System and method for isolating and analyzing cells

• US 10,391,490 B2
• Filed: 02/24/2017
• Issued: 08/27/2019
• Est. Priority Date: 05/31/2013
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Celsee Diagnostics, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Handique, Kalyan, Gogoi, Priyadarshini, Sepehri, Saedeh Javdani, Gleason, Kyle
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/442,222
• Publication Number:
  US 20170157610A1
• Time in Patent Office: 914 Days
• Field of Search: 506 9
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0445 Natural or forced convection
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1023 Microstructural devices for non-optical measurement
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 2800/52 : Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/57415 : of breast
• CPC Class Codes: G01N 33/57423 : of lung

Abstract

A system and method for isolating and analyzing single cells, comprising: a substrate having a broad surface; a set of wells defined at the broad surface of the substrate, and a set of channels, defined by the wall, that fluidly couple each well to at least one adjacent well in the set of wells; and fluid delivery module defining an inlet and comprising a plate, removably coupled to the substrate, the plate defining a recessed region fluidly connected to the inlet and facing the broad surface of the substrate, the fluid delivery module comprising a cell capture mode.

Claims

1. A system for isolating and analyzing single cells, comprising:

a substrate having a broad surface;

a set of wells defined at the broad surface of the substrate, each well of the set of wells comprising;

a base surface defined within the substrate,

an open surface directly opposing the base surface and coplanar with the broad surface,

a wall, extending between the base surface and the open surface in a direction substantially perpendicular to the broad surface of the substrate, the wall, base surface, and open surface cooperatively defining an open volume, and

a set of channels, defined by the wall, that fluidly couple each well to at least one adjacent well in the set of wells;

a fluid delivery module defining an inlet and comprising a plate, the plate defining a recessed region fluidly connected to the inlet and facing the broad surface of the substrate, the fluid delivery module comprising a cell capture mode operable to provide a fluid layer between the recessed region and the broad surface of the substrate, through which a sample containing a set of single cells flows in a direction perpendicular to the broad surface, the cell capture mode further operable to deliver at least one of the set of single cells into the open volume of at least one of the set of wells from the direction substantially perpendicular to the broad surface, wherein the fluid delivery module is further operable in a reagent delivery mode operable to deliver reagent fluid, through the inlet, and laterally in a second direction parallel to the broad surface, to the set of wells; and

a heating plate coupled to the plate of the fluid delivery module within the recessed region, the heating plate at least partially defining the fluid layer through which a convective flow provided by the heating plate can flow in the second direction parallel to the broad surface, wherein, with the heating plate, the system is operable in a diffusion mode that provides diffusion transport between the convective flow and the set of wells.

2. The system of claim 1, wherein the fluid delivery module further comprises a set of fluid pathways, wherein each fluid pathway of the set of fluid pathways fluidly connects the inlet to a corresponding well of the set of wells, and wherein each fluid pathway of the set of fluid pathways is of a substantially identical length.

3. The system of claim 1, wherein the wall of each of the set of wells gradually reduces a characteristic dimension of each of the set of wells from the open surface to the base surface of each of the set of wells.

4. The system of claim 1, wherein the wall of each of the set of wells defines a hexagonal footprint about its base surface.

5. The system of claim 1, wherein wells of the set of wells are arranged in a hexagonal close-packed configuration.

6. The system of claim 5, wherein the set of wells is divided into subsets of wells, wherein each well of each subset is fluidly connected by its set of channels to each of its surrounding wells of the subset of wells, and wherein the walls of each well of each subset prevent fluid communication between each subset and its surrounding subsets of wells.

7. The system of claim 6, wherein the subsets of wells are grouped in a hexagonal close-packed configuration.

8. The system of claim 1, wherein the substrate is optically transparent, and each of the set of wells is optically accessible from the direction perpendicular to the broad surface at a first side of the substrate.

9. The system of claim 8, further comprising a cell retrieval subsystem operable in an extraction mode that extracts at least one of the set of single cells from a well of the set of wells along a direction normal to the base surface of the well, in coordination with decoupling of the plate of the fluid delivery module from the substrate.

