In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with CellAdhereTM Type I Collagen, human (StemCell Technologies, Cat# 07005, § mg/ml) 0.01 N HCl (sterile filtered).

For preparation of 100 ml 0.01 N HCl mix 50 ml HyPure Cell Culture Grade Water (HyClone, Cat# SH30529), 133,3 µl 25 % HCl, Emsure (Merck, Cat# 1.00316.1011) and 50 ml HyPure Water to get 0.01 N HCl. Sterile filter the solution with a 0.22 µm filter.

For preparation of Collagen I stock solution (3 mg/ml, 200 x) add 5 ml of 0.01 N HCl to 15 mg of lyophilized Collagen I powder,  mix gently until everything has been solubilized

For coating of a T25 roux flask, transfer 2 ml 0.01 N HCl (80 µl/cm²) to a sterile tube, add 10 µl Collagen I (3mg/ml) and transfer the coating solution to a T25 roux flask. Completely wet the surface of the culture flask and incubate at room temperature for at least 1 hour.

Before introducing cells, remove excess of coating solution, wash the flask once with PBS (160 µl/cm²), remove PBS completely and use culture flask immediately for seeding of cells. The surface must not dry out!For passaging the cells, remove and discard the culture medium, wash the cells once with PBS (160 µl/cm²), remove PBS completely.For detachment of the cells, add Trypsin-EDTA and Trypsin 0.25% (10 µl/cm² for each solution), make sure that all cells have been in contact with the solutions and incubate the culture flask at 37°C for approximately 10-15 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 same amount Trypsin-Inhibitor (20 µl/cm²) and growth medium (about 160 µl/cm²). 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 Collagen I pre-coated culture
flasks supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:4 – 1:6 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 1:1 Trypsin-EDTA and Trypsin 0.25%
solution as described in protocol Passaging of TB/SVTERT350.

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 10^5 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 T25 roux flask with Collagen I coating as described in protocol
Passaging of TB/SVTERT350 cells.

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 cell 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 as described in protocol Passaging of TB/SVTERT350 cells.

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