HFF-1 (ATCC® SCRC-1041)

Organism: Homo sapiens, human  /  Cell Type: Fibroblast  /  Tissue: Skin; foreskin  /  Disease: Normal

Organism Homo sapiens, human
Tissue
Skin; foreskin
Cell Type Fibroblast
Product Format frozen
Morphology fibroblast
Culture Properties Adherent
Biosafety Level 1
Disease Normal
Age Newborn
Gender male
Applications
Can be used to produce feeder cells
Storage Conditions Liquid nitrogen vapor phase
Derivation
The cell line was established by ATCC in 2003 from normal human foreskin pooled from two individuals.
Clinical Data
male
Comments
The growth of these cells should be arrested before being used as a feeder layer. ATCC has successfully irradiated (SCRC-1041.1) and treated the cells with Mitomycin C (SCRC-1041.2) for use as a feeder layer. If the HFFs are being used as a feeder layer for ES cells, it is not recommended to use them past passage no. 50 (P50). It is recommended that the feeder cells be plated 24 hours before use at 5X10cells/cm2 in order to obtain a supportive monolayer for stem cell growth.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium:
  • fetal bovine serum to a final concentration of 15%

  • This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended).
    Subculturing

    Procedure:
    To insure the highest level of viability, be sure to warm media and Trypsin/ EDTA to 37ºC before using it on the cells.

    Cells should be split when they reach confluency. A split ratio of 1:5 to 1:7 is recommended. Volumes used in this protocol are for 225 cm2 (T225); proportionally reduce or increase amount of dissociation medium for culture flasks of other sizes.

    1. Remove and discard culture medium.
    2. Briefly rinse the cell layer with 1X PBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
    3. Add 5 mL of Trypsin-EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to flask and incubate for 1 minute, gently tapping the flask observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).
    4. Add 6.0 to 8.0 mL of complete growth medium and rinse surface of the flask to detach all cells. Gently pipetting up and down will break cell clumps.
    5. Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 xg for 5 minutes.
    6. Remove and discard the supernatant
    7. Add 10 mL complete growth medium to cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
    8. Add more complete growth medium to cell suspension as needed to plate cells at approximately 5x106/T225 flask.
    9. Place flasks in incubator @ 37°C with a 5% CO2 in air atmosphere.

    Flask/Plate 

     

    Growth Area (cm2

     

    1xPBS (mL) 

     

    Trypsin/EDTA (mL) 

     

    Equal vol. Complete Growth Medium (mL)

     

    Growth Medium (mL) 

     

    T225 

     

     225

     

    10 ± 0.2 

     

    6 ± 0.2 

     

     6 ± 0.2

     

     30

     

     75

     

     75

     

    5 ± 0.1 

     

     3 ± 0.1

     

     3 ± 0. 1

     

     12

     

     T25

     

     25

     

    3 ± 0.1 

     

    1.5 ± 0.1 

     

     1.5 ± 0.1

     

     6

     

     6 well

     

     9.5

     

     1 ± 0.1

     

    1 ± 0.1 

     

     1 ± 0.1

     

     3

     

    Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 5th edition, published by Alan R. Liss, N.Y., 1994.  

    Subcultivation Ratio: A subcultivation ratio of 1:5 to 1:7 is recommended
    Medium Renewal: Twice a week or as pH decreases
    Cryopreservation
    Complete growth medium supplemented with an additional 40% FBS and 10% (v/v) DMSO
    Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
    Culture Conditions
    Atmosphere: Air, 95%; carbon dioxide (CO2), 5%
    Temperature: 37°C
    Name of Depositor ATCC
    Year of Origin 2003
    References

    Amit M, et al. Human feeder layers for human embryonic stem cells. Biol. Reprod. 68: 2150-2156, 2003. PubMed: 12606388

    Hovatta O, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18: 1404-1408, 2003. PubMed: 12832363

    Andrews P, et al. Human embryonic fibroblast feeder cells. International Patent Application WO 03/078611 A1

    Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

    Basic Documentation
    References

    Amit M, et al. Human feeder layers for human embryonic stem cells. Biol. Reprod. 68: 2150-2156, 2003. PubMed: 12606388

    Hovatta O, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18: 1404-1408, 2003. PubMed: 12832363

    Andrews P, et al. Human embryonic fibroblast feeder cells. International Patent Application WO 03/078611 A1

    Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.