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Q and A Best of Both Worlds – hTERT-immortalized Primary Cells

  1. Will we be able to download the presentation?

    This presentation will be available to watch on demand on the ATCC website

  2. How does ATCC verify that hTERT cells have been immoralized?

    To confirm that cells have been immortalized, we perform longevity testing for at least 25 population doublings (PDLs). We also test the cells for cell type-specific performance (immunocytochemistry markers and/or biofunctional test), genetic stability (to verify that the immortalization process did not adversely affect the karyology), and growth characteristics after 25 PDLs. We have observed that some hTERT-immortalized primary cells grow in excess of 50 PDLs without any change in cell performance.

  3. How are the cells stably selected?

    We use antibiotic selection, usually puromycin. For each cell type we provide specific growth conditions, which usually include keeping the cells under a low level of selective antibiotic pressure for routine growth to ensure that the hTERT gene modification is maintained.

  4. Do you need special media to culture the RPTEC/TERT1-OAT1, RPTEC/TERT1-OCT2, and RPTEC/TERT1-OAT3 cell?

    You can use the same medium as the parental RPTEC/TERT1 cell line (ATCC® CRL-4031™), simply add hTERT-immortalized RPTEC Growth Kit (ATCC® ACS-4007™) to Dulbecco’s Modified Eagle’s Medium (DMEM: F12; ATCC® 30-2006™). If you want to culture the cells continuously, we suggest that you add 0.3ug/mL puromycin for selection pressure.

  5. How long can the RPTEC/TERT1-OAT1, RPTEC/TERT1-OCT2, and RPTEC/TERT1-OAT3 be passaged?

    RPTEC/TERT1-OAT1, RPTEC/TERT1-OCT2, and RPTEC/TERT1-OAT3 were confirmed at ATCC to be functional at passage 10.

  6. Have you used any other substrate to validate your RPTEC SLC transport cells?

    We are planning to perform that analysis. We used fluorescence-based assays as the starting point because the assay itself is simple to perform, did not require sophisticated instruments, and is technically less challenging.

  7. Does ATCC have the control RPTEC/TERT1 cell line (ATCC® CRL-4031™) as well?

    Yes, the cell line is available on the ATCC website under the catalog number ATCC® CRL-4031™.

  8. Have you looked at other transporter such as OCTN1, OCTN2, OATP4C1?

    When we investigated OATP4C1 expression in the RPTEC/TERT1 cells, we found that the gene is expressed at a lower level than that of primary cells. We have not yet analyzed cells for OCTN1 and OCTN2 expression.

  9. How is this model different from the OAT1 HEK 293T/17 (ATCC® CRL-11268G-1™) cell model?

    The major difference is that OAT1 HEK 293T/17 was derived from an embryonic kidney cell line (HEK 293T/17; ATCC® CRL-11268™) that was transformed by an adenovirus. In contrast, the RPTEC/TERT1 (ATCC® CRL-4031™) cell line that was featured in the webinar was derived from adult kidney proximal tubule cells. In mammalian physiology, the renal tubule is where the actual organic anion and cation uptake occurs. Therefore, the RPTEC/TERT1 transporter models will be more relevant to the in vivo situation and will have more predictability for clinical testing than embryonic cell-based models.

  10. Do these cells express plasma membrane receptors such as megalin, cubilin, and amnionless?

    We have not stained for these receptors yet.

  11. Is the level of OAT1 comparable to endogenous OAT1 in primary proximal tubule cells? Does the overexpression of OAT1 affect the response to drugs and molecules?

    We have not performed this comparison yet. However, our available drug testing data has indicated that the overexpression of OAT1 does not affect the drug response.

  12. Can these cells be cultured in a polarized way with a clear separation of apical vs. basolateral markers?

    The parental line RPTEC/TERT1 displays clear separation between apical and basolateral markers. We are planning to perform the same experiments in RPTEC/TERT1-OAT1, RPTEC/TERT1-OCT2, and RPTEC/TERT1-OAT3 cells.

  13. Do you have transepithelial electrical resistance (TEER) data for these two cell lines? Have you tried expressing OAT1, OCT2, and OAT3 in the same cell?

    We have not performed the TEER experiments yet. In addition, we have not co-expressed OAT1, OCT2, and OAT3 in the same cell.

  14. Do you need a special surface to grow theRPTEC/TERT1-OAT1, RPTEC/TERT1-OCT2, and RPTEC/TERT1-OAT3 cells? What media do you recommend using to culture them?

    These cells will attach and grow on cell culture dishes, flasks, or plates treated for adherent cells. We recommend culturing the cells in DMEM: F12 (ATCC® 30-2006™) supplemented with the hTERT RPTEC Growth Kit (ATCC® ACS-4007™).