Preliminary Reconfigurable Nerve-Artery Microphysiological System for 3D Cell Culture and Immunostaining

Tissue engineered microphysiological systems (MPS), also known as organs-on-chips, have recently emerged as a more patient-relevant and cost-effective alternative for disease modeling and drug discovery compared to traditional cell culture and animal models. Despite their increased functional relevance, most current MPS lack capabilities for analysis of individual components or layers through biological assays. Furthermore, current MPS rarely use 3D cell cultures to accurately mimic biological structures.<br/><br/>Here, we present a preliminary nerve-artery MPS. This novel MPS includes three removable layers: a three-chamber glass-bottom 3D cell culture layer which allows the contact of different cell types with GelPins (previously published by our lab) [1], with the middle 3D chamber in contact with a 2D cell culture layer through a semi-permeable membrane, and a removable media reservoir at the top of the assembly. The MPS also includes separate pipetting access ports to each chamber, which allows the specific addition and removal of cell culture medium, treatments, or reagents. This design allows specific endpoint analyses for each chamber, including immunostaining for the 3D hydrogel layer, and western blotting, flow cytometry, PCR, and immunostaining for the 2D layer. Furthermore, the glass bottom of the 3D hydrogel layer allows for high-resolution light and fluorescent microscopy of both the 2D and 3D layers, with the layers assembled or separated.<br/><br/>The manufacturing and assembly of the MPS include the use of stereolithography (SLA) 3D printing, laser cutting, and layer-by-layer assembly (as previously published by our lab) [2]. First, an SLA-printed clear resin piece is attached to a glass coverslip to form the bottom of the assembly. To form the three-chamber hydrogel cell culture layer, a Viton rubber gasket and modified GelPin [1] made of polyethylene terephthalate (PET) are laser cut and assembled with 3M 966 tape, and attached to the glass coverslip. Above the 3D cell culture layer is the 2D cell culture layer, which includes a laser-cut semi-permeable PET membrane. Above this 2D layer is the removable media reservoir layer, which includes a laser-cut Viton rubber gasket attached to a laser-cut polymethyl methacrylate (PMMA) piece. The 3D layer, 2D layer, and media reservoir are held together using flat head screws and nuts. Together, the three layers form a biomimetic structure to simultaneously model a nerve and artery: neurons can be cultured in the outer 3D hydrogel chambers to form the nerve component, and smooth muscle cells and endothelial cells can be cultured in the middle hydrogel chamber and the single-chamber 2D layer, respectively, to form the arterial component. To our knowledge, this design is the only nerve-artery model that includes 3D cell culture or an MPS.<br/><br/>As a proof of concept, F11 (mouse neuroblastoma - rat dorsal root ganglion) cells were encapsulated in 5% gelatin methacrylate (GelMA) hydrogel and seeded in the outer hydrogel chambers in the 3D layer of the MPS. The encapsulated cells were cultured in differentiation media for 2 weeks, fixed, immunostained for NeuN and actin, and imaged with an inverted fluorescent microscope. This study demonstrates that the MPS can be successfully used for longer-term 3D cell culture, immunostaining, and imaging experiments. Furthermore, the simple fabrication methods used to assemble the MPS can rapidly be modified to model other tissues or include other endpoint analyses, flow channels, or probes. Overall, this novel MPS has shown promising results for use as a nerve-artery model, and its applications and uses can be expanded with further research and development.<br/>_______________<br/>[1] Soucy et al., 2020. <i>Advanced Biosystems</i>. https://doi.org/10.1002/adbi.202000133<br/>[2] Hosic et al., 2020. <i>ACS Biomaterials Science & Engineering</i>. https://doi.org/10.1021/acsbiomaterials.0c00190