Methods for assaying biological cells in a microfluidic device
Inventors
Bronevetsky, Yelena • Mocciaro, Annamaria • Stadler, Guido K. • Beemiller, Peter J. • Marks, Natalie C. • Smith, Duane • Pai, Vincent Haw Tien • McEwen, Jason M. • GOODSELL, Amanda L. • Tenney, John A. • Vetterli, Thomas M. • KIM, Hansohl E.
Assignees
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Publication Number
US-12345701-B2
Publication Date
2025-07-01
Expiration Date
Abstract
Methods are provided for the assay of secreted biomolecules using automated detection and characterization of micro-objects in a microfluidic device. The biomolecules can be secreted by cells, particularly immunological cells, such as T cells. The biomolecules being assayed can include cytokines, growth factors, and the like. Methods are also provided for assaying the cytotoxicity of a cell with respect to another, target cell. Also provided are kits and non-transitory computer-readable media in which programs are stored for causing a system comprising a computer to perform automated methods for detecting secreted biomolecules and/or cytotoxicity in a microfluidic device.
Core Innovation
The invention provides a method of assaying for antigen-specific cytotoxicity of a T lymphocyte (T cell) in a microfluidic device. The method disposes the T cell within a microfluidic device that includes a flow region and a chamber opening to the flow region. A target cell is disposed in proximity to the T cell, where the target cell is configured to capture a secreted biomolecule from the T cell after a period of exposure in proximity to the T cell.
The secreted biomolecule captured by the target cell is detected using image acquisition of a region of interest. A first image and one or more second images of the region of interest are received, and each of the one or more second images is optically transformed to align with the first image. Pixel data in the first image is processed using a machine learning algorithm to detect target cells present in the region of interest, including identifying a boundary of each target cell and detecting a signal located within the boundary of each detected target cell in each of the one or more second images.
The method further determines the viability of the target cell after the period of exposure in proximity to the T cell. The kit form provides a microfluidic device, a cytotoxicity detection reagent configured to detect viability of a target cell, and an image acquisition unit configured to detect the secreted biomolecule captured by the target cell. In one kit embodiment, the microfluidic device has an isolation region that is an unswept region with a single opening and a connection region fluidically connecting the isolation region to the flow region.
Claims Coverage
The independent claims are directed to a method and a kit. Across these claims, five inventive feature areas are recited: microfluidic proximity between a T cell and a capture-configured target cell, optical alignment of images, machine-learning-based detection of target cells including boundary identification, signal detection within those boundaries, and target cell viability determination or reagent-based viability detection.
Proximal microfluidic cytotoxicity assay with secreted biomolecule capture
Disposing the T cell within the microfluidic device and disposing a target cell in proximity to the T cell, wherein the target cell is configured to capture a secreted biomolecule from the T cell after a period of exposure in proximity to the T cell.
Optical alignment of subsequent images to a first image
Receiving a first image and one or more second images of a region of interest, and transforming each of the one or more second images to optically align with the first image.
Machine learning detection of target cells with boundary identification
Processing pixel data in the first image using a machine learning algorithm to detect target cells present in the region of interest, the detection comprising identifying a boundary of each target cell.
Signal detection within detected target cell boundaries and viability determination
Detecting a signal located within the boundary of each detected target cell in each of the one or more second images; and determining the viability of the target cell after a period of exposure in proximity to the T cell.
Kit for microfluidic antigen-specific cytotoxicity assay with imaging and viability reagent
A kit comprising a microfluidic device with a flow region and a chamber opening, where the chamber comprises an isolation region having a single opening and a connection region fluidically connecting the isolation region to the flow region, the isolation region being an unswept region; a cytotoxicity detection reagent configured to detect viability of a target cell; and an image acquisition unit configured to detect a secreted biomolecule captured by the target cell.
Overall, the claims cover a microfluidic antigen-specific cytotoxicity assay where T cell activity is read out via secreted biomolecule capture by a nearby target cell, and where aligned image acquisition and machine learning are used to detect target cells by identifying boundaries so that a signal within those boundaries can be measured, with target cell viability determined in the method or provided via a kit including device, reagents, and an image acquisition unit.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Assaying antigen-specific cytotoxicity of a T lymphocyte (T cell) in a microfluidic device.
Using a kit to assay antigen-specific cytotoxicity by a T lymphocyte (T cell) in a microfluidic device.
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