In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with human collagen I. Therefore, the culture flasks are pre-treated with collagen I solution (80 µl/cm²) at 37°C for at least 30 min.

Before introducing cells, remove excess of collagen I solution and rinse flask once with PBS (160 µl/cm²). For detachment of the cells remove and discard the culture medium and wash the cells twice with PBS. Remove PBS completely. Then, add 0.05% Trypsin-EDTA (1x) 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 3 – 4 min. Observe cell detachment under an inverted microscope. As soon as all cells are detached (if necessary, agitate the cells by gently hitting the flask), add growth medium (about 160 µl/cm²) and aspirate cells by pipetting. Add appropriate aliquots of the cell suspension to collagen I pre-coated culture vessels supplemented with growth medium (final volume of 240 µl/cm²). Cells should be split every 3-4 days (after having reached not more that 80 % confluence) with a split ratio of 1:4 to 1:6. Never allow the culture to become confluent!

Cryopreservation

Freezing of cells

Detach the cells from the culture vessel by using Trypsin-EDTA solution (Protocol passaging of PODO/TERT256).

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 (tempered to 4°C) to reach a cell density of about 7 x 10e5 cells /ml (for thawing in a 25 cm² culture flask). Add 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 rouxflask

Pre-coat a 25 cm² culture flask with collagen I (Protocol passaging of PODO/TERT256). Add 6 ml of growth medium to the pre-coated 25 cm² culture flask and place the culture flask in the incubator for at least 20 min to allow the medium to reach its normal pH. Take a vial of frozen cells, rinse it 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 cell pellet in the remaining droplet. Add 1 ml of pre-warmed medium to the cell suspension, transfer the cells 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 70-80 % confluent at this point, they have to be passaged.

Schlichenmaier N, Zielinski A, Beneke S, Dietrich DR. PODO/TERT256 – A promising human immortalized podocyte cell line and its potential use for in vitro research at different oxygen levels. Chem Biol Interact. 2024 Jan 5;387:110813. doi: 10.1016/j.cbi.2023.110813. 

Yoshioka T, Goda M, Kanda M, Itobayashi S, Sugimoto Y, Izawa-Ishizawa Y, Yagi K, Aizawa F, Miyata K, Niimura T, Hamano H, Sakurada T, Zamami Y, Ishizawa K. Valproic acid treatment attenuates cisplatin-induced kidney injury by suppressing proximal tubular cell damage. Clin Transl Sci. 2023 Nov;16(11):2369-2381. doi: 10.1111/cts.13638. 

Gludovacz E, Schuetzenberger K, Resch M, Tillmann K, Petroczi K, Schosserer M, Vondra S, Vakal S, Klanert G, Pollheimer J, Salminen T A, Jilma B, Borth N, Boehm T. Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical. ELife, 10, e68542. 2021 Sep https://doi.org/10.7554/eLife.68542

Gludovacz E, Schuetzenberger K, Resch M, Tillmann K, Petroczi K, Vondra S, Vakal S, Schosserer M, Virgolini N, Pollheimer J, Salminen T A, Jilma B, Borth N, Boehm T. Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N -glycans but by heparan sulfate proteoglycan interactions. Glycobiology, 31(4), 444–458. 2020 Sep. https://doi.org/10.1093/glycob/cwaa090

Stevens M, et al.  The natural drug DIAVIT is protective in a type II mouse model of diabetic nephropathy. PLoS One. 2019; 14(3): e0212910. 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415805/

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