2025 Volume 48 Issue 1 Pages 23-32
A 3-dimensional (3D) cell culture is now being actively pursued to accomplish the in vivo-like cellular morphology and biological functions in cell culture. We recently obtained nano-fibrillated bacterial cellulose (NFBC). In this study, we developed a novel NFBC-based 3D cell-culture system, the OnGel method, and the Suspension method. HepG2 human liver cancer cells were cultured via these methods and formed spherical formulations in the optimized condition, 1.0% (w/v) of NFBC in the OnGel method, and 0.06–0.10% (w/v) of NFBC in the Suspension method. Non-cancerous cells such as human-induced pluripotent stem (iPS) cells and human mesenchymal stem cells (MSCs) also formed spherical formulations. It is noteworthy that both the size and cell viability of spheroids prepared via these methods were comparable to those cultured using commercially available 3D cell-culture systems. Both OnGel and Suspension methods are less complicated than the existing 3D cell-culture systems, which is an invaluable advantage for the preparation of cancer spheroids. The NFBC-based 3D cell-culture systems introduced here show great promise as a tool to prepare cultures for cell-derived spheroids for the progress of both in vitro and in vivo studies of the biological functioning of cells.
Cell culture is an essential technique in biology and drug discovery to elucidate molecular mechanisms and drug sensitivity.1,2) A classical 2-dimensional (2D) cell culture involves a monolayered cell culture on flat and rigid plastic culture vessels such as dishes, flasks, and well plates, and is well recognized as a process that is simple to operate to promote growth, proliferation, and differentiation of cells.3,4) However, in vitro 2D cell culture is known to interfere with the morphology and biological functions of cells compared with those in in vivo tissues or organs because in vitro 2D cell culture cannot establish the multidirectional cell-to-cell and cell-to-extracellular matrix (ECM) interactions that are present in the in vivo microenvironment.5,6) To dispel such issues, the use of a 3-dimensional (3D) cell-culture technique to prepare spheroids or organoids is beginning to attract much attention.
In general, spheroids or organoids could be prepared via a liquid-based 3D cell-culture system,7–9) a scaffold-based 3D cell-culture system,10–12) and by using other techniques such as microfluidic platforms13,14) or bioprinting.15,16) A 3D cell culture replicates the cellular heterogeneity of cancer cells in the in vivo ECM and tumor microenvironments.17) Because of the transport gradient of oxygen and nutrients, cancer spheroids with a diameter of more than 500 μm form a stratified structure with a proliferating cell population in the periphery and a non-dividing/necrotic cell population in the core, which is consistent with in vivo tumors.18) In addition, 3D cell cultures partially replicate the cell-to-cell or cell-to-ECM interactions by restoring the expressions of cell-adhesion molecules such as e-cadherin, which results in chemoresistance.19,20) The spherical structure of either spheroids or organoids prepared via a 3D cell-culture system better reflects the in vivo tumor organization.
We recently developed nano-fibrillated bacterial cellulose (NFBC) by culturing a novel strain of Gluconacetobacter intermedius (NEDO-01) in a culture medium supplemented with either carboxymethylcellulose (CMC) or hydroxypropyl cellulose (HPC) to prepare either hydrophilic CM-NFBC or amphiphilic HP-NFBC, respectively.21,22) NFBC is composed of uniformed cellulose nanofibers (CNFs) that form a 3D cellulose network in a medium. We further reported the utility of NFBC as a drug-delivery vehicle for anticancer agents such as hydrophobic paclitaxel23) and amphiphilic doxorubicin,24) which are used to treat peritoneally disseminated tumors. Notably, an anticancer agent-embedded NFBC produced no adverse events following intraperitoneal injection,23,24) which suggests that NFBC possesses a high level of biocompatibility with the body while exerting low levels of cytotoxic effects on cells. In addition, NFBC possesses hydrophilic properties and a well-developed network based on long fibers that can hold large amounts of water, which mirrors the properties of hydrogel scaffold-type 3D culture materials.25,26) Based on the above findings, 1 hypothesis suggests that NFBC could be applied to a 3D cell-culture system as a scaffold- or liquid-based 3D cell-culture material.
