3D-Printed Organ-on-a-Chip Models: Easily Customizable and Scalable Platforms for Study the Neurovascular Unit

Introduction Please briefly frame your work. The Neurovascular Unit (NVU) regulates cerebral blood flow and substance exchange with the Central Nervous System[1]. However, due to the inadequacy of current models, our understanding of cell-cell interactions remains limited. Organ-on-a-Chip (OoC) models are promising avenue to address the limitations of conventional preclinical models [2]. Materials and Methods Please describe your procedures. The custom-designed microfluidic platforms were created using Autodesk Fusion 360. Each platform layer was printed using Formlabs AmusedTerrier Form3B+ printer with BioMedClear V1 resin. Primary endothelial and neuronal cells were used as cellular models. We performed live imaging and immunofluorescence (IF) measurements to assess cellular morphology andfunctionality. Results Please describe your main results. (text can continue in the next page) Here we present two biocompatible 3D printed microfluidic OoC platforms for recapitulating the Neurovascular unit. The first OoC platform was developed in order to evaluate drugs kinetics targeting CNS. It consists of two chambers separated by a porous PET membrane. Each chamber has an inlet and outlet allowing connection to a peristaltic pump. The top and bottom of the OoC were sealed with two round glass coverslips for easy cell imaging. The biocompatibility of the chip was validated by seeding endothelial cells on the top and neuronal cells on the bottom of the platform, and then evaluated through live calcium imaging and IF measurements. Moreover, to evaluate endothelial barrier integrity, silver electrodes were incorporated into the chip to perform TEER measurement. The second platform developed is composed of a single round open chamber presenting an open channel shape and an inlet and outlet to connect the chip to a flow system. On the bottom of the chamber a PET membrane is attached by using bio resin. This OoC allows the possibility to culture three different cell types: endothelial cells on the top of the membrane, pericyte on the bottom of the membrane and neurons on the well plate in which the OoC is positioned. Results (continues from page 1) The biocompatibility of the chip has been validated by culturing endothelial cells on the membrane and performing IF as well as live imaging. The goal of this chip is to expose cells to a controllable flow in order to investigate how flow alterations can impact cell-cell interactions. To achieve this, the chip was designed to enable different flow conditions, allowing the study of the effects of shear stress on endothelial cells and their interactions with different NVU cell types. Discussion The most common material in use to fabricate OoC is PDMS, which is biocompatible, transparent, and gas permeable. However, its hydrophobic nature can lead to the absorption of hydrophilic substances. Furthermore, chip fabrication can take several days and requires specialized facilities [3]. To overcome this challenge, the OoCs presented here were fabricated using stereolithography 3D printing, a faster and more precise technique that does not require multi-step fabrication. Furthermore, the modularity of 3D printing facilitates the integration of different sensors, such as TEER electrodes, allowing in situ on-line analysis. Conclusions We have described the design, fabrication and application of easy assembled 3D printed OoCs. We demonstrated the modularity of our platforms and their ability to support different cell types growth and maturation. Additionally, we demonstrate that our platforms can be used with different techniques, including in-situ online analysis, which are crucial for physiological and drug development studies, particularly for assessing barrier permeability.