A fetus was isolated from the amniotic membrane of a Jeju black pig (JBP) at 16 and 17 weeks of pregnancy and transferred to the laboratory. Muscles were taken from the femur skeletal muscle of the newborn pig’s hind leg thighs. Muscle samples were washed 3-4 times with phosphate buffered saline (PBS, Gibco, Carlsbad, CA, USA) containing 10% penicillin-streptomycin (PS, Gibco, Carlsbad, CA, USA). Connective tissues, blood vessels, and adipose tissues were removed. And muscle tissues were cut into small sizes. Chopped muscles were then dissociated and disaggregated with collagenase D (2 mg/ml, Roche, Indianapolis, IN, USA), dispase II (1 U/ml, Roche, Indianapolis, IN, USA), and 0.25% trypsin-EDTA (TE, Gibco, Carlsbad, CA, USA) in DMEM/F12 (Gibco, Carlsbad, CA, USA) supplement with 10% PS at 37 ℃ for 1 hour. After digestion, the mixture was filtered through a 70 μm cell strainer. DMEM/F12 containing 15% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) was then added to the mixture to finish the digestion process. The suspension was centrifuged at 1000 ×g for 5 min at 4℃ and incubated with erythrocyte lysis buffer (ACK buffer) for 5 min on ice. After discarding the supernatant, cells were resuspended in culture medium (DMEM/F12) supplemented with 15% FBS and 1% PS. The cell suspension was seeded into a 100 mm cell culture dish and incubated at 37℃ with 5% CO2 atmosphere. To separate and purify satellite cells, cell suspension was transferred to new plates 1 h later. These cells were labelled as P0 generation. Cultures were continued until cells reached about 90% confluency. Cells were then washed with PBS and dissociated with 0.25% TE for subculture on new plates.
To optimize culture conditions for JBP muscle cells, cells at passage 6 (P6) were collected and reseeded at a density of 1.5 x 105 cells per well in a 6-well plate under the following four different culture conditions: 1) culture condition Ⅰ (DMEM + 15% FBS + 1% penicillin-streptomycin + 1% L-glutamine); 2) culture condition Ⅱ (DMEM + 15% FBS + 1% penicillin-streptomycin + 1% L-glutamine + 10 ng/mL EGF + 10 ng/mL bFGF); 3) culture condition Ⅲ (DMEM/F12 + 15% FBS + 1% penicillin-streptomycin + 1% L-glutamine); and 4) culture condition Ⅳ (DMEM/F12 + 15% FBS + 1% penicillin-streptomycin + 1% L-glutamine + 10 ng/mL EGF + 10 ng/mL bFGF). Every culture condition was repeated three times.
Cell proliferation analysis
To analyze proliferation rate of JBP muscle cells, these muscle cells at P6 were seeded into 6-well plates at a density of 1.5 x 105 cells per well and cultured for 3 days. After counting the number of cells in each well, cells were reseeded at a density of 1.5 x 105 cells per well. These processes were repeated three times until P10.
Protein extraction and Western blotting
Total protein was extracted from JBP muscle cells. Briefly, harvested muscle cells were mixed with radio immune precipitation assay buffer (RIPA buffer, Biosesang, Sungnam, Korea) containing protease inhibitors and incubated for 40 min on ice. Cells were then centrifuged at 15,000 rpm for 30 min at 4℃ to collect supernatant. Protein concentration was determined using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). The same amount of protein extract was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gels. Separated proteins were transferred to PVDF membranes. Membranes were incubated with 5% skim milk in TBST (20mM Tris, 137mM NaCl, 5mM KCl, and 0.05% Tween 20) at room temperature for 1 h 30 min. They were then incubated with primary antibodies against beta-actin (1:5000, polyclonal, ab8227, Abcam, Cambridge, MA, USA) and MyoD (1:1000, polyclonal, 18943-1-AP, Proteintech, Rosemont, IL, USA) at 4℃ overnight. After washing with TBST, membranes were incubated with secondary antibodies for 1 h 30 min at room temperature. Protein expression levels were detected using an ECL kit (SuperSignal WestPico Plus, Thermo Fisher, San Jose, CA, USA) and exposed with iBright CL100 Imaging System (Thermo Fisher, San Jose, CA, USA).
For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After cells were washed with PBS, they were treated with PBS containing 3% bovine serum albumin and 0.03% Triton X‐100 for 1 hour at room temperature. Cells were then incubated with primary antibodies anti‐MyoD (MyoD; polyclonal, 1:200, Proteintech, Rosemont, IL, USA) and anti‐Pax7 (Pax7; monoclonal, 1:50, DSHB, Iowa, IA, USA). For detection of primary antibodies, fluorescently labeled (Alexa Fluor 488 or 568; Molecular Probes, Eugene, OR, USA) secondary antibodies were used according to specifications of the manufacturer.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
JBP muscle cells were collected at passages of P6 to P10. Total RNAs were extracted from these cells using an AccuZol Total RNA extraction kit (Bioneer, Daejeon, Korea). Quantity and purity of RNAs were determined using a spectrophotometer (μDrop plate, Thermo Fisher Scientific, San Jose, CA, USA). One microgram of total RNA was reverse‐transcribed to cDNA with a cDNA synthesis kit (Bioneer, Daejeon, Korea). Quantitative real-time polymerase chain reaction assays were performed using 1 uL of cDNA and 19 uL of stock solution containing AMPIGENE® qPCR Green Mix (Enzo, San Diego, CA, USA), UltraPure distilled water (Invitrogen, Carlsbad, CA, USA), and primer solution containing both sense and antisense custom-designed primers on a CFX96™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Samples were denatured at 95℃ for 5 minutes and cycled 40 times at 95℃ (for denaturing) for 5 seconds and 60℃ (for annealing and extension) for 30 seconds. Gene-specific primer sequences are listed in Table 1. qRT-PCR results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene to calculate the expression of each target gene.
Statistical analysis was carried out using SAS 9.4 software program (SAS Institute Inc., USA). Statistical differences were evaluated with Student’s t-test or analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test for post hoc comparisons. All data are expressed as mean ± standard error (SE).