In this study, we tested two methods for applying NFBC to a 3D cell-culture system: The first involves seeding cells onto an NFBC-based scaffold layer on the bottom of culture vessels (the OnGel method), and the second amounts to suspending cells in a medium that disperses the NFBC (the Suspension method). We optimized the NFBC concentration for preparing cancer spheroids and cultured several types of cancer cells to accommodate a 3D cell culture when using either the OnGel or Suspension methods. We further evaluated the diameters and capacities for cell viability of the resultant spheroids using the OnGel and the Suspension methods and compared the use of each with a commercially available 3D cell-culture system.
3D-NanoFibGrow-I, which contains 1% (w/v) HP-NFBC, was purchased from Nano T-Sailing, LLC. (Tokushima, Japan). CM-NFBC was manufactured by Kusano Sakko, Inc. (Hokkaido, Japan). Matrigel® was purchased from Corning (NY, U.S.A.). FCeM® was purchased from Nissan Chemical Corporation (Tokyo, Japan). All other reagents were of analytical grade.
CellsHepG2 human liver cancer cells (RCB1886), Colon26 mouse colorectal cancer cells (RCB2657), MCF-7 human breast cancer cells (RCB1904), Panc-1 human pancreatic cancer cells (RCB2095), and human-induced pluripotent stem (iPS) cells (HPS0063) were purchased from RIKEN BioResource Center (Ibaraki, Japan). B16 mouse melanoma cells (JCRB0202) and human bone marrow-derived mesenchymal stem cells (BM-MSCs) (JCRB1136) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank (Osaka, Japan). HepG2 and MCF-7 cells were maintained in Dulbecco’s modified Eagle medium (D-MEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% of heat-inactivated fetal bovine serum (FBS) (COSMO BIO, Tokyo, Japan), 100 units/mL penicillin, and 100 μg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation). Colon26, Panc-1, and B16 cells were maintained in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% of heat-inactivated FBS (COSMO BIO), 100 units/mL penicillin, and 100 μg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation). iPS cells were maintained using the Cellartis® DEF-CSTM 500 Culture System (TaKaRa Bio, Shiga, Japan). MSCs were maintained using Cellartis® MSC Xeno-Free Culture Medium (TaKaRa Bio). All cells were cultured in a 5% CO2/air incubator at 37°C.
NFBC-Based 3D Cell-Culture System Cell-Culture at the Interface between NFBC Gel and Medium (the OnGel Method)A conceptual diagram of the OnGel method appears in Fig. 1A. HP-NFBC at a concentration of 0.2–1.0% (w/v) was added onto the bottom surface of a 25 cm2 cell-culture flask (2 mL/flask), and the surface of NFBC gel was flattened by gently tapping the flask. The NFBC-gel layer was acclimated by incubation at 37°C for 30 min. Cells were suspended in a medium (1 × 106 cells/8 mL/flask) and gently added to the prepared NFBC-layered flask where they were cultured at 37°C. The culture medium was changed to a fresh medium every 48 h by replacing half the volume of the culture medium (4 mL) with fresh medium. Cells were observed using a Primovert optical microscope (Carl Zeiss, Oberköchen, Germany) and imaged using a DS-3500WB J-scope microscopic camera (Sato Shouji Inc., Tokyo, Japan).
