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Best of Both Worlds: hTERT-immortalized Primary Cells

May 31, 2018 at 12:00 PM ET

Abstract

Two of the major challenges that many scientists experience when developing a cell-based assay include obtaining cells with high biological relevance and then producing or procuring enough cells to run the assay without introducing cell variability. hTERT-immortalized primary cells address both issues. These cells are genetically modified such that the cells exhibit the growth characteristics of a continuous cell line but maintain the physiology of a primary cell. In this webinar, ATCC scientists will discuss our broad portfolio of hTERT-immortalized primary cells and provide some application data to illustrate how these cell models can easily be incorporated into your workflow. Special emphasis will be placed upon our new kidney transporter models for predictive toxicology (RPTEC/TERT1 OAT1, RPTEC/TERT1 OCT2, and RPTEC/TERT1 OAT3).

Key Points

  • There is a lack of in vitro models that durably and correctly recapitulate in vivo physiology
  • hTERT-immortalized primary cells solve the problem of limited biological relevancy in cell-based assays
  • hTERT-immortalized primary cells exhibit the growth characteristics of a continuous cell line but maintain the physiology of a primary cell
  • ATCC has created kidney cell models using a well-characterized hTERT-immortalized RPTEC that stably overexpress the OAT1, OCT2, or OAT3 gene; our data show that these modified cell lines are very useful tools that provide kidney tissue-relevant results, improved consistency over time, and predictability for clinical trials

Presenter

Kevin Grady, headshot.

Kevin Grady, BS

Senior Product Line Business Manager, ATCC

Kevin Grady is the Senior Product Line Business Manager for Cell Biology at ATCC. He has been with ATCC for 8 years; prior to ATCC, he held positions at Lonza as Worldwide Product Manager and Director of Scientific Support. Kevin has a long history in the life science industry additionally serving as Director of Scientific Support at Amaxa and Manager of Technical Support at Life Technologies. Mr. Grady has always found great satisfaction in helping researchers learn how to use available products and tools to be more productive and successful in reaching their research goals.

Chaozhong Zou, headshot.

Chaozhong Zou, PhD

Senior Scientist, ATCC Cell Systems

Dr. Chaozhong Zou is a Senior Scientist and the Group Leader of the Immortalized Cells group at ATCC Cell Systems. Dr. Zou has extensive experience in cell-related product development such as immortalized cell lines, stable reporter cell lines, and cell-based assays development. Dr. Zou was a research scientist at NorthShore University HealthSystem, University of Chicago Medical School and conducted postdoctoral research at Northwestern University and Tufts University.

Questions and Answers

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.

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

We have not stained for these receptors yet.

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.

Do you need a special surface to grow the RPTEC/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).

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.

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.

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.

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.

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.

How does ATCC verify that hTERT cells have been immortalized?

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.

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.

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.

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.