An oxygen-permeable spheroid culture chip (Oxy chip) promotes osteoblastic differentiation of mesenchymal stem cells

Sensors and Actuators B 232 (2016) 75–83

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

An oxygen-permeable spheroid culture chip (Oxy chip) promotes osteoblastic differentiation of mesenchymal stem cells Takuo Kamoya a,b , Takahisa Anada a,∗ , Yukari Shiwaku a , Teruko Takano-Yamamoto b , Osamu Suzuki a,∗ a b

Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan Orthodontics and Dentofacial Orthopaedics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

a r t i c l e

i n f o

Article history: Received 13 December 2015 Received in revised form 12 February 2016 Accepted 22 March 2016 Available online 23 March 2016 Keywords: Cell culture device Spheroid Mesenchymal stem cells Oxygen concentration Osteoblasts

a b s t r a c t Mesenchymal stem cells (MSCs) are being clinically used for bone and cartilage regeneration. However, the preparation of MSCs for implantation is still costly and time consuming, and controlling the differentiation of stem cells remains a challenge. Although much attention has been paid to three-dimensional cultures in the fields of tissue engineering and regenerative medicine, adequate oxygen supply remains a challenge for growing thicker and larger cellular constructs. To solve this problem, we have developed an oxygen-permeable spheroid culture device (Oxy chip) that enables direct oxygen supply to the cells. The aim of this study was to examine the effect of a three-dimensional culture and oxygenation to the cells on the differentiation of mouse MSC strain D1 cells. Our data demonstrated that MSCs grown in the Oxy chip differentiated into osteoblasts more quickly and efficiently than those grown in the conventional non-oxygen permeable chip and monolayer culture. DNA array and energy metabolism analyses revealed that the Oxy chip facilitated osteoblastic differentiation and aerobic glycolysis, rather than chondrogenic differentiation and anaerobic glycolysis. Together, we revealed for the first time that the oxygenation by the Oxy chip was effective on the osteoblastic differentiation and survival of three-dimensional cultured MSCs. This chip is useful for preparing differentiated cells and controlling the direction of differentiation of MSCs. Moreover, this approach may be useful for transitioning spheroid cultures as a therapy in regenerative medicine. © 2016 Elsevier B.V. All rights reserved.

1. Introduction A multicellular spheroid system is known to better reflect in vivo physiology compared to a conventional monolayer culture [1]. Some types of cells, such as hepatocytes or chondrocytes, gradually lose their cellular function and differentiation in monolayer cultures. In contrast, cells in spheroids are more functional and retain their differentiated phenotype through cell–cell interactions. Numerous reports have shown that hepatocytes [2,3], pancreatic cells [4,5], chondrocytes [6], and osteoblasts [7,8] exhibit alterations in function and cell behavior when grown in a threedimensional (3-D) culture system. Spheroid cultures also mimic the early avascular stage of solid tumors. Spheroids have been widely used for assessing hypoxia responses of tumor cells and screening therapeutic compounds

∗ Corresponding authors. E-mail addresses: [email protected] (T. Anada), [email protected] (O. Suzuki). http://dx.doi.org/10.1016/j.snb.2016.03.107 0925-4005/© 2016 Elsevier B.V. All rights reserved.

in order to reduce experimental animal use. However, one of the biggest obstacles to the application of spheroids in regenerative medicine as a building block of 3-D tissue is the occurrence of cell death at the center of large spheroids due to a lack of oxygen [9]. In order to overcome this problem, we have developed a spheroid culture system that supplies oxygen through a highly oxygen-permeable device [10]. In our previous study, we demonstrated that the device maintains not only metabolic functions of hepatoma HepG2 cells, but also dramatically prevents hypoxia and subsequent central necrosis of relatively large hepatoma spheroids. Oxygen tension influences the survival and differentiation of mesenchymal stem cells (MSCs). There are a number of reports on the effect of oxygen tension on osteogenic differentiation of MSCs cultured as a monolayer [11–13]. Oxygen tension has a much greater influence on 3-D cell aggregates compared to cells cultured as a monolayer due to the highly dense conditions. The oxygen concentration in spheroids depends on the balance between the amount of oxygen supply and consumption by cells. The effect of oxygen supply on the osteogenic differentiation of 3-D cultured MSCs has not been examined in any detail.

