Fig 1: Development of a 3D biomimetic PN platform. (A) Illustration of the biofabrication process, depicting its two main phases. The first phase (left) is the formation of nociceptor neurospheres via iPSCs differentiation within an agarose mold containing 400 µm microwells. The whole process takes 18 days from the day cells are seeded (at a density of 200 cells per well) until the spheres are ready to be harvested. The differentiation process itself takes 14 days. On day 11, primary SCs are seeded and cultured on an aligned microfibrous scaffold (at a density of 100 × 103 cells per scaffold). In the second phase (right), the neurospheres are harvested and placed on the SC-seeded scaffold (one neurosphere per scaffold). A fibrin hydrogel embedding is also added, following neurosphere attachment. The co-culture is maintained for up to 21 days to allow compact myelination to occur. (B) At 7 days of co-culture, vast, 3D, and aligned neurite outgrowth was observed, illustrated by the 3D reconstruction of a micrograph showing immunostaining to ßIII-tubulin (red). (C) At 21 days of co-culture, neurites maintain their anisotropy and show signals of myelination as evidenced by the 3D reconstruction of a micrograph, correlating ßIII-tubulin+ (red) with MBP+ segments (green). For (C) and (D), the scale bars represent 100 µm. (D) TEM micrograph of the platform cross section showing the presence of multiple, compact myelin rings surrounding the neurites (average thickness is 89.1 ± 17.6 nm from 11 measurements). The scale bar represents 100 nm.