I am looking into protocols for culturing primary rat hippocampal neurons (no feeder layers). The standard protocol from ThermoFisher advises to replace 50% of the media every 2-3 days. However, several other sources indicate to change less of the media and less frequently. What is the best procedure for culturing previously cryopreserved neurons?
Hello Ashlee
Primary neuronal cultures are fragile and susceptible to oxidative damage, and they are known not to survive for no reason at all. They are extremely prone to changes when culture medium is frequently changed. So, there should be minimum interference as far as neuronal cultures are concerned. I suggest you change the medium after 5 to 6 days and only exchange half the medium each time. Do not expose neurons to air.
Good Luck.
Similar to what Malcolm mentioned, do not exchange all the media and do not do it too often. To give an example, in long term primary cultures, I culture neurons on PLL-coated coverslips in 6 cm dishes with 5 ml of neuronal media (N2) on top of a glial feeder layer (Banker's protocol). In these conditions I refresh 1-1.5 ml of media every 3 days and cultures make it very well for over 30 days.
Cheers,
Hi Ashlee, we just made the (maybe anecdotal) experience that it might depend on the medium used for culturing. In our hands, hippocampal and cortical neurons grown in Neurobasal-based medium tolerate changing 50% of the medium every 3.4 days. Recently, we introduced medium changes to cortical neurons grown in MEM-based medium (changes of 50% medium every 3.4 days), however they seemed to enjoy handling this much less than their Neurobasal-grown fellows.
It basically depends on the amount (mls) of cells introduced in a plate or dish aswell as the size of dish/plate you are using. Usually changing media can be done every two days and the guiding principle is the cell growth and health. I wish you well.
If you change media, do use media without Glutamine. It is excitotoxic for Neurons starting div 6 when they for Synapses. (actually ThermoFisher suggests this for Neurobasal Media: For plating L-Glutamine is needed but do not add it into the Media that you use for feeding you neurons later) We in fact do not change the media for up to 2-3 weeks and Neurons are healthy and happy. We do not use Neurobasal media anymore. We have had bad experience with the viability. This depended a lot on the Lot of B27. Very unreliable. The Neurons are also not active (tested: Neurobasal Plus, Neurobasal E, StemCell, Lonza). Only in the Lonza media they were active.
PNGMTM Primary Neuron Growth Medium BulletKitTM, CatNo: CC-4461
Here the same: For plating use L-glutamine containing media but do not add L-Glutamine in the media for feeding.
Cheers
Edda
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