In vitro propagation

Passaging of cells

For detachment of the cells remove and discard the culture medium and wash the cells once with PBS (about 160 µl/cm²). Remove PBS completely.

Then, add Trypsin-EDTA solution (20 µl/cm²), make sure that all cells have been in contact with this solution and incubate the culture flask at 37°C for approximately 2 – 5 min. Observe the cell detachment under an inverted microscope. As soon as all cells are detached (if necessary, agitate the cells by gently hitting the flask), add Trypsin-Inhibitor (20 µl/cm²). Resuspend the cells in growth medium (about 160 µl/cm²) and centrifuge at 170 g for 5 min. Discard the supernatant, resuspend the cell pellet in the remaining droplet and add growth medium (about 160 µl/cm²). Then, add appropriate aliquots of the cell suspension to new culture vessels supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:2 to 1:3 twice a week is recommended (after having reached about 95 % confluence). Cells should be passaged when near confluent. Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation

Freezing of cells

Detach the cells from the culture vessel by using Trypsin and Trypsin-Inhibitor (Protocol passaging of RPTEC/TERT1).

Resuspend the detached cells in growth medium and centrifuge at 170 g for 5 min. Discard the supernatant, resuspend in the remaining droplet and add freezing medium (4°C) to reach a cell density of about 1,5 – 2 x 10^6 cells/ml (for thawing in a 25 cm² culture flask). Transfer 1 ml of this cell suspension to each pre-cooled cryovial and immediately transfer the cells to -80°C. After 24 hours transfer the vials to the liquid nitrogen tank.

Thawing of cells

Original Evercyte cells are to be thawed in a T25 roux flask

Add 6 ml of growth medium to a 25 cm² culture flask and place the culture flask in the incubator for at least 30 min. Take a vial of frozen cells, rinse outside with Ethanol and pre-warm in hand until one last piece of frozen cells is seen. Then, immediately transfer the content of the vial to a 15 ml centrifugation tube pre-filled with 9 ml of medium pre-cooled to 4°C and centrifuge for 5 min at 170 g. Discard the supernatant and resuspend the cells in the remaining droplet. Add 1 ml of pre-warmed medium to the cells, transfer the cell suspension to the prepared culture flask and incubate at 37°C in a suitable incubator. Perform a medium change 24 hours after thawing. If the cells are already confluent at this point, they have to be passaged. Typically, RPTEC/TERT1 cells can be split 2-3 days after thawing when having reached about 95% confluence.

Product data sheet – certificate of analysis

is available upon request | Please contact us indicating the respective LOT numbers

Protocols

Data on Markers and Functions

Selected publications

Carta G, van der Stel W, Scuric EWJ, Capinha L, Delp J, Bennekou SH, Forsby A, Walker P, Leist M, van de Water B, Jennings P. Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells. Cell Biol Toxicol. 2023 Dec;39(6):3031-3059. doi: 10.1007/s10565-023-09816-7. 

Schreiber P, Friedrich AK, Gruber G, Nusshag C, Boegelein L, Essbauer S, Uhrig J, Zeier M, Krautkrämer E. Differences in the Susceptibility of Human Tubular Epithelial Cells for Infection with Orthohantaviruses. Viruses. 2023 Jul 31;15(8):1670. doi: 10.3390/v15081670. 

Namazian Jam N, Gottlöber F, Hempel M, Dzekhtsiarova Y, Behrens S, Sonntag F, Sradnick J, Hugo C, Schmieder F. Microphysiological Conditions Do Not Affect MDR1-Mediated Transport of Rhodamine 123 above an Artificial Proximal Tubule. Biomedicines. 2023 Jul 20;11(7):2045. doi: 10.3390/biomedicines11072045. 

Pearson A, Haenni D, Bouitbir J, Hunt M, Payne BAI, Sachdeva A, Hung RKY, Post FA, Connolly J, Nlandu-Khodo S, Jankovic N, Bugarski M, Hall AM. Integration of High-Throughput Imaging and Multiparametric Metabolic Profiling Reveals a Mitochondrial Mechanism of Tenofovir Toxicity. Function (Oxf). 2022 Dec 24;4(1):zqac065. doi: 10.1093/function/zqac065. 

