Cell capture system and method of use

• US 9,103,754 B2
• Filed: 07/25/2012
• Issued: 08/11/2015
• 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, Javdani, Saedeh Sepehri, Gogoi, Priyadarshini, Siemer, Christopher P
• Primary Examiner(s): Wecker, Jennifer
• Application Number:
  US13/557,510
• Publication Number:
  US 20130190212A1
• Time in Patent Office: 1,112 Days
• Field of Search: 422/73, 422500-507, 506/37, 506/40
• 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 cell capture system for capturing cells in single-cell format, comprising:

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

a plurality of parallel pores, each pore in the plurality of parallel pores linearly displaced from at least one other pore in the plurality of parallel pores, comprising;

a chamber, defined by a maximum width at an upstream end, configured to accept and hold a single cell, and

a pore channel fluidly coupled to the chamber at a downstream end of the chamber, the pore channel defined by a pore channel width, smaller than the upstream width of the chamber, and 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 parallel pores;

an outlet channel fluidly coupled to each pore channel of the plurality of parallel pores, wherein flow from the inlet channel reaches the outlet channel only by way of the plurality of parallel pores and provides a volumetric flow rate within inlet channel that is substantially equal to the volumetric flow rate within the outlet channel;

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 cell capture system of claim 1, wherein each pore in the plurality of parallel pores, the inlet channel, and the outlet channel are each defined by recesses on a singular broad face of the substrate, wherein the inlet channel is partially defined by a self-sealing wall oriented perpendicular to the broad face of the substrate, and wherein the system further includes a cell removal tool configured to penetrate the self-sealing wall in a direction parallel to the broad face of the substrate in order to retrieve a single captured cell.

3. The cell capture system of claim 1, wherein all pores in the plurality of parallel pores of the array are substantially identical.

4. The cell capture system of claim 1, wherein the system further comprises a second array substantially identical to the array, a second inlet channel substantially identical to the inlet channel and fluidly coupled to the second array, a second inlet manifold fluidly coupled to the second inlet channel, a second outlet channel fluidly coupled to the second array, and a second outlet manifold fluidly coupled to the second outlet;

wherein the array and the second array are fluidly independent of each other.

5. The cell capture system of claim 1, wherein the system comprises a set of arrays, each substantially identical to the array;

wherein the system comprises a single inlet manifold fluidly connecting the inlet channels of each array in the set of arrays in parallel.

6. The cell capture system of claim 5, wherein the system comprises a set of outlet manifolds, wherein each outlet channel of the set of arrays is independently fluidly coupled to a respective outlet manifold of the set of outlet manifolds.

7. The cell capture system of claim 5, wherein the outlet manifold is fluidly coupled to the outlet channels of the set of arrays in parallel.

8. The cell capture system of claim 7, wherein the outlet manifold comprises a plurality of outlet sub-manifolds fluidly connected in parallel, wherein each outlet sub-manifold fluidly connects a subset of the outlet channels of the set of arrays in parallel.

9. The cell capture system of claim 1, wherein the system comprises a second array fluidly coupled in series with the array, the second array including a second inlet channel and a second outlet channel coupled to a second set of pores defining a second pore channel width smaller than the pore channel width of the array, wherein the inlet channel of the array is fluidly coupled to the inlet manifold, wherein the outlet channel of the array is directly fluidly coupled to the second inlet channel of the second array, wherein the second outlet channel of the second array is fluidly coupled to the outlet manifold;

and wherein flow from the second inlet channel reaches the second outlet channel only by way of the second set of pores.

10. The cell capture system of claim 1, wherein each pore in the plurality of parallel pores comprises a set of fluidly coupled chambers having chambers of differing chamber widths, wherein the set of fluidly coupled chambers is linearly arranged by decreasing width, such that the chamber of the set of fluidly coupled chambers with the largest width is proximal the inlet channel of the array, and the chamber of the set of fluidly coupled chambers with the smallest width is proximal the outlet channel for a respective pore.

11. The cell capture system of claim 1, wherein the array, the inlet manifold, and the outlet manifold are cooperatively defined between a broad face of the substrate and a top layer coupled to the broad face of the substrate.

