Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size.

Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 µm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 µm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria.

Cyanobacteria can be utilized as a potential biocatalyst for the production of biofuels and biochemicals directly from CO2. Useful mutants of cyanobacteria, which can grow rapidly or are resistant to specific metabolic products, are essential to improve the productivity of biofuels. In this study, we developed a single cell culture system to effectively screen mutant cyanobacteria using magnetite nanoparticles and magnetic force. Lens culinaris Agglutinin (LCA) was selected as a lectin, which binds to the surface of Synechococcus elongatus PCC7942 cells and the LCA-conjugated magnetite cationic liposomes (MCLs) were developed for magnetic labeling of PCC7942 cells. The MCL-labeled PCC7942 cells were magnetically patterned at a single cell level by using 6,400 iron pillars of the pin-holder device. The device enabled 1,600 single cells to be arrayed in one square centimeter. We cultured the patterned cells in liquid medium and achieved higher colony-forming ratio (78.4%) than that obtained using conventional solid culture method (4.8%). Single cells with different properties could be distinguished in the single cell culture system depending on their growth. Furthermore, we could selectively pick up the target cells and subsequently perform efficient isolation culture. The ratio of successful isolation culture using the developed method was 13 times higher than that of the conventional methods. Thus, the developed system would serve as a powerful tool for screening mutant cyanobacteria.

Enhanced angiogenesis by transplantation of mesenchymal stem cell sheet created by a novel magnetic tissue engineering method.

OBJECTIVE: Therapeutic angiogenesis with cell transplantation represents a novel strategy for severe ischemic diseases. However, some patients have poor response to such conventional injection-based angiogenic cell therapy. Here, we investigated a therapeutic potential of mesenchymal stem cell (MSC) sheet created by a novel magnetite tissue engineering technology for reparative angiogenesis. METHODS AND RESULTS: Human MSCs incubated with magnetic nanoparticle-containing liposomes were cultured, and a magnet was placed on the reverse side. Magnetized MSCs formed multilayered cell sheets according to magnetic force. Nude mice were subjected to unilateral hind limb ischemia and separated into 3 groups. For the control group, saline was injected into ischemic tissue. In the MSC-injected group, mice received magnetized MSCs by conventional needle injections without sheet formula as a control cell group. In the MSC-sheet group, MSC sheet was layered onto the ischemic tissues before skin closure. Blood flow recovery and the extent of angiogenesis were assessed by a laser Doppler blood flowmetry and histological capillary density, respectively. The MSC-sheet group had a greater angiogenesis in ischemic tissues compared to the control and MSC-injected groups. The angiogenic and tissue-preserving effects of MSC sheets were attributable to an increased expression of vascular endothelial growth factor and reduced apoptosis in ischemic tissues. In cultured MSCs, magnetic labeling itself inhibited apoptosis via a catalase-like antioxidative mechanism. CONCLUSIONS: MSC sheet created by the novel magnetic nanoparticle-based tissue engineering technology would represent a new modality for therapeutic angiogenesis and tissue regeneration.

Selective concentration of antimicrobial peptides to heat-treated porous silica gel using adsorption/desorption.

Heat-treated porous silica gel (HT silica gel) previously developed by our group has selectively adsorbed cationic peptides at a pH of 7. Therefore, we focused on the use of antimicrobial peptides (AMPs) as bioactive peptides (BPs). First, 32 AMPs and 32 randomly designed peptides were generated using Fmoc solid synthesis, and their adsorption ratio to HT-silica gel was investigated. Thirty two AMPs showed a relatively higher adsorption ratio of 58.8% compared to that of randomly designed peptides, which was 35.3%. Desorption conditions were investigated using Amyl-1-18 antimicrobial peptides. Next, pepsin hydrolysate from rice endosperm protein (REP) powder was prepared by ourselves. The REP hydrolysate containing dry matter (7.5 mg) was applied to the adsorption/desorption (AD) procedure using HT silica gel to obtain 1.6 mg of AD hydrolysate. When the two hydrolysates were subjected to mass spectrometry, 305 concentrated peptides were obtained. In total, 26 peptides with high content and high enrichment ratios were listed and synthesized. When the antimicrobial activity of these 26 peptides was evaluated using Cutibacterium acnes, five peptides consisting of 12-27 amino acids were identified as novel AMPs. Two of these peptides, which were derived from rice glutelin, showed antimicrobial activity against all four microbes, including Porphyromonas gingivalis, Escherichia coli, and Streptococcus mutans. In the present study, we showed that AMPs could be easily enriched from protein hydrolysate using HT silica gel. The adsorption/desorption procedure using HT silica gel was confirmed to be a useful tool for convenient BP separation.

