[CANCERRESEARCH
37, 3639-3643,October1977]
A Simplified Method for Production and Growth of
Multicellular Tumor Spheroids
John M. Yuhas, Albert P. LI, Andrew 0. MartInez, and Aaron J. Ladman1
CancerResearchand TreatmentCenter(J.M. Y.,A. P. L., andA. 0. M.Jand Departmentsof Radiology(J.M. Y.,A. P. L.J,andAnatomy(A.J. L.J,Universityof
New Mexico, Albuquerque, New Mexico 87131
SUMMARY
A new technique, based on the growth of tumor cells in
liquid media over an agambase, has been developed for the
research, we initiated attempts to develop a simple method
forproducingand growing MTS, and our resultsare me
ported below.
formation and growth of multicellular tumor spheroids. All
of the 11 transformed cell lines tested formed multicellular
tumor spheroids, while none of the 8 normal cell types
tested did so. The advantages of the present technique over
olden methods include its simplicity, generality, and expeni
mental flexibility.
INTRODUCTION
MTS2 offer many of the characteristics of in vivo tumors,
which are unavailable in monolayer or suspension culture
(8). These include intimate cell-cell contacts (2), chronically
hypoxic cell populations (7), and cycle times that range
from comparable to exponential monolayer rates through
essentially nondividing (3). In brief, they combine the rele
vance of organized tissues with the accuracy of in vitro
methodology.
Of the method proposed for their production and growth,
the most adequate is the spinner flask method (9). In this
method, tumor cells are maintained in spinner flasks, and
the constant movement prevents their attachment to the
walls of the vessel and allows them to attach to each other
and grow. While this spinner flask method overcame many
of the limitations [e.g., the diffusion limitations of colonies
grown in semi-solid agar (5)], it has not been used by a large
number of investigators, nor has it been used in areas of
cancer research other than tumor radiobiobogy (9). Presum
ably, the reason for the lack of general interest in MTS is
primarily a technical problem. The technique is difficult,
and those who have mastered it are primarily interested in
tumor radiobiobogy. Furthermore, the empirical methods (9)
required to adapt any given tumor to this method have
limited the array of tumors that are available for study.
Finally, the need for large volumes of reagents, the inability
to study individual MTS for prolonged periods, and other
requirements of the system make it ill adapted for certain
types of investigations.
Since MTS could prove useful in many areas of cancer
I
Supported
by
the
Division
of
Cancer
Centers
and
Resources,
National
Cancer Institute, through Grant 1-P30-C-21074.01.
2 The
abbreviations
used
are:
MTS,
multicellular
tumor
sphcroids;
Eagle's basal medium; HBSS, Hanks' balanced salt solution.
Received April 11, 1977; accepted June 30, 1977.
EBME,
MATERIALS AND METHODS
Cells. A total of 19 different cell types were used in the
present investigations, 8 normal and 11 transformed (Table
1). Cells were classified as normal unless they produced
tumors in appropriate hosts, formed colonies in soft agam,
or lacked contact inhibition in monolayer. All normal cell
samples were obtained from apparently normal tissues in
vivo, whereas transformed cells were obtained from ob
vious tumors.
Monolayer cultures were maintained (100% relative hu
midity; 95% aim + 5% CO2; 37°)in either EBME or F-12,
supplemented with 10% fetal calf serum, 50 units of penicil
bin per ml, and 50 @g
per ml of streptomycin (Grand Island
Biological Co., Grand Island, N.Y.). For the data presented
below, all cultures were harvested by mild trypsinization
(0.25% w/v, 3 to 5 mm at 37°),but similar results can be
obtained by scraping the cells off the surface.
The single primary normal tissue studied (adult lung from
a C3H mouse) and both transplanted tumors (FSA and Line
1) were harvested by mincing in 0.9% NaCI solution, fol
bowed by cell dissociation with a Teflon and glass tissue
grinder. The single-cell suspension was pelleted and resus
pended either in 0.9% NaCI solution for transplant or com
plete EBME for attempted production of MTS.
