Research Report
Simulated Microgravity Culture System for a
3-D Carcinoma Tissue Model
BioTechniques 33:1068-1076 (November 2002)
K. Nakamura, H. Kuga, T.
Morisaki, E. Baba, N. Sato,
K. Mizumoto, K. Sueishi, M.
Tanaka, and M. Katano
Kyushu University, Fukuoka,
Japan
ABSTRACT
An in vitro organotypic culture model is
needed to understand the complexities of
carcinoma tissue consisting of carcinoma
cells, stromal cells, and extracellular matri-
ces. We developed a new in vitro model of
carcinoma tissue using a rotary cell culture
system with four disposable vessels
(RCCS-4D) that provides a simulated mi-
crogravity condition. Solid collagen gels
containing human pancreatic carcinoma
NOR-P1 cells and fibroblasts or minced hu-
man pancreatic carcinoma tissue were cul-
tured under a simulated microgravity condi-
tion or a static 1g condition for seven days.
NOR-P1 cultures subjected to the simulated
microgravity condition showed greater
numbers of mitotic, cycling (Ki-67-posi-
tive), nuclear factor-κ B-activating cells,
and a lower number of apoptotic cells than
were shown by cultures subjected to the sta-
tic 1g condition. In addition, human pancre-
atic carcinoma specimens cultured under
the simulated microgravity condition main-
tained the heterogeneous composition and
cellular activity (determined by the cycling
cell ratio and mitotic index) of the original
carcinoma tissue better than static culture
conditions. This new 3-D rotary cell culture
system with four disposal vessels may be
useful for in vitro studies of complex pan-
creatic carcinoma tissue.
1068 BioTechniques
INTRODUCTION
Patients with pancreatic cancer have
a low survival rate (20). Although adju-
vant radiochemotherapy and new bio-
logical agents, such as angiogenesis in-
hibitors and matrix metalloproteinase
inhibitors currently in clinical trials (2,
20), provide modest prolongation of sur-
vival time, currently, surgical resection
is the only potentially curative treatment
(2). Pancreatic cancer differs from other
types of carcinoma in that it is highly in-
vasive and shows poor neovasculariza-
tion (32). For an in-depth understanding
of the complexities of pancreatic carci-
noma tissue, which consists of carcino-
ma cells, stromal cells, and extracellular
matrices, the establishment of an in vit-
ro organotypic culture system is needed.
Excess shear stress (>5 dyn/cm2)
may prevent the establishment of in-
tercellular junctional contacts (17).
The spatial collocation of cultured
cells and their assembly into tissue-
like organoids are achieved concomi-
tant with a reduction in fluid shear
stress to less than 0.5 dyn/cm2 (17).
Thus, several types of static collagen
gel matrix cultures in which shear
stress is 0 dyn/cm2 have been devel-
oped to establish organotypic 3-D cul-
ture models. The 3-D cultures display
both morphological and biochemical
characteristics that mimic those of in
vivo carcinoma tissue (10,15). Howev-
er, the cell density in gel matrix cul-
tures is severely limited by the diffu-
sion of nutrients and wastes through
the matrix (15).
Recent studies suggest that cells may
sense mechanical stress and that micro-
gravity influences cellular functions
such as proliferation (13,21), signal
transduction (3,11), and gene expres-
sion (4,23,31). A new 3-D culture sys-
tem that simulates microgravity (about
10-2g) on earth was developed at the
NASA Johnson Space Center (17). This
rotary cell culture system with four dis-
posable vessels (RCCS-4D; Synthe-
con, Houston, TX, USA) involves hori-
zontally rotating wall vessels (RWVs)
that provide controlled supplies of oxy-
gen and nutrients, with minimal turbu-
lence and extremely low shear stress
(8). In many studies that used RWV
bioreactors, cells were cultured on car-
rier beads (1,5,6,9,10,19, 24–26,30).
Using carrier beads to provide anchor-
age for minced tissue, some investiga-
tors have been successful in establishing
primary cultures under simulated mi-
crogravity conditions (10). Because the
RCCS-4D system uses microcarriers, it
has a high surface area/volume ratio that
facilitates nutrient and waste transfer.
However, carrier beads are not essential
for culture in RWV bioreactors (14). In
addition, the first report of an in vitro
culture in the RWVs of fresh human
melanoma specimens showed that the
RWV culture system provides a unique
model of human melanoma that mimics
the in vivo tumor environment much
more closely than do current culture
methods (18). Here we show that the
RCCS-4D system preserves the cellular
architecture, heterogeneous composi-
tion, and biological characteristics of
the original pancreatic tissue.
We hypothesized that the RCCS-4D
system can maintain the biological
characteristics of cancer cells in the
original tumor tissue. This is the first
report on the use of the RCCS-4D sys-
Vol. 33, No. 5 (2002)
tem for the in vitro culture of a conven-
tional 3-D model consisting of human
pancreatic carcinoma cells and human
fibroblasts embedded in type I collagen
gel. This microgravity-based RCCS-4D
system may provide new information
and an in-depth understanding of pan-
creatic carcinoma cell biology that is
difficult to elucidate in currently avail-
able embedded gel systems.
MATERIALS AND METHODS
Cells and Reagents
from metastatic subcutaneous tumor
tissue of a patient with advanced pan-
creatic carcinoma (28). The cells were
grown in tissue culture flasks; TIG-1-
20 cells were grown in DMEM (Invit-
rogen, Carlsbad, CA, USA), and NOR-
P1 cells were grown in RPMI 1640
(Invitrogen). Both culture media were
supplemented with 10% FCS (Invitro-
gen), 100 U/mL penicillin, and 100
µg/mL streptomycin. Aqueous type I
cow tendon collagen was purchased
from Koken (Tokyo, Japan).
3-D Collagen Gel Culture
pensed into 48-well plates at 400
µL/well and allowed to gel for approxi-
mately 30 min at 37°C before the addi-
tion of complete culture medium (Fig-
ure 1A, Step 1). After RPMI 1640
medium with 10% FCS was added, the
3-D models were incubated at 37°C
overnight in a humidified atmosphere of
5% CO2. The collagen gel cultures
showed slight contraction (Figure 1A,
Step 2). The solid gels were cultured un-
der either a static 1g condition or a sim-
ulated microgravity condition at 37°C
for seven days in a humidified atmos-
phere of 5% CO2 (Figure 1A, Step 3).

