System for isolating and analyzing cells in a single-cell format

• US 10,512,914 B2
• Filed: 07/30/2018
• Issued: 12/24/2019
• 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:
  US16/049,173
• Publication Number:
  US 20180333723A1
• Time in Patent Office: 512 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 a population of target particles from a biological sample in single-particle format, comprising:

a substrate having a broad surface, wherein the substrate defines a set of wells arranged in a hexagonal close-packed configuration, wherein each well in the set of wells is configured to receive and retain a single target particle of the population of target particles and comprises;

a base surface defined within the substrate;

an open surface directly opposing the base surface; and

a well cavity defined by the base surface and the open surface, the well cavity having a longitudinal axis arranged normal to the broad surface of the substrate; and

a fluid delivery module comprising a fluid reservoir arranged superior to the broad surface of the substrate and directly fluidically connected to each well simultaneously in the set of wells at the open surface of each well, wherein a fluid delivery cavity between the fluid delivery module and the substrate defines a flow pathway configured for flow of a fluid volume received into the fluid reservoir, to the set of wells in a direction parallel to the broad surface of the substrate.

2. The system of claim 1, wherein the biological sample is a blood sample, and wherein the population of target particles is a subpopulation of circulating tumor cells contained within the blood sample.

3. The system of claim 1, wherein the well cavity defines a prismatic volume.

4. The system of claim 3, wherein the well cavity defines a hexagonal prism.

5. The system of claim 1, wherein each well further comprises a set of walls extending between the base surface and the open surface, wherein each wall in the set of walls has a wall thickness that is equal to or less than five microns.

6. The system of claim 5, wherein the set of walls of each well defines a hexagonal footprint about the base surface of each well.

7. The system of claim 5, wherein the set of walls of each well defines a hexagonal perimeter about the open surface of each well.

8. The system of claim 1, wherein the substrate defines the set of wells at a region having a total surface area of at least 144 square millimeters, and wherein the set of wells comprises at least 250,000 individual wells within the total surface area.

9. The system of claim 1, wherein the substrate defines the set of wells at a region having a total surface area, wherein more than 50% of the total surface area comprises the open surfaces of each well in the set of wells.

10. The system of claim 1, wherein each well in the set of wells defines a characteristic dimension that is less than 50 microns.

11. The system of claim 1, wherein the set of wells comprise a plurality of subsets of wells, wherein the wells in the subset of wells are fluidly connected to one another by a set of fluidic channels.

12. The system of claim 11, wherein each subset of wells includes six peripheral wells arranged about a central well within the plane of the broad face of the substrate, and each peripheral well is fluidly connected to the central well.

13. The system of claim 12, wherein a peripheral well of the subset of wells includes at least a first fluidic channel fluidly connecting the peripheral well to the central well, and a second fluidic channel fluidly connecting the peripheral well to a first and second adjacent peripheral well.

14. The system of claim 11, wherein the set of fluidic channels defines a characteristic dimension that is approximately five microns.

15. The system of claim 1, wherein the fluid delivery module includes an upper plate reversibly attached to the substrate, wherein the upper plate spans at least a portion of the broad surface of the substrate, and is arranged superior to the set of wells.

16. The system of claim 15, wherein the upper plate has a recess facing the broad face of the substrate and, upon attaching the upper plate to the substrate, the recess and the broad surface cooperatively define the fluid reservoir against the set of wells.

17. The system of claim 15, wherein the fluid delivery module further comprises a heating plate coupled to the recess of the upper plate and in thermal communication with the fluid delivery cavity, wherein a lateral portion of the heating plate is fluidly connected with a top surface of fluid within the fluid reservoir permitting convective flow of reagents through the reservoir.

18. The system of claim 1, wherein the fluid delivery module further comprises an inlet and an outlet fluidly coupled by and arranged on opposing sides of the fluid reservoir, and wherein the inlet and the outlet are fluidly coupled to a 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 the outlet of the fluid delivery module.

19. The system of claim 1, further comprising an imaging subsystem including an optical assembly positioned at a first side of the substrate and oriented in a direction perpendicular to the broad surface of the substrate;

and a particle extractor operable to access the set of wells from a direction normal to the broad surface of the substrate, wherein the imaging subsystem is operable in cooperation with the particle extractor to identify and extract a selected particle from a selected well of the set of wells.

20. The system of claim 1, further comprising a thermal control module arranged below the set of wells and adjacent to a bottom surface of the substrate directly opposing the broad surface of the substrate.