Methods, systems and devices for multiple single-cell capturing and processing using microfluidics

• US 9,304,065 B2
• Filed: 02/28/2013
• Issued: 04/05/2016
• Est. Priority Date: 02/29/2012
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Fowler, Brian, Kimball, Jake, Maung, Myo Thu, May, Andrew, Norris, Michael C., Toppani, Dominique G., Unger, Marc A., Wang, Jing, West, Jason A. A.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Kingan, Timothy G
• Application Number:
  US13/781,307
• Publication Number:
  US 20130295602A1
• Time in Patent Office: 1,132 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/043 magnetic forces
• 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 2400/088 by specific surface properties
• 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 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• 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 1/34 : Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 15/1484 : microstructural devices

Abstract

Methods, systems, and devices are described for multiple single-cell capturing and processing utilizing microfluidics. Tools and techniques are provided for capturing, partitioning, and/or manipulating individual cells from a larger population of cells along with generating genetic information and/or reactions related to each individual cell. Different capture configurations may be utilized to capture individual cells and then processing each individual cell in a multi-chamber reaction configuration. Some embodiments may provide for specific target amplification, whole genome amplification, whole transcriptome amplification, real-time PCR preparation, copy number variation, preamplification, mRNA sequencing, and/or haplotyping of the multiple individual cells that have been partitioned from the larger population of cells. Some embodiments may provide for other applications. Some embodiments may be configured for imaging the individual cells or associated reaction products as part of the processing. Reaction products may be harvested and/or further analyzed in some cases.

Claims

1. A method for multiple single-cell capturing and processing using microfluidics, the method comprising:

a) loading a plurality of cells into a microfluidic device;

b) flowing the plurality of cells to a first capture configuration of the microfluidic device;

c) capturing a first single cell from the plurality of cells in the first capture configuration, wherein the first capture configuration comprises one or more physical barriers configured to capture a single cell from the plurality of cells and wherein the first capture configuration is coupled to a first multi-chamber reaction configuration;

d) flowing a first remaining plurality of cells from the plurality of cells through one or more first bypass channels to a second capture configuration wherein the second capture configuration comprises one or more physical barriers configured to capture a single cell from the plurality of cells and wherein the second capture configuration coupled to a second multi-chamber reaction configuration;

e) capturing a second single cell from the first remaining plurality of cells in the second capture configuration;

f) contacting the first and second single cells with a lysis reagent to release one or more cell constituents of each respective captured cell;

g) flowing the one or more constituents of the first captured cell into the first multi-chamber reaction configuration and flowing the one or more constituents of the second captured cell into the second multi-chamber reaction configuration; and

h) processing the one or more constituents of the first captured single cell in the first multi-chamber reaction configuration and processing the one or more constituents of the second captured single cell in the second multi-chamber reaction configuration to produce harvest products with respect to at least the first captured single cell and the second captured single cell,

wherein the multi-chamber reaction configurations each comprise;

a first reaction chamber coupled to the capture configuration and a second reaction chamber coupled to the first reaction chamber, wherein the capture configuration, the first reaction chamber, and the second reaction chamber are arranged so that fluid transported from the capture configuration to the second reaction chamber must pass through the first reaction chamber, and wherein the first and second reaction chambers are coupled though two non-overlapping fluid flow paths,

valves positioned to fluidically isolate the first and second reaction chambers from each other when closed, and,

a valve positioned to fluidically isolate the capture configuration from the multi-chamber reaction configuration when closed,

wherein steps (a)-(h) are carried out in the microfluidic device.

2. The method of claim 1, wherein the first and second capture configurations each comprise a capture nest comprising one or more physical barriers configured to form a concave surface and sized to hold only a single cell, coupled with a drain channel wherein a cell that enters the capture nest stops flow through the drain channel, but allows other cells from the plurality of cells to flow through one or more bypass channels into the output channel and enter a downstream capture configuration.

3. The method of claim 1, wherein the processing in step (h) comprises active mixing between chambers.

4. The method of claim 3, further comprising:

i) flowing a second remaining plurality of cells from the first remaining plurality of cells through-second bypass channels to a single output channel that delivers the cells to a third capture configuration.

5. The method of claim 1, wherein the released cell constituents comprise RNA, DNA and proteins.

6. The method of claim 1, wherein

the first capture configuration includes:

a pair of bypass channels coupled with a first input channel and a first output channel;

a first drain channel coupled with the first input channel and the first output channel; and

a first capture nest coupled with the first drain channel and configured to capture an individual cell from the plurality of cells; and

the second capture configuration includes;

a pair of bypass channels coupled with a second input channel and a second output channel, wherein the second input channel is coupled with the first output channel of the first capture configuration;

a second drain channel coupled with the second input channel and the second output channel; and

a second capture nest coupled with the second drain channel and configured to capture an individual cell from the first remaining plurality of cells.

7. The method of claim 3, wherein the processing of at least the first captured single cell and the second captured single cell to produce respective harvest products with respect to at least the first captured single cell and the second captured single cell on the microfluidic device comprises:

performing a thermal cycling procedure while flowing the one or more constituents through multiple chambers of a respective multi-chamber reaction configuration of the microfluidic device.

8. The method of claim 1, wherein performing the multistage processing of at least the first captured single cell and the second captured single cell to produce respective harvest products with respect to at least the first captured single cell and the second captured single cell on the microfluidic device comprises:

washing, in the microfluidic device, each respective captured cell with one or more reagents.

9. The method of claim 1, wherein performing the multistage processing of at least the first captured single cell and the second captured single cell to produce respective harvest products with respect to at least the first captured single cell and the second captured single cell on the microfluidic device comprises:

dosing, in the microfluidic device, each respective captured cell with one or more reagents.

10. The method of claim 1, further comprising:

imaging the respective captured cells within the microfluidic device.

11. The method of claim 1, wherein processing the one or more constituents of the first and second cells comprises:

performing a preamplification process within the microfluidic device.

12. The method of claim 1, wherein processing the one or more constituents of the first and second cells comprises:

a primer extension reaction, reverse transcription of mRNA and/or amplification of genomic DNA or cDNA.

13. The method of claim 1, further comprising:

harvesting the harvest products from a plurality of harvest wells of the microfluidic device.

14. The method of claim 13, further comprising:

processing the harvest products outside of the microfluidic device.

15. The method of claim 1, wherein capturing at least the first single cell or the second single cell comprises:

capturing at least the first single cell or the second single cell utilizing;

a capture compartment; and

a binding partner covering a discrete region of the capture compartment, where the discrete portion is sized so that only a single cell binds to the discrete region.

16. The method of claim 1, wherein capturing at least the first single cell or the second single cell comprises:

capturing at least the first single cell or the second single cell utilizing one or more capture supports, wherein each capture support comprises a binding partner distributed over at least a portion of the capture support.

17. The method of claim 16, wherein the one or more capture supports comprise one or more bead structures.

18. The method of claim 16, further comprising:

a capture feature configured to capture the capture support.

19. The microfluidic device of claim 1 wherein the second reaction chamber is adjacent to the first reaction chamber.

20. The microfluidic device of claim 1 wherein the multi-chamber reaction configurations each comprise a third reaction chamber that is coupled to the second reaction chamber though through two nonoverlapping fluid flow paths, and valves positioned to fluidically isolate the second and third reaction chambers from each other.