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 4-5 min. Observe cell detachment under an inverted microscope. Cells typically detach as a cell layer which decomposes when resuspended in medium. As soon as the cells are detached, add Trypsin-Inhibitor (20 µl/cm²), resuspend the cells in growth medium (about 160 µl/cm²), aspirate the cells by pipetting, transfer to a tube 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 1:6-1:8 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 from the culture vessel by using Trypsin-EDTA and Trypsin-Inhibitor (see Protocol passaging of hTCEpi).

Resuspend the detached cells in KGM™️ Gold Keratinocyte 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 T5 rouxflask.

Add 6 ml of complete KGM Gold Keratinocyte Growth Medium 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 about 60-70 % 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

Raghunathan V, Edwards SG, Leonard BC, Kim S, Evashenk AT, Song Y, Rewinski E, Marangakis A, Hoehn A, Chang C, Reilly CM, Muppala S, Murphy CJ, Thomasy SM.  Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo. Exp Eye Res. 2021 Jan;202:108362.  https://pubmed.ncbi.nlm.nih.gov/33220237/

Kasus-Jacobi A, Land CA, Stock AJ, Washburn JL, Pereira HA.  Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9.  Invest Ophthalmol Vis Sci. 2020 Apr 9;61(4):16.  https://pubmed.ncbi.nlm.nih.gov/32298435/

Alekseev O, Donegan WE, Donovan KR, Limonnik V, Azizkhan-Clifford J. HSV-1 Hijacks the Host DNA Damage Response in Corneal Epithelial Cells through ICP4-Mediated Activation of ATM.  Invest Ophthalmol Vis Sci. 2020 Jun 3;61(6):39.  https://pubmed.ncbi.nlm.nih.gov/32543665/

Kim S, Gates B, Leonard BC, Gragg M, Pinkerton KE, Winkle LV, Murphy CJ, Pyrgiotakis G, Zhang Z, Demokritou P, Thomasy SM. Engineered metal oxide nanomaterials inhibit corneal epithelial wound healing in vitro and in vivo. NanoImpact. 2020 Jan;17:100198.  https://pubmed.ncbi.nlm.nih.gov/32154443/

Robertson DM, Li L, Fisher S, Pearce VP, Shay JW, Wright WE, Cavanagh HD, Jester JV. Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line. Invest Ophthalmol Vis Sci. 2005 Feb;46(2):470-8. https://pubmed.ncbi.nlm.nih.gov/15671271/

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