One challenge in using neural 3D microtissues for translational research is the ability to perform real-time measurements of neuronal activity in a multi-well plate format. The currently available technologies for in vitro measurement of neural activity including electrophysiology require high cellular density and are restricted by fixed pre-fabricated sensors mounted on the bottom surface of the plate well, limited by either spatial or temporal resolutions, and are not useful for 3D gel-matrix cellular support scaffolds.

In vitro 3D microtissues are being developed to model human tissues and organs and as assay platforms for therapeutics development. Examples of 3D microtissues include, but are not limited to spheroids, organoids, 3D bioprinted tissue, and organ-on-a chip systems. 3D microtissues attempt to recapitulate the morphology and physiology of native human tissues and organs by including relevant cell types, creating spatial organizations, cell-to-cell and cell-to-extracellular matrices (ECM) interactions, mechanical stresses, and vascularization. These models are assembled from human induced pluripotent stem cells (iPSCs) and have the ability to reproduce healthy and disease phenotypes, as well as predict drug responses more accurately than 2D cell culture models. 3D in vitro microtissue models have been particularly useful and applicable in creating in vitro neural microtissues.

The 3D microenvironment enhances growth of mature neurons, facilitates the rewiring of networks, supports synaptic connections, and promotes the outgrowth of long neurites for the formation of complex circuitry, as well as the synaptic release of neurotransmitters.

To address limitations in real-time measurement of 3D microtissues, scientists at the National Center for Advancing Translational Sciences (NCATS) invented a novel device and method Dual Fiber Photometry and Multielectrode Electrophysiology System (DF-PHOTOMES) to take functional measurements of intracellular calcium or neurotransmitter dynamics via genetically encoded fluorescence biosensors and extracellular electrical signals such as action potentials or local field potentials in 3D in vitro microtissues in real-time with optogenetics activation. The instrument utilizes a top-down probe(s) insertion method for the precise spatial positioning and interchangeable probes for signal acquisition from 3D microtissue models. To increase experimental throughput and reproducibility, the probes were integrated with a semi-automated robotic platform providing micron-scale spatial (X-Y-Z) positioning, guided by a user interface and long-working-distance microscopy inside a clean-air Faraday enclosure, compatible for spheroids, organoids, assembloids, scaffold-based constructs, layered tissues, and suspended tissue models, this platform supports drug screening, disease modeling, and advanced live functional assays.

The application of this technology is not limited by cellular type, density, design of bio fabrication, or spatial arrangement of multicellular models; the device retains flexibility by culture plate-type and format, including trans-well and micro-chip platforms.

Competitive Advantages of DF-PHOTOMES:

1. Multi-modal integration: Tailored for intracellular photometry, extracellular electrical signal acquisition from 3D in vitro New Approach Methodologies (NAMs) models with optogenetics and pharmacological assay designing compatibilities.
2. Compatibility with any 3D matrix: Signal fidelity high due to direct tissue penetration with top-down probe insertion approach irrespective of any extracellular matrix type, height, or density.
3. Dynamic probe(s) navigation: Automated maneuvering of probe(s) with micron-label spatial (X-Y-Z) resolution in multiwell platform.
4. Signal quality: High-quality reproducibility of robust biologically-relevant signal acquisition from low density 3D cellular constructs.
5. Throughput and format: The instrument delivers medium- to high-throughput screening with semi-automated plate and probe maneuvering capabilities, regardless of culture plate-type or format

Potential Applications: In vitro research use to measure extracellular electrophysiological activity and fluorescence biosensor-based intracellular dynamic signals and network functions in 3D microtissues.