Isolation and Long-Term Culture of Diploid
Mammalian
Cell Lines
JEAN FERGUSONANDANN WANSBROUGH
(Commonwealth
Serum Laboratories,
Parkville,
Victoria, Australia)
SUMMARY
Cells have been established in continuous culture from human and animal tissues,
both normal and carcinomatous. From 197 specimens, 105 could be maintained in vitro
for varying lengths of time, 27 for more than 6 months. In all cases the cells were found
to be diploid. Cell lines with both epithelial and fibroblastic morphology have been
isolated in medium 199 with added fetal calf serum, but all lines maintained over long
periods have eventually become fibroblast-like. Following the additional use of other
synthetic media, cells with epithelial morphology have retained their characteristic
shape for up to 3 months.
Cells derived from mammalian tissue have been
used for biochemical and cytological studies, par
ticularly with respect to cultural characteristics
of the rapidly multiplying
"continuous"
cell
lines (2,4,5). These heterogeneous and heteroploid
cell populations are a valid analytical tool within
certain limitations. Cloned and characterized lines
are being widely used but give little basis for com
parison with the normal cell in vivo (J8, 20).
In recent work, lines of fibroblast-like
cells
from normal tissues have been isolated and propa
gated in vitro without apparent alteration of their
morphology or cultural behavior (15, 22), and in
addition these cells have retained the diploid
chromosome number of the original tissue (16).
Technics for the cultivation
of normal cells
permit studies on the morphology and behavior of
different cell types. Recent improvements in media
and definition of the factors affecting cell morphol
ogy in vitro will extend the application of such
technics. In this paper the methods for establish
ing fibroblast-like
cell lines with morphological
and biochemical characteristics
of primary tissue
outgrowth are described. Preliminary
investiga
tions into the propagation
of diploid epitheliallike cells are also reported.
MATERIALS
Biochemicals
Corporation
(1:300)
in P.B.S.)
L Y medium
199 medium
858 medium
N.C.T.C. 109
N. 16
Additives.—
Sera:
Human pooled, individual and cord
Rabbit
Calf, fetal calf
Horse, pooled
Monkey, rhesus and cynomolgus.
After collection all sera are passed through
Corning ultra-fine sintered glass filters. Calf serum
and individual human sera are prepared weekly
and stored at 4°C. for periods not exceeding 1
month; all other sera and extracts
—30°C. until required.
are stored at
Before use individual human sera and fetal calf
serum batches are tested on sensitive cell lines for
capacity to support growth and for toxicity.
AND METHODS
Solutions.—
Phosphate-buffered
saline
(P.B.S.)
Trypsin,
0.25 per cent (Nutritional
(11)
(14)
(13)
( 6)
(16)
(3)
Extracts.—
Chick embryo : modification of method
of Bryant el al.
Beef embryo
Fowl plasma: freeze-dried and stored
at 4°C.
Fetuin:
modification
Fisher, Puck, and
Received for publication September 5, 1961.
of method
Sato
(1)
of
(10)
556
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FERGUSONANDWANSBROUGH—Culture
of Diploid Mammalian Cell Lines
Tissue samples.—
Human :
Skin—Pinch
biopsies, adult and child
(from inside of forearm).
Pieces taken at operation (normal skin
taken from border of wound).
Foreskin taken at circumcision.
Amnions—from normal deliveries.
Cervical samples—biopsies taken at clin
ical examination for carcinoma.
Normal and tumor tissue removed at
operation.
Animal:
Normal tissues taken aseptically from labo
ratory animals.
Culture vessels.—Pyrex screw-capped
bottles
(160 ml.), tubes (160 X 25 mm.), and Petri
dishes (60 mm.) were used.
Initiation of cell cultures.—Various procedures
for isolating cultures have been used.
Three methods were compared for small tissue
specimens (less than 2 gm.).
A. Plasma clot method : Minced tissue fragments
moistened with chick embryo extract were allowed
to clot on the plasma-coated
surface of tubes or
Petri dishes, and culture-medium
was added.
B. Trypsinization : The tissue was minced as
finely as possible in a 60-mm. Petri dish and incu
bated for 10-15 minutes at 37°C. with an aliquot
of trypsin determined by the size of the sample.
The suspension from a small biopsy (<2 mg.)
had 0.8 ml. of serum added. The suspension from
larger samples was divided among several dishes,
each moistened with 0.8 ml. serum. The final
volume of the trypsin solution should be less than
1:10 of the volume in the dish. When the frag
ments had settled, 3.2 ml. of basal medium was
added gently without disturbing the tissue, and
the dish was incubated for 72 hours before the
medium was changed.
