Developing Human iPSC-Derived Motor Nerve Organoids Using a Microfluidic Chip

Take human induced pluripotent stem cells, or iPSCs, in a nutrient-rich stem cell medium with Rho-kinase inhibitors.

The inhibitors enter the cells and block Rho-kinase enzymes, preventing apoptotic cell death.

The cells utilize the nutrients to proliferate and spontaneously aggregate to form a three-dimensional cell cluster called a spheroid.

Gradually, replace the medium with a neural induction medium. Signaling modulators from the medium drive the differentiation of stem cells into neural cells.

Replace with a growth-factor-rich neural medium to differentiate the spheroid cells into motor neurons and form a motor neuron spheroid or MNS.

Transfer these MNS to a polymer-coated microfluidic chip containing a maturation medium.

The nerve growth factors in the medium stimulate the axons or longer projections of neurons to grow from the spheroid and extend through the microchannel of the chip.

This forms a motor nerve organoid, a three-dimensional model of motor nerve tissue.

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