MTS Production.Arguingthat the lackof an appropriate
surface for cell attachment might promote MTS formation,
just as constant agitation does in the spinner flask system
(9), we compared 3 methods for producing MTS, all of
which involved stationary plates maintained in a standard
tissue culture incubator (100% relative humidity; 95% air +
5% CO2; 37°).Approximately 10@cells in 10 ml of EBME (as
above) were added to 100-mm plastic Petri dishes (Falcon
Plastics, Oxnard, Calif.)that (a) had not been treated for cell
attachment, henceforth referred to as bacteriological
plates; (b) had been base-coated (2 to 3 mm) with 0.5%
Noble agar (Difco Laboratories, Inc., Detroit, Mich.) in
HBSS, henceforth referred to as agar-HBSS plates; or (C)
had been base-coated (2 to 3 mm) with 0.5% Noble agar in
complete EBME, henceforth referred to as agar-EBME
plates. The plates were then returned to the incubator and
observed for up to 30 days. No agitation or mocking was
used in any of the experiments.
OCTOBER1977
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3639
J. M. Yuhas et a!.
MTS Growth.MTS (n = 24) were harvestedfrom9- to 14- tested were fewer than 100 MTS produced within the 1st 3
day-old agar-EBME plates and were transferred individually
into 16-mm agar-EBME wells (Costar, Cambridge, Mass.)
containing 1 ml of EBME. All MTS, within a group, were the
same size at the time of harvest: MCa-11 , 140 @m;
FSA, 224
@m;line 1, 168 @m.Subsequent experiments (data not
shown) have demonstrated that estimated growth matesdo
not vary as a function of size at harvest over the mangeof 100
to 600 @m.For this growth study, media were changed
daily, at which time the MTS were sized on a dissecting
microscope (x40).
weeks with an inoculum of 106 cells.
Fig. 1A is a scanning electron micrograph of a 420-sm
MTS derived from the highly malignant line 1 lung carci
noma (10). Individual cells possess multiple microvilli and
are loosely packed within the MTS. A further description of
the surface morphology of this and other types of MTS will
be provided elsewhere.
Figs. lB through iF are autoradiographs of sections
taken through the center of line 1 MTS of increasing size
(280 to 840 sm). At the smallest size shown (Fig. 18, 280
The s.c. TumorGrowth.Approximately10@
tumorcells(in pm), virtually all of the nuclei are labeled, indicating that all
0.2 ml of 0.9% NaCl solution) from 3 tumor lines were of the cells are in cycle and had passed through DNA syn
injected s.c. into the night leg of 16-week-old, female, syn thesis during the 24 hr of exposure of [3H]thymidmne. As
geneic hosts (BALB/c mice for line 1 and MCa-11 and C3H MTS size increases (Fig. 1, C to F), a nondividing but viable
for FSA). Tumors arose within 7 to 8 days and were sized central region develops followed by, with further growth, a
with vernier calipers through the 32nd day posttnansplant.
central necrotic come.As shown in Fig. 1C, the viable por
Tumor size is expressed as the average of the 2 perpendicu
tion of the MTS can be divided into 3 general areas: (a) the
lamdiameters.
outermost shell in which almost all cells are labeled; (b)
Autoradlography. Individual MTS were placed in 16-mm immediately beneath that a shell in which approximately
agar-EBME wells containing 1 ml of EBME + 2 @Ciof 50% of the cells are labeled; and (C) the innermost viable
[3H]thymid me (Amersham-Searle, Arlington Heights, III.; shell, immediately adjacent to the necrotic core, in which
specific activity, 6.7 Ci/mmole). Twenty-four hr later the none of the cells are labeled. As suggested elsewhere (1),
MTS were fixed and processed according to standard tech
we interpret this depth dependence for percentage of cells
niques.