(A) Conceptual diagram of 3D cell culture by the OnGel method. (B, C) HP-NFBC at a concentration of 0.2–1.0% (w/v) was added onto the bottom surface of a 25 cm2 cell culture flask (2 mL/flask). Cell suspension in medium (8 mL/flask) was gently added onto the NFBC-layered flask (1 × 106 cells/flask) and cultured at 37°C. The culture medium was changed to a fresh medium every 48 h. Spheroids were observed using a microscope with low magnification (B) or high magnification (C) and imaged. Scale bars represent 100 μm. (D) Size of spheroids from the images on Day 4 with different HP-NFBC concentrations was measured, and the number of spheroids in each spheroid size was counted. Spheroids were counted up to a total of 60 numbers from 5 to 8 different images in each HP-NFBC concentration, and the number of spheroids in each spheroid size, for example, the size of spheroids from 40 to 60 was counted as “40,” was graphed. (E) HP-NFBC at a concentration of 0.2–1.0% (w/v) was added onto the bottom surface of a 25 cm2 cell culture flask (2 mL/flask). Cell suspension in medium (1 × 106 cells/8 mL/flask) was gently added onto the NFBC-layered flask and cultured at 37°C. The culture medium was changed to a fresh medium every 48 h. On Day 4, after starting to culture cells, the spheroids were collected and lysed with RIPA buffer, and then protein concentration in the cell lysate was measured. Values show a protein amount (μg/well) and represent as means ± S.D. (n = 3, *** p < 0.001 vs. 0.2% [w/v], ###p < 0.001 vs. 0.4% [w/v], $$$p < 0.001 vs. 0.6% [w/v]). HP-NFBC: hydroxypropyl nano-fibrillated bacterial cellulose; RIPA: radioimmunoprecipitation assay.
A conceptual diagram of the Suspension method is featured in Fig. 2A. HP-NFBC at a concentration of 1.0% (w/v) was mixed with culture medium to prepare the 0.04–0.2% (w/v) HP-NFBC-dispersing medium, and this then was mixed with an equal volume of cell suspension medium (2 × 105 cells/mL). The final concentration of HP-NFBC was 0.02–0.1% (w/v), and the final cell concentration was 1 × 105 cells/mL (the Suspension medium). The Suspension medium was added into a 25 cm2 cell-culture flask (10 mL/flask), and cultured at 37°C. The culture medium was changed to fresh medium as follows: Forty-eight hours after the initial incubation, the 0.02–0.1% (w/v) HP-NFBC-dispersing medium (10 mL) was gently added onto the Suspension medium; every 48 h of incubation, the upper layer of the HP-NFBC-dispersing culture medium (10 mL) was removed and an equal volume of the 0.02–0.1% (w/v) HP-NFBC-dispersing medium was gently added. The cultured cells were observed using a Primovert optical microscope (Carl Zeiss) and imaged using a DS-3500WB J-scope microscopic camera (Sato Shouji, Inc.).
(A) Conceptual diagram of 3D cell culture by the Suspension method. (B, C) HP-NFBC at a concentration of 1.0% (w/v) was mixed with culture medium to prepare the Suspension medium (0.02–0.1% [w/v] HP-NFBC and 1 × 105 cells/mL). The Suspension medium was added into a 25 cm2 cell culture flask (10 mL/flask) and cultured at 37°C. The culture medium was changed to a fresh medium every 48 h. Spheroids were observed using a microscope with low magnification (B) or high magnification (C) and imaged. Scale bars represent 100 μm. (D) Size of spheroids from the images on Day 4 with different HP-NFBC concentrations was measured, and the number of spheroids in each spheroid size was counted. Spheroids were counted up to a total of 60 numbers from 5 to 8 different images in each HP-NFBC concentration, and the number of spheroids in each spheroid size, for example, the size of spheroids from 40 to 60 was counted as “40,” was graphed. (E) The Suspension medium (0.02–0.1% [w/v] HP-NFBC and 1 × 105 cells/mL) was added into a 25 cm2 cell culture flask (10 mL/flask) and cultured at 37°C. The culture medium was changed to a fresh medium every 48 h. On Day 4, after starting to culture cells, the spheroids were collected and lysed with RIPA buffer, and then protein concentration in the cell lysate was measured. Values show a protein amount (μg/well) and represent as means ± S.D. (n = 6, ***p < 0.001 vs. 0.02% [w/v], ###p < 0.001 vs. 0.04% [w/v]). HP-NFBC: hydroxypropyl nano-fibrillated bacterial cellulose; RIPA: radioimmunoprecipitation assay.
