Primary Dermal Fibroblast Normal; Human, Neonatal (HDFn)

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. D-PBS (ATCC 30-2200)
2. Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) containing 0.05% Trypsin and 0.02% EDTA. Note: Do not use other trypsin-EDTA concentrations with ATCC PCS-201-010.
3. Trypsin Neutralizing Solution (ATCC PCS-999-004)

1. Fibroblast Growth Kit–Serum-Free (ATCC PCS-201-040) containing each of the following growth supplements: L-glutamine, hydrocortisone hemisuccinate, HLL supplement (human serum albumin, linoleic acid, lecithin), rh FGF β, rh EGF / TGF β-1 supplement, rh insulin and ascorbic acid.
2. Fibroblast Growth Kit–Low Serum (ATCC PCS-201-041) containing each of the following growth supplements: L-glutamine, hydrocortisone hemisuccinate, rh FGF β, rh insulin, ascorbic acid and Fetal Bovine Serum.

1. Gentamicin-Amphotericin B Solution (ATCC PCS-999-025)
2. Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002)
3. Phenol Red (ATCC PCS-999-001)

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from this lot of ATCC PCS-201-010.
2. Using the total number of viable cells reported, determine how much surface area can be inoculated to achieve an initial seeding density of 2,500 to 5,000 cells per cm².
3. Prepare the desired combination of flasks. Add 5mL of complete growth medium 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.
4. While the culture flasks equilibrate, remove one vial of ATCC PCS-201-010 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).
5. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point onward should be carried out under strict aseptic conditions.
6. Add the appropriate volume of complete growth media [volume = (1 mL x number of flasks to be seeded) - 1 mL] 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.
7. Transfer 1 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Cultures. Pipette several times, then cap and gently rock each flask to evenly distribute the cells.
8. Place the seeded culture flasks in the incubator at 37°C, 5% CO₂ atmosphere. Incubate at least 24 hours before processing the cells further.

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.

1. Before beginning, pre-warm complete growth media in a 37°C water bath. This will take between 10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less), warm only the volume needed in a sterile conical tube. Avoid warming complete growth media multiple times.
2. 24 hours after seeding, remove the cells from the incubator and view each flask under the microscope to determine percent cellular confluence.
3. Carefully remove the spent media without disturbing the monolayer.
4. Add 5 mL of fresh, pre-warmed complete growth media per 25 cm² of surface area and return the flasks to the incubator.
5. After 24 to 48 hours, view each flask under the microscope to determine percent cellular confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have reached 80% to 100% confluence, and are actively proliferating (many mitotic figures are visible), it is time to subculture. Fibroblasts are not a contact inhibited cell type.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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