[CANCER RESEARCH 41. 611-618,
0008-5472/81
/0041-OOOOS02.00
February 1981]
Growth Characteristics of Primary Tissue Cultures from Heterotransplanted
Human Colorectal Carcinomas in Serum-free Medium1
Jürgenvan der Bosch,2 Hideo Masui, and Gordon Sato
Department of Biology, University of California, San Diego, La Jolla, California 92093
ABSTRACT
Procedures are described for the preparation of reproducible
primary cultures from human colorectal tumors transplanted in
the athymic nude mouse and for the quantitative evaluation of
growth by means of counting suspensions of nuclei from these
cultures with a Coulter counter. Growth curves of primary
cultures from 11 colorectal tumors in serum-free medium are
shown and discussed with respect to the in vitro conditions to
be met for the propagation of in vivo stem cell populations.
INTRODUCTION
During the past few years, a number of cell lines originating
from human adenocarcinomas of the colon and rectum have
been established in culture (4-9, 11, 12). Phenotype and
ploidy of these lines show a wide spectrum of differing char
acteristics, as do individual tumors of the colon and rectum
from different patients. However, long periods of in vitro ad
aptation, ranging from several weeks to more than a year
without significant net growth, regularly precede the emer
gence of these cell lines, which are characterized by high
growth rates and apparently unlimited in vitro life span.
The processes leading to the emergence of such a tumor cell
line are not well understood at present, and thus the outcome
of any of the presently available establishing procedures is
unpredictable. This is especially true in serum-containing me
dia, which favor the overgrowth by fibroblasts. It remains un
clear, for example, whether a minor subpopulation is selected
or whether adaptational genetic and/or epigenetic changes
must occur in order to yield an in vitro cell line.
A careful analysis of the population development in primary
cultures, as well as the elaboration of in vitro procedures and
conditions which allow for survival and growth of cells repre
sentative of the original in vivo population and for control of the
above-mentioned adaptational changes, seems highly desira
ble not only regarding the significance of tumor cell cultures
for cancer diagnosis and therapy but also for a more funda
mental understanding of tissue culture systems in general.
For this purpose, an approach has been put forward in this
laboratory during the past years to develop serum-free culture
media containing exclusively defined components which selec
tively favor the survival and growth of experimentally desired
cell types (1 -3). These media counteract the tendency toward
overgrowth of primary cultures by fibroblasts or other unde-
1 This work was supported
meinschaft (BO 500/3). To whom requests for reprints should be addressed.
Received August 15. 1980; accepted November 4. 1980.
1981
MATERIALS
AND METHODS
Explantation and Seeding
After the host animal is killed by cervical dislocation, the
tumor is immediately excised and placed in a Petri dish. All
work is done with sterile instruments, solutions, and laboratory
ware. Unless stated otherwise, all solutions used are prewarmed to 37°.If possible, the collagenous capsule surround
ing the tumor is peeled off with the help of 2 pairs of forceps.
After the tumor is washed with PBS,3 2 to 4 ml are cut with a
scalpel into slices about 1 mm thick. The material is then cut
into a fine mince with the help of a special tool, consisting of a
handle with an array of 18 parallel razor blades placed 0.75
mm apart from each other by washers. The mince, consisting
of pieces 100 to 500 firn in diameter and small debris, is
suspended in 50 ml of PBS by pipeting and is centrifuged for
2 to 3 min in a clinical centrifuge at half-maximal speed. The
supernatant is discarded and the top layer of the sediment,
consisting of small debris or mucoid material which is devoid
of live cell aggregates, is removed by aspiration. Depending on
the individual tumor, this procedure must be repeated with the
remaining sediment once or twice until a top layer of debris or
mucoid substance is no longer detectable after centrifugation,
as determined by microscopic examination of the sediment.
The last sediment, not exceeding a volume of 3 ml, is
suspended in 8 ml of trypsin solution and incubated in a 10-cm
tissue culture dish on a warm plate at 37°.Then, consecutively,
the following additions are made: 2 ml collagenase solution; 2
ml Dispase solution; 5 ml hyaluronidase solution. Each addition
is mixed thoroughly with the contents of the dish by pipeting.
in part by National Cancer Institute Grants CA
23052 and CA 19731 and also by American Cancer Society Grant C D65.