Human embryonic pulmonary fi-
broblast TIG-1-20 (JCRB501) cells
were obtained from Science Research
and Resource Bank (Osaka, Japan).
Poorly differentiated human pancreatic
carcinoma NOR-P1 cells were obtained
TIG-1-20 and NOR-P1 cells were
used at 2 × 107 and 1 × 107 cells/mL, re-
spectively. Cells suspended in RPMI
1640 medium with 20% FCS were
mixed with the same volume of chilled
type I collagen. Each mixture was dis-
Pancreatic Carcinoma Specimens
Carcinoma specimens were ob-
tained during surgery from patients
with advanced pancreatic carcinoma.
Each specimen was rinsed three times

Figure 1. Scheme of culture system and macroscopic features of reconstituted carcinoma tissue models. (A) Reconstituted carcinoma tissue gel or pan-
creatic carcinoma specimen was transferred onto either a plastic plate containing complete medium (static 1g condition) or a rotary-wall vessel (simulated mi-
crogravity condition) and cultured at 37°C for seven days. (B) Reconstituted carcinoma tissue models on Day 7 of culture. Scale bar represents 1 mm. The mean
of three independent experiments showed similar results.

Vol. 33, No. 5 (2002) BioTechniques 1069

Research Report
with PBS and minced with a sterile
scalpel into pieces of 1–2 mm3. The
pieces were rinsed again with RPMI
1640 medium to eliminate single or ag-
gregated cells. Pieces of approximately
2 mm3 were selected and cultured un-
der a static 1g or simulated microgravi-
ty condition (Figure 1).
Culture Under Static 1g Condition
Solid collagen gels containing
NOR-P1 and TIG-1-20 cells or tumor
pieces were transferred to plastic plates
filled with 55 mL culture medium and
cultured for seven days under the static
1g condition.
Culture Under Simulated
Microgravity Condition
The NASA bioreactor used for the
study is described as a rotary cell cul-
ture system with four disposable ves-
sels (RCCS-4D) (Figure 1A). RCCS-
4D cultures were started as previously
described (30). Briefly, collagen gel
containing NOR-P1 and TIG-1-20 cells
or a 2-mm3 piece of pancreatic carcino-
ma was placed into a 55-mL volume
disposable RWV. The gel or tumor
piece was cultured in RPMI 1640
medium supplemented with 10% FCS.
The bioreactor was rotated on a rotary
cell culture system power supply (mod-
el PS-4; Synthecon) at an initial speed
of 20 rpm. The rotation speed was ad-
justed so that the gels or tumor pieces
remained in a state of free fall as they
changed in size; they remained in place
in the vessel and did not touch the ves-
sel wall. Bubbles were removed from
the vessels daily so that the chamber re-
mained completely filled with fluid.
Immunohistochemistry
The 3-D carcinoma blocks and tu-
mor pieces were fixed with buffered
formalin (10% methanol, 4% formalde-
hyde) and embedded into paraffin for
immunohistochemical analysis. Paraf-

Figure 2. Effect of simulated microgravity condition on proliferation. (A) H&E stain on Day 7 for the reconstituted carcinoma tissue model and original tu-