C. Trypsinization
and plasma: A combination
of methods A and B was attempted.
The tissue
suspension in trypsin was dropped on the plasmacoated surface of the culture vessel and allowed to
settle before the culture fluid was added.
Large tissue specimens were treated in the
following ways to produce single cell suspensions.
D. Amnion trypsinization : Following the meth
od of Ferguson and Tobin (9), the membrane was
given one 3- to 4-hour extraction with trypsin, and
the cell yield was inoculated to give a final con
centration of 0.3-0.4 X IO6 cells per ml. of culture
medium.
E. Mass trypsinization:
A cell suspension was
obtained from tissues of more than 2 gm. by the
557
method of Youngner (23), with trypsin used for
repeated extraction of the chopped tissue (10- to
20-mg. pieces). Cells were inoculated to give a
concentration
of 0.2 X IO6 cells per ml. of culture
medium.
F. Liver extraction: After laparotomy the he
patic artery was ligated. The inferior vena cava
was pierced, and the liver was perfused liberally
with normal saline through a cannula inserted into
the portal vein. The inferior vena cava was then
clamped off and medium 199 injected forcibly
through the cannula with the aid of a 50-ml. syr
inge. The volume of medium used was varied
between 50 and 150 ml. per experimental animal.
The well inflated liver was removed, chopped, and
extracted by agitation in medium 199 or trypsin
diluted to 0.125 per cent with medium 199, thus
producing a single cell suspension.
^Maintenance and propagation of cultures.—
Petri dish cultures: A humidified incubator with
an atmosphere of 5 per cent CO2 in air was used to
maintain a pH of 7.1 in the medium. After the
first 3 days the medium was replaced 3 times
weekly. Cells which had become attached to the
glass might stretch and begin multiplying up to a
fortnight after trypsinization.
The time taken for
a continuous cell sheet to form depended on the
original amount of tissue and varied from a few
days to a month. Subculture was carried out when
the surface of the vessel was about 50 per cent
covered, the medium being withdrawn and 0.20.5 ml. trypsin added. After 1 or 2 minutes the
cells were gently rubbed free from the surface with
the tip of a pipette and suspended in the trypsin
solution. The cell suspension from the first sub
culture might be left in the original dish and fresh
medium added. Rapid multiplication
followed;
the culture became confluent and could be divided
between two dishes every few days by the method
described above. After three passages in Petri
dishes the culture was well enough established to
be transferred to bottles or stored at —65°
C. (7).
Bottle cultures : Cells obtained from large speci
mens by methods D, E, and F can be established
in bottles directly. The medium was replaced on
the 4th day. After primary inoculation at the
optimum concentration
the cell sheet should be
confluent within a week. Subculture was carried
out by removing the medium, washing the cell
sheet once with P.B.S., and incubating the bottle
at 37°C. with trypsin for 15-30 minutes (1-2 ml/
160-ml bottle). The cell suspension obtained was
divided between two bottles containing fresh cul
ture medium.
Growth media.—All components used are listed
above. Fibroblast lines were initiated and propa-
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558
Cancer Research
gated in medium 199 with fetal calf serum. Some
established lines could be maintained
with calf
serum.
Liver and kidney cultures have been grown in
medium 199 or 858 with fetal calf serum (5-15 per
cent) and with or without N.C.T.C. 109 (5 per
cent) added. Amnion cultures were grown in fetal
calf or human serum (cord or adult pooled).
Storage of tissue samples.—Although specimens
were generally cultured as soon as possible, they
could be stored in medium 199 before use. Amnions were washed and left overnight while steril
ity was confirmed (9). Operation specimens re
ceived in the afternoon were washed, chopped into
1- to 2-gm. pieces and left overnight in fresh
medium at 4°C. Chloramphenicol
(20-50 mg/ml
medium) was added if infection was suspected.
Skin biopsies have been stored in a few ml. of
medium at 4°C. or room temperature
for up to
Vol. 22, June 1962
dish cultures in a CO2 incubator was used, cells
from small tissue samples could not be successfully
propagated. Later use of methods A, B, and C all
gave satisfactory results, but because of its con
venience method B was mostly emploved (see
Table 2).
Serial passage of cells derived from normal ani
mals was attempted
before the use of the Petri
dish system of culture. As shown in Table 1 in no
case was a line established.
Nutritional requirements.—In early attempts to
propagate cells in primary culture biological ad
ditives and various media and sera were used.
Embryo and yeast extracts gave variable results.
Horse, rabbit, and monkey sera were often toxic.
Amnion cultures did not grow significantly better
in LY medium than in 199. Puck's medium N.16
and lower concentration
of trypsin gave similar
results to 0.25 per cent trypsin and 199.