HepG2 cells (1 × 106 cells/flask) were cultured via either the OnGel method or the Suspension method using different concentrations of NFBC. On Day 4 of the OnGel method, the cells, or spheroids, were collected by gentle pipetting on the surface of the NFBC-gel layer and were then centrifuged (10000 × g, 4°C, 15 min) to precipitate the cells/spheroids as a pellet. After washing with phosphate-buffered saline (PBS)(–) 3 times, the cells or spheroids were lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (ProteoGuardTM, TaKaRa Bio). In the Suspension method, the Suspension medium was centrifuged (10000 × g, 4°C, 15 min) to precipitate the cells/spheroids and NFBC as a pellet. After washing with PBS(–) 3 times, RIPA buffer containing protease inhibitor cocktail (ProteoGuardTM, TaKaRa Bio) was added to the pellet and was vigorously pipetted to lyse the cells/spheroids. After incubation for 30 min on ice and further centrifugation (10000 × g, 4°C, 5 min), the supernatant was collected as a cell lysate. The protein concentration in the cell lysate was measured using a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.).
Measuring the Size Distribution of the Prepared SpheroidsHepG2 cells (5 × 104 cells/well, 24-well plate) were cultured using either the OnGel method or the Suspension method. Also, HepG2 cells (5 × 104 cells/well, 24-well plate) were cultured using commercially available 3D cell-culture materials, Matrigel® and FCeM®, according to the manufacturer’s instructions. After 96 h of incubation, the spheroids were observed using a Primovert optical microscope (Carl Zeiss) and imaged using a DS-3500WB J-scope microscopic camera (Sato Shouji Inc.). The diameters of the prepared spheroids were calculated using ImageJ software, which is an open-source Java-written program provided by NIH (Bethesda, MD, U.S.A.).
Evaluating the Viability of the Prepared SpheroidsHepG2 cells (5 × 104 cells/well, 24-well plate) were cultured using the OnGel and Suspension methods. Also, HepG2 cells (5 × 104 cells/well, 24-well plate) were cultured using commercially available 3D cell-culture materials, Matrigel® and FCeM®, according to the manufacturer’s instructions. After 96 h of incubation, the cells were collected using either the OnGel method—gently pipetting the medium to suspend the cells and then centrifuging the cell suspension (300 × g, 4°C, 5 min)—or by the Suspension method—mixing the Suspension medium (0.5 mL) with PBS(–) (3 mL) and then centrifuging the cell suspension (300 × g, 4°C, 5 min). The cells were gently resuspended with PBS(–) (1 mL) and were stained with Calcein-AM (Dojindo Laboratories, Kumamoto, Japan) and propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, U.S.A.) according to the manufacturer’s instructions. For fluorescent microscopic observation, the live cells, as manifested by testing Calcein-AM positive (λex = 490 nm, λem = 515 nm), and the dead cells, as manifested by testing PI positive (λex = 530 nm, λem = 620 nm), were imaged using an LSM700 confocal laser-scanning microscope (Carl Zeiss). For flow cytometric analysis, the Calcein-AM-positive live cells and the PI-positive dead cells were analyzed using the CytoFLEX flow cytometric system (Beckman Coulter, Brea, CA, U.S.A.).
Statistical AnalysisStatistical differences between the groups were evaluated via ANOVA using the Tukey post hoc test with Prism 8 software (GraphPad Software, San Diego, CA, U.S.A.). All values are reported as the mean ± standard deviation (S.D.). The levels of significance were set at *p < 0.05, **p < 0.01, and ***p < 0.001.
