Before starting the isolation of porcine muscle satellite cells, the following reagents need to be purchased, including Dulbecco’s Modified Eagle Medium, high glucose (DMEM-HG) (Invitrogen, Grand Island, USA. cat. no. 11995-065), fetal bovine serum (FBS) (Invitrogen, Carlshad, CA, USA. cat. no. 12664-025), horse serum (HS) (Invitrogen, New Zealand, cat. no. 26050-070), 2-[4-(2-hydroxyethyl)-1-piperazine]-1-ethanesulfonic acid buffer (HEPES buffer, 1 M) (Life Technologies, Grand Island, NY, USA. cat. no. 15630106), Phosphate-buffered saline (PBS) (Life Technologies, Grand Island, NY, USA. cat. no. 10010-023), 0.25% Trypsin-ethylenediaminetetraacetic acid (Life Technologies, NY, Grand Island, USA. cat. no. 25200056), Protease from Streptomyces griseus (Sigma, St. Louis, MO, USA. cat. no. P8811, 3.5 U/mg); Collagenase from Clostridium histolyticum (Sigma, St. Louis, MO, USA. cat. no. C7657), 100×Penicillin-Streptomycin (10,000 U/mL) (Invitrogen, Carlsbad, CA, USA. cat. no. 15140-122) and 100% ethanol (GB 678-2002, Beijing, China).

Subsequently, the cell culture reagents need to be prepared carefully. The 100×penicillin-streptomycin solution was diluted to 1 fold with PBS to prepare the 1×penicillin-streptomycin PBS (+), and the final concentration of penicillin and streptomycin was 100 U/mL and 100 μg/mL, respectively. The PBS (+) was stored at 4°C until using. The 20% FBS proliferation medium (PM) was prepared with DMEM-HG and FBS according to the ratio of 4:1, e.g. 100 mL 20% FBS PM was prepared with 80 mL DMEM-HG and 20 mL FBS. The PM was stored at 4°C until using or prepared freshly. The 100×penicillin-streptomycin solution was diluted to 1 fold with PM to prepare 20% FBS proliferation medium+ (PM+), the final concentration of penicillin and streptomycin was 100 U/mL and 100 μg/mL, respectively. The PM+ was stored at 4°C until using (not too long before using) or prepared before using. The 2% HS differentiation medium (DM) was prepared with DMEM-HG and HS according to the ratio of 50:1, and the DM was stored at 4°C until using (not too long before using). 1.5 mg proteases (3.5 U/mg) was added to 1 mL preheated PBS including 1.0% HEPES (37°C) to prepare 1.5 mg/mL proteases from streptomyces griseus solution, the solution was gently mixed and filtered through 0.22 μm filter, and stored at −20°C. The collagenase type XI of 1.5 mg was added to 1 mL preheated DMEM-HG containing 5% FBS (37°C) to prepare 1.5 mg/mL collagenase type XI from clostridium histolyticum solution containing 5% FBS, the solution was filtered through 0.22 μm filter, and stored at −20°C. The freezing medium was prepared with dimethyl sulfoxide (DMSO) and FBS according to the ratio of 1:5, e.g. 1 mL freezing medium was prepared with 200 μL DMSO and 800 μL FBS, and then gently mixed. The freezing medium was prepared before using. The ethanol of 75% and 70% was prepared with 100% ethanol and sterilized ultrapure water according to the ratio 3:1 and 7:3, respectively. E.g. 100 mL 75% ethanol was prepared with 75 mL 100% ethanol and 25 mL sterilized ultrapure water, and the solution was gently mixed.

The following instruments, including water bath, centrifuge, incubator, light microscope, confocal microscopy, ice maker, are required for the isolation of porcine muscle satellite cells. In addition, the following consumables also need to be prepared, including scalpels, razor blades, forceps, scissors, cell strainers (pore size 40 μm, 70 μm, 100 μm), plastic petri dishes, polypropylene centrifuge tubes (15 mL, 50 mL), culture flask (T75, T25), Cryo tube vials, 6-wells culture dishes. All the consumables must be sterile and then can be directly used for cell culture.

