Cell capture system and method of use

• US 9,513,195 B2
• Filed: 01/28/2015
• Issued: 12/06/2016
• Est. Priority Date: 08/01/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Denovo Sciences, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Handique, Kalyan, Gogoi, Priyadarshini, Siemer, Christopher, Javdani, Saedeh Sepehri
• Primary Examiner(s): Wecker, Jennifer
• Application Number:
  US14/607,918
• Publication Number:
  US 20150204766A1
• Time in Patent Office: 678 Days
• US Class Current: 1/1
• 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 2015/149 : Sorting 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 system for capturing cells in single-cell format, comprising:

an array defined on a broad face of a substrate, comprising;

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 configured to accept a single cell, and a pore channel, fluidly coupled to the chamber at a downstream end of the chamber, defined by a pore channel width configured to block egress of the single cell, wherein each chamber of the plurality of parallel pores is physically coextensive with an adjacent chamber by a barrier configured to substantially block fluid flow in a direction parallel to fluid flow through the pore channel;

an inlet channel directly fluidly coupled to the upstream end of each chamber of the plurality of pores; and

an outlet channel fluidly coupled to each pore channel of the plurality of pores, wherein flow from the inlet channel is configured to reach the outlet channel only by way of the plurality of pores;

an inlet manifold, defined on the broad face of the substrate, fluidly coupled to the inlet channel of the array; and

an outlet manifold, defined on the broad face of the substrate, fluidly coupled to the outlet channel of the array.

2. The system of claim 1, wherein the inlet channel is partially defined by a self-sealing wall oriented perpendicular to the broad face of the substrate.

3. The system of claim 2, further including a cell removal tool that penetrates the self-sealing wall in a direction parallel to the broad face of the substrate in order to retrieve a single captured cell from one of the plurality of pores.

4. The system of claim 3, wherein the cell removal tool comprises a cannula defining a perforating tip at a first end configured to penetrate the inlet channel by way of the self-sealing wall, an aperture through a longitudinal wall of the cannula, wherein the aperture is configured to transmit a negative pressure from the cannula to retrieve the single captured cell, and a lumen configured to be fluidly connected to a cell collection volume at a second end of the cannula.

5. The system of claim 1, wherein fluid coupling between the inlet channel and the outlet channel of the array, only by way of the plurality of pores, provides a volumetric fluid flow rate within inlet channel that is substantially equal to the volumetric fluid flow rate within the outlet channel.

6. The system of claim 1, further comprising a second array having a second plurality of pores, a second inlet channel fluidly coupled to the second array, and a second outlet channel fluidly coupled to the second inlet channel only by way of the second plurality of parallel pores.

7. The system of claim 6, wherein outlet manifold is fluidly coupled to the second inlet channel of the second array, thereby coupling the array and the second array in series.

8. The system of claim 7, wherein pores of the plurality of pores have a first pore channel width and pores of the second plurality of pores have a second pore channel width smaller than the first pore channel width.

9. The system of claim 1, wherein each pore in the plurality of pores comprises a set of fluidly coupled chambers having chambers of differing chamber widths, wherein the set of fluidly coupled chambers is linearly arranged according to decreasing width from a large width to a small width, such that a first chamber of the set of fluidly coupled chambers with the large width is proximal the inlet channel of the array, and a last chamber of the set of fluidly coupled chambers with the small width is proximal the outlet channel for a respective pore.

10. The system of claim 1, wherein the substrate further comprises a 90 degree illumination element disposed within the substrate, between the broad face of the substrate and a directly opposing face to the broad face of the substrate, adjacent the array, wherein the 90 degree illumination element is separated from the inlet channel by an air gap, configured to reflect incident light at a 90 degree angle longitudinally into each pore of the plurality of pores.

11. A system for capturing cells in single-cell format, comprising:

an array defined on a broad face of a substrate, comprising;

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 configured to accept a single cell, and a pore channel, fluidly coupled to the chamber at a downstream end of the chamber, defined by a pore channel width configured to block egress of the single cell, wherein each chamber of the plurality of parallel pores is physically coextensive with an adjacent chamber by a barrier configured to substantially block fluid flow in a direction parallel to fluid flow through the pore channel;

an inlet channel, directly fluidly coupled to the upstream end of each chamber of the plurality of pores, partially defined by a self-sealing wall oriented perpendicular to the broad face of the substrate; and

an outlet channel fluidly coupled to each pore channel of the plurality of pores, wherein flow from the inlet channel is configured to reach the outlet channel only by way of the plurality of pores;

an inlet manifold, fluidly coupled to the inlet channel of the array; and

an outlet manifold, fluidly coupled to the outlet channel of the array.

12. The system of claim 11, further including a cell removal tool that penetrates the self-sealing wall in a direction parallel to the broad face of the substrate in order to retrieve a single captured cell from one of the plurality of pores.

13. The system of claim 12, wherein the cell removal tool comprises a cannula defining a perforating tip at a first end configured to penetrate the inlet channel by way of the self-sealing wall, an aperture through a longitudinal wall of the cannula, wherein the aperture is configured to transmit a negative pressure from the cannula to retrieve the single captured cell, and a lumen configured to be fluidly connected to a cell collection volume at a second end of the cannula.

14. The system of claim 11, wherein flow from the inlet channel reaches the outlet channel only by way of the plurality of pores, and wherein fluid coupling between the inlet channel and the outlet channel of the array, only by way of the plurality of pores, provides a volumetric fluid flow rate within inlet channel that is substantially equal to the volumetric fluid flow rate within the outlet channel.

15. The system of claim 11, further comprising a second array having a second plurality of pores, a second inlet channel fluidly coupled to the second array, and a second outlet channel fluidly coupled to the second inlet channel only by way of the second plurality of parallel pores, wherein outlet manifold is fluidly coupled to the second inlet channel of the second array, thereby coupling the array and the second array in series.

16. The system of claim 11, further including an isolation module comprising an isolation inlet fluidly coupled between a first end of the inlet channel and a first end of the outlet channel at an inlet end of the array, and an isolation outlet fluidly coupled between a second end of the inlet channel and a second end of the outlet channel at an outlet end of the array, wherein the isolation module is configured to facilitate transfer of an isolation material into each chamber of the array, thereby isolating captured single cells in individual chambers of the array.

17. A method for capturing cells in single-cell format, comprising:

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 configured to accept a single cell, and a pore channel, fluidly coupled to the chamber at a downstream end of the chamber, defined by a pore channel width configured to block egress of the single cell, wherein each chamber of the plurality of parallel pores is physically coextensive with an adjacent chamber by a barrier configured to substantially block fluid flow in a direction parallel to fluid flow through the pore channel;

an inlet channel directly fluidly coupled to the upstream end of each chamber of the plurality of pores; and

an outlet channel fluidly coupled to each pore channel of the plurality of pores;

providing flow from the inlet channel to the outlet channel only by way of the plurality of pores;

receiving a sample of cells at the inlet channel; and

delivering the sample through the inlet channel, into the plurality of pores, and into the outlet channel, thereby capturing cells in single-cell format.

18. The method of claim 17, wherein providing the plurality of pores comprises providing the plurality of pores defined on a broad face of a substrate, and wherein the method further comprises providing a self-sealing wall oriented perpendicular to the broad face of the substrate, the self-sealing wall at least partially defining the inlet channel.

19. The method of claim 18, further comprising receiving a cell removal tool into the inlet channel, the cell removal tool penetrating the self-sealing wall in a direction parallel to the broad face of the substrate.

20. The method of claim 19, further comprising delivering a single captured cell from at least one pore of the plurality of pores and into an aperture of the cell removal tool.