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Fig. 1. An illustration of a cross-sectional view of the oxygen-permeable chip (Oxy chip) (A) and non-oxygen-permeable chip (non-Oxy chip) (B).

The present study was designed to investigate whether a 3-D culture and continuous oxygenation to MSC spheroids using an oxygen-permeable chip made with polydimethylsiloxane (PDMS) effectively promotes osteoblastic differentiation. PDMS is widely used for the fabrication of various microdevices due to its high transparency, biocompatibility, low production cost, and high diffusivity and solubility of oxygen (oxygen permeability) [14]. To demonstrate the potential application of the oxygen permeable device for bone regeneration, we compared the osteoblastic differentiation of the MSC strain D1 grown on the Oxy chip to those grown on a conventional non-oxygen permeable chip as well as a monolayer culture. In the present study, we examined the effect of the Oxy chip on differentiation of MSCs for relatively short periods of time, since faster in vitro development would be preferable for potential future therapeutic applications.

2. Experimental 2.1. Fabrication of spheroid culture chips We prepared a spheroid culture chip as previously reported [10]. We previously reported that the spheroid culture device (prototype device) consisted of a sealed chamber and a deformable thin PDMS membrane [15]. A PDMS negative mold was replicated from the prototype culture device utilizing the thin PDMS membrane deformation by applying negative pressure [15]. A PDMS (Silpot 184, Dow Corning Toray, Co. Ltd., Tokyo, Japan) prepolymer was prepared by mixing the base and curing agent at a ratio of 10:1, respectively. The negative mold (25 × 25 × 8 mm) was treated with oxygen plasma for 3 min in a small plasma etch system (PIB10 Ion Bombarder, Vacuum Device, Ibaraki, Japan). After plasma treatment, the mold was immersed in 4% Pluronic F-127 (SigmaAldrich, St. Louis, MO, USA) solution for 24 h to facilitate wetting of the surface of the mold and to prevent PDMS-to-PDMS adhesion. The PDMS negative mold was wiped with paper towels to remove excess Pluronic F-127 solution. PDMS prepolymer (base and curing agent were mixed at a ratio of 10:1) was poured into the PDMS negative mold and cured at 70 ◦ C for 1 h. The PDMS replica was peeled off from the mold and used in the cell culture in the present study (Oxy chip, Fig. 1A). For the non-Oxy chip, the PDMS replica, which was fabricated using a very similar method as described above, was inlaid into a custom-made acrylic resin tray (Fig. 1B). The Oxy chip and non-Oxy chip were designed to be comprised of multicavities (512 wells, 1.00 mm in diameter, 1.05 mm pitch, 1.06 mm in depth) in a triangular arrangement on a 25 × 25 mm section of the cell