Assembly of skeletal muscle cells on a Si-MEMS device and their generative force measurement.

We have fabricated a simple Si-MEMS device consisting of a microcantilever and a base to measure active tension generated by skeletal muscle myotubes derived from murine myoblast cell line C2C12. We have developed a fabrication process for integration of myotubes onto the device. To position myotubes over the gap between the cantilever and the base without damage due to mechanical peeling or the use of an enzymatic reaction, we cultured myotubes on poly-N-isopropylacrylamide (PNIPAAm) as a sacrifice layer. By means of immune staining of alpha-actinin, it was confirmed that a myotube micropatterned onto the device bridged the gap between the cantilever and the base. After 7d differentiation, the myotube was actuated by electrical stimulation. The active tension generated by the myotube was evaluated by measuring the bending of the cantilever using image processing. On twitch stimulation, the myotube on the device contracted and generated active tension in response to the electrical signals. On tetanus tension measurement, approximately 1.0 microN per single myotube was obtained. The device developed here can be used in wide area of in vitro skeletal muscle studies, such as drug screening, physiology, regenerative medicine, etc.

Oxygen plasma-treated thermoresponsive polymer surfaces for cell sheet engineering.

Although cell sheet tissue engineering is a potent and promising method for tissue engineering, an increase of mechanical strength of a cell sheet is needed for easy manipulation of it during transplantation or 3D tissue fabrication. Previously, we developed a cell sheet-polymer film complex that had enough mechanical strength that can be manipulated even by tweezers (Fujita et al., 2009. Biotechnol Bioeng 103(2): 370-377). We confirmed the polymer film involving a temperature sensitive polymer and extracellular matrix (ECM) proteins could be removed by lowering temperature after transplantation, and its potential use in regenerative medicine was demonstrated. However, the use of ECM proteins conflicted with high stability in long-term storage and low cost. In the present study, to overcome these drawbacks, we employed the oxygen plasma treatment instead of using the ECM proteins. A cast and dried film of thermoresponsive poly-N-isopropylacrylamide (PNIPAAm) was fabricated and treated with high-intensity oxygen plasma. The cells became possible to adhere to the oxygen plasma-treated PNIPAAm surface, whereas could not to the inherent surface of bulk PNIPAAm without treatment. Characterizations of the treated surface revealed the surface had high stability. The surface roughness, wettability, and composition were changed, depending on the plasma intensity. Interestingly, although bulk PNIPAAm layer had thermoresponsiveness and dissolved below lower critical solution temperature (LCST), it was found that the oxygen plasma-treated PNIPAAm surface lost its thermoresponsiveness and remained insoluble in water below LCST as a thin layer. Skeletal muscle C2C12 cells could be cultured on the oxygen plasma-treated PNIPAAm surface, a skeletal muscle cell sheet with the insoluble thin layer could be released in the medium, and thus the possibility of use of the cell sheet for transplantation was demonstrated.

Tissue suction-mediated gene transfer to the beating heart in mice.

We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that -75 kPa and -90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at -75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.

Application of a cell sheet-polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability.

We have succeeded in fabricating a cell sheet-polymer film complex involving a temperature-sensitive polymer that has enough mechanical strength that can be manipulated even by forceps. The polymer film can be removed by lowering the temperature after transplantation, demonstrating its potential use in regenerative medicine. Recently, tissue engineering involving cell sheets was developed, tissues being fabricated by layering of these cell sheets. This technique promises high density cell packing, which is important for native cell functions, and successful heart therapy using cardiac cell sheets has been reported. On the other hand, the fabrication of a large tissue using cell sheets is difficult because of fragility of the cell sheets. Here, we have developed a novel method in which cells are attached to a temperature-sensitive poly-N-isopropylacrylamide film mixed with laminin and collagen IV, and report that the cell sheet-polymer film complex can be manipulated with forceps. A cell sheet can be removed from the polymer film by lowering the temperature after the manipulation. We have utilized this technique for the primary myocardium and fabricated a physiologically active multi-layered cardiac cell sheet. By applying a micropattern to this polymer film, we have succeeded in making a skeletal muscle cell sheet in which myotubes are oriented in the desired direction. Overall, we showed that this method is useful for cell sheet manipulation, morphogenesis, and transplantation.

