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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.

SUBMITTER: Jang M

PROVIDER: S-EPMC8764040 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:It has been shown from the isolation and characterization of exosomes from cell culture media supplemented with fetal bovine serum that both their quality and purity are affected. The high abundance of serum proteins, including bovine cell derived exosomes, is also a potential source of contaminants, which may result in appreciable yields of impure exosomes, thereby leading to artifacts. Isolation and characterization of exosomes from cells maintained under serum‑free conditions should therefore ensure the high quality necessary for medical applications. To meet this end, the present study aimed to characterize exosomes released from THP‑1 macrophages cultured in serum‑free, ultra‑centrifuged medium upon infection with the human pathogen Mycobacterium tuberculosis (Mtb). Macrophages differentiated from the human cell line THP‑1 were infected at a multiplicity of infection (MOI) of 5. Macrophages were cultivated in CellGenix® GMP DC serum‑free ultra‑centrifuged medium for 4, 24 and 48 h at 37˚C in a humidified atmosphere with 5% CO2. Total exosome isolation reagent was used to extract the exosomes from the cell culture supernatants of naïve and Mtb‑infected THP‑1 macrophages. The size and purity of the exosomes isolated were subsequently assessed by various methods, including nanoparticle tracking analysis, flow cytometry, MACSPlex exosome analysis, and western blotting. The serum‑free, ultra‑centrifuged medium was found to support the proliferation of the THP‑1 cells successfully. The nanoparticle tracking analysis data revealed that the majority of the isolated particles were within the size range of exosomes (i.e., 30‑150 nM). The MACSPlex exosome analysis confirmed the expression of the exosomal markers, CD9, CD63 and CD81. Furthermore, western blot analysis of the isolated exosomes indicated the presence of CD9, CD63, CD81 and lysosomal associated membrane protein‑1 (LAMP‑1), and also confirmed the absence of Mtb proteins. Taken together, these data provide evidence that serum‑free, ultra‑centrifuged CellGenix® GMP DC medium is suitable for application in exosome research, and may significantly advance such studies. Therefore, the use of serum‑free medium for exosome isolation purposes could offer considerable advantages, and constitute a significant improvement in the growing field of extracellular vesicle research. The use of more sensitive methods represents an advance that will enable researchers to rule out the presence of Mtb pathogenic proteins in exosomes isolated from infected serum‑free cell cultures.

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

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