This article describes a method for creating micro-tissue engineered neural networks (micro-TENNs) using neuronal cell aggregates. The process involves seeding neuronal aggregates into micro-columns and incubating them to promote axonal growth and network formation.
Begin with a dish containing a micro-column. The micro-column has a hydrogel outer shell and an extracellular matrix, or ECM core.
Add drops of culture medium to the same dish.
Transfer the neuronal cell aggregates into the dish, then place them into one of the medium droplets.
Observe under a stereomicroscope and insert cell aggregates at both ends of the micro-column.
Then, transfer the seeded micro-column into the medium for cellular viability.
Incubate to allow the cell attachment to the ECM.
Using the stereomicroscope, confirm cell attachment. Then, add culture medium to cover the dish and incubate.
The attached neurons begin to extend their axons through the ECM.
Regularly replace the half-medium with a fresh medium to maintain the nutrient levels.
Over time, the axons grow and span the entire length of the micro-column, forming neural networks.
The developed micro-tissue engineered neural network is ready to reconstruct the damaged neural circuitry.
Proceed to cell seeding immediately after incubation. Transfer approximately 10 to 20 microliters of culture medium to two free areas in the Petri dishes holding the micro columns. Use a micropipette to collect the neuronal aggregates individually, and transfer them to the Petri dish containing the constructs. Move the aggregates with forceps to one of the small pools of culture medium to preserve cell health.
While observing under a stereomicroscope, use forceps to insert an aggregate at one end of the micro columns for unidirectional micro-TENNs or at each end for bidirectional architecture. Then, move the seeded micro column to the other small pool of culture medium to avoid dehydration and to preserve aggregate health. When all micro columns have been loaded, incubate at 37 degrees Celsius and 5% carbon dioxide for 45 minutes to allow the aggregates to adhere to the ECM.
After incubation, verify that the aggregates remain at the ends of the micro columns, using the stereomicroscope. Lastly, carefully flood the Petri dishes containing the micro-TENNs with culture medium using a pipette. Place the dishes in an incubator at 37 degrees Celsius and 5% carbon dioxide for long-term culture.
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