LF/TERT309 Human lung fibroblast Cell Line (Nonprofit use only)

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 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 aspirate the cells by pipetting. Add appropriate aliquots of the cell suspension to new culture vessels supplemented with growth medium (final volume of 240 µl/cm²). A split ratio of 1:4 – 1:6 twice a week is recommended (after cells have reached about 70-90 % 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 solution (Protocol passaging of LF/TERT309)

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.

Add 6 ml of complete 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 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. 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

Protocols

 

Data on Markers and Functions