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Neural Engineering

Neural Engineering - Poster Session C & D

Y22 - Electrical Recording Thresholds of 3D Neural Tissue Constructs

Friday, October 10, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Countless neurological disorders lack efficient treatments due to mechanistic knowledge gaps of neuronal network processing. The native cerebral cortex manages "higher-order" cognitive functions, i.e. memory and learning, via action potential propagation along axons between cortical neurons inducing structural and functional changes. However, the driving forces behind selective strengthening/rewiring of specific neurons resulting in said changes, remain unclear. Recent advancements to investigate this challenge include 2D neural cultures seeded atop high-definition microelectrode array (HD-MEA) chips to record neuronal activity and apply user-defined electrical stimuli, mimicking native “learning” experiences in vitro. However, standard protocols for neural 2D-HD-MEAs result in sporadic, variable seeding across both active and non-active regions of chips, limiting spatiotemporal control. In response, polydimethylsiloxane (PDMS) microstructures confine both neurons and axonal projections specifically to active regions, such that single neuron interactions may be studied. This method strictly examines small neuronal information processing in 2D, unlike native information flow. Thus, our group aims to build upon prior PDMS-microstructure work to elucidate larger, 3D neural network information processing. Our microstructures seeded with higher neuron:astrocyte co-culture densities show clear microchannel delineations, and preliminary astrocyte-seeded gelatin methacrylate (GelMA) biocasted constructs depict clear neural cell morphology and viability. Combinatorially, we will biocast neuron:astrocyte GelMA constructs within said microstructures, and hypothesize to observe similar electrical recording thresholds of mean firing rate, spike amplitude, and active electrode areas. By expounding tissue construct electrical recording resolution, this approach upgrades 2D small neural network analysis into 3D, unlocking future capacities for sophisticated in vitro larger neural network processing.

Materials and

Methods: : MaxWell Biosystems (MXWB) 2D-HD-MEA chips were wiped with 100% isopropyl alcohol (IPA), rinsed with deionized water (diH2O), and pressurized with nitrogen. Photolithography-manufactured polydimethylsiloxane (PDMS) microstructures were dipped in 100% IPA and diH2O ultrasonic baths. Using an oxygen plasma asher, HD-MEAs with PDMS microstructures were subject to plasma, strategically aligned via methanol to prevent microchannel collapse. Dried chips were plated with co-culture of E18 Primary Rat Astrocyte & Neurons (Gibco), maintaining a 1 astrocyte:8 neurons ratio, and split into: n=4 control, with normal cell seeding density of ~5000 cells/mm^2, and n=4 experimental group with doubled concentration. Chips underwent neuronal media changes 3x/week and electrically recorded activity and network scans 2x/week via MXWB Scope software.

Co-culture tissue constructs will be biocasted based on preliminary data, firstly by synthesizing 5% (w/v) gelatin methacrylate (GelMA) via stepwise methacrylation of PBS-dissolved Type A porcine gelatin, 1-week dialyzation, and LAP photoinitiator crosslinking via UV. Using aforementioned co-culture and ratio: we will generate 20,000 astrocytes + 160,000 neurons/5 µL droplets (based on literature values) to be dispensed and UV-cured directly atop 2D-HD-MEAs inside PDMS pockets, subject to same media change and electrical recording regiments as aforementioned. Scope-exported 3D-bottom-level neuronal activity data will be statistically compared to 2D-PDMS culture values on the same chips using unpaired t-test, and a two one-sided t-test (TOST) will test for equivalence. Spike sorting and clustering algorithms will normalize activity-electrode patterns and filter extraneous noise, respectively. Live/dead and lineage-specific immunocytochemistry confocal imaging will confirm cell viability and specific neural processes via dipping objective.

Results, Conclusions, and Discussions:: Preliminary results suggest that high seeding densities of primary rat cortical neurons lead to clearer, robust fine processes and axonal projections via polydimethylsiloxane (PDMS) microchannels between pockets/groups of neurons. PDMS neuronal activity is clearly output via MaxWell Biosystems Scope recording software. We observe robust microchannel delineations via mean firing rate, spike amplitude, and active electrode activity scans, as shown by the high concentrations of activity within the microchannels. Preliminary astrocyte-seeded gelatin methacrylate (GelMA) constructs confirmed via Brightfield microscopy that distinct neural cell morphologies within gels can be observed. Moreover, gels underwent live/dead viability/cytotoxicity assays, and depicted via confocal microscopy high astrocyte survivals, suggesting that a sufficient cell seeding density, w/v % GelMA, and droplet volume was used. Thus, with the proposed neuron:astrocyte GelMA constructs, we aim to observe similar fine processes and confined axonal projections inside PDMS microchannels between tissue constructs inside PDMS pockets. However, due to cells being randomly dispersed within the gels, we do not necessarily expect to see an “increase” in activity. Given the small volume dispensation, we expect to see similar/lower thresholds of activity, as literature reports the minimum neuron-electrode distance threshold to be 50-70 µm to detect activity.

These proposed results align with recent literature that uses PDMS molds to cast 3D neuronal hydrogel constructs via micropatterning directly atop HD-MEAs, the immunostaining and neural activity recording of which shows incredible promise and feasibility for our alternative approach. Moreover, our method improves upon said recent advancements, as confining axonal projections/fine processes within PDMS microchannels promotes possibilities for larger network stimulation studies with increased topology and spatiotemporal control, retaining enhanced signal isolation capacities of 2D-PDMS neural microstructures while investigating 3D-tissue mechanic effects (notably 5% (w/v) GelMA having reportedly closer Young’s modulus to native brain tissue (3.1 KPa) while optimizing neural cell growth/adherence). By encapsulating neural cells in 3D tissue microenvironments, recorded electrical activity will depict more cytoarchitecturally relevant patterns, leading to more sophisticated experimental outcomes. This novel proposed approach bridges the gap between 2D small network analyses and 3D extracellular-matrix effects on neuronal activity, coming closer to solving mechanistic knowledge gaps in neuronal connectivity and communication.

Acknowledgements and/or References (Optional): : We would like to acknowledge the following labs and their contributions from the University of Maryland, College Park: Tissue Engineering & Biomaterials Laboratory (TEBL) at the Fischell Department of Bioengineering for their assistance in gelatin methacrylate synthesis, photocrosslinking, and curing (Amal Shabazz, Dr. John Fisher); Integrated Biomorphic Information Systems Lab (IBIS) for their assistance in both PDMS microstructure fabrication and electrical recording (Lauren Gomes, Kasun Pathirage, Dr. Pamela Abshire); Computational Sensorimotor Laboratory (CSSL) for their assistance with electrical recording spike sorting analyses (Nathaniel Renegar, Dr. Timothy Horiuchi). Furthermore, we acknowledge the NSF EFRI BEGIN-OI grant (2422352) “Spatiotemporal Learning in 3D Neuronal Organoids”, for funding this project.

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