culture area. The bottom thickness of the culture chip was adjusted to 1.5 mm. 2.2. Cell culture Mouse bone marrow-derived mesenchymal stem cells (D1 ORL UVA [D1]) were obtained from ATCC (Rockville, MD, USA). The cells were maintained in minimum Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO) and 1% penicillin/streptomycin (PS, Invitrogen-Gibco, Carlsbad, CA) at 37 ◦ C in a 5% carbon dioxide environment. The PDMS chips were sterilized in an oven (160 ◦ C, 2 h). Before use, the PDMS chips were incubated with 2 ml of 4% Pluronic F-127 solution for 6 h. The polymer is adsorbed on the surface of the PDMS and prevents cell attachment [16]. The chips were then rinsed three times with DMEM to remove excess Pluronic F-127. The indicated number of D1 cells (25 × 104 , 50 × 104 , 100 × 104 , 200 × 104 , 400 × 104 , and 800 × 104 cells/chip) was added to chips in 2 ml of osteogenic differentiation medium (DMEM supplemented with 10% FBS, 1% PS, 50 ␮g/ml ascorbate 2-phosphate, 10 mM ␤-glycerophosphate, and 100 nM dexamethasone). After allowing the cells to settle for 2 h, 1 ml of the medium was added to the culture chips. Cells in a monolayer culture were seeded on 6-well plates (Corning) at a cell density of 25 × 104 cells/well in 3 ml medium and used as a control group. This group is referred to as the “plate”. All cells were cultured for 7 days at 37 ◦ C, 5% CO2 , and 95% air in humidified incubators. The culture medium was changed every two days. 2.3. Spheroid diameter measurement and analysis by histochemistry Cells were cultured on each chip and 6-well plates (n = 3). To evaluate changes in spheroid diameter, spheroids were photographed with a photomicroscope (Leica DFC300 FX, Leica Microsystems Japan, Tokyo, Japan). Spheroid diameters were analyzed using an image analysis program for Windows (Image-Pro Plus 7.0, Media Cybernetics Inc., Bethesda, MD, USA). A minimum of 30 spheroids on each chip was photographed and diameters measured. Spheroid diameter was defined as the average length of diameters measured at two-degree intervals joining two outline points and passing through the centroid. Cells (100 × 104 cells/chip) were cultured in the Oxy chip and non-Oxy chip for 7 days, as described above. Cells were rinsed three times with PBS buffer. The spheroids collected from the culture chips were fixed in 3.7% paraformaldehyde in PBS for 2 h and embedded in paraffin. Serial sections (4 ␮m) were mounted onto silane-coated slides and stained with hematoxylin-eosin (HE). Pho-

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Fig. 2. Light-microscopic images of spheroid formation on the Oxy chips (A) and non-Oxy chips (B). Initial cell seeding density was 100, 400, 800 × 104 cells/chip. Bar = 1 mm. (C) Time-lapse images of spheroid formation on the Oxy chip. Initial cell seeding density was 100 × 104 cells/chip. Bar = 0.5 mm.

Fig. 3. (A) Changes in the spheroid diameter on both chips. Initial cell seeding density was 25, 50, 100, 200, 400, 800 × 104 cells/chip. For each time point, n = 30. (B) Changes in the DNA concentration on both chips. Initial cell seeding density was 100 × 104 cells/chip. *p < 0.05, **p < 0.01. (C) HE staining of the spheroids in both chips at day 7. Initial cell seeding density was 100 × 104 cells/chip. The mean diameter of spheroids in the medium at day 7 was 170 ± 11, and 172 ± 8 ␮m for on the Oxy chip and non-Oxy chip, respectively.

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tographs were taken with a photomicroscope (Leica DFC300 FX, Leica Microsystems Japan, Tokyo, Japan). 2.4. Measurement of DNA content and evaluation of osteogenic differentiation of D1 cells Cells on culture chips or 6-well plates were rinsed three times with phosphate buffered saline (PBS). Spheroids were then retrieved from culture chips by washing them out with PBS using a pipette. The collected spheroids were suspended in 0.5 ml of 0.2% Triton X-100 solution and sonicated in an ice bath. DNA concentration in cell lysate was measured using a Quant-iT PicoGreen dsDNA kit (Invitrogen). Alkaline phosphatase (ALP) activity was measured using a commercially available kit (Wako Pure Chemical Industries, Ltd.). The ALP activity was normalized using DNA amounts as determined with the Pico Green kit. RNA was isolated using a TRIZOL reagent (Invitorogen, Carlsbad, CA, USA). DNase treatment was performed using RQ1 RNase-free DNase (Promega). Reverse transcription (RT) was performed with 1 ␮g of RNA using a SuperScript III Reverse Transcriptase (Invitrogen). The mRNA expression levels were determined for osteopontin (OPN) and glyceraldehyde phosphate dehydrogenase (GAPDH) using real-time TaqMan PCR analysis. Optimal oligonucleotide primers and TaqMan probes were designed using the Roche Assay Design Center (Roche Diagnostics, Basel, Switzerland) for murine OPN and GAPDH sequences. The primer sequences and Universal Probes used for the qPCR were as follows: OPN: 5 -cccggtgaaagtgactgatt-3 ,

