An Organ-on-a-Chip Model to Study Neuroinflammatory and Neurodegenerative Diseases

Introduction:: Neuroinflammatory and neurodegenerative diseases impact millions of individuals worldwide, and that number is only expected to increase as populations continue to age. Therefore, there is a critical need to better understand the mechanisms driving neuroinflammatory and neurodegenerative disease progression. One such technology that can aid in this discovery are organ-on-a-chip platforms, which strike a balance between recapitulating relevant cell-cell interactions found in vivo, while reducing the complexity of the system to allow for more controlled mechanistic studies. Here, we present our group’s progress in developing an organ-on-a-chip model to study neuroinflammatory and neurodegenerative diseases; with a particular focus on modeling how these disease states are able to propagate to synaptically connected, but anatomically remote regions of the brain.

Materials and Methods:: Primary cortical cells from P0-P2 rat pups were cultured using in serum-free media designed to support a tri-culture of neurons, astrocytes, and microglia [1]. The overall health of the tri-culture was compared to neuron-astrocyte co-cultures, and the response of the two culture types to common neuroinflammatory stimuli, including toll-like receptor (TLR) activation, mechanical trauma, and glutamate induced excitotoxicity were compared. The geometry of axon guidance microchannels in the organ-on-a-chip platform was optimized via theoretical and computational models, and the silicone-based devices embedded with microelectrode arrays were fabricated. We maintained both tri- and co-cultures in these devices and neural activity was recorded for 60 days from axons crossing between the two cell populations using custom fabricated microelectrode arrays. To demonstrate the capability to monitor electrophysiological response of the cultures, we spiked them individually at DIV 21 with 5 μg/mL lipopolysaccharide (LPS) and acquired extracellular activity for 10 minutes at various timepoints for a week. For the analysis, for each active electrode we compared the change in number of spikes during the 10-min window of treatment to that for baseline (pre-treatment) activity.

Results, Conclusions, and Discussions:: We are able to maintain a physiologically relevant population of neurons, astrocytes, and microglia for at least 14 days in vitro (DIV) and show that the continuous presence of microglia may improve the overall health of the culture (increased % of live cells and reduced caspase 3/7 activity) [1]. Additionally, the tri-culture responds appropriately to several neuroinflammatory stimuli (Figure 1), with a significant neurotoxic response to TLR2, TLR3, & TLR4 activation and mechanical trauma (increased caspase 3/7 activity, significantly reduced % live cells, astrocyte hypertrophy, and the secretion of pro-inflammatory cytokines) [2]. Conversely, the microglia in the tri-culture play a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy, when compared to neuron-astrocyte co-cultures [1]. LPS exposure decreases electrophysiological activity starting with 6 hours after exposure for the tri-culture configuration while having no effect on the co-culture configuration [3], highlighting the presence of microglia playing a critical role in shaping the electrophysiological response (Figure 2).

Theoretical and COMSOL modeling indicate that microchannel height is the key factor in minimizing mass transport between the two cell populations, and reducing microchannel height significantly reduces the number of cells that cross through the microchannels, while having minimal effect on the ability for axons to cross between the chambers [4]. Additionally, we demonstrate that we can maintain two distinct cell populations that are chemically isolated, but synaptically connected (Figure 3). Finally, we show that neurons in these microchannel devices exhibit spontaneous neural activity for at least 60 DIV and that the spatial confinement within the microchannels enhance electrophysiological recordings (~17-fold increase in SNR in the microchannels vs. chamber).

We demonstrated an organ-on-a-chip platform enabled by the combination of a novel primary cortical tri-culture model, microfluidic neural compartmentalization, and microelectrode arrays. We used this platform to study progression of neuroinflammatory and neurodegenerative diseases and condensed the findings into a set of practical design rules to establish long-term neural cultures.

Acknowledgements (Optional): : We gratefully acknowledge the support from the National Institutes of Health via NINDS/NIA R03-NS118156 and NCCIH R21-AT010933, and from the National Science Foundation via DMR-2003849. This project also benefited from the resources of the Center for Nano/Micro-Manufacturing facility at University of California, Davis.

References (Optional): :

1. Goshi, N., Morgan, R.K., Lein, P.J., Seker, E., “A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation,” Journal of Neuroinflammation 17:155 (2020).


2. Goshi, N., Kim, H., Seker, E., “Primary Cortical Cell Tri-Culture-Based Screening of Neuroinflammatory Response in Toll-like Receptor Activation,” Biomedicines 10:2122 (2022).


3. Goshi, N., Kim, H., Girardi, G., Gardner, A., Seker, E., “Electrophysiological Activity of Primary Cortical Neuron-Glia Mixed Cultures,” Cells 12:821 (2023).


4. Goshi, N., Girardi, G., da Costa Souza, F., Gardner, A., Lein, P.J., Seker, E., “Influence of Microchannel Geometry on Device Performance and Electrophysiological Recording Fidelity During Long-Term Studies of Connected Neural Populations,” Lab on a Chip 22:3961 (2022).