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. 2019 Oct 24;13(5):054111. doi: 10.1063/1.5125650

Boyden chamber-based compartmentalized tumor spheroid culture system to implement localized anticancer drug treatment

Shohei Kaneda 1,2,3,1,2,3,1,2,3,a),b), Jiro Kawada 1,b), Marie Shinohara 1, Momoko Kumemura 2,4,2,4,c), Ryohei Ueno 2, Tomoaki Kawamoto 3, Kenji Suzuki 3, Beomjoon Kim 2,4,2,4, Yoshiho Ikeuchi 5,6,5,6, Yasuyuki Sakai 1,7,1,7, Dominique Collard 2, Hiroyuki Fujita 4, Teruo Fujii 1,a)
PMCID: PMC6932857  PMID: 31893010

Abstract

In anticancer drug development, it is important to simultaneously evaluate both the effect of drugs on cell proliferation and their ability to penetrate tissues. To realize such an evaluation process, here, we present a compartmentalized tumor spheroid culture system utilizing a thin membrane with a through-hole to conduct localized anticancer treatment of tumor spheroids and monitor spheroid dimensions as an indicator of cell proliferation. The system is based on a commercialized Boyden chamber plate; a through-hole was bored through a porous membrane of the chamber, and the pre-existing 0.4 μm membrane pores were filled with parylene C. A HepG2 spheroid was immobilized onto the through-hole, separating the upper and lower compartments. Fluorescein (to verify the isolation between the compartments) and tirapazamine (TPZ; to treat only the lower part of the spheroid) were added to the upper and lower compartments, respectively. Since the transportation of fluorescein was blocked during treatment, i.e., the upper and lower compartments were isolated, it was confirmed that localized TPZ treatment was successfully conducted using the developed system. The effect of localized TPZ treatment on cell proliferation was estimated by measuring the maximum horizontal cross-sectional areas in the upper and lower parts of the spheroid by microscopic observations. This system can, thus, be used to perform localized anticancer drug treatment of tumor spheroids and evaluate the effect of drugs on cell proliferation.

I. INTRODUCTION

Multicellular in vitro tumor models1–13 play important roles in drug testing and studies of tumor biology, as they reflect many of the properties of solid tumors, including the development of an extracellular matrix; gradients in the concentrations of nutrients, oxygen, and drugs; and cell proliferation from the exterior to the interior. Multicellular models can be categorized broadly into two types, i.e., planar and spherical models.

Multilayered cell cultures (MCCs) are composed of a planar multilayer of tumor cells growing on a porous membrane support [Fig. 1(a)], which allows them to be used to evaluate both the effects of anticancer drugs on cell proliferation and their ability to penetrate the tissue.1,3–6,12,14 The effect on cell proliferation is evaluated by measuring the thickness of the MCC in histological sections.4,6,14 A conventional and simple drug penetration assessment method determines the distribution of fluorescent- or radio-labeled drug molecules in histological sections.1,15–17 However, the drugs available for such visualization are limited. MCCs can offer an assessment method for drug penetration without the need to visualize the drug molecule because the MCC separates the culture system into donor and receptor compartments for the drug of interest, allowing the performance of localized anticancer drug treatment on one side of the MMC [Fig. 1(a)]. One assessment method involves quantifying the drug concentration in the medium of the receptor compartment using chemical analysis,6,12 and another involves the comparison of the effect of the drug (e.g., on DNA synthesis) on both sides of the MCC.4 One issue for the format of MCCs is that they are incompatible with microscopic observation. Consequently, to measure the thickness of MCCs using microscopy, the collection of the MCC from its culture system is required. This is usually followed by the preparation of histological sections to assess drug penetration. Tumor spheroids are spherical aggregates of tumor cells that are most commonly used as a multicellular in vitro tumor model due to their versatility and easiness to culture.2,7–11,18–22 Incubation of a tumor spheroid in a culture medium containing a drug, followed by monitoring of the change in its size (e.g., diameter), allows the effect of drugs on cell proliferation to be estimated.

FIG. 1.

FIG. 1.