10. A system for isolating and analyzing single cells, comprising:

a chip comprising;

a substrate having a broad surface;

a set of wells defined at the broad surface of the substrate, each of the set of wells comprising a base surface defined within the substrate, an open surface directly opposing the base surface and coplanar with the broad surface of the substrate, and a wall extending between the base surface and the open surface; and

a fluid delivery module defining an inlet and an outlet, and fluidly connecting the inlet and outlet to each well in the set of wells;

an imaging subsystem, including an optical assembly positioned at a first side of the chip and oriented in a direction perpendicular to the broad surface;

a cell removal subsystem comprising a cell extractor, the cell removal subsystem positioned at a second side of the chip opposing the first side; and

a flow control subsystem fluidly coupled to the inlet and outlet of the chip, the flow control subsystem operable in a flow mode that applies one of a positive pressure gradient and a negative pressure gradient between the inlet and outlet of the fluid delivery module, wherein the flow mode further promotes a flow of a sample containing a single cell in a direction substantially parallel to the broad surface of the substrate, and captures the single cell within the wall of a well of the set of wells from a direction normal to the base surface of the well.

11. The system of claim 10, wherein the imaging subsystem is operable in an identification mode that generates an identification of a well of the set of wells that is retaining a single cell;

and wherein the cell removal subsystem is operable in an extraction mode that extracts the single cell from the well based on the identification.

12. The system of claim 11, wherein the imaging subsystem comprises a fluorescence microscope, and wherein the identification mode comprises detecting a fluorescence signal from the single cell.

13. The system of claim 10, wherein the fluid delivery module comprises a set of fluid pathways, wherein each fluid pathway of the set of fluid pathways fluidly connects the inlet to a corresponding well of the set of wells, and wherein each fluid pathway of the set of fluid pathways is of a substantially equal length.

14. The system of claim 10, wherein the wall of each of the set of wells reduces a characteristic dimension of each of the set of wells from the open surface to the base surface of each of the set of wells in a series of discrete steps.

15. The system of claim 10, wherein the wall of each well of the set of wells defines a set of channels, the set of channels fluidly connecting each well to at least one adjacent well of the set of wells.

16. The system of claim 15, wherein the set of wells is divided into subsets of wells, wherein each well of each subset is fluidly connected by its set of channels to each of its surrounding wells of the subset of wells, and wherein the walls of each well of each subset prevent fluid communication between each subset and its surrounding subsets of wells.

17. The system of claim 16, wherein the cell extractor of the cell removal subsystem comprises a repositionable capillary tube.

18. The system of claim 17, wherein each subset of wells is circumscribed by a closed curve in a plane parallel to the broad surface, and wherein an inner diameter of the capillary tube is smaller than a largest chord of the closed curve.

19. A system for isolating and analyzing single cells, comprising:

a chip comprising;

a substrate having a broad surface;

a set of wells defined at the broad surface of the substrate, each of the set of wells comprising a base surface defined within the substrate, an open surface directly opposing the base surface and coplanar with the broad surface of the substrate, and a wall extending between the base surface and the open surface, wherein the wall of each of the set of wells reduces a characteristic dimension of each of the set of wells from the open surface to the base surface of each of the set of wells in a series of discrete steps;

a fluid delivery module defining an inlet and an outlet, and fluidly connecting the inlet and outlet to each well in the set of wells;

an imaging subsystem, including an optical assembly positioned at a first side of the chip and oriented in a direction perpendicular to the broad surface; and

a cell removal subsystem comprising a cell extractor, the cell removal subsystem positioned at a second side of the chip opposing the first side.

20. A system for isolating and analyzing single cells, comprising:

a chip comprising;

a substrate having a broad surface;

a set of wells defined at the broad surface of the substrate, each of the set of wells comprising a base surface defined within the substrate, an open surface directly opposing the base surface and coplanar with the broad surface of the substrate, and a wall extending between the base surface and the open surface, wherein the wall of each well of the set of wells defines a set of channels, the set of channels fluidly connecting each well to at least one adjacent well of the set of wells, and wherein the set of wells is divided into subsets of wells, wherein each well of each subset is fluidly connected by its set of channels to each of its surrounding wells of the subset of wells, and wherein the walls of each well of each subset prevent fluid communication between each subset and its surrounding subsets of wells; and

a fluid delivery module defining an inlet and an outlet, and fluidly connecting the inlet and outlet to each well in the set of wells;

an imaging subsystem, including an optical assembly positioned at a first side of the chip and oriented in a direction perpendicular to the broad surface; and

a cell removal subsystem comprising a cell extractor, the cell removal subsystem positioned at a second side of the chip opposing the first side, wherein the cell extractor of the cell removal subsystem comprises a repositionable capillary tube.