2 Supported by a Habilitandenstipendium
from the Deutsche Forschungsge
FEBRUARY
sired cells and allow for the serial cultivation of primary cell
populations which preserve their original characteristics.
In combination with a collection of human tumors, serially
transplanted in the athymic (nude) mouse as a reproducible
source of in vivo tissue (10), this approach should definitely
resolve the culture problems stated above.
In the present paper, we report on the growth characteristics
of primary cultures from 11 transplanted colon tumors in a
defined serum-free medium, described earlier (9), and in mod
ifications of this medium. Also, general procedures are de
scribed for the preparation of reproducible primary seedings,
representative of the tumors in vivo, yielding 50 to 200 uniform
parallel cultures per explantation, as well as for the quantitative
evaluation of growth in these cultures.
3 The abbreviations used are: PBS. phosphate-buffered saline; F-12. Ham's
F-12 medium; DME, Dulbecco's modification of Eagle's medium; HEPES, 4-<2hydroxyethyl-1-piperazineethanesulfonic
acid; EGF, epidermal growth factor.
611
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J. van der Bosch et al.
For rather sensitive tumors, incubations are allowed to proceed
for 2 min with each solution. For more resistant tumors, 5 min
are allowed for each step (Table 1).
The suspension is then centrifuged, and the resulting sedi
ment is freed of debris by aspiration. The sediment is resuspended in 200 ml of F-12:DME which is stirred magnetically.
This suspension is filtered through a nylon screen of 100-jum
mesh size. Permeation through this screen must be facilitated
by scraping back and forth with a spatula on the screen;
otherwise, an unnecessarily large amount of valuable material
is frequently withheld on the screen. The filtrate is centrifuged,
resuspended in 50 ml of culture medium, once more centri
fuged, and freed from a top layer of debris.
This last sediment is suspended in an appropriate amount of
culture medium at room temperature. Immediately after the
addition of acid-solubilized collagen (30 ^g/ml), the suspen
sion is plated onto tissue culture dishes (1.5 ml/35-mm dish)
and incubated at 37°in a humidifed atmosphere of 5% CO2 in
air. Plating should be performed from a stirred suspension by
means of a hand-driven dispenser ("repipeter")
with no less
than 105 cells/plate in order to keep differences in cell numbers
between plates seeded from the same suspension below 10%.
In order to prevent significant sedimentation in the dispensor
between the platings of consecutive dishes, attached Teflon
tubings should be as short as possible, and their inner diameter
should not exceed 2 mm. With the amount of material de
scribed, this procedure yields a suspension of cell aggregates
containing between 5 x 106 and 2 x 10s cells, depending on
the individual tumor. The time required for the explantation,
from the moment of killing the host animal to the moment of
transferring the primary cultures to the incubator, should not
exceed 1.5 hr. Medium changes (2 ml/35-mm dish) are per
formed every 2 days with culture medium containing collagen
(6 jug/ml).
Secondary cultures are produced by applying the same
enzymatic procedures on the primary cultures as described
here for the preparation on primary cultures from in vivo tissue.
Solutions for Tissue Disintegration
PBS (pH 7.3)
NaCI
KCI
KH2PO.
8.0 g
0.2 g
0.2g
Na2HPO4
D-Glucose
H20
1.15g
4.0 g
to 1000 ml
Characteristics
Trypsin Solution (adjust pH 7.5 with NaOH)
Trypsin
EDTA
HEPES
50 mg/liter
2 x 10~4M
Trypsin (21 7 units/mg)
pore.
1.6 x 10"'
NaCI
KCI
D-Glucose
10~2M
10~2
M
M
2.2 X 10~2M
was obtained from Worthington/Milli-
Collagenase Solution (pH 7.5)
Collagenase
2 g/liter
1.25 x 10~
CaCI2
HEPES, NaCI, KCI, D-glucose as given for trypsin solution.
Collagenase (type CLS III, 125 units/mg) was obtained from
Worthington/Millipore.
Dispase Solution (pH 7.5)
Dispase
2 g/liter
CaCI2
2.5 x 10"" M
HEPES, NaCI, KCI, D-glucose as given for trypsin solution.
