We are hoping to show that Axol human cerebral cortical neurons can easily be adapted to grow in a microfluidic culture device allowing us to separate cell bodies from the extended axons and we will be blogging our progress along the way. This culturing method has many potential applications including investigating biochemical analysis of pure axonal fractions, studying axonal injury and studying axonal regeneration.
The first step in this process was to adapt existing protocols that have been demonstrated to work using primary rat cortical neurons for use with Axol's iPS cell-derived human cerebral cortical neurons (hyCCNs) .
To get set up for the study of axonal biology, we ensured we had the following reagents and equipment:
Neuronal Microfluidic Device with 450mm microgroove barrier
24mm x 40mm No.1 cover glass
Axol Sure Bond+ Kit (ax0041+)
Axol Human Cerebral Cortical Neurons Kit (hyCCNs) ( ax0026F )
Axol Neural Maintenance Medium Kit ( ax0031 )
Sterile dH 2 O
PBS (no magnesium, no calcium)
60mm tissue culture dish
Water bath sonicator
37C 5% CO 2 tissue culture incubator
Preparing and Assembling the Microfluidic Device
Before we considered thawing our cerebral cortical neurons , we needed to set up the microfluidic device to prepare for cell culture. We did this by following the protocol described below.
Preparing the cover glass for device attachment
Note to self: It is advisable to prepare extra cover glasses in case of breakage during the device bonding process (Day 2).
1. Use a water bath sonicator to clean the cover glass to be used for growing neurons. This can be done by placing the cover glass in a rack filling it with dH 2 O and sonicating for 30min.
2. Transfer the cleaned cover glass to the tissue culture hood to maintain sterility and rinse the cover glass with 70% Ethanol.
3. Rinse the cover glass 3 X with sterile dH 2 0.
4. Submerge the cover glass in Axol ReadySet solution and leave to coat overnight.
5. Thaw the required reagents that will be needed for coating and plating of the thawed Axol hyCCNs (they will be needed in the morning). This includes Axol Sure Bond and Axol Neural Maintenance Medium Supplement ( ax0031a ).
See Day 2 for what happened next!