Download Development of an in Vitro Multicellular Tumor Spheroid Model

Biotechnol. Prog. 2005, 21, 1289−1296
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Development of an in Vitro Multicellular Tumor Spheroid Model
Using Microencapsulation and Its Application in Anticancer Drug
Screening and Testing
Xulang Zhang,†,‡ Wei Wang,† Weiting Yu,† Yubing Xie,† Xiaohui Zhang,†
Ying Zhang,† and Xiaojun Ma*,†
Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, 457 Zhongshan Road, Dalian 116023, China, and Graduate School of the Chinese Academy of Sciences,
19 Yuquan Road, Beijing 100039, China
In this study, an in vitro multicellular tumor spheroid model was developed using
microencapsulation, and the feasibility of using the microencapsulated multicellular
tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated.
Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate
(APA) microcapsules, and a single multicellular spheroid 150 µm in diameter was
formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy,
BrdU-labeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated
that the MMTS was viable and that the proliferating cells were mainly localized to
the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7
MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times
that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was
demonstrated clearly by the reduction in cell spheroid size and the decrease in cell
viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at
concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture
was used as control. Similar to monolayer culture, the cell viability of MMTS was
reduced after treatment with anticancer drugs. However, the inhibition rate of cell
viability in MMTS was much lower than that in monolayer culture. The MMTS was
more resistant to anticancer drugs than monolayer culture. The inhibition rates of
cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in
monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively.
MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU
and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid
in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo threedimensional (3-D) cell growth pattern.
1. Introduction
With the development in anticancer drug discovery and
new drug synthesis, it is of paramount importance to
develop an efficient and high-throughput drug screening
and testing system that will faithfully mimic tumors in
vivo.
Conventionally, suspension and monolayer cultures of
tumor cells were applied to screen chemotherapeutic
drugs in vitro. However, cells cultured in monolayer
cannot substitute for cells in vivo properly. Monolayer
culture consists of cells generally growing in a nutrientrich liquid environment or on agar surface with plenty
of oxygen, which forms a nearly homogeneous colony. An
in vivo mature tumor with an extensive vasculature has
* To whom correspondence should be addressed. Tel: +86-41184379139. Fax: +86-411-84379139. E-mail: [email protected].
† Dalian Institute of Chemical Physics, Chinese Academy of
Sciences.
‡ Graduate School of the Chinese Academy of Sciences.
10.1021/bp050003l CCC: $30.25
a very complex structure, generally consisting of regions
of regularly dividing cells, hypoxic cells, and necrosis, at
increasing distances from blood vessels with a threedimensional (3-D) pattern bearing structural heterogeneity (1). To mimic the 3-D structure of tumors, the
multicellular tumor spheroid (MTS) model was developed
by Sutherland and collaborators in the early 1970s (2,
3). It has been applied to therapeutics-oriented investigations, including radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy (4-9).
MTS represents a level of complexity intermediate
between that of cells growing as a monolayer in vitro and
that of solid tumor in vivo. It has been demonstrated that,
in comparison to monolayer culture, cells in MTS culture
more closely resemble the in vivo situation with regard
to cell shape and cellular environment (10, 11), which
can regulate the gene expression and hence the biological
behavior of the cells. However, the MTS system has
certain limitations, including poor accessibility to manipulation of cultures growing in semisolid media and
© 2005 American Chemical Society and American Institute of Chemical Engineers
Published on Web 06/18/2005
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Biotechnol. Prog., 2005, Vol. 21, No. 4
Figure 1. (a) Microencapsulated human MCF-7 breast cancer cell spheroid was observed by inverted phase contrast microscopy
after 7 days of cultivation. Cells grew in a 3-D pattern. One or two cells stuck to the inner wall of the microcapsule. Scale bar: 100
µm. (b) Schematic diagram shows the similarities between solid in vivo tumor and the in vitro microencapsulated multicellular
tumor spheroid (MMTS) model.
the open pattern where media contact the outer layer
cells of the spheroid directly without simulating the
barrier (e.g., blood vessel) between cells and nutrients.
In addition, the size of the cell spheroid in MTS culture
is hard to control.
With the advance in tissue engineering, biomaterials
have been employed to guide cell development into 3-D
organization to approximate the in vivo tissue as closely
as possible. For example, collagen gels and semipermeable hollow fibers have been tried in tumor biology study.
