Three‐dimensional spherical gelatin bubble‐based scaffold improves the myotube formation of H9c2 myoblasts

Abstract

Microenvironmental factors including physical and chemical cues can regulate stem cells as well as terminally differentiated cells to modulate their biological function and differentiation. However, one of the physical cues, the substrate's dimensionality, has not been studied extensively. In this study, the flow‐focusing method with a microfluidic device was used to generate gelatin bubbles to fabricate highly ordered three‐dimensional (3D) scaffolds. Rat H9c2 myoblasts were seeded into the 3D gelatin bubble‐based scaffolds and compared to those grown on 2D gelatin‐coating substrates to demonstrate the influences of spatial cues on cell behaviors. Relative to cells on the 2D substrates, the H9c2 myoblasts were featured by a good survival and normal mitochondrial activity but slower cell proliferation within the 3D scaffolds. The cortical actin filaments of H9c2 cells were localized close to the cell membrane when cultured on the 2D substrates, while the F‐actins distributed uniformly and occupied most of the cell cytoplasm within the 3D scaffolds. H9c2 myoblasts fused as multinuclear myotubes within the 3D scaffolds without any induction but cells cultured on the 2D substrates had a relatively lower fusion index even differentiation medium was provided. Although there was no difference in actin α 1 and myosin heavy chain 1, H9c2 cells had a higher myogenin messenger RNA level in the 3D scaffolds than those of on the 2D substrates. This study reveals that the dimensionality influences differentiation and fusion of myoblasts.

The Effects of Mechanical Loading Variations on the Hypertrophic, Anti-Apoptotic, and Anti-Inflammatory Responses of Differentiated Cardiomyocyte-like H9C2 Cells

“…In addition, we found that the progression of H9C2 cardiomyoblast differentiation was also accompanied by a gradual increase in p38 activation over time, which has been shown to inhibit the activation of ERK1/2 and to induce cell cycle arrest in adult mammalian cardiomyocytes [42]. Furthermore, and in accordance with previous studies, our study documented the increased phosphorylation of Akt in the differentiating H9C2 cells, which is an important mediator of protein synthesis, a critical process for the completion of myogenic differentiation [40,41] (Figure 2C-E).…”

“…Myogenic differentiation is a highly orchestrated process driven by multiple signal transduction and mechanotransduction pathways, which coordinate the balance between muscle growth and atrophy [24,36,[39][40][41]. Cell cycle arrest and morphological alterations appear to accompany the differentiation process of the cardiomyocyte-like H9C2 cells [3].…”

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts

“…Myoblasts were used to generate spheroids as such a culture provides an environment that facilitates myogenic differentiation due to a 3D structure improving physical interactions, differentiation and fusion of myoblasts Chiron et al, 2012 ; Corona et al, 2012 ; Mei et al, 2019 ; Kim et al, 2020 . One million myoblasts per genotype and per mouse were pelleted at 300 g for 5 min in 15 mL centrifuge tubes before pellets were transferred into individual wells of a six-well plate containing 50% proliferation medium (20% foetal bovine serum, Gibco 10500–064; 10% horse serum, Gibco 26050–088; 0.5% chicken embryo extract, Seralab CE-650-J; penicillin / streptomycin, Sigma P0781; in DMEM) and 50% DMEM (Gibco 31885–023) and cultured for 24 hr.…”

Microfluidic technologies to engineer mesenchymal stem cell aggregates—applications and benefits

“…Three-dimensional cell culture approaches can be classified as scaffold-based and scaffold-free strategies. In general, scaffold-free cell delivery can be divided into three basic methods which are single-cell delivery (Mao et al, 2017;Kamperman et al, 2017a;Lienemann et al, 2017;Qiu et al, 2018;Carvalho et al, 2015;Mei et al, 2019), cell sheet engineering, and microtissue technology (Kelm and Fussenegger, 2010). Microtissues are cell aggregates with a spheroidal shape and diameters between 100 and 500 μm.…”