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. 2019 Jun 15;10(6):399.
doi: 10.3390/mi10060399.

Effect of Cyclic Stretch on Tissue Maturation in Myoblast-Laden Hydrogel Fibers

Shinako Bansai  1 , Takashi Morikura  2 , Hiroaki Onoe  3 , Shogo Miyata  4
Affiliations

Effect of Cyclic Stretch on Tissue Maturation in Myoblast-Laden Hydrogel Fibers

Shinako Bansai et al. Micromachines (Basel). .

Abstract

Engineering of the skeletal muscles has attracted attention for the restoration of damaged muscles from myopathy, injury, and extraction of malignant tumors. Reconstructing a three-dimensional muscle using living cells could be a promising approach. However, the regenerated tissue exhibits a weak construction force due to the insufficient tissue maturation. The purpose of this study is to establish the reconstruction system for the skeletal muscle. We used a cell-laden core-shell hydrogel microfiber as a three-dimensional culture to control the cellular orientation. Moreover, to mature the muscle tissue in the microfiber, we also developed a custom-made culture device for imposing cyclic stretch stimulation using a motorized stage and the fiber-grab system. As a result, the directions of the myotubes were oriented and the mature myotubes could be formed by cyclic stretch stimulation.

Keywords: core-shell hydrogel fiber; cyclic stretch; engineered muscle; myoblast; skeletal muscle.

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Conflict of interest statement

H.O is a stockholder and a board member of Cellfiber Inc. which has licenses for certain cell fiber-related technologies and patents from The University of Tokyo.

Figures

Figure 1
Figure 1
Schematic for fabrication of core-shell hydrogel microfibers. The C2C12 cell-laden core-shell hydrogel microfiber was formed by the double co-axial laminar flow.
Figure 2
Figure 2
Cell-laden core-shell hydrogel microfiber culture under 2%, 7%, and 10% horse serum (HS) and 10% fetal bovine serum (FBS) conditions. (a) Phase-contrast images and (b) the change in the diameter of cell-laden core along with culture time. Scale bar: 200 μm.
Figure 3
Figure 3
Custom-made cell culture device for “cell fiber” to impose the cyclic stretch. (a) Schematic of the culture device, (b) photograph of the gripper for hydrogel fibers, and (c) procedure to grab the fibers using the collagen gel and two stainless rods.
Figure 4
Figure 4
Fluorescent images of rhodamine-phalloidine/DAPI counterstaining to visualize the actin cytoskeleton of the C2C12 cells. (a) Monolayer culture and (b) three-dimensional culture using a hydrogel microfiber culture of the C2C12 cells. Scale bar: 50 μm.
Figure 5
Figure 5
(a) Phase-contrast and (b) fluorescent images (calcein-AM staining) of C2C12-cell laden hydrogel fibers subjected to cyclic stretch. Scale bar: 100 μm.
Figure 6
Figure 6
Fluorescent images of rhodamine-phalloidine/DAPI counterstaining to visualize the actin cytoskeleton of C2C12 cells in (a) control and (b) cyclic stretch groups. Scale bar: 100 μm.
Figure 7
Figure 7
(a) Image-based classification of cells (blue: Mature, green: Immature, and red: Undifferentiated) and (b) the ratio of three types of C2C12 cells in the cell laden microfibers. A * indicate a significant difference (p < 0.05) between control and cyclic stretch groups. Scale bar: 100 μm.
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