The Duroc×Yorkshire×Landrace newborn piglets less than one week old were provided by Jiangpu Farm of Nanjing Agricultural University. The piglets were slaughtered at College of Animal Science and Technology of Nanjing Agricultural University according to the Animal Care and Use Statute of China, and all the sample collections were specifically approved by the ethics committee of Nanjing Agricultural University.

After slaughter, the body surface of the piglets was sterilized with 75% ethanol, and then transferred in to the cell culture laboratory for muscle tissue collection. The hair on hind legs was removed and the skin of the hind legs sterilized with 75% ethanol, then the whole semitendinosus (ST) and semimembranosus (SM) muscles on the right and left were dissected using scalpels, respectively.

Firstly, the collected muscles were quickly sterilized with 70% ethanol in a plastic petri dish, and then immediately transferred into a new plastic petri dish with cold PBS (+) and rinsed 3 to 4 times with 4 fold volume cold PBS (+) in a new plastic petri dish. Visible adipose and connective tissues on the muscle mass were removed with a scalpel in laminar-flow hoods or Biological safety cabinets.

Secondly, the whole ST or SM muscles were excised and cut into small pieces using scissors under the cold PBS (+) containing 1% HEPEs in a plastic petri dish and the shredded muscle pieces were transferred into 15 mL polypropylene centrifuge tubes, centrifuged at 1,000 g for 5 min to separate the supernatant and shredded muscle pellet. The supernatant was again centrifuged at 2,000 g for 5 min to collect a second pellet. Then 2 to 4 folds volume 1.5 mg/mL Protease solution was added to the pooled collected pellets (ensuring the muscle pieces were covered by protease solution).

Thirdly, the 15 mL polypropylene centrifuge tubes containing the muscle pieces were digested in a water bath at 37°C for 1 h with 10 min shaking (a continuous shaking water bath would be better) and the digested muscle pieces were aspirated back and forth for 10 to 15 times with 5 or 25 mL pipettes to gently dissociate the cells, and centrifuged for 5 min at 200 g to separate the supernatant containing the cell pellet and the underlying tissue fragments pellet. While the underlying tissue fragment pellets were temporarily kept on ice for subsequent collagenase type XI digestion. Then, the supernatant was transferred into 15 mL polypropylene centrifuge tubes and was centrifuged for 5 min at 2,000 g to collect the cell pellet, and the cell pellet was further resuspended with 10 mL PM+, and then centrifuged for 10 min at 2,000 g to collect the cell pellet again. Afterwards, the cell pellet was resuspended again with 5 mL PM+, and the cell suspension was filtered through 100 μm and 70 μm cell strainers, successively. Then, the filtered cell suspension was centrifuged for 10 min at 2,000 g to collect the cell pellet (Pellet I), and the cell pellet was resuspended with 1 mL PM+ again and kept on ice.

Fourthly, the underlying tissue fragment pellet derived from the third step was further digested for 1 h with 2 to 4 folds pre-prepared collagenase type XI solution. Similarly, the tissue fragments were aspirated for 10 to 15 times with 5 or 25 mL pipettes to gently dissociate the cells and filtered through a 100 μm cell strainer, Then, the filtrated cell suspension was centrifuged for 5 min at 2,000 g to separate the supernatant and the cell pellet, and the supernatant centrifuged for 10 min at 2,000 g to collect the residual cell pellet, and both collected cell pellets were mixed. Next, the cell pellet was resuspended with 10 mL PM+ in 15 mL polypropylene centrifuge tubes and the cell suspension was filtered through a 70 μm cell strainer, and then centrifuged for 5 min at 2,000 g to collect the cell pellet (Pellet II). The cell pellet was resuspended with 1 ml PM+ and kept on ice.

