The Development of Biomimetic Aligned Skeletal Muscles in a Fully 3D Printed Microfluidic Device

Human skeletal muscles are characterized by a unique aligned microstructure of myotubes which is important for their function as well as for their homeostasis. Thus, the recapitulation of the aligned microstructure of skeletal muscles is crucial for the construction of an advanced biomimetic model aimed at drug development applications. Here, we have developed a 3D printed micropatterned microfluid device (3D-PMMD) through the employment of a fused deposition modeling (FDM)-based 3D printer and clear filaments made of biocompatible polyethylene terephthalate glycol (PETG). We could fabricate micropatterns through the adjustment of the printing deposition heights of PETG filaments, leading to the generation of aligned half-cylinder-shaped micropatterns in a dimension range from 100 µm to 400 µm in width and from 60 µm to 150 µm in height, respectively. Moreover, we could grow and expand C2C12 mouse myoblast cells on 3D-PMMD where cells could differentiate into aligned bundles of myotubes with respect to the dimension of each micropattern. Furthermore, our platform was applicable with the electrical pulses stimulus (EPS) modality where we noticed an improvement in myotubes maturation under the EPS conditions, indicating the potential use of the 3D-PMMD for biological experiments as well as for myogenic drug development applications in the future.

How Does a Cell Change Flow Direction Due to a Micro Groove?

The change in direction of a cell flowing over an oblique micro groove has been analyzed in vitro. The micro flow-channel (0.05 mm height x 1 mm width x 25 mm length) with oblique micro grooves (4.5 μm depth) was manufactured on a polydimethylsiloxane (PDMS) disk by the micromachining technique. The angle between the main flow direction and the longitudinal axis of the groove is 45 degrees. The effect of variation of the groove width (0.03 mm, 0.04 mm, and 0.05 mm) was studied. Myoblasts (C2C12: mouse myoblast cell line) were used in the test. The main flow velocity (0.02 mm/s < vx < 0.23 mm/s) of the medium was controlled by the pressure difference between the inlet and the outlet. The shape of each flowing cell was tracked on a movie recorded by the camera attached to the eyepiece of the microscope. The experimental results show that the change of the direction of each cell by each groove depends on the shape of the cell, which depends on both the shape of the cell and the width of the groove.

Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.

Movement of Cell Flowing Over Oblique Micro Grooves in Flow Channel

A micro flow-channel with bottom-microgrooves has been manufactured by photolithography technique for cell sorting. The movement of each cell passing over microgrooves has been analyzed in relation to cell deformation and alignment in vitro. The flow path (height 0.05 mm × width 1 mm × length 25 mm) between the two transparent PDMS disks has rectangular microgrooves (4.5 μm deep, 0.2 mm long) on the bottom. Variations are made in groove widths (0.03 mm, 0.04 mm, and 0.05 mm). The angle between the flow direction and the longitudinal axis of the groove is 45 degrees. Myoblasts (C2C12: mouse myoblast line) were used in the flow test. The main flow velocity of the medium (0.02 mm/s &lt; vx &lt; 0.23 mm/s) was controlled by the pressure difference between the inlet and the outlet. The shape of each flowing cell was tracked in a movie recorded by a camera attached to the eyepiece of the microscope. Experimental results show that the movement perpendicular to the main flow direction in the micro-groove can distinguish cells in relation to smaller deformations and larger alignment changes.

Movement of Myoblast Flowing Through Electric Field Perpendicular to Flow Channel

The sorting technology with little invasion to cells would be applied to regenerative medicine and diagnosis. In this study, dielectrophoresis is focused on. The dielectrophoretic effect on the flowing myoblasts was maximized by adjusting several parameters: the shape of the electrodes, the amplitude and frequency of the alternating current. The suspension of C2C12 (mouse myoblast cell line) was injected into the channel, and the movement of each flowing cell was analyzed at the microscopic movie image. A pair of titanium-coated (200 nm thick) asymmetric surface electrodes (a triangular electrode with a tip angle of 0.35 rad and a rectangular reference electrode with a flat edge) was manufactured by photolithography technique. With the alternating square cyclic wave at the frequency of 3 MHz and the amplitude of current of ± 7.5 mA, 70 μm movement along the electric field (perpendicular to the main flow direction) of the cell was obtained. The movement along the electric field is governed by several parameters of the cell: the diameter, the deformation ratio, and the direction of the major axis. The method can be applied to cell sorting.