Dispase was obtained from Boehringer Mannheim (Grade II).
Hyaluronidase
Hyaluronidase
EDTA
600 mg/liter
2 x 10"" M
Solution (pH 4.0)
2-(N-morpholinojethanesulfonic acid
3 x 10~2 M
NaCI, KCI, D-glucose as given for trypsin solution. Hyaluroni
dase (bovine testes; type I-S, 270 NF units/mg) was obtained
from Sigma Chemical Co.
Culture Medium I
This is a modification of the medium described by Murakami
and Masui (9). All further supplements mentioned in "Results"
are added to this medium immediately before plating.
DME
6.2 g
EGFNa2SeO3NaSiO3NiSO«SnCI2MnSO4(NH4)6Mo,O2
jig5
F-12
NaHCC-3
HEPES
Penicillin G, K* salt
Streptomycin sulfate
Ampicillin
Insulin
Transferrin
Triiodothyronine
10-9M8
X
4.9 g
M8X 10'"
1.1g
M8 X
3.6g
120 mg
270 mg
25 mg
2 mg
2 mg
2 X 10"'°l
IO'"
M1.7
X 10""
10-'°1.7
X
10^'°8
X
MMM1.7
X 10~'°
10~8Mto
X
1 000
ml
Insulin, transferrin, triiodothyronine, and EGF are added sep
arately immediately before plating as small volumes of concen
trated stocks. After suspending cells for plating in this medium
at room temperature, add 30 mg of acid-solubilized collagen
Table 1
of the tumors described in this work
For passage from one host animal to another, tumors were explanted, minced, and washed as described for primary
cultures in "Materials and Methods." The washed and centrifuged mince was injected (s.c. 0.2 to 0.5 ml/animal) in the
dorsal part of the thorax, using 18-gauge needles. Tumors were passaged, when reaching a volume of 3 to 5 ml. Details
of enzymatic treatment for obtaining primary cultures from colon carcinomas are given in "Materials and Methods."
TumorT
Cessationulation-doupopgrowthbling
Pronounced
of
within
mousepassage 7vitro time in multinucleano.34-3725-2924-2717-1912-157-85-74-63-52-32-3Minimal
vitro22.52.544.5
(days)
tion in vitro days in
of enzymatic
(min)after
treatment
additionof
each
enzyme to cul
ture dish55522525222
84T219T
183T
157T
245T
+2.52-4
348T
+7
+
362T
347T
+73.52
+
380T
379T
-Time
398OriginColonColonColonRectumColonColonColonColonColonColonColonNude
612
CANCER
RESEARCH
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41
Primary Tissue Cultures of Human Colon Carcinomas
per liter of medium and plate immediately. DME and F-12 were
obtained from Grand Island Biological Company. Purified acidsoluble bovine dermal collagen in acetic acid (pH 3) was
purchased from Collagen Corporation, Palo Alto, Calif.
Extracts from Tumors and Mouse Embryos
Tumors and embryos were minced as described previously.
The minces were stirred for 30 min with 2 volumes of PBS and
then centrifuged at 50,000 x g for 3 hr. The supernatant was
frozen in 1-ml portions in an acetone:dry ice mixture and stored
at -20°. For experiments, 2 to 4 ml of extract were added per
100 ml of medium. Then, the medium was filtered sterile (pore
size, 0.2 ¡im).
Quantitative Evaluation of Growth
Cell numbers in tissue cultures from tumors of epithelial
origin are frequently difficult to evaluate, since enzymatic treat
ment of these cultures either gives rise to a polydisperse
suspension of cell aggregates, which often contain cells of
varying size with unknown size frequency distribution, or, under
more drastic conditions, leads to single-cell suspensions at the
expense of loss of an unpredictable percentage of cells. A way
out of this dilemma is the procedure outlined below. This
procedure gives the details for quantitative preparation and
counting of nuclei from such tissue cultures and was adapted
for the special needs of these tissues from a method described
for microfluorimetric determination of nuclear DNA contents
(13).
