I grown LNCaP cells in an RPMI media with 20% FBS. They are all tested and negative for any mycoplasma. They are being grown in a new incubator. It has been fully filled with water, 11 gallons on Thurs. Feb 20. The level of CO2 is 5% and was calibrated with a Fyrite calibrator on Wed. Mar 4. The temperature is set to 38C under ATCC recommendations and is calibrated with a thermometer that reads the same value. there is a medium sized water basin that is maintained at about half full and check every Friday. The cells present no signs of contamination, with the hood being in its own isolated room, opposite of the entrance into the lab, and is only ever accessed by myself, since I am in a two person lab. I sterilize all materials that enter the hood with 70% EtOH, wear a lab coat and gloves when in the hood room. I am not sure why the cells will not stretch and grow, but instead have decided to have a drop in the population.
Please let me know what I am doing wrong and what I could do to get these cells to grow. They were all growing great and working fine in the old incubator, but due to moving from one lab room to another, the new incubator has just been unable to reproduce the same conditions and are not allowing these cells to grow.
Hi Ethan,
Sorry your cells are not growing up to par, I will offer a few thoughts from my lab's optimizations with LNCaP in the hopes that it will help. It sounds like you are taking many technical precautions to avoid myco contamination, etc. and that is great. A few other things that may also affect growth are:
(1) Composition of medium. We grow our cells in RPMI 1640 supplemented with 10% FBS (make sure it isn't charcoal-stripped or dextran-treated, as LNCaP cells are androgen dependent and will not grow well in hormone-depleted serum) and PS. Also watch out for bicarb content in the medium as this can affect cellular CO2. Our cells are currently growing great in RPMI from Genessee Sci (cat#25-506).
(2) Passage number. LNCaP cells grown past 20-ish passages tend to slow down and have a bit more trouble attaching after passage. At that point, going back to a low-passage cryovial or purchasing new stocks from ATCC are possibly better options (also good ideas to prevent confounding in-culture changes).
(3) Other culture conditions. LNCaP (and VCaP) cells tend to be more "finicky" than, say PC3 and DU145, regarding culture technique; excessive pipeting, time to adherence, removal of trypsin during passage, etc. can affect viability during culture. Feel free to reach out to my post-doc Shannalee René Martinez if you would like additional tips/discussion.
All the best!
Carlos
It might be plastic. In my experience efficiency of attachment significantly varies between different plastic providers.
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