Cell lab HTH
Cell culturing muscle cells
In the Bioactuator project, we use C2C12 myoblasts. For more information about C2C12 myoblasts click on the following link: https://en.wikipedia.org/wiki/C2C12#:~:text=C2C12%20is%20an%20immortalized%20mouse%20myoblast%20cell%20line.&text=C2C12%20cells%20are%20used%20to,to%20explore%20mechanistic%20biochemical%20pathways.
We use the following cell culture protocol for the C2C12 cells:
C2C12 cells protocol
1. The base medium for this cell line Dulbecco's Modified Eagle's Medium (DMEM, Sigma D6429) with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin.
2. The seeding density should be 5.0 X103viable cells/cm2
3. IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.
Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.
Myotube formation is enhanced when the medium is supplemented with 10% horse serum instead of fetal bovine serum.
II-Trypsinizing or passaging the cells
1. Remove and discard culture medium. Rinse the cells with PBS twice.
2. Add 2.0 mL of Trypsin-EDTA solution to flask and put it back in the incubator and wait about 5 minutes. Tap the flask and observe cells under an inverted microscope until cell layer is dispersed.
3. Add 8.0 mL of complete growth medium and dispense cells by gently pipetting.
4. Tranfer the cell suspension into 5a 0 ml tube.
5. Centrifuge the cell suspension to make cell pellet (300g, 3 min). Remove the supernatant and re-suspend the cells in 10 ml of culture medium.
6. Add appropriate aliquots of the cell suspension to new culture vessels.Inoculate at a cell concentration between 1.5 X 105 and 1.0 X 106 viable cells/75 cm2 bottle.(Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
7. Incubate cultures at 37°C.
8. Medium Renewal: Every two to three days (see the medium colour)