Bone tissue engineering with human mesenchymal stem cell sheets constructed using magnetite nanoparticles and magnetic force.

An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.

Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering.

Bone tissue engineering has been investigated as an alternative strategy for autograft transplantation. In the process of tissue engineering, cell seeding into three-dimensional (3-D) scaffolds is the first step for constructing 3-D tissues. We have proposed a methodology of cell seeding into 3-D porous scaffolds using magnetic force and magnetite nanoparticles, which we term Mag-seeding. In this study, we applied this Mag-seeding technique to bone tissue engineering using bone marrow stromal cells (BMSCs) and 3-D hydroxyapatite (HA) scaffolds. BMSCs were magnetically labeled with our original magnetite cationic liposomes (MCLs) having a positive surface charge to improve adsorption to cell surface. Magnetically labeled BMSCs were seeded onto a scaffold, and a 1-T magnet was placed under the scaffold. By using Mag-seeding, the cells were successfully seeded into the internal space of scaffolds with a high cell density. The cell seeding efficiency into HA scaffolds by Mag-seeding was approximately threefold larger than that by static-seeding (conventional method, without a magnet). After a 14-d cultivation period using the osteogenic induction medium by Mag-seeding, the level of two representative osteogenic markers (alkaline phosphatase and osteocalcin) were significantly higher than those by static-seeding. These results indicated that Mag-seeding of BMSCs into HA scaffolds is an effective approach to bone tissue engineering.

Using size-controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice.

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane-based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor-bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 µm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 µm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin ß1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 104 colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor-bearing mouse models.

Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles.

To engineer functional tissues, a large number of cells must be successfully seeded into scaffolds. We previously proposed a methodology for tissue engineering using magnetite nanoparticles and magnetic force, which we termed "Mag-TE." In the present study, we applied the Mag-TE technique to a cell seeding process and have termed the technique "Mag-seeding." The cell-seeding efficiency of NIH/3T3 fibroblasts (FBs) by Mag-seeding was investigated using six types of commercially available scaffolds (5 collagen sponges and 1 D,D-L,L polylactic acid sponge) having various pore sizes. FBs were magnetically labeled with our original magnetite cationic liposomes (MCLs), which have a positive surface charge, to improve adsorption onto the cell surface. FBs labeled with MCLs were seeded onto a scaffold, and a magnet (4 kG) was placed under the scaffold. Mag-seeding facilitated successful cell seeding into the deep internal space of the scaffolds. Cell-seeding efficiency increased significantly in all scaffolds when compared to those without magnetic force. Moreover, when a high-intensity magnet (10 kG) was used, cell-seeding efficiency was significantly enhanced. These results suggest that Mag-seeding is a promising approach for tissue engineering.

Magnetic force-based mesenchymal stem cell expansion using antibody-conjugated magnetoliposomes.

Recently, there has been an accumulation of evidence indicating that human mesenchymal stem cells (MSCs, multipotent cells resident in the bone marrow) are useful for autologous cell transplantation. However, only small numbers of MSCs have been obtained in bone marrow aspirates. We have developed a novel methodology for enriching and proliferating MSCs from bone marrow aspirates using antibody-conjugated magnetoliposomes (AMLs). The AMLs are liposomes conjugated to anti-CD105 antibody (immunoliposomes) and contain magnetite nanoparticles (diameter 10 nm). In the present study, the AMLs were added to a small volume (1 mL) of human bone marrow aspirate. After a 1-h incubation period, the bone marrow aspirates containing AMLs were seeded into 10-cm tissue culture dishes, and a disk-shaped magnet (diameter 2.2 cm; height 1 cm; 4000 Gauss) was positioned under the dish to enrich MSCs by magnetic force. The MSCs proliferated, forming colonies at the site where the magnet was positioned. In contrast, no colonies and very few viable cells were observed in ordinary culture based on plastic-adherent tendencies of cells without use of AMLs. These results suggest that this AML culture method can rapidly and efficiently expand a small number of MSCs into numbers suitable for clinical application.

Transplantation of insulin-secreting multicellular spheroids for the treatment of type 1 diabetes in mice.

