Effects of Hypoxia on Proliferation and Differentiation in Belgian Blue and Hanwoo Muscle Satellite Cells for the Development of Cultured Meat

Abstract

Among future food problems, the demand for meat is expected to increase rapidly, but the production efficiency of meat, which is a protein source, is very low compared to other foods. To address this problem, research on the development and production of cultured meat as an alternative meat source using muscle stem cells in vitro has recently been undertaken. Many studies have been conducted on myosatellite cells for medical purposes, but studies on alternative meat production are rare. In vitro cell culture mimics the in vivo environment for cell growth. The satellite cell niche is closer to hypoxic (2% O2) than normoxic (20% O2) conditions. The aim of this study was to investigate the efficient oxygen conditions of myosatellite cell cultures for the production of cultured meat. The bovine satellite cell counts and mRNA (Pax7, Myf5 and HIF1α) levels were higher in hypoxia than normoxia (p < 0.05). Through Hoechst-positive nuclei counts, and expression of Pax7, MyoD and myosin protein by immunofluorescence, it was confirmed that muscle cells performed normal proliferation and differentiation. Myoblast fusion was higher under hypoxic conditions (p < 0.05), and the myotube diameters were also thicker (p < 0.05). In the myotube, the number of cells was high in hypoxia, and the expression of the total protein amounts, differentiation marker mRNA (myogenin, myosin and TOM20), and protein markers (myosin and TOM20) was also high. The study results demonstrated that the proliferation and differentiation of bovine myosatellite cells were promoted more highly under hypoxic conditions than under normoxic conditions. Therefore, hypoxic cultures that promote the proliferation and differentiation of bovine myosatellite cells may be an important factor in the development of cultured meat.

Effect of Serum and Oxygen on the In Vitro Culture of Hanwoo Korean Native Cattle-Derived Skeletal Myogenic Cells Used in Cellular Agriculture

“…Isolation of pure skeletal muscle satellite cells, muscle stem cells, and myoblasts is required for precise research in muscle regeneration medicine, and most studies have used the following two major isolation methods: sorting using specific cell surface antibodies to obtain integrin beta-7 + CD31 − CD45 − CD29 + CD56 + cells [ 3 , 11 ] and the pre-plating isolation method using a difference in cell adhesion rates [ 14 , 15 ]. However, for cell-cultured meat, which requires a large number of cells to be productive, these two methods are inadequate.…”

“…There is little doubt that physiological hypoxia, rather than normoxia, is beneficial for maintaining and proliferating myoblasts, although there are contradictory studies on the influence of physiological hypoxia on myoblast/muscle satellite cell differentiation [ 10 , 12 , 23 ]. Regarding myoblast differentiation, the negative effect of physiological hypoxia is mostly observed in C2C12 cells, an immortalized mouse myoblast cell line; however, its positive effects were primarily confirmed in primary myoblasts from animals [ 10 , 11 , 12 , 13 ]. Physiological hypoxia and the culture medium of the composition are factors that have a major influence on the in vitro differentiation of myoblasts [ 7 , 10 , 24 ].…”

“…The oxygen concentration of skeletal muscle in the body, which contains skeletal muscle stem cells, is reportedly between 1 and 10% (physical oxygenation and physiological hypoxia) [ 8 , 9 ]. Therefore, some studies sought to culture myocytes under various hypoxic conditions to induce high cell proliferation in vitro [ 9 , 10 , 11 ]. However, previous research on hypoxia and normoxia during myocyte development has reported inconsistent results [ 12 ].…”

Investigating Proliferation and Differentiation Capacities of Hanwoo Myosatellite Cells at Different Passages for Developing Cell-Cultured Meat

“…The membranes stained with antibodies were exposed to Clarity Western ECL Substrate (Bio-Rad). Blots were visualized using a charge-coupled device (CCD) camera and IQ800 control software (Cytiva) 29 .…”

Decoding cultured meat production

“…70,71 Similarly, physical parameters such as temperature or oxygen levels may strongly influence cell proliferation. 72,73 Additional considerations must be taken into account when transitioning from culture flasks to bioreactors. To accommodate the exponentially increasing number of cells during the proliferative phase, increasing volumes need to be employed, either by bioreactor parallelisation or by upscaling vessel sizes.…”