Micropatterning contractile C₂C₁₂ myotubes embedded in a fibrin gel

Abstract

Contractile C(2)C(12) myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C(2)C(12) myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish.

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain

“…Thus, scientists still seek feasible strategies to obtain mature, long, functional muscle fibers in volumetric constructs, with proper vascularization and innervation of the tissue. Proposed approaches to engineer muscle (-like) tissue range from scaffold anchoring [10,40,58] or micropatterning of biomaterials for guided myotube formation [39,60,61] to electrical [36][37][38][39]62] or mechanical stimulation in bioreactors in 2D [29,31,[63][64][65] and 3D settings [6,9,11,14,27,[66][67][68]. However, while there is a clear consensus in the field concerning the necessity for mechanical stimulation in 3D skeletal muscle tissue engineering, not all available systems offer programmable and individually adjustable control over strain parameters.…”

“…With regard to cellular alignment, micropatterned polymer surfaces offering precise control over scaffold nano-or microtopology have been used to promote guided myotube formation [33][34][35]. In terms of muscle maturity, electric stimulation has been demonstrated to enhance the contractile force of engineered muscle in two-and three-dimensional settings [36][37][38][39]. In the past, diverse bioreactor systems enabling mechanical stimulation of cell-seeded scaffolds have been developed, serving as platforms that promote myotube alignment as well as muscle maturation.…”

Engineered Contractile Skeletal Muscle Tissue on a Microgrooved Methacrylated Gelatin Substrate

“…13,29,43,44 For example, Flaibani et al studied the effect of long-term electrical stimulation on myoblasts cultured on a 50-mm groove-50-mm ridge micropatterned polylactic acid and found a greater rate of myoblast differentiation compared to the control system; however, no significant changes in myotube alignment was observed. 29 In this work, for the electrically stimulated myotubes in the wide-ridge micropatterns, the coverage area significantly increased when compared with those in the nonpatterned samples ( p < 0.05, Fig.…”

“…Previously, many attempts at creating 3D contractile muscle tissues were made on a natural material, such as, collagen or fibrin, or were assembled on an anchored muscle sheet. 13,16,23 The properties of natural materials promote cell proliferation and differentiation. However, they are mechanically weak, which makes it hard to control the properties of the final tissue construct, for example, geometry, elasticity, stiffness, and biodegradability.…”

“…9,10 In addition, recent developments in micropatterning and self-assembly have further enhanced the promise of hydrogels for tissue engineering and drug delivery. [11][12][13][14] Micropatterning approaches can be used to alter the behavior and fate of cells such as elongation, 15,16 differentiation, 17 and cell-cell contact and signaling. 18 Therefore, by combining micropatterning technologies and hydrogel microarchitecture, mechanical properties of scaffolds can be tuned to mimic those of the desired tissues.…”