System for isolating cells

• US 9,856,535 B2
• Filed: 05/28/2014
• Issued: 01/02/2018
• Est. Priority Date: 05/31/2013
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Denovo Sciences, Inc. (Bio-Rad Laboratories Incorporated)
• Inventors: Handique, Kalyan, Gogoi, Priyadarshini, Zhou, Yi, Sepehri, Saedeh, Siemer, Christopher
• Primary Examiner(s): DINES, KARLA A
• Application Number:
  US14/289,155
• Publication Number:
  US 20140357511A1
• Time in Patent Office: 1,315 Days
• Field of Search: 506 9
• 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 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• 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 23/12 Well or multiwell plates C12M25/04 takes precedence
• 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 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• 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 cells, comprising: a substrate having a broad surface; an array comprising a set of wells defined at the broad surface of the substrate, each well including: a base surface, an open surface directly opposing the base surface, defined at the broad surface of the substrate, and configured to receive one of a single cell and a single cluster of cells from a direction perpendicular to the broad surface of the substrate, and a set of channels that fluidly couple each well to at least one adjacent well; wherein the set of wells includes an interior subset and an exterior subset fluidly coupled to and surrounding the interior subset by way of the set of channels; and a fluid delivery module surrounding the array and fluidly coupled to each well in the set of wells.

Claims

1. A system for isolating cells in at least one of single cell format and single-cluster format, comprising:

a substrate having a broad surface;

an array comprising a set of wells defined at the broad surface of the substrate, each well of the set of wells including;

a base surface,

an open surface directly opposing the base surface, defined at the broad surface of the substrate, and sized to receive one of a single cell and a single cluster of cells from a direction perpendicular to the broad surface of the substrate, and

a first set of channels that fluidly couple each well to at least one adjacent well in the set of wells;

wherein the set of wells includes an interior subset and an exterior subset fluidly coupled to and surrounding the interior subset by way of the first set of channels; and

an encapsulation module removably coupled to the substrate at an interface, wherein the encapsulation module and the substrate cooperatively isolate each well of the set of wells to form an encapsulated array, the encapsulated array comprising the set of wells and an encapsulation layer of the encapsulation module;

a fluid delivery module surrounding the array and fluidly coupled to each well in the set of wells, the fluid delivery module comprising an inlet fluidically coupled to each of the set of wells by a second set of channels, wherein each of the second set of channels comprises an identical length; and

a perimeter channel directly fluidly coupled to the inlet and to each well of the exterior subset of the set of wells, the exterior subset of the set of wells positioned at an outermost edge of the array.

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

3. The system of claim 1, wherein at least two of the set of wells are separated by a channel of the first set of channels having a characteristic dimension less than the diameter of a single cell such that fluid flow between the two wells is unrestricted and single cell transport between the two wells is restricted.

4. The system of claim 1, further comprising a cell removal module that retains at least one of a single cell and a single cluster of cells away from the subset of the set of wells upon application of a pressure force by a pump.

5. The system of claim 4, the cell removal module comprising a cell removal tool including a tip comprising a first hollow needle and a second hollow needle, the tip configured to interface with a well of the set of wells in producing a first seal between the first hollow needle and an inlet channel of the well, and a second seal between the second hollow needle and an outlet channel of the well, the first and second hollow needle forming a unidirectional fluidic pathway in combination with the well.

6. The system of claim 1, wherein the encapsulation layer forms a boundary that isolates each well of the set of wells from each other well of the set of wells upon coupling of the encapsulation layer to the broad surface of the substrate.

7. The system of claim 6, further comprising a second encapsulation layer, wherein the second encapsulation layer is positioned between the set of wells and the substrate such that the second encapsulation layer defines the base surface of each of the set of wells, and wherein the encapsulation layer, the set of wells, and the second encapsulation layer cooperatively form an encapsulation volume.

8. The system of claim 1, wherein the encapsulation module further comprises an encapsulation matrix fluidly positioned between the encapsulation layer and the set of wells to form an encapsulation volume spanning the set of wells and the set of channels for each well in the set of wells.

9. The system of claim 8, wherein the encapsulation matrix is operable between a flow state and a set state, wherein in the flow state, the encapsulation matrix is substantially viscous and the encapsulation volume is distinct from the interior volume of the set of wells, and wherein in the set state, the encapsulation matrix is substantially rigid and the encapsulation volume occupies at least a portion of the interior volume of the set of wells.

10. The system of claim 9, wherein the encapsulation matrix is operable between the flow state and the set state by at least one of a photochemical reaction, a phase transition, a thermochemical reaction, and a polymerization reaction.

11. The system of claim 1, wherein the base surface of each of the set of wells is characterized by a surface morphology having a characteristic porosity, a characteristic concavity, and a cell retaining surface treatment.

12. The system of claim 1, wherein the array is separated from the broad surface of the substrate by a void, and wherein the set of wells are mutually fluidically coupled by the void.

13. The system of claim 1, wherein the open surface of each of the set of wells further comprises a lip with a characteristic dimension smaller than a characteristic dimension of the base surface.

14. The system of claim 1, wherein each of the set of wells includes a wall, each well separated from each other well by a corresponding wall, wherein each corresponding wall partially extends between the base surface and the open surface in defining a void between the wall of each of the set of wells and the base surface, each of the wells fluidically coupled to at least one other of the set of wells by way of the void.

15. The system of claim 1, wherein the set of wells comprises a plurality of subsets of wells, wherein the wells of each subset of the set of wells comprises a corresponding characteristic dimension, and wherein the corresponding characteristic dimension of each subset differs.

16. The system of claim 15, wherein a first subset of the set of wells is enclosed by a second subset of the set of wells, wherein the characteristic dimension of the first subset is larger than the characteristic dimension of the second subset such that the characteristic dimension decreases in a radial direction defined between a central region and an edge region of the array.

17. The system of claim 1, wherein a characteristic dimension of each well in the set of wells at least one of increases and decreases along a direction substantially aligned between the base surface and the open surface.