Piossek F et al. Physiological Oxygen and Co-Culture with Human Fibroblasts Facilitate in Vivo-like Properties in Human Renal Proximal Tubular Epithelial Cells. Chemico-Biological Interactions, vol. 361, 2022 Jul. https://doi.org/10.1016/j.cbi.2022.109959.

Merches K, Breunig L, Fender J et al. The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro. Arch Toxicol 96, 2341–2360. 2022 May. https://doi.org/10.1007/s00204-022-03306-1

Jarzina S, Di Fiore S, Ellinger B, Reiser P, Frank S, Glaser M, Wu J, Taverne FJ, Kramer NI and Mally A Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study. 4:864441. 2022 Apr. doi: 10.3389/ftox.2022.864441

Rajasekar S, Lin D S Y, Zhang F, Sotra A, Boshart A, Clotet-Freixas S, Liu A, Hirota JA, Ogawa S, Konvalinka A, Zhang B. Subtractive manufacturing with swelling induced stochastic folding of sacrificial materials for fabricating complex perfusable tissues in multi-well plates. Lab on a Chip, 22(10), 1929–1942. 2022 Mar. https://doi.org/10.1039/D1LC01141C

Nunes C et al. An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat. Toxicology in Vitro, 81, 105333. 2022 Jun. https://doi.org/10.1016/j.tiv.2022.105333

Glykofridis IE, et al. (2021) Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells. eLife. 2021; 10: e61630 www.ncbi.nlm.nih.gov/pmc/articles/PMC7899648/

Delp J, et al. (2021) Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors. Arch Toxicol. 2021; 95(2): 591–615. www.ncbi.nlm.nih.gov/pmc/articles/PMC7870626/Megan L, et al. (2021) Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model. J Am Soc Nephrol. 2020 Jan; 31(1): 67–83. www.ncbi.nlm.nih.gov/pmc/articles/PMC6934997/Krebs A, et al. (2020) The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods. Arch Toxicol. 2020; 94(7): 2435–2461. www.ncbi.nlm.nih.gov/pmc/articles/PMC7367925/Spinu N, et al. (2020) Quantitative adverse outcome pathway (qAOP) models for toxicity prediction. Arch Toxicol. 2020; 94(5): 1497–1510. www.ncbi.nlm.nih.gov/pmc/articles/PMC7261727/van der Stel W, et al. (2020) Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals. Arch Toxicol. 2020; 94(8): 2707–2729. www.ncbi.nlm.nih.gov/pmc/articles/PMC7395062/Shrestha S, et al. (2020) Characterization and determination of cadmium resistance of CD133+/CD24+ and CD133−/CD24+ cells isolated from the immortalized human proximal tubule cell line, RPTEC/TERT1. www.ncbi.nlm.nih.gov/pmc/articles/PMC6766375/Obaidi I, et al. (2020) Curcumin Sensitizes Kidney Cancer Cells to TRAIL-Induced Apoptosis via ROS Mediated Activation of JNK-CHOP Pathway and Upregulation of DR4. Biology (Basel) 2020 May; 9(5): 92. www.ncbi.nlm.nih.gov/pmc/articles/PMC7284747/Pirklbauer M, et al. (2020) Empagliflozin Inhibits Basal and IL-1β-Mediated MCP-1/CCL2 and Endothelin-1 Expression in Human Proximal Tubular Cells. Int J Mol Sci. 2020 Nov; 21(21): 8189. www.ncbi.nlm.nih.gov/pmc/articles/PMC7663377/Wieser M, et al. (2019) CD46 knock-out using CRISPR/Cas9 editing of hTERT immortalized human cells modulates complement activation. PLoS One. 2019; 14(4): e0214514. www.ncbi.nlm.nih.gov/pmc/articles/PMC6453361/Limonciel A, et al. (2018) Comparison of base-line and chemical-induced transcriptomic responses in HepaRG and RPTEC/TERT1 cells using TempO-Seq. Arch Toxicol. 2018; 92(8): 2517–2531. www.ncbi.nlm.nih.gov/pmc/articles/PMC6063331/Kramer N.I., et al. (2015) Biokinetics in repeated-dosing in vitro drug toxicity studies. Toxicol In Vitro. 2015 Sep8. pii: S0887-2333(15)00218-0. https://pubmed.ncbi.nlm.nih.gov/26362508/Slyne J, et al., (2105) New developments concerning the proximal tubule in diabetic nephropathy: in vitro models and mechanisms. Nephrol Dial Transplant. 2015 Aug;30. https://pubmed.ncbi.nlm.nih.gov/26209740/

