Skeletal muscle cell culture experiment

Summary

Skeletal muscle cell culture experiments are those in which skeletal muscle cells are cultured under in vitro conditions.

Principle

The basic principle of the culture experiment of skeletal muscle cells is that in vitro culture, adding fetal bovine serum to the culture medium and inoculating the cells on gelatin can cause the skeletal muscle mononuclear precursor cells in muscle tissue to further differentiate to mature skeletal muscle cells on the basis of proliferation, i.e., a large number of fusions between the cells take place, there are transverse stripes formed inside the cells, and the cells can undergo spontaneous contraction, etc., and as a result a large number of Skeletal muscle cells.

Operation method

Skeletal muscle cell culture experiment

Principle

The basic principle of the culture experiment of skeletal muscle cells is that in vitro culture, adding fetal bovine serum to the culture medium and inoculating the cells on gelatin can cause the skeletal muscle mononuclear precursor cells in muscle tissue to further differentiate to mature skeletal muscle cells on the basis of proliferation, i.e., a large number of fusions between the cells take place, there are transverse stripes formed inside the cells, and the cells can undergo spontaneous contraction, etc., and as a result a large number of Skeletal muscle cells.

Materials and Instruments

Equipment:
① Newborn 1～2d rats
② Culture bottle
③ Straws and caps
④ Petri dish
⑤ Dissecting scissors
⑤ Dissecting scissors ⑥ Ophthalmic scissors
⑦ Ophthalmic forceps
⑧ Sterile sand net
⑨ Centrifuge
⑩ Inverted microscope
⑪ Ultra-clean bench
⑫ CO2 incubator
Tubes
Reagents
① D-Hanks liquid
① D-Hanks liquid ② DMEM medium (containing 10% fetal bovine serum)
③ Collagenase solution
④ 0.01% gelatin, etc.

Move

The basic procedure for the culture of skeletal muscle cells can be divided into the following steps:
(1) Collection of materials
A. Take a rat 1~2 days old, and kill it by decapitation.
B. Wipe the whole body of the rat with 70% ethanol for 3 times.
C. Under aseptic conditions, use dissecting scissors to cut open the skin of the thighs, and strip off the skin hair.
D. Use ophthalmic scissors to take off the muscle of the thighs, and put it into a petri dish containing D-Hanks liquid.
E. Remove the adherent fat tissue on the muscle tissue, and remove the bone in the muscle. F. Wash the muscle tissue with D-Hanks liquid again 3 times, and then put it into a dish containing D-Hanks liquid. Remove the adherent fatty tissue from the muscle tissue and remove the bone from the muscle.
F. Wash the muscle tissue with D-Hanks liquid for 3 more times and place it in a petri dish with culture medium.
(2) Digestion
A. Use ophthalmic scissors to cut the muscle tissue into 1 mm³ pieces, put them into a conical flask containing collagenase solution (2000 U/ml), and digest them at 37 ℃ for about 24 h.
B. Blow the digested pieces of muscle tissue vigorously with a pipette, filter the residual pieces of tissue after blowing with a sterile mesh net, and then collect the cell suspension in a 10-ml centrifuge tube.
C. Centrifuge the muscle tissue for 5 min at 800 r/min, discard the supernatant. min, and discard the supernatant.
D. Add medium containing 10% fetal bovine serum, and blow the precipitated cells to prepare cell suspension.
E. Inoculate the cells in Petri dishes covered with 0.01% gelatin, and the inoculation volume can be increased to 2 x ¹⁰⁶ cells/dish.
F. Place the Petri dishes in a 37 ℃ incubator and culture, and a large number of skeletal muscle cells can be obtained after 4~5 d. The cells can be collected in a 10 ml centrifuge tube.

Caveat

1. Because the number of skeletal muscle mononuclear precursor cells is higher in growing muscle tissue, the tissue material used for culture should be taken from animal larvae or embryos as much as possible in order to improve the success rate of culture.

2. because myoblasts are slower to attach to the wall than non-myoblasts, the method of repeated attachment can be used to reduce the contamination of non-myoblasts in primary culture.

3. Good culture conditions, such as adding fetal bovine serum to the culture medium and inoculating the cells on gelatin, etc., can prevent the cultured adult myoblasts from undergoing dedifferentiation and promote their transformation to mature skeletal muscle cells, i.e., the formation of multinucleated cells by massive fusion of the cells and the spontaneous contraction of the cells.

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