Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Dissection of Embryos and Dissociation of Tissue

1. House adult female rats (200-250 g) at a constant temperature (22 ± 1 °C) and relative humidity (50%) under a regular light–dark schedule (light-on 7 AM–7 PM) in the animal facility. Ensure that food and water are freely available.
2. Anesthetize a pregnant female rat after 18 days of development (E18) using 3% isoflurane. Then, sacrifice the animal by cervical dislocation.
3. Remove hippocampi from each rat embryo and place them into ice cold Hank's balanced salt solution without Ca²⁺ and Mg²⁺. At day 18 of development, hippocampus and cortex tissues are very soft and do not need to be cut into small pieces. Further details can be found.
4. Dissociate the tissue in 0.125% of Trypsin/Hank's solution containing 0.05% of DNAse for 18-20 min at 37 °C.
5. Remove the supernatant solution with a Pasteur pipette and stop the enzymatic digestion by adding medium with 10% fetal bovine serum (FBS) for 5 min.
6. Remove the medium with FBS and wash once with the medium with its supplement, 1% L-glutamine, gentamicin 10 µg/ml. Remove again the medium with a Pasteur pipette and refill it again with a small amount (500 µl) of growth medium with its supplement, 1% L-glutamine, gentamicin 10 µg/ml.
7. Dissociate the tissue pellet mechanically with a narrow Pasteur pipette until a milky suspension of cells is apparent. It is not necessary to centrifuge the cell suspension.
8. Dilute the small volume of cell suspension with the growth medium to obtain a final volume of 2.0 ml. Count the obtained cellular concentration with a hemocytometer chamber. Dilute this concentration at 1:5 in order to obtain the desired cell concentration of 600-700 cells/µl.

2. Cell Plating

1. Plate cells at a density of about 2,000 cells/mm² onto the active area of the MEA, defined by the PDMS constraint to create a 2D neuronal network.
   NOTE: Each hippocampus contains about 5 x 10⁵ cells, (1 x 10⁶ for a single embryo). Dissect 6 embryos to get a total amount of 6 x 10⁶ cells in 2 ml, and an estimated concentration of 3,000 cells/µl. Dilute this concentration at 1:5 in order to obtain the desired cell concentration and plate about 600-700 cells/µl. If the MEA area delimited by the PDMS constraint is about 7.065 mm², and the total number of plated cells 600 cells/µl x 24 µl = 14,400 cells, the final density of 2,038 cells/mm².
2. Place the MEA devices into the incubator with humidified CO₂ atmosphere (5%) at 37 °C.
3. Distribute 160 µl of the suspension with a cell concentration of 600-700 cells/µl (about 100,000 cells) onto the surface of the microbeads monolayer positioned inside the multiwell plates to complete the preliminary step for constructing three-dimensional culture. Put the multiwell plates in the incubator with humidified CO₂ atmosphere (5%) at 37 °C.

3. 3D Neuronal Network Construction

1. 6-8 hr after the plating, transfer the suspension (microbeads with neurons) from the multiwell plates very carefully inside the area delimited by the PDMS constraint by using a pipette set for a volume of about 30-40 µl. After each transfer, wait for about half a minute, to allow the microbeads to self-assemble in a hexagonal compact structure.
2. Once all the layers are deposited and spontaneously assembled, refill with a large drop of about 300 µl of medium the top of the area delimited by the PDMS constraint.
3. Put the 3D structure coupled to the MEA in incubator (T = 37.0 °C, CO₂ = 5%) for 48 hr before adding a final volume (about 1 ml) of growth medium culture with its supplement.
   NOTE: Consider the total number of beads on the multiwell plates with membrane insert (30,000) and the number of beads on a single layer onto the MEA device (6,000). The resulting 3D structure is composed of 5 layers of microbeads and cells. Considering that the 3D neuronal network is not geometrically perfect, the resulting 3D structure could comprise 5-8 layers.
4. The day after plating, carefully add the final volume of the medium (about 900 µl) inside the MEA ring. Maintain the 3D cultures in a humidified CO₂ atmosphere (5%) at 37 °C for 4-5 weeks. Replace half of the medium once a week.