Cultures are washed 4 times with 5 ml Solution A and 4 times
with Solution B, the washes with Solution B following each
other in rapid succession. All solutions are applied at room
temperature. Volumes given are for tissue cultures on 35-mm
plates. Immediately after the last wash, 0.8 ml detergent Solu
tion C is applied per plate. Under this condition, cells swell and
lyse. In order to facilitate cell lysis and detachment, it is useful
to swirl the dishes several times gently. After 5 to 20 min, the
swollen nuclei can be observed under the microscope floating
singly and in aggregates. In order to stabilize the nuclei against
prolonged action of the detergent and mechanical rupture
during pipeting, fixation is effected now by the addition of 3.2
ml formaldehyde Solution D. At this stage, contents of the
culture plate must not be pipeted. Addition of Solution D is
performed by a single stroke from a dispenser with a wide bore
opening, ensuring gentle and thorough mixing with the contents
of the culture plate.
After 10 to 20 hr, the contents (4 ml) of the culture plates
are transferred to counting vessels. In order to adjust to an
ionic strength appropriate for counting with the Coulter counter,
4 ml of Solution E are added. Aggregates of nuclei are dis
persed into single nuclei at this stage by gentle pipeting. For
further dilution, Solution F is used. With appropriate settings of
current, amplification, and threshold, the nuclei in such a
suspension can correctly be counted with a Coulter counter,
and good agreement can be obtained with nuclei counts in a
microscopic counting chamber (2). The stability of such nuclear
suspensions is quite satisfactory; counts taken 3 hr after the
first measurement do not deviate significantly from the original
values (2). Two culture dishes were evaluated per data point.
Differences between duplicates rarely exceeded 10%.
FEBRUARY
1981
Solutions
Solution A, 0.9% NaCI solution:10 HIM HEPES, pH 7.5;
Solution B, 2 HIM EDTA:1 HIM HEPES, pH 7.5; Solution C,
0.1% Nonidet P-40:2 rriM EDTA:1 mw HEPES, pH 7.5; Solution
D, 3 row HEPES (pH 7.5):10% formaldehyde solution (37%
formaldehyde: 10% methanol in H2O); Solution E, 1.8% NaCI
solution:20 mw HEPES, pH 7.4; Solution F, 0.9% NaCI solu
tion:! 0 HIM HEPES, pH 7.4. Nonidet P-40 was obtained from
BDH Biochemicals, Ltd., Poole, England.
Sources of Substances
Glucagon, transferrin, insulin, triiodothyronine,
hydrocortisone, acetylcholine chloride, ascorbic acid, histamine, sero
tonin, Substance P, neurotensin, linoleic acid, oleic acid, ethanolamine, and phosphoethanolamine
were obtained from
Sigma Chemical Co. (St. Louis, Mo.). Fatty acid-free albumin,
methionyl-enkephalin were from Miles Laboratories, Inc. (Elkhart, Ind.). EGF was from Collaborative Research, Inc. (Waltham, Mass.). Gastrin was from United States Biochemical
Corp. (Cleveland, Ohio).
RESULTS
The 10 tumors of the colon and 1 of the rectum investigated
here (Table 1) display a variety of diverging properties in vivo
as well as in vitro. For example, sensitivity to disintegrating
actions of several enzymes; contents of stroma, necrotic de
bris, and mucoid material; as well as spatial organization vary
widely among the different tumors. Nevertheless, the methods
of tissue disintegration and quantitation of growth by means of
counting cell nuclei, as described above, yield satisfactory
results with all these tumors.
Single-cell suspensions prepared from tumors by prolonged
enzymatic treatment underwent cellular disintegration during
the first few days of culture and did not give rise to growth to
any extent. In fact, the primary plating of cell aggregates, as
described here, has an advantage over single-cell seedings in
that it diminishes selection due to differing sensitivities among
the cells of a tumor against the disintegration procedure. Fur
thermore, the original cellular arrangement in the tumors is
maintained in these aggregates.
Quantitative attachment of the cell aggregates to the culture
plates occurs during 10 to 20 hr after seeding (Fig. 1). Spread
ing of the cell aggregates on the culture plates occurs within
the following 30 to 50 hr (Fig. 2).
