Hi everyone,
so, we always had good working primary cortical neurons preps (embryonic) and no issue with culturing them to DIV10-14. However, the animal facility now moved to a different location and instead of being next door it's now a 10min cycle ride away. Interestingly the issue of sudden neuronal cell death at around DIV10 started around the same time. Has anyone experienced something similar before?
We also started adding AraC on DIV4 at 2uM (final conc.) to the cells as we experienced overgrowth of non-neuronal cells but according to the literature this is a well accepted concentration by the neurons. I leave it on until the next media change which happens 3days later.
I use the new B-27™ Plus Neuronal Culture System from ThermoFisher with 50% media change every 4days. Plating (4-5h) is done in DMEM, 10% FBS, P/S.
Hi Verena,
if we have to keep embryonic tissue alife for several hours (i.e. to do genotyping of the embryos before plating the hippocampal neurons) we keep the tissue (NOT the dissociated cells) in Hibernate E media.
Thermo Fisher Scientific , CatNo: A1247601
You can even keep it in this media for days in the fridge an still get viable healthy neurons.
We never add AraC, it is really bad for neurons. An actually you do not need this for embryonic neurons.
One additional info regarding the media:
We have tested Neurobasal, Neurobasal Plus, Media from the company StemCell, and Lonza media. The Neurons are ONLY active in the media of "Lonza". We had a lot of trouble with Neurobasal media ....our Cells died very often depending on the B27 Lot. B27 is very inconsistent....and totally unreliable. And even in the NeurobasalPlus Media the neurons were dead or not active...
This is the info for the Lonza Media:
PNGM Primary Neuron Growth Medium BulletKit Catalog #: CC-4461
It will make an amazing difference.
And one more info ;) :
If you change media (no matter which media you use): from div 6 on use Media without Glutamine. Glutamine is excitotoxic. In fact, we do not change the Media for 3 weeks......but sometimes you have to to add a substance etc. Then use Media without the Glutamine (which you usually add when you make fresh media).
Hope it helps
Cheers
Edda
Hi Edda,
thanks a lot for your reply and all the suggestions. I tested 3 different batches of B27 and one of them was really bad and I had neurospheres in all my wells 3 days after plating so I will definitely give the Lonza media a go!
We normally transport the embryonic tissue in KREBS media and also do the dissections in it and we never had an issue with this until the animal facility moved.
I will do some testing and let you know how it goes.
Thanks,
Verena
Hi Verena Zyka
I agree with Edda in NOT dissociating tissue that needs to be transported. If you can do dissection in your local building it will greatly help with survival.
Lot-testing your neural supplements is always something to check. NeuroCult SM1 from STEMCELL technologies undergoes testing to ensure lot-to-lot consistency in performance.
Thanks
Jason