mor specimen of NOR-P1 cells (original). Representative pictures (400×). (B) Results are expressed as the –x ± SD of three independent experiments. (C) The mi-
crograph shows mitotic cells (arrows) under simulated microgravity condition (400×). Mitotic index (M.I.), total number of mitotic cells in three fields/total
number of cells in three fields. (D) Immunohistochemistry for Ki-67 expression. Representative pictures (200×; arrowhead, Ki-67 positive cells). (E) Results are
expressed as the –x ± SD of three independent experiments.

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Research Report
fin sections were deparaffinized, rehy- Table 1. Results from the Comparative Analysis of Static 1g Condition and Simulated Micro-
drated, incubated in antigen retrieval gravity Condition
solution (citrate buffer, pH 6.0), and SC SMC Original
heated in a high-pressure steam steriliz-
Cancer Tissue Model
er at 120°C for 20 min. The slides were
washed in PBS, and endogenous perox- Cell proliferation (cells/field)a
idases were quenched in 3% H2O2 Surface of tissue model 42 ± 10 215 ± 25 163 ± 5
methanol for 30 min. Primary antibod- Center of tissue model 17 ± 6 39 ± 7 154 ± 9
ies, anti-Ki-67 (1:50; Dako, Glostrup, TUNEL-positive cells (cells/field)
Denmark) and anti-NF-κB p65 (1:50; Apoptotic cellsb 18 ± 2 2±2 1±2
Santa Cruz Biotechnology, CA, USA), Immunohistochemistry
were diluted with PBS supplemented
Ki-67-positive cells (%)c 3±1 69 ± 4 46 ± 4
with 0.01% NaN3 and 1% BSA and ap-
plied to sections, which were incubated NF-κB nuclear translocation (%)d 0±0 16 ± 2 19 ± 2
overnight at 4°C. After being washed in SC SMC Fresh
PBS, secondary antibodies were ap-
Fresh Human Carcinoma Specimene
plied according to the specificity of the
primary antibody: Ki-67 for Histofine Cell proliferation (cells/field)
Simple Stain MAX PO (mouse) Surface of tissue model 4±2 72 ± 4 76 ± 8
(Nichirei, Tokyo, Japan) and NF-κB Center of tissue model 2±2 36 ± 8 64 ± 4
p65 for Histofine Simple Stain MAX Immunohistochemistry
PO (rabbit) (Nichirei). After the addi- Ki-67-positive cells (cells/field) 3±0 33 ± 6 35 ± 5
tion of secondary antibodies, the slides aas in Figure 2B
were incubated for 30 min at room tem- bas in Figure 3B
perature. Reaction sites were visualized
cas in Figure 2E
with diaminobenzidine as the chro-
dResults are expressed as –x ± SD of three independent experiments.
mogen, and nuclei were counterstained
with hematoxylin and eosin (H&E). ePancreatic carcinoma specimen was obtained during surgery and minced into
% of Ki-67-positive cells = (total pieces of 2 mm3. Small pieces were cultured for seven days under static 1g
number of Ki-67-positive cells in three condition or simulated microgravity condition. Fresh, surgically resected
fields/total number of cells in three carcinoma specimen was immediately fixed with buffered formalin. SC, static 1g
fields) ×100. Three fields were selected condition; SMC, simulated microgravity condition; original, original carcinoma
at random under high-power light mi- specimen of NOR-P1 cells.
croscopy (200×).

