Functionalized Hydrogel Scaffold for Guiding Axonal Connections Between Cortical Organoids

Introduction: : Neurodevelopmental disorders (NDDs), such as autism spectrum disorder, affect up to 8% of children (Yang, et al. 2022) but the origins of these complex disorders remain ill-defined. Functional MRI studies examining brain connectivity suggest that NDDs are associated with disrupted communication between different brain regions. However, systematically investigating relationships between NDDs and brain connectivity requires accessible and perturbable human-relevant model systems. Human stem cell-derived brain organoids revolutionized our ability to understand the development of single brain regions and short-range connectivity via assembloids. However, existing models have failed to recapitulate long-range connectivity between organoids. To address this need, we previously developed a microfluidic device that perfuses organoids to maintain their viability in static culture. (Fig. 1A). To connect organoids in this device, our current goal is to develop a hydrogel scaffold that both stimulates axon extension and prohibits cell migration. To achieve this, we plan to fabricate a microporous annealed particle (MAP) scaffold from gelatin methacryloyl (GelMA) functionalized with a laminin-mimetic peptide (YIGSR). We are using MAP scaffolds to control pore size such that soma will be excluded but neurites will be permitted. We are utilizing GelMA as the hydrogel backbone because it is inert to neural tissues. Thus, through functionalization with YIGSR or other specific peptides, we can provide controllable neurite guidance between brain organoids (Fig. 1B). Our ultimate goal is to develop a modular system for inducing axon extensions from specific cell populations, such as those implicated in NDDs, to establish the molecular origins of tissue-level dysfunctions (Fig. 1C).

Materials and

Methods: : We synthesized YIGSR peptide with a terminal cysteine residue for labeling with a maleimide-linked fluorophore and modified it with a methacrylate group for free radical conjugation to GelMA (Fig 1D). For validation, GelMA-coated plastic coverslips were incubated with fluorescent methacrylated YIGSR and exposed to UV light under a photomask. Coverslips were imaged with a fluorescent microscope and pixel intensity was quantified (Fig 1E).
Next, we examined neurite extension in an immortalized cell line. SH-SY5Y neuroblastoma cells were selected because of their widespread adoption in studies of neurological disorders, as they are capable of differentiation to neuron-like cells. We crosslinked methacrylated YIGSR to GelMA-coated plastic coverslips as lanes with 10μm width on the labeled side of the GelMA. We then differentiated SH-SY5Y cells on these coverslips over 11 days (Fig. 1G). We immunostained for the neuronal differentiation marker beta-III-tubulin, as well as actin (phalloidin) and nuclei (DAPI), and observed neurite alignment to the peptide lanes using brightfield and fluorescence microscopy (Fig. 1H,I).
Finally, we fabricated a microfluidic flow-focusing droplet generator using standard PDMS soft lithography techniques to form GelMA microbeads. To verify that the scaffold would create micropores on the scale of neurite bundles, we altered the flow rate ratios of oil and GelMA through the droplet generator to modify the droplets’ size inside the microfluidic device (Fig. 1J). The beads were then collected from the oil using density separation, coated with methacrylated peptide, and crosslinked together with a radical chain reaction initiated by UV light (Fig 1L).

Results, Conclusions, and Discussions:: Fluorescence microscopy confirmed successful functionalization of the GelMA hydrogel with methacrylated YIGSR, demonstrating high signal-to-background and indicating precise spatial control over the placement of the peptide (Fig. 1F). Undifferentiated SH-SY5Y cells cultured on YIGSR-patterned 2D GelMA surfaces adhered and exhibited neurite extension over an 11-day differentiation period, aligning with the patterned peptide lanes (Fig. 1H,I). This initial finding suggests the bioactivity and spatial specificity of the surface-bound peptides in promoting directed neurite outgrowth.
We also demonstrated control over the diameter of GelMA droplets, achieving a size range suitable for creating 10 μm pores, a dimension intended to restrict cell migration (Fig. 1K). Stable 3D GelMA scaffolds were successfully formed within our multi-organoid culture device using bead-to-bead photocrosslinking. The incorporation of methacrylated peptides within these 3D scaffolds (Fig. 1L) suggests the potential to induce and guide neurite extension within a three-dimensional environment relevant to organoid cultures.
Our preliminary results establish a functional GelMA hydrogel platform capable of presenting spatially defined cues for neurite extension in both 2D and 3D settings. The demonstrated control over peptide presentation and scaffold architecture within our microfluidic device lays the groundwork for future investigations into targeted axon guidance in more complex organoid models. Ongoing work will focus on quantifying the extent and directionality of peptide-induced neurite extension on 2D hydrogels. Subsequently, we will translate this technology to our multi-organoid platform to investigate its capacity to facilitate controlled axonal connections, offering a novel tool for studying the cellular mechanisms underlying neurodevelopmental disorders.

Acknowledgements and/or References (Optional):: We would like to acknowledge our funding sources, including the NIH-R01MH136351, the NSF-RECODE CBET 2034495, and the USC Office of Research & Innovation Alternative Methods to Animal Research Award. We would also like to thank the Di Carlo lab at UCLA for their generous assistance with design specifications for a microfluidic droplet generator.