HepG2-ACTT
Cell Line Designation: Hep G2 ATCC Catalog No. HB-8065?
Table of Contents:
?Cell Line Description
? Biosafety Level
? Use Restrictions
?Handling Procedure for Frozen Cells
?Handling Procedure for Flask Cultures
? Subculturing Procedure
? Medium Renewal Procedure
?Complete Growth Medium
? Cryoprotectant Medium
? References
? Replacement Policy
Cell Line Description
Organism: Homo sapiens ( human)
Tissue: liver; hepatocellular carcinoma
Age: 15 years
Gender: male
Ethnicity: caucasian
Morphology: epithelial
Growth properties: adherent
Tumorigenic: the cells were not tumorigenic in immunosuppressed mice, but did form colonies in semisolid medium.
Cellular Products: alpha-fetoprotein (alpha fetoprotein); albumin; alpha2 macroglobulin (alpha-2-macroglobulin); alpha1 antitrypsin (alpha-1-antitrypsin); transferrin; alpha1 antichymotrypsin; (alpha-1-antichymotrypsin); haptoglobin; ceruloplasmin; plasminogen;
complement (C4); C3 activator; fibrinogen; alpha1 acid glycoprotein (alpha-1 acid glycoprotein); alpha2 HS glycoprotein (alpha-2-HS-glycoprotein); beta lipoprotein (beta-lipoprotein); retinol binding protein (retinol-binding protein)
Receptors expressed: insulin; insulin-like growth factor II (IGF II)
Depositors: Wistar Institute
Comments: The cells express 3-hydroxy-3-methylglutaryl-CoA reductase and hepatic triglyceride lipase activities. The cells demonstrate decreased expression of apoA-I mRNA and increased expression of catalase mRNA in response to gramoxone (oxidative stress). There is no evidence of a Hepatitis B virus genome in this cell line
Karyotype: Modal number = 55 (range = 50 to 60); has a rearranged chromosome 1.
Note: Cytogenetic information is based on initial seed stock at ATCC. Cytogenetic instability has been reported in the literature for some cell lines.
Purified DNA from this line is available as ATCC HB-8065D? (10μg) Biosafety Level: 1
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 4th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 1999. The entire text is available online at https://www.wendangku.net/doc/1f13869541.html,/od/ohs/biosfty/bmbl4/bmbl4toc.htm Use Restrictions
These cells are distributed for research purposes only. Cell lines and hybridomas deposited for patent purposes are not always screened for contamination, antibody production or characterized by the ATCC. Release of a culture, the use of which may be claimed in a patent, from the ATCC during the effective term of any such patent is not meant to carry with it, and does not grant any license, express or implied, under any patent, or the right to use a culture in any process described in a patent.
The above culture was deposited in the ATCC in connection with a patent application. Copies of U.S. Patents may be obtained from the Commissioner of Patents, U.S. Patent and Trademark Office, Box 9, Washington, D.C. 20231.
This material is cited in a U.S and/or other Patent and
may not be used to infringe the patent claims.
Handling Procedure for Frozen Cells
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at
–70°C. Storage at –70°C will result in loss of viability. SAFETY PRECAUTION: ATCC highly recommends that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
1. Thaw the vial by gentle agitation in a 37°C water bath.
To reduce the possibility of contamination, keep the O-
ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the
contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing
9.0 ml complete culture medium. and spin at
approximately 125 xg for 5 to7 minutes.
4. Resuspend cell pellet with the recommended complete
medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a
25 cm2 culture flask. It is important to avoid excessive
alkalinity of the medium during recovery of the cells. It is
suggested that, prior to the addition of the vial contents,
the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0
to 7.6). pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator. A
5% CO2 in air atmosphere is recommended if using the
medium described on this product sheet.
Handling Procedure for Flask Cultures
The flask was seeded with cells (see specific batch information) grown and completely filled with medium at ATCC to prevent loss of cells during shipping.
1. Upon receipt visually examine the culture for
macroscopic evidence of any microbial contamination.
Using an inverted microscope (preferably equipped with phase-contrast optics), carefully check for any evidence of microbial contamination. Also check to determine if the majority of cells are still attached to the bottom of the
flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and
become suspended in the culture medium (but are still viable).
2. If the cells are still attached, aseptically remove all but
5 to 10 ml of the shipping medium. The shipping
medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO2 in air atmosphere until they are ready to be subcultured.
3. If the cells are not attached, aseptically remove the
entire contents of the flask and centrifuge at 125 xg for 5
to 10 minutes. Remove shipping medium and save.
Resuspend the pelleted cells in 10 ml of this medium and add to 25 cm2 flask. Incubate at 37°C in a 5% CO2
in air atmosphere until cells are ready to be subcultured. Subculturing Procedure
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-
0.53mM EDTA solution to remove all traces of serum,
which 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 6.0 to 8.0 ml of complete growth medium and
aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new
culture vessels.
Subcultivation Ratio: 1:4 to 1:6.
