General cell culture protocol

Cell culture protocol introduction:

1. Initiate cell culture from frozen cells:
- Warm up appropriate cell culture media for the cell line in 37c waterbath for at least 30 min.
- Take screw-cap vial containing cells in frozen medium from nitrogen tank and place immediately in a warm 37c waterbath with constant agitation to rapidly thaw cells.
- Wipe the vial with 70% ethanol before openning it in cell culture hood.
- Dilute cells in 10-fold volume of warm culture medium and seed in culture dishes or flasks. Change medium to remove DMSO after few hours or overnight after cells completely attached on the bottom of culture dish. (You will see cell morphology change from round shape to irregular shape after attachment)
Note: After initial cell cultivation, it is recommended to freeze 3-4 vials for each vial of cells thawed at soon as possible (use early passages).

2. Culture and passage cells
- Medium change should be done every few days depending on growth curve of the cell.
- Monitor cell growth everyday under an phase-contrast microscopy. Pay close attention on any sign of possible cell culture contamination by bacterium and fungi, such as medium color and clarity changes due to pH change caused by bacterium growing. Mycoplasm contamination is not easy to recognize and have to be determined by PCR and DNA staining etc.
- Split cells every few days when cells reach 100% confluent.

3. Freezing cells
- Trypsinize cells and centrifuge to remove medium.
- Resuspend cells with freezing medium to 2-4 million cells per ml.
- Transfer cells to sterile cryovial (2-4 millions/ml) on a freezing rack. Label clearly.
- Store at -70c freezer (<1 week) and then transfer to liquid nitrogen tank for permant storage.

Composition of a Typical Medium Suitable for the Cultivation of Mammalian Cells
Some Commonly Used Cell Lines
Some Landmarks in the Development of Tissue and Cell Culture

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Last update 08-Sep-2006, Rating Good of 3 votes.