Isolation and culture of single muscle fibers from skeletal muscle
Isolation and culture of single muscle fibers from skeletal muscle
Grind whole skeletal muscle treated with collagen to disperse into single muscle fibers. Cultivate single muscle fibers on Matrigel and collect the migrating cells.
Operation method
Scheme 23.15 Isolation and culture of single muscle fibers from skeletal muscle
Principle
Grind whole skeletal muscle treated with collagen to disperse into single muscle fibers. Cultivate single muscle fibers on Matrigel and collect the migrating cells.
Materials and Instruments
Matrigel DMEM L-glutamine Type I collagen Move 1. Remove the muscle immediately after execution of the animal. Take care to clamp the tendon to minimize damage to the muscle fibers. For more product details, please visit Aladdin Scientific website.
Penicillin and streptomycin mixture Chicken embryo extract Horse serum Fetal bovine serum Inoculation medium Proliferation medium
Petri Petri dishes Modified Pasteur pipettes 24-well plates Diamond pens Translucent stereo dissecting microscopes
2. Incubate the muscle in 0.2 % collagenase type I solution (W/V, prepared with DMEM) in a shaking water bath at 35°C for 60-90 min. Larger muscles require longer digestion time, in general, the extensor digitorum longus muscle taken from 6-8 weeks old rats needs to be digested for 60 min.
3. Prepare Petri dishes containing muscle fibers, i.e., wash the dishes with horse serum to prevent the muscle fibers from sticking to the bottom wall of the dishes. Then, add 20 ml of DMEM.
4. After digestion, transfer the muscle into Petri dishes containing DMEM.
5. Under a transilluminated stereoscopic dissecting microscope, a modified Pasteur pipette is used (cut the Pasteur pipette and make the opening larger with a diamond pen). The jagged edges are then smoothed with fire to avoid damaging the muscle fibers) to grind the muscle and disperse the single muscle fibers.
Dip the pipette in 10 % horse serum (prepared in DMEM) to avoid adhesion of the muscle fibers to the pipette.
6. As the muscle fibers are dispersed, the diameter of the muscle becomes smaller. Gradually use Pasteur pipettes with a smaller diameter.
7. When 20-30 muscle fibers have been separated, transfer the muscle to another Petri dish and continue until enough muscle fibers have been separated.
8. Larger muscles require a second digestion to disperse the centrally located muscle fibers. After the superficial muscle fibers have been separated, the remaining muscle is placed in collagenase solution for further digestion.
9. Place the muscle fibers into a Petri dish coated with Matrigel (1 mg/ml, prepared in DMEM).
(a) Place a single muscle fiber in the center of the dish so that it adheres to the Matrigel. in order to culture pure single muscle fibers, only undamaged muscle fibers are placed in, and the more crumpled portions are removed. These sections may have contaminated adherent cells, mostly microvascular segments;
(b) Slowly add the inoculation medium (DMEM with 10% horse serum, 0.5% chicken embryo extract, 4 mmol/L L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin), taking care not to move the inoculated muscle fibers.
10. Place the petri dishes in an incubator and incubate at 37°C and 5 % CO2.
Proliferation
After 12-24 h of incubation, satellite cells begin to migrate out of the myofibers. By 3-4 days, there are 50-300 satellite cells around each myofiber. The exact number of cells depends on a number of factors such as the muscle from which the myofibers originate and the weekly age of the mice [ Bockhold et al., 1998 ].
11. When many satellite cells have migrated out of the myofibers, the myofibers are aspirated with a Pasteur pipette. The tip of the pipette should be thinned over a flame.
12. Continue to culture the remaining cells.
13. If desired, passaging culture can be performed by inoculating the cells with proliferation medium (DMEM with 20% FBS, 10% horse serum, 1% chicken embryo extract, 4 mmol/L L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin).