Cell Biologics Company - 2201 West Campbell Park Drive, Chicago, IL 60612
website: http://www.cellbiologics.com - e-mail:
[email protected] - Phone: 1-
312-226-8198
Expansion of Cultured Primary Cells
1. Remove culture medium and wash the cells with room temperature 1X phosphate buffered saline solution
(without calcium and magnesium).
2. Incubate the cells with pre-warmed (37°C) 0.05% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6915)
for 3-5 minutes. Use 3.0 ml of Trypsin-EDTA solution for a T75 flask and 2 ml for a T25 flask, respectively.
As soon as the cells have detached (a few firm gentle taps on side of flask may help detachment), add 5-10
ml of Cell Biologics’ Cell Culture Medium to the flask (the FBS will neutralize the trypsin) and gently pipette
up and down a few times.
3. Seed the cells in fresh flasks/plates precoated with Gelatin, and then return flasks/plates into a humidified,
37°C, 5% CO
2
incubator.
4. Change culture medium the following day and then every 24-48 hours afterwards to remove non-adherent
cells and replenish nutrients. Please note that the medium should be changed or add more medium every
day when cells are >70-80% confluent.
5. Cells should be checked daily under a microscope for cell confluence and appropriate cell morphology.
6. Cells can be pre-washed with 1X PBS whenever replacing the medium (optional).
We recommend splitting primary cells at the following ratio
• The recommended split ratio for primary cells is 1:2.
Procedure for Freezing Cells
Materials:
1. 1X Phosphate Buffered Saline (PBS-1X)
2. 0.05% Trypsin-EDTA (1X) solution (Cell Biologics, Catalog No. 6915)
3. Tissue Culture Media
4. Cold Freezing Media (10% DMSO, 50% FBS and 40% culture medium, Cell Biologics, Catalog No. 6916).
5. Labeled Cryovials
6. Confluent Cells
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1. Remove and discard the cell culture media from the flask.
2. Flush the adherent layer 2 times with 7 ml sterile PBS (1X, without calcium and magnesium) to remove
residual medium.
3. Incubate the cells with pre-warmed (37°C) 0.05% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6915)
for 3-5 minutes. Use 3.0 ml of Trypsin-EDTA solution for a T75 flask and 2 ml for a T25 flask, respectively.
As soon as the cells have detached (a few firm gentle taps on side of flask may help detachment), add 5 ml
of Cell Biologics’ Cell Culture Medium to the flask (the FBS will neutralize the trypsin) and gently pipette up
and down 1-2 times.
4. Transfer the cell suspension to a centrifuge tube,
5. Centrifuge cells at 200 x g for 5 minutes.
6. Carefully remove supernatant with sterile Pasteur pipette.
7. Quickly resuspend the cell pellet by adding 1 ml freezing media per vial to be frozen.
8. Place vials in Nalgene "Mr. Frosty" freezing container containing100% isopropyl alcohol at -80 °C for 6-12h.
9. Transfer vials to liquid nitrogen tank for indefinite storage.
We recommend freezing primary cells at the following ratio
• A confluent primary endothelial cells grown in a T75 flask may be frozen in 2-3 cryovials.
• A confluent primary endothelial cells grown in a T25 flask may be frozen in 1 cryovial.
• Please send us light microscope cell images (Cell confluence >70-80%) if you have any questions with
cultured cells.