Figure 3. Apoptosis and NF-κ κB activation in cells. (A) TUNEL assay. Representative pictures (400×; arrows, TUNEL-positive cells). (B) Results are expressed
as the –x ± SD of three independent experiments. (C) Immunohistochemistry for NF-κB. Representative pictures (400×; arrows, nuclear translocation of NF-κB/p65).

1072 BioTechniques Vol. 33, No. 5 (2002)

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NF-κB nuclear translocation (%) =
(total numbers of cells showing NF-κB
nuclear translocation in three fields/to-
tal number of cells in three fields)
(×100). Three fields were selected at
random under high-power light mi-
croscopy (400×).
TUNEL Assay
Cell apoptosis was evaluated by in
situ TUNEL labeling (7), which detects
extensive DNA degradation, a charac-
teristic event that often occurs in the
early stage of apoptosis. The slides
were deparaffinized in xylene and de-
creasing concentrations of ethanol, and
then rehydrated in PBS. Endogenous
peroxidase activity was quenched by
incubation in 3% hydrogen peroxide
(30% H2O2 stock solution; Sigma, St.
Louis, MO, USA) for 15 min, followed
by a washing in PBS. The sections
were digested with 20 µg/mL pro-
teinase K for 15 min at room tempera-
ture and were then washed and incubat-
ed with the TUNEL reaction mixture
(enzyme solution and labeling solution;
Takara Bio, Tokyo, Japan) for 60 min at
Figure 4. Effect of culture under the simulated microgravity condition on the surgically resected, fresh specimen of human pancreatic carcinoma tissue.
(A) Microscopic features (400×; arrowheads, Ki-67-positive cells). (B) The frequency of Ki-67-positive cells. Results are expressed as the –x ± SD of three inde-
pendent experiments.
1074 BioTechniques
37°C in a humidified atmosphere. Re-
action sites were visualized with the
same method used for the immunohis-
tochemistry protocol.
Apoptotic cells (TUNEL-positive
cells)/field = mean number of TUNEL-
positive cells per field. Three fields
were selected at random under high-
power light microscopy (400×).
Statistical Analysis
The statistical significance of differ-
ences between the static 1g condition
and the simulated microgravity condi-
tion data was determined by a Student’s t
test. P < 0.05 was considered significant.
RESULTS AND DISCUSSION
The viability, proliferation, cell cy-
cling, and activation of the NF-κB of
pancreatic carcinoma cells embedded
in collagen gel that had been cultured
under the simulated microgravity con-
dition were well maintained for seven
days, with results similar to those of the
pancreatic carcinoma cells of primary
carcinoma surgery specimens.
The collagen gel containing NOR-
P1 and TIG-1-20 cells cultured under
the simulated microgravity condition
contracted more than the gel cultured
under the static 1g condition, which
suggests that a higher gel density is as-
sociated with the simulated microgravi-
ty condition (Figure 1B). We know
from experience that the rapid growth
of cells embedded in collagen gel re-
sults in the contraction of the gel
(12,16). Our results indicate that cells
cultured under the simulated micro-
gravity condition proliferate better than
those cultured under the static 1g con-
dition. In fact, NOR-P1 cells embedded
in a gel cultured under the simulated
microgravity condition showed this im-
proved proliferation (Figure 2A).
NOR-P1 cells were found at signifi-
cantly greater numbers than at the sur-
face area (from surface to 100 µm in
depth) or at the center of the gel under
the simulated microgravity condition
(Figure 2B). Mitotic NOR-P1 cells
were found at a higher frequency in
gels cultured under the simulated mi-
crogravity condition than in those cul-
Vol. 33, No. 5 (2002)
tured under the static 1g condition (Fig- [i.e., NF-κB (27)]. In most unstimulat- and cycling cell ratio, were compared
ure 2C). Thus, the proliferation of ed cells, p65 proteins are sequestered in with those of the fresh, original carcino-
NOR-P1 cells may be accelerated un- the cytoplasm (27). The stimulation of ma specimen obtained during surgery
der the simulated microgravity condi- the cells allows for translocation of p65 (Figure 4, A and B). Carcinoma speci-
tion, or apoptosis of NOR-P1 cells may to the nucleus (nuclear translocation) mens cultured under the static 1g condi-