labeled as being the product of declining oxygen concen
trations between the periphery and center of the MTS. Di
rect evidence supporting this conclusion will be provided
RESULTS
elsewhere.3
MTS versusin Vivo Growth Rates. One of the major
MTS Production and Morphology. Normal cells (Table 1),
advantages of MTS is that they simulate in vivo tumors
when added to bacteriological, agar-HBSS, or agar-EBME
momphobogically and hopefully should simulate them func
plates, formed cellular aggregates (slOO sm), which failed
to grow and broke apart within 72 hr. These normal cell tionally. To test this possibility, we compared the in vivo
growth matesof line 1, FSA, and MCa-11 with their growth
clumps contained viable cells through 48 hm,as evidenced
matesas MTS. Chart 1 is a plot of the mean diameter of the 3
by the ability of these aggregates to reestablish monolayer
tumors as a function of time after s.c. transplantation. As
cultures when placed in Petmidishes that had been treated
pointed out elsewhere (11) the growth matesof FSA and line
for cell attachment. By 72 hm,the number of surviving non
mal cells in the few clumps remaining was insufficient to 1 are indistinguishable and averaged 0.56 ±0.07 mm/day
reestablish monolayer growth under appropriate condi
(Chart 1) in spite of the fact that they differ markedly in their
tions.
immunogenicity, with the former (12) being far more immu
nogenic than the latter (6). The growth mateof MCa-11 (0.23
Similar results were obtained with tumor cells in the bac
teriological
plates,
but intheagam-HBSS platesQT-A31,K- ±0.04 mm/day) is far bower. Chart 2 is a plot of mean MTS
A31 , MCa-11, FSA, and line 1 formed cellular aggregates diameter as a function of time for the same 3 tumor cell
lines. In this experiment, media were changed daily in order
(Fig . 1A) that continued
to grow, while SV-A31 and BP-A31
formed aggregates that grew slowly then broke apart. With to avoid the possibility of nutrient limitation. Subsequent
experiments (data not shown) have demonstrated that me
the agar-EBME plates, all 11 tumor lines formed cellular
aggregates that continued to grow, while repeated testing dia can be changed as infrequently as once per week with
of the 8 normal cell types yielded consistently negative out reducing the growth rate for most of the MTS studied.
As was the case for the tumors growing in vivo (Chart 1), the
results (Table 1). We refer to these growing cellular aggre
growth matesfor line 1 and FSA (Chart 2), when grown as
gates,therefore,
as MTS.
The MTS that developed do not attach to the agar-EBME MTS in vitro, were indistinguishable and averaged 83 ±3
base but are freely movable. For the tumor lines listed in pm/day, while the growth matefor MTS derived from MCa
11 was far lower at 32 ±5 pm/day (Chart 2). Monolayer
Table 1, MTS (100
@m)appear within3 to 14 days and
growthmatesforthese3 lines
do notcorrelate
witheither
the
continue to appear through the time (3 to 7 days) at which
subcultuming into new agam-EBME plates is required due to in vivo on MTS growth mates,since all 3 lines show doubling
media exhaustion. In general, large cell inocula (10@to 106) times of 16 to 18 hr.
This lack of correspondence between monolayer and
result in more rapid MTS development, while lower num
bers yield a greater number of MTS per cell inoculated, but MTS growth matesis a reflection of the fact that not all cells
they take longer to appear. For 1 tumor, at least, the line 1
lung carcinoma, MTS can be produced with as few as 100 Oxic and Hypoxic Cells in the Presence of Both Aadioprotective and Radio
cells/100-mm agan-EBME dish. In none of the tumor lines sensitizing Drugs, submitted for publication to Radiation Research.
3 J.
3640
M.
Yuhas,
and
A.
P.
Li.