However, the introduction
of fetal calf serum
1 week.
Storage of cells.—Cells were stored in ampoules
was found to be an important factor for the con
at —65°C., each ampoule containing
2X IO6 tinued multiplication
of a culture after transfer.
cells in 1 ml. of growth medium, with 10 per cent
Apart from amnion cultures, which grew similarly
glycerol added (7). After thawing, the cell sus
in human, human cord, or fetal calf serum, all
pension was not washed but added directly to primary cultures grew consistently better in fetal
calf serum.
fresh growth medium in a fresh Petri dish or bottle.
Chromosome enumeration.—After
three pas
Morphology and chromosome number.—Although
a mixture of cell types often appeared in primary
sages mitoses were frequent, and the chromosome
cultures, all the lines we established were fibronumber of a culture could be determined
(8).
blast-like in appearance, but morphology differed
RESULTS
slightly according to the tissue of origin. The
Cell lines established.—A variety of normal and
chromosome
number of several of these stock
lines was checked at intervals during their estab
carcinomatous
tissues from human sources and
lishment (Figs. 1, 2). The normal diploid number
normal animal tissues were investigated.
Exclud
ing those contaminated
with microorganisms,
of was found in all cases investigated, each line hav
ing been in continuous
culture for at least 6
197 specimens cultured 170 showed a primary
months. In no case was a cell line found with other
growth of viable cells, judged by their stretched
than the diploid number (Table 3). In addition, no
appearance
on the glass. In total, 105 cultures
alteration of chromosome morphology was noted
which were still multiplying after the third trans
during cultivation of these lines. Cells tested after
fer in vitro were regarded as successful isolations.
storage at —65°
C. were also found to retain their
Of these, 27 lines were propagated in vitro for more
diploid configuration.
than 6 months and were considered established
Storage of cells.—Stock fibroblast lines have
lines; 35 others were stored before 6 months had
been
stored at —65°C. and later tested for via
elapsed. The remainder were lost, due to inade
bility. After a period ranging from 1 to 9 months
quate media in the case of epithelial lines, toxicity
of storage twenty cell lines were tested for capacity
of serum or contamination.
These results are sum
to grow. All except three cultures yielded viable
marized in Table 1.
Following the system of Foley et al. (12), a new cells which were propagated and re-stored. In some
cell line was not considered to be 'isolated' until
cases the number of viable cells recovered from
three serial passages had been made, and this line the ampoule has been counted and recorded as a
was not considered to be 'established' until it had
percentage
of the original (Table 4). Cultural
were found to be unaltered, and
been carried in culture in the laboratory for 6 characteristics
the condition of the cells at the time of storage
months.
Culture methods.—The cells obtained from trypwould appear to be the most important
factor
sinization of a large tissue specimen rarely failed to affecting the recovery rate.
Growth from tissue samples stored for up to 7
form a culture which would multiply for at least
days at various temperatures
was compared with
a few weeks in vitro. Before the system of Petri
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TABLE 1
ORIGINOFISOLATEDANDESTABLISHED
CELLLINES
celllinesisolated7142135411017133741113214105No.
celllinesestablished62131102227
originNormal
Tissue of
humanSkin:
Biopsy, normalfor
investigationOperation
adultForeskinThyroidKidneyCervixSpleenBladderAumionMalignant
specimen,
humanCervical
biopsyCarcimona,
vaginalungbreastbladdercolonrectumkidneyNormal
animalMonkey,
kidneylungspleenthyroidliverHorse,
kidneyRabbit,
kidneyliverRat,
kidneyskindiaphragmlungBeef,
skinTotal:No.specimenscultured925417615111114212425537621624521214197No.specimensshowingprimarygrowth82031451411039122154
embryo
TABLE 2
METHODSOFCCLTURING
SMALLTISSUESAMPLES
SHOWINGPRIMARY
GROWTHUn-»tretchedcells2
SHOWINGSUBSEQUENT
GROWTHMultiplicationaftertransfer
METHODA.
propaga
tion
aftertransfer
cells2678
115
Expiant
B. Trypsinization
101
23
C. Trypsinization+plasmaNo.CULTURESATTEMPTED28
14NO.
4Stretched
10No.
60
5Serial
31
55
5
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560
Cancer Research
controls of the same samples cultured when re
ceived. The samples tested were a carcinoma of
the rectum, two pieces of adult skin taken at
operation, and nine foreskin specimens. Each was
divided in four to nine parts; two parts were cul
tured immediately as controls and the rest stored
and cultured on subsequent days. Apart from the
exaggeration of previous microbial contamination,
the difference in successful isolation and multipli
cation of cultures from control and stored samples
was not significant for samples stored for 7 days
either at room temperature
or at 4°C. Of two
samples each stored at 37°C. for 3 and 5 days,
one was successfully cultured on the 3d day only.
