RPTEC/TERT1 OAT3 (ATCC® CRL-4031-OAT3)

Organism: Homo sapiens, human  /  Tissue: kidney: renal cortex; proximal tubules, epithelium  /  Disease: normal

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Organism Homo sapiens, human
Tissue kidney: renal cortex; proximal tubules, epithelium
Product Format frozen 1.0 mL
Morphology epithelial
Culture Properties adherent
Biosafety Level 2  [Cells contain SV40 viral DNA sequences]

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
Applications kidney drug toxicity screening and research; transport studies
Storage Conditions liquid nitrogen vapor phase
Karyotype normal diploid; abnormal pseudodiploid; abnormal near-diploid
Comments

RPTEC/TERT1 cell line stably expressing SLC transporter hOAT3(SLC22A8)

  • Marker Testing (ICC) - > 75% positive for OAT3, E-Cadherin, CD13
  • Dome formation – positive, comparable to parental line
  • RT PCR OAT3 Expression Assay – one band at 1.6 kb
  • Telomerase Activities (TRAP Assay) – Positive, > 4 repeats
  • OAT3 copy number confirmed via ddPCR – 7 copies (parental line has 2 copies)
Complete Growth Medium

The base medium for this cell line is DMEM/F-12 (ATCC 30-2006). To make the complete medium add the following components: 

  • hTERT Immortalized RPTEC Growth Kit (ATCC ACS-4007) with Supplement A (5 ml) and Supplement B (8ml)
  • 0.3 µg/mL puromycin
Subculturing
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 Phosphate Buffered Saline (PBS; ATCC 30-2200) solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) 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. 0.1% Soybean Trypsin Inhibitor (ATCC 30-2104) is then used to neutralize the trypsin. After neutralization, aspirate cells by gently pipetting.
  5. Remove Trypsin Inhibitor via centrifugation.
  6. Resuspend the cells with complete growth medium.
  7. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 1.0 x 104 and 2.0 x 104 viable cells/cm2.
  8. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 1.1X 104 and 1.5 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation complete growth media + 10% DMSO (ATCC 4-X)
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 1 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: X,Y
CSF1PO: 11
D13S317: 11,13
D16S539:11,12
D5S818: 9,11
D7S820: 10
THO1: 9,9.3
TPOX: 8,11
vWA: 16,18
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human immunodeficiency virus: None detected
Epstein-Barr virus: None detected
Human papillomavirus: None detected
Functional Tests Functionality was validated during product development using both a 5-CF uptake assay and a 6-CF uptake assay. For the uptake assays, the cells were seeded on 96 well plate, 24 hours later, increasing concentrations of 5-CF or 6-CF were added in the HBSS buffer and incubated at 37°C for 20 minutes. The uptake was stopped by washing the wells with ice-cold HBSS, after 4 washes with HBSS, 100 µL of lysate buffer was added to each well, fluorescence intensity was measured using the Promega GloMax®-Multi Detection System Uptake ratio for both substrates was > than 5.
Population Doubling Time

Approximately 70 hours/PD.

We have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. In addition, it has been verified that no gross changes are observed in karyotype and morphology during the first 10 population doublings.

Name of Depositor ATCC
Year of Deposit 2017
References

Wieser M, et al. hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am. J. Physiol. Renal Physiol. 295: 1365-1375, 2008. PubMed: 18715936

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

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.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103F™).

Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development.

If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.

References

Wieser M, et al. hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am. J. Physiol. Renal Physiol. 295: 1365-1375, 2008. PubMed: 18715936

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

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.