The efficacy of cell-based therapy depends on the function and survival of transplanted cells, which have been suggested to be enhanced by spheroid formation. However, few attempts at spheroid generation from insulin-secreting cells, which may be used to treat type 1 diabetes, have been reported. We therefore developed spheroids from the mouse insulinoma cell line NIT-1 by using polydimethylsiloxane (PDMS)-based microwells with a coating of poly(N-isopropylacrylamide) (PNIPAAm). The prepared NIT-1 spheroids or dissociated NIT-1 cells were transplanted into the subrenal capsule in streptozotocin-induced diabetic mice. NIT-1 spheroids prepared using the PNIPAAm-coated PDMS-based microwells had a uniformly sized spherical structure with a diameter of 200-300µm. The PNIPAAm coating increased cell survival in the spheroids and the recovery of the spheroids from the microwells. In diabetic mice, the transplanted NIT-1 spheroids reduced blood glucose levels to normal values faster than dissociated NIT-1 cells did. Additionally, survival was higher among NIT-1 cells in spheroids than among dissociated NIT-1 cells 24h after transplantation. These results indicate that insulin-secreting NIT-1 spheroids prepared using PNIPAAm-coated PDMS-based microwells are more effective for the treatment of type 1 diabetes than dissociated cells in suspension.

Poly(N-isopropylacrylamide)-coated microwell arrays for construction and recovery of multicellular spheroids.

Microwell arrays that have many micro-sized cavities on the device have been employed to form multicellular spheroids. However, methods to efficiently harvest the constructed spheroids from the microwell arrays have not been thoroughly investigated. We evaluated the effects of poly(N-isopropylacrylamide) (PNIPAAm) for constructing and harvesting spheroids from microwell arrays. Microwell arrays were coated with ethanol containing 1%, 2.5%, 5%, or 10% PNIPAAm by a solvent-casting method and then dried. Spheroids formed using the coated microwell arrays were harvested. Highly efficient and rapid recovery of NIH3T3 mouse fibroblast spheroids were achieved for the 5% and 10% coated wells (93.2% ± 1.6% and 93.6% ± 1.1% at 60 s, respectively), whereas recovery was not efficient for 0%, 1%, and 2.5% coated wells (0.2% ± 0.2%, 1.1% ± 0.6%, and 7.8% ± 4.0% at 60 s, respectively). Because PNIPAAm is a thermoresponsive polymer that exhibits a lower critical solution temperature (LCST) of 32°C, we examined the effects of temperature on the recovery rate. The recovery rates at 4°C (below LCST) were equivalent to or higher than those at 37°C (above LCST) for all four cell types examined. Functional assessment suggests that the PNIPAAm microwell arrays are not toxic to the formed spheroids. The PNIPAAm microwell array developed in the present study will be useful for constructing and harvesting spheroids.

In vivo site-specific transfection of naked plasmid DNA and siRNAs in mice by using a tissue suction device.

We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.

Micropatterning of single myotubes on a thermoresponsive culture surface using elastic stencil membranes for single-cell analysis.

We have developed a micropatterning procedure for single myotubes and demonstrated recovery of patterned myotubes without the use of methods that might cause damage to the cells. Since skeletal muscle is a highly ordered tissue mainly composed of myotubes, analysis of single myotubes is one of the promising approaches for studying the various diseases related to skeletal muscle tissues. However, the analysis of single myotubes is quite complicated because of the difficulty in distinguishing individual myotubes differentiated on a normal cell culture surface. In the present study, thin polydimethylsiloxane (PDMS) membranes, which have rectangular holes (30, 50, 100, and 200 microm in width; 500, 750, and 1000 microm in length) through them, were fabricated by using a photolithography technique and used for single myotube micropatterning. A bovine serum albumin-coated (BSA-coated) stencil membrane was placed on a cell culture surface and C2C12 myoblasts were seeded on it. Since the cells could not attach to the surface of the stencil membrane, the cell proliferated and differentiated into myotubes in the hole areas specifically. By peeling off the membrane, a micropattern of myotubes was obtained. It was revealed that the optimum width of rectangular holes for a micropattern of single myotubes was between 30 to 50 microm. Furthermore, by placing a membrane on a thermoresponsive culture surface, recovery of the micropatterned myotubes was possible by lowering the temperature. This method involving the stencil membranes and a thermoresponsive culture surface is useful for analyzing subcellular or single myotubes.