Primary Dermal Fibroblast Normal; Human, Neonatal (ATCC® PCS-201-010)

Organism: Homo sapiens, human  /  Tissue: Foreskin  /  Cell Type: Fibroblasts

Organism Homo sapiens, human
Tissue Foreskin
Cell Type Fibroblasts
Morphology Spindle-shaped; cells are bipolar and refractile.
Growth Properties Adherent
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease Normal
Age Neonatal
Gender Male
Ethnicity Batch-specific
Applications
Response to pathogens, skin aging, wound healing, gene delivery, skin diseases (e.g., scleroderma)
Product Format frozen 1 mL
Storage Conditions -130°C or below
Comments
Serum-free medium supports excellent growth curves and normal morphology, as well as serum-free (not animal-free) experimental conditions. The presence of 2% fetal bovine serum in the Fibroblast Growth Kit-Low serum (ATCC PCS-201-041) supports more prolific growth.
Complete Growth Medium
  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.
  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.

 

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 mg/mL

     

     

    HLL Supplement

     

     

    1.25 mL

     

     

    HSA 500 mg/mL

    Linoleic Acid 0.6 mM

    Lecithin 0.6 mg/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 mg/mL

     

     

    Ascorbic acid

     

     

    0.5 mL

     

     

    50 mg/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 mg/mL

     

     

    Hydrocortisone Hemisuccinate

     

     

    0.5 mL

     

     

    1 mg/mL

     

     

    rh Insulin

     

     

    0.5 mL

     

     

    5 mg/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 each optional component 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

     

     

     

    Subculturing
    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.
    Volume 1 mL
    Sterility Tests
    Bacteria: Negative
    Yeast: Negative
    Mycoplasma: Negative
    Viral Testing
    Hepatitis B: Negative
    Hepatitis C: Negative
    HIV: Negative
    Viability ≥ 50% when thawed from cryopreservation
    Population Doubling Capacity ≥ 10 in complete growth medium
    C of A
    Certificate of Analysis
    Certificate of Analysis
    Basic Documentation
    References

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    Du Z, Yang C, Rothschild M, Ross J. Novel microRNA families expanded in the human genome. BMC Genomics 14: 98, 2013. PubMed: 23402294

    Erazo-Oliveras A, et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nat. Methods 11(8): 861-867, 2014. PubMed: 24930129