The characterization of HP-NFBC, including transmission electron microscopy (TEM) images, distribution of CNFs diameters, Fourier transform IR spectroscopy (FT-IR) spectra, and wide-angle X-ray diffraction analysis (WAXD) patterns, was evaluated in the previous publication.21) A conceptual diagram of the 3D cell-culture via the OnGel method appears in Fig. 1A. Spheroids cultured by the OnGel method with different HP-NFBC concentrations were imaged using a microscope with low magnification (Fig. 1B) or high magnification (Fig. 1C), and the size of each spheroids on Day 4 was measured (Fig. 1D). The HepG2 cells formed a spherical formulation in all concentrations of HP-NFBC. They increased in spheroid size when the incubation time was increased. At higher concentrations of HP-NFBC, such as 0.8% (w/v) and 1.0% (w/v), a larger size was frequently observed for the spheroids on Day 4 compared with those at lower concentrations, such as 0.2% (w/v) and 0.4% (w/v). The average spheroid size was 91.0 ± 31.8 μm in 0.2% (w/v), 98.9 ± 22.7 μm in 0.4% (w/v), 154.8 ± 35.4 μm in 0.6% (w/v), 185.5 ± 36.9 μm in 0.8% (w/v), and 210.4 ± 53.4 μm in 1.0% (w/v). The amount of protein extracted from the cultured cells and spheroids on Day 4 increased with an increase in the HP-NFBC concentration (Fig. 1E), which is clearly consistent with the microscopic images of the cultured cells and spheroids (Figs. 1B and 1C). In addition, various numbers of HepG2 cells were added onto a well of a 24-well plate coated with 1.0% (w/v) HP-NFBC and cultured by the OnGel method; the cells successfully formed spherical formulation in any initial numbers of cells, and notably the spheroids grew up earlier and bigger in larger numbers of cells (Supplementary Fig. 1). These suggest that the OnGel method could be used to construct the HepG2 spheroids at higher concentrations of HP-NFBC.
3D Cell-Culture of HepG2 Cells via the Suspension MethodA conceptual diagram of the 3D cell culture via the Suspension method appears in Fig. 2A. In the Suspension method, HP-NFBC was diluted and mixed well with the medium, and then the cells were suspended in HP-NFBC-dispersing medium. Therefore, to create spaces in the cellulose networks for culturing cells in suspension, the final concentration of HP-NFBC in the medium was set lower when using the Suspension method (0.02% (w/v) to 0.10% (w/v)) compared with that in the OnGel method (0.2% (w/v) to 1.0% (w/v)). Spheroids cultured by the Suspension method with different HP-NFBC concentrations were imaged using a microscope with low magnification (Fig. 2B) or high magnification (Fig. 2C), and the size of each spheroid on Day 4 was measured (Fig. 2D). The HepG2 cells successfully gathered in spherical formulations at all concentrations of HP-NFBC. They increased in size from 50 to 100 μm on Day 4. When using the Suspension method, higher concentrations of HP-NFBC, such as 0.08% (w/v) and 0.10% (w/v), tended to produce a bit larger spheroids on Day 4 compared with those for lower concentrations, such as 0.02% (w/v) to 0.06% (w/v). The average spheroid size was 62.6 ± 11.5 μm in 0.02% (w/v), 61.2 ± 9.4 μm in 0.04% (w/v), 61.9 ± 11.1 μm in 0.06% (w/v), 64.2 ± 10.1 μm in 0.08% (w/v), and 66.3 ± 8.3 μm in 0.10% (w/v). The amount of protein in collected cells/spheroids on Day 4 was much higher in HP-NFBC concentrations of 0.06% (w/v) to 0.10% (w/v) compared with that at either 0.02% (w/v) or 0.04% (w/v) (Fig. 2E). In addition, HepG2 cells at a various cell concentration were cultured by the Suspension method with 0.1% (w/v) HP-NFBC; while cells successfully formed spherical formulation in any initial concentration of cells, a lot of several spheroids were observed notably in higher cell concentration (Supplementary Fig. 2). This result was consistent with the microscopic images of cells or spheroids (Figs. 2B and 2C). These results suggest that the Suspension method using HP-NFBC could produce a spherical formulation of HepG2 cells.