In
Vitro
Studies
on
the
Radioresistance
of
CANCER RESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Spheroid Growth
Table1
Capabilities of normal and transformed cells' from 3 species to form MTS. None of the normal cells formed MTS, while all of the
transformed cells formed them readily
A. Normal cells
PropagationRemarksBALB/c
SourceCell
typeDesignationb
2BALB/cmouseEmbryonic
lungAL-iMCTested
2BALB/cmouseEmbryoAL-2MTested
2C3H mouseAdult
lungAL-3MTested
2C3HmouseLung
fibroblastsAL-4MTested
mouseAdult
lungPBALB/c
mouse3T3,
A31A31MHuman,
clone
at passages0, 1, and
at passages0, 1, and
at passages0, 1, and
at passages0, 1, and
newbornFibroblast75-69MHuman,
fetusFibroblast75-86
cellsMSourceOriginal
RemarksC3H
B. Transformed
cell typeTransforming
tion
agentDesignationPropaga
mouseConnective
MTSC3H
tissueSpontaneousL-cellsM
mouseFibroblastsMethylcholanthreneFSAMC3H
mouseFibroblastsMethylcholanthreneFSATPBALB/c
mouseType
1MBALB/c
II lung alveolar cellSpontaneousLine
mouseType
ITPBALB/c
II lung alveolar cellSpontaneousLine
mouseMammary
epitheliumRadiationMCa-11MBALB/c
mouse
clone A31
BALB/c mouse3T3,
MBALB/c
3T3, clone A31Methylcholanthrene
SV4OQT-A31
mouse3T3,
clone A31Kirsten
virusK-A31MBALB/c
muninesarcoma
mouse3T3,
A31BenzopyreneBP-A31MChinese
clone
hamster
All formed
SV-A31M
Chinese
hamsterFibroblast Ovarian epitheliumMethylcholanthrene
MHumanCervical
SpontaneousBi4-150
CHO-K,M
epitheliumSpontaneousHeLaM
a Cells
were
classified
as
normal,
unless
they
produced
tumors
in animals,
grew
in soft
1 , MCa-1
1 , and
agar,
or
showed
a piled
up
morphology
in
monolayer culture.
b Cell
lines
were
derived
in
our
own
laboratory
(AL-i
, AL-2,
AL-3,
AL-4,
Line
primary
C3H
lung)
or
were
a gift
from
Dr.
G.
Martin, Universityof Washington(75-69,75-86,L, Bi4-150, and HeLa);Dr. A. Tennantand Dr. A. W. Hsie, Oak Ridge National Laboratory
(A31, QT-A31,SV-A31,K-A31,and BP-A3i); or Dr. H. A. Withers, M. 0. Anderson Hospital and Tumor Institute (FSA).
C M,
monolayer;
P,
primary;
TP,
transplant.
in the MTS are in division just as is the case in vivo, in
addition to possible differences in the growth rate of the
respective individual cells.
L-15, and Dulbecco's Modified Eagle's Medium), and using
at least 2 sources of agan, Noble agar and special Noble
agam(Difco). The reason for our success is not only the lack
of a surface for attachment; but it must also include nutri
tional factors from the agam-EBME combination, since bac
DISCUSSION
temiobogical plates did not support growth of the tumor cell
aggregates that formed. We point out the range of expemi
The data presented above have demonstrated 3 points: (a) mental conditions that have allowed MTS production in
order to emphasize the fact that successful use of this
MTS can be produced very simply from a variety of tumor
method does not require unique conditions.
cell sources; (b) 8 normal cell types do not form cellular
aggregates that are capable of growth; and (C) growth rates
The experimental flexibility of the agar-EBME (or agar
of MTS show a better correlation with in vivo tumor growth
media) system is readily apparent, as is its adaptability to
rates than do monolayer cultures.
