3D printed organ-on-chip device and neural tissue engineering of spheroid and organoid cultures

Abstract

With the increased relevance of 3D culture techniques, such as spheroids and organoids, additional equipment and techniques are needed to accommodate growing tissue and measure physiological functions. Organ-on-Chip devices have increasingly been employed thanks to their perfusion and gradient capabilities, aiding in controlling the microenvironment surrounding the growing culture. Here, we describe an easy-to-use 3D printed neuron-on-chip device able to create sustainable diffusion gradients in a large hydrogel chamber. The device consists of a 3x3x11mm3 culture area, flanked by two parallel media channels. The nutrient/waste exchange created by the diffusion of the two media channels creates a microenvironment suitable for different cell types, including: cancer, embryonic, and fibroblasts. The drug delivery capabilities of this device seem to enhance pharmacokinetics, with an increased effect of perfused substances. Experiments with perfused paclitaxel showed a decrease in mobility and viability of the spheroids only achieved in controls with a 50% higher concentration of the drug. Taking advantage of the perfusion capabilities of the two media channels, a dual gradient can be formed within the hydrogel creating a complex microenvironment within the culture chamber. This versatile, proof-of-concept device holds great promise by enabling a wide range of experiments with 3D cultures. Additionally, we describe a cost-effective method of creating embryoid bodies (EB) from human embryonic stem cells (ESC). Our method combines covering the concave bottom well-plates with a commercially available anti-adherence solution and force-aggregating the cells through centrifugation. This method can be performed minutes before EB formation, instead of days of plate preparation using other ad hoc methods. More importantly, the use of this method, with either U-bottom or V-bottom well plate, provides reproducible EB’s with low variability in diameters and differentiation iwhen comparable to the commercially available plates. Lastly, we further differentiate our EBs into hippocampal organoids to develop a physiologically relevant model for neurodegenerative diseases, such as Alzheimer’s. Our organoids express markers for all the hippocampal regions including the HuB for CA1-CA3 and the PROX1 marker for the dentate gyrus. Voltage sensitive dyes allowed for a minimally invasive method of studying the electrophysiological activity of the neurons, which revealed mature synchronization by day in vitro (DIV) 60. Exposure to Amyloid-β (Aβ) showed a direct correlation between concentration and neural damage, demonstrating the potential for future disease modeling. Overall, our organoid differentiation suggest a fully formed hippocampal organoid able to assist in uncovering hippocampal developmental data and disease model.