In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with gelatin. Therefore, the culture flasks are treated with 0.1 % gelatin solution (60 µl/cm²) at 37°C for at least 10 min (10 – 60 min). Before introducing cells, remove excess of gelatin solution.

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

Add appropriate aliquots of the cell suspension to gelatin pre-coated culture vessels supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:8 twice a week is recommended (after having reached about 90-95 % 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 HUVEC/TERT2).

Resuspend the detached cells in endothelial cell 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 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 roux flask

Before thawing the original vial containing Evercyte cells, pre-coat a 25 cm² culture flask with gelatin (Protocol passaging of HUVEC/TERT2). Add 6 ml of complete endothelial cell 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 near confluent at this point, they have to be passaged.

Product data sheet – certificate of analysis Leaflet

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

Protocols

Data on Markers and Functions

Selected publications

Kim BK, Lee SJ, Kim TW, Kim H, Lee H, Hong EY, Cho YJ, Kwon S, Kim SH. EGT022, an RGD-containing recombinant disintegrin, inhibits the VEGF-induced angiogenic process by targeting integrin β3 in endothelial cells. Am J Transl Res. 2023 Mar 15;15(3):1831-1841. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10086907/

Di Matteo A, Belloni E, Pradella D, Chiaravalli AM, Pini GM, Bugatti M, Alfieri R, Barzan C, Franganillo Tena E, Bione S, Terenzani E, Sessa F, Wyatt CDR, Vermi W, Ghigna C. Alternative Splicing Changes Promoted by NOVA2 Upregulation in Endothelial Cells and Relevance for Gastric Cancer. Int J Mol Sci. 2023 Apr 30;24(9):8102. doi: 10.3390/ijms24098102. 

Capinha L, Zhang Y, Holzer AK, Ückert AK, Zana M, Carta G, Murphy C, Baldovini J, Mazidi Z, Grillari J, Dinnyes A, van de Water B, Leist M, Commandeur JNM, Jennings P. Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models. Arch Toxicol. 2023 Feb;97(2):523-545. doi: 10.1007/s00204-022-03436-6. 

Böttcher B, Pflieger A, Schumacher J, Jungnickel B, Feller K-H. 3D bioprinting of prevascularized full-thickness gelatin-alginate structures with embedded co-cultures. Bioengineering, 9(6), 242. 2022 May. https://doi.org/10.3390/bioengineering9060242

Radziwon A, Bhangu SK, Fernandes S, Cortez-Jugo C, De Rose R, Dyett B, Wojnilowicz M, Laznickova P, Fric J, Forte G, Caruso F,  Cavalieri F. Triggering the nanophase separation of albumin through multivalent binding to glycogen for drug delivery in 2D and 3D multicellular constructs. Nanoscale, 14(9), 3452–3466. 2022 Jan https://doi.org/10.1039/D1NR08429A

Moser D, Leitner P, Filipek P A, Hussain S, Rainer M, Jakschitz T, Rode B M,  Bonn G K. Quantification and cytotoxicity of degradation products (Chloropropanols) in sucralose containing e-liquids with propylene glycol and glycerol as base. Toxicology and Applied Pharmacology, 430, 115727. 2021 Nov. https://doi.org/10.1016/j.taap.2021.115727Gludovacz 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, Resch M, Schuetzenberger K, Petroczi K, Maresch D, Hofbauer S, Jilma B, Borth N,  Boehm T. Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants. Glycobiology, 32(5), 404–413. 2020 Jan. https://doi.org/10.1093/glycob/cwab122De Martin S, et al. (2021) Refill liquids for electronic cigarettes display peculiar toxicity on human endothelial cells. Toxicol Rep.  26;8:456-462. 2021 Feb https://pubmed.ncbi.nlm.nih.gov/33717998/Gludovacz E, et al. (2020) 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. https://pubmed.ncbi.nlm.nih.gov/32985651.

Noël JC, et al. Quantification of selected aroma compounds in e-cigarette products and toxicity evaluation in HUVEC/Tert2 cells. Biomed Chromatogr. 2020 Mar;34(3):e4761. https://pubmed.ncbi.nlm.nih.gov/31758585

Angiolini F, Belloni E, Giordano M, Campioni M, Forneris F, Paronetto M P, Lupia M, Brandas C, Pradella D, Di Matteo A, Giampietro C, Jodice G, Luise C, Bertalot G, Freddi S, Malinverno M, Irimia M, Moulton J D, Summerton J, … Ghigna C. A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing. ELife, 8, e44305. 2019 Mar https://doi.org/10.7554/eLife.44305Bashyal B, Li L, Bains T, Debnath A,  LaBarbera D V. Larrea tridentata: A novel source for anti-parasitic agents active against Entamoeba histolytica, Giardia lamblia and Naegleria fowleri. PLOS Neglected Tropical Diseases, 11(8), e0005832. 2017 Aug. https://doi.org/10.1371/journal.pntd.0005832Mohr T, Haudek-Prinz V, Slany A, Grillari J, Micksche M, Gerner C. Proteome profiling in IL-1β and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis. PLOS ONE, 12(6), 2017 Jun https://doi.org/10.1371/journal.pone.0179065 Thul P J, Åkesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, Alm T, Asplund A, Björk L, Breckels L M, Bäckström A, Danielsson F, Fagerberg L, Fall J, Gatto L, Gnann C, Hober S, Hjelmare M, Johansson F, … Lundberg E. A subcellular map of the human proteome. Science, 356(6340). 2017 May. https://doi.org/10.1126/science.aal3321 

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