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Complete Growth Medium
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The base medium for this cell line is Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-030). To make the complete growth medium, add Mesenchymal Stem Cell Growth Kit (ATCC PCS-500-040) for Adipose and Umbilical-derived MSCs - Low Serum Components and G418 to the base medium as the following:
482 mL of basal medium (PCS-500-030)
10 mL of MSC supplement (2% FBS, 5 ng/mL rh FGF basic, 5 ng/mL rh FGF acidic, 5 ng/mL rh EGF)
6 mL of L-Alanyl-L-Glutamine (2.4 mM, final concentration)
2 mL of 50 mg/mL G418 (0.2 mg/mL, final concentration)
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Subculturing
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Protocol:
1. Passage immortalized adipose-derived MSCs when the culture has reached approximately 80% confluence.
2. 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 the complete growth medium to 37°C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
5. Add prewarmed trypsin-EDTA solution (1 to 2 mL for every 25 cm2) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.
7. 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.
8. When the majority of cells appear to have detached, quickly add an equal volume of the 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.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the culture flask.
10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that might have been left behind.
11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA- dissociated cells.
12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13. Centrifuge the cells at 270 x g for 5 minutes.
14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, prewarmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm2.
16. Place newly seeded flasks in a 37°C, 5% CO2 incubator for at least 24 to 48 hours before processing the cells further.
Cell seeding density: 5,000 viable cells per cm2
Medium renewal: every 2 to 3 days
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Cryopreservation
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Freeze medium: 90% Complete growth media; DMSO, 10%
Storage temperature: liquid nitrogen vapor phase
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Culture Conditions
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Temperature: 37.0°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
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