In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with porcine gelatin. Therefore, the culture flasks are treated with gelatin solution (80 μl/cm2) at 37°C for at least 4 hours (up to one week). Before introducing cells, remove excess of gelatin solution. Use the pre-coated flasks immediately for seeding of cells, the surface must not dry out.

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

Then, add 0.025 % 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. Thereafter, resuspend the cells in growth medium (about 160 µl/cm²) and aspirate the cells by pipetting, centrifuge at 170 g for 5 min. Centrifuge at 170 g for 5 min. Then, discard the supernatant, resuspend the cell pellet in the remaining droplet and add growth medium (about 160 µl/cm²). Transfer appropriate aliquots of the cell suspension to gelatin-coated 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 80-90 % 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 (Protocol passaging of HBEC3-KT).

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 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 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. Before thawing the original vial containing Evercyte cells, pre-coat a 25 cm2 culture flask with gelatin (Protocol passaging of HBEC3-KT). Add 6 ml of growth medium to the prepared 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 about 80-90 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

Li Z, Jella KK, Jaafar L, Moreno CS, Dynan WS. Characterization of exosome release and extracellular vesicle-associated miRNAs for human bronchial epithelial cells irradiated with high charge and energy ions. Life Sci Space Res (Amst). 2021 Feb;28:11-17. https://pubmed.ncbi.nlm.nih.gov/33612174/

Bianchi M, Sivarajan R, Walles T, Hackenberg S, Steinke M. Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions. Innate Immun. 2021 Jan;27(1):89-98. https://pubmed.ncbi.nlm.nih.gov/33317363/Skuland T, Maslennikova T, Låg M, Gatina EM, Serebryakova MK, Trulioff AS, Kudryavtsev IV, Klebnikova N, Kruchinina I, Schwarze PE, Refsnes M. Synthetic hydrosilicate nanotubes induce low pro-inflammatory and cytotoxic responses compared to natural chrysotile in lung cell cultures. Basic Clin Pharmacol Toxicol. 2020 Apr;126(4):374-388. https://pubmed.ncbi.nlm.nih.gov/31628893/Nakauchi M, Nagata N, Takayama I, Saito S, Kubo H, Kaida A, Oba K, Odagiri T, Kageyama T. Propagation of Rhinovirus C in Differentiated Immortalized Human Airway HBEC3-KT Epithelial Cells. Viruses. 2019 Mar 4;11(3):216. https://pubmed.ncbi.nlm.nih.gov/30836639/

Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J, Peyton M, Zou Y, Kurie JM, Dimaio JM, Milchgrub S, Smith AL, Souza RF, Gilbey L, Zhang X, Gandia K, Vaughan MB, Wright WE, Gazdar AF, Shay JW, Minna JD. Immortalization of human bronchial epi-thelial cells in the absence of viral oncoproteins. Cancer Res. 2004 Dec 15;64(24):9027-34. https://pubmed.ncbi.nlm.nih.gov/15604268/

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