Behavior of Cell Passing Through Micro Slit Between Micro Machined Plates

Deformation of each cell, as it passes through the micro-slit in the flow channel, has been investigated in vitro. A slit with a rectangular cross section (height 10 μm, width 0.4 mm, length 0.1 mm) was made in the center of the flow path by photolithography technique. Myoblasts (C2C12: mouse myoblast cell line) were used for the test. The flow rate of the medium, in which the cells were suspended, was controlled by a pressure head between the inlet and the outlet. Deformation of each cell passing through the micro-slit was observed with an inverted phase contrast microscope. Using the contour of the image of each cell passing through the slit intermittently, several parameters were analyzed: the two-dimensional projected area, the degree of deformation by ellipse approximation, and the deformation direction. The experimental results show that elongation of the cell in the slit tends to decrease the area of the cell.

Bioinspired electrospun dECM scaffolds guide cell growth and control the formation of myotubes

While skeletal muscle has a high capacity for endogenous repair in acute injuries, volumetric muscle loss can leave long-lasting or permanent structural and functional deficits to the injured muscle and surrounding tissues. With clinical treatments failing to repair lost tissue, there is a great need for a tissue-engineered therapy to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) with tunable physicochemical properties to control mouse myoblast growth and myotube formation. The material properties as well as cell behavior – growth and differentiation – were assessed in response to modulation of crosslinking and scaffold architecture. The fabrication of a bioactive dECM-based system with tunable physicochemical properties that can control myotube formation has several applications in skeletal muscle engineering and may bring the field one step closer to developing a therapy to address these unmet clinical needs.

Zebrafish Melanophores Suggest Novel Functions of Cell Chirality in Tissue Formation

Several types of cells show left–right asymmetric behavior, unidirectional rotation, or spiral movements. For example, neutrophil-like differentiated HL60 (dHL60) cells show leftward bias in response to chemoattractant. Neurons extend neurites, creating a clockwise spiral. Platelet cells shows unidirectional spiral arrangements of actin fibers. In the microfabricated culture environment, groups of C2C12 cells (mouse myoblast cell line) were autonomously aligned in a counter-clockwise spiral pattern, and isolated C2C12 cells showed unidirectional spiral pattern of the actin skeleton. This biased directionality suggested that these cells have inherent cell chirality. In addition to these cells, we recently found that melanophores of zebrafish also have an intrinsic cellular chirality that was shown by their counter-clockwise self-rotation. Although this cell chirality is obvious, the function of the cell chirality is still unclear. In this review, we compare the cell chirality of melanophores of zebrafish with other cell chirality and consider the function of cell chirality in morphogenesis.

IL-1β and TNF-α Modulation of Proliferated and Committed Myoblasts: IL-6 and COX-2-Derived Prostaglandins as Key Actors in the Mechanisms Involved

In this study, we investigated the effects and mechanisms of the pro-inflammatory cytokines IL-1β and TNF-α on the proliferation and commitment phases of myoblast differentiation. C2C12 mouse myoblast cells were cultured to reach a proliferated or committed status and were incubated with these cytokines for the evaluation of cell proliferation, cyclooxygenase 2 (COX-2) expression, release of prostaglandins (PGs) and myokines, and activation of myogenic regulatory factors (MRFs). We found that inhibition of the IL-6 receptor reduced IL-1β- and TNF-α-induced cell proliferation, and that the IL-1β effect also involved COX-2-derived PGs. Both cytokines modulated the release of the myokines myostatin, irisin, osteonectin, and IL-15. TNF-α and IL-6 reduced the activity of Pax7 in proliferated cells and reduced MyoD and myogenin activity at both proliferative and commitment stages. Otherwise, IL-1β increased myogenin activity only in committed cells. Our data reveal a key role of IL-6 and COX-2-derived PGs in IL-1β and TNF-α-induced myoblast proliferation and support the link between TNF-α and IL-6 and the activation of MRFs. We concluded that IL-1β and TNF-α induce similar effects at the initial stages of muscle regeneration but found critical differences between their effects with the progression of the process, bringing new insights into inflammatory signalling in skeletal muscle regeneration.