Passaging of cells - need to remove in-activated trypsin? - (Jun/04/2007 )

I have been doing cell culture for a couple of years and while passaging my cells, have always spun them down after trypsinisation and resuspended in fresh media before plating out. However I have just started with primary cell culture and on the protocol I inherited from someone who has since left the lab I noticed he does not do this - after trypsinising, he just adds serum-containing DMEM to inactivate and then plated out.

Would this have any effect on the cells? They seem to be fine when visualised several hours after plating out and seem to adhere and grow normally. Would it have any effect on frozen cell stocks? It doesn't seem like a big deal but the primary cell culture facility I am using does not have a centrifuge capable of handling the 14mL Falcon tubes I use when passaging and it would be great if I didn't need to leave the room to spin my cells down to avoid any contamination issues etc.

Thanks!

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For primary neurons, we prefer to spin them and remove most of the serum as they are cultured in serum free media. If this is not a problem for you primary cells, I dont see why it should be a problem to seed them after adding serum containing media.

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There will not have any effect on the cells if the final concentration contains less than 5% trypsin



As long as the DMSO in the final concentration is less than 1%.


Hope this may help.

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I was also taught to spin down the cells after trypsinisation but many posts in Bioforum have written that it is not absolutely necessary.

Thank U Minne.

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Hi,

I think there was a looong discussion on this subject a while ago. Pinned somewhere but no, you do not need to remove it. Just add the serum containing media and it will work just fine. Many people do this routinely and their cells are healthy. Spinning cells down on the other hand may kill some percent of your population...

D

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Dear All,

I have been doing cell culture for 30 years now and always spin down my cells to get rid of the trypsin, this was taught to me by cell culture experts in the 1970's who were pioneers of the art. The ATCC (guide to cell line subculture) advises to centrifuge the suspension gently (125g for 5 minutes). This removes Trypsin and PBS that IS THERE that will dilute your culture media i.e PBS washing of the monolayer will result in a couple of mls left in the flask. You can only remove this completely by having the flask upright for a long period. This is not good as the cell monolayer will dry out. If you then add trypsin to this mix it will further dilute your media.
Gentle centrifugation of cell lines will have no effect on viabilty. It also gets rid of DEAD cells as these do not pellet at 125g.

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Someone told me that less manipulation in the tissue culture... less chance of contamination.
By adding serum-containing DMEM to inactivate trypsin...and skip one step of centrifuge...will minimize contamination.

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i do the following :-

1 Defrost T/E aliquots, Trypsin – EDTA (2ml or 5ml)
2 aspirate old media from cell culture flask
3 wash with PBS (aspirate)
4 add 2ml trypsin per flask, leave in incubator for 5-10 min
5 knocking of flask may aid removal of cells
6 neutralise trypsin with 5ml DMEM (+FCS,P/S)
7 transfer to universal
8 use another 3ml DMEM (+FCS,P/S) to wash out remaining cells
9 centrifuge for five min at 1000 rpm (use 10ml balance)
10 aspirate, add 5ml DMEM as a wash, aspirate
11 add 5ml DMEM, resuspend cells
12 seed flasks (about 1.5-2ml suspension, 10ml DMEM(+FCS,P/S))
13 replace in incubator


and have no problems with contamination (even when i dont use the p/s penicillin,streptomycin)
i'm of the opinion that if you can remove it you should remove it

dom

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depends on trypsin solution; some recombinant trypsins such as TrypLE do not need to be inactivated by serum;

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