A microwell array specifically designed for culturing organoids is provided along with a system to enable automated imaging, identification, and isolation of individual organoids. The microwells of the microarray include a releasable cellraft that enables the automated release and transfer of selected organoids present on the cellrafts to a separate collection plate. Organoids grown on the microarray can be reliably tracked, imaged, and phenotypically analyzed by the instrument system in brightfield and fluorescence as they grow over time, then released and transferred fully intact for use in downstream applications. The use of the system is demonstrated using mouse hepatic and pancreatic organoids for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis.
A microwell array specifically designed for culturing organoids is provided along with a system to enable automated imaging, identification, and isolation of individual organoids. The microwells of the microarray include a releasable cellraft that enables the automated release and transfer of selected organoids present on the cellrafts to a separate collection plate. Organoids grown on the microarray can be reliably tracked, imaged, and phenotypically analyzed by the instrument system in brightfield and fluorescence as they grow over time, then released and transferred fully intact for use in downstream applications. The use of the system is demonstrated using mouse hepatic and pancreatic organoids for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis.
The Trustees of Columbia University in the City of New York (USA)
Inventor
Duvall, Jacquelyn
Thompson, Brandon
Sims, Peter Alan
Yuan, Jinzhou
Liu, Zhouzerui
Mizrak, Dogukan
Gebhart, Steven C.
Boone, Peter Glyn
Abstract
Systems and methods for associating single cell imaging data with RNA transcriptomics. Single cells are isolated into microwells with a microbead having oligonucleotides conjugated on its surface. Each oligonucleotide includes a cell identifying optical barcode that is unique to that bead and binding sequence for RNA capture after cell lysis. The system is configured for loading single cells into the microarray and for flowing cell lysis buffers and other reagents into the microarray for performing RNA library sample preparation. The system is also configured for lowing optical hybridization probes that are complementary to the cell identifying optical barcodes and optically labeled onto the microwell array and for obtaining images of the microwells in response to the probes. The system and unique cell identifying optical barcodes and complementary optical hybridization probes facilitate a link between phenotypic imaging of cells resident on the microwell array with single cell whole transcriptome sequencing.
A microwell array specifically designed for culturing organoids is provided along with a system to enable automated imaging, identification, and isolation of individual organoids. The microwells of the microarray include a releasable cellraft that enables the automated release and transfer of selected organoids present on the cellrafts to a separate collection plate. Organoids grown on the microarray can be reliably tracked, imaged, and phenotypically analyzed by the instrument system in brightfield and fluorescence as they grow over time, then released and transferred fully intact for use in downstream applications. The use of the system is demonstrated using mouse hepatic and pancreatic organoids for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis.
A microwell array specifically designed for culturing organoids is provided along with a system to enable automated imaging, identification, and isolation of individual organoids. The microwells of the microarray include a releasable cellraft that enables the automated release and transfer of selected organoids present on the cellrafts to a separate collection plate. Organoids grown on the microarray can be reliably tracked, imaged, and phenotypically analyzed by the instrument system in brightfield and fluorescence as they grow over time, then released and transferred fully intact for use in downstream applications. The use of the system is demonstrated using mouse hepatic and pancreatic organoids for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis.
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK (USA)
Inventor
Duvall, Jacquelyn
Thompson, Brandon
Sims, Peter, Alan
Yuan, Jinzhou
Liu, Zhouzerui
Mizrak, Dogukan
Gebhart, Steven, C.
Boone, Peter, Glyn
Abstract
Systems and methods for associating single cell imaging data with RNA transcriptomics. Single cells are isolated into microwells with a microbead having oligonucleotides conjugated on its surface. Each oligonucleotide includes a cell identifying optical barcode that is unique to that bead and binding sequence for RNA capture after cell lysis. The system is configured for loading single cells into the microarray and for flowing cell lysis buffers and other reagents into the microarray for performing RNA library sample preparation. The system is also configured for lowing optical hybridization probes that are complementary to the cell identifying optical barcodes and optically labeled onto the microwell array and for obtaining images of the microwells in response to the probes. The system and unique cell identifying optical barcodes and complementary optical hybridization probes facilitate a link between phenotypic imaging of cells resident on the microwell array with single cell whole transcriptome sequencing.
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK (USA)
CELL MICROSYSTEMS, INC. (USA)
Inventor
Duvall, Jacquelyn
Thompson, Brandon
Sims, Peter Alan
Yuan, Jinzhou
Liu, Zhouzerui
Mizrak, Dogukan
Gebhart, Steven C.
Boone, Peter Glyn
Abstract
Systems and methods for associating single cell imaging data with RNA transcriptomics. Single cells are isolated into microwells with a microbead having oligonucleotides conjugated on its surface. Each oligonucleotide includes a cell identifying optical barcode that is unique to that bead and binding sequence for RNA capture after cell lysis. The system is configured for loading single cells into the microarray and for flowing cell lysis buffers and other reagents into the microarray for performing RNA library sample preparation. The system is also configured for lowing optical hybridization probes that are complementary to the cell identifying optical barcodes and optically labeled onto the microwell array and for obtaining images of the microwells in response to the probes. The system and unique cell identifying optical barcodes and complementary optical hybridization probes facilitate a link between phenotypic imaging of cells resident on the microwell array with single cell whole transcriptome sequencing.
The University of North Carolina at Chapel Hill (USA)
Inventor
Gebhart, Steven C.
Mcclellan, Robert W.
Trotta, Nicholas C.
Allbritton, Nancy L.
