Methods for multiple single-cell capturing and processing using micro fluidics

• US 9,952,126 B2
• Filed: 02/28/2013
• Issued: 04/24/2018
• 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, Unger, Marc A., Wang, Jing, West, Jason A. A.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/781,313
• Publication Number:
  US 20130302807A1
• Time in Patent Office: 1,881 Days
• Field of Search: 435 912
• 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 of preamplification utilizing a microfluidic device configured to capture and to process individual cells from a plurality of cells, the method comprising:

priming the microfluidic device utilizing one or more solutions;

flowing the plurality of cells through channels of the microfluidic device such that individual cells from the plurality of cells are captured at individual capture sites of the microfluidic device, wherein each said individual capture site is configured to capture a single cell and is coupled with an individual microfluidic multi-chamber reaction configuration;

lysing the plurality of captured individual cells at the individual capture sites of the microfluidic device thereby releasing RNA from each lysed captured individual cell into the corresponding individual multi-chamber reaction configuration;

performing reverse transcription of the released RNA of each lysed captured individual cell within the multi-chamber reaction configuration to produce reverse transcription products associated with each respective individual cell; and

performing preamplification, within the multi-chamber reaction configuration, on the respective reverse transcription products associated with each respectively lysed individual cell to produce preamplification products associated with each individual captured cell,

wherein the multi-chamber reaction configuration comprises a first reaction chamber, a second reaction chamber, a third reaction chamber, and a fourth reaction chamber fluidically linked in series such that a solution can flow from the capture site into the first reaction chamber, from the first reaction chamber into the second reaction chamber, and then into the third reaction chamber, and then into the fourth reaction chamber; and

the

solution can flow from the first reaction chamber to any one of the second, third and fourth reaction chambers without flowing through any other of the second, third and fourth reaction chambers;

solution can flow from the second reaction chamber to the third reaction chamber without flowing through either of the first or fourth reaction chambers and solution can flow from the second reaction chamber to the fourth reaction chamber without flowing through the first or third reaction chambers;

solution can flow from the third reaction chamber to the fourth reaction chamber without flowing through either of the first or second reaction chambers.

2. The method of claim 1, further comprising:

exporting the preamplification products from each multi-chamber reaction configuration through a harvest inlet associated with the specific individual configuration from a plurality of harvest inlets of the microfluidic device.

3. The method of claim 1, further comprising loading the one or more solutions into the microfluidic device.

4. The method of claim 1, wherein the one or more solutions includes at least one or more reagents or one or more buffers.

5. The method of claim 1, further comprising loading the plurality of cells into the microfluidic device.

6. The method of claim 1, further comprising imaging one or more of the captured individual cells on the microfluidic device.

7. The method of claim 1, further comprising:

loading at least one or more lysis reagents, one or more reverse transcription reagents, or one or more preamplification reagents into the microfluidic device.

8. The method of claim 2, further comprising:

removing one or more protective layers of one or more harvesting inlets; and

harvesting the preamplification products from each respective harvest inlet from the plurality of harvest inlets of the microfluidic device.

9. The method of claim 1, further comprising staining the one or more individual capture cells on the microfluidic device.

10. The method of claim 9, further comprising determining whether the one or more individual captured cells are alive or dead based on the staining.

11. The method of claim 6, further comprising determining whether the one or more individual captured cells are alive or dead based on the imaging.

12. The method of claim 1, wherein the microfluidic device comprises:

a plurality of capture configurations coupled in series, each respective capture configuration comprising;

two or a plurality of bypass channels coupled with an input channel and an output channel;

a capture nest coupled with said bypass channels and with a drain channel,

wherein the capture nest is configured to capture an individual cell from the plurality of cells,

wherein a cell that enters the capture nest stops flow though the drain channel;

and wherein the capture nest and bypass channels are configured such that, when the capture nest is occupied by a captured cell, other cells of the plurality are diverted into the bypass channel(s) and flow to a downstream capture configuration;

a plurality of multi-chamber reaction configurations, wherein each respective multi-chamber reaction configuration is coupled with a respective capture configuration from the plurality of capture configurations and is configured for single-cell processing.

13. A method of preamplification utilizing a microfluidic device configured to capture and to process individual cells from a plurality of cells, the method comprising:

loading one or more solutions into the microfluidic device;

priming the microfluidic device utilizing the one or more solutions;

loading the plurality of cells into the microfluidic device;

flowing the plurality of cells through the microfluidic device such that individual cells from the plurality of cells are captured at individual capture sites of the microfluidic device;

imaging one or more of the captured individual cells on the microfluidic device;

determining for each individual capture site the absence of a cell, the presence of one cell, or the presence of two cells, and then;

loading at least one or more lysis reagents, one or more reverse transcription reagents, or one or more preamplification reagents into the microfluidic device;

lysing the plurality of captured individual cells at the individual capture sites of the microfluidic device;

performing reverse transcription, within the microfluidic device, on the plurality of individual lysed cells to produce reverse transcription products associated with each respective individual cell;

performing preamplification, within the microfluidic device, on the respective reverse transcription products associated with each respectively lysed individual cell to produce preamplification products associated with each individual capture cell;

delivering the preamplification products associated with each individual capture cell to a respective harvest inlet from a plurality of harvest inlets of the microfluidic device;

removing one or more protective layers of one or more harvesting inlets; and

harvesting the preamplification products from each respective harvest inlet from the plurality of harvest inlets of the microfluidic device.