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 0.05 % 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 3 min. 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 1:4 twice a week is recommended (after cells have reached about 90 % confluence).

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-EDTA solution (Protocol passaging of fHDF/TERT166)

Resuspend the detached cells in growth medium and centrifuge at 170 g for 5 min. Discard the supernatant, resuspend the resulting cell pellet in the remaining droplet and add freezing medium (tempered to 4°C) to reach a cell density of about 5 x 10^5 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 roux flask

Add 6 ml of complete growth medium to a 25 cm² culture flask and place the culture flask in the incubator for at least 30 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 the 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 the pre-warmed medium to the cells, transfer them 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 80 % confluent at this point, they have to be passaged.

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

Meijer, T., Naderlinger, E., Jennings, P., & Wilmes, A. (2023). Differentiation and Subculturing of Renal Proximal Tubular-like Cells Derived from Human iPSC. Current protocols, 3(8), e850. https://doi.org/10.1002/cpz1.850

Wu, X., Chen, J., Sun, W., Hart, D. A., Ackermann, P. W., & Ahmed, A. S. (2023). Network proteomic analysis identifies inter-alpha-trypsin inhibitor heavy chain 4 during early human Achilles tendon healing as a prognostic biomarker of good long-term outcomes. Frontiers in immunology, 14, 1191536. https://doi.org/10.3389/fimmu.2023.1191536

Chen, J., Wang, J., Hart, D. A., Zhou, Z., Ackermann, P. W., & Ahmed, A. S. (2023). Complement factor D regulates collagen type I expression and fibroblast migration to enhance human tendon repair and healing outcomes. Frontiers in immunology, 14, 1225957. https://doi.org/10.3389/fimmu.2023.1225957

Chen, J., Wang, J., Wu, X. et al. eEF2 improves dense connective tissue repair and healing outcome by regulating cellular death, autophagy, apoptosis, proliferation and migration. Cell. Mol. Life Sci. 80, 128 (2023). https://doi.org/10.1007/s00018-023-04776-x

Falquet M, Prezioso C, Ludvigsen M, Bruun J-A, Passerini S, Sveinbjørnsson B, Pietropaolo V, Moens U. Regulation of Transcriptional Activity of Merkel Cell Polyomavirus Large T-Antigen by PKA-Mediated Phosphorylation. International Journal of Molecular Sciences. 2023; 24(1):895. https://doi.org/10.3390/ijms24010895

Piossek F, Beneke S, Schlichenmaier N, Mucic G, Drewitz S, Dietrich D R. Physiological oxygen and co-culture with human fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells. Chemico-Biological Interactions, 361, 109959. 2022 Jul. https://doi.org/10.1016/j.cbi.2022.109959

Rasheed K, Moens U, Policastro B, Johnsen J I, Koljonen V, Sihto H, Lui W-O,  Sveinbjørnsson B. The merkel cell polyomavirus t-antigens and il-33/st2-il1racp axis: Possible role in merkel cell carcinoma. International Journal of Molecular Sciences, 23(7), 3702. 2022 Mar. https://doi.org/10.3390/ijms23073702Yang N, Lode K, Berzaghi R, Islam A, Martinez-Zubiaurre I,  Hellevik T. Irradiated tumor fibroblasts avoid immune recognition and retain immunosuppressive functions over natural killer cells. Frontiers in Immunology, 11, 602530. 2021 Jan. https://doi.org/10.3389/fimmu.2020.602530Abdulsalam I, Rasheed K, Sveinbjørnsson B, Ehlers B,  Moens U. Promoter activity of Merkel cell Polyomavirus variants in human dermal fibroblasts and a Merkel cell carcinoma cell line. Virology Journal, 17(1), 54. 2020 Apr. https://doi.org/10.1186/s12985-020-01317-x

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