trypsinization in what temperature will disturb less the cells? - (Sep/11/2008 )

hello,

I dont understand why there is different temperature recommended for trypsinization to detach the adherent cells? Many cell lines from ATCC, they recommend trypsinize under microscope in room temperature for 10-15 min and observe up to seeing cell detach. Others cell producer recommend trypsinize in 37 degree for 3-5 min. I dont know which way is better considering the protect of cells.

thank you very much

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As a rule cells the best way to protect the cells from damage is to only trypsinise the cells for a small amount of time.

trypsinising cells at 37 C is fine for the majority of cells as this is the temperature they normally grow at,

plus cells will normally detach alot quicker when trypsinising at 37 C (in comparison to room temp which is around 25 C )as the trypsin works better at 37 c meaning shorter exposure time.

i recomend when trypsinising your cells, place them in the 37 C incubator and remove them every 2 mins to see if they have deteached. when they have fully detached use this amount of time for all future trypsinisations with your cells.

Some slight tapping of the flask/plate can aid detachment if they are stubborn to detach but be careful not to tap them too hard as this can also damge them or if using a plate such as a 96 well plate can contaminate adjacent wells as media splashes up.

hope this helps.

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Thanks a lot for your time and your valuble recommendation.

I also think it is reasonable. But what make me confused is that I also used cells purchased from Promocell like HUVEC or SMC and from ATCC like L132 or MC3T3 cell line, both recommended in their technical book that DO NOT TRYPSINIZE AT 37 DEGREE. Of course what I can do is follow the corresponding instruction to their product. But just want to really understand such different preference stand on what point.

Still wait for the help from you and other experienced colleagues.

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Dear Lilylille,

I always trypsinise at RT. Most cells will start to detach after 1-2 minutes. If they take longer either:

Wash more times with PBS (W/O Ca2+/Mg2+)
Wash in EDTA as well as PBS (as above)

If you leave the cells longer the trypsin may be pinocytosed and therefore can degrade intracellular proteins. If this happens you WILL see a loss in viability.

I recently went to a class II cabinet manufacturer who were thinking of producing a heated stainless work surface to aid in trypsinisation. In my view for most cells this would not help either in the trypsin process or keeping the cells nearer 37oC while in the cabinet.

Hope this is useful.

Rhombus

p.s. to wind DOM up this is just my opinion, reached over nearly 30 years of tissue and cell culture.

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Rhombus, another practice is pre-warming EDTA/Trypsin to 37degC before using. Is that recommendable or should it also be at RT?

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Dear Rhombus.

thanks a lot,

I agree with you, even I not yet have long-time experience to base on. I will try to not trypsinize the cells at 37 degree.

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Your quite correct, all PBS, Trypsin and media should be pre-warmed to 37oC.

Regards

Rhombus

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