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

• US 9,506,845 B2
• Filed: 02/28/2013
• Issued: 11/29/2016
• 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): HORLICK, KENNETH R
• Application Number:
  US13/781,318
• Publication Number:
  US 20130296196A1
• Time in Patent Office: 1,370 Days
• Field of Search: 435/6.12, 435/91.2
• 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 of amplifying mRNA sequences from individual eukaryotic cells using a microfluidic device configured to capture and to process individual cells from a plurality of cells, the method comprising:

a) priming the microfluidic device by flowing one or more solutions through a plurality of capture configurations of the device;

b) flowing a cell suspension comprising the plurality of cells in solution through channels of the microfluidic device such that individual cells from the plurality of cells are captured at individual capture sites of the plurality of capture configurations, wherein each capture configuration comprises

a capture site comprising one or more physical barriers configured to capture a single cell, an input channel through which cells can flow towards the capture site, an output channel through which cells can flow away from the capture site, a drain channel, and one or more bypass channels, and the physical barriers, drain channel, and bypass channel(s) are configured so that when capture site is unoccupied by a cell solution flows past the one or more physical barriers into the drain channel, and from the drain channel into the output channel, and when the capture site is occupied by a captured cell, flow through the drain channel is blocked by the captured cell and cells that are not captured are diverted into a bypass channel;

c) lysing the plurality of captured individual cells at the individual capture sites of the microfluidic device thereby releasing RNA from the individual captured cells at the individual capture sites;

d) performing separate individual reverse transcription reactions, within the microfluidic device, on the RNA released from the individual lysed cells to produce reverse transcription products associated with each respective individual captured cell; and

e) performing PCR, within the microfluidic device, on the respective reverse transcription products associated with each respectively lysed individual cell to produce PCR products associated with each individual capture cell.

2. The method of claim 1, further comprising:

delivering the PCR products associated with each individual captured cell to a respective harvest inlet 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 PCR reagents into the microfluidic device.

8. The method of claim 1, further comprising:

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

harvesting the PCR 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, further comprising:

preparing one or more libraries utilizing the PCR products associated with each individual captured cell.

13. The method of claim 12, wherein preparing the one or more libraries comprises:

determining a cDNA concentration from each respective harvest products associated with each individual cell; and

diluting each respective cDNA concentration to within a pre-determined concentration range.

14. The method of claim 13, further comprising:

preparing the diluted cDNA concentration for tagmentation to produce tagmentation products.

15. The method of claim 14, further comprising:

performing PCR amplification on the tagmentation products to produce PCR products.

16. The method of claim 1, wherein the microfluidic device comprises a plurality of capture configurations coupled in series, each respective capture configuration comprising:

a plurality of bypass channels

a capture nest comprising a concave surface formed by said one or more physical barriers, situated proximal to a junction of the input channel and the plurality of bypass channels and coupled with the drain channel,

wherein the capture nest is configured to capture an individual cell from the plurality of cells such that a remaining plurality of cells is diverted into at least one of the plurality of bypass channels when the individual cell is captured in the capture nest; and

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 configured for single-cell processing.

18. The method of claim 16 wherein performing PCR comprises active mixing between multiple chambers of the multi-chamber reaction configuration.

19. The method of claim 1 wherein the microfluidic device comprises a plurality of multi-chamber reaction configurations for performing PCR, wherein each respective multi-chamber reaction configuration is coupled with a respective capture configuration from the plurality of capture configurations.

20. The method of claim 19 wherein performing PCR comprises active mixing between multiple chambers of the multi-chamber configuration.

23. The method of claim 1 comprising exposing captured individual cells to one or more biological or chemical modulator(s) before lysis.

24. The method of claim 1 comprising the step of confirming the presence of a single captured cell by distinguishing the presence of a single cell from absence of a cell or the presence of two cells.

25. The method of claim 1, wherein the one or more bypass channels comprise a first bypass channel and a second bypass channel.

26. The method of claim 25, wherein the first bypass channel and the second bypass channel are symmetrically configured.

27. The method of claim 26, wherein the symmetrically configured first bypass channel and second bypass channel comprise a first wing configuration and a second wing configuration.

17. A method of mRNA processing 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 at least one or more lysis reagents or one or more reverse transcription reagents, into the microfluidic device;

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;

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 polymerase-mediated amplification within the microfluidic device, on the respective reverse transcription products associated with each respectively lysed individual cell to produce PCR products associated with each individual capture cell;

delivering the amplification 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 amplification products from each respective harvest inlet from the plurality of harvest inlets of the microfluidic device.

21. The method of claim 17 wherein the polymerase-mediated amplification is PCR.

22. The method of claim 17 wherein active mixing between multiple chambers of the multi-chamber reaction configuration occurs during the reverse transcription and/or polymerase-mediated amplification steps.