During many repeated experiments, the primary in vitro
growth characteristics as shown in Charts 1 to 11 have proven
to be reproducible properties of individual tumors. Growth is
observed to start in cultures of all 11 tumors at the first day
after seeding with minimal population-doubling
times ranging
from 2 to 7 days (Table 1). At the same time, abundant cell
death is observed in these cultures, an in vivo characteristic of
normal intestinal epithelium as well as of tumors originating
from this tissue. The presence of morphologically differing cells
in these cultures as well as of 3-dimensional glandular struc
tures, increasing in size and number with culture age, suggests
that differentiation and morphogenesis take place similarly to
processes characterizing the in vivo tissues.
In 7 cases, continuous growth in defined medium has been
followed for up to 24 days (Charts 1 to 7). One tumor (T 380)
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J. van der Bosch et al.
and growth rates were comparable to the ones observed in
primary cultures. This is true even in cases in which primary
cultures had entered a phase of constant or decreasing cell
number before being subcultured (Chart 1).
T 84
5-10'
T 183
2-10«
IO1
5-10s
10*
5-10»
2-10s
12
16
20
24
DAYS AFTER PLATING
Chart 1. T 84 primary cultures were plated in Culture Medium I (/). Supple
ments" 2 to 10 were added from Day 1 on. Õ,Culture Medium I; 2, EA, PEA, LA,
2'10s
A; 3, G;4, EE 11: 5. EE 12; 6, EE 18; 7, TE 84; 8, TE 183; 9, TE 219; i O, FBS.
Concentrations
Table 2
of supplements as used in this work
10»
8
medium0.5
in
SupplementsAlbuminAcetylcholineAscorbic
mg/ml10/ig/ml20
acidEthanolamineMethionylenkephalinFetal
/ig/ml2
M2X 10~8
10~'M5
X
serumGastrinGlucagonHistamineHydrocortisoneLinoleic
bovine
/¿I/ml200
.ig/ml200
ng/ml5
fig/ml2
12
16
20
24
DAYS AFTER PLATING
Chart 2. Growth of T 183 primary cultures in Culture Medium I.
10'
T 348
10~8M2.5/ig/ml10~7
X
acidNeurotensinOleic
M2.5^9/ml10"'
acidPhosphoethanolamineSerotoninSubstance
M5/ig/ml10~7M
PAbbreviationAAchAseEAEnkFBSGGlueHHCLANTOAPEASSPConcentration
Embryo extracts
11-day embryo
12-day embryo
18-day embryo
19 day embryo
19-day embryo + placenta
20-day embryo
EE
EE
EE
EE
EE
EE
11
12
18
19
19420
20/il/ml
20/il/ml
20/il/ml
20/il/ml
20/il/ml
20/il/ml
E
10«
Tumor extracts
T 84
T 183
T219
T 245
T 347
T 362
T 380
TE 84
TE 183
TE219
TE 245
TE 347
TE 362
TE 380
20/il/ml
20 ni/ml
20 (il/ml
20/il/ml
40 /il/ml
20 ni/ml
grew continuously only in the presence of a tumor extract
(Chart 8). Subcultures have been prepared from 5 primary
cultures up to now (T 84, T 183, T 348, T 219, and T 398).
Growth was observed to occur in all of them and was followed
quantitatively in 3 cases (Charts 1 to 3). Growth in secondary
cultures was resumed with a lag period not exceeding 1 day
4 Abbreviations
and concentrations
ends are explained in Table 2.
614
of supplements
mentioned in chart leg
10*
8
12
16
20
DAYSAFTERPLATING
Chart 3. T 348 primary cultures were plated in Culture Medium I ( 1). Supple
ments 2 to 12 were added from Day 1 on. 2, HC; 3, Glue; 4, S; 5, G; 6, EA, PEA;
7, EA, PEA, LA, A; 8, EA, PEA, OA, A; 9, H; 10, HC, Glue, S, G; /1, S, G; T2. EE
18.
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41
Primary Tissue Cultures of Human Colon Carcinomas
even after appreciable
cell number decrease
had occurred
(Fig. 3). In the third case (T 245), the cells did not undergo
T 157
5-105
such obvious changes. They displayed
a rather normal cellular
morphology
(Fig. 4) comparable
to tumors, which did not stop
growing during the time of observation
[e.g., T 379 (Fig. 5)].