many areas of cancer research. With relative ease 11 differ
The technique is far simpler than any of the other meth
ent tumors are now available as MTS, but this does not
ods (5, 9) presently used to produce MTS, and it is far more necessarily mean that all solid tumors will form MTS in our
adaptable to a variety of problems in cancer research. We system. In fact, we are presently attempting to select both in
have successfully produced MTS using agam-EBME Petmi vitro and in vivo for variants of MTS-forming tumors that can
dishes, ranging in size from 35 to 100 mm, and made of no longer do so, in much the same way that Fidler (4) has
glass (Pyrex) or plastic, from a number of manufacturers
selected for greater metastatic potential. In brief, this
(Falcon; Linbro Chemical Co., New Haven, Conn.; and Cos
method allows the long-term study (30 to 60 days) of individ
tar), using fetal calf serum from various batches and sup
ual MTS from a variety of solid tumors in medium volumes
pliers (Grand Island Biological Co. ; Microbiological Associ
as small as 1 ml. Hopefully, the combination of simplicity
ates, Inc., Bethesda, Md.; Pacific Biobogicals, Richmond, and experimental flexibility will allow the use of this method
Calif. ; Kansas City Biological, Lenexa, Kans.), using cell for a variety of experimental procedures.
concentrations of 102to 10' per 100 mm agar-EBME plate,
The fact that the 8 normal cell types studied could not
using media from various formulations (EBME, F-12, F-10, form aggregates that were capable of growth suggests that
OCTOBER1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
3641
J_ M. Yuhas et a!.
I
a basic difference exixts between normal and transformed
I
S. C. TUMORS
20 -
cells and that our MTS system relies on this difference for its
function. Whether or not other methods can produce MTS
from normal cells is not the problem, however, since the
agar-EBME system appears able to discriminate between
the 2 types of cells.
The last point concerns the proportionality between tu
FSA /
/
I
E
.!
momgrowth rates in vivo (Chart 1) and the growth of the
same tumors as MTS (Chart 2). The growth rates in the 2
l@
LU
w
4
0
:D
I-
z
4
w
@-ll
5
systems are not identical but proportional, suggesting that
the in vivo factors that affect growth rate (non-tumor cell
infiltration, stromal elements, growth factors, mateof dead
cell clearance, immunological inhibition, etc.) operate simi
lamlyon all 3 tumors. The fact that MTS grow at a rate that is
proportional to their in vivo growth rate, while monolayers
do not, further suggests that the organized nature of MTS
allows for greater expression of inherent growth character
istics and lessendependence on the particulars of the media
used. In addition,
I
I
I
I
20
10
I
I
30
growth in the MTS system is a function
of
not only cellular doubling times but also the size of the
growth fraction. Preliminary data indicate that the size of
the growth fraction varies widely among MTS from different
tumors. These data are presently being expanded and will
be reported elsewhere.
DAYS
Chart 1. The s.c. tumor diameter as a function of time after transplanta
tion for 3 murine tumor lines. Five x 10' tumor cells were transplanted s.c. in
the right leg, and tumors were measured (2 perpendicular diameters) with
vernier calipers. Line I and MCa-1I were grown in 4-month-old BALB/c
females, while FSA cells were grown in similar C3H mice.
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E
w
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4
z
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Chart 2. MTS diameter as a function of time for 3 murine tumor lines.
Individual MTS were harvested from 100-mm production plates, and placed,
along with 1 ml of EBME, In 16-mm agar-undcrlayed wells. Twelve MTS were
used per line, and the media were changed daily.
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in MiceBearingtheWeakly
Immunogenic
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Fig. 1. Line 1 alveolar cell carcinoma MTS. A, scanning electron microscopic view of a 420-g.m MTS. X 200. B to F, midline autoradlographic sections of
MTS that had been exposed to (‘Hjthymidine,
2 @Ci/ml,
for 24 hr prior to fixation. X 250. B, 280-sm MTS. C, 420-@&m
MTS. 0, 560-sm. MTS: 1, denselylabeled
outer shell; 2, lightly labeled intermediate shell; 3, nonlabeled innermost shell; NC, necrotic area. E, 700-sm MTS. F, M0-@m MTS.
3642
CANCER RESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
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OCTOBER1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
3643
A Simplified Method for Production and Growth of Multicellular
Tumor Spheroids
John M. Yuhas, Albert P. Li, Andrew O. Martinez, et al.
Cancer Res 1977;37:3639-3643.
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