5 -ttcttcagaggacacagcattc-3 , and universal probe #82; GAPDH: 5 -caatgaatacggctacagcaac-3 , 5 -ttactccttggaggccatgt-3 , and universal probe #77. Real-time TaqMan PCR was performed using a LightCycler 1.5 (Roche Applied Science, Mannheim, Germany). All PCR reactions were performed in duplicate and OPN signals were normalized to the GAPDH signal in the same reaction. 2.5. Gene expression analysis by DNA chip D1 cells (100 × 104 cells/well) were cultured on the Oxy chip, non-Oxy chips, and 6-well culture plates as a monolayer in the osteogenic medium for 7 days. After incubation, total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). DNA microarray analysis was performed using the Genopal Mouse Bone Metabolism Chip (BONM-MX, Mitsubishi Rayon, Tokyo, Japan). 2.6. Measurement of the partial pressure of oxygen in the medium A sensor chip, which consists of a glass disc (4 mm in diameter) coated by a fluorescent dye, was placed at the bottom of the culture chips (medium height: 4.5 mm). Molecular oxygen causes a quenching of the fluorescence and the signal, which is dependent on the partial pressure of oxygen in the medium, and was measured using the Fibox3 single-channel fiber-optic oxygen meter (Presens, Regensburg, Germany). Data were collected by accompanying software (OxyView PST3, PreSens). Measurements were performed 24 h after medium exchange.

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Fig. 5. DNA microarray analysis related to bone metabolism. Initial cell seeding density was 100 × 104 cells/chip. (A) Significantly promoted genes in the Oxy chip compared to the non-Oxy chip and plate. (B) Significantly promoted genes in the non-Oxy chip compared to the Oxy chip and plate. *p < 0.05, **p < 0.01.

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2.7. Measurement of lactate production and the rate of oxygen consumption Lactate concentrations in culture medium were measured using a lactate assay kit (Biovision, CA, USA). Lactate concentrations in the culture medium over 24 h were measured after 7 days in culture, and their values were normalized with the amount of DNA as determined by the Pico Green kit. The oxygen consumption rate (OCR) was measured using a Seahorse Bioscience extracellular flux analyzer (XF24). D1 cells were seeded in Oxy and non-Oxy chips at a density of 100 × 104 cells per chip. After 7 days, spheroids were transferred into 24-well culture plates for measuring basal OCR using an XF24. 2.8. Statistical analysis Results were expressed as the mean ± standard deviation (SD). All experiments were performed at least three times and showed reliable reproducibility. Statistical differences among specimens were evaluated by Tukey-Kramer multiple comparison analysis. A value of p < 0.05 was regarded as statistically significant. 3. Results 3.1. Spheroid formation and cell proliferation on culture chips Fig. 2A and B show light microscopy images of spheroid formation on culture chips and changes in spheroid morphology over time. In both chips, cells were dropped into cavities, resulting

in the formation of a single spheroid in each. Both culture chips generated well-rounded spheroids in each cavity. Fig. 2C shows the time-lapse images of spheroid formation for 100 × 104 cells/chip on the Oxy chip. D1 cells inoculated onto culture chips formed spheroids within 4 h, with no difference in the time required for spheroids to form regardless of the number of cells initially inoculated. Fig. 3A shows changes in spheroid diameter over time. Spheroid diameters increased depending on the initial cell density at day 1. When 800 × 104 and 400 × 104 cells were inoculated on the culture chip, the mean spheroid diameter at day 7 was smaller at early culture time points. For 200 × 104 , 100 × 104 , 50 × 104 , and 25 × 104 cells, spheroids were close to a constant size during the culture period. Spheroid diameters increased as the initial cell density increased. In addition, the diameters of the spheroids of each initial cell density group were not significantly different between the Oxy chip and non-Oxy chip after 7 days in culture. At the end of the culture period, the cells were retrieved from the culture chip by washing them out with PBS or culture medium without physical abrasion or trypsinization. These results indicate that the culture chip generated spheroids with a narrow size distribution. As shown in Fig. 3B, the cell proliferation on the Oxy chip was higher after 7 days of culture compared to cells with non-Oxy chip. Cells on the non-Oxy chip significantly decreased at 7 days of culture compared to those at 3 days. The survival of cells in the spheroids was observed by HE staining as shown in Fig. 3C. Histochemical analysis demonstrated that the spheroids cultured on the non-Oxy chip revealed cell necrosis in the core of spheroids. On

T. Kamoya et al. / Sensors and Actuators B 232 (2016) 75–83

and plate decreased with culture time. At day 7, the pO2 of the non-Oxy chip and plate decreased by approximately 70 mmHg.