None of the supplements
added
thus far to the basic defined
8
12
2-105
T 379
E
5-105
E
5-10'
2-105
a
o
2-10'
8
12
16
24
20
10«
0
16
20
DAYS AFTER PLATING
Chart 4. T 157 primary cultures were plated in Culture Medium I ( 7). Supple
ments 2 and 4 to 12 as in Chart 3.
DAYS AFTER PLATING
Chart 6. Growth cf T 379 primary cultures in Culture Medium I.
10'
T219
T 398
5.10'
2-10"
E
I
I
10«
10*
5-10s
2-10!
0
4
8
12
DAYS AFTER PLATING
Chart 7. Growth of T 398 primary cultures in Culture Medium I.
to>
T 380
12
16
20
2-10s
DAYSAFTERPLATING
Charts. T 219 primary cultures were plated in Culture Medium I (7) at 2
different densities. Supplements 2 to 10 were added from Day 1 on. 2. G: 3. EE
11; 4. EE 12; 5, EE 18; 6, TE 84; 7, TE 183; 8. TE 219; 9. TE 362; IO, FBS.
In primary cultures from 3 tumors (Charts 9 to 11 ), net growth
ceased within 7 days after seeding and was followed by a
period of constant or decreasing cell numbers. In 2 of these
cases (T 347 and T 362), a pronounced increase of multinucleated cells, accompanied by the development of large vacu
oles, took place in some ranges of these cultures during the
first week after seeding (Fig. 3). Other areas in the same
cultures were still populated by cells of original morphology,
FEBRUARY
/. l 3.
4-10'
8
12
16
DAYS AFTERPLATING
Chart 8. T 380 primary cultures were plated in Culture Medium 1(7). Supple
ments 2 to 4 were added from Day 1 on. 2, G; 3. G. S; 4. TE 347.
1981
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615
J. van der Bosch et al.
medium (containing insulin, transferrin, EGF, triiodothyronine,
and trace elements in a 1:1 mixture of DME and F-12 medium)
had a striking effect on the net growth rates observed. In only
one case [T 380 (Chart 8)] could a supplement (extract from
Tumor T 347) prevent the discontinuation of growth observed
in the basic medium. This same addition did not affect the
growth cessation observed in primary cultures of 3 other tu
mors (T 245, T 347, T 362).
Differences of population development due to different sup
plementation become frequently visible only in late phases of
primary cultures, characterized by different rates of cell number
decrease (Charts 1,4,5,10,
and 11 ), or in secondary cultures,
characterized by different initial growth rates (Chart 1). An
exception to this rule is observed following the addition of
linoleic acid bound to serum albumin. This supplement causes
a net cell number decrease during the initial phase of primary
cultures of some tumors [T 157 (Chart 4); T 347 (Chart 9)],
whereas others remain completely unaffected [T 84 (Chart 1);
T 348 (Chart 3); T 183 (not shown)].
An appreciable influence of the average cell density on the
population development in primary cultures is demonstrated in
Chart 5. At low density, a T 219 population continues to grow
for more than 16 days, whereas a pronounced cell number
decrease can be observed at 10 times higher cell density from
Day 12 on.
A correlation between the number of animal passages of the
10"
-
T362
5-10s
2-10s
4
8
12
16
DAYS AFTERPLATING
Chart 10. T 362 primary cultures were plated in Culture Medium I ( T). Suppplements 2 to 9 were added from Day 1 on. 2, HC; 3, Glue; 4, HC, Glue; 5, EA,
PEA; 6, Glue, HC, EA, PEA; 7, TE 183; 8. TE 362; 9, EE 18.
3-10s
T245
2-10«
HI1'
12
16
20
DAYSAFTERSEEDING
Chart 11. T 245 primary cultures were plated in Culture Medium I (Curve a),
CMI plus EGF (40 /ig/liter) (Curve o), and CMI plus EGF (400 /ig/liter) (Curve c),
and kept under these conditions throughout the entire experiment.
tumors investigated here and in vitro properties like populationdoubling time or development of multinucleation could not be
detected during the limited period of time covered by this
investigation (Table 1).
