Hello, I am working with cell culture and I need some advice. I usually use trypsin to detach the cells from the flask, but I wonder if I can stop the trypsin reaction with serum-free medium instead of serum-containing medium. Is this possible or will it affect the cell viability and growth? How do you subculture your cells with trypsin? Thank you for your help.
You will need to either add a protein sich as BSA (bovine serum albumi) or try soybean trypsin inhibitor. Not all cells will tolerate these treatment. https://www.thermofisher.com/order/catalog/product/17075029
Hello Botong Liu
I usually subculture my adherent cells using trypsin for cell detachment. The protocol consists of trypsinizing the cells for 5 mins at 37 degree C followed by inactivation of trypsin using serum containing media.
After trypsin treatment, tap the flask gently to remove loosely adherent cells so that most of the cells get detached from the substratum and add serum containing medium. Centrifuge cell suspension at 1500rpm for 10 mins. Discard supernatant, tap cell pellet. Take cell count using hemocytometer and subculture at the required cell density in a fresh flask with fresh culture medium.
This is a standard procedure followed for subculturing adherent cells.
FYI: You need serum to inactivate trypsin. Serum-free medium is unable to inactivate trypsin. As a result the cells will be overexposed to trypsin leading to cell death, further reducing cell viability and growth.
So, I suggest you follow the standard protocol mentioned above. The cell viability percent by using this procedure is around 90-95% and my cells really look quite happy on subculturing and they keep growing well.
Best.
I've seen serum free media that has as much protein as serum-containing media. Compare the amount of total protein in your media to serum-containing media. If they are similar then it's probably fine. If not, try EDTA to dislodge the cells.
Yes, you can.
Even if there is nothing to neutralize trypsin, dilution with media or PBS heavily will decrease the activity of trypsin, and then you can centrifuge cells to remove all trypsin solution.
Usually, I just rinse adherent cells with trypsin and pour out trypsin before cells are detached. Then I can skip centrifugation after adding cell culture medium.
Well, I will agree with Malcolm.
@ Botong, you may just have to follow the established protocol except you are exploring a novel procedure which can be quite interesting! Good luck
Considering the necessity of growth factors for cells even for a few minutes, I usually use medium with fbs 2%, but serum-free medium is not recommended.
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