Hideaki Fujita

Alignment of skeletal muscle myoblasts and myotubes using linear micropatterned surfaces ground with abrasives.

Alignment of cells plays a significant key role in skeletal muscle tissue engineering because skeletal muscle tissue in vivo has a highly organized structure consisting of long parallel multinucleated myotubes formed through differentiation and fusion of myoblasts. In the present study, we developed an easy, simple, and low‐cost method for aligning skeletal muscle cells by using surfaces with linear microscale features fabricated by grinding. Iron blocks were ground in one direction with three kinds of abrasives (9 µm diamond suspension, #400 sandpaper, and #150 sandpaper) and then used as molds to make micropatterned polydimethylsiloxane (PDMS) substrates (type I, type II, and type III). Observation of the surface topography revealed that the PDMS substrates exhibited different degree of mean roughness (Ra), 0.03 µm for type I, 0.16 µm for type II, and 0.56 µm for type III, respectively. Murine skeletal muscle cell line C2C12 myoblasts were cultured and differentiated on the patterned PDMS substrates, and it was examined whether the alignment of C2C12 myoblasts and myotubes was possible. Although the cell growth and differentiation on the three types of patterned substrates were similar to those on the flat PDMS substrate as a control, the alignment of both C2C12 myoblasts and myotubes was obviously observed on types II and III, but not on type I or the control substrate. These results indicate that surfaces ground with abrasives will be useful for fabricating aligned skeletal muscle tissues. Biotechnol. Bioeng. 2009;103: 631–638.

Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique

Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering.

Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation

Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3u2005V/mm amplitude, 4u2005ms width, and 1u2005Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50–60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.

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. Biotechnol. Bioeng. 2010;106: 303–310.

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. Biotechnol. Bioeng. 2009;103: 370–377.

Designing of a Si-MEMS device with an integrated skeletal muscle cell-based bio-actuator

With the aim of designing a mechanical drug delivery system involving a bio-actuator, we fabricated a Micro Electro Mechanical Systems (MEMS) device that can be driven through contraction of skeletal muscle cells. The device is composed of a Si-MEMS with springs and ratchets, UV-crosslinked collagen film for cell attachment, and C2C12 muscle cells. The Si-MEMS device is 600xa0μmu2009×u20091000xa0μm in size and the width of the collagen film is 250u2009~u2009350xa0μm, which may allow the device to go through small blood vessels. To position the collagen film on the MEMS device, a thermo-sensitive polymer was used as the sacrifice-layer which was selectively removed with O2 plasma at the positions where the collagen film was glued. The C2C12 myoblasts were seeded on the collagen film, where they proliferated and formed myotubes after induction of differentiation. When C2C12 myotubes were stimulated with electric pulses, contraction of the collagen film-C2C12 myotube complex was observed. When the edge of the Si-MEMS device was observed, displacement of ~8xa0μm was observed, demonstrating the possibility of locomotive movement when the device is placed on a track of adequate width. Here, we propose that the C2C12-collagen film complex is a new generation actuator for MEMS devices that utilize glucose as fuel, which will be useful in environments in which glucose is abundant such as inside a blood vessel.

Fabrication of scaffold-free contractile skeletal muscle tissue using magnetite-incorporated myogenic C2C12 cells.

We have fabricated a functional skeletal muscle tissue using magnetite‐incorporated myogenic cell line C2C12 and a magnetic field. Magnetite‐incorporated C2C12 cells were patterned linearly on a monolayer of fibroblast NIH3T3 cells, using a magnetic field concentrator. After induction of differentiation, the C2C12 cells fused and formed multi‐nucleated myotubes. The 3T3 layer became detached in a sheet‐like manner after cultivation in differentiation medium for 5–8 days. When two separate collagen films were placed on a culture dish as tendon structures, a cylindrical construct was formed. Histological observation of the fabricated cylindrical tissue revealed the presence of multinucleate cells within it. Immunofluorescence staining of the construct showed the presence of sarcomere structures within the construct. Western blot analysis showed that muscle proteins were expressed in the construct. When the construct was stimulated with electric pulses, it exhibited active tension of approximately 1 µN. These results demonstrate that functional skeletal muscle tissue was formed through magnetic force‐based tissue engineering. This is the first report of fabrication of skeletal muscle tissue with active tension‐generating capability using magnetic force‐based tissue engineering. The scaffold‐free skeletal muscle tissue engineering technique presented in this study will be useful for regenerative medicine, drug screening or use as a bio‐actuator. Copyright

