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This line was the first human diploid cell line to be used in human vaccine preparation.

The 8th passage ampule from which this freeze was derived was found to contain a bacterial contaminant (a micrococcus). The cell line was subsequently cured by several passages in the presence of antibiotics.

Growth of the cells is enhanced by addition of tumor necrosis factor alpha (TNF alpha) to the medium.

This cell line is negative for reverse transcriptase.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning^® T-75 flasks (catalog #430641) are recommended for subculturing this product.

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 cell suspension to new culture vessels
6. Place culture vessels in incubators at 37°C.

Sugarman BJ, et al. Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro. Science 230: 943-945, 1985. PubMed: 3933111

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Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37: 614-636, 1965. PubMed: 14315085

Hayflick L, et al. Preparation of poliovirus vaccines in a human fetal diploid cell strain. Am. J. Hyg. 75: 240-258, 1962. PubMed: 13905660

First International Conference on Vaccines against Viral and Rickettsial Diseases of Man. Pan Am. Health Organ. Off. Doc. 147: 581, 1967.

Tomasek JJ, et al. Gelatinase A activation Is regulated by the organization of the polymerized actin cytoskeleton. J. Biol. Chem. 272: 7482-7487, 1997. PubMed: 9054450

Hoppe HC, et al. Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells. Infect. Immun. 65: 3896-3905, 1997. PubMed: 9284169

Landers JE, et al. Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer Res. 57: 3562-3568, 1997. PubMed: 9270029

Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc. Natl. Acad. Sci. USA 93: 136-140, 1996. PubMed: 8552591

Debant A, et al. The multidomain protein trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains. Proc. Natl. Acad. Sci. USA 93: 5466-5471, 1996. PubMed: 8643598

Zhu X, et al. Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc. Natl. Acad. Sci. USA 93: 6091-6095, 1996. PubMed: 8650224

Biological evaluation of medical devices. Part 5: Tests for in vitro cytotoxicity. Sydney, NSW, Australia:Standards Australia;Standards Australia AS ISO 10993.5-2002.

Biological evaluation of medical devices--Part 5: Tests for in vitro cytotoxicity. Geneva (Switzerland):International Organization for Standardization/ANSI;ISO ISO 10993-5:1999.

Nucleotide (GenBank) : AA762049 EST01 Human Lung cDNA Library Homo sapiens cDNA clone 3I35 5', mRNA sequence.