MEA

Neural Organoid Procedure for MEA

Prepare the MEA plate and Matrigel solution

1. Prepare MEA plate according to standard orgnoid protocol (PEI-coated surface with standard washing and overnight drying steps)
2. Add laminin to final concentration (10 ug/mL) into media containg organids
3. Prepare enough 100% Martigel to add at least 15 uL to each well of the MEA plate that will contain an organoid

Collect the organoids

1. Using a wide-bore pipette tip or a pipette tip that has been cut to a wide-bore size, gently aspirate some media from the organoid prep wells, and then remove one orgaiod via pipetting

Deposit the organoids

1. Hold the pipette vertically, allowing the organoid to fall to the bottom of the tip
2. Place the organoid in the center of a well in the MEA plate by loding the pipette vertically tip: use your other hand to stabilize the pipette tip during the dispense
3. The MEA plates should be flat on the surface during this transfer step. Repeat steps 4-6 untill all wells in the plate have an organoid placed

Position the organoids

1. Using the pipette of choice (10uL or 200uL), gently aspirate midia from the region surrounding each organic, using that suction to reposition the organoid in the well (e.g., if the organoid is placed too far to the right use suction form the left to reposition the organoid toward the center). Note: the organoids will be sitting in only a small volume of media, but this should be sufficient to maintain hydration for the duration of the plating
2. Once the Matrigel is at the appropirate temperature, use a 200 uL pipette tip to aspirate 15 uL. Hold the pipette vertically directly over the organoid and drip the Matrigel onto the organoid Note: 15 uL is optimal for 2-3 mm organoids, but this volume can be adjusted based on size.

Incubate the organoids

1. Once Matrigel has been added on top of all plated organoids, close the plate lid and transfer to a 37C/5%CO2 incubator for at least 2 hours, allowing Martigel to solidify
2. After 2 hours, transfer the plate back to the biosafety cabinet, and add half volumes of media to each side of the well. If this is a 6-well plate, use 500 uL on either side, paying careful attention to add the meida to the well of the conial feature in the well, allowing the media to gently flow into the well and not disturb the organoids. The plate may be tilted at this stage to assist in media addition

Troubleshooting detachment

If an organoid becomes detached, which can occur, simply aspirate the midia and repeat from step 8. Consider incubation with Marigel for longer OR using a larger volume of matigel if the organoid is larger.

Maintenace

Once the wells on the plate have media added and organs are secure, you may proceed with maintaing the culuture by performing half media changes regularly. Record from the plate via the Maestro perodically to observe culture maturation over time. You may be able to observe signals within hours of plating the orgaoids on the MEA, however, this experimence may very based on the maturity of the organoids, the differentiation protocol used, and, in general, time on the MEA.

Recording activity

While some organoids may show activity shortly after plating, orgnoids typically require 1-2 weeks of culture on the MEA plates before showing robust activity. Begin monitoring after 24 hours to identify the optimal analysis window when peak activity and stability is observed.