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Compartmentalized tumor spheroid culture using a through-hole membrane for localized treatment with an anticancer drug. (a) Multilayered cell culture. (b) Concept of compartmentalized tumor spheroid culture. (c) Through-hole fabricated on a porous PET membrane. (d) Fabrication of the through-hole membrane. (e) and (f) Scanning electron micrographs of membrane surfaces without (e) and with (f) parylene deposition. Scale bars: 200 μm (c); 5 μm (e) and (f).

Here, we present a newly developed tumor spheroid culture system using a through-hole membrane that enables localized anticancer drug treatment [Fig. 1(b)] and evaluation of the effect of an anticancer drug on cell proliferation through measurement of the spheroid size. In this culture system, the tumor spheroid is immobilized by hydrostatic pressure into a through-hole on the membrane separating the upper and lower compartments. Only the lower part of the spheroid is then exposed to the anticancer drug. The concept of a cell aggregate culture with a through-hole on a PDMS (polydimethylsiloxane) membrane was previously reported by our group and validated by inducing spatially patterned differentiation of an embryoid body derived from mouse-induced pluripotent stem cells.23 The system described here uses a Boyden chamber; the through-hole was fabricated on a porous membrane made of polyethylene terephthalate (PET), and pre-existing submicrometer-sized pores on the membrane were filled with parylene to block molecular transport.24,25 Because the system is implemented with a commercially available Boyden chamber plate, it is intrinsically compatible with microscopic observation. Using this system, we performed fluorescence labeling of a tumor spheroid to estimate the spatial confinement of molecules in the two compartments. We examined the long-term stability of the tumor spheroid culture and localized anticancer drug treatment for 48 h using fluorescein as a model cell-impermeable small molecule26,27 and tirapazamine (TPZ)28–30 as a model drug. We also investigated the effect of localized anticancer drug treatment on cell proliferation, which was estimated by measuring the maximum horizontal cross-sectional areas of the upper and lower parts of the spheroids.

II. MATERIALS AND METHODS

A. Materials

Boyden chambers with a porous (0.4 μm) PET membrane (Corning Inc., Corning, USA) and an associated 24-well plate (Corning Inc.) were used as the platform for our system. The thickness and diameter of the membrane were 11 μm and 6.4 mm, respectively, and the density of the 0.4 μm pores was 1.6 × 106 pores/cm2. The height of the chamber and the distance from the porous membrane to the bottom of the well were 17.5 mm and 0.8 mm, respectively. The areas of the porous membrane and well were 30 mm2 and 200 mm2, respectively. Parylene C (DPX-C; Specialty Coating Systems, Indianapolis, USA) was used to fill the 0.4 μm pores.

B. Fabrication process

The through-hole diameter was designed to be smaller than that of tumor spheroids used in this study. A through-hole was bored into the PET porous membrane by laser processing with a 7 μm spot size [Fig. 1(c)] by a contract manufacturing service (CSTEC Co., Osaka, Japan). The semimajor and semiminor axes of the resultant elliptical through-hole were (mean ± s.d.) 269 ± 3 μm and 254 ± 3 μm, respectively (n = 12). Some microdebris remained along the through-hole edge. The pre-existing pores of the membrane were filled with parylene using a vacuum deposition system (PDS 2010; Specialty Coating Systems, Inc., Indianapolis, USA) after the through-hole was fabricated [Fig. 1(d)]. Figures 1(e) and 1(f) show the porous membrane before and after deposition of parylene, which had a thickness ranging from 1.6 to 1.9 μm. Both sides of the through-hole membrane were sterilized by combining three sprays of 70% ethanol with ultraviolet light irradiation (253.7 nm, 130 μW/cm2) for 20 min.

C. Cell culture

HepG2 human hepatocellular carcinoma cells (HB-8065; American Type Culture Collection, Manassas, USA) were maintained in a Cancer Stem Cell Medium (PromoCell GmbH, Heidelberg, Germany) on an untreated 6-well plate at 37 °C under 5% CO2. The cells were subcultured for 10 passages before tumor spheroid formation.

