1. Passage normal neonatal fibroblasts when the cells have reached approximately 80% to 100% confluence and are actively proliferating.
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 two times with 3 to 5 mL of D-PBS per 25 cm2 of surface area (ATCC 30-2200)
to remove any residual traces of serum. Rinse the cell layer one time with 3 to 5 mL of D-PBS if serum-free culture conditions are used.
5. Add pre-warmed 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 about 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 to each flask, a volume of the Trypsin Neutralizing Solution (ATCC PCS-999-004)
equal to the volume of trypsin-EDTA solution used previously. 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 150 x g for 3 to 5 minutes.
14. Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
15. Count the cells and seed new culture flasks at a density of 2,500 to 5,000 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. Refer to Maintenance
for guidelines on feeding.