Towards Modeling Human Brain Connectivity with a Multi-Organoid-on-a-Chip

Introduction: Understanding the formation of long-range neural connections is key to better characterizing neurodevelopmental disorders such as autism, intellectual disability, and attention-deficit hyperactivity disorder. These are a large set of behaviorally-defined syndromes for which crosstalk between brain regions is implicated, but the mechanisms for this crosstalk have not yet been established.1 Human pluripotent stem cell (hPSC)-derived cortical organoids recapitulate aspects of human fetal brain development on the microscale.2,3 For example, after 30 days of differentiation, hPSC-derived cortical organoids develop neuronal progenitor populations that give rise to spontaneously-active neurons by 60 days.4 However, individual organoids are limited in their capacity to replicate regional connections that are characteristic of later neurodevelopmental architecture, impairing their use for understanding long-range connections implicated in neurodevelopmental disorders. To induce axonal projections from cortical organoids, they have been embedded into biomaterial hydrogels.5,6 However, connected organoids must be maintained in static culture to preserve their delicate architecture, which impairs growth and differentiation. To overcome this, our goal is to develop a microfluidic device that can both maintain the viability and function of human brain organoids while in static culture and induce long-range neural connections between organoids. To further this goal, we have first engineered a microfluidic device for housing immobilized organoids for up to 60 days that maintains their viability and supports the development of spontaneously active neurons.

Materials and

Methods: To culture and ultimately connect organoids, we built a system to constantly perfuse pairs of immobilized organoids with fresh media. We designed a polydimethylsiloxane (PDMS) culture chamber that consists of two rings of pillars to house the organoids, bridged by a central sub-chamber. The central sub-chamber is designed to hold a hydrogel that would provide a scaffold for axon extension between the organoids. (Fig. 1A). The pillars allow for distributed flow around the tissue to sustain the organoids (Fig. 1B). We fabricated the device using cleanroom-free digital light processing (DLP) 3D-printed molds and PDMS soft lithography. The device was connected to a syringe pump and ran at a low flow rate of 1.25 mL/hr to conserve media. Two-month-old human pluripotent stem cell-derived cortical organoids were differentiated and then cultured for an additional two months in the microfluidic device and compared to no flow (static) conditions (negative control) and spinner flask bioreactors (positive control). To investigate organoid survival in the devices, the size of the necrotic core was measured with terminal dUTP-ase nick-end labeling (TUNEL) staining on immunohistological sections. Immunohistological staining of SOX2+ neural progenitors and SATB2+ upper-layer neurons was used to characterize the differentiation patterns. To assess functional capabilities of the microfluidics-cultured organoids, spontaneous calcium handling was characterized by transfecting organoids with an adeno-associated virus carrying a fluorescent calcium labeling cassette (GCaMP). As a proof of concept, pairs of organoids were adjoined with Matrigel in the central sub-chamber and cultured for two months.

Results, Conclusions, and Discussions: We successfully maintained immobilized human cortical organoids in microfluidic culture for 60 days. Brightfield imaging of organoids revealed that 1 month in microfluidic culture led to increased organoid size relative to static conditions. There were no significant differences in size between the microfluidic device and the spinner flask bioreactor (Fig. 1C). TUNEL staining revealed that organoids cultured in microfluidic devices had comparable sizes of apoptotic cores to those cultured in spinner flasks, while static culture increased apoptosis and resulted in wide-spread cell death (Fig. 1D,E). Expression of differentiation markers SOX2+ and SATB2+ demonstrated that organoids in microfluidic devices were progressing towards neuronal states at 1 month in culture (Fig. 1F,G) At 2 months, fluorescent imaging of spontaneous calcium activity revealed that neuronal populations in the microfluidic device demonstrated a higher percent of neurons firing relative to static conditions and a comparable percent of neurons firing to organoids grown in spinner flask culture. (Fig 1H) Cortical organoids formed cellular connections after 6 weeks through cell migration into the central sub-chamber. (Fig.1I)

In summary, we designed, fabricated, and tested a microfluidic device for long-term culture of immobilized cortical organoids. In our next experiments, we plan to enhance the imaging capabilities of this device, expand into higher-throughput experiments, and engineer a scaffold for targeted inter-organoid axonal connections. Our future work will leverage this device to investigate the mechanisms of long-range connection formation in hPSC-derived connected organoids. Multiregional brain organoid systems such as this can provide a platform for screening new molecular therapies or neuromodulation treatments and provide new insights into brain pathophysiology.

References:
1. Urenda, et al. (2023) Biol. Psychiatry
2. Gordon, et al. (2021) Nat. Neurosci
3. Trujillo, et al. (2019) Cell Stem Cell
4. Dixon, et al. (2023) Mol. Psychiatry
5. Kirihara, et al. (2019) iScience
6. O’Grady, et al. (2020) ACS Biomater. Sci. Eng.

Acknowledgements (Optional): Funding for this work was provided by NSF grants RECODE (2034495) and CAREER (1944734), and the Viterbi School of Engineering/Graduate School Fellowship for Incoming Students