Influence of co-culturing muscle satellite cells with preadipocytes on the differentiation of adipocytes and muscle cells isolated from Korean native cattle

Abstract

The present study was done to investigate the effect of co-culturing muscle satellite cells (MSCs) and intramuscular preadipocytes (IPs) on the differentiation of adipocytes and muscle cells isolated from Korean native cattle. MSCs and IPs were single-cultured in 10% fetal bovine serum/Dulbecco's modified Eagles medium (FBS/DMEM) for 48 h followed by culturing in 5% FBS/DMEM as the growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without any additives for the single- or co-culture of muscle cells and intramuscular adipocytes to induce the differentiation of both cell types. Cell differentiation was measured by morphological investigation and cytosolic enzyme analysis of glycerol-3-phosphate dehydrogenase (GPDH) for the adipocytes and creatine kinase (CK) for the muscle cells. In the morphological test, the presence of muscle cells did not stimulate adipocyte differentiation showing more differentiation of the adipocytes in the single-culture compared to the co-culture condition. However, the differentiation of muscle cells was promoted by adipocytes in the co-culture. The results of the enzymatic analysis were highly associated with the morphological results with a statistically higher GPDH activity (p < 0.05) appearing in the single-culture than in the co-culture, whereas the opposite was true for the CK activity of the muscle cells (p < 0.05). By manipulating in vivo the milieu using a co-culture, we could detect the difference in the rate of cell differentiation and suggest that a co-culture system is a more reliable and precise technique compared to a single-culture. Further studies on various co-culture trials including supplementation of differentiating substances, gene expression analysis, etc. should be done to obtain practical and fundamental data.

Fig. 1. Effect of different culture techniques on morphological changes of intramuscular adipocytes and muscle cells. Intramuscular preadipocytes (IP) and muscle satellite cells (MSC) obtained from Hanwoo were grown at 37℃ under a humidified atmosphere of 5% CO₂. Upon reaching approximately 70% of confluency, the growth medium was replaced with 2% fetal bovine serum/Dulbecco's modified Eagles medium (FBS/DMEM) for both IP and MSC. Intramuscular adipocytes and muscle cells were co-cultured using a 0.4 μm insert membrane. Black arrows indicate lipid droplets for adipocytes and myotubes for muscle cells, respectively. Magnification = 100 x.

Table 1. Effect of different culture techniques on differentiation of muscle cells and intramuscular adipocytes isolated from Korean native cattle^z.