This protocol describes the fabrication of micro-tissue engineered neural networks or micro-TENNs, which are miniature three-dimensional constructs comprised of long aligned axonal tracts spanning discrete neuronal populations within the lumen of a tubular hydrogel. Micro-TENNs replicate the general systems level anatomy of the brain connectome, specifically, functionally similar groups of neurons connected by long axonal tracts. Because they are pre-grown to emulate the cytoarchitecture of brain pathways, micro-TENNs may be applied for the targeted reconstruction of neural circuitry lost due to trauma or neurodegenerative disease and as biofidelic models for neurobiological studies.
One of the most challenging aspects of this protocol is working with the micron scale form factor, which is ultimately necessary to allow for a minimally invasive delivery into the brain. Begin by manually breaking glass capillary tubes into 2 to 2.5-centimeter fragments, and insert one acupuncture needle into each fragment. Transfer one milliliter of three percent agarose in DPBS to the surface of an empty petri dish.
Holding the capillary tube in the introduced needle, place one end of the tube in contact with the liquid agarose pool to fill it by capillary action. When the liquid ceases to rise, remove the capillary tube from the pool, and place it horizontally on the surface of a petri dish. Next, place the thumb and index finger on either side of the tube and grasp tightly.
Use the other hand to quickly pull the needle out while the thumb and index finger prevent the micro-column itself from sliding out of the tube. Then insert a 30-gauge needle into the capillary tube to slowly push the micro-column out into a dish containing DPBS. Carefully transfer one micro-column from DPBS to an empty dish using fine forceps.
Add 10 microliters of DPBS to the top of the micro-column with a micropipetted to prevent drying. While working under a stereomicroscope, trip the micro-column with a micro scalpel to shorten it to the desired length. Then use fine forceps to transfer the trimmed micro-column to a different petri dish containing DBPS.
Sterilize the micro-columns in the DPBS-containing petri dishes under ultraviolet light for 30 minutes. Use a drill bit to puncture 16 holes as a 4-centimeter 4 by 4 array with a 1-centimeter separation into the lid of a 10-centimeter petri dish. Inside a chemical fume hood, use the bottom piece of a petri dish to weigh 27 grams of PDMS and three grams of curing agent.
Stir with a micro spatula to distribute the agent evenly. Then cover the PDMS curing agent with the punctured lid. Connect one end of a hose to the vacuum port of the fume hood, and insert the other end into the stem of a suitably sized funnel.
Place a 1-milliliter pipette bulb on a 1-milliliter tip into the hose, and pull the hose upwards until the bulb seals the hose into the stem of the funnel. Next, place the funnel on the punctured dish lid and secure the hose with an available sturdy support. Open the vacuum valve for five minutes to suction and bring the air bubbles to the surface.
After five minutes, close the valve, remove the funnel, and hit the petri dish against the surface of the fume hood a few times to burst any remaining air bubbles. Place a pyramidal well 3D printed mold into each of the wells in a 12-well culture plate with the pyramids pointing up. Then pour the PDMS curing agent on top of the molds until each well of the plate is filled.
Transfer the covered plate to an oven for one hour at 60 degrees Celsius to dry. After sterilizing the PDMS arrays by autoclaving, and working in a biosafety cabinet, insert one micro-well array into each well of a sterile 12-well plate. To form the neuronal aggregates, use a micropipetted to add 12 microliters of cell suspension into each micro-well of the PDMS array.
Centrifuge the plate at 200 times g for five minutes to force the aggregation of cells at the bottom of the micro-wells. Then carefully add approximately two milliliters of culture medium to the top of each PDMS array to cover all the seeded microwells. Incubate the plate for 12 to 24 hours at 37 degrees Celsius in 5%carbon dioxide.
To begin CEM core fabrication, mix Type 1 collagen, laminin, and culture medium in a micro centrifuge tube and adjust the pH to 7.2 to 7.4. Then place the tube containing CEM on ice. Use sterile forceps to transfer the micro-columns to empty 35 or 60-millimeter petri dishes.
Then, while working under a stereomicroscope, use a 10-microliter tip attached to a 1000-microliter tip to extract residual DPBS and air bubbles from the lumen. Quickly draw up four to five microliters of CEM in a micropipetted, then place the tip of the micropipetted at one end of a micro-column, and discharge enough CEM to fill the lumen. To prevent dehydration, add two microliters of CEM around each micro-column.
Incubate the hydrogel CEM micro-columns in petri dishes at 37 degrees Celsius in 5%carbon dioxide for 15 minutes. 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 micropipetted 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 bi-directional 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 in 5%carbon dioxide for 45 minutes to allow the aggregates to adhere to the CEM. 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 in 5%carbon dioxide for long-term culture. These phase contrast images show unidirectional 2-milliliter long micro-TENNs up to eight days in-vitro.
In these images, notice the neuronal aggregates at the extremes of the micro-column while the aligned axonal tracts project across the lumen. As seen here, axonal tracts extend almost the entire length of the micro-column by five days in-vitro. For 5-millimeter bi-directional micro-TENNs, axonal tracts populate the length of the micro-column at 5 days in-vitro, and this architecture is maintained at least until 10 days in-vitro.
The following two images can help troubleshoot problems with the procedure. This image shows a completely dehydrated dried hydrogel micro-column as a result of the complete removal and/or evaporation of DPBS, CEM, or culture medium during fabrication. This image shows a micro-column with the lumen lining the outer wall due to the lack of concentric alignment of the acupuncture needle with the capillary tube.
Finally, this confocal image shows a bi-directional micro-TENN at 28 days in-vitro, stained for cell nuclei with Hoechst in blue, and with axons labeled with tudge one in red, and pre-synaptic boutons labeled with synapsin 1 in green. Cell migration from the aggregates along the axonal tracts is seen in the presence of pre-synaptic terminals as suggested. Micro-TENNs are self-contained, anatomically inspired constructs that emulate key aspects of connectome cytoarchitecture;therefore, micro-TENNs may provide a means to replace lost neural pathways upon implantation into the brain.
Crucial components of this method include the tubular biomaterial encasement scheme, which allows for longitudinal axonal growth, and the aggregate method, which ensures obtaining the proper cytoarchitecture of cell bodies restricted to the ends of axonal tracts along the interior. Following mastery of these methods, the principles of this technique may be applied to build micro-TENNs with other cell phenotypes and biomaterial components, according to the specific application.