6. Incubate cultures at 37°C.
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. Medium Renewal
Fluid change twice weekly
Complete Growth Medium
The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium:
?fetal bovine serum to a final concentration of 10% This medium is formulated for use with a 5% CO2 in air atmosphere.
ATCC tested fetal bovine serum is available as ATCC Catalog No. 30-2020 and ATCC Catalog No. 30-2021
(100ml).
Cryoprotectant Medium
Complete growth medium described above supplemented with 5% (v/v) DMSO. Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
Additional Information
Additional product and technical information can be obtained from the catalog references and the ATCC Web site at https://www.wendangku.net/doc/1f13869541.html,, or by e-mail at tech@https://www.wendangku.net/doc/1f13869541.html,. References
(additional references are available in the catalog at https://www.wendangku.net/doc/1f13869541.html,)
Knowles BB et al. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209: 497-499, 1980 PubMed: 80236294
Knowles BB and Aden DP. Human hepatoma derived cell line, process for preparation thereof, and uses therefor. U.S. Pat. 4,393,133 dated July 12, 1983
Schardt C et al. Characterization of insulin-like growth factor II receptors in human small cell lung cancer cell lines. Exp. Cell Res. 204: 22-29, 1993 PubMed: 93106097 Aden DP et al. Controlled synthesis of HBsAg in a differentiated human liver carcinoma- derived cell line. Nature 282: 615-616, 1979 PubMed: 81012119
Busch SJ et al. Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3- methylglutaryl-CoA reductase gene expression in a human hepatoma cell
line, HepG2. J. Biol. Chem. 265: 22474-22479, 1990 PubMed: 91093095
Darlington GJ et al. Growth and hepatospecific gene expression of human hepatoma cells in a defined medium. In Vitro Cell. Dev. Biol. 23: 349-354, 1987 PubMed: 87222055
Cuthbert C et al. Regulation of human apolipoprotein
A-I gene expression by gramoxone. J. Biol. Chem. 272: 14954-14960, 1997 PubMed: 97313474
Deleersnyder V et al. Formation of native hepatitis C virus glycoprotein complexes. J. Virol. 71: 697-704, 1997 PubMed: 97138375
Benn J et al. Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases. J. Virol. 70: 4978-4985, 1996 PubMed: 96357020
Goodrum FD et al. Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells. J. Virol. 70: 6323-6335, 1996 PubMed: 96323154
Kolanus W et al. alphaLbeta2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1 a cytoplasmic regulatory molecule. Cell 86: 233-242, 1996 PubMed: 96319726 Lewis W et al. Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts. Proc. Natl. Acad. Sci. USA 93: 3592-3597, 1996 PubMed: 96195016
Jang SI et al. Activator protein 1 activity is involved in
the regulation of the cell type-specific expression from
the proximal promoter of the human profilaggrin gene. J. Biol. Chem. 271: 24105-24114, 1996 PubMed: 96394543 Roesler WJ et al. The alpha-isoform of the CCAAT/enhancer-binding protein is required for mediating cAMP responsiveness of the phosphoenolpyruvate carboxykinase promoter in hepatoma cells. J. Biol. Chem. 271: 8068-8074, 1996 PubMed: 96215198
Lee JH et al. The proximal promoter of the human transglutaminase 3 gene. J. Biol. Chem. 271: 4561-4568, 1996 PubMed: 96224044
Lieber A et al. Recombinant adenoviruses with large deletions generated by cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo. J. Virol. 70: 8944-8960, 1996 PubMed: 97126100
Dubuisson J and Rice CM. Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. J. Virol. 70: 778-786, 1996 PubMed: 96135186
Yamaguchi Y et al. Biochemical characterization and intracellular localization of the Menkes disease protein. Proc. Natl. Acad. Sci. USA 93: 14030-14035, 1996 PubMed: 97098515
Kounas MZ et al. Cellular internalization and degradation of antithrombin III-thrombin, heparin cofactor II-thrombin, and alpha1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein. J. Biol. Chem. 271: 6523-6529, 1996 PubMed: 96198123
Klemm DJ et al. Adenovirus E1A proteins regulate phosphoenolpyruvate carboxykinase gene transcription through multiple mechanisms. J. Biol. Chem. 271: 8082-8088, 1996 PubMed: 96215200
Wu X et al. Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of ApoB-containing lipoproteins. J. Biol. Chem. 271: 10277-10281, 1996 PubMed: 96215326
Ostlund RE Jr et al. A sterospecific myo-inositol/D-chiro-inositol transporter in HepG2 liver cells. J. Biol. Chem. 271: 10073-10078, 1996 PubMed: 96215295
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
ATCC Warranty
The viability of ATCC products is warranted for 30 days from the date of shipment. If you feel there is a problem with this product, contact Technical Services by phone at 800-638-6597 (U.S., Canada, and Puerto Rico) or 703-365-2700 (elsewhere) or by e-mail at tech@https://www.wendangku.net/doc/1f13869541.html,. . Or you may contact your local distributor.
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Please see the enclosed Material Transfer Agreement (MTA) for further details regarding the use of this product. The MTA is also available on our Web site at https://www.wendangku.net/doc/1f13869541.html,.
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