A skin biopsy taken at autopsy 24 hours after
death was successfully cultured after a further 2
days at room temperature.
Comparison of growth from normal and malig
nant sites.—Cells grown from tissues from malig
nant sites have shown a similar morphology to
those grown from normal tissues. Isolations from
malignant tissues were more difficult to achieve,
but subsequent
establishment
of cell lines was
TABLE 3
CHROMOSOME
No. OFCELLLINESISOLATED
FROMHUMAN
TISSUES
Vol. 22, June 1962
found to occur with greater frequency (see Table
5). Although epithelial-like cells were present in
the initial stages of a large number of cultures
(Fig. 3), they did not survive propagation,
since
apparently the method and medium we had chosen
tended to favor fibroblast-like cells (Fig. 4). At
tempted propagation in the same medium of epi
thelial-like colonies isolated from earlier skin and
carcinoma cultures resulted in initial multiplica
tion of the cells followed by elongation and decline
in the rate of metabolism over one or two trans
fers. Our experience with trypsinized
cells from
predominantly
epithelial tissues such as kidney
and amnion was the same—the epithelial-like cells
multiplied during a few passages, then degenerated
or became overgrown by fibroblasts. The epithe
lial-like cell type grown from trypsinized thyroid
appeared to elongate during its initial passages
and then behaved as a fibroblast-like cell. Four of
seven rectal or colon carcinomas received over a
6-month period produced cultures of short bi
polar cells which, in later passages, constantly ap
peared as a mixture of distinct epithelioid and
fibroblast-like cells.
Epithelial-like cell lines.—The last part of our
investigation
was concerned with propagation in
the diploid state of epithelial-like cells from norTABLE 5
AFTER25 NO.
PASSAGESNo.lineatested97No.lineswith
LINES
ESTAB
LISHED910CHROMOSOME
ORIGIN
CELLNormal
OF
ESTABLISHMENT
OFCELLLINESFROMTISSUE
FROMNORMAL
ANDMALIGNANT
SITES
lineswith
chro
chro
mosomeno.
mosomeno. otherthan
4697No.
of
4600
skinCa.
cervixNo.
oforiginNormal
Tissue
specimenscultured9064No.isolations5724No.
linesestablished1814
humanMalignant
humanNo.
TABLE 4
SURVIVALOF DIPLOID CELLLINES AFTERSTORAGEAT —65°
C.
TlBSCE
OFORIGINOF
AFTERSTORAGE
CENT CELLS SURVIVING
MONTHS)1402515t47381958V4V56V7VVVVV8VV0VVVREMARKSOf
TIME (iN
LISERectal
carcinomaColon
carcinomaCervical
biopsyCervical
biopsyCervical
viableat
9 other lines tested 6 were still
months7
9
biopsyThyroidSpleenSkinPER
lines tested
V = viable cells present but not counted.
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FERGUSONANDWANSBROUGH—Culture
of Diploid Mammalian Cell Lines
mal tissues. Rabbit liver and human kidney were
chosen for preliminary experiments.
Cultures from human kidney consisted of uni
form, small epithelial cells. With 199 as the basal
medium the concentration of 5 per cent fetal calf
serum proved to be optimal, giving the highest
multiplication rate over the first few passages. After
this period of rapid growth the cultures entered a
phase where cell enlargement continued without cell
division, the cells becoming granular and vacuolated and eventually degenerated. Eleven cultures
were studied in this medium, and apart from one
which gave rise to a fibroblast line requiring a
higher serum concentration the latest transfer
followed by multiplication occurred 50 days after
primary trypsinization. Following the recommen
dation of Puck et al. (16), 5 per cent N.C.T.C. 109
was added to a mixture of 199 and 5 per cent fetal
calf serum. Under these conditions the initial
growth rate improved, and the onset of cell de
generation was delayed. Comparisons were made
between 858 and 199 as basal media for each of
two kidney cultures. A marked difference was
noted after the first two transfers. Control bottles
in 199 and 5 per cent fetal calf serum showed
degeneration of the cells by 40 days, whereas those
in 858 with the serum concentration increased to
15 per cent were still small, multiplying epithelial
cells after 85 days and nine transfers in the first
case, 74 days and five transfers in the second.
Rabbit liver cultures showed a similar reaction
to more complex basal media. Four cultures were
initiated by the inflation trypsinization technic.
