Harvesting traumatized muscle derived mesenchymal progenitor cells (MPCs)
1. The muscle tissue was dissected without contaminating by granulation, adipose or fibrous tissues.
2. The excised muscle was transferred into a dish containing primary cell culture system and minced until the slurry could easily passed through the tip of a 25 mL serological pipette.
3. The minced muscle tissue was then transferred to a 50 mL conical tube containing primary cell culture system and 0.5 mg/mL Collagenase Type 2, and incubated at 37°C for 2 hours with gentle agitation.
4. At the end of the digestion, the suspension was vortexed briefly and passed through a 5 mL serological pipette to mechanically break down any tissue remnants.
5. The digestate was strained through a 40 μm sieve into a new 50 mL conical tube and centrifuged for 5 minutes at 200g.
6. After aspirating the supernate, the pellet was resuspended in primary cell culture system supplemented with 10% fetal bovine serum (FBS) and 5 units/mL of penicillin, streptomycin and fungizone (PSF).
7. The cell suspension was plated in a
T175 tissue culture flask and incubated for 2 hours at 37°C, and then
the cells were washed extensively with Hank’s Buffered Saline Solution
(HBSS) to remove any cells that did not adhere to the tissue culture
plastic.
8. The adherent cells were cultured in primary cell culture system supplemented with 10% FBS and 3 units/mL of PSF.
9. On each of the first three days, the cells were washed with primary cell culture system and the medium was replaced.
10. The cells were trypsinized and subcultured into new flasks after tightly packed colony forming units (CFUs) were observed and maintained in primary cell culture system.
11. Subsequent subcultures were performed when the cells were approximately 85% confluent.
Harvesting bone marrow derived MSCs
1. Bone marrow derived MSCs were harvested.
2. The remaining marrow space was washed by inserting 28G needle and perfusing with primary cell culture system, and the resulting slurry was transferred to a 50 mL conical tube and vortexed briefly.
3. The tissue slurry was passed through a 40 μm cell strainer into a new 50 mL conical tube and centrifuged for 5 minutes at 200g.
4. After aspirating the supernate, the pellet was resuspended in primary cell culture system supplemented with 10% fetal bovine serum and 1 unit/mL of PSF, and the cell suspension was plated in T175 tissue culture flasks.
5. The medium in the flask was changed once a week, and the cells were subcultured once tightly packed CFUs are observed.
6. Subsequent subcultures were performed when the cells were approximately 85% confluent.
References
1. Nesti L, Jackson W,
Shanti R, Koehler S, Aragon A, Bailey J, Sracic M, Freedman B, Giuliani
J, and Tuan R. Differentiation potential of multipotent progenitor cells
derived from war-traumatized muscle tissue. J Bone Joint Surg Am. 2008;
90: 2390
2. Jackson WM, Aragon AB, Djouad F, Song Y, Koehler SM, Nesti LJ, Tuan RS. Mesenchymal progenitor cells derived from traumatized human muscle. J Tissue Eng Regen Med. 2009; 3:129.