T 347
10'
5% FCS
DISCUSSION
The most remarkable observation of the present study is the
fact that all 11 tumors display substantial growth during the
initial phase of primary cultivation. This is good evidence for
the presence of an appreciable, viable "stem cell population"
10s
18
2-10«
12
16
20
DAYS AFTER PLATING
Chart 9. T 347 primary cultures were plated in Culture Medium I (/) at 3
different densities, a, fa, and c. Supplements 2 to 20 were added from Day 1 on
with medium changes every 2 days (a). In b and c. medium changes were
performed daily and 5% FBS (FCS) was added on Day 7. 2. EE 11 ; 3, EE 12; 4,
EE 18; 5, EE 19; 6, EE 19 + ; 7, EE 20; 8, TE 84; 9, TE 183; 10. TE 219; 11, TE
245; 12. TE 347; 73, TE 362; 14, TE 380; 75, G; 16, S; 17, EA, PEA; Õ8,EA,
PEA, LA, A; 79, FBS; 20, G, Asc, Ach, S, SP, NT, Enk.
616
in the primary expiants. Furthermore, this stem cell population
seems to constitute a rather constant fraction of the expiants,
inasmuch as initial growth rates show good reproducibility in
different explantations of the same tumor. However, quantita
tive and/or qualitative (progression-like) changes of the stem
cell fraction, which may take place during multiple animal
passages of the tumors, are not in the scope of the present
investigation, which has been carried out over a period of time,
not exceeding 4 consecutive animal passages in any case.
The growth rates of secondary cultures, adjusting them
selves readily to the values observed in primary cultures
(Charts 1 to 3), indicate, that, in the serum-free medium used
here, a substantial and intact part of the stem cell fraction
survives even stationary phases or phases of net cell loss of
primary cultures (Chart 1), as well as the procedures of de
tachment and reseeding. This observation and the fact that
total cell number increases (primary and secondary cultures)
by factors of up to 90 have been recorded in the present study
show that the defined medium used actively supports the
growth of these cells.
The phenomenon of multinucleation observed in primary
cultures of 2 tumors (T 347, T 362), in coincidence with an
initial increase of the number of nuclei per culture, suggests
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Primary Tissue Cultures of Human Colon Carcinomas
that replication mechanisms, in these cells are unperturbed up
to the phase of mitosis. Multinucleation is invariably accom
panied by pronounced enlargement of the cellular surface of
attachment to the culture dish. Thus, the reason for multinucleation might be a difficulty of detachment during mitosis.
Therefore, further work is aimed at the elucidation of the
influence of different culture substrates on the development of
these primary cultures.
We would like to stress that the quantitative investigation of
primary cultures of cells of epithelial origin, as described in the
present work, has become possible only as a result of the
development and use of serum-free culture media, designed
for the specific needs of these cells. Such media prevent
completely the growth of fibroblasts and thus overcome one of
the most irritating obstacles in the way of obtaining reproduci
ble primary cultures, which are representative of the in vivo
tissue and can be subjected to quantitative in vitro measure
ments.
On the basis of this progress, it is planned to characterize
the tumorigenicity of colon tumor expiants in the nude mouse
quantitatively and to investigate changes of this parameter
occurring during in vitro culture.
It is hoped that the approach described here will allow
evaluation of primary cultures of human tumors taken directly
from patients and provide a basis for clinical investigation of
individual cases.
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617
Fig. 1. T 84 primary culture in serum-free culture medium 1 day after seeding. Fixed with methanol. Stained with Giemsa's. a, x 120; b, x 400.
Fig. 2. T 219 primary culture in serum-free medium 3 days after seeding. Fixed with methanol. Stained with Giemsa's. x 400.
Fig. 3. T 347 primary culture in serum-free medium 16 days after plating, showing simultaneously areas of multinucleation (a) and of normal morphology
Fixed with methanol. Stained with Giemsa's. x 400.
Fig. 4. T 245 primary culture in serum-free medium 23 days after plating. Fixed with methanol. Stained with Giemsa's. x 400.
Fig. 5. T 379 primary culture in serum-free medium 23 days after plating. Fixed with methanol. Stained with Giemsa's.
x 400.
618
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(b).
Growth Characteristics of Primary Tissue Cultures from
Heterotransplanted Human Colorectal Carcinomas in
Serum-free Medium
Jürgen van der Bosch, Hideo Masui and Gordon Sato
Cancer Res 1981;41:611-618.
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