Bone-exempt muscle-derived myogenic myoblasts are capable of culturing myoblasts isolated from the skeletal muscle of several adult animals, and can be used for (1) structural studies of myoblasts (2) studies of myoblast function (3) research on myoblast-related topics (4) clinical cell transplantation.
Operation method
Isolation of cultured cells
Principle
Bone-excluded muscle-derived myoblasts are capable of culturing myoblasts isolated from skeletal muscle of several adult animals. Under culture conditions, myoblasts continue to express a number of differentiation features. Myoblasts are called satellite cells and the early steps of differentiation are very similar to those of skeletal muscle. Myoblasts proliferate and migrate on a culture substrate, then align in a straight line and eventually fuse to form multicellular nuclear myotubes. In single myofiber culture, skeletal muscle is taken as a whole block. Incubation with collagen solution was used to digest the connective tissue. Then, slight mechanical grinding separates the myofibers. Myofibers and their satellite cells can be isolated from skeletal muscle. Primary cells grow readily in Ham's F12 cultures supplemented with 20% (fetal bovine serum, FBS). Without changing the culture conditions, these cells proliferated and differentiated, fusing to form myotubes with multiple nuclei. This demonstrates the myogenic nature of cultured cells
Materials and Instruments
Biopsy Tissue Blocks Move I. Materials Maintaining Cultures Caveat Remaining tissues and used test tubes, pipettes, and petri dishes should be treated with hypochlorite before being thrown in the trash.Cultures need to be opened only in a sterile environment to avoid contamination. Common Problems 1. The early steps in the differentiation of adult myoblasts, called satellite cells, are very similar to those of skeletal muscle. Myoblasts proliferate and migrate on a culture substrate, then align themselves in a linear pattern and eventually fuse to form multicellular nuclear myotubes. Although myoblasts can be passaged for 3 to 4 generations by trypsin digestion, they cannot be passaged once they have differentiated (fused). 2. Characteristics of myoblasts: easy to obtain, widely available, easy to isolate and proliferate in vitro, and easy to receive exogenous genes during the culture phase. For more product details, please visit Aladdin Scientific website.
Ham F12 FBS D-PBSA
Scalpel Long scissors Petri Petri dishes Centrifuge tubes Hematocrit plates
aseptic
1. Transfer medium: Ham F12 (Seromed 08101), without NaHCO3, containing 20 mmol/LHEPES (Serva 25245), 75 ml.
2. growth medium: Ham F12, with 20% FBS (Gibco 011-06290), 200 ml
3. Streptomyces protease solution: 0.15% Streptomyces protease (Sigma P-6911), 0.03% EDTA (Merck 8418), 20IU/ml Penicillin and 20ug/ml Streptomycin (0.4% Eurobio PES300) in 100 ml of Ham F12 or D-PBSA.
4. D-PBSA: 100 ml
5. Nylon mesh: 100um pore size
6. Scalpel
7. Long, sharp scissors
8. Petri dish: 90 mm in diameter for cutting tissue blocks
9. Petri dishes: 25 cm2, 4 pieces
10. 20 ml centrifuge tubes or general containers, 5 pieces
Non-sterile
1. Hematocrit plates
II. Operating Procedure:
1. Separation: Excise the non-muscle tissue from the biopsy tissue block with a scalpel and then soak it in D-PBSA.
2. prior to weighing, the excised biopsy tissue is sliced parallel to the muscle fibers and rinsed with D-PBSA.
3. Place the tissue slices parallel to each other in the Petri dish lid, cut into thin columnar tissue blocks, and then into 1 mm3 pieces. Do not squeeze the tissue. Tissue can also be shortened into small pieces in the test tube with long scissors; avoid squeezing the tissue.
4. Wash the block with D-PBSA, let it stand and aspirate the supernatant.
5. Digest the blocks with Streptomyces protease solution for 1 h at 37 ℃. 15 ml of Streptomyces protease solution should be used for every 1~3 mg of tissue block. Shake the tube gently every 10-15 min.
6. Incubate and then grind the blocks with a pipette. The culture medium will gradually become turbid as more and more cells are detached.
7. Allow the tissue mass to sink to the bottom of the test tube to form a settled tissue mass (P1) and supernatant (S1).
8. Filter S1 into a 20 ml centrifuge tube using a 100um pore size nylon mesh. Gently shake or blow the supernatant with a pipette to mix the cells.
9. Centrifuge (350 g, 8-10 min) and aspirate the supernatant.
10. Accurately add 10 ml of growth medium and suspend the cells very slightly with a pipette with a rubber tip. Then, count the cells with a hemocyte counting plate.
11. Dilute the cell suspension with the growth medium and inoculate the cells in culture flasks at a cell density of approximately 1. 5x104 cells/ml. Approximately 1~2x105 cells are obtained from 1 g of healthy donor biopsy tissue.
12. Add 15 ml of digestive solution to P1 and incubate the tissue block in a 37°C water bath for 30 min with regular shaking.
13. Blow the cell suspension with a pipette to disperse the cells. Then, filter the cell suspension through a nylon mesh. Wash the mesh with 20 ml of growth medium.
14. After centrifugation (350 g, 8~10 min), count the cells and inoculate the cells as described above.
15. Transfer the culture flasks to a humidified incubator and culture the cells at 37℃ and 5%CO2.
Safety Tips
Remaining tissue and used test tubes, pipettes, and petri dishes should be treated with hypochlorite before disposal in the trash.
16. Change the culture medium very slightly 24 h after inoculation, and then every 3~4 d thereafter. The cells mainly grew towards differentiation. The growth of human myoblasts is divided into 3 phases, with the peak of proliferation at 4~6d after culture; 8d when the cells are arranged in rows; and 10~12d when the cells are fused to form more myotubes. However, it must be kept in mind that some cells may differentiate earlier, and some cells are still proliferating at the time that the majority of cells differentiate.
Passage culture
17. Aspirate a small amount of EDTA immediately after gently injecting it on top of the cells.
18. Add 0.25% trypsin solution, enough to form a thin layer on top of the cells.
19. When the cells are de-attached, add 5 to 10 ml of complete growth medium, blow the cells very slightly with a pipette, and centrifuge (350 g, 10 min).
20. Count the cells and plant the cells at a certain density.
Source: Animal Cell Culture: A Guide to Basic Techniques (5th Edition).