3D Cell-Culture of Various Cell Lines via the OnGel and Suspension MethodsCancer cells, including HepG2, Colon26, MCF-7, Panc-1, and B16 cells, were cultured using either the OnGel method or the Suspension method, and imaged on Day 4 following the beginning of the cell culture (Fig. 3A). By comparison with the cells in a 2D culture, the cells among all cell lines tested resulted in spherical formulations when using either the OnGel or Suspension methods. The sizes of the prepared spheroids among all cell lines tested tended to be larger when using the OnGel method than the Suspension method. Non-cancerous cell lines such as human iPS cells (Fig. 3B) and human MSCs (Fig. 3C) were cultured using the OnGel or Suspension methods. The iPS cells clearly resulted in a spherical formulation when using the NFBC method compared with the cells in the 2D culture, and the MSCs established a spherical formulation when using the NFBC method. These results indicate that both the OnGel method and the Suspension method could be applied to various types of cell lines to prepare spherical formulations.
(A) HepG2 human liver cancer cells, Colon26 mouse colorectal cancer cells, MCF-7 human breast cancer cells, Panc-1 human pancreatic cancer cells, or B16 mouse melanoma cells were cultured for 4 d by the conventional 2D culture, the OnGel method, or the Suspension method. (B) Human iPS cells were cultured for 9 d by the 2D culture or the OnGel method. (C) Human MSCs were cultured for 9 d by the 2D culture or the Suspension culture. In the OnGel method, 1.0% (w/v) HP-NFBC was added to the well of a 24-well plate (0.25 mL/well), and cell suspension in medium (0.5 mL) was gently added onto the prepared HP-NFBC-layered well (5 × 104 cells/well). In the Suspension method, the Suspension medium (0.1% [w/v] HP-NFBC and 1 × 105 cells/mL) was added to the well of a 24-well plate (0.5 mL/well) and cultured at 37°C. Spheroids were observed using a microscope and imaged. The scale bars represent 100 μm. HP-NFBC: hydroxypropyl nano-fibrillated bacterial cellulose.
HepG2 cells were cultured via the OnGel method. Also, the cells were cultured on Matrigel® (Fig. 4A), one of the most popular 3D cell-culture scaffold materials in basal studies.27) HepG2 spheroids derived by the OnGel method had a size distribution ranging from 120 to 260 μm with an average size of 164.1 ± 32.7 μm, which is comparable to those cultured on Matrigel® (100–260 μm in size distribution and 153.3 ± 34.3 μm for the average size) (Fig. 4A). There were no significant differences between these average spheroid sizes. Next, HepG2 cells were cultured via either the Suspension method or FCeM® (Fig. 4B). FCeM® was selected as a reference system for the Suspension method. FCeM® is composed of gellan gum (FP001), one of the glycolic polymers. It enables the preparation of spheroids by suspension culture.28) HepG2 spheroids derived by the Suspension method had a size distribution that ranged from 60 to 160 μm with an average size of 87.4 ± 17.8 μm, which is comparable to, or perhaps even a bit larger without statistical significance than, those derived via the FCeM® system (size distribution of 40–140 μm and an average size of 80.0 ± 22.5 μm) (Fig. 4B). In addition, HepG2 spheroids were larger in average size when cultured using the OnGel method compared with those produced via the Suspension method (Figs. 4A and 4B), which is clearly consistent with the spheroid size distribution at HP-NFBC concentration of 1.0% (w/v) in the OnGel method (Fig. 1D) and 0.10% (w/v) in the Suspension method (Fig. 2D). These were clearly certified by time-lapse imaging using a BIOREVO BZ-X800 microscopic system (Keyence, Osaka, Japan) equipped with a time-lapse module (Supplementary Fig. 3). The cells cultured by the OnGel method migrated on the surface of an NFBC layer and then gathered to form larger spheroids, in contrast, the cells cultured by the Suspension method grew up to form spheroids without any aggressive migration. These results suggest that the systems reviewed here—NFBC-based 3D cell culture using the OnGel and the Suspension methods—could be used to prepare spheroids of cancer cells that are comparable to, or even better than, those produced using commercially available 3D cell-culture systems such as Matrigel® and FCeM®.
