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hTERT RPE-1; Retinal Epithelium; Human (Homo sapiens) Item ID: ATCCRL4000

Permits and Restrictions

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Tissue Retina, eye; pigmented epithelium
Cell Type Epithelial cells immortalized with hTERT
Product Format frozen
Morphology Epithelial-like
Culture Properties Adherent
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Disease Normal
Gender Female
Storage Conditions Liquid nitrogen vapor phase
Karyotype This is a near-diploid human cell line of female origin with a modal chromosome number of 46 that occurred in 90% of the cells counted. The sex chromosomes consist of a karyotypically normal X-chromosome and a derivative X-chromosome with additional chromosomal material at the terminal end of the q-arm. The derivative X-chromosome was present in all of the cells analyzed.
Derivation

The hTERT-immortalized retinal pigment epithelial cell line, hTERT RPE-1, was derived by transfecting the RPE-340 cell line with the pGRN145 hTERT-expressing plasmid (ATCC MBA-141). 

Cells were cultured in medium containing hygromycin B until stable clones were selected [Pubmed: 9454332, 9916802].
Antigen Expression The cells express the Ep-16 antigen as determined by flow cytometry using the Ep-16 monoclonal antibody (ATCC HB-155), and cytokeratins as determined by immunocytochemistry using a pan-cytokeratin antibody.
Complete Growth Medium The base medium for this cell line is ATCC-formulated DMEM:F12 Medium Catalog No. 30-2006.To make the complete growth medium, add the following components to the base medium:
  • fetal bovine serum to a final concentration of 10%
  • 0.01 mg/ml hygromycin B
Subculturing Volumes used in this protocol are for 75 cm2 flasks; 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 Hanks-Balanced Salt Solution (HBSS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
  3. Add 3.0 to 5.0 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 10.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 4 x 103 to 6 x 103 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C.
Subcultivation Ratio: 1:5 to 1:10 twice weekly Medium Renewal: Every 2 days (or as needed) 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
Cryopreservation Complete growth medium supplemented with an additional 60% fetal bovine serum and 10% DMSO. Store in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.
Culture Conditions Temperature: 37°C Atmosphere: air, 95%; carbon dioxide (CO2), 5% Note: Subculture when cell concentration reaches between 2 X 104 and 4 X 104 cells/cm2.
STR Profile Amelogenin: X CSF1PO: 12,14 D13S317: 11,12 D16S539: 11 D5S818: 11 D7S820: 10,11 THO1: 9 TPOX: 8 vWA: 17,18
Population Doubling Level (PDL) As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation.
Name of Depositor Geron Corporation
References

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Jiang XR, et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet. 21: 111-114, 1999. PubMed: 9916802

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

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