Page 7 - Cell Culture Training Hand Book - A4 Edited 20.1.2020

CELL TRYPSINIZATION / CELL PASSAGING

Reagents
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1. DPBS 1x (Dulbecco’s Phosphate Buffered Saline) (- Ca - Mg )
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2. Trypsin EDTA 0.25%
3. Culture media (DMEM with 10% FBS)

Protocol
1. Remove media from the flask using 5 ml serological pipette.
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+2
2. Wash cells with 5 ml of DPBS (- Ca - Mg ) for 2 times.
3. Add 2 ml of trypsin to detach the cells from flask wall.

4. Incubate the culture for 5 minutes at 37°C.
5. Tap the flask gently, then observe the culture under microscope to ensure cell detachment

.
6. Add 4 ml of culture media to neutralise trypsin. Dislodge cells from the wall a few times.
7. Transfer the mixture into fresh 15 ml falcon tube.
8. Observe the flask under microscope to check if cells have been removed completely.
9. Harvest the collected mixture by centrifugation at 1,500 rpm for 5 minutes.
10. Remove the supernatant. Tap tube gently to loosen pellet and resuspend the pellet (at least
50 x, and avoid bubbles) with 1 ml of culture media. Volume of culture media is in
accordance with cell pellet.
11. Perform cell counting using trypan blue exclusion assay.
12. Calculate appropriate volume of culture media required to culture the cells. Add sufficient
volume of media such that 1 ml of cell suspension can be transferred into each fresh culture

flask .
13. Seed the cell suspension into new culture flask with seeding density of 6 x 10 – 7 x 10
5
5
cells/flask .
14. Spread the cells evenly.
15. Incubate the cell cuture in the 5% CO 2 incubator at 37 °C. Change media every 2-3 days.

Tips
1. Proteolytic enzyme which breaks down protein, to dissociate adherent cells from flask wall.
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2. Appropriate quantity of trypsin is 0.5 ml/ 10 cm .
3. Incubation time depends on cell type.
4. If less than 90% of cells are detached, incubate the culture for another 2 minutes. Prevent cell exposure to trypsin solution
>10mins. Long term incubation will damage cells by stripping cell surface protein and kill the cells.
5. 2:1 of culture media to trypsin is a common practise to neutralise trypsin solution. Otherwise, 1:1 (equal volume) is suffcient.
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6. Common practise: 5-10 ml vol. of media for 25 cm flask & 10-30ml vol. of media for 75 cm flask.
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5
7. Depending on cell type. 6-7 x 10 is a common practise.

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