To avoid single-cell attachment to the glass bottom, the microfluidic device was incubated with 4% Pluronic F-127 (Sigma) in the incubator for 1 hour, rinsed with distilled water, and dried. Human iPSC-skeletal muscle cells were injected into the right compartment of the microfluidic device with a mixture of porcine skin type I collagen gel (Nitta Gelatin, 2.4 mg/ml) and 10% Matrigel (BD Biosciences) via gel injection port no. 1 (Fig. 1, A and B). A 3D muscle fiber bundle was formed within 24 hours, with both sides of the muscle attached to the pillar structures. The muscle fiber bundle was then cultured and differentiated using 2% horse serum and IGF-1. After 13 days of differentiation, predifferentiated MN spheroids (normal and ALS) were injected into the left compartment (Fig. 1B) via gel injection port no. 2 with collagen without Matrigel. For the cocultured MN spheroids and skeletal muscle fiber bundles in microfluidic devices, StemPro hESC medium supplemented with RA (50 μM), BDNF (10 ng/ml), and GDNF (10 ng/ml) was poured into the left medium reservoir (close to the MN spheroids), and DMEM, 10% horse serum, human recombinant IGF-1 (50 ng/ml), and 1% penicillin/streptomycin were poured into the right medium reservoir (close to the skeletal muscle fiber bundles). Two types of culture medium maintained segregating at least for 6 hours. To study the effects of drug application, bosutinib (100 μM) and rapamycin (200 nM) were applied to recover the ALS-derived motor unit muscle contraction force at day 4 with MN medium, and then muscle contraction was tested at day 7. The muscle apoptosis assay, PCR, and immunostaining of TDP-43 were performed at day 14.

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