General Protocol for Recovering or Freezing Primary Cells

All cell culture procedures must be conducted in a bio-safety cabinet.

Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.

Use aseptic technique to prevent microbial contamination.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival.

Medium

Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements).

Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells and when changing media or splitting cells.

Coating of flasks or dishes

Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950) for 10-30 min and then aspirate the excess solution before seeding cells.

Expansion of cultured primary cells

·       Remove and discard the cell culture media from the flask.

·       Flush the adherent layer 2 times using a 5 ml sterile pipette with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.

·       Remove and discard the wash solution from the flask.

·       Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 8-10 ml of Cell Biologics’ Cell Culture Medium supplemented with 5-10 % FBS to a T25 or T75 flask (the FBS will neutralize the trypsin).

·       Plate cells in fresh flasks or plates precoated with Gelatin-Based Coating Solution in a humidified, 5%-CO₂ incubator at 37°C.

·       Change culture media the following day to remove non-adherent cells and replenish nutrients.

·       Cells should be checked daily under a microscopy to verify appropriate cell morphology.

·       Change culture medium every 24-48 hours. Please note that the medium should be changed every day when cells are >70% confluent to remove non-adherent cells and replenish nutrients. Pre-wash cells with 1X PBS 1-2 times whenever replacing the medium.

·       We recommend splitting primary cells at the follow ratio

·       The recommended split ratio for primary murine cells is 1:2.

·       Confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Procedure for Freezing Cells

Materials:

·       1X Phosphate Buffered Saline (PBS-1X)

·       0.05% or 0.25% Trypsin-EDTA (1X) solution (Cell Biologics, Catalog No. 6914. 6915)

·       Tissue Culture Media

·       Cold Freezing Media (10% DMSO, 50% FBS and 40% culture medium, Cell Biologics, Catalog No. 6916).

·       Labeled Cryovials

·       Confluent cells

------------------------------------------------------------------

·       Remove and discard the cell culture media from the flask.

·       Flush the adherent layer with a 5 ml sterile pipette 2 times with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.

·       Remove and discard the wash solution from the flask.

·       Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of 0.25% Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 10 ml of Cell Culture Medium supplemented with 5-10 % FBS to the flask (the FBS will neutralize the trypsin).

·       Centrifuge the cell suspension at 200 g for 5 minutes.

·       Remove supernatant with sterile Pasteur pipette.

·       Quickly re-suspend pellet by adding 1 ml freezing media per vial to be frozen.

·       Place vials in Nalgene "Mr. Frosty" freezing container containing100% isopropyl alcohol at -70-80 °C for 24 h.

·       Transfer vials to liquid N₂ tank for indefinite storage.

·       We recommend freezing primary cells at the follow ratio

·       Confluent primary endothelial cells grown in a T75 flask may be frozen in 2 cryovials. Confluent primary endothelial cells grown in a T25 flask may be frozen in 1 cryovial.