D. Tumor spheroid formation

HepG2 cells were seeded in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Waltham, USA) containing 10% fetal bovine serum (FBS; Nichirei Bioscience, Tokyo, Japan) and 1% penicillin (100 U/ml) and streptomycin (100 μg/ml) (Thermo Fisher Scientific) at 103 cells per 100 μl in a V-shaped well of a 96-well plate (PrimeSurface 96V plate; Sumitomo Bakelite Co., Tokyo, Japan) with an ultralow cell adhesion surface and cultured at 37 °C under 5% CO2. HepG2 spheroids were cultured until day 11, and the medium was changed on days 4 and 8.

E. Immobilization of the tumor spheroid on the through-hole

On day 11, a HepG2 spheroid was collected from the 96-well plate using a pipetter with a wide-orifice tip and transferred to the through-hole membrane in the chamber, which was placed in the associated 24-well plate. A 200-μl aliquot of medium added to the chamber generated flow induced by hydrostatic pressure resulting from the difference in water levels above and below the through-hole membrane. The flow caused the spheroid to spontaneously move toward and become immobilized onto the through-hole (Movie S1 in the supplementary material). For preculture, a 200-μl aliquot of medium was then added to the well, followed by incubation at 37 °C under 5% CO2 for at least 19 h before experimentation. After 3 h of preculture, the chambers were examined to confirm that the spheroid could be maintained in the through-hole; approximately 83.3% of the chambers kept the spheroid in the through-hole.

F. Visualization of the treated part of the spheroid

To confirm the sealing of the through-hole by the spheroid, the lower part of the immobilized HepG2 spheroid (below the through-hole membrane) was fluorescently labeled with calcein AM (Dojindo Molecular Technologies, Kumamoto, Japan). The preculture medium was replaced with 400 μl of fresh medium after the chamber was positioned in an empty well of the 24-well plate. After washing with the serum-free medium containing 1% penicillin-streptomycin, 400 μl of 4μM calcein AM in the serum-free medium was added to the well to label the lower part of the immobilized spheroid, followed by incubation at 37 °C and 5% CO2 for 15 min. The calcein AM-containing medium was replaced with the same medium used for spheroid formation to maintain spheroid viability before observation using a confocal microscope (FV1000; Olympus Corp., Tokyo, Japan) and an inverted fluorescence microscope (BZ-X700; Keyence Corp., Osaka, Japan). The labeled spheroid was collected from the through-hole membrane by dipping the chamber into a well containing 800 μl of the same medium used for spheroid formation and by gently shaking the chamber up and down in the well. To distinguish the part of the spheroid labeled with calcein AM, confocal images (512 × 512 pixels for the X–Y plane, 60 sections) were obtained at 5 μm intervals along the Z axis. Both bright-field and fluorescence images (960 × 720 pixels for the X–Y plane, 160 and 70 sections for the immobilized and collected spheroid, respectively) were obtained at 2.5 μm intervals along the Z axis using the inverted fluorescence microscope, yielding an image stack for the spheroid.

G. Localized treatment with anticancer drug and stability of compartmentalized spheroid culture

The potential for localized treatment of spheroids with an anticancer drug was evaluated using TPZ (Sigma-Aldrich, St. Louis, USA). TPZ is reductively metabolized to a cytotoxic free radical intermediate under hypoxic conditions that induces DNA strand breaks and results in cell death.6,30 Fluorescein (Thermo Fisher Scientific), as a model cell-impermeable molecule,26,27 was used to assess the stability of compartmentalization during localized drug treatment. The lower part of the immobilized HepG2 spheroid (below the through-hole membrane) was exposed to 10μM TPZ for 48 h.8 A 400-μl aliquot of 1μM fluorescein-containing medium (with 10% FBS, 1% penicillin-streptomycin, and 0.2% ethanol) was added to the chamber, followed by 400 μl of 10μM TPZ-containing medium (with 10% FBS, 1% penicillin-streptomycin, and 0.1% dimethylsulfoxide).

The stability of compartmentalization (i.e., isolation between the compartments by sealing of the through-hole by the spheroid) during the localized treatment was estimated by observing fluorescein transport at 0 and 48 h. Chambers with the native porous membrane without and with parylene coating were used as negative and positive controls, respectively. Chambers with HepG2 spheroids immobilized onto the through-holes without drug treatment served as the untreated controls.