HepG2 cells (5 × 104 cells/well, 24-well plate) were cultured by the OnGel method, the Suspension method, or using either Matrigel® or FCeM®. After 96-h incubation, spheroids were observed using a microscope and imaged. The diameter of spheroids in four images that were taken from different four wells of the 24-well plate was calculated using ImageJ software. Values show cumulative numbers of spheroids in each spheroid diameter.
Both live and dead cells among the HepG2 spheroids were evaluated using Calcein-AM to stain live cells and PI for dead cells. Under a fluorescent microscope, the cells cultured in both the OnGel and Suspension methods were mostly stained with Calcein-AM and not with PI (Fig. 5A). The cells cultured on Matrigel® or FCeM® were fully stained with Calcein-AM, and the results were comparable to those of either the OnGel or Suspension methods (Fig. 5A). In flowcytometric analysis, the cells produced using both the OnGel and Suspension methods were composed mostly of life (>98%) Calcein-AM-stained cells (Fig. 5B) with less than 5% recognized as PI-stained dead cells (Fig. 5C). As aforementioned, cancer spheroids in large size, more than 500 μm, form a stratified structure with growing cells in periphery and non-dividing/necrotic cells in core because of transporting gradient of oxygen and nutrients.18) In this study, meanwhile, we prepared HepG2 spheroids in size of 164.1 ± 32.7 μm by the OnGel method and 87.4 ± 17.8 μm by the Suspension method (Fig. 4), which may result in the preparation of spheroids with apparent overall surviving cells (>98%) and dead cells (<5%). These results indicate that both the OnGel and Suspension methods could be used to prepare HepG2 spheroids with high levels of cell viability and showing no apoptotic/necrotic cells.
(A) HepG2 cells (5 × 104 cells/well, 24-well plate) were cultured by the OnGel method, the Suspension method, or using either Matrigel® or FCeM®. After 96-h incubation, the spheroids were collected and stained with Calcein-AM and PI. Live cells (green) and dead cells (red) were imaged using a confocal laser-scanning microscope. Scale bars represent 100 μm. (B, C) HepG2 cells (5 × 104 cells/well, 24-well plate) were cultured by the OnGel method, the Suspension method, or using either Matrigel® or FCeM®. After 96-h incubation, the spheroids were collected and stained with Calcein-AM and PI. Live cells, as manifested by Calcein-AM positive (λex = 490 nm, λem = 515 nm) (B), and dead cells, as manifested by PI positive (λex = 530 nm, λem = 620 nm) (C), were analyzed by flow cytometry. Data show a positive area (%) with Calcein-AM or PI and are represented as means ± S.D. (n = 3). PI: propidium iodide.
To date, several types of 3D cell-culture systems have been established. However, most 3D cell-culture systems require cumbersome steps such as gelation coating, creating cross-linking formations, and using ice for control.29) In this study, we successfully developed a novel and simple 3D cell-culture system using NFBC. With the OnGel method, cells were seeded onto the interface between an HP-NFBC layer and the culture medium (Fig. 1). In the Suspension method, cells were simply suspended in an HP-NFBC-dispersing medium (Fig. 2). These NFBC-based 3D cell-culture systems produced preferential spheroids of several types of cancer cells (Fig. 3A) and also non-cancerous cells such as iPS cells (Fig. 3B) and MSCs (Fig. 3C). In addition, the average sizes of the resultant spheroids were comparable to, or even better than, those prepared using commercially available 3D cell-culture systems such as Matrigel® and FCeM® (Fig. 4). The NFBC-based systems produced mostly living cells with very few dead cells (Fig. 5), which in this regard is comparable to results using either a 2D cell-culture or the commercially available 3D cell-culture system (Fig. 5).
