Electrical pulse stimulation-induced tetanic exercise simulation increases the secretion of extracellular vesicles from C2C12 myotubes.

Extracellular vesicles (EVs) released into the blood during exercise mediate its whole-body health effects. The differentiation of EVs released by skeletal muscle cells in vivo from those released by other cells is challenging, therefore, it is unclear whether exercise increases the number of EVs secreted by skeletal muscle cells. In this study, we investigated whether exercise affects the quantity of EVs released from skeletal muscle cells using in vitro exercise models. C2C12 myotubes were cultured on a gel layer with 1 or 30 Hz electrical pulse stimulation (EPS) to induce contractions as an artificial simulating exercise. We found that tetanic contraction induced by 30 Hz EPS increased the number of secreted EVs. MicroRNA (miRNA)-seq analysis revealed that 30 Hz EPS altered the miRNA in the secreted EVs. Furthermore, expression analysis of genes related to the biogenesis and transport of EVs revealed that the expression of ALG-2 interacting protein X (Alix) was increased in response to 30 Hz EPS, and the peak value of intracellular Ca2+ in myotubes at 30 Hz EPS was higher than that at 1 Hz, indicating that the increase in intracellular Ca2+ concentration may be related to the increased secretion of EVs in response to 30 Hz EPS.

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons.

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

Screening of anti-atrophic peptides by using photo-cleavable peptide array and 96-well scale contractile human skeletal muscle atrophy models.

Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti-atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti-atrophic peptides using photo-cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96-well system and established a screening process with less variability. Dexamethasone-induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti-atrophic activity. These results indicate that the combinatorial use of the photo-cleavable peptide array technology and 96-well screening system could comprise a powerful approach to obtaining anti-atrophic peptides, and suggest that the 96-well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.

Embryoid Body-Explant Outgrowth Cultivation from Induced Pluripotent Stem Cells in an Automated Closed Platform.

Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system "P 4C S" for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 10(7). There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells.

Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering.

The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering.

Effects of B-cell lymphoma 2 gene transfer to myoblast cells on skeletal muscle tissue formation using magnetic force-based tissue engineering.

Tissue-engineered skeletal muscle should possess a high cell-dense structure with unidirectional cell alignment. However, limited nutrient and/or oxygen supply within the artificial tissue constructs might restrict cell viability and muscular functions. In this study, we genetically modified myoblast cells with the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene and evaluated their function in artificial skeletal muscle tissue constructs. Magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of skeletal muscle bundles by applying a magnetic force. Bcl-2-overexpressing muscle bundles formed highly cell-dense and viable tissue constructs, while muscle bundles without Bcl-2 overexpression exhibited substantial necrosis/apoptosis at the central region of the bundle. Bcl-2-overexpressing muscle bundles contracted in response to electrical pulses and generated a significantly higher physical force. These findings indicate that the incorporation of anti-apoptotic gene-transduced myoblast cells into tissue constructs significantly enhances skeletal muscle formation and function.

Construction of cardiac tissue rings using a magnetic tissue fabrication technique.

Here we applied a magnetic force-based tissue engineering technique to cardiac tissue fabrication. A mixture of extracellular matrix precursor and cardiomyocytes labeled with magnetic nanoparticles was added into a well containing a central polycarbonate cylinder. With the use of a magnet, the cells were attracted to the bottom of the well and allowed to form a cell layer. During cultivation, the cell layer shrank towards the cylinder, leading to the formation of a ring-shaped tissue that possessed a multilayered cell structure and contractile properties. These results indicate that magnetic tissue fabrication is a promising approach for cardiac tissue engineering.

Genetically engineered angiogenic cell sheets using magnetic force-based gene delivery and tissue fabrication techniques.

A major limitation in tissue engineering is the insufficient formation of blood vessels in implanted tissues, resulting in reduced cell density and graft size. We report here the fabrication of angiogenic cell sheets using a combination of two magnetic force-based techniques which use magnetite cationic liposomes (MCLs), magnetofection and magnetic cell accumulation. A retroviral vector encoding an expression cassette of vascular endothelial growth factor (VEGF) was labeled with MCLs, to magnetically attract the particles onto a monolayer of mouse myoblast C2C12 cells, for gene delivery. MCL-mediated infection increased transduction efficiency by 6.7-fold compared with the conventional method. During the fabrication of the tissue constructs, MCL-labeled cells were accumulated in the presence of a magnetic field to promote the spontaneous formation of a multilayered cell sheet. VEGF gene-engineered C2C12 (C2C12/VEGF) cell sheets, constructed using both magnetic force-based techniques, were subcutaneously transplanted into nude mice. Histological analyses revealed that on day 14 the C2C12/VEGF cell sheet grafts had produced thick tissues, with a high-cell density, and promoted vascularization. This suggests that the method described here represents a powerful strategy in tissue engineering.