To investigate the effect of localized drug treatment on cell proliferation, bright-field images of the spheroid treated with TPZ were obtained, and the maximum horizontal cross-sectional areas of the upper and lower parts of the spheroid were measured. Bright-field and fluorescence images (960 × 720 pixels for the X–Y plane) of the spheroid were obtained at 2.5 μm intervals along the Z axis (200 sections) using the inverted fluorescence microscope; the image stack was used to measure the maximum horizontal cross-sectional areas as an indicator of cell proliferation. During image acquisition, the system was placed in an incubation chamber (CK-150HA-KD; BLAST, Kanagawa, Japan) with a CO2 control unit (CB-701; BLAST) and incubated at 37 °C under 5% CO2.

H. Image and histological analyses

A raster graphics editor (Photoshop CS5; Adobe Systems, San Jose, USA) was used to measure the maximum horizontal cross-sectional areas in the upper and lower parts of the HepG2 spheroids in the bright-field image stack. Frozen sections of the spheroids were cut at a thickness of 16 μm using a microtome (Retoratome REM-710; Yamato Kohki Industrial Co., Saitama, Japan) and stained with hematoxylin and eosin.

III. RESULTS AND DISCUSSION

A. Visualization of the treated portion of the spheroid

Confocal imaging revealed that the parts of the spheroid below the through-hole membrane were labeled with calcein AM [Fig. 2(a) and Movie S2 in the supplementary material]. The labeled area was larger than the through-hole [Fig. 2(b)]. Because this was not the case immediately after immobilization (data not shown), we concluded that the increase in size resulted from cell proliferation during the preculture period before calcein AM labeling. The collected spheroid was mushroom-shaped [Fig. 2(c) and Fig. S1 in the supplementary material]. Although the manner in which the spheroid was divided using a pipetter could be improved, we found that it was possible to selectively collect the lower or upper parts of the spheroid (Fig. S2 in the supplementary material).

FIG. 2.

FIG. 2.

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HepG2 spheroid partly labeled with calcein AM after preculturing. The lower part of the tumor spheroid below the membrane was exposed to calcein AM-containing medium. (a) Series of confocal images. The position in the Z axis from the bottom of the lower parts of spheroid is indicated in the upper right of each image. (b) and (c) Images of partly labeled spheroid immobilized onto the through-hole (b) and of the collected spheroid (c). Scale bars: 200 μm.

B. Localized treatment with anticancer drug and stability of compartmentalized spheroid culture

Fluorescence images of Boyden chambers in the compartmentalized culture without or with local TPZ treatment are shown in Fig. 3. In the latter condition, the lower part of the HepG2 spheroid was exposed to the medium containing 10μM TPZ. Fluorescein transport from the upper to the lower compartments (i.e., from above to below the membrane) was not observed for the positive control (lane 2, porous membrane with parylene deposition), untreated control (lane 3), and TPZ-treated sample (lane 4), in contrast to the negative control (lane 1, native porous membrane). Because fluorescein is a cell-impermeable small molecule, these results indicate that mass transport between upper and lower compartments was limited only to cell-permeable molecules via the tumor spheroid. TPZ is an intrinsically cell-permeable anticancer drug; thus, it was assumed that TPZ passed through the tumor spheroid in the upper compartment. The quantity of TPZ present would, therefore, reflect upon its ability to penetrate the tumor spheroid. However, we found that higher concentrations of TPZ (e.g., 50μM, data not shown) induced cell death, resulting in displacement of the spheroid from the through-hole and consequent loss of isolation between the compartments.

FIG. 3.

FIG. 3.

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Fluorescence micrographs of chambers during compartmentalized culture with partial TPZ treatment. Boyden chambers with native porous membrane (lane 1) and those with parylene-coated porous membrane (lane 2) served as negative and positive controls, respectively. The lower parts of HepG2 spheroids immobilized in chambers were exposed to a 10μM TPZ-containing medium (partially TPZ treated; lane 4) or left untreated as a control (lane 3). Each sample has four independent chambers. Scale bars: 5 mm. White dotted and white circles indicate the edge of wells and membranes of chambers, respectively. Arrows indicate spheroids immobilized on the through-holes.