be decelerated under the simulated mi- (27); therefore, NF-κB activation was tion showed a remarkable decrease in
crogravity condition. evaluated on the basis of nuclear the numbers of carcinoma cells and fi-
In the present study, proliferation translocation of p65. As shown in Fig- broblasts, and only a few cells showed
speed was estimated indirectly by de- ure 3C, the nuclear translocation of p65 cell cycling. In comparison to cells un-
termining the ratio of cycling cells to occurred significantly more often in der the static 1g condition culture, spec-
total cells. The expression of the human NOR-P1 cells cultured under the simu- imens cultured under the simulated mi-
Ki-67 protein is thought to be strictly lated microgravity condition than in crogravity condition well maintained
associated with cell proliferation (29). NOR-P1 cells cultured under the static the histopathological and biological
Ki-67 is present in the nuclei of cells in 1g condition. This suggests that NF-κB characteristics of fresh carcinoma tis-
the G1, S, and G2 phases of the cell cy- activation is related to the fewer apop- sue. The overall data obtained in this
cle and in mitosis (29). Quiescent or totic NOR-P1 cells found under the study (Table 1) indicate that the simulat-
resting cells in G0 phase do not express simulated microgravity condition. ed microgravity condition mimics the in
the Ki-67 antigen (29). Thus, we used Finally, we cultured minced human vivo microenvironment more closely
Ki-67 antigen to detect cycling cells. As pancreatic carcinoma specimens under than the conventional static 1g condi-
shown in Figure 2D, more Ki-67-posi- either the simulated microgravity condi- tion. Although it is still unclear what
tive cells were found in gels under the tion or the static 1g condition for seven factors provide the advantages we de-
simulated microgravity condition than days, and histopathological and biologi- tected in the simulated microgravity
in gels under the static 1g condition. cal characteristics, such as 3-D architec- condition, the RCCS-4D system may be
The ratio of Ki-67-positive cells to total ture, cellular composition, cell viability, useful for establishing highly organ-
cells was also higher under the simulat-
ed microgravity condition (Figure 2E).
We then estimated the NOR-P1 cell
death speed by counting apoptotic cells.
Apoptosis is a critical control mecha-
nism in the morphogenesis and normal
cell turnover of adult tissues (33).
Apoptosis of carcinoma cells can be in-
duced in a variety of ways, including
withdrawal of growth factors (22), treat-
ment with DNA damaging agents (22),
and exposure to ionizing radiation (22).
DNA fragmentation is considered the
most characteristic feature of apoptosis
(7). The TUNEL method is based on di-
rect, specific labeling of DNA breaks in
nuclei in situ (7). Therefore, we used
the TUNEL method to detect apoptotic
cells in the collagen gel. Although the
total cell number was significantly low-
er under the static 1g condition than un-
der the simulated microgravity condi-
tion (Figure 2, A and B), apoptotic cells
were more numerous under the static 1g
condition (Figure 3, A and B).
To investigate why our cultures un-
der the simulated microgravity condi-
tion showed few apoptotic cells, we fo-
cused on the NF-κB activation in
NOR-P1 cells. Rel/NF-κB is a family
of dimeric transcription factors that
control the expression of numerous
genes involved in cell growth and regu-
lation of apoptosis (27). The most com-
mon dimer is the p65/p50 heterodimer

Vol. 33, No. 5 (2002)

Research Report
otypic culture models that can increase
our understanding of the complexities
of pancreatic carcinoma tissue.
ACKNOWLEDGMENTS
We thank Hiroshi Fujii, Takaaki
Kanemaru, and Kaori Nomiyama for
their skillful technical assistance.
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Received 9 July 2002; accepted 5
September 2002.
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Graduate School of Medical Sciences
Kyushu University
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e-mail: mkatano@tumor.med.kyushu-u.ac.jp
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Vol. 33, No. 5 (2002)