Fifthly, the cell suspension (Pellet I and Pellet II) were mixed in 15 mL polypropylene centrifuge tubes and centrifuged for 5 min at 2,000 g to recover the Pellet I and Pellet II. Next, the cell pellet was resuspended with 10 mL cold PBS (+) and centrifuged for 10 min at 2,000 g to collect the cell pellet. Subsequently, the cell pellet was resuspended with 5 mL PM+ again and the cell suspension was filtered through a 40 μm cell strainer. Then, the cell suspension was adjusted to 10 mL with PM+, centrifuged for 10 min at 700 g to collect the cell pellet.

Finally, the cell pellet was resuspended in 10 mL 37°C preheated PM+ and the cell suspension was pre-plated in T75 cell culture flask and incubated at 37°C under 5% CO₂ for 1 h. The fibroblasts become quickly adherent to the bottom of cell culture flask, while the skeletal muscle satellite cells will remain in the supernatant. Then, the supernatant containing the skeletal muscle satellite cells was collected to 15 mL polypropylene centrifuge tube and centrifuged for 10 min at 500 g, and the cell pellet was resuspended with 10 mL PBS+, centrifuged for 10 min at 500 g to collect the skeletal muscle satellite cells pellet. Afterwards, the skeletal muscle satellite cells pellet was resuspeneded with 10 mL 37°C preheated PM+ and plated in T75 cell culture flask and incubated at 37°C under 5% CO₂.

To characterize the porcine skeletal muscle satellite cells, the isolated cells were plated into covered 6-well plates at a density of 10⁵ cells/mL per well and cultured in an incubator at 37°C with 5% CO₂ until to 70% to 80% confluent. Subsequently, immunofluorescence staining with specific antibodies was performed to identify the porcine skeletal muscle satellite cells. Briefly, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature, and then washed three times for 5 min each with PBS again, followed by permeabilization with 0.5% Triton X-100 for 30 min at room temperature. Afterward, the cells were blocked with 1% bovine serum albumin (BSA)-supplemented PBS for 1 h and incubated overnight at 4°C or 4 h at room temperature with anti-Pax7 primary antibody (Santa Cruz Tech, Santa Cruz, CA, USA) and anti-Desmin primary antibody (Santa Cruz Tech, USA). After three washes for 5 min each time in PBS, the cells were labeled with appropriate fluorescein-labeled secondary antibody for 1 h in the dark at room temperature, and followed by washing three times in PBS again (5 min each time). Meanwhile, the cell nuclei were counterstained with 4′, 6-diamidino-2-phenylindole for 5 min, and then the samples were mounted on glass slides and examined on confocal laser scanning microscope (Zeiss LSM 700 META, Jena, Germany).

Proliferative properties of the porcine skeletal muscle satellite cells were detected using cell counting kit-8 (CCK-8) (Dojindo, Tokyo, Japan). The isolated cells were seeded into 96-well plates at a density of 2×10³ cells/well with 100 μL culture medium. After 1, 2, 3, 4, 5, 6, and 7 d of incubation at 37°C with 5% CO₂, respectively, 10 μL CCK-8 solution was added to each well and incubated in 5% CO₂ at 37°C for 4 h. Finally, absorbance was measured at 450 nm using a Multiskan GO Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA). All tests were carried out in ten replicates and the data were presented as means±standard error. All statistical analyses were performed using SPSS version 18.0. Differences between two adjacent groups were evaluated using unpaired-sample t-test. A p value <0.05 is shown as *, p<0.01 is shown as **, and p<0.001 is shown as ***.

To validate the differentiation characteristics of skeletal muscle satellite cells, the isolated cells were seeded into the 6-well plates at a density of 10⁴ cells/mL per well and were cultured with DMEM supplemented with 10% FBS in an incubator at 37°C with 5% CO₂ until 50% confluent. Then, the cells were cultured with DMEM containing 2% HS to induce differentiation and the cell morphology was observed with an inverted microscope (Leica fluorescence microscope, Mannheim, Germany). Moreover, the differentiation characteristics of muscle satellite cells were confirmed with differentiation biomarker protein anti-myogenin (Santa Cruz, USA) using immunofluorescence staining. The protocol was performed according to the method mentioned above.