Images of HepG2 spheroids at 0 and 48 h in the compartmentalized culture with or without localized TPZ treatment are shown in Fig. 4(a). The maximum horizontal cross-sectional areas in both upper and lower parts of spheroids (i.e., above and below the through-hole membrane) were measured as an indicator of cell proliferation. There was no difference between the untreated control and the TPZ-treated sample in terms of the maximum horizontal cross-sectional area of the upper part at 0 and 48 h [Fig. 4(b)]. However, the maximum horizontal cross-sectional area of the lower part of TPZ-treated spheroids was markedly smaller than that of untreated controls at 48 h [P < 0.001; Fig. 4(c)]. Histological analysis confirmed that the TPZ-treated lower part was smaller than the untreated lower part [Figs. 4(d) and 4(e)]. These results indicate that TPZ suppressed cell proliferation only in the lower part exposed to the drug.

FIG. 4.

FIG. 4.

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Maximum horizontal cross-sectional areas of upper and lower parts of spheroids locally treated with 10μM TPZ. (a) Representative images of spheroids. Black and white dotted lines indicate the maximum horizontal cross-sectional area in the upper and lower parts of spheroids, respectively (i.e., above and below the membrane, respectively). (b) and (c) Average maximum horizontal cross-sectional areas in upper (b) and lower (c) parts. Error bars indicate ±s.d. (n = 4). Statistical significance was analyzed with Student's t test (NS, not significant; ***P < 0.001, n = 4). (e) and (d) Hematoxylin and eosin-stained frozen sections of a spheroid locally treated with 10μM TPZ (e) or untreated (d). Scale bars: 200 μm [(a), (d), and (e)].

IV. CONCLUSIONS

In this study, we developed a novel cell culture system using a through-hole membrane that enables the compartmentalized tumor spheroid culture for localized anticancer drug treatment and is able to evaluate the effect on cell proliferation by measuring spheroid size. Our system is based on a commercially available Boyden chamber plate, is highly stable and yields reproducible results, is compatible with microscopic observation, and requires only a conventional pipetter and an aspirator in addition to standard cell culture materials and apparatus. Because transport of cell-impermeable molecules is limited in the system by the through-hole membrane with the immobilized spheroid, it potentially can be used to assess drug penetration without visualization of the drug by comparing the effect of the drug on cell proliferation in the upper compartment or quantifying drug concentration in the medium of the upper compartment, which is not possible with a conventional spheroid culture system.

In the next step, we will expand the application fields of this system to construct an in vitro 3D tumor tissue model that reflects the geometrical situation between blood vessels and tumor cells by considering the upper compartment as the tumor tissue side and the lower compartment as the blood vessel side. For example, by using a low nutrient and hypoxic medium for the upper compartment, the effect of the distribution of the concentrations of nutrient and oxygen in the tumor tissue on drug effectiveness can be studied. Moreover, the present Boyden chamber format of the developed system would easily allow coculture with other cell types such as epithelial cells or vascular endothelial cells on the through-hole membrane to understand its interactions with tumor cells.

SUPPLEMENTARY MATERIAL

See the supplementary material for a video showing an immobilization process of a HepG2 spheroid on the through-hole (Movie S1 in the supplementary material), a confocal micrograph of a HepG2 spheroid partly labeled with calcein AM (Movie S2 in the supplementary material), and micrographs of HepG2 spheroids partly labeled with various fluorescent dyes and divided spheroid (Fig. S1 in the supplementary material).

ACKNOWLEDGMENTS

We are grateful to Professor S. Takeuchi and M. Onuki at the Institute of Industrial Science, the University of Tokyo for their help with the parylene deposition on the membranes. This work was supported by JSPS KAKENHI under Grant No. JP 17K14985.

Contributor Information

Shohei Kaneda, Email: .

Teruo Fujii, Email: .

REFERENCES

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

See the supplementary material for a video showing an immobilization process of a HepG2 spheroid on the through-hole (Movie S1 in the supplementary material), a confocal micrograph of a HepG2 spheroid partly labeled with calcein AM (Movie S2 in the supplementary material), and micrographs of HepG2 spheroids partly labeled with various fluorescent dyes and divided spheroid (Fig. S1 in the supplementary material).

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