication 21 July 1972
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The ability of cells from tissues of several species of animals to survive in
primary culture without serum was tested. Of the species tested, cells from the
kidneys of Macaca mulatta (rhesus) and Cercopithecus aethiops (vervet)
monkeys and chicken embryo cells not only survived under these conditions, but
indeed developed into confluent monolayer cultures. The addition of either
serum or its globulin or albumin fraction enhanced the development of cell
monolayers and permitted those cells unable to survive in the absence of-serum
to do so. Certain specific protein trypsin-inhibitors not of serum origin were
unable to provide conditions necessary for cell survival or growth when used in
place of serum proteins.
In most mammalian cell culture systems, number of animal species to cultural conditions
serum protein is incorporated into the medium devoid of serum.
to insure optimal cell growth. The protein may
be in the form of either whole serum or serum MATERIALS AND METHODS
components (e.g., protein growth factor [13], Preparation of serum fractions and growth
alpha globulins [11], fetuin [201 and albumin media. A fetal calf serum with demonstrated growth-
[15, 25D, or both. Although continuous cell promoting properties was used as the control serum.
lines have been used extensively in estab- A portion of the serum was separated into its
lishing nutritional requirements, little use albumin and globulin fractions by two successive
precipitations with 50% saturated ammonium sul-
has been made of primary cell cultures in this fate. The salt was removed from the fractions by
regard. Healy and Parker (10) were able to grow dialysis against Hanks balanced salt solution
newly explanted mouse embryo cells in a se- (HBSS). The protein solutions were either concen-
rumless chemically defined basal medium. Ru- trated by evaporation or diluted with HBSS back to
bin and Hatie (23) and Levinthal and Rubin the original volume of the serum. All operations were
(12) noted that chicken embryo cells grown performed aseptically at 4 C.
without serum did not exhibit the increase in The serum protein fractions and 1% (w/v) solutions
cell size nor the formation of numerous polyri- of egg-white albumin and soybean trypsin inhibitors
bosomes and differential cytoplasmic struc- in HBSS were used as substitutes for 10% whole
serum in Eagle minimum essential medium
tures, fibrils, and microtubules as did serum- (MEME), except as noted.
stimulated cells. Rappaport (21) and Wallis et The undiluted solutions of fetal calf serum, globu-
al. (26) have reported the successful cultivation lin, albumin, soybean trypsin inhibitor, and egg-
of primary cultures of monkey kidney cells in white albumin contained 34.49, 9.13, 15.75, 6.56, and
the absence of serum. The latter investigators 6.19 mg of protein per ml, respectively, by Kjeldahl
(26) suggest that one role of serum proteins in determinations.
cell culture medium is that of inhibiting the Preparation of primary cell cultures. Cells were
action of tryptic enzymes, synthesized by the dispersed from the kidneys of guinea pigs, young
cells themselves and released into the culture rabbits and hamsters, juvenile Macaca mulatta
medium. (rhesus) and Cercopithecus aethiops (vervet) mon-
Because of these and other observations, it keys, and from whole embryos of chickens, mice, and
hamsters by a method previously described (17). The
became of interest to examine more closely the cells were washed several times with Dulbecco saline
ability of cells to survive at 37 C in the absence after removal from the trypsin solution by centrifuga-
of serum. This communication describes our tion. Washed cells were counted and diluted appro-
results in subjecting primary cells from a priately in MEME or saline without serum and
4950 ORR, BAKER, AND CHEESMAN APPL. MICROBIOL.
inoculated into 2-oz (ca 0.06 liter) prescription bot- fraction benefited about equally well all of the
tles containing the particular fluid under study. primary cells. However, trypsin inhibitors not
Inoculated cell cultures were incubated at 37 C for of serum origin, when used in place of serum
2 to 8 days as indicated in the different experiments. proteins, failed to provide any beneficial effects
After removal of the fluids, the cultures were rinsed
briefly with saline, and the cells were dispersed with for cells unable to survive in serumless me-
0.25% trypsin. Viable cells were counted in a hemocy- dium.
tometer using the trypan blue dye-exclusion method. The observation that certain primary cells
In certain experiments, samples of incubated cul- appeared capable of being propagated in
tures were taken for determining the number of MEME without serum (Fig. 1) led us to exa-
viable cells every 2nd day as described above. In mine further this growth potential by experi-
other experiments, cells were allowed to remain ments designed to separate cell attachment
undisturbed before they were dispersed, counted, and spreading from actual cell multiplication.
and subcultured at a split ratio of 1: 2.
