Primary Epidermal Keratinocytes; Normal, Human, Neonatal Foreskin (HEKn)

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. Obtain one Keratinocyte Growth Kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.
3. Obtain one bottle of Dermal Cell Basal Medium (485 mL) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component, as indicated in "A" below, to the bottle of basal medium using a separate sterile pipette for each transfer.

1. Keratinocyte Growth Kit Components
   • Bovine Pituitary Extract (BPE), 2.0 mL (Final concentration 0.4%)
   • rh TGF-a, 0.5 mL (Final concentration 0.5 ng/mL)
   • L-Glutamine, 15.0 mL (Final concentration 6 mM)
   • Hydrocortisone Hemisuccinate, 0.5 mL (Final concentration 100 ng/mL)
   • rh Insulin, 0.5 mL (Final concentration 5 mg/ml)
   • Epinephrine, 0.5 mL (Final concentration 1.0 mM)
   • Apo-Transferrin, 0.5 mL (Final concentration 5 mg/ml)

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components (GA solution or PSA solution) to be added to the complete growth media is summarized below.


2. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional):
   • Gentamicin-Amphotericin B Solution, 0.5 mL (Final concentration Gentamicin: 10 µg/mL, Amphotericin B: 0.25 µg/mL).
   • Penicillin-Streptomycin-Amphotericin B Solution, 0.5 mL (Final concentration Penicillin: 10 Units/mL, Streptomycin: 10 µg/mL, Amphotericin B: 25 ng/mL).
   • Phenol Red, 0.5 mL (Final concentration 33 µM)

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.

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-200-010.
3. Trypsin Neutralizing Solution (ATCC PCS-999-004)

1. Refer to the batch specific information for the total number of viable cells recovered from this lot of ATCC PCS-200-010.
2. Using the total number of viable cells, determine how much surface area can be inoculated to achieve an initial seeding density between 2,500 and 5,000 cells per cm².
3. Prepare the desired combination of flasks. Add 5 mL 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-200-010 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 medium [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 1.0 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 Culture. 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 for at least 24 hours before processing the cells further.

1. Passage normal keratinocytes when the culture has reached approximately 70% to 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 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 3 to 6 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface. If cells are difficult to detach, incubate each flask containing cells and the trypsin-EDTA solution at 37°C to facilitate dispersal.
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 150 x g for 3 to 5 minutes.
14. Aspirate 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 medium 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 approximately 80% confluence, and are actively proliferating (many mitotic figures are visible), it is time to subculture. Keratinocytes will begin to terminally differentiate once they become 100% confluent.

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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