The former has been used for the study of brain tumor
biology, but with difficulty in culturing primary tumor
cells free from stromal cells (12). The latter has been used
to study the effects of anticancer drugs on cell cultures
in vitro and in vivo (13, 14). It has been speculated that
one of the greatest impacts of tissue engineering in the
coming decade might be the design of in vitro physiological models to develop molecular therapeutics (15).
Microencapsulation is one of the promising strategies
for tissue engineering and cellular therapy to develop a
3-D growth pattern of cells to mimic tissue in vivo.
Microcapsules were originally used for cell transplantation as an immunoisolation device (16). A microcapsule
is spherical, with a diameter that can be controlled in
the range of 200-1500 µm and a biocompatible semipermeable membrane, which allows the bidirectional
diffusion of nutrients, oxygen, secreted therapeutic product, and waste but prevents the penetration of high
molecular weight substances from the microcapsule, such
as antibodies and immunocytes. Transplantation of microencapsulated cells has emerged as a promising therapeutic strategy to treat a wide range of diseases from
endocrine disorders and central nervous system diseases
to cancer (17, 18).
In addition to cell transplantation, we hypothesize that
tumor cells enclosed within microcapsules can form a
microencapsulated multicellular tumor spheroid (MMTS)
as drug testing model (Figure 1a and b). In contrast to
conventional suspension and monolayer culture, cultivation as spherical aggregates restores morphological and
functional features of the original tissue (19). Thus,
MMTS can mimic the avascular tumor nodules or microregions of solid in situ tumors in terms of growth
kinetics with discrete cell populations consisting of
proliferating, quiescent, and necrotic cells and the nonuniform microenvironment such as oxygen, glucose, and
pH gradients that exist within solid tumors. For example,
cells located in the spheroid periphery are comparable
to those tumor cells situated close to capillaries in vivo
remaining active in the cell cycle. In contrast, the
innermost cells stop cycling and eventually die as the
spheroid enlarges beyond the critical size of approximately 200-300 µm, which mimics the tumor necrotic
core away from vessels. Tannock (20) reported the
effective diffusion distance of nutrients such as oxygen
and glucose was 100-150 µm in a nodular tumor, beyond
which necrosis appeared. One of the advantages of
MMTS compared with MTS is that MTTS has a selectively semipermeable membrane around the tumor cells
spheroid, which can mimic blood wall and tumor envelopment in structure as well as function partially, such
as infiltration of nutritions and drugs. The permeability
of the semipermeable membrane can be controlled through
monitoring the designed parameters of microencapsulation. Similarly, cells grown on the surface of semipermeable membrane with a Transwell chamber have been
used to quantify the penetration of anticancer drugs
through solid tissue (21, 22). In addition, the size of cell
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spheroids is easy to control by the size of microcapsule,
and the culture of MMTS is easier to handle than that
of MTS. So far, MMTS has only been used in in vivo
cancer therapeutic studies using human tumor cells
grown in immunodeficient or immunocompetent animals
(23, 24), which suggested that the in vivo tumor microencapsulation assay is promising for new drug development.
In this study, we test the feasibility of applying
microencapsulation to in vitro anticancer drug screening
using human MCF-7 breast cancer cells as a model,
taking the advantage of controlled tumor spheroid size,
3-D cell growth, and membrane-controlled mass transfer
in microcapsules with semipermeable membranes.
2. Materials and Methods
2.1. Cell Culture. MCF-7 human breast cancer cells
(kindly offered as a gift from Professor Kong Li at Dalian
Medical University, Dalian, China) were used in this
study. The cells were cultured in RPMI 1640 medium
supplemented with 2.2 g/L sodium bicarbonate, 10% fetal
bovine serum, 100 U/mL penicillin, and 100 µg/mL
streptomycin, at 37 °C in a humidified incubator with
5% CO2 and 95% air.
2.2. Preparation of APA Microencapsulated Cells.
APA microcapsules were prepared as described previously with modification (25). Briefly, cells (6 × 106 cells/
mL) were suspended in 1.5% w/v sterilized sodium
alginate (Sigma, St. Louis, MO) and extruded into 100
mM CaCl2 using an electrostatic droplet generator to
form calcium alginate gel beads. The gel beads were
incubated with 0.1% w/v poly-L-lysine (Mw 65,000; Sigma,
St. Louis, MO) to form alginate-poly-L-lysine membrane
around the surface. The membrane-enclosed gel beads
were further suspended in 55 mM sodium citrate to
liquefy the alginate gel core. The resulted APA microcapsules were 250-350 µm in diameter. The microcapsules with MCF-7 cells were cultured at 37 °C in 5% CO2
in RPMI-1640 medium supplemented with 10% FBS.
