Primary Vaginal Epithelial Cells

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Using the total number of viable cells, determine how much surface area can be inoculated to achieve an initial seeding density of between 5000 cells per cm².
2. Prepare the desired combination of flasks. Add 5 mL of complete growth media per 25 cm² of surface area. Place the flasks in a 37°C, 5% CO₂, humidified incubator and allow the media to pre-equilibrate to temperature and pH for 30 minutes prior to adding cells.
3. While the culture flasks equilibrate, remove one vial of ATCC PCS-480-010 from storage and thaw the cells by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 1 to 2 minutes).
4. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by spraying with 70% ethanol. All operations from this point onward should be carried out under strict aseptic conditions.
5. Add 5 mL of complete growth – into a sterile conical tube. Using a sterile pipette, transfer the cells from the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not centrifuge.
6. Count the cells. Plate 5,000 cells per cm2 into each of the pre-equilibrated culture flasks prepared in steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Gently rock the culture vessel from side to side and front to back to evenly distribute the cells within the vessel.
7. Place the seeded culture flasks in the incubator at 37°C with a 5% CO₂ atmosphere. Incubate for at least 48 hours before processing the cells further.

1. Passage normal vaginal epithelial cells when culture has reached approximately 85 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 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. 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.
5. Add pre-warmed trypsin-EDTA solution (2 to 3 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.
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 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.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.
10. Add 3 to 5 mL Trypsin Neutralizing Solution to the flask to collect any remaining dissociated cells. Transfer remaining cells into the centrifuge tube.
11. Repeat steps 10 as needed until all cells have been collected from the flask.
12. Centrifuge the cells at 150 x g for 3 to 5 minutes.
13. 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.
14. Count the cells and seed new flasks at a density of 5,000 cells per cm2.
15. 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.

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