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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Dissection of Embryos and Dissociation of Tissue
- 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.
- Anesthetize a pregnant female rat after 18 days of development (E18) using 3% isoflurane. Then, sacrifice the animal by cervical dislocation.
- 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.
- Dissociate the tissue in 0.125% of Trypsin/Hank's solution containing 0.05% of DNAse for 18-20 min at 37 °C.
- 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.
- 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.
- 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.
- 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
- 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.
- Place the MEA devices into the incubator with humidified CO2 atmosphere (5%) at 37 °C.
- 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
- 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.
- 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.
- 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.
- 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.