Developing Human iPSC-Derived Motor Nerve Organoids Using a Microfluidic Chip
Trascrizione
To induce 3D iPS cell differentiation into motor neurons, seed 4 x 104 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 Y27632.
The next day, replace the supernatant in each well with 100 microliters of KSR medium supplemented with 10 micromolar SB-431542 and 100 nanomolar LDN-193189. On days 2 and 3, replace the supernatants with 100 microliters of KSR medium supplemented with 10 micromolar SB-431542, 100 nanomolar LDN-193189, 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and one micromolar smoothened agonist. On days 4 and 5, replace the supernatants with a mixture of 75% of KSR medium and 25% of N2 medium, supplemented with 10 micromolar SP-431542, 100 nanomolar LDN-193189, 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist.
On days 6 and 7, replace the supernatants with a mixture of 50% of KSR medium and 50% of N2 medium, supplemented with 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist. On days 8 and 9, replace the supernatants with a mixture of 25% of KSR medium and 75% of N2 medium, supplemented with 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 micromolar smoothened agonist. On days 10 and 11, replace the supernatants with N2 medium supplemented with 5 micromolar DAPT, 5 micromolar SU-5402, 1 micromolar retinoic acid, and 1 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 micropipette tip to gently add motor neuron 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.
