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 all cells are detached (if necessary, agitate the cells by gently hitting the flask), add Trypsin-Inhibitor (20 µl/cm²). Thereafter, resuspend the cells in growth medium (about 160 µl/cm²) and aspirate the cells by pipetting, centrifuge at 170 g for 5 min. A split ratio of 1:16 twice a week is recommended (after cells have reached about 85-95 % confluence). Perform a medium change after 2-3 days if cells have not reached required cell density. 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 and Trypsin-Inhibitor (Protocol passaging of HCEC-1CT).

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 1-2 x 106 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 it 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

Muys BR, Shrestha RL, Anastasakis DG, Pongor L, Li XL, Grammatikakis I, Polash A, Chari R, Gorospe M, Harris CC, Aladjem MI, Basrai MA, Hafner M, Lal A. Matrin3 regulates mitotic spindle dynamics by controlling alternative splicing of CDC14B. Cell Rep. 2023 Mar 28;42(3):112260. doi: 10.1016/j.celrep.2023.112260.

Liu, C., Rokavec, M., Huang, Z. et al. Salicylate induces AMPK and inhibits c-MYC to activate a NRF2/ARE/miR-34a/b/c cascade resulting in suppression of colorectal cancer metastasis. Cell Death Dis 14, 707. Oct 2023. https://doi.org/10.1038/s41419-023-06226-9

Liu, C., Rokavec, M., Huang, Z. et al. Curcumin activates a ROS/KEAP1/NRF2/miR-34a/b/c cascade to suppress colorectal cancer metastasis. Cell Death Differ 30, 1771–1785. May 2023. https://doi.org/10.1038/s41418-023-01178-1

Di Fusco, D., Di Grazia, A., Di Maggio, G. et al. A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer. Cell Death Dis 14, 243. Apr 2023. https://doi.org/10.1038/s41419-023-05772-6

Laudisi F, Pacifico T, Maresca C, Luiz-Ferreira A, Antonelli S, Ortenzi A, Colantoni A, Di Grazia A, Franzè E, Colella M, Di Fusco D, Sica GS, Monteleone I, Monteleone G, Stolfi C. Rafoxanide sensitizes colorectal cancer cells to TRAIL-mediated apoptosis. Biomed Pharmacother. 2022 Nov;155:113794. doi: 10.1016/j.biopha.2022.113794.

Di Grazia A, Di Fusco D, Franzè E, Colella M, Strimpakos G, Salvatori S, Formica V, Laudisi F, Maresca C, Colantoni A, Ortenzi A, Stolfi C, Monteleone I, Monteleone G. Hepcidin Upregulation in Colorectal Cancer Associates with Accumulation of Regulatory Macrophages and Epithelial-Mesenchymal Transition and Correlates with Progression of the Disease. Cancers (Basel). 2022 Oct 27;14(21):5294. doi: 10.3390/cancers14215294.

Laudisi F, Stolfi C, Bevivino G, Maresca C, Franzè E, Troncone E, Lolli E, Marafini I, Pietrucci D, Teofani A, Di Grazia A, Di Fusco D, Colantoni A, Ortenzi A, Desideri A, Monteleone I, Monteleone G. GATA6 Deficiency Leads to Epithelial Barrier Dysfunction and Enhances Susceptibility to Gut Inflammation. J Crohns Colitis. 2022 Feb 23;16(2):301-311. doi: 10.1093/ecco-jcc/jjab145. PMID: 34374415.

Scavo MP, Rizzi F, Depalo N, Fanizza E, Ingrosso C, Curri ML, Giannelli G. A Possible Role of FZD10 Delivering Exosomes Derived from Colon Cancers Cell Lines in Inducing Activation of Epithelial-Mesenchymal Transition in Normal Colon Epithelial Cell Line. Int J Mol Sci. 2020 Sep 3;21(18):6705. https://pubmed.ncbi.nlm.nih.gov/32933173/

Woelflingseder L, Adam G, Marko D. Suppression of Trichothecene-Mediated Immune Response by the Fusarium Secondary Metabolite Butenolide in Human Colon Epithelial Cells. Front Nutr. 2020 Aug 6;7:127. https://pubmed.ncbi.nlm.nih.gov/32850941/Granofszky N, Lang M, Khare V, Schmid G, Scharl T, Ferk F, Jimenez K, Knasmüller S, Campregher C, Gasche C. Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer. Carcinogenesis. 2018 Feb 9;39(2):146-157. https://pubmed.ncbi.nlm.nih.gov/ 29106440/Warth B. et al (2016), Identification of a novel human deoxynivalenol metabolite enhancing proliferation of intestinal and urinary bladder cells. Sci Rep. 2016 Sep 23;6:33854. https://pubmed.ncbi.nlm.nih.gov/27659167/Roig AI, Eskiocak U, Hight SK, Kim SB, Delgado O, Souza RF, Spechler SJ, Wright WE, Shay JW. (2010), Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro. Gastroenterology 138(3):1012-21. https://pubmed.ncbi.nlm.nih.gov/19962984/

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 1700

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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