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.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:6 is recommended
Medium Renewal: Every 2 to 3 days
Flask Coating
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.
Cryopreservation Freeze medium: Complete growth medium, 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions Temperature: 37°C
STR Profile Amelogenin: X
CSF1PO: 11,12
D13S317: 13
D16S539: 11,12
D5S818: 13
D7S820: 8,10
THO1: 8,9.3
TPOX: 6,9
vWA: 17,18
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
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
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
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
