3D Neuron Spheroid Optical Assays for Consistent High-Throughput Screening
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Solution Overview
Problem
Current high-throughput assays using two-dimensional cell formats in which the cells do not always fill the wells uniformly or attached with high fidelity, leading to inconsistent data generation in 2D cell formats, particularly in 96, 384, and 1536 well plates, which affects the reliability and validity of the results.
Innovation Solution
An optical assay, specifically a functional FLIPR assay or high content high magnification optical microscopy, of 3D human cell spheroids, particularly mixed population human cell neuron spheroids, which are cultured for 4 to 16 weeks to induce robust synchronized synaptic networks, allowing predictable and consistent synaptic firing for long periods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If 2D cell formats are used in high-throughput assays, then the assay setup is simple and cells can be easily added to microplate wells, but the cells do not fill the wells uniformly or attach with high fidelity, leading to inconsistent data generation
Solution Approach 1:
The patent transitions from two-dimensional (2D) cell culture formats to three-dimensional (3D) spheroid cell cultures. This dimensional change allows cells to self-organize into uniform spherical structures that consistently fill microplate wells, thereby improving data consistency while maintaining ease of assay setup through automated dispensing systems
2Reliability
If 3D spheroid cell cultures are used, then data consistency and uniformity are improved, but the assay complexity and difficulty of setup increase
Solution Approach 1:
The patent employs self-assembling 3D spheroid cell cultures that automatically form uniform spherical structures without requiring complex manual manipulation. The cells spontaneously organize into spheroids when dispensed into microplate wells, eliminating the need for complex assembly procedures while maintaining high data consistency
3Reliability
If 3D spheroid cell cultures are used, then uniformity and data consistency are improved, but the assay throughput and automation capability are reduced
Solution Approach 1:
The patent segments the 3D spheroid formation process into discrete, automated steps: single-cell suspension preparation, automated dispensing into individual microplate wells, and incubation. This segmentation allows each step to be performed by automated liquid handling systems, maintaining high throughput while ensuring uniform spheroid formation for consistent data
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enables the detection of the effect of compounds on spheroids by monitoring and the resulting data is quite consistent, e.g., spheroid neuron synapses fired predictably and consistently for long periods of time, enabling high throughput optical assays with FLIPR and calcium uptake fluorescence oscillations.
Implementation Method 1
Typically, the argon laser excites a suitable fluorescent dye. The resultant emitted light is detected by a cooled CCD camera that acts as an integrated detector
Implementation Method 2
The system generates real-time kinetic data by stimulating and reading all 96 wells in 1-second intervals. The cooled CCD camera accumulates signal over the duration of the exposure
Implementation Method 3
Sensitivity is further enhanced by the FLIPR's optical scheme, which limits the depth of field of the CCD camera to a few hundred microns on the bottom of each well, or essentially at the level of the cell monolayer. This technique reduces the background fluorescence from extracellular dye by about an order of magnitude
Implementation Method 4
enabling high throughput optical assays with FLIPR and calcium uptake fluorescence oscillations
Data Source
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AI summary
The present disclosure provides a method of performing a functional assay on human spheroids, e.g., three-dimensional human cell spheroids using, in one embodiment, a fluorometric imaging plate reader.