Isolation of Skeletal Muscle Satellite Cells for In Vitro Myogenesis Studies

We developed an optimized protocol to isolate viable satellite cells from neonatal and adult mouse skeletal muscle. Existing methods yield variable purity, low neonatal efficiency, and stress-induced cell loss. Our method preserves viability, high purity, as well as reproducibility.

To begin, obtain skeletal muscle tissue from mice and wash it first with PBS containing 1%penicillin and streptomycin and a few drops of Betadine. Then rapidly wash the muscles twice in fresh PBS containing penicillin and streptomycin to thoroughly rinse off the Betadine. Next, using autoclave scissors, mince the washed tissue finely into fragments measuring approximately one to two millimeters.

Transfer the minced muscles into tissue dissociation tubes containing 0.2%collagenase in DMEM. Incubate the tubes at 37 degrees Celsius for 30 minutes to initiate enzymatic digestion. Using a mechanical tissue dissociator, run the predefined program to facilitate enzymatic digestion while preserving cell viability.

Apply the program twice to enhance mechanical disruption of the tissue. Then place the samples back into the incubator at 37 degrees Celsius for an additional 30 minutes to complete the digestion process. Repeat mechanical dissociation once more to ensure thorough dissociation.

Confirm that the digestion medium becomes cloudy with few or no visible tissue fragments remaining. Next, place a 100-micrometer cell strainer on top of a 50-milliliter conical tube. Using a pipette, pre-wet the strainer with one milliliter of pre-warmed neutralizing medium.

Pour the digested muscle suspension over the pre-wetted 100-micrometer strainer. Then rinse the strainer with an additional one milliliter of neutralizing medium to remove residual debris. After straining the suspension over 70 and 40-micrometer cell strainers, pre-wet a 30-micrometer cell strainer placed on a 15-milliliter conical tube with one milliliter of neutralizing medium.

Then pass the filtrate through the strainer and wash again with one milliliter of neutralizing medium to maximize cell recovery. Centrifuge the collected cell suspension at 300 g for 15 minutes at room temperature. Under a sterile biosafety cabinet, carefully remove and discard the supernatant.

Confirm the presence of a white cell pellet at the bottom of the tube. To perform magnetic-activated cell sorting, first, suspend the cell pellet in freshly prepared MACS buffer using 80 microliters of buffer per gram of original tissue. Add 20 microliters of magnetic-labeling reagent.

Then gently mix and incubate for 15 minutes at two to eight degrees Celsius on ice. Adjust the total volume to 500 microliters with sorting buffer for samples up to five grams of tissue. For larger tissue amounts, divide the suspension equally into multiple tubes.

Place a separation column in the magnetic field of a magnetic separator. Wash the column with three milliliters of sorting buffer to equilibrate it. Apply the labeled cell suspension onto the column.

Collect the eluant containing the satellite cell-enriched population. Then wash the column twice with one milliliter portions of sorting buffer and combine the collected fractions. Centrifuge the combined suspension at 300 for 10 minutes at room temperature to pellet the isolated satellite cells.

Dilute Type I rat collagen in 20 millimolar acetic acid to a final concentration of 50 microgram per milliliter. Add the solution to the culture plates and incubate at room temperature for one hour. Carefully aspirate the collagen solution from the plates.

Then rinse the plates three times with sterile PBS using equal volumes to remove residual acid. Next, prepare myoblast proliferating medium for adult mouse myoblasts or neonatal mouse myoblasts. Resuspend the cell pellet in 10 milliliters of myoblast proliferating medium, pre-warmed to 37 degrees Celsius.

Seed adult myoblasts onto the collagen-coated plates. Then incubate the plates at 37 degrees Celsius in a carbon dioxide incubator for 24 hours. After the optimized pre-plating step, most fibroblasts and other non-myogenic cells remained adherent to the initial collagen-coated surface, whereas myoblasts stayed in suspension and subsequently attached to the Matrigel-coated plates.

Within 72 hours of culture in myoblast proliferation medium, small, round spindle-shaped myoblasts with a high nuclear cytoplasmic ratio predominated. A minor population of large, flattened fibroblast-like cells remained in culture, typically representing less than 3%of the total cells. More than 97%of the cells isolated from adult and neonatal mice were myogenic progenitors, as indicated by PAX7 positive expression.

Neonatal myoblasts achieved high purity with or without magnetic-activated cell sorting. After switching to differentiation medium at 80 to 90%confluency, myoblasts fused to form elongated multinucleated myotubes within 48 to 72 hours. Differentiated myotubes were confirmed by immunostaining for myosin heavy chain.

The presence of organized sarcomeric structures in differentiated myotubes indicated structural maturation under these culture conditions. This method enables direct observation of satellite cell behavior, fusion parameters, marker expression, and responses to environmental or chemical stimuli. The main challenge is controlling digestion, plating density, and fiberblast removal to ensure healthy, high purity myogenic cells.

Isolated cells can support research on muscle regeneration, disease modeling, drug testing, and mechanisms controlling satellite cell differentiation and self-renewal.