The cell suspension appeared to contain four cell
types—large irregular cells with a granular cyto
plasm, large ovoid cells with a displaced distinct
nucleus, and granular cytoplasm and small regular
cells of varying diameters with and without gran
ules. In primary culture occasional large ovoid or
irregular cells could be identified attached to the
glass, but these did not multiply. Thus, the cell
types which formed a cell sheet appeared to fall
into four groups ranging from epithelial-like to
fibroblast-like cells, the ratio depending on the
medium used. Further study of these cell types,
their origin and behavior in cultured media, is in
progress.
DISCUSSION
Cultures grown from a variety of tissues have
shown that cells may be propagated which are
initially epithelial in appearance. These cells will,
in most cases, stretch out to give a fibroblast-like
appearance with variation of the culture medium
not affecting the result.
Fibroblast lines have been reported to decline
in growth rate after a period in vitro (16, 22). Three
561
of our lines have been kept growing continuously
in vitro for longer than 12 months, the remainder
being stored satisfactorily. One of these lines has
shown no decline in growth rate; the others have
been poor cultures since their accidental exposure
to toxic serum.
We have found that diploid lines can be initiat
ed and propagated under conditions of pH and
temperature similar to, but less stringent than,
those recommended by Puck et al. (16). However,
conditions of culture do have to be very carefully
controlled if only a few cells are available. Sanford
et al. (19) have shown that there is a critical ratio
between the volume of growth medium and the
size of the cell population. When their number
falls below a certain level in the growth medium,
cells do not appear to be able to adjust the fluid
sufficiently for their survival and proliferation.
Carcinomatous tissues have also yielded fibro
blast-like cells in culture which are sensitive to
cultural conditions and are more difficult to propa
gate than cells derived from normal tissue. The
relationship between these cultured euploid cells
and the carcinoma cell is difficult to ascertain,
especially since epithelial-like cells are also fre
quently present.
The experience of Puck et al. (16) is that euploid
cells from any source adopt an elongated config
uration in culture. Our studies with other tissues
have underlined the importance of the variable
medium requirements of different cell types. It
would appear that factors in fetal calf serum and
embryo extract do aid fibroblast-like growth. Cell
density is also an important factor. When the con
centration of cells is high the medium require
ments are less stringent.
Several workers have noted the appearance in
their cultures after a few weeks in vitro of a very
rapidly multiplying cell of compact appearance.
This cell rapidly overgrows all the other cells and
continues to maintain this high rate of division
indefinitely. These 'transformed' cell lines are
found to be heteroploid, with chromosome num
bers almost double the diploid number of the orig
inal tissue. Probably the most widely known of
such lines is the HeLa cell, which was isolated by
Scherer, Syverton, and Gey in 1953 (21).
During the serial passage of their amnion cul
tures Zitcer and Dunnebacke (24) noted the regu
lar appearance of a rapidly multiplying epitheliallike cell which could be maintained in continuous
culture over long periods. Pulvertaft et al. (17)
cultured thyroids showing various pathological
conditions and were able to demonstrate the ap
pearance of a fast growing cell type morphologi
cally similar to HeLa cells.
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However, sudden transformations have not
been noted in our cell systems, and it is thought
that this is due to the stringent cultural conditions.
It is possible that in a system which is lacking
some cultural requirement only a particular cell
will be able to survive prolonged cultivation. This
cell may be able to adapt to this particular en
vironment better than any of the other cells, or,
alternatively, such a cell could have arisen from a
chance mutation.
ACKNOWLEDGMENTS
We would like to thank Dr. M. Sherlock for arranging the
collection of the human tissue samples. Several Melbourne hos
pitals kindly provided us with these specimens, and, in particu
lar, we are indebted to Dr. J. D. Hicks and Mr. E. S. R.
Hughes of the Royal Melbourne Hospital and to Dr. J. C.
Laver and the late Mr. G. G. Godfrey of the Royal Women's
Hospital. We are also grateful to Dr. P. L. Bazeley and Mr.
John \Vhite for their interest in the project.
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FIG. 1.—Chromosome preparation from culture derived
from normal skin, 3d passage. Mag. X 2,000.
FIG. 2.—Chromosome preparation from culture derived
from same skin sample as above but at 26th passage. Mag.
X 2,500.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
Fio. 3.—Epithelial-like cells derived from human kidney,
3d passage. Mag. X400.
FIG. 4.—Fibroblast-like cells derived from human embryo
skin, 18th passage. Mag. X400.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
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Isolation and Long-Term Culture of Diploid Mammalian Cell
Lines
Jean Ferguson and Ann Wansbrough
Cancer Res 1962;22:556-562.
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