This protocol facilitates investigation of the mechanisms underlying the development and the disease of axon bundles using motor nerve organoid derived from the human iPS cells outside of the bodies. By simply culturing neuronal spheroid and custom-made microculture chips, unidirectional axon bundles can be spontaneously generated and be used for various downstream experiments. Motor nerve organoid can facilitate the screening and testing of the drugs for motor neuron diseases, including LS, by providing a more physiological model that are previous in vitro systems.
In a fume hood, and wearing the appropriate PPE, dispense three milliliters of SU-8 2100 onto an acetone cleaned silicon wafer and place the wafer in the center of a spin coater. Fix the wafer by vacuum and spin the wafer at 500 revolutions per minute for 10 seconds to coat the SU-8 uniformly across the wafer surface, then spin the wafer at 1, 500 revolutions per minute for 30 seconds with an acceleration of 300 revolutions per second to obtain a 150 micron thick layer of photoresist on the wafer. After soft baking, set the photomask to the mask aligner and expose the wafer to ultraviolet light for 60 seconds.
After baking on a hot plate, develop the wafer for 10 to 20 minutes in photoresist developer with agitation on an orbital shaker, changing the developing solution once during the process. To prepare the bottom layer of the chip for tissue culture, pour freshly prepared PDMS onto the wafer to the desired thickness and degas the mixture in a vacuum chamber. Cure the PDMS in an oven for at least three hours at 60 degrees Celsius.
After cooling, use a blade to cut the cured PDMS from the wafer, then use uncured PDMS with baking to bond a medium reservoir to the PDMS bottom layer to obtain the assembled PDMS tissue culture chip. To prepare the PDMS chip for motor nerve organoid formation, sterilize the chip and a 76 by 52 millimeter microscope glass in a Petri dish containing 70%ethanol for at least one hour. At the end of the incubation, place the chip on the wet microscope glass.
After overnight drying the chip should adhere to the glass. Place a 30 microliter droplet of diluted basement membrane matrix in DMEM/F12 on one side of the inlet of the channel and aspirate the solution from the other side of the inlet to coat the surface of the microchannel with the matrix. Then incubate the PDMS chip with the coating solution in a Petri dish for one hour at room temperature.
To induce 3D iPS cell differentiation into motor neurons, seed four times 10 to the fourth iPS cells per well into the appropriate number of wells of a 96-well U-bottom plate in 100 microliters of feeder and serum-free cell culture medium supplemented with 10 micromolar Y-27632. The next day, replace the supernatant in each well with 100 microliters of KSR medium supplemented with 10 micromolar SB431542 and 100 nanomolar LDN-193189. On days two and three, replace the supernatants with 100 microliters of KSR medium supplemented with 10 micromolar SB431542, 100 nanomolar LDN-193189, five micromolar DAPT, five micromolar SU5402, one micromolar retinoic acid, and one micromolar smoothened agonist.
On days four and five, replace the supernatants with a mixture of 75%of KSR medium and 25%of N2 medium supplemented with 10 micromolar SB431542, 100 nanomolar LDN193189, five micromolar DAPT, five micromolar SU5402, one micromolar retinoic acid, and one micromolar smoothened agonists. On days six and seven, replace the supernatants with a mixture of 50%of KSR medium and 50%of N2 medium supplemented with five micromolar DAPT, five micromolar SU5402, one micromolar retinoic acid, and one micromolar smoothened agonist. On days eight and nine, replace the supernatants with a mixture of 25%of KSR medium and 75%of N2 medium supplemented five micromolar DAPT, five micromolar SU5402, one micromolar retinoic acid, and one micromolar smoothened agonist.
On days 10 and 11, replace the supernatants with N2 medium supplemented with five micromolar DAPT, five micromolar SU5402, one micromolar retinoic acid, and one micromolar smoothened agonist. On day 12, replace the supernatants in each well with 100 microliters of maturation medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor per well. To induce motor nerve organoid formation, replace the coating solution in the PDMS chip with 150 microliters of maturation medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and use a wide-bore micropipet tip to gently add motor neurons spheroids from one well of the 96-well plate into the inlet of the microchannel of the chip.
Place a small reservoir of sterile water near the tissue culture chip in the dish to prevent medium evaporation and place the dish in a 37 degrees Celsius and 5%carbon dioxide incubator. Every two to three days, replace half of the exhausted culture medium from the center of the medium reservoir with fresh maturation medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor. Axons will grow from the motor neuron spheroid into the channel, spontaneously assembling into a single bundle over a period of two to three weeks to form a motor nerve organoid.
Motor neurons differentiate within 12 to 14 days in 3D differentiation procedures. Importantly, more than 60%of the cells express the motor neuron marker HB9 during the differentiation and approximately 80%of the cells in the motor neuron spheroid are SMI32 positive. With the microchannel serving as physical guides, axons elongate from the motor neuron spheroid to form a bundled by axo-axonal interaction within 24 hours of introduction to the spheroid.
The axons reached the center of the microchannel within the next three to four days, extending to the other end of the microchannel after an additional 10 days. Motor nerve organoids can be collected from the chip for biological analysis by detaching the PDMS from the microscope glass. The axon bundles and cell bodies can then be dissected and isolated under a microscope using a surgical knife or tweezers.
In axon bundles of motor nerve organoids, dendritic marker proteins are not detected by Western blotting. It is important to make sure that the spheroids are completely dropped to the bottom within the culture chip. We showed examination of the sites and the bottom can help determine the location of the spheroid in the chip.
The isolated axon bundles can be examined for various additional methods. A large quantity of axons can be obtained and used for biochemical assays that requires substantial materials.
