The muscle stem cells of domestic animals are of interest to researchers in the food and biotechnology industries for the production of cultured meat
March 11, 2021
The muscle stem cells of domestic animals are of interest to researchers in the food and biotechnology industries for the production of cultured meat. tissue debris and myofibers. The producing cell population contains various types of cells such as somatic cells, blood cells, stromal cells, and muscle mass stem cells. Therefore, numerous sorting methods have been developed to obtain highly purified muscle mass stem cells based on their physical, biological, and molecular features. Density gradient centrifugation and preplating are widely used methods for sorting muscle mass stem cells because no special devices are required. The density gradient centrifugation separates cells based on their density. Because the muscle mass stem cells and other somatic cells have different densities, the stem cells can be isolated from your mixed populations via centrifugation using a solution with a density gradient made of dense substrates (Bischoff, 1997). Because muscle mass stem cells and fibroblasts prefer laminin and collagen as an adherent niche, respectively Synephrine (Oxedrine) (Khl et al., 1986), the preplating technique divides the cell populations using this difference in adhering ability onto the culture Synephrine (Oxedrine) plate or the substrates. At 40C60 min after seeding around the collagen-coated culture plate, the stem cell populace can be obtained by harvesting the supernatant, since most of the fibroblasts and epithelial cells remain attached to the culture plate (Rando and Blau, 1994; Richler and Yaffe, 1970). However, density gradient centrifugation and the preplating technique reportedly show wide variations and low fidelities (Ding et al., 2017). Improvements in molecular biology allow us to analyze and individual the cells based on their molecular features. Fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) systems isolate the muscle mass stem cells using fluorescence and magnetic microbead-conjugated antibodies against the marker genes of the stem cells, respectively (Blanco-Bose et al., 2001; Liu et al., 2015). FACS and MACS are considered to be more precise methods for isolating muscle mass stem cells compared to the aforementioned methods (Ding et al., 2017). Every cell in the body has its markers that it exclusively expresses compared to Synephrine (Oxedrine) other cells, and FACS and MACS analyze and sort the cells through a acknowledgement of markers using antibodies. To date, numerous markers, including cluster of EIF4EBP1 differentiation 29 (CD29; integrin 1), CD34, CD56 (neural cell adhesion molecule, NCAM), C-X-C chemokine receptor 4 (CXCR4), vascular cell adhesion Synephrine (Oxedrine) molecule (VCAM), integrin 7, and SM/C-2.6, have been used for the sorting of muscle mass stem cells (Liu et al., 2015). Both antibody-based methods have shown a consistently high efficiency for isolating muscle mass stem cells. While FACS allows us to conduct a more precise analysis using circulation cytometry, MACS especially is relatively less harmful to the cells during a sorting process and is more suitable for scale-up. For generating cultured meat, it is crucial for muscle mass stem cells to be efficiently isolated and stably managed at a large level. In a previous study, we optimized the culture conditions to maintain the stemness of pig muscle mass stem cells for an expanded period (Choi et al., 2020). For the purification of pig muscle mass stem cells, the density gradient centrifugation and preplating techniques have been widely used in pig studies. However, only a few protocols using FACS and MACS for pig muscle mass stem cells have been reported (Ding et al., 2017). Accordingly, in the present study, we aimed to develop a scalable method for the enrichment of pig muscle mass stem cells using the MACS system. Materials and Methods Animal care The care and experimental use of pigs were approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University (approval no. SNU-180612-2). The experiments were conducted according to the standard protocol of the Institute of Laboratory Animal Resources at Seoul National University or college. Isolation and culture of pig muscle mass stem cells Pig muscle mass stem cells were isolated from your muscles were collected and washed with Dulbeccos phosphate-buffered saline (DPBS; Welgene, Gyeongsan, Korea) made up of 2antibiotic-antimycotic (AA; Gibco, Gaithersburg, MD, USA), after which the excessive connective tissues and blood vessels were removed. The 30 g of collected tissues was minced by a meat grinder and digested with 0.8 mg/mL Pronase (Sigma-Aldrich, St. Synephrine (Oxedrine) Louis, MO, USA) for 40 min at 37C with vortexing every 10 min. The resultant combination was harvested by centrifugation at 1,200g for 15 min and resuspended in minimum essential medium (MEM) made up of 10% fetal bovine serum (FBS, Gibco). For separation of the undigested tissues from your digested cells made up of the muscle mass stem cell populace, the digested muscle tissues were centrifuged at 300g for 5 min and the supernatant was.