MCF-7 cells in the microcapsules began to aggregate
after 24 h and formed a single multicellular tumor
spheroid up to 150 µm in diameter after 5 days of
cultivation, which was used for the following experiments.
2.3. Drug Treatment. Microencapsulated MCF-7
multicellular tumor spheroids were tested with adriamycin (ADM, Pharmacia & Upjohn S.P.A., Milan, Italy),
mitomycin C (MC, Kyowa Hakko Kogyo Co. Ltd., Tokyo,
Japan), and 5-fluorouracil (5-FU, Jinyao, Tianjin, China).
Peak plasma concentration (ppc), the highest level of drug
that can be obtained in the blood usually following
multiple doses, of ADM, MC, and 5-FU was 0.4, 3.0, and
10 µg/mL, respectively (26). Concentrations of 0.1, 1, and
10 ppc were chosen for the drug treatment experiments,
i.e., 0.04, 0.4, and 4.0 µg/mL for ADM; 0.3, 3.0, and 30
µg/mL for MC; and 1, 10, and 100 µg/mL for 5-FU. Briefly,
190 µL of microencapsulated MCF-7 cells in medium was
put in each well of a 96-well plate. Next, 10 µL of the
drug at each concentration was added and incubated at
37 °C for 24, 48, and 72 h. The cytotoxicity was measured
using a MTT assay. MCF-7 cells cultured in monolayer
were used as control. Quadruplicate samples were performed, and experiments were repeated three times.
2.4. MTT Assay. MTT [3-(4, 5-dimethylthiazol-2-vl)2, 5-diphenyl tetrazoliumbromide] assay was performed
as described previously with modification (27). Briefly,
at the conclusion of drug treatment, 20 µL of MTT
solution (5 mg/mL; Sigma) was added and incubated at
37 °C for 4 h. The medium was removed and replaced
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with DMSO to solubilize the MTT tetrazolium dye. The
absorbance (A) was determined at 570 nm using a plate
reader (Wellscan MK3, Labsystems, Finland). All MTT
assays were repeated three times. The cytotoxicity was
expressed in the form of the inhibition rate of viability
using the following formula:
[
inhibition rate (%) ) 1 -
]
Atreated - Ablank
× 100
Acontrol - Ablank
where Atreated is the average absorbance in wells treated
with drugs, Ablank is the average absorbance in wells
without cells but with DMSO, and Acontrol is the average
absorbance in wells with natural saline.
2.5. Live/Dead Staining. The live/dead staining
working solution was prepared by diluting 2 mM ethidium homodimer-1 (ED-1; Sigma) in D-PBS to give 4 µM
ED-1 solution, followed by adding 4 mM calcein AM stock
solution (Sigma) to result in 2 µM calcein AM. After
exposure to MC at concentrations of 0, 0.1, 1, and 10 ppc
in 96-well plates for 24, 48, or 72 h, microencapsulated
MCF-7 cell spheroids were incubated with live/dead
staining working solution at 37 °C for 25 min in the dark.
MCF-7 monolayer cultured on a glass coverslip was used
as control. Samples were observed under confocal laser
scanning microscope (TCS-SP2, Leica, Germany). Live
cells were labeled with calcein AM producing green
fluorescence at excited wavelength 485 ( 10 nm, and
dead cells were labeled with ED-1 emitting red fluorescence at 530 ( 12.5 nm.
2.6. Histological Procedures and Morphological
Evaluation. After microencapsulated MCF-7 multicellular tumor spheroids were treated with MC at concentrations of 0, 0.1, 1, and 10 ppc for 24, 48, or 72 h, the
samples were fixed in 4% formalin, embedded in paraffin,
and sectioned (5 µm thick). Sections were stained with
hematoxylin and eosin (H&E) according to standard
protocol (28). MCF-7 monolayer culture was used as
control and fixed with cold acetone. The samples were
examined under a light microscope (CK40, Olympus,
Japan).
