Before You Begin
1. Read this entire manual before using cells or microelectrode arrays.
2. Check the Axion Maestro system for correct performance. Contact Axion at firstname.lastname@example.org with any issues.
Note: Human cortical neurons cultured using this protocol should show spike activity by day 7 in vitro!
MEA Surface Pre-Treatment
1. Wipe the packaged and sealed MEA plate with 70% EtOH, then place the MEA in a biosafety cabinet.
2. Pull the MEA from the sealed package and wipe the top, bottom and sides of the plate with a Kimwipe soaked in 70% EtOH.
3. Add 6 mL of sterile deionized water to the area surrounding the wells (MEA reservoirs) of the MEA plate to prevent substrate evaporation. Do not allow water into the wells of the MEA.
4. Add a 5 μL droplet of Axol ReadySet solution over the MEA electrode area in a biosafety cabinet. Incubate for 1 hour at 37ºC in a cell culture incubator. See Figure 1 for placement.
5. Rinse Axol ReadySet from the culture surface with 200 μL of sterile de-ionized water 4 times.
6. Air dry the MEA plate in a biological safety cabinet overnight.
7. Prepare fresh Axol Sure Bond (50X) working solution in Complete Axol Neural Maintenance Medium. Prepare fresh Axol Sure Bond (50X) working solution in complete Axol Neural Maintenance Medium.
8. Add a 5 μL droplet of Axol Sure Bond working solution over the MEA electrode area in a biosafety cabinet. See Figure 1 for placement.
9. Incubate for 2 hours at 37ºC. Do not allow the Axol Sure Bond droplet to dry.
Warning: Prepare the Axol Sure Bond fresh from frozen aliquots every time. Never reuse Axol Sure Bond!
Thawing Cryopreserved Axol hyCCNs for MEA attachment
1. Remove the cryopreserved hyCCNs from the liquid nitrogen storage container.
2. Hold the cryovial (avoid submerging above the cap) in a 37ºC water bath for exactly 1.5 minutes.
3. Quickly remove the cryovial from the water bath. Spray the outside with 70% ethanol, wipe dry and place in a biosafety cabinet.
Top Tip: Avoid repeatedly pipetting the thawed neurons and be very gentle to promote cell viability.
4. Carefully transfer the contents of the cryovial to a centrifuge tube using a 1 mL pipettor.
5. Wash the inside of the cryovial with 1 mL of room temperature Complete Axol Neural Maintenance Medium to recover what residual cells are left in the vial. Add this 1 mL of media from the cryovial drop-wise (~1 drop/sec) to the centrifuge tube with the neural cell suspension. Gently swirl the tube while also adding the medium to completely mix the solution and to limit the chances of osmotic shock to the thawed cells.
Top Tip: Drop-wise transfer of Complete Axol Neural Maintenance Medium is critical in this step. It limits the osmotic shock and maximizes viability and attachment to the MEA.
6. Add an additional 8 mL of Complete Axol Neural Maintenance Medium.
7. Carefully mix the contents of the centrifuge tube by inverting it 2-3 times. Careful mixing is key to ensuring maximal viability. Take care to avoid any vigorous shaking or vortexing of the cell suspension.
8. Concentrate the neurons by centrifuging at 380 x g for 5 minutes.
9. Resuspend the cell pellet in Complete Axol Neural Maintenance Medium supplemented with Axol Sure Boost (1000X stock solution; 1 μL per mL of medium).
Top Tip: After centrifugation be mindful of the pellet as the final volume will be very small to accommodate the 5 μL droplet. Resuspending the cortical neurons in this small-scale volume will require some finesse and careful pipetting up and down.
Seeding Axol hyCCNs onto the MEA
1. Determine the total number of cells in suspension via hemocytometer count.
2. Remove, from a single row or column, most of the Axol Sure Bond on the MEA surface, but do not let the MEA surface dry before seeding the cells onto the surface (the surface will dry in ~2-3 minutes).
3. Seed a 5 μL droplet of the suspension (60,000 neurons) directly over the array of electrodes in each pre-treated well. See Figure 1 for an example of drop placement.
4. Repeat the previous 2 steps until all rows or columns have been seeded.
5. Incubate the MEA with seeded neurons in a cell culture incubator at 37ºC, 5% CO2 for 1 hour.
6. Remove the MEA plate after 1 hour and carefully add 150 μL of Complete Axol Neural Maintenance Medium to each well using a multi-channel pipette in a biosafety cabinet. Addition of the medium too quickly will detach the adhered neurons.
7. Repeat the previous step a second time to reach a volume of 300 μL per well.
8. Incubate the MEA plate in a cell culture incubator at 37ºC, 5% CO2.
Top Tip: The timing of medium addition is critical as performance of the cortical neurons degrades if the droplets begin to dry (~1-2 minutes).
Maintaining Axol hyCCNs on MEAs
1. Pre-warm Complete Axol Neural Maintenance Medium in a 37ºC water bath immediately before use.
2. A day after seeding the hyCCNs on the MEA, replace the medium with fresh Complete Axol Neural Maintenance Medium supplemented with Axol Neural Enhance (1000X stock solution; 1 μL per mL medium) and Axol Sure Mix (500X stock solution; 2 μL per mL medium).
3. Culture the hyCCNs for two days, after which the medium should be replaced with Complete Axol Neural Maintenance Medium supplemented with Axol Sure Mix.
4. Feed cells every 2 days by replacing approximately half of the medium. Continue to culture the cells in a cell culture incubator at 37ºC, 5% CO2.
Top Tip: Axol Sure Mix should be added once every 4 days during a media change to prevent neuronal clumping.
5. Perform the initial baseline MEA recording on day 7 after seeding, and conduct any compound testing after DIV 34.
Top Tip: Using larger cell densities, such as 60,000 hyCCNS per well, has been shown to elicit increasing firing responses from the cortical neurons on the MEA. See Figure 2 for more information.
Data Acquisition & Analysis
Axion AxIS Software is used to record raw voltage data and detect spikes for rate analysis. Product manuals can be downloaded at http://www.axionbiosystems.com for more comprehensive instructions. To characterize the use of Axol hyCCNs, several densities were tested to determine a minimal cell density required for robust spike activity. The data displayed in Figure 2 demonstrates activity at 60,000 cells per well. Figure 3 shows the morphology of hyCCNs in a 12-well MEA when plated at different densities.
Figure 2: Neural spike activity at 5 weeks on the MEA for hyCCN cells plated at 60,000 per well.
Left) An activity map from the AxIS Software suite showing positions of spike activity for each well of the MEA.
Right) Extracted waveform for each raster and electrode referenced by the color arrows, and color coded to those arrows as examples of activity on plate. Raster plots of spike activity recorded from the plate at the time shown in the activity map.