Neuronal Cell Culture Woes - Not even making it out of the gate! (Mar/04/2011 )

So I have recently been beginning to work with primary neuronal cells (mouse cortex) and am in need of some insight and advice. The cultures had been going along great for a couple of months with only some minor hiccups. I've been optimizing my manipulation protocols etc. and all was well for the most part. However, over the past few weeks I have not been able to get ANY of my four cultures to work (before this I had a very high success rate), and I'm tearing my hair out trying to figure out what is going on.

I dissect out cortex, put in three cortices per plastic tube, treat them with papain for 40 min, wash several times and triturate with fire-polished glass pipettes. I plate 2 million cells per 35 mm dish in neurobasal + B27 + L-glutamine + pen/strep. An hour after I plate I replace the media (and if I have enough hibernate-A I give 'em a brief wash in that to remove any floating debris and dead cells). Lately my neurons show essentially no extension of processes after one day and are basically dead after a couple of days (incubated @ 37C, 5% CO2). Just yesterday I opened a new bottle of media and a new bottle of B27 (which I aliquot into 1 mL aliquots and store at -20C). I even tried plating cells in new media + previous B27, old media + new B27, new media + new B27 in a different incubator. All crapola.

Other things to consider are the L-glutamine (which I doubt, it's stored at -20C and I add it fresh every time I make up media) or the pen/strep, but I don't see how that would stop my cells from growing as there are no obvious signs of infection as best I can tell. One other possibility is that I recently got in a shipment of new sterile plastic tubes (not your typical Fisher 15 mL falcon tubes, but some Sterilin brand tubes w/ a round bottom) I use for the enzyme digestion and trituration steps.

I would VERY MUCH appreciate ANY insight. This is my primary experimental system and things were going so well! I am at a complete loss as to what else there could be! Help!

Thanks

MM

Maybe check the calibration of the CO2 on your incubator--we sometimes have this issue where the monitor will read 5% but then if we check it manually with a fyrite (?sp?) that it is off and that can affect cell growth--especially new cultures--where they seem to just detach and die with no other clues.

Thanks for your response...I'll see if we have that equipment. If not, at the very least I could probably stand to zero the incubator as I don't know when that was done last.

MM

Well I haven't had a chance to check the CO2, but the temp in the incubator reads over 38C (about 38.2, 38.3 on an alcohol thermometer)....anyone have any idea how sensitive neurons are to temperature increases? Any insight would be grand!

Thanks

MM

Well if anyone out there is curious, it turns out the incubator was not off for temperature (I guess my thermometers suck) as an engineer came out today and verified temperature and CO2 (he was in fixing a centrifuge and I grabbed him to check the incubator). Although I have to say, I'm still a little skeeved about the incubator so I've set it to 36.5C just to be safe....but the other thing I switched up was my Pen/Strep. I had been storing it at 4C for too long apparently, and I guess it has some toxic breakdown products (?). So now my neurons are FINALLY back up and running! Holy shit, almost makes me want to go back to doing behavior!

Hi,

I have just started now as well with adult mice neuronal cultures..do you do adult mouse cortex or embrional?
I am asking because i have lots of problems during the purification (i do not have just nice round neurons when i am plating them like with embrional cerebellar neurons for example, but also other ugly aggregates of smaller cells) and also the neurons do not grow much after a few days...
any suggestion? have you had these problems as well?

marco

marcop on Fri Apr 8 14:55:46 2011 said:

Hi Mighty Mouse,

I'm having similar problems to you as well. I just can't seem to get my neurons to grow, and it seems that I have got them to grow just fine in the past. Do you think you could post your isolation and culture protocols so I can try them out. We are doing P0 mouse hippocampus, but may be moving to cortical. Any help would be greatly appreciated.

PRH182