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In addition, rhesus and vervet monkey kidney cells
Trypsinized primary cells were inoculated as
were seeded in several synthetic or chemically de-
previously described into culture vessels con-
fined media (Table 1) both with and without the taining saline as well as MEME. Some of the
addition of serum for 5 days. Finally rabbit kidney vessels in each group contained 2% serum.
and chicken embryo cells were used to examine the Inoculated cells were allowed 48 hr to settle and
influence of inoculum size on their ability to be adhere to the vessel surface before being re-
sustained in serum-free medium. moved by decanting the fluids. Those cells that
RESULTS
had attached themselves to the vessels were
rinsed with saline, and some cultures were used
Primary cells from the several species of for determining the number of attached viable
animals tested varied in their capacity to cells. Photomicrographs were taken also at this
survive in the absence of serum (Fig. 1). It time. The remaining cultures were refed with
appeared that cultures of chicken embryo and the respective fluids and further incubated for
both species of monkey kidney cells developed an additional 6-day period. Photomicrographs
to about the same degree in Eagle medium with again were taken and compared with those
or without serum. Most of the cells from the taken earlier. In addition, the number of viable
other species of animals were less consistent in cells in the cultures were counted.
their ability to grow in the absence of serum. Representative results from these experi-
Hamster embryo, hamster kidney, and guinea ments are shown in Fig. 2-4. In all instances,
pig kidney cells were never successfully main- under the most optimal conditions employed,
tained in serumless medium, and maintenance more than 50% of the primary cells failed to
of rabbit kidney cells under such conditions attach to the vessel surface in 4.8 hr. Moreover,
was poor and erratic. Medium containing ei- it is apparent that not only does MEME
ther whole serum or its globulin or albumin without serum enhance cell survival over that
TABLE 1. Percent of inoculated cells attached and viable after 5 days incubation in various synthetic mediaa
Vervet monkey kidney cells Rhesus monkey kidney cells
Medium Reference With 2% fetal Or~~~~~~Wih22% fetal
With Withu serum
calf serum calf serum
MEME .................3 77±1.4 69 + 3.0 99 4.1 59 1.5
Waymouth MAB87/3 with-
out insulin ............ 6 86 X 3.0 24 0.9 88 3.8 24 0.5
Waymouth MAB87/3 with
insulin ............... 6 83 2.6 25 0.7 46 2.5 18 0.3
Medium 199 ............ 19 71 1.7 28 1.0 37 1.3 15 0.3
RPMI 1640 .............. 18 59 1.7 27 1.0 45 2.5 16 0.2
NCTC 109 .............. 4 48 ±0.4 19 ± 0.9 67 ± 4.1 13 0.1
Ham's F-12 ............. 8 56 1.5 18 0.7 41 1.9 10 0.1
HEPESb Hanks BME ... 27 53 ± 2.6 12 0.1 16 1.0 5 ±VOL. 25, 1973 SURVIVAL OF CELLS WITHOUT SERUM 51
2.0
FoJ III-,. IInn 1 n 1z
o)
L
0D_
a 0 XIPtii 5ruert
ct
3.0 _
~HAMSTER
KIDNEY
El
GUINEA PIG
KIDNEY
I
RABBIT
KID NEY
P
HAMSTER
EMBRYO
1=1~~~~~~~~~
Serum
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CHICK EMBRYO VERVET MONKEY RHESUS MONKEY
KIDNEY KIDNEY
FIG. 1. Inoculum multiplication indexes of primary cell cultures from seven species of animals after 7 days
in MEME without serum, with 10% serum, or with trypsin inhibitors. Quadruplicate bottles were each
inoculated with 7 ml of suspension containing 3 x 106 cells/mI. Indexes of inoculum multiplication were
determined
140 from the expression /C,/C1: where C,. = final cell count; C, = initial cell count.