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Culture time (day) Fig. 6. Changes in the partial pressure of oxygen (pO2 ) in culture medium using the Oxy chip, non-Oxy chip, and plate. Initial cell seeding density was 100 × 104 cells/chip. n = 3 chips.

the other hand, viable nucleated cells were seen even in the core of spheroids cultured on the Oxy chip. 3.2. Cell differentiation on the chips Fig. 4A and B shows the influence of the initial cell density on the ALP enzymatic activity of D1 cells after 3 and 7 days of culture, respectively. At day 3, ALP activity was significantly higher in the spheroid culture than the monolayer culture. The activity of 400 × 104 cells/chip in the Oxy chip was 119-fold higher than that of the plate culture. ALP activity of D1 cells in the Oxy chip was significantly higher than that in the non-Oxy chip, except when cells were seeded at a density of 25 × 104 cells/chip and grown for 3 days in culture. At day 7, ALP activity was significantly higher in the spheroid culture in the Oxy chip compared to the non-Oxy chip and monolayer culture. The activity of cells grown at a density of 400 × 104 cells/chip in the Oxy chip was 9-fold higher than for cells grown as a monolayer culture. We further examined the effect of the initial numbers of cells and oxygen supply on osteoblastic cell differentiation of D1 cells by measuring mRNA levels of osteopontin. We performed a TaqMan real-time PCR analysis after 7 days of culture (Fig. 4C). The osteopontin mRNA expression level decreased as the initial cell density increased. The osteopontin mRNA expression level in cells grown at a density of 100 × 104 cells/chip in the Oxy chip was 2.5-fold and 12-fold higher than that of cells grown at a density of 400 × 104 cells/chip and 800 × 104 cells/chip in the Oxy chip, respectively. Based on these results, the initial cell density of 100 × 104 cells/chip was evaluated further. Fig. 5 shows the results of a DNA microarray analysis using a Genopal chip, which has 214 genes related to bone metabolism and housekeeping genes. The analysis indicated that the Oxy chip significantly promoted the expression of Col1a, Sparc/Osteonectin, Spp1/Osteopontin, Mgp, Bmp4, Tgf␤1, Tgf␤r1, Smad9, Adipor1, Ctgf, Nfatc3, and Col10a compared with the non-Oxy chip and plate culture. On the other hand, the non-Oxy chip promoted the expression of Col2a, Smad3, Mmp13, Tgf␤3, Ppar␥, and Adipoq compared with the Oxy chip and monolayer culture. 3.3. Measurement of the partial pressure of oxygen in culture medium Fig. 6 shows the changes of the partial pressure of oxygen (pO2 ) in culture medium of each culture condition. The pO2 of the Oxy chip was maintained at approximately 130 mmHg for up to 7 days of culture. In contrast to the Oxy chip, the pO2 of the non-Oxy chip

