In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with NutriCoat™ Attachment Solution (Biological Industries/SATORIUS, Cat# 05-760-1-15) following the instructions of the manufacturer. Briefly, dilute the substrate 1:500 in Ringer Solution and transfer the diluted substrate to the culture flasks (72 µl/cm²), incubate at least 1 hours at 37°C.

Before introducing cells, remove excess of coating solution and immediately use the flask for seeding of cells. For detachment of the cells, add CTS TrypLE Select Enzyme 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 growth medium (about 160 µl/cm²) and centrifuge at 180 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 70-80 % confluence).

Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation

Freezing of cells

Detach the cells after having reached about 70-80 % confluence from the culture vessel by using CTS TrypLE Select Enzyme solution (Protocol passaging of P-MSC/TERT308).

Resuspend the detached cells in growth medium and centrifuge at 180 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 10⁵ 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.

Pre-coat a cm² roux flask with NutriCoat™ Attachment Solution (Protocol passaging of P-MSC/TERT308).

Add 6 ml of complete growth medium to a pre-coated 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 180 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. Passage the cells the day after thawing.

Product data sheet – certificate of analysis

Protocols

Data on Markers and Functions

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