I am setting up cell culture facility in my lab. Initially, I am taking off with HeLa cells. I am ready with media and all other preparations. As a part of scientific community, I hereby ask everyone to share your suggestions regarding setting up of culture facility.
I think the best thing you can do is train everyone who works for you. Ethanol in, ethanol anything going in, and if you are unsure use some more ethanol. The hood should be wiped down after every usage. Contamination has to be met with swift opposition - remake your media, throw out your trypsin stock, eliminate the sources. Always keep cryo stocks of important cell lines so if something goes wrong you have a backup. I think that one of the most common mistakes is overgrowing cells - it is generally bad to let cell lines get to 100% confluency between passages, and overgrowth will eventually kill many cell lines. Don't passage too many times before getting a new stock. If you are doing genetics or receptor work make sure to serum starve cells before critical experiments (for example, if you want to study EGFR, serum will deplete the majority of it from the cell surface, etc). If you need to thaw a new stock, do it a week before your experiment as you need to passage at least once to have everything ready - thawing stocks late can cause significant delays. Use trypan blue when you count cells before experiments - if half of your cells are dead, trusting the data is probably a bad idea in most cases (unless you expect some death and the control is fine).
Go here and see whether you get the info you need: http://histolii.ugr.es/Postgrado/2008-2009/trabajos/Celis,_Volume_1,_Chapter_1-Setting_up_a_Cell_Culture_Laboratory.pdf
I think the best thing you can do is train everyone who works for you. Ethanol in, ethanol anything going in, and if you are unsure use some more ethanol. The hood should be wiped down after every usage. Contamination has to be met with swift opposition - remake your media, throw out your trypsin stock, eliminate the sources. Always keep cryo stocks of important cell lines so if something goes wrong you have a backup. I think that one of the most common mistakes is overgrowing cells - it is generally bad to let cell lines get to 100% confluency between passages, and overgrowth will eventually kill many cell lines. Don't passage too many times before getting a new stock. If you are doing genetics or receptor work make sure to serum starve cells before critical experiments (for example, if you want to study EGFR, serum will deplete the majority of it from the cell surface, etc). If you need to thaw a new stock, do it a week before your experiment as you need to passage at least once to have everything ready - thawing stocks late can cause significant delays. Use trypan blue when you count cells before experiments - if half of your cells are dead, trusting the data is probably a bad idea in most cases (unless you expect some death and the control is fine).
Water baths are a horrific issue with contamination- they're expensive, but an investment in lab armour beads or glad beads for the water bath can save a lot of hassle.
Silly question, but would using EDTA 0.05M sans the trypsin reduce issues with too many passages? Many breast cell lines seem to detach quite well that way (though MCF7 could be stubborn). Never used HeLa, though.
My suggestion is to use disposable plastic where ever possible rather than autoclaving glass bottles of media etc. May seem expensive and not particularly 'green' but it makes sense to let someone else do all the QA/QC !
Above suggestions are good. I add a few: Rewarming the same media, trypsin (kept at 4degC) is a bad idea. Trypsin will slowly destroy itself, and growth factors serum in media will loose potency. Using the same batch of serum for a set of expts is helpful To get reproducible data, its also important to find the growth curve of your cell line. The , response to (cheap) toxics like H2O2 can be standardized. Later, if you want to test effects of a new drug on new batch/passage of your cells, its good to verify if your IC50 values with H2O2 repeats. Hope this helps!
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