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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.

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.

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.

A culture submitted to the ATCC in January of 1993 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with mycoplasma removal agent (MRA).

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.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

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-53mM EDTA solution (GIBCO cat# 25300-054) 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 with 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.

1. Prepare a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin (BSA) dissolved in culture medium. Store pre-prepared Coating Solution at 4°C in cold room for up to 3 months. 
2. For a growth area of 75 cm², add 4.5 mL of the fibronectin/collagen/BSA solution and rock gently to coat the entire surface. 
3. Incubate the freshly coated vessel(s) in a 37°C incubator overnight (it is preferable to use tissue culture vessels with tightened, plug-seal caps to prevent evaporation during the coating process).
4. Store coated flasks with solution at room temperature, light protected, up to 1 month. Suction off solution before plating cells.

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

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