The results are in agreement with changes occurring in muscle cells during differentiation, when a high glycolytic state in the myoblasts transforms into intensive oxidative phosphorylation in mature muscle cells [62]. embryo limb bud were treated with GO and CEME. Cell morphology and differentiation were observed using different microscopy methods. Cytotoxicity and viability of cells were measured by lactate dehydrogenase and Vybrant VX-661 Cell Proliferation assays. Gene expression of myogenic regulatory genes was measured by Real-Time PCR. Our VX-661 results demonstrate that CEME, VX-661 independent of the culture surface, was the main factor influencing the intense differentiation of muscle progenitor cells. The present results, for the first time, clearly demonstrated that the cultured tissue-like structure was capable of inducing contractions without externally applied impulses. It has been indicated that a small amount of CEME in media (about 1%) allows the culture of pseudo-tissue muscle capable of spontaneous contraction. The study showed that the graphene oxide may be used as a niche for differentiating muscle cells, but the decisive influence on the maturation of muscle tissue, especially muscle contractions, depends on the complexity of the applied growth factors. 0.05). The key result was the spontaneous contraction activity of myotubes in the cultures with extract supplementation (Video S1). One to three contractions per minute were noted, an average of 1.7 contraction per minute. Rhythmic contractions of primary muscle fibers were observed under the influence of CEME, both in the group without the GO VX-661 nanofilm as well as with the GO nanofilm. Moreover, there was no difference in contractions between the cells of the CEME and GO + CEME groups. There were no contractions in the control and GO groups. Thus, only the growth factor cocktail (CEME), not the substrate characteristics, generated the physiological activity of cells involved in myogenesis. 2.6. Cytotoxicity and Viability of Muscle Cells To evaluate the cytotoxicity of GO nanofilm and the addition of the extract, the lactate dehydrogenase (LDH) assay was performed after 48 and 96 h of culture (Figure 5A,B). The test was based on the enzymatic reduction of NAD+ by LDH released from damaged cells into the culture media. LDH levels reflect the integrity of the cell membrane. Compared to the control group, the presence of GO nanofilm slightly elevated LDH release from cells after 48 h of culture, but there were no significant differences after 96 h. Open in a separate window Figure 5 Lactate dehydrogenase (LDH) release (A,B) and cell viability (C,D) were determined using LDH and MTT assays, VX-661 respectively. Tests were performed after 48 and 96 h of primary culture. Negative control for LDH maximum release (Triton X), control group (CTRL), cells cultured on graphene oxide nanofilm (GO), cells cultured with addition of the extract (CEME), and cells cultured on GO nanofilm with addition of the extract (GO + CEME). The error bars represent standard deviations. Different letters (a, b, c, d) above the columns indicate statistically significant fallotein differences between the groups ( 0.05). To compare the effect of GO nanofilm and CEME on cell viability, the ability of cells to reduce tetrazolium salt (MTT) and produce insoluble formazan crystals was tested. MTT reduction was measured after 48 and 96 h of culture (Figure 5C,D). Compared to the control group, GO nanofilm slightly affected cell viability but the differences were negligible. 2.7. Expression of Genes Changes in gene expression at the mRNA level were examined using the Real-Time PCR method. Compared to the control group, GO nanofilm had no significant effect on the expression of genes related to basic.