To begin, generate virus labeled assembloids using fate-specific brain organoids. To prepare reagents for MEA surface pre-treatment, dissolve 0.5 grams of detergent enzyme powder in 50 milliliters of deionized water. Vortex the mixture thoroughly until the powder is fully dissolved and sterilize the solution using a sterile tube top vacuum filter inside the biosafety cabinet.
Dilute 500 microliters of the 7%polyethylenimine, or PEI stock solution with 49.5 milliliters of 1X borate buffer to prepare a 0.07%PEI solution. To sterilize the solution, filter it through a 0.22 micrometer filter unit inside the biosafety cabinet. Next, dispense one milliliter of the 1%detergent enzyme solution to each well of a sterile MEA plate and incubate it at 37 degrees Celsius for two hours.
Then remove the detergent enzyme solution from each well and wash the wells five times with 1.5 milliliters of sterile deionized water per well. Fill each well with one milliliter of culture media for preconditioning. Place the MEA plates back into a 37 degrees Celsius, 5%carbon dioxide incubator for two days.
After incubation, remove the culture media from the wells and add 50 microliters of 0.07%PEI solution to cover the MEA electrodes for primary coating. Incubate the plate at 37 degrees Celsius for one hour. Then aspirate the PEI solution completely from each well.
Wash each well three times with one milliliter of sterile deionized water. After aspirating the water, dry the plates at room temperature for 15 minutes in the biosafety cabinet with the lid open. Now cover the electrode area in each well with 50 microliters of one to 20 volume to volume basement membrane matrix diluted in culture media for secondary coating.
Place the MEA plates back into the 37 degrees Celsius incubator overnight. The next day, aspirate the diluted matrix solution, leaving a thin layer on the surface. If a thick layer forms, spray the electrode contact area forcefully with culture media using a P1000 pipette tip.
Then using a P1000 tip with a wide cut, collect an assembloid from the dish and carefully place it on top of the electrodes in a well while transferring as little media as possible. Place the plate under a dissection microscope in a laminar flow hood and using a sterile P 20 tip, carefully push the assembloid to the desired location. Use a P 20 tip to remove excess liquid around the assembloid.
After incubation, remove the plate from the incubator. Then observing under a dissection microscope, apply two to three small drops of one to 50 basement membrane matrix diluted in culture media on top of the assembloids, and return the plate to the incubator for another 30 minutes. Afterward, carefully add three drops of culture media close to the assembloids using a dissection microscope.
The next day, check if the assembloids have settled and stabilized under a dissection microscope. Add 750 microliters of culture media to bring the total volume to one milliliter per well and leave the plate undisturbed for at least two days in the incubator. Every week, carefully remove 500 microliters of culture media from each well, and introduce fresh 700 microliters of neurophysiological basal media to each well.
Increased network bursting duration was observed on day 92 compared to day 78, likely due to neuron maturation. The cellular and network activity slowly faded by day 125.
