System and method for retrieving and analyzing particles

• US 10,466,160 B2
• Filed: 11/16/2017
• Issued: 11/05/2019
• Est. Priority Date: 08/01/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Celsee Diagnostics, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Handique, Kalyan, Payne, Austin, Sharma, Vishal, Gleason, Kyle, Gogoi, Priyadarshini, Ganesan, Karthik, Boniface, Brian, Chow, Will
• Primary Examiner(s): Wecker, Jennifer
• Application Number:
  US15/815,532
• Publication Number:
  US 20180088023A1
• Time in Patent Office: 719 Days
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0848 Specific forms of parts of containers
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/021 Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• 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: G01N 1/20 for flowing or falling materials G01N1/2035 G01N1/12, G01N1/14 take precedence
• CPC Class Codes: G01N 1/28 Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 1/4077 : by other techniques involving separation of suspended solids
• 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 15/1433 : using image recognition
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 2035/00158 : Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/1039 : Micropipettes, e.g. microcapillary tubes
• CPC Class Codes: G01N 35/00029 : provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: G01N 35/1011 : Control of the position or alignment of the transfer device
• CPC Class Codes: G06V 10/141 : Control of illumination
• CPC Class Codes: G06V 10/145 : Illumination specially adapted for pattern recognition, e.g. using gratings
• CPC Class Codes: G06V 10/147 : Details of sensors, e.g. sensor lenses fingerprint or palmprint sensors G06V40/13; vascular sensors G06V40/145; eye sensors G06V40/19
• CPC Class Codes: G06V 20/69 : Microscopic objects, e.g. biological cells or cellular parts

Abstract

A system and method for isolating and analyzing single cells, including: 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 cell retrieval, comprising:

a cell capture substrate comprising a first broad face and second broad face opposing the first broad face, the cell capture substrate defining;

a substrate body bounded by a plane, the plane coplanar with the first broad face;

a set of wells within the substrate body, each well of the set defining;

an aperture at the plane;

a base arranged between the aperture and the second broad face; and

a wall extending from the aperture to the base; and

a plurality of channels defined within the substrate body, wherein each well of the set is fluidly coupled to an adjacent well of the set by a respective channel of the plurality;

an imaging subsystem configured to sample image data of a target well of the set;

a cell retriever subsystem comprising;

a cell extractor comprising an extractor tip and an outlet, wherein;

the cell extractor defines a void between the extractor tip and the outlet; and

the extractor tip is positioned proximal the plane; and

a pump fluidly coupled to the void via the outlet;

an actuator configured to translate the cell extractor with respect to the cell capture substrate along an axis substantially normal the plane, the actuator operable to place the extractor tip for fluid communication with the target well; and

a control subsystem configured to;

receive the image data from the imaging subsystem;

based on the image data, control a motor of the actuator to position the extractor tip for fluid communication with the target well; and

in response to achieving positioning of the extractor tip for fluid communication with the target well, control the cell retriever to extract a cell from the target well.

2. The system of claim 1, wherein the imaging system comprises:

an optical sensor;

a filter module comprising;

an excitation filter and an emission filter;

a light source configured to transmit light through the excitation filter to reach the target well;

a lens defining an optical axis substantially normal the plane, wherein the lens;

focuses light from the excitation filter onto the cell; and

transmits light from the cell, through the emission filter, toward the optical sensor; and

a focus actuator configured to translate the lens substantially along the optical axis.

3. The system of claim 2, wherein:

the filter module further comprises a dichroic mirror configured to bisect intersecting planes defined by the excitation filter and the emission filter; and

the system further comprises a mirror configured to reflect light from the light source into the filter module, such that light from the light source is reflected by 90°

twice before reaching the cell capture substrate.

4. The system of claim 1, wherein the pump is a syringe pump fluidly coupled to the void by a flexible tube defining a dead-volume less than 20 microliters.

5. The system of claim 1, wherein the cell extractor is a capillary tube.

6. The system of claim 5, wherein the void defines an aperture at the extractor tip, the aperture defining a width between 10 microns and 50 microns.

7. The system of claim 1, further comprising a cell receptacle, wherein the extractor tip is operable to move from within the target well to within the cell receptacle.

8. The system of claim 1, further comprising a containment subsystem configured to prevent communication of an ambient environment with the set of wells.

9. The system of claim 1, wherein:

the set of wells is divided into subsets of wells, wherein each well of each subset is fluidly connected, by channels of the plurality, to each adjacent well of the subset of wells; and

the walls of each well of each subset prevent fluid communication between each subset and the adjacent subsets of wells.

10. The system of claim 9, wherein:

each subset of wells is circumscribed by a closed curve in the plane; and

an inner diameter of the extractor tip is smaller than a largest chord of the closed curve.

11. The system of claim 1, wherein the cell capture substrate further comprises a manifold defining an inlet and an outlet, wherein the inlet and outlet are fluidly coupled to each well in the set of wells via an interior of the manifold.

12. A system for cell retrieval, comprising:

a cell capture substrate comprising a broad face, the cell capture substrate defining;

a substrate body bounded by a plane, the plane coplanar with the broad face;

a set of wells within the substrate body, each well of the set defining;

an aperture at the plane;

a base within the substrate body; and

a wall extending from the aperture to the base; and

a manifold defining an inlet and an outlet, wherein the inlet and outlet are fluidly coupled to each well in the set of wells via an interior of the manifold;

an optical assembly opposing the plane across the cell capture substrate, the optical assembly defining an optical axis substantially normal the plane;

a cell retriever comprising;

a capillary tube positioned proximal the plane; and

a pump fluidly coupled to the capillary tube;

an actuator configured to translate the cell extractor with respect to the cell capture substrate along an axis substantially normal the plane, the actuator operable to place the extractor tip for fluid communication with the target well; and

a control subsystem configured to;

receive the image data from the imaging subsystem;

based on the image data, control a motor of the actuator to place the extractor tip for fluid communication with the target well; and

in response to achieving extractor tip positioning for fluid communication with the target well, control the cell retriever to extract a cell from the target well.

13. The system of claim 12, wherein the cell capture substrate further comprises a plurality of channels defined within the substrate body, wherein each well of the set is fluidly coupled to an adjacent well of the set by a respective channel of the plurality.

14. The system of claim 13, wherein:

the set of wells is divided into subsets of wells, wherein each well of each subset is fluidly connected, by channels of the plurality, to each adjacent well of the subset of wells; and

the walls of each well of each subset prevent fluid communication between each subset and the adjacent subsets of wells.

15. The system of claim 12, further comprising a substrate actuator configured to translate the cell capture substrate along an axis substantially parallel the plane.

16. The system of claim 15, further comprising a cell receptacle mechanically coupled to the cell capture substrate, wherein:

the cell receptacle moves together with the cell capture substrate during actuation of the substrate actuator;

the substrate actuator is operable to position the cell receptacle along a central axis of the capillary tube in a receiving configuration; and

in the receiving configuration, the cell retriever is operable to deposit contents of the capillary tube into the cell receptacle.

17. The system of claim 12, wherein the capillary tube defines an inner diameter between 10 microns and 50 microns.

18. The system of claim 12, wherein the optical assembly comprises a fluorescence microscope and an image sensor configured to sample image data from the fluorescence microscope.

19. The system of claim 12, wherein the set of wells contain a plurality of cells in single-cell format.