4. Discussion The significance and originality of this paper lies in the fact that oxygenation by the Oxy chip is effective on the osteoblastic differentiation and survival of the 3-D cultured MSCs. One of the most important issues in tissue engineering is the regulation of stem cell differentiation. The present study suggests that the oxygen concentration could regulate cell lineage determination of the 3-D cultured MSCs. We have previously reported that a spheroid culture device developed in our laboratory, Oxy chip, effectively maintains the cellular function of hepatoma HepG2 cells [10]. The most important feature of the Oxy chip is its composition of gas-permeable PDMS. The oxygen concentration in PDMS (2 mM) is 10 times higher than that in culture medium [17,18]. These studies support our results that the cells in the Oxy chip were continuously supplied with oxygen. Since hepatocytes exhibit a highly metabolic nature and oxygen demand [17], conventional cell culture conditions, in particular high cell density in a 3-D environment, tend to cause a hypoxic environment. In fact, the viable diameter of a hepatocyte spheroid is limited to a maximum of 200 ␮m due to depletion of oxygen [19,20]. Use of the Oxy chip in HepG2 cultures dramatically prevents hypoxia and subsequent central necrosis. It has been reported that oxygen consumption by MSCs is lower than that by hepatocytes [13], but several studies have shown the effects of oxygen tension on MSC differentiation. Although the effects of oxygen tension on osteoblastic differentiation have remained controversial, some studies have reported that spheroid cultures promote osteoblastic differentiation of MSCs [7,8]. Our data in the present study indicate that MSC strain D1 cells form spheroids within several hours after seeding (Fig. 2C). The size of D1 spheroids in the Oxy chip and non-Oxy chip was almost identical after 7 days of culture when the same numbers of cells were inoculated into the chips. A previous study found that the 3D growth of HepG2 cells is significantly increased when the cells are grown on the Oxy chip compared to the non-Oxy chip [10]. On the other hand, changes in D1 spheroid size were negligible over the culture period when 200 × 104 , 100 × 104 , 50 × 104 , or 25 × 104 cells were inoculated on the chips. We postulate that the 3-D growth is dependent on the inherent properties of the cells. This size analysis revealed that 3-D growth of spheroids on both chips in the case of 100 × 104 cells/chip was equivalent. However, DNA quantification analysis and histological analysis revealed that cell number on the non-Oxy chip at 7 days culture decreased compared to that of 3 days culture (Fig. 3B), and this reduction could be caused by cell necrosis in the core of spheroids (Fig. 3C). These results demonstrated that the Oxy chip has an advantage over the cell survival of the 3-D cultured MSCs. The spheroid culture greatly improved ALP activity compared to the monolayer culture. These results are consistent with results reported by other groups [7,8]. Since the Oxy chip conditions pro-

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moted greater ALP activity than the non-Oxy chip conditions, the oxygen supply to the MSC spheroids was effective during the early stages of osteoblastic cell differentiation. In the non-Oxy chip at day 7, ALP activity decreased as the size of the spheroid increased (Fig. 4B). This result might reflect the oxygen depletion in the relatively large spheroids. Similarly, the size effect on osteoblastic differentiation was observed in cells grown on the Oxy chip (Fig. 4). Based on these results, we focused on the initial cell density of 100 × 104 cells/chip and performed a DNA array analysis focused on bone metabolism in order to confirm the effect of oxygen concentration on D1 spheroids (Fig. 5). The DNA array analysis revealed that growth on the Oxy chip led to an increase in the expression of typical osteogenic markers, such as Col1a, Sparc/Osteonectin, and Spp1/Osteopontin. Furthermore, several factors were significantly upregulated in cells grown on the Oxy chip compared to the non-Oxy chip and monolayer culture, including the following: Bmp4, which is a member of the bone morphogenetic protein family and an essential factor during skeletal formation [21]; Tgf␤ and its receptor Tgf␤r1, known to promote proliferation and early differentiation of an osteoprogenitor [22–25]; Smad9, which is a transcriptional regulator for TGF␤ family signaling [26]; Mgp, which is a matrix protein in bone and cartilage [27]; Adipor1, which regulates osteoblast differentiation through GSK-3␤ and ␤catenin signaling pathway [28]; Ctgf, which is thought to play a role in osteoblast development and function [29,30]; Nfatc3, which induces RANKL expression in osteoblasts [31]; Col10a, which is known to be expressed in hypertrophic chondrocytes as well as during osteoblast differentiation [32]. In cells grown on the non-Oxy chip, the following genes were upregulated: Col2a1, which is a typical chondrocyte marker gene, Smad3, which is known to enhance the transcriptional activity of SOX9 and Col2a1 expression during chondrogenesis [33], and Mmp13, which is expressed during chondrogenesis of MSCs [34]. Moreover, Ppar␥, which is a typical adipogenic marker gene, and Adipoq, which encodes an adipocyte-specific secreted protein and is induced during adipocyte differentiation [35], were upregulated in cells grown on the non-Oxy chip compared to the Oxy chip and monolayer culture. These results suggest that the Oxy chip tended to accelerate osteoblastic differentiation of MSCs in the 3-D environment, whereas the non-Oxy chip tended to promote chondrogenic and adipogenic differentiation of MSCs. To understand this phenomenon, we measured partial pressure of oxygen (pO2 ) in the culture medium (Fig. 6). As expected, the pO2 in the Oxy chip was significantly higher than that in the non-