2.7. BrdU Incorporation Assay. The proliferation
status of cells within microcapsules was determined by
measurement of 5-bromo-20-deoxy-uridine (BrdU) incorporation in DNA using a peroxidase-based immunohistochemical assay. After the microencapsulated MCF-7
cell spheroids were treated with MC at concentrations
of 0, 0.1, 1, and 10 ppc for 24, 48, or 72 h, the samples
were incubated with BrdU (10 mM) for 12 h followed by
histological procedure. The paraffin-embedded tissue
sections were completely dewaxed and rehydrated with
PBS followed by microwave antigen retrieval. Thereafter
they were incubated in 0.3% H2O2 in methanol for 10 min
to inhibit endogenous peroxidase activity and rinsed in
PBS. After blocking with serum at room temperature,
the sections were incubated with primary anti-BrdU
antibody (Sigma, St. Louis, MO) overnight at 4 °C in a
humidified chamber followed by washing in PBS. The
negative control was incubated with PBS. After being
incubated with secondary antibody (biotin-conjugated
rabbit anti-mouse IgG, Sigma, St. Louis, MO) for 1 h at
room temperature and washed in PBS, the sections were
incubated with HRP-linked streptavidin at room temperature for 1 h and washed with PBS. The sections were
developed with DAB and examined under a light microscope.
2.8. Size Measurement. To study the growth of
microencapsulated MCF-7 cell spheroids treated with
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various concentrations of MC, the average diameter of
individual cell aggregates in microcapsules was measured
every 24 h as described by Hiroshi (29). Using a phase
contrast microscope with a calibrated reticule eyepiece,
the mean diameter of 50 cell spheroids was obtained. The
mean diameter of the spheroid was calculated according
to (a + b)/2, where a represents the long half axis and b
the short half axis of the tumor cell spheroid.
2.9. Statistical Analysis. Quadruplicate samples
were performed for each analysis. All quantitative results
were expressed as the mean ( SE. Statistical analysis
was performed using Student’s t-test to compare the
difference between selected groups. p < 0.05 was considered significant. Each experiment was repeated three
times.
3. Results
3.1. Characterization of MMTS. MCF-7 cells were
microencapsulated in APA microcapsules. A stable 3-D
structure was formed within 24 h of cultivation and a
single cell spheroid about 150 µm in diameter was formed
after being cultured for 5 days. The microcapsules
retained the spherical shape with intact membrane. The
phase contrast micrography showed that the cell spheroid
was bright with a clear outline (Figure 2a). The MMTS
was composed of viable cells as determined by MTT stain,
which produced dark blue crystals in the circumference
(Figure 2b). To reveal the proportion and distribution of
live and dead cells inside the microcapsule, the MCF-7
MMTS was dual-labeled with calcein AM (labeled live
cells in green) and ED-1 (labeled dead cells in red) and
examined under a confocal laser scanning microscope.
The live cells were mainly localized to the periphery of
the cell spheroid and the most of the dead cells were in
the center (Figure 2c). After more than 5 days of culture,
the cross-section of MMTS with size over 200 µm was
further stained with H&E to reveal the cell distribution,
which showed well-compact outlayers of cells and a
necrotic core (Figure 2d). The cross-section was further
labeled with BrdU and revealed that the proliferating
cells with nuclei stained in brown were restricted to the
outmost two to three cell layers (Figure 2e).
3.2. Cytotoxicity in Drug-Treated Microencapsulated Cells. Cell viability after drug treatment is one of
the indexes of cytotoxicity. Viability of MCF-7 MMTS was
examined after being treated with MC at 10 ppc for 72
h. The size of the cell aggregates reduced dramatically
(compare Figure 2f to 2a) and fewer cells showed MTT
viability (compare Figure 2g to 2b). The dual labeling of
calcein AM and ED-1 revealed that most of cells in the
MC-treated MMTS were dead (stained in red) and the
size of spheroids was reduced (compare Figure 2h to 2c).
H&E stain showed that the cells within microcapsules
tended to apoptosis or necrosis after MC treatment
(compare Figure 2i to 2d). The striking effect of 10 ppc
of MC was a remarkable increase in BrdU-labeled cells
with nuclei stained in brown (compare Figure 2j to 2e).
The increased BrdU incorporation likely resulted from
drug-induced recruitment of cells to the proliferating pool
and unscheduled DNA synthesis resulting from DNA
repair pathways.