Another valid consideration is that the conventional 3D cell-culture system has several shortcomings and requires cumbersome steps for crosslinking components in the system for culturing cells. Matrigel® is an ECM hydrogel that helps grow cells and aids in the development of spherical formulations of cultured cells. Also, Matrigel® has an inconsistent composition that includes concentrations of growth factors and other biological components, which often results in batch-to-batch variability,30) which could cause poor reproducibility of experiments. In addition, Matrigel® is a soluble basement membrane extract that is derived from an animal epithelial tumor,30) and thus cannot be applied for human use. Gellan gum, a polysaccharide component in the FCeM® system, is an anionic extracellular microbial fermentation product that is secreted from bacteria,31) and its use requires the formation of a bridging structure via carboxyl groups in the presence of divalent ions,32) to suspend a 3D cell-culture. On the other hand, our NFBC is composed of a network structure of CNFs with no additional cross-linking reaction and is characterized by high levels of water dispersibility.21,22) Accordingly, our NFBC-based 3D cell-culture system requires no cumbersome procedures to form a cellulose network structure before starting to culture the cells (Figs. 1 and 2). In particular, the Suspension method makes it easy to scale up the volume of the Suspension medium for the preparation of spheroids because our Suspension method can be applied to all culture vessels, such as flasks for a liter-scale cell-culture and a fermenter for a 100 to 1000 liter-scale cell-culture. Collectively, our NFBC-based 3D cell-culture systems could be used to prepare spheroids derived from cancer cells via a simple procedure that is easily scaled up.
The sizes of the resultant spheroids differed substantially between the OnGel and Suspension methods (Fig. 4). The spheroids derived using the OnGel method were much larger than those produced using the Suspension method. In the OnGel method, cells were cultured on the interface between the NFBC gel and the culture medium, suggesting that the cells could easily migrate on such an interface to form larger sizes of spheroids. On the other hand, with the Suspension method, CNFs uniformly disperse in the culture medium, and spheroids form in such spaces within cellulose networks. Therefore, the cells could not migrate among the spaces formed within an NFBC suspension to expand in size under the present culture conditions when using the Suspension method. The stiffness of 3D culture hydrogel is reported to greatly contribute to the motility and the spherical formation of cultured cells.33,34) These researchers presumed that the interface of NFBC gel in the OnGel method could produce stiffness suitable to allow cells to migrate and form spheroids. To reveal such relationships between the spherical formation of cells and the surface properties of NFBC, further mechanochemical and rheological analyses would be required. In addition, to prepare larger sizes of spheroids using the Suspension method, any other artificial options to allow the suspended cells to migrate, such as the use of an aeration stirred culture system, could be required.
In the present study, when CM-NFBC, instead of HP-NFBC, was used, HepG2 cells could not form sufficiently solid spheroids in either the OnGel or Suspension methods (Supplementary Fig. 4). The CM-NFBC was prepared by culturing NEDO-01 in a medium containing hydrophilic CMC and was consistently dispersed in a water solution, albeit not in organic solvents.22) On the other hand, HP-NFBC was prepared by culturing NEDO-01 in a medium containing amphiphilic HPC and was consistently dispersed in not only a water solution but also in organic solvent solutions such as methanol, acetone, and isopropyl alcohol.21) The phospholipid molecules in the plasma membrane have an amphiphilic nature containing both hydrophilic parts such as a head of phospholipid and oligosaccharides and hydrophobic parts such as a hydrophobic lipid bilayer and membrane proteins.35) Therefore, the amphiphilic HP-NFBC easily disperses cells due to their weak interaction with the cells in the NFBC networks, which promotes the spherical formation of cancer cells.
We succeeded in developing a novel and simple 3D cell-culture system using HP-NFBC and both the OnGel and Suspension methods, which enable a series of cancer cells to form spheroids via a simple procedure. Our new systems using HP-NFBC could become a valuable tool for preparing spherical formulations of spontaneously proliferating cells, which are useful for in vitro and in vivo research into the morphology and biological functioning of cells.
This work was partially supported by a research program at Tokushima University that is focused on the development of an intelligent Tokushima artificial exosome (iTEX). The authors are grateful to Mr. James L. McDonald for his helpful advice in developing the English manuscript.
Hidenori Ando and Tatsuhiro Ishida are company members of Nano T-Sailing, LLC. The authors declare that there are no conflicts of interest that could appear to influence this work.
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