Oxy chip and monolayer culture during the culture period. Cells grown on the Oxy chip tended to have an increased OCR compared to those grown on the non-Oxy chip (Fig. 7B). In contrast, glycolytic lactate production of cells grown on the Oxy chip was decreased (Fig. 7A), suggesting that these cells relied more on oxidative phosphorylation than glycolysis for energy production. A few studies to date have evaluated the energy metabolism of MSCs during osteoblastic or chondrogenic differentiation. Chen et al. found that oxygen consumption of MSCs increased during osteoblastic differentiation [36]. Pattappa et al. reported that MSCs cultured in 3-D differentiated into chondrocytes, had significantly reduced oxygen consumption, and relied predominantly on glycolytic metabolism. In contrast, MSCs cultured as a monolayer in the osteogenic culture conditions underwent greater oxidative metabolism compared to those grown in chondrogenic culture conditions [13]. Sasaki et al. reported that MSC spheroids with a relatively large size (over 1 mm in diameter) differentiated into a chondrogenic lineage due to hypoxic conditions present in the core of the spheroids, despite the use of typical osteogenic differentiation medium [37]. These previous studies support our current findings and suggest that providing an aerobic environment to MSCs may promote differentiation toward an osteoblastic lineage rather than a chondrogenic lineage.

5. Conclusions In this study we developed an oxygen permeable spheroid culture device that consists solely of PDMS. We demonstrated that the chip facilitated formation of MSC spheroids with a narrow size distribution. We showed greater ALP activity and bone-related gene expression in the spheroids than in the monolayer culture. Additional striking results of the present study are that the oxygenated spheroid culture system improved the cell viability and osteoblastic differentiation and inhibited alternate differentiation pathways compared to the conventional spheroid culture system. These results suggest that although spheroid formation is effective for the osteoblastic differentiation of MSCs, the development of central necrosis may contribute to suppressing the differentiation toward osteoblasts. These results may provide a guide for bone regenerative therapy using MSCs to control the differentiation of MSCs, shorten the culture time needed prior to implantation, and design bioreactors for 3-D cultures [38]. Importantly, we believe that our culture system has potential utility in a wide variety of applications, such as tissue engineering and drug screening.

T. Kamoya et al. / Sensors and Actuators B 232 (2016) 75–83

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Biographies Takuo Kamoya received his DDS in 2011 and is now under examination for his PhD degree in Dentistry by Tohoku University. His current research interests are cell and tissue engineering in mineralized tissues. Takahisa Anada is an associate professor at the Graduate School of Dentistry, Tohoku University, Japan. He obtained his PhD in engineering from Kyushu University, Japan. His research has focused on biomaterials and cell culture devices for tissue engineering. Yukari Shiwaku is an assistant professor at the Graduate School of Dentistry, Tohoku University, Japan. She received her DDS in 2007 and her PhD degree in Dentistry from Tohoku University in 2012. Her current research interests are bone tissue engineering and bone metabolism. Teruko Takano-Yamamoto is Professor of Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Japan. Her research area is mainly mechanobiology of bone, cartilage, and tooth. She obtained her Ph.D degree from Osaka University Graduate School of Dentistry, Japan. Osamu Suzuki is Professor of Tohoku University Graduate School of Dentistry, Japan. He is mainly engaged in the research fields of engineering and science of biomaterials of calcium phosphate ceramics and bone regeneration. He obtained his Master’s degree in polymer materials science from the Faculty of Engineering, Yamagata University Graduate School Engineering and Ph.D degree in medical sciences from Tohoku University Graduate School of Medicine, Japan.