THLE-2 (ATCC® CRL-2706)

Organism: Homo sapiens, human  /  Cell Type: epithelial cells transformed with SV40 large T antigen  /  Tissue: liver/left lobe  / 

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Organism Homo sapiens, human
Tissue liver/left lobe
Cell Type epithelial cells transformed with SV40 large T antigen
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2 [Cells contain SV40 viral DNA sequences]
Age adult
Applications

These immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma. ­


Storage Conditions liquid nitrogen vapor phase
Karyotype near diploid
Derivation
TThe THLE-2 (ATCC CRL-10149 and the THLE-3 (ATCC CRL-11233) cell lines were derived from primary normal liver cells by infection with SV40 large T antigen. [RF84749] The virus was generated by introducing a retroviral vector containing the of Bgl I-Hpa I fragment of SV40 T antigen into the amphotropic packaging cell line PA317.
Tumorigenic No
Effects
No, nude mice
Comments

[RF84750] THLE-2 and THLE-3 cells express phenotypic characteristics of normal adult liver epithelial cells. They are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. [RF84750] THLE-2 and THLE-3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. [RF84750] Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells.

[RF84750]  A culture submitted to the ATCC in May of 1989 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cycline. The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative. The cured cell line is available as ATCC CRL-2706. The original patent deposit is available as ATCC CRL-10149

Complete Growth Medium BEGM from Lonza/Clonetics Corporation, Walkersville, MD 21793 (BEGM Bullet Kit; CC3170). The kit includes 500 mL basal medium and separate frozen additives from which we discard the gentamycin/ Amphotericin (GA) and Epinephrine and to which we add extra 5 ng/mL EGF, 70 ng/mL Phosphoethanolamine and 10% fetal bovine serum.
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: The flasks used should be precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in BEBM medium. 

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin-0.53% (w/v) EDTA solution (GIBCO cat# 25300-054) to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 0.1% Soybean Trypsin inhibitor and aspirate cells by gently pipetting. 
  5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes
  6. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new coated culture vessels. 
  7. Place culture vessels in incubators at 37°C.

Subculture Ratio: 1:3 to 1:6
Medium Renewal: Two to three times a week.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Cryopreservation
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Growth Conditions: The flasks used should be precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in BEBM medium.
Name of Depositor National Cancer Institute
References

Harris CC, et al. Human liver epithelial cells. US Patent 5,759,765 dated Jun 2 1998

Pfeifer AM, et al. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. Proc. Natl. Acad. Sci. USA 90: 5123-5127, 1993. PubMed: 7685115

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
References

Harris CC, et al. Human liver epithelial cells. US Patent 5,759,765 dated Jun 2 1998

Pfeifer AM, et al. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. Proc. Natl. Acad. Sci. USA 90: 5123-5127, 1993. PubMed: 7685115