Primary Dermal Fibroblast; Normal, Human, Adult (ATCC^® PCS-201-012^™)

1. Obtain one growth kit from the freezer; make sure that the caps of all containers 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 Fibroblast Basal Medium (480 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 volume of each growth kit component, as indicated in Table 1 or 2, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. If using the Fibroblast Growth Kit–Serum-Free (ATCC^® PCS-201-040), add the indicated volume for each component in the order shown. 

Component	Volume	Final Concentration
L-glutamine	18.75 mL	7.5 mM
Hydrocortisone Hemisuccinate	0.5 mL	1 µg/mL
HLL Supplement	1.25 mL	HSA 500 µg/mL Linoleic Acid 0.6 µM Lecithin 0.6 µg/mL
rh FGF b	0.5 mL	5 ng/mL
rh EGF / TGF  b-1 Supplement	0.5 mL	5 ng/mL 30 pg/mL
rh Insulin	0.5 mL	5 µg/mL
Ascorbic acid	0.5 mL	50 µg/mL

 

       Table 2. If using the Fibroblast Growth Kit–Low Serum (ATCC^® PCS-201-041), add the indicated volume for each of the following components:

Component	Volume	Final Concentration
rh FGF b	0.5 mL	5 ng/mL
L-glutamine	18.75 mL	7.5 mM
Ascorbic acid	0.5 mL	50 µg/mL
Hydrocortisone Hemisuccinate	0.5 mL	1 µg/mL
rh Insulin	0.5 mL	5 µg/mL
Fetal Bovine Serum	10.0 mL	2%

 

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 in Table 3.

 

Table 3. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional)

Component	Volume	Final Concentration
Gentamicin-Amphotericin B Solution	0.5 mL	Gentamicin: 10 µg/mL Amphotericin B: 0.25 µg/mL
Penicillin-Streptomycin-Amphotericin B Solution	0.5 mL	Penicillin: 10 Units/mL Streptomycin: 10 µg/mL Amphotericin B: 25 ng/mL
Phenol Red	0.5 mL	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. Passage normal adult 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. 

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