Reduction in tumor size represents one of the positive
effects of an anticancer drug. Therefore, the diameter of
MCF-7 cell spheroid in MMTS, exposed to MC at concentrations 0.1, 1, and 10 ppc for 24, 48, or 72 h was
measured. The MMTS treated with physiological saline
was used as control. The size of the control increased
during the first 24 h and then decreased. The size of cell
Figure 2. The morphology and viability of MCF-7 MMTS
cultured for 5 days without treatment (a-e) or treated with 10
ppc of MC for 72 h (f-j). (a, f) Phase-contrast micrograph of
MMTS showing a single cell spheroid formed in the microcapsule. (b, g) Images of MTT staining showing viability of MMTS
(dark blue crystal). (c, h) Images of live-dead stained MMTS
with ED-1 (red, dead) and calcein AM (green, live) observed by
confocal laser scanning microscopy. Scale bar: 80 µm. (d, i)
Images of H&E stained cross-section of MMTS showing the cell
spheroid in the microcapsule composed of well-compact outlayers
of cells and a necrotic core. (e, j) BrdU-labeled cross-section of
MMTS showeing the proliferating cells with nuclei in brown.
The capsule’s irregular shape is the result of dehydration during
histological processing.
spheroids in MMTS treated with MC was reduced with
the extending of exposure time and the increasing of drug
concentration (Figure 3). The effect was prominent when
treated with MC at concentration of 10 ppc for 72 h.
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Figure 3. The effect of MC treatment on the size of MCF-7
cell spheroids in MMTS. The diameter of the MCF-7 cell
spheroid reduced with an increase in exposure time (24, 48, or
72 h) and concentration (0.1, 1, or 10 ppc) of MC. Each point
was described as mean ( standard error (SE) of 50 MMTS (p <
0.05).
3.3. Comparison of Cytotoxicity in Drug-Treated
MMTS and Monolayer Culture. The MCF-7 MMTS
and monolayer culture were treated with MC at concentrations of 0.1, 1, and 10 ppc for 24, 48, or 72 h. The cell
viability was inhibited by MC treatment. The inhibition
rate of cell viability for MMTS was lower than that for
the monolayer at 10 ppc (Figure 4a and b). With MC
treatment from 24 to 72 h, there was no change in
inhibition rate for MMTS at 1 ppc and there was no
inhibition effect on cell viability of MMTS at 0.1 ppc (data
not shown). In both monolayer and microencapsulated
cell spheroid, the inhibition rate of MC enhanced with
the increase in the concentration of MC when treated for
72 h (Figure 4b).
Finally, the MCF-7 MMTS and monolayer culture was
treated with anticancer drugs MC, ADM, and 5-FU at
concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h.
The cytotoxicity was assessed by the inhibition rate in
cell viability. The inhibition rate was increased with the
increase in time of treatment and drug concentration in
both MMTS and monolyer culture (Figure 5a and b), but
the inhibition rate in MMTS was much lower than that
in monolayer. This has demonstrated that MMTS was
more drug-resistant than monolayer culture. There was
almost no inhibition effect in cell viability after being
treated with MC, ADM, and 5-FU at 0.1 ppc or with ADM
at 1 ppc for 72 h (data not shown). The inhibition rates
of cell viability were 68.1%, 45.1%, and 46.8% in MMTS
and 95.1%, 86.8%, and 91.6% in monolayer culture
treated with MC, ADM, and 5-FU at 10 ppc for 72 h,
respectively (Figure 5a and b). Altogether, MC showed
the strongest cytotoxicity in both MMTS and monolayer,
followed by 5-FU and ADM.
4. Discussion
Originally, monolayer culture was proposed for anticancer drug study (30, 31). However, it becomes more and
more apparent that monolayer culture of tumor cells
cannot completely represent the characteristics of 3-D
solid tumors with non-homogeneous diffusion of nutrients
in vivo. To mimic the physiological behavior of in vivo
tumors, e.g., cell-cell and cell-extracellular matrix
(ECM) interactions, some 3-D in vitro tumor models
including MTS were developed to screen and analyze the
Figure 4. Comparison of cytotoxicity in anticancer-drug-treated
MCF-7 MMTS (S) and monolayer culture (M). (a) Inhibition rate
after treatment with MC at 10 ppc for 24, 48, and 72 h. (b)
Inhibition rate after treatment with MC at 0.1, 1, and 10 ppc
for 72 h. The inhibition rate of cell viability was increased with
the increase in exposure time and concentration. The MMTS is
more drug-resistant than the monolayer culture. Each point
represents the mean of four wells and each assay was performed
in triplicate. Errors bars are standard deviation of the mean
(SEM) and are shown when greater than the size of the symbols
(p < 0.05).
effect of chemotherapeutics or gene therapy (32). Indeed,
it is recognized that 3-dimensional cell culture systems
better reflect the in vivo behavior of most cell types.
