(M-494) Microfluidic Device for Immobilizing, Perfusing, and Functionally Connecting Cortical Organoids in Long-term Culture

Introduction:: Cortical organoids derived from human induced pluripotent stem cells are fueling many new discoveries in human brain development. Cortical organoids are especially powerful for modeling neurodevelopmental disorders, such as autism, which are uniquely human conditions associated with complex phenotypes and misexpression of multiple genes, warranting patient-specific models. Due to their large size and metabolic rate, cortical organoids are typically cultured in spinner flasks, which are relatively high throughput but also cause collisions between organoids, contributing to variability in organoid size and morphology. Organoids cultured in spinner flasks also cannot be continuously monitored via assays that require the organoids to be immobilized, such as imaging or electrophysiology. Additionally, organoids cultured in spinner flasks cannot extend neurites due to the lack of scaffolding, precluding the generation of multi-organoid systems for modeling communication between different regions of the brain in healthy and neuropathic backgrounds. Given these limitations, our goal is to engineer a culture device that: (1) immobilizes organoids while simultaneously supplying them with continuous media perfusion to support viability and differentiation, and (2) induces axonal connections between organoids representing different brain regions to investigate long-range connections and synaptic formation or dysfunction. To achieve this, we are designing and testing a multi-chamber microfluidic device for housing two organoids and inducing axonal connections between them by providing a hydrogel scaffold.

Materials and Methods:: Self-gasketing microfluidic devices were designed in 3D CAD software. Negative templates were fabricated in photocrosslinkable resin using a dynamic light processing (DLP) 3D printer. Final components of the devices were cast in polydimethylsiloxane (PDMS) using soft lithography. 57-day old cortical organoids were cultured for 30 days in the microfluidic device, spinner flask (positive control), or static culture (negative control). In the device, fresh media was constantly perfused at a rate of 1.25 mL/hour. Organoids were cryosectioned and stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), where fragmented DNA is tagged with a fluorescent marker to quantify apoptosis. Proliferation was quantified by staining sections with DAPI to mark nuclei and antibodies against phospho-histone H3 (pHH3) to mark exposed histones of mitotic cells. Differentiation was quantified by staining for Sox2, an early neural progenitor marker; Tbr2, an intermediate progenitor marker; and Satb2 and Ctip2, markers for upper and lower cortical neurons, respectively.

Results, Conclusions, and Discussions:: We designed and fabricated a microfluidic device to immobilize two organoids within rings of pillars to allow fresh media and waste to flow around the organoids (Fig 1A,B). Within the device, the two organoids are connected by a channel designed to be filled with a hydrogel scaffold to support axonal connections. 57-day old organoids cultured in microfluidic devices and spinner flasks for 30 days grew larger than those in static culture (1C,D). Furthermore, TUNEL staining revealed that organoids cultured in microfluidic devices had comparable sizes of apoptotic cores to those cultured in spinner flasks, while static culture demonstrated increased apoptosis and more wide-spread cell death (Fig. 1E,F). Phosphorylated histone 3 (pHH3) staining did not reveal a difference in mitotic activity across conditions (Fig 1G). Differentiation markers revealed higher levels of early progenitors (Sox2) in microfluidic devices and static culture organoids than spinner flasks, but no distinguishable difference for intermediate progenitors (Tbr2) or deep-layer neurons (Ctip2) (Fig 1H,I,K) Organoids cultured in microfluidic devices also expressed higher percentages of markers for upper-layer neurons (Satb2) (Fig 1J). To understand the phenotypic impact of these different expression patterns , we will measure spontaneous calcium activity as a marker for functional activity.

In summary, our fluidic device was successful for culturing immobilized pairs of organoids for 30 days and achieving viability comparable to traditional spinner flask culture. In future studies, we plan to integrate a granular hydrogel for recruiting and stabilizing axonal connections between the cortical organoids while also enabling higher perfusion than bulk hydrogels. Collectively, these approaches will lead to new models for understanding human neurodevelopment and disease from the molecular to the tissue level.