NeuroMeshoids: a scalable 3D nylon mesh-based platform for hiPSC-derived neural aggregates and projection-bearing cell cultures

Conventional 2D neural monolayer cultures exhibit limited structural complexity and functional connectivity. 3D neural organoid models overcome some of these issues but can present practical challenges related to mechanical fragility during handling, reliance on hydrogel encapsulation, and storage. To complement existing approaches, we developed NeuroMeshoids, a 3D culture platform that utilizes nylon mesh scaffolds to support the differentiation, maturation, and long-term maintenance of human induced pluripotent stem cell (hiPSC)-derived neural aggregates. hiPSCs were differentiated into neural stem cells using dual SMAD inhibition and matured into mixed neural cultures containing neurons and cells expressing glial/astrocyte-associated markers using a growth factor cocktail. After 14 days, aggregated cultures were replated onto Geltrex-coated nylon mesh (40 and 70 μm pore sizes) to yield NeuroMeshoids. Morphological and functional characterization included immunofluorescence microscopy, scanning electron microscopy, cell viability assessment, qPCR, calcium imaging, MEA recording, and skeleton-based neurite analysis. NeuroMeshoids were compared with 2D cultures using gene expression, synaptic density, and spontaneous neuronal activity. Platform applicability beyond hiPSC-derived neural aggregates was evaluated using glutamatergic neurons, neural spheroids, fibroblasts, and hiPSC-derived myotubes. NeuroMeshoids exhibited robust attachment, extensive neurite outgrowth, and long-term structural stability with continued neurite elaboration through 210 days in culture. Confocal microscopy and SEM revealed complex, fasciculated neurite networks spanning multiple z-planes and extending over long distances. qPCR showed upregulation of neural ( MAPT , MAP2 , TUBB3 ) and synaptic ( SYN1 , DLG4 , SLC17A6 ) markers in NeuroMeshoids relative to 2D cultures. Image-based analyses indicated progressive increases in field-level neurite-network measures during long-term NeuroMeshoid culture and increased synaptic puncta density relative to 2D cultures. Colocalization of presynaptic and postsynaptic markers demonstrated robust synaptic-marker organization. Calcium imaging revealed spontaneous activity in numerous neurons, and HD-MEA recordings confirmed genuine neuronal action potentials, with biphasic waveforms and refractory-compliant inter-spike intervals, organized into recurrent bursts across multiple NeuroMeshoids. Functional coordination between electrodes decayed steeply with inter-electrode distance and was confined to the cellular scale (approximately 35–50 μm), reproducibly across three hiPSC lines, with limited coordination detected at longer network scales at the day-28 timepoint. NeuroMeshoids also exhibited preservation of morphology and function after recovery from cryopreservation. In addition, the platform supported robust attachment of glutamatergic neurons, neural spheroids, fibroblasts, and functional hiPSC-derived myotubes, suggesting that beyond neural aggregates, the mesh-based system may be adaptable to diverse ‘Meshoid’ culture formats. NeuroMeshoids represent a robust and scalable substrate-based 3D culture platform supporting extended biocompatibility, long-term structural stability and neurite elaboration, and synaptic marker organization in hiPSC-derived neural aggregates. The mechanically stable nylon mesh architecture facilitates physical manipulation, advanced imaging, functional analyses, and reliable cryopreservation-recovery workflows. While primarily developed for neural cultures, the platform’s compatibility with additional projection-bearing and structurally complex cell types suggests broader utility for “off-the-shelf” culture in research and high-throughput screening applications.