9Eg ht
0~~~~~~~~/~1
220
MEME
+ 2%/FCS / 1C
200 _/
/ 160
60
260
3: / 1 14 | X _ L
~20 /
(1)~ / SAIN +2%C MEMMEdys
KIDNEY
KISALI
0 2 8iFIG. 3. Survival of cells in medium and in saline
TIME (days) with and without serum. Maintenance of vervet
FIG. 2. Survival of cells in medium and in saline monkey kidney cells. See legend to Fig. 2.
with and without serum. On day 2, all cells which
had not adhered werexremoved by decanting the found in saline, but indeed allowed some cell
fluids, after which fresh fluids of the respective type mu.lt
were added to the cultures. Numbers represent ilcto oocree nteasneo
averages from three cultures. Maintenance of mouse serum.
embryo cells. Triplicate bottles were each inoculated The growth potential of all primary cells kept
with 7ml ofcell suspension containing atotal of18 x in serum-free conditions for 7 days was ex-
106 cells. amined further by transferring the same num-52 ORR, BAKER, AND CHEESMAN APPL. MICROBIOL.
TABLE 2. Relationship between inoculum size and
survival of primary chicken embryo and rabbit
kidney cells in medium with or without seruma
No. of viable cells recovered after 5 days
No. of growth in medium with:
viable cells 5% Fetal calf serum No serum
MEME inoculated
+ 2% FCS > x 106/ml Rabbit Chicken Rabbit Chicken
kidney embryo kidney embryo
5
7.0 9.5 ± 1.1 7.7 ± 1.3 0.9 ±VOL. 25, 1973 SURVIVAL OF CELLS WITHOUT SERUM 53
Some earlier findings on the role of serum in and clearer demonstration of the presence of
tissue culture systems have been reviewed (14). adventitious agents.
Recently, Wallis and co-workers (26) postu- In addition, the omission of serum from cell
lated that one role of serum in the growth of culture medium denies cholesterol-requiring
monkey kidney cell cultures is to inhibit pro- mycoplasmas their optimal growth conditions
teolytic enzymes synthesized by the cells them- (22) and eliminates one source of contaminat-
selves. Shodell and Rubin (24) found that ing mycoplasmas (1) and viruses (16) from
serum was needed to stimulate mitotic activity tissue culture systems.
in chicken embryo cells. Our results partially
confirm and extend the observations of Wallis ACKNOWLEDGMENTS
et al. and may disagree with those of Shodell We are grateful to the Laboratory of Pathology, Division
and Rubin in that we found that primary cells of Biologics Standards, National Institutes of Health, for
providing the tissues and to Joseph P. Davis of our section for
of monkey kidneys and chicken embryos do not technical assistance.
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require serum for initial growth in vitro. How-
ever, the protease claimed by Wallis and co- LITERATURE CITED
workers to be responsible for cell sloughing and 1. Barile, M. F., and J. Kern. 1971. Isolation of Myco-
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In addition to whole serum, either the globu- protein requirements of mouse fibroblast cells (strain
lin or albumin fraction was sufficient to facili- LS) in culture. J. Cell Sci. 5:135-142.
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serially for at least 12 passages. Although Fioramonti, K. K. Sanford, B. B. Westfall, and W. R.
serum in cell culture medium is thought to Earle. 1956. Studies of nutrient media for tissue cells
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tems, in our experience the absence of serum defined medium for long-term cultivation of strain
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Birch and Pirt (2), working with continuous Schilling. 1964. Chemically-defined media for cultiva-
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not grow in suspension. The lytic enzyme Biol. Med. 133:1257-1258.
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13. Lieberman, I., and P. Ove. 1958. A protein growth factor
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(21). It is thus suggested that primary cells 15. Maysuya, Y., and I. Yamane. 1968. Serial culture of
grown without serum may provide a more rapid54 ORR, BAKER, AND CHEESMAN APPL. MicRoBIoL.
Syrian hamster fibroblasts in albumin fortified me- 21. Rappaport, C. 1956. Monolayer cultures of trypsinized
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Med. 72:1-8. 26. Wallis, C., B. Ver, and J. L. Melnick. 1969. The role of
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199. in the culture of animal cells. J. Gen. Virol. 2:309-312.Survival of Animal Tissue Cells in Primary
Culture in the Absence of Serum
HENRY C. ORR, JAMES BAKER, AND JUDY 0. CHEESMAN
Cell Biology Section, Laboratory of Virology and Rickettsiology, Division of Biologics Standards, National
Institutes of Health, Bethesda, Maryland 20014
Volume 25, number 1, p. 53, column 1, line 9: Delete "partially." Lines 14 and 15: Change
"However, the protease claimed by Wallis..." to read "We further confirm that the protease
claimed by Wallis.. ."
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