Single cell capture with polymer capture films

• US 9,995,662 B2
• Filed: 10/06/2015
• Issued: 06/12/2018
• Est. Priority Date: 06/12/2014
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Husain, Syed A., Griswold, Bradley L., Slater, Michael, Espinoza, Patricio, Dunne, Jude, Hein, Glenn, Srinivasan, Maithreyan
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US14/876,365
• Publication Number:
  US 20160033378A1
• Time in Patent Office: 980 Days
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/165 Specific details about hydrophobic, oleophobic surfaces
• CPC Class Codes: B01L 2400/049 vacuum
• 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: B01L 3/50857 using arrays or bundles of open capillaries for holding samples
• CPC Class Codes: G01N 1/405 by adsorption or absorption

Abstract

The present invention provides methods, systems, assemblies, and articles for capturing single cells with a polymer capture film. In certain embodiments, the polymer capture films comprise a plurality of individual channels with top and bottom openings, where the channels are dimensioned such that a single cell is: i) is captured inside the channel, partially or substantially occluding the channel, when negative pressure is provided to the bottom opening; or ii) is captured by the top opening, but does not enter the channel, when negative pressure is provided to the bottom opening. In some embodiments, the channels of the polymer capture film align with the wells of a multi-well chip such that the cell, or the contents of the single cell, may be transferred to a corresponding well.

Claims

1. A method of isolating single cells comprising:

a) contacting a cell suspension with a cell-isolating system, wherein said cell-isolating system comprises;

i) a hydrophobic polymer capture film comprising a planar top surface, a bottom surface, and a plurality of individual holes extending through said polymer capture film, wherein each of said individual holes;

A) has a top opening in said top surface of said polymer capture film,

B) has a bottom opening in said bottom surface of said polymer capture film, and

C) is dimensioned such that one cell, and only said one cell, from said cell suspension is captured by said top opening on said planar top surface, when negative pressure is provided to said bottom opening; and

ii) a pneumatic device that provides said negative pressure, and

b) incubating said cell suspension with said cell-isolating system, wherein said pneumatic device provides said negative pressure, such that at least a portion of said plurality of individual holes in said polymer capture film captures one cell from said cell suspension on said planar top surface of said polymer capture film;

c) washing said polymer capture film to remove non-captured cells to generate a captured-cell polymer film comprising a plurality of captured cells;

d) bringing together said captured-cell polymer film and a multi-well device to generate an assembly, wherein said multi-well device has a plurality of well-openings, and wherein said top openings of said plurality of individual holes of said captured-cell polymer film matches one-for-one, and aligns with, said plurality of well openings in said multi-well device; and

e) treating said assembly such that one of said captured cells, or partial contents of one of said captured cells, for each of said portion of said plurality of individual holes is transferred into the corresponding well opening of said multi-well device.

2. The method of claim 1, wherein, prior to said bringing together and said treating, at least some of said plurality of wells of said multi-well device comprises reagents such that, after said bringing together and said treating, said one captured cell, or contents of said one captured cell, are modified by said reagents.

3. The method of claim 2, wherein said reagents are selected from the group consisting of:

a buffer, a lyse buffer, a polymerase, sequencing reagents, proteinase, and nucleotides.

4. The method of claim 2, further comprising sequencing nucleic acid from said captured cell or contents of said captured cell.

5. The method of claim 1, wherein said cell isolating system further comprises a porous layer comprising a porous material that allows liquid, but not cells, to pass therethrough, wherein said porous layer is in contact with said bottom surface of said polymer capture film such that said bottom opening of each of said plurality of individuals holes is covered by said porous material.

6. The method of claim 1, wherein said cell isolating system further comprises a film support component comprising a substrate with a plurality of individual holes therethrough which align one-for-one with said plurality of individual holes in said polymer capture film, wherein said film support component is in contact with, and supports, said bottom surface of said polymer capture film.

7. The method of claim 1, further comprising at least one film guide component attached to, or integral with, said polymer capture film, wherein said film guide component allows alignment of said plurality of individual holes in said polymer capture film one-for-one with wells in a multi-well device and/or holes in a polymer film support plate.

8. The method of claim 1, wherein said polymer capture film comprises at least 500 individual holes.

9. The method of claim 1, wherein said polymer capture film has a thickness between 10 μ

m and 50 μ

m, and wherein said top surface has an area between 1 cm² and 30 cm².

10. The method of claim 1, wherein each of said individual holes has a diameter of about 2-6 μ

m.

11. The method of claim 1, wherein said cell isolating system further comprises a film support component and a housing base, wherein said housing base contains said polymer capture film and said film support component.

12. The method of claim 11, wherein said housing base comprises at least one base feature selected from the group consisting of:

a vacuum connection port, at least two guide pins for aligning said polymer capture film and said film support component, a film support plate recess, a liquid reservoir, a gasket, and at least one connection rod for connection to a housing top.

13. The method of claim 11, further comprising a housing top, wherein said housing top is connected to a housing base to enclose said polymer capture film.

14. The method of claim 13, wherein said housing top comprises at least one top feature selected from the group consisting of:

an inlet port, an outlet port, a slot array, a guide pin receiver, and a connection rod receiver.

15. The method of claim 11, wherein said cell isolating system further comprises a component selected from the group consisting of:

a vacuum pump that provides said negative pressure, a centrifuge motor configured to spin said housing base, a diaphragm pump, a chip alignment component, a swing arm attached to a swing arm support rod, a sample delivery component, a user interface display, and at least one waste container.

16. The method of claim 1, wherein said one captured cell is selected from the group consisting of:

a platelet, a red blood cell, a neutrophil, a lymphocyte, a fibroblast, and a macrophage.

17. The method of claim 1, wherein said bringing together comprises attaching said captured-cell polymer film to said multi-well device with double-sided adhesive having holes formed therein corresponding to said well-openings in said multi-well device.