Ranninger C., et al. (2015) Nephron Toxicity Profiling via Untargeted Metabolome Analysis Employing a High
Performance Liquid Chromatography-Mass Spectrometry-based Experimental and Computational Pipeline. J BiolChem. 2015 Jul 31;290(31):19121-32. https://pubmed.ncbi.nlm.nih.gov/26055719/

Simon-Friedt BR, et al. (2015) The RPTEC/TERT1 Cell Line as an Improved Tool for In Vitro Nephrotoxicity
Assessments. Biol Trace Elem Res. 2015 Jul;166(1):66-71. https://pubmed.ncbi.nlm.nih.gov/25893367/

Maschmeyer I, et al. (2015) A four-organ-chip for inter-connected long-term co-culture of human intestine, liver, skin and kidney equivalents. Lab Chip. 2015 Jun 21;15(12):2688-99.https://pubmed.ncbi.nlm.nih.gov/25996126/

Fliedl L, et al. (2015) Optimisation of a quantitative PCR based method for Plasmid Copy Number Determination in Human Cell Lines. N Biotechnol. 2015 Mar 18. pii: S1871-6784(15)00046-1. https://pubmed.ncbi.nlm.nih.gov/25796475/Limonciel A., et al. (2015) Transcriptomics hit the target: Monitoring of ligand-activated and stress response
pathways for chemical testing. Toxicol In Vitro. 2015 Jan 13. pii: S0887-2333(14)00251-3. https://pubmed.ncbi.nlm.nih.gov/25596134/Crean D., et al. (2014) Development of an in vitro renal epithelial disease state model for xenobiotic toxicity testing. Toxicol In Vitro. 2014 Dec 20. pii: S0887-2333(14)00239-2. https://pubmed.ncbi.nlm.nih.gov/25536518/

Aschauer L, et al. (2015) Application of RPTEC/TERT1 cells for investigation of repeat dose nephrotoxicity: A
transcriptomic study. Toxicol In Vitro. 2015 Dec 25;30(1 Pt A):106-16. https://pubmed.ncbi.nlm.nih.gov/25450743/Wilmes A, et al. (2015) Mechanism of cisplatin proximal tubule toxicityrevealed by integrating transcriptomics, proteomics, metabolomics and biokinetics. Toxicol In Vitro. 2015 Dec 25;30(1 Pt A):117-27. https://pubmed.ncbi.nlm.nih.gov/25450742/

Wilmes A, et al. (2014) Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water channel. Toxicol Appl Pharmacol. 2014 Sep 1;279(2):163-72. https://pubmed.ncbi.nlm.nih.gov/24907557/

Simon BR, et al. (2014) Cadmium alters the formation of benzo[a]pyrene DNA adducts in the RPTEC/TERT1 human renal proximal tubule epithelial cell line. Toxicol Rep. 2014 Jul 14;1:391-400. https://pubmed.ncbi.nlm.nih.gov/25170436/

Fliedl L, Wieser M, Manhart G, Gerstl MP, Khan A, Grillari J, Grillari-Voglauer R. (2014) Controversial role of gammaglutamyl transferase activity in cisplatin nephrotoxicity. ALTEX. 2014;31(3):269-78. doi: Epub 2014 Mar 21. https://pubmed.ncbi.nlm.nih.gov/24664430/

Aschauer L, et al. (2013) Delineation of the Key Aspects in the Regulation of Epithelial Monolayer Formation. Mol Cell Biol. 2013 Jul;33(13):2535-50. https://pubmed.ncbi.nlm.nih.gov/23608536/

Radford R, Slattery C, Jennings P, Blacque O, Pfaller W, Gmuender H, Van Delft J, Ryan MP, McMorrow T. (2012) Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle. Am J Physiol Renal Physiol. 2012 Apr 15;302(8):F905-16. doi: 10.1152/ajprenal.00427.2011. Epub 2012 Jan 18. https://pubmed.ncbi.nlm.nih.gov/22262483/

Wilmes A, Limonciel A, Aschauer L, Moenks K, Bielow C, Leonard MO, Hamon J, Carpi D, Ruzek S, Handler A, Schmal O, Herrgen K, Bellwon P, Burek C, Truisi GL, Hewitt P, Di Consiglio E, Testai E, Blaauboer BJ, Guillou C, Huber CG, Lukas A, Pfaller W, Mueller SO, Bois FY, Dekant W, Jennings P. (2013) Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress. J Proteomics. 2013 Feb 21;79:180-94. https://pubmed.ncbi.nlm.nih.gov/23238060/

