Cell capture system and method of use

• US 10,408,736 B1
• Filed: 05/22/2019
• Issued: 09/10/2019
• 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
• Primary Examiner(s): Wecker, Jennifer
• Application Number:
  US16/419,254
• Publication Number:
  US 20190271634A1
• Time in Patent Office: 111 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 15/0272 : with screening; with classification by filtering B01D takes precedence
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 15/1436 : the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 15/1463 : using image analysis for extracting features of the particle
• CPC Class Codes: G01N 15/1475 : using image analysis for extracting features of the particle
• 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 2015/1486 : Counting the particles
• CPC Class Codes: G01N 2015/1497 : Particle shape
• CPC Class Codes: G01N 2035/00158 : Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 33/48728 : Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10
• CPC Class Codes: G06V 20/69 : Microscopic objects, e.g. biological cells or cellular parts

Abstract

A cell capture system including an array, an inlet manifold, and an outlet manifold. The array includes a plurality of parallel pores, each pore including a chamber and a pore channel, an inlet channel fluidly connected to the chambers of the pores; an outlet channel fluidly connected to the pore channels of the pores. The inlet manifold is fluidly connected to the inlet channel, and the outlet channel is fluidly connected to the outlet channel. A cell removal tool is also disclosed, wherein the cell removal tool is configured to remove a captured cell from a pore chamber.

Claims

1. A method for capturing and processing cells in single-cell format, the method comprising:

providing a microfluidic network comprising an inlet channel at an upstream end, one or more outlet channels at a downstream end, and a set of structures in fluid communication with the inlet channel and the outlet channel, wherein flow from the inlet channel is configured to reach at least one of the one or more outlet channels only by way of the set of structures;

transmitting a fluid sample comprising a set of target cells into the microfluidic network;

capturing and partitioning the set of target cells in single-cell format, by way of the set of structures, wherein capturing and partitioning comprises isolating each of the set of target cells in single-cell format with an isolation material within the microfluidic network; and

transmitting a subportion of the set of target cells from the microfluidic network in a viable state.

2. The method of claim 1, wherein capturing and partitioning the set of target cells comprises separating each target cell from adjacent target cells with a set of barriers configured to fluidly isolate each target cell from adjacent target cells.

3. The method of claim 1, wherein the microfluidic network comprises channels with a characteristic dimension of less than 100 micrometers.

4. The method of claim 1, wherein transmitting the fluid sample comprises transmitting the fluid sample from the inlet channel to the outlet channel with a pressure difference of less than 10,000 Pascals.

5. The method of claim 1, wherein capturing and partitioning comprises segregating each of the set of target cells in single-cell format in a spatially arranged array within the microfluidic network.

6. The method of claim 5, wherein providing the microfluidic network comprises providing a plurality of pores, each pore in the plurality of pores displaced from at least one other pore in the plurality of pores and comprising:

a chamber having an upstream end, and a pore channel, fluidly coupled to the chamber at a downstream end of the chamber, having a pore channel width configured to block egress of a particle,

wherein the inlet channel is directly fluidly coupled to the upstream end of each chamber of the plurality of pores,

wherein the outlet channel is directly fluidly coupled to each pore channel of the plurality of pores, and

wherein flow from the inlet channel is configured to reach the outlet channel only by way of the plurality of parallel pores.

7. The method of claim 1, further comprising receiving a reagent fluid from a reagent container into the inlet channel, and performing a single-cell analysis of a target cell with the reagent.

8. The method of claim 7, wherein performing the single-cell analysis comprises performing at least one of:

a molecular reaction, immunostaining, single cell proteomic analysis, nucleic acid analysis, and genomic sequencing individually for each target cell.

9. The method of claim 8, wherein performing the single-cell analysis comprises performing a polymerase chain reaction (PCR) process individually for nucleic acids of the target cell.

10. The method of claim 1, wherein the set of target cells is co-captured with a set of functionalized microspheres, thereby forming a set of complexes, and wherein capturing and partitioning the set of target cells in single-cell format comprises capturing and partitioning the set of complexes in single-complex format.

11. The method of claim 1, wherein transmitting the subportion of the set of target cells from the microfluidic network comprises establishing communication between the microfluidic network and a cell removal tool, and delivering the subportion of the set of target cells into the cell removal tool.

12. The method of claim 11, wherein the cell removal tool comprises a tip configured to penetrate the microfluidic network, extract at least one target cell from the microfluidic network, and be reversibly retracted from the microfluidic network with at least one target cell.

13. A method for capturing and processing cells in single-cell format, the method comprising:

providing a microfluidic network comprising an inlet channel at an upstream end, an outlet channel at a downstream end, and a set of structures in fluid communication with the inlet channel and the outlet channel;

transmitting a fluid sample comprising a set of target cells into the microfluidic network;

capturing and partitioning the set of target cells in single-cell format, by way of the set of structures, wherein capturing and partitioning comprises delivering an isolation material into an isolation inlet of the microfluidic network, retaining the isolation material between the isolation inlet and an isolation outlet, and isolating each of the set of target cells in single-cell format with the isolation material within the microfluidic network; and

transmitting a subportion of the set of target cells from the microfluidic network in a viable state.

14. The method of claim 13, wherein delivering the isolation material comprises delivering oil into the isolation inlet.

15. The method of claim 14, wherein the microfluidic network comprises channels with a characteristic dimension of less than 100 micrometers.

16. The method of claim 15, wherein capturing and partitioning comprises segregating each of the set of target cells in single-cell format in a spatially arranged array within the microfluidic network, using the isolation material.

17. The method of claim 16, wherein the set of target cells is co-captured with a set of functionalized microspheres, thereby forming a set of complexes, and wherein capturing and partitioning the set of target cells in single-cell format comprises capturing and partitioning the set of complexes in single-complex format.

18. The method of claim 17, wherein capturing and partitioning the set of target cells comprises separating each target cell from adjacent target cells with a set of barriers configured to fluidly isolate each target cell from adjacent target cells, wherein the set of barriers block fluid flow between adjacent target cells.

19. The method of claim 18, further comprising delivering, by way of a fluid handling subsystem, a reagent fluid from a reagent container into the inlet channel, and performing a single-cell analysis of a target cell with the reagent, wherein the single-cell analysis comprises a molecular reaction.

20. The method of claim 13, wherein providing the microfluidic network comprises providing a plurality of pores, each pore in the plurality of pores displaced from at least one other pore in the plurality of pores and comprising:

a chamber having an upstream end, and a pore channel, fluidly coupled to the chamber at a downstream end of the chamber, wherein the inlet channel is directly fluidly coupled to the upstream end of each chamber of the plurality of pores, wherein the outlet channel is directly fluidly coupled to each pore channel of the plurality of pores, and wherein flow from the inlet channel is configured to reach the outlet channel only by way of the plurality of parallel pores.