Cell-patterning using poly (ethylene glycol)-modified magnetite nanoparticles.

Development of cell-patterning techniques is a major challenge for the construction of functional tissues and organs in tissue engineering. Recent progress in surface chemistry has enabled spatial control of cell adhesion onto cultural substrates by varying hydrophilicity, for example, by using poly (ethylene glycol) (PEG). In the present study, we developed a novel cell-patterning procedure using PEG-modified magnetite particles (PEG-Mags) and magnetic force. Using an array-patterned magnet, PEG-Mags were magnetically patterned on the surface of a tissue culture dish. The resultant substrate surface consisted of two regions: the PEG-Mag surface that acts as a cell-resistant region and the native substrate surface that promotes cell adhesion. When human keratinocyte HaCaT cells were seeded onto the PEG-Mag-patterned surface, cells adhered only to the native substrate surface, resulting in cell-patterning on the tissue culture dish. The patterned PEG-Mags were then washed away to expose the native substrate surface, and thereafter, when mouse myoblast C2C12 cells were seeded to the dish, cells adhered to the exposed substrate surface, resulting in a patterned coculture of heterotypic cells. Moreover, it is worth noting that the magnetic force-based cell-patterning procedure is not limited by the property of cultural substrate surfaces, and that cell-patterning of mouse fibroblast NIH3T3 cells on a monolayer of HaCaT cells was successfully achieved using PEG-Mags and magnetic force. These results indicate that this procedure provides a novel concept for cell-patterning and may be useful for tissue engineering and cell biology.

Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique.

We describe the fabrication of three-dimensional tissue constructs using a magnetic force-based tissue engineering technique, in which cellular organization is controlled by magnetic force. Target cells were labeled with magnetite cationic liposomes (MCLs) so that the MCL-labeled cells could be manipulated by applying a magnetic field. Line patterning of human umbilical vein endothelial cells (HUVECs) labeled with MCLs was successfully created on monolayer cells or skin tissues using a magnetic concentrator device. Multilayered cell sheets were also inducible on a culture surface by accumulating MCL-labeled cells under a uniform magnetic force. Based on these results, we attempted to construct a complex multilayered myoblast C2C12 cell sheet. Here, patterned HUVECs were embedded by alternating the processes of magnetic accumulation of C2C12 cells for cell layer formation and magnetic patterning of HUVECs on the cell layers. This technique may be applicable for the fabrication of complex tissue architectures required in tissue engineering.

Magnetic force-based cell patterning using Arg-Gly-Asp (RGD) peptide-conjugated magnetite cationic liposomes.

Micropatterning of target cells is highly desired for tissue engineering and cell biology. Although recent progress in surface chemistry has enabled the spatial control of cell adhesion onto substrates, conventional methods usually require specialized devices and time-consuming processes to fabricate the substrate. In this study, we demonstrate a simple and rapid cell-patterning procedure using magnetite nanoparticles and magnetic force. To label the target cells magnetically, magnetite nanoparticles were encapsulated in cationic liposomes (magnetite cationic liposomes; MCLs). To promote cell attachment, an Arg-Gly-Asp (RGD)-motif-containing peptide was coupled to the phospholipid of MCLs (RGD-MCLs). A human keratinocyte cell line, HaCaT, which has a high anchorage dependency, was used as a model. The RGD-MCLs were added to an ultralow-attachment plate, whose culture surface is modified with a covalently bound hydrogel layer that is hydrophilic and neutrally charged, and then HaCaT cells were seeded to the plates. The RGD-MCLs induced cell adhesion, spreading, cytoskeletal organization, and fibronectin expression. When steel plates with a 200 microm width placed on a magnet were set under a culture surface, magnetically labeled cells aligned on the surface where the steel plate was positioned, resulting in cell patterning. Furthermore, various cell patterns using a computer-aided design were successfully fabricated. These results suggest that cell patterning using RGD-MCLs is a promising approach to tissue engineering and studies in cell biology.