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 2-3 min. Observe cell detachment under an inverted microscope. As soon as the cells are detached, add Trypsin-Inhibitor (20 µl/cm²), resuspend the cells in growth medium (about 160 µl/cm²) and aspirate the cells by pipetting. Pass cell suspension through an EASY 100 µM cell strainer 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, transfer 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 – 1:6 twice a week is recommended (after cells have reached about 60-70 % confluence). Never allow the culture to become confluent! Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation

Freezing of cells

Detach the cells (60-70 % confluence) from the culture vessel by using Trypsin-EDTA and Trypsin-Inhibitor (Protocol passaging of NHEK/SVTERT3-5).

Resuspend the detached cells in growth medium and centrifuge at 170 g for 5 min. 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

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 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.Recovery from cryopreservation will take a few days. Perform a medium change 24 hours after thawing. If the cells are already about 60-70 % confluent at this point, they must 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

Publications

Hakoi H, Miki Y, Nomura S, Nakajima K, Terashima-Murase C, Takeichi T, Sano S, Akiyama M, Sakasegawa SI, Murakami M, Yamamoto K. Lysophospholipase D from Thermocrispum limits psoriatic inflammation by hydrolyzing epidermal lysoplasmalogen produced by group IIF secreted phospholipase A₂. Biochimie. 2023 Dec;215:75-87. doi: 10.1016/j.biochi.2023.09.027.

Ota A, Morita H, Naganuma T, Miyamoto M, Jojima K, Nojiri K, Matsuda J, Kihara A. Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides. J Biol Chem. 2023 Apr;299(4):104603. doi: 10.1016/j.jbc.2023.104603. Epub 2023 Mar 11.

Wei Z, Liu X, Ooka M, Zhang L, Song MJ, Huang R, Kleinstreuer NC, Simeonov A, Xia M, Ferrer M. Two-Dimensional Cellular and Three-Dimensional Bio-Printed Skin Models to Screen Topical-Use Compounds for Irritation Potential. Front Bioeng Biotechnol. 2020 Feb 21;8:109. doi: 10.3389/fbioe.2020.00109. 

Weinmuellner R, Kryeziu K, Zbiral B, Tav K, Schoenhacker-Alte B, Groza D, Wimmer L, Schosserer M, Nagelreiter F, Rösinger S, Mildner M, Tschachler E, Grusch M, Grillari J, Heffeter P. Long-term exposure of immortalized keratinocytes to arsenic induces EMT, impairs differentiation in organotypic skin models and mimics aspects of human skin derangements. Arch Toxicol. 2018 Jan;92(1):181-194. https://pubmed.ncbi.nlm.nih.gov/28776197/

Wagner T, Gschwandtner M, Strajeriu A, Elbe-Bürger A, Grillari J, Grillari-Voglauer R, Greiner G, Golabi B, Tschachler E, Mildner M. Establishment of keratinocyte cell lines from human hair follicles. Sci Rep. 2018 Sep 7;8(1):13434. https://pubmed.ncbi.nlm.nih.gov/30194332/

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 1900

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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 2700

Annual license fee R&D: royalty based