Attayek, Peter Joseph
Armistead, Paul Michael
Abstract
Embodiments of the disclosed subject matter provide an automated method and system to isolate and collect cells using computerized analysis of images of cells and their surroundings obtained from a digital imaging device or system. Embodiments of the disclosed subject matter make use of a “microwell array,” which can comprise a formed, elastomeric grid of indentations or “wells.” Many, most, or all of the wells in a microwell array can contain a releasable, microfabricated element, which can be referred to as a “raft.” Embodiments of the disclosed subject matter provide a system and method for cell collection that includes computerized identification and collection of rafts with isolated single cells or a specific group or groups of cells, eliminating the need for continuous human identification and selection.
The University of North Carolina at Chapel Hill (USA)
Inventor
Gebhart, Steven C.
Mcclellan, Robert W.
Trotta, Nicholas C.
Allbritton, Nancy L.
Attayek, Peter Joseph
Armistead, Paul Michael
Abstract
Embodiments of the disclosed subject matter provide an automated method and system to isolate and collect cells using computerized analysis of images of cells and their surroundings obtained from a digital imaging device or system. Embodiments of the disclosed subject matter make use of a “microwell array,” which can comprise a formed, elastomeric grid of indentations or “wells.” Many, most, or all of the wells in a microwell array can contain a releasable, microfabricated element, which can be referred to as a “raft.” Embodiments of the disclosed subject matter provide a system and method for cell collection that includes computerized identification and collection of rafts with isolated single cells or a specific group or groups of cells, eliminating the need for continuous human identification and selection.
The University of North Carolina at Chapel Hill (USA)
Cell Microsystems, Inc. (USA)
Inventor
Allbritton, Nancy L.
Wang, Yuli
Sims, Christopher E.
Dobes, Nicholas C.
Gebhart, Steven C.
Trotta, Nicholas C.
Abstract
An array of magnetic or paramagnetic micro-elements comprised of polysilsesquioxane is described having ultra-low-autofluorescence and other optical properties to improve microscopic imaging of cells or other objects present on the array. These materials are also amenable to chemical modification allowing surface attachment of affinity capture moieties or chemical reporters for selective binding or analysis of cells, macromolecules or other targets.
The invention discloses diagnostic techniques based on single cell genomics, consisting of obtaining a blood sample, enriching a sub-population of cells present in the blood sample, sequestering individual cells or group of cells from the blood sample, obtaining sequencing data from the sequestered cells or group of cells, using genetic variant information to determine the provenance of the cells, and genetically analyzing the cells of the correct provenance to provide a diagnostic readout. Using the cell-based testing techniques of the invention, the number of false positives is greatly reduced when compared to cell-free DNA (cfDNA) based traditional testing techniques. The invention may be effectively employed for non-invasive prenatal (NIPT) diagnostics, oncological testing and other diagnostic procedures.
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL (USA)
Inventor
Gebhart, Steven C.
Mcclellan, Robert W.
Trotta, Nicholas C.
Allbritton, Nancy L.
Attayek, Peter Joseph
Armistead, Paul Michael
Abstract
Embodiments of the disclosed subject matter provide an automated method and system to isolate and collect cells using computerized analysis of images of cells and their surroundings obtained from a digital imaging device or system. Embodiments of the disclosed subject matter make use of a "microwell array," which can comprise a formed, elastomeric grid of indentations or "wells." Many, most, or all of the wells in a microwell array can contain a releasable, microfabricated element, which can be referred to as a "raft." Embodiments of the disclosed subject matter provide a system and method for cell collection that includes computerized identification and collection of rafts with isolated single cells or a specific group or groups of cells, eliminating the need for continuous human identification and selection.
G01N 35/02 - Automatic analysis not limited to methods or materials provided for in any single one of groups Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
13.
AUTOMATED COLLECTION OF A SPECIFIED NUMBER OF CELLS
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL (USA)
Inventor
Gebhart, Steven, C.
Mcclellan, Robert, W.
Trotta, Nicholas, C.
Allbritton, Nancy, L.
Attayek, Peter, Joseph
Armistead, Paul, Michael
Abstract
Embodiments of the disclosed subject matter provide an automated method and system to isolate and collect cells using computerized analysis of images of cells and their surroundings obtained from a digital imaging device or system. Embodiments of the disclosed subject matter make use of a "microwell array," which can comprise a formed, elastomeric grid of indentations or "wells." Many, most, or all of the wells in a microwell array can contain a releasable, microfabricated element, which can be referred to as a "raft." Embodiments of the disclosed subject matter provide a system and method for cell collection that includes computerized identification and collection of rafts with isolated single cells or a specific group or groups of cells, eliminating the need for continuous human identification and selection.
G01N 35/02 - Automatic analysis not limited to methods or materials provided for in any single one of groups Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
C12M 1/32 - Inoculator or sampler multiple field or continuous type
A mount suitable to be mounted to a microscope objective is described with a first member; a second member engaged with the first member; a first configuration where the second member has a first effective diameter; and a second configuration where the second member has a second effective diameter less than the first effective diameter for securably engaging an outer surface of a microscope objective. Methods of using the mount are also disclosed.
The invention discloses diagnostic techniques based on single cell genomics, consisting of obtaining a blood sample, enriching a sub-population of cells present in the blood sample, sequestering individual cells or group of cells from the blood sample, obtaining sequencing data from the sequestered cells or group of cells, using genetic variant information to determine the provenance of the cells, and genetically analyzing the cells of the correct provenance to provide a diagnostic readout. Using the cell-based testing techniques of the invention, the number of false positives is greatly reduced when compared to cell-free DNA (cfDNA) based traditional testing techniques. The invention may be effectively employed for non-invasive prenatal (NIPT) diagnostics, oncological testing and other diagnostic procedures.
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