Limonciel A, Wilmes A, Aschauer L, Radford R, Bloch KM, McMorrow T, Pfaller W, van Delft JH, Slattery C, Ryan MP, Lock EA, Jennings P. (2012) Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells. Arch Toxicol. 2012 Nov;86(11):1741-51. doi: 10.1007/s00204-012-0897-0. Epub 2012 Jul 4. https://pubmed.ncbi.nlm.nih.gov/22760423/

Jennings P, Weiland C, Limonciel A, Bloch KM, Radford R, Aschauer L, McMorrow T, Wilmes A, Pfaller W, Ahr HJ, Slattery C, Lock EA, Ryan MP, Ellinger-Ziegelbauer H. (2012) Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model. Arch Toxicol. 2012 Apr;86(4):571-89. doi: 10.1007/s00204-011-0780-4. Epub 2011 Nov 29. https://pubmed.ncbi.nlm.nih.gov/22124623/

Sarkozi R. et al. (2011) Oncostatin M is a novel inhibitor of TGF-β1-induced matricellular protein expression. Am J Physiol Renal Physiol 2011 Nov, 301(5):F1014-F1025. https://pubmed.ncbi.nlm.nih.gov/21816755/

Limonciel A, Aschauer L, Wilmes A, Prajczer S, Leonard MO, Pfaller W, Jennings P. (2011) Lactate is an ideal noninvasive marker for evaluating temporal alterations in cell stress and toxicity in repeat dose testing regimes. Toxicol In Vitro. 2011 Dec;25(8):1855-62. doi: 10.1016/j.tiv.2011.05.018. Epub 2011 May 24. https://pubmed.ncbi.nlm.nih.gov/21635945/

Ellis JK, Athersuch TJ, Cavill R, Radford R, Slattery C, Jennings P, McMorrow T, Ryan MP, Ebbels TM, Keun HC. (2011) Metabolic response to low-level toxicant exposure in a novel renal tubule epithelial cell system. Mol Biosyst. 2011 Jan;7(1):247-57. doi: 10.1039/c0mb00146e. Epub 2010 Nov 19. https://pubmed.ncbi.nlm.nih.gov/21103459/

Wieser M, Stadler G, Jennings P, Streubel B, Pfaller W, Ambros P, Riedl C, Katinger H, Grillari J, Grillari-Voglauer R. (2008) hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am J Physiol Renal Physiol. 2008 Nov;295(5):F1365-75. doi: 10.1152/ajprenal.90405.2008. Epub 2008 Aug 20. https://pubmed.ncbi.nlm.nih.gov/18715936/

Licence Conditions

The business concept of Evercyte is to out-license telomerized cells to our customers. The license conditions depend on whether the contract partner is a for profit or a nonprofit organization and the intended use of the cells.

Nonprofit organizations

Evercyte grants licenses for an unlimited period to academic or nonprofit-organizations, whereby the use of Evercyte cell lines is restricted to research & development purposes and non-commercial use. The cells are not intended for human use. The customers have to agree to the conditions described in our material transfer agreement as well as accept our general terms and conditions.

On time payment for unlimited use: EUR 1600

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Profit organizations

Pharmaceutical – chemical – cosmetic industries

Evercyte grants licenses for commercial organizations, whereby we offer an initial testing phase for a flat fee that allows our customers to test our cells in their laboratories for a period of 6 months. Thereafter, annual license fees fall due, depending on the cell line of interest. Besides offering cell lines for research & development purposes, we also have established cell factories that qualify for production of clinical grade extracellular vesicles for human application. The customer has to agree to the conditions described in our license agreements.

Contract research organizations (CRO)

Evercyte grants licenses for contract research organizations, whereby we offer an initial testing phase for a flat fee that allows our customers to test our cells in their laboratories. Thereafter, we would negotiate a royalty based long-term license agreement individually. The use of the cells during these phases is restricted to research & development purposes. The cells are not intended for human use. The customers have to agree to the conditions described in our material transfer agreement and accept our general terms and conditions.

Initial license fee for 3 months: EUR 2500

Annual license fee R&D: royalty based