Day 2: Preparing and Assembling the Microfluidic Device Continued
1. Rinse the cover glass several times with sterile dH2O in the tissue culture hood. Leave the cover glass in dH2O as you sterilize the microfluidic device. Then remove from water and allow the cover glass to air dry in the hood at the same time as the device.
2. Clean and sterilize the microfluidic device with 70% Ethanol using a spray bottle. Allow the microfluidic device to air dry in the tissue culture hood for 1hr with feature side up.
3. After both the microfluidic device and the cover glass are dry they can be bonded together.
4. The microfluidic device needs to be assembled in the tissue culture hood. For efficient bonding the cover glass needs to be place on flat sterile/clean surface. Press the device on top of the cover glass with the feature side down. Be careful not to break the glass. You might be able to use the back of a pair of tweezers to press the device onto the cover glass. Pay attention to key areas around the main channels, microgrooves and wells as the proper bonding is needed.
5. Transfer the bonded microfluidic device on the cover glass (take care to lift by the edge of the cover glass and do not lift by the device itself) to a 60mm dish and check bonding under the microscope.
6. Prepare 1 mL of Axol Sure Bond working solution following the standard Axol Sure Bond+ protocol.
7. In order to coat the inside of the device and wells with Axol Sure Bond, first add 150 μL of PBS to the top left well of the microfluidic device using a P200 pipette allowing the liquid be pushed into the microgrooves. Check under the microscope to make sure the liquid has entered the grooves of the microfluidic device.
8. Remove the majority of PBS from the top left well of the device and add 150μl of Axol Sure Bond solution. Then add 100μl of Axol Sure Bond to the bottom left, top right and bottom right wells.
9. Place the 60mm plate with the device in the 37ºC incubator for 1hr.
Thawing Axol hyCCNs
While the device is being coated with Axol Sure Bond prepare Complete Axol Neural Maintenance Medium and thaw cells!
1. Follow the thawing hyCCNs section of the Culture of Axol Human Cerebral Cortical Neurons (Frozen Young hCCNs) protocol up to the resuspension of the cell pellet. For cell pellet resuspension make sure that you resuspend the cerebral cortical neurons to a concentration range of 2 to 3 million cells per mL in Complete Axol Neural Maintenance Medium supplemented with Axol Sure Boost.
2. Remove the Axol Sure Bond coating solution from each well at the end of the hour incubation.
3. After the Axol Sure Bond has been removed from each well, load 20 μL of cell suspension using a p20 pipette into the top left well by placing the pipette tip to the channel opening. Check under the microscope that the cortical neurons are flowing through the left channel of the device.
4. Return the microfluidic device to the incubator for 10 minutes to allow the cerebral cortical neurons to attach.
5. After 10 minutes, take the plate out of the incubator and add 150μl of Complete Axol Neural Maintenance Medium supplemented with Axol Sure Boost to each well starting with the top left well where the cells were loaded.
6. Check under the microscope that the neurons are not flowing through the channel of the microfluidic device or the microgroove to the right side of the device.
7. Return the microfludic device to the incubator.
8. Come back tomorrow for changing medium!
Figure 2. Brightfight image of the Microfluidic culture device loaded with Axol hyCCNs (ax0026F) taken at 4X magnification.
The thawed human cerebral cortical neurons were loaded in the channel on the left side of the microfluidic device. As we ensured, the cerebral cortical neurons have not travelled into the right side channel of the device or the 450mm microgrooves.
Day 3: Investigating axonal biology using cerebral cortical neurons in a microfluidic culture device.