Resources: Posters

Concurrent live-imaging and single-cell transcriptome analysis of intact functional neuronal networks on a highly parallel scale

April 1, 2025

GRC | Gordon Research Conference: Functional Neurogenomics

Olaia F Vila, Ph.D.

Keywords: Neuronal networks, Functional neurobiology, Live-cell imaging, Single-cell transcriptomics, Synaptic plasticity, R3200 Platform, CellCage™ technology

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Leveraging the R3200 Platform and CellCage™ technology, this research enables the concurrent live-imaging and transcriptomic analysis of intact, functional neuronal networks. By maintaining the structural integrity of these networks on a highly parallel scale, the study reveals how complex cell-cell interactions and connectivity patterns correlate with underlying gene expression. This multimodal approach provides a powerful new framework for studying neurodevelopment, synaptic plasticity, and disease mechanisms in a preserved physiological context.

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Case Study: Functional Profiling of Microglia in Neuroinflammation

Link microglial behavior to gene expression at single-cell resolution, for insight into neuroinflammation, drug response, and immune dysfunction in CNS disease.

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Phagocytosis assay in CellCage enclosures - (click to expand image).

Researchers used the Cellanome R3200 to enclose individual microglia with fluorescent particles and track phagocytosis over 12 hours via fluorescent imaging. Each cell’s transcriptome was then sequenced, linking activity levels to gene expression.

What they found:

High-activity microglia upregulated genes in complement signaling, lipid metabolism, and lysosomal function–key pathways in neuroinflammation and repair.

Why it matters:

This approach overcomes key limitations in standard assays by capturing phagocytic function and gene expression in the same individual cells without dissociation, pooling, or inference. It enables a direct, scalable readout of immune heterogeneity, and reveals the transcriptional programs driving effective or impaired microglial responses.

‍What’s next:

Extend to co-cultures by layering enclosed microglia over intact neuronal networks. Study how cell-cell interactions shape phagocytic behavior and fate. Combine with cytokines, CRISPR libraries, or immunotherapies to generate time-resolved, multi-modal datasets that can be used for MoA analysis, early biomarker discovery, and AI-guided modeling in CNS disease.

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Case Study: Modeling Synapse Formation and Developmental Trajectories in 3D

Track development, function, and gene expression in intact neurospheres, a human-relevant 3D model increasingly vital as regulators move away from animal studies.

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Neurospheres in windowed cages that allow them to reach out to form a synapse - (click to expand image).

Stem-cell-derived neurospheres offer a robust 3D model of early brain development, but standard assays disrupt their structure and miss critical dynamics.

Approach:

Using the Cellanome R3200, the research team explored,

Hundreds of intact neurospheres (100–200 cells each) were cultured inside individual CellCage™ enclosures.
Axon extension, synapse formation and calcium activity were tracked over multiple days.
End-point RNA-Seq was linked back to each neurosphere’s functional behavior.
UMAP clustering revealed lineage-specific gene programs, validated by fluorescent markers.

‍What's next:

This lays the groundwork for CRISPR-based multimodal screens to probe mechanisms of development, degeneration, and repair within preserved 3D architecture.

‍Why it matters:

As the FDA and others move to reduce reliance on animal models, human-relevant in vitro systems like this are increasingly essential.

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FAQ's

How does the R3200 Platform support the study of intact neuronal networks?

The R3200 Platform utilizes a highly parallel architecture to monitor multiple neuronal networks simultaneously. By capturing high-resolution, longitudinal imaging data across these networks before moving to sequencing, the platform ensures that the functional connectivity and spatial orientation of the cells are fully documented and linked to their individual transcriptomes.

What is the benefit of "concurrent" imaging and transcriptome analysis in neuroscience?

Concurrent analysis allows researchers to link real-time physiological activity, such as synchronized firing patterns or network-level signaling, directly to the molecular state of the participating cells. This eliminates the need for inference and provides a definitive map of the gene programs driving specific network behaviors.

How does CellCage™ technology preserve functional neuronal networks?

CellCage™ technology provides a non-destructive microenvironment that secures neurons and their established connections in place. Unlike traditional single-cell methods that require dissociation—which severs axons and dendrites—CellCage™ enclosures protect the intact network, allowing for the study of complex cellular architectures without sacrificing molecular resolution.

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