System and method for isolating and analyzing cells

• US 10,449,543 B2
• Filed: 07/30/2018
• Issued: 10/22/2019
• 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:
  US16/049,240
• Publication Number:
  US 20180353962A1
• Time in Patent Office: 449 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 method for analyzing a population of target cells from a biological sample, comprising:

providing a substrate comprising a set of wells, wherein each well in the set of wells has a hexagonal open surface defined at the broad surface of a substrate, a base surface directly opposing the open surface, and a set of walls extending between the base surface and the open surface;

distributing the biological sample containing the population of target cells across the hexagonal open surface of each well of the set of wells,

capturing the population of target cells through the hexagonal open surface of each well into the set of wells in single-cell format, wherein exactly one of a single target cell settles into a single well of the set of wells in a direction perpendicular to the broad face of the substrate;

flowing a reagent in a direction parallel to the broad surface of the substrate at a predetermined flowrate between 1 milliliter per second and 1 milliliter per minute through a fluid reservoir laterally superior and directly fluidly connected to the set of wells;

receiving the reagent through the hexagonal open surface of each well into the set of wells, without egressing the population of target cells from the set of wells by means of the predetermined flowrate; and

analyzing the population of target cells in single-cell format.

2. The method of claim 1, further comprising coupling the substrate to an upper plate having a recess facing the broad surface of the substrate, wherein the recess of the upper plate and the broad surface of the substrate define the fluid reservoir.

3. The method of claim 2, wherein coupling the substrate to the upper plate further comprises aligning the recess of the upper plate directly above the set of wells.

4. The method of claim 2, further comprising, upon coupling the substrate to the upper plate, forming a hermetic seal at the interface of a region of the recess surrounding the set of wells and the substrate.

5. The method of claim 2, wherein coupling the substrate to the upper plate applies a force on the reagent in the fluid reservoir to form a uniform fluid layer of the reagent against the set of wells.

6. The method of claim 2, further comprising, upon flowing the reagent through the fluid reservoir, applying heat to the reagent with a heating element embedded within the recess of the upper plate, thereby permitting convective flow of the reagent through the fluid reservoir along a fluid path.

7. The method of claim 1, further comprising rotating the substrate about an axis of rotation parallel to and offset from the broad surface of the substrate, at an angular velocity less than 2000 revolutions per minute, thereby increasing capture efficiency of the population of target cells to the set of wells.

8. The method of claim 1, further comprising delivering reagent from an inlet to a first end of the fluid reservoir through a set of fluidic pathways, wherein each fluid pathway of the set of fluid pathways is of a substantially identical length, permitting uniform distribution of reagents across the fluid reservoir.

9. The method of claim 1, wherein receiving the reagent into the set of wells comprises receiving a lysing reagent for extracting intracellular content of the population of target cells.

10. The method of claim 9, further comprising binding a probe to intracellular content of the population of target cells.

11. The method of claim 1, further comprising transmitting heat to the set of wells with a thermal control module, wherein each well of the set of wells receives substantially equivalent heat, and wherein the thermal control module is arranged below the set of wells and adjacent to a bottom surface of the substrate directly opposing the broad surface of the substrate.

12. The method of claim 11, wherein transmitting heat to the set of wells facilitates polymerase chain reaction (PCR) for intracellular content of the population of target cells.

13. The method of claim 1, further comprising removing heat from the set of wells.

14. The method of claim 1, wherein the sum total area of the open surfaces of the set of wells is greater than 50% of the total area of the region of the substrate at which the set of wells are defined.

15. The method of claim 14, wherein the sum total area of the open surfaces of the set of wells is greater than 80% of the total area of the region of the substrate at which the set of wells are defined.

16. The method 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.

17. The method of claim 1, wherein the well cavity of each well defines a hexagonal prism, wherein the base surface of the well cavity defines a hexagon directly opposing and parallel the hexagonal open surface.

18. The method of claim 1, wherein the set of walls of each well have a wall thickness that equal to or less than five microns.

19. The method of claim 1, wherein each well in the set of wells has a characteristic dimension less than 50 microns.

20. The method of claim 1, wherein the set of wells is arranged in a hexagonal close-packed configuration.