Novel method for fabrication of skeletal muscle construct from the C2C12 myoblast cell line using serum‐free medium AIM‐V

We have fabricated muscle tissue from murine myoblast cell line C2C12 by modifying the previously reported method. Fabrication of skeletal muscle tissue has been performed in many ways including the use of a biodegradable scaffold, a collagen gel‐embedded culture, or cell sheet tissue engineering, but the extent of tension generation remains low. Recently, a new skeletal muscle tissue engineering technique involving self‐dissociation of a cell sheet from a laminin‐coated polydimethylsiloxane surface was reported which mostly involved a primary cell culture or co‐culture of C2C12 and 10T1/2 cells. In this study, we succeeded in fabricating muscle tissue using C2C12 cells alone by enhancing cell–cell attachment by the use of serum‐free medium AIM‐V. C2C12 cells were seeded on to a laminin‐coated PDMS surface in a 35 mm culture dish with two silk sutures of 5 mm in length each pinned at two places 18 mm apart. Then, cells were allowed to differentiate in AIM‐V, and the cells started to dissociate in a sheet‐like manner after 5–8 days of differentiation. The cells remained attached to the silk sutures, and tissue having a cylindrical morphology was fabricated. After the cylindrical morphology had been obtained, the medium was changed to DMEM supplemented with 2% horse serum, followed by culture for an additional 5–8 days for maturation. Tissue fabricated using this method was excitable with electric pulse stimulation and the generated active tension was approximately 1.4× greater than that reported previously for a co‐culture of C2C12 and 10T1/2 cells. Immuno‐fluorescence study revealed the presence of a sarcomere structure within the fabricated tissue, and Western blotting confirmed the expression of muscle specific‐proteins. The increased active tension generation compared to that with the previously reported method is probably attributable to the increased proportion of myogenic cells in the tissue. Myooid fabricated from mono‐culture of C2C12 will be useful in the muscle study, especially in the area where gene modification is needed. Biotechnol. Bioeng. 2009;103: 1034–1041.

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.

Novel method for measuring active tension generation by C2C12 myotube using UV-crosslinked collagen film

We have developed a novel method for measuring active tension generated by cultured myotubes using UV‐crosslinked collagen film. Skeletal myoblasts cell line C2C12 or human primary skeletal myoblasts were seeded onto a thin (35u2009µm) collagen film strip, on which they proliferated and upon induction of differentiation they formed multinucleated myotubes. The collagen film–myotube complex contracted upon electric pulse stimulation which could be observed by light microscope. When collagen film–myotube complex were attached to force transducer, active tension generation was observed upon electric pulse stimulation. Measurement of active tension was possible for multiple times for more than 1 month with the same batch of collagen film–myotube complex. Active tension generation capability of C2C12 myotubes increased with progression of differentiation, reaching maximal value 6 days after induction of differentiation. Using this method, we measured the effect of artificial exercise induced by electric pulse on active tension generation capability of C2C12 myotubes. When the electric pulses of 1u2009Hz were continuously applied to induce artificial exercise, the active tension augmentation was observed. After 1 week of artificial exercise, the active tension reached ∼10× of that before the exercise. The increased active tension is attributable to the formation of the sarcomere structure within the myotubes and an increased amount of myotubes on the collagen film. The increased amount of myotubes is possibly due to the suppressed atrophy of myotubes by enhanced expression of Bcl‐2. Biotechnol. Bioeng. 2010; 106: 482–489.