12. The cell capture system of claim 11, wherein the substrate and top layer comprise optically transparent materials.

13. The cell capture system of claim 12, 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 parallel pores.

14. The cell capture system of claim 13, wherein the system further comprises a cell removal tool configured to penetrate the inlet channel in a direction parallel to the broad face of the substrate, extract a captured cell from a pore, and be reversibly retracted from the inlet channel with the captured cell.

15. The cell capture system of claim 14, wherein the cell removal tool comprises a cannula defining a sealed perforating tip at a first end configured to penetrate the inlet channel, an aperture through a longitudinal wall of the cannula, wherein the aperture is configured to transmit a negative pressure from the cannula to the pore, and a lumen configured to be fluidly connected to a cell collection volume at a second end of the cannula, and wherein the inlet channel is partially defined by a self-sealing wall oriented perpendicular to the broad face of the substrate.

18. The cell capture system of claim 1, wherein the chamber of each pore in the array is configured to capture a circulating tumor cell, wherein the maximum width of the chamber is substantially between 25 and 50 micrometers, wherein the chamber has a height substantially between 30 micrometers and 50 micrometers, wherein the pore channel width of the pore channel is substantially between 7 and 10 micrometers, and wherein the pore channel has a length of substantially between 5 and 50 micrometers.

19. The cell capture system of claim 1, 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.

16. A cell sorting apparatus for capturing cells in single cell format, comprising:

a set of parallel arrays defined on a substrate broad face, each array in the set of parallel arrays comprising;

a plurality of parallel pores, each pore in the plurality of parallel pores linearly displaced from at least one other pore in the plurality of parallel pores and comprising;

a chamber, defined by a maximum width at an upstream end, configured to accept and hold a single cell, and

a pore channel coupled to the chamber at a downstream end of the chamber, the pore channel defined by a pore channel width, smaller than the upstream width of the chamber, and configured to block egress of the single cell,

wherein each chamber 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 parallel pores;

an outlet channel fluidly coupled to each pore channel of the plurality of parallel pores , wherein flow from the inlet channel reaches the outlet channel only by way of the plurality of parallel pores and provides a volumetric flow rate within inlet channel that is substantially equal to the volumetric flow rate within the outlet channel;

an inlet manifold, defined on the substrate broad face, that is fluidly coupled to each inlet channel of the set of parallel arrays;

an inlet, defined through the substrate thickness, that is fluidly connected to the inlet manifold;

an outlet manifold, defined on the substrate broad face, that is fluidly coupled to each outlet channel of the set of parallel arrays; and

an outlet, defined through the substrate thickness, that is fluidly connected to the outlet manifold.

17. The cell sorting apparatus of claim 16, wherein the inlet and outlet extend perpendicularly through the substrate thickness from a second substrate broad face to the inlet and outlet manifolds, respectively, wherein the second substrate broad face is parallel to and opposes the substrate broad face.

20. The cell sorting apparatus of claim 17, wherein the chamber of each pore is configured to capture a circulating tumor cell, wherein the maximum width of the chamber is substantially between 25 and 50 micrometers, wherein the chamber has a height substantially between 30 micrometers and 50 micrometers, wherein the pore channel width of the pore channel is substantially between 7 and 10 micrometers, and wherein the pore channel has a length of substantially between 5 and 50 micrometers.

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

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

a plurality of parallel pores, each pore in the plurality of parallel pores linearly displaced from at least one other pore in the plurality of parallel pores, comprising;

a chamber, defined by a maximum width at an upstream end, configured to accept and hold a single cell, and

a pore channel fluidly coupled to the chamber at a downstream end of the chamber, the pore channel defined by a pore channel width, smaller than the upstream width of the chamber, and 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 parallel pores, wherein the inlet channel is partially defined by a self-sealing wall oriented perpendicular to the broad face of the substrate;

an outlet channel fluidly coupled to each pore channel of the plurality of parallel pores, wherein flow from the inlet channel reaches the outlet channel only by way of the plurality of parallel pores and provides a volumetric flow rate within inlet channel that is substantially equal to the volumetric flow rate within the outlet channel;

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

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

a cell removal tool configured to penetrate the self-sealing wall in a direction parallel to the broad face of the substrate in order to retrieve a single captured cell.