Advances in tissue engineering are in demand to avoid
large-scale and cost-intensive animal testing. However,
the 3-D culture system established using tissue engineering strategies has not yet been incorporated into mainstream drug development operation.
Microencapsulation has the potential to form multicellular tumor spheroids for tumor study. Our data
showed that MCF-7 human breast cancer cells formed a
single multicellular spheroid in alginate-poly-L-lysinealginate (APA) microcapsule after being cultured in vitro.
In contrast to monolayer culture where cells are stretched
out on a two-dimensional surface, cells inside the microcapsule grew in a 3-D pattern and organized into a single
spheroid about 150 µm in diameter. Compared to the
current 3-D tumor models, such as MTS, collagen gel,
and hollow fiber systems, microencapsulation has advantages such as improved simulation of cell-cell and
cell-matrix interactions in in vivo conditions and welldefined spherical geometry that allows the correlation
of structure with function and even heterogeneity. In
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Figure 5. The anticancer effect of MC, ADM, and 5-FU
screened in MMTS (a) and monolayer culture (b). The effect of
MC on inhibition rate of cell viability was more significant than
that of ADM and 5-FU with treatment for 72 h. Each point
represents the mean of four wells and each assay was performed
in triplicate. Errors bars are standard deviation of the mean
(SEM) and are shown when greater than the size of the symbols
(p < 0.05).
addition, the microcapsule membranes can mimic barriers such as the blood vessel wall and tumor envelope
in a certain way and serve as diffusion-limited tissue
models for their similarity with the initial avascular
growth stage of malignancies, micrometastases, and
intracapillar tumor microregions. The selective permeable microcapsule environment has been shown to support cellular metabolism, proliferation, differentiation,
and cellular morphogenesis. We acknowledge that there
are structural and functional differences between the
endothelial layer of a blood vessel and the simple alginate-poly-L-lysine membrane, which suggest that the
membrane permeability and even the transport mechanisms may be profoundly different, but they both serve
as a barrier between tumor cells and nutrient or drug.
The membrane permeability can be controlled by the
regulation of many factors (polymer molecular weight,
strength, thickness, chemical structure, formation of
membrane laminates) and process factors (membrane
polymer solution concentration, reaction time, pH, additives). It will be our next step to modify the microcapsulated multicellular tumor spheroid to approximate a
tumor in vivo after we test the feasibility of using
microencapsulation to develop multicellular tumor spheroids for anticancer drug testing.
Biotechnol. Prog., 2005, Vol. 21, No. 4
Most anticancer drugs as well as assay reagents such
as MTT and its solute of formazan, ED-1, and calcein AM
in DMSO are small molecules with low molecular weight,
which can penetrate through the microcapsule membrane
freely (23, 33). MC, ADM, and 5-FU, general chemotherapeutic drugs in the treatment of cancer whose chemotherapeutic efficacy are different due to pathological style
and individual differences, were chosen as model drugs
in this study. MC is a natural cytotoxic agent used in
clinical anticancer chemotherapy that alkylates DNA
through covalent linkage of its C1 position with the
guanine N2 amino group, ultimately forming a cross-link
adduct with the adjacent guanines at a CpG step. ADM
forms noncovalent complexes with DNA using interactions via intercalation (34). In this study, we found the
drug-induced effect on cell morphology in MMTS confirmed anticancer drugs had permeated through microcapsule membrane, killing or inhibiting the viability of
cells.
