Proliferation method of myogenic stem cells
A muscle-derived, stem cell technology, applied in the field of cell proliferation, can solve the problems of small cell volume, low purity, and easy pollution costs, and achieve the effects of strong differentiation ability, increased number of cultures, and low cost
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Embodiment 1
[0135] Example 1 mouse skeletal muscle tissue collection
[0136] 2-week-old C57BL / 6 mice were used in the experiment.
[0137] Step a): prepare ultra-clean table equipment materials, soak equipment in 75% alcohol, and ventilate for 15 minutes after ultraviolet irradiation for half an hour;
[0138] Step b): Put 10ml of skeletal muscle tissue culture solution in a 50ml centrifuge tube for use;
[0139] Step c): with C0 2 20 mice were killed by suffocation, and the tails of the mice were lifted with ophthalmic forceps and soaked in 75% alcohol, and the soaked mice were placed on the ultra-clean bench;
[0140] Step d): Use ophthalmic tweezers to hold the mouse along the longitudinal axis of the hind limbs, cut the skin at the base of the tail of the mouse with ophthalmic scissors, and separate the skin from here, carefully cut off the fatty tissue around the muscles, and pay attention to separating the nerves and blood vessels;
[0141] Step e): cutting the mouse limb skelet...
Embodiment 2
[0144] Example 2 Enzyme digestion and cell extraction of skeletal muscle tissue
[0145] Step a): After aseptically sealing the centrifuge tube containing the skeletal muscle tissue, it is transferred to a cell laboratory;
[0146] Step b): Place the centrifuge tube in a centrifuge and centrifuge at 1000rpm / 5min, suck off the supernatant, add 5ml of 0.2% type I collagenase solution to reach the working concentration of 0.1% type I collagenase solution, and place at 37°C In a shaker, shake and digest at 100rpm for 1-2h, during which the digestion status of the tissue is observed;
[0147] Step c): Centrifuge at 1000rpm / 5min, suck off the supernatant, add 5ml of 2.4units / ml neutral protease solution, adjust to the working concentration of 1.2units / ml neutral protease solution, pipette to disperse the precipitate, and place at 37°C Digest with 100rpm shaking in a shaker for 45 minutes, during which the digestion status of the tissue is observed;
[0148] Step d): Centrifuge at ...
Embodiment 3
[0151] Example 3 Separation of muscle-derived stem cells and satellite cells
[0152] The schematic flow chart of the separation test of muscle-derived stem cells and satellite cells in this example is as follows Figure 5 shown. Specific steps are as follows:
[0153] Step a): Place the cell suspension obtained in step f of Example 2 in a 100mm cell culture dish, and put it in a constant temperature incubator at 37°C;
[0154] Step b): After 2 hours, the suspension cells were collected, centrifuged at 1000rpm / 5min, the supernatant was sucked off, and the fibroblast culture medium (FbCM: b-FGF 12.5ng / ml, EGF 0.4ng / ml, IGF 20ng / ml, 20 %FBS, 1% double antibody, IMDM) 8ml, resuspended by gently blowing with a pipette, inoculated into a new dish, placed in 37°C, 5% CO 2 A constant temperature incubator to obtain the first suspension cells and the first adherent cells;
[0155] Step c): Centrifuge the first suspended cells at 1000rpm / 5min, suck off the supernatant, add muscle-der...
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