Complete Growth Medium
Complete Expansion Medium: One bottle of Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-030) plus one Primary Skeletal Cell Muscle Growth Kit (ATCC PCS-950-040)
Complete Differentiation Medium: Primary Skeletal Differentiation Tool (ATCC PCS-950-050); a standalone media with no additional supplements required.
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
- Passage normal skeletal muscle cells when culture has reached approximately 80% to 90% confluence, and are actively proliferating.
- Warm both the Trypsin-EDTA for Primary Cells (ATCC® PCS-999-003) and the Trypsin Neutralizing Solution (ATCC® PCS-999-004) to room temperature prior to dissociation. Warm complete growth medium to 37°C prior to use with the cells.
- For each flask, carefully aspirate the spent media without disturbing the monolayer.
- Briefly rinse the cell layer with 3 to 5 mL DPBS (ATCC® 30-2200) to remove residual traces of serum and then aspirate and discard the DPBS.
- Add pre-warmed trypsin-EDTA solution (2 to 3 mL for every 25 cm2) to each flask.
- Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells.
- Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.
- When the majority of cells are detached, quickly add an equal volume of Trypsin Neutralizing Solution (ATCC® PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
- Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.
- Add 3 to 5 mL Trypsin Neutralizing Solution to the flask to collect any remaining dissociated cells. Transfer remaining cells into the centrifuge tube.
- Repeat steps 10 as needed until all cells have been collected from the flask.
- Centrifuge the cells at 150 x g for 3 to 5 minutes.
- Carefully aspirate the neutralized dissociation solution from the cell pellet and re-suspend the cells in 5 to 8 mL fresh, pre-warmed, complete growth medium.
- Count the cells and seed new flasks at a density of 2,500 to 5,000 cells per cm2.
- Place freshly seeded flasks in a 37°C, 5% CO2 incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance for guidelines on feeding.