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BJ-5ta; Foreskin Fibroblast; Human (Homo sapiens) Item ID: ATCCRL4001

Permits and Restrictions

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Tissue Foreskin
Cell Type Fibroblast immortalized with hTERT
Product Format frozen
Morphology Fibroblast-like
Culture 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
Storage Conditions Liquid nitrogen vapor phase
Karyotype This is a diploid human cell line of male origin with a modal chromosome number of 46 that occurred in 90% of the cells counted. The sex chromosomes, X and Y are both karyotypically normal.
Images
Derivation

The hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, was derived by transfecting the BJ foreskin fibroblast cell line with the pGRN145 hTERT-expressing plasmid (ATCC MBA-141) at population doubling 58. Cells were cultured in medium containing hygromycin B until stable clones were selected [Pubmed: 9454332]. 
Antigen Expression

Antigen expression: Positive for fibroblast surface protein; Homo sapiens, expressed (fibroblast surface protein (FSP) was assayed by flow cytometry.).

Negative for the pan-cytokeratin epithelial marker; Homo sapiens (cytokeratins were assayed by immunocytochemistry using a pan-cytokeratin antibody).
Subculturing Volumes are given for a 75 cm2 flask. Recommended use of Corning® T-75 flasks (catalog #430641). Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
  1. Remove and discard culture medium.
  2. Add 3.0 to 5.0 mL of 0.25% trypsin-0.53 mM EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).
    3. Note: To avoid clumping do not hit or shake the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  3. Add 6.0 to 10.0 mL of complete growth medium and aspirate cells by gently pipetting. 
  4. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 3 x 103 to 5 x 103 viable cells/cm2 is recommended. Maintain cultures at a cell concentration between 8 x 103 and 1 x 104 cells/cm2.
  5. Subcultivation ratio: 1:2 to 1:3 twice weekly
  6. Incubate cultures at 37°C.
Subcultivation Ratio: 1:2 to 1:3 twice weekly Medium Renewal: every 2 to 3 days 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 Wiley-Liss, N.Y., 2005.
Cryopreservation Freeze medium: culture medium, 30%; fetal bovine serum, 60%; DMSO, 10% Storage temperature: liquid nitrogen vapor phase
Culture Conditions Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37°C Growth Conditions: Subculture when cell concentration reaches between 8 X 103 and 1 X 104 cells/cm2.
STR Profile Amelogenin: X,Y CSF1PO: 10,12 D13S317: 8,9 D16S539: 9,13 D5S818: 12 D7S820: 11,12 THO1: 7,8 TPOX: 10,11 vWA: 16,18
Population Doubling Level (PDL) As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation.
Name of Depositor Geron Corporation
References

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

Jiang XR, et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet. 21: 111-114, 1999. PubMed: 9916802

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