Serum-free cultures of C2C12 cells show different muscle phenotypes which can be estimated by metabolic profiling

Abstract

In vitro skeletal muscle cell production is emerging in the field of artificial lab-grown meat as alternative future food. Currently, there is an urgent paradigm shift towards a serum replacement culture system. Surprisingly, little is known about the impact of serum-free culture on skeletal muscle cells to date. Therefore, we performed metabolic profiling of the C2C12 myoblasts and myotubes in serum-free mediums (B27, AIM-V) and compared it with conventional serum supplementation culture. Furthermore, cell morphology, viability, and myogenic differentiation were observed for 7 days of cultivation. Intriguingly, the metabolic difference is more dominant between the cell status than medium effects. In addition, proliferative myoblast showed more distinct metabolic differences than differentiated myotubes in different culture conditions. The intracellular levels of GL3P and UDP-GlcNAc were significantly increased in myotubes versus myoblast. Non-essential amino acids and pyruvate reduction and transamination showed significant differences among serum, B27, and AIM-V cultures. Intracellular metabolite profiles indicated that C2C12 myotubes cultured in serum and B27 had predominant glycolytic and oxidative metabolism, respectively, indicating fast and slow types of muscle confirmed by MHC immunostaining. This work might be helpful to understand the altered metabolism of skeletal muscle cells in serum-free culture and contribute to future artificial meat research work.

H3K18 lactylation marks tissue-specific active enhancers

“…MT display higher OXPHOS and similar glycolytic rates compared to MB [ 23 ]. Nevertheless, MT have higher lactate levels compared to MB (Supplementary Figure 1 A and [ 24 ]). Similarly, muscles are known to be metabolically highly active and producing high amounts of lactate [ 25 ].…”

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

“…The concentration of extracellular glucose and lactate were measured from the culture medium according to the previous publication from our group ( Jang et al, 2022 ). Briefly, 2 ml of the medium was collected every day for 1 week from individual three T25 flasks prepared for monitoring of cell growth.…”

“…Metabolomics is a superior technique for providing a comprehensive profile of metabolites and is widely acknowledged as the field of omics closest to the phenotype ( Guijas et al, 2018 ). Previously, our group has reported changes in the central carbon metabolism - particularly the alteration of non-essential amino acids and pyruvate metabolism—in the C2C12 mouse muscle cell line when cultured in serum-free media, based on targeted metabolomics by mass spectrometry ( Jang et al, 2022 ).…”

Switching ubiquitous and muscle‐specific isoforms of mitochondrial respiratory complex IV in skeletal muscle fine‐tunes complex IV activity

“…We further tried to differentiate C2C12 cells in 1% B27, an alternative differentiation medium used to generate myotubes with an oxidative metabolic phenotype. 60 As shown in Figure S1A, the B27 supplement promoted the isoform switching expression of Cox6a2, but only had a limited effect on Cox7a1 and Cox8b mRNA levels. Elevated individual CIV and CIV-containing RSCs levels, as well as enhanced CIV enzymatic activity, were observed when C2C12 cells were differentiated in 1% B27 (Figure S1B-D).…”