Our results demonstrated that the cytotoxities of drugs
have a positive relationship with the concentration and
exposure time to cells both in monolayer culture and
MMTS, but the inhibition rate of cell viability was
different. This is consistent with the report that multicellular spheroid was generally more resistant than
corresponding monolayer cultures to a given dose of many
anticancer drugs (35, 36). The reason lies in that a
mature tumor with an extensive vasculature is a very
complex structure, generally consisting of regions of
regularly dividing cells, hypoxic cells, and necrosis, at
increasing distances from (viable) blood vessels. MMTS
of MCF-7 revealed a necrotic core with diameter greater
than about 200 µm, where only the outermost layers were
actively proliferating with quiescent cells inside in accord
with conditions in vivo. Chemotherapeutic drugs, delivered via the vasculature, readily attack cells that are in
the vicinity of a blood vessel; these cells, being wellnourished and undergoing mitosis regularly, are generally more sensitive to the drugs, so that treatment on
them is often successful. There is experimental evidence
that both molecular diffusion and vascular transfer are
the lowest near the center of the tumor, increasing to
maximal levels at the tumor periphery (37, 38). However,
limitations to the effectiveness of such forms of therapy
result from the survival of the hypoxic cells, which is
enhanced as a result of their reduced sensitivity to many
drugs and the limited penetration of effective quantities
of drugs deep into the tumor, as drug transport is mainly
restricted to diffusion (39). It concluded drug resistance
in solid tumors representing a complex and interconnected series of resistance pathways that are both
inherent and acquired (40).
MC induced a significant reduction of diameter and
thus the size of microencapsulated cell spheroid after 72
h of incubation, compared to the original MMTS. The
decrease in cell spheroid size was due to shedding of cells,
either the necrotic/apoptotic cells or cells at the periphery
loosely associated with the cell aggregates. Jackson and
co-workers addressed that the spheroid expands or
shrinks at a rate that depends on the balance between
cell growth and division or cell death within the tumor
volume and presented a mathematical model to describe
the reduction in volume of a vascular tumor in response
to specific chemotherapeutic administration strategy (41).
Cell populations are most sensitive to chemotherapeutic regimes targeted against some aspect of DNA replication according to BrdU incorporation. Our results show
the proportion of proliferating cells relatively increased
after being incubated with MC for 72 h in microencap-
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sulated MCF-7 cells. The increased BrdU incorporation
likely resulted from drug-induced recruitment of cells to
the proliferating pool and unscheduled DNA synthesis
resulting from DNA repair pathways. It had similarity
with that of Halla (42), but Glenn indicated cells at the
center of the spheroid exhibited lower Ki-67 labeling than
cells at the surface of the spheroid at the effect of
radiation on proliferation markers (43). What causes the
establishment of quiescent cell populations within the
3-D spheroid was complex. However, several investigations demonstrated that hypoxia and glucose deficiency
were not the primary causative agents (44, 45). More
possible causes include cell-cell, and cell-matrix interactions, growth factor availability, and expression of
growth factor receptors (44).
Altogether, MMTS is a promising drug screen and test
system. First, it is feasible to use microencapsulation to
set up a rapid and feasible drug-screening test on a small
scale. After simply encapsulating tumor cells in the
microcapsule, culturing for a certain period of time, and
picking up a certain number of cells to distribute in a
96-well plate or even a 384-well plate, a MMTS drug test
system is ready to use. The amount of cell spheroids can
be determined by counting the number of microcapsules,
and the size of the cell spheroids can be controlled by
the size of the microcapsule. The small scale allows the
use of small amounts of drug, which is especially essential for new drug synthesis. Second, it is possible to
selectively deliver protein drugs to MMTS because the
permeability of the microcapsule can be controlled by the
parameters of microencapsulation. Further study will
focus on the small-scale drug test and selective delivery
of certain protein drugs to tumor spheroids using microencapsulation.
In conclusion, MCF-7 cells encapsulated in APA microcapsule were able to grow in a 3-D pattern and
organized into a single multicellular spheroid after being
cultured in vitro. The MMTS can be used as an in vitro
cell-tissue level model system for drug testing that is
superior to monolayer cell culture. Since it is practical
to microencapsulate cells with controlled size on a large
scale, microencapsulated multicellular tumor spheroids
have great potential for application in a high-throughput
tissue-like level of drug screening and testing, which can
fill the gap between animal models and monolayer
culture.
Acknowledgment
The authors thank Dr. Li Xiu-Hua for providing
anticancer drugs used in these studies and are grateful
to the staff of the Department of Biomedical Material
Engineering, Dalian Institute of Chemical Physics for
supporting this study. Contract grant sponsor: National
Natural Science Foundation, People’s Republic of China,
contract grant number 20236040; National Basic Research Program of China, contract grant number
2002CB713804.
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Accepted for publication May 11, 2005.
BP050003L