U.S. patent application number 14/909195 was filed with the patent office on 2016-07-28 for system and method for laser lysis.
The applicant listed for this patent is Laimonas BAUSKAS, Shih-Hui (Joseph) CHAO, Weimin GAO, Andrew HATCH, Jeff HOUKAL, Deirdre MELDRUM, David Wayne RICHARDSON, Thai TRAN. Invention is credited to Shih-Hui (Joseph) Chao, Weimin Gao, Andrew Hatch, Jeff Houkal, Laimonas Kelbauskas, Deirdre Meldrum, David Richardson, Thai Tran.
Application Number | 20160215254 14/909195 |
Document ID | / |
Family ID | 52744386 |
Filed Date | 2016-07-28 |
United States Patent
Application |
20160215254 |
Kind Code |
A1 |
Meldrum; Deirdre ; et
al. |
July 28, 2016 |
SYSTEM AND METHOD FOR LASER LYSIS
Abstract
The present invention provides a system and method for lysing
individual cells in situ, including the steps of capturing a tissue
sample comprising a cellular content, subjecting the tissue sample
to a stream of continuous fluid flow, lysing a selected area of the
tissue sample with a laser, thereby releasing at least a portion of
the cellular content from the tissue sample, recovering at least
one target molecule from the cellular content in the stream, and
processing the at least one target molecule.
Inventors: |
Meldrum; Deirdre; (Phoenix,
AZ) ; Chao; Shih-Hui (Joseph); (Phoenix, AZ) ;
Tran; Thai; (Phoenix, AZ) ; Kelbauskas; Laimonas;
(Gilbert, AZ) ; Houkal; Jeff; (Los Angeles,
CA) ; Hatch; Andrew; (Tempe, AZ) ; Gao;
Weimin; (Chandler, AZ) ; Richardson; David;
(Chandler, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MELDRUM; Deirdre
CHAO; Shih-Hui (Joseph)
TRAN; Thai
BAUSKAS; Laimonas
HOUKAL; Jeff
HATCH; Andrew
GAO; Weimin
RICHARDSON; David Wayne |
Phoenix
Phoenix
Tempe
Gilbert
Los Angeles
Tempe
Chandler
Chandler |
AZ
AZ
AZ
AZ
CA
AZ
AZ
AZ |
US
US
US
US
US
US
US
US |
|
|
Family ID: |
52744386 |
Appl. No.: |
14/909195 |
Filed: |
September 23, 2014 |
PCT Filed: |
September 23, 2014 |
PCT NO: |
PCT/US14/56960 |
371 Date: |
February 1, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61883739 |
Sep 27, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/0668 20130101;
C12M 47/06 20130101; B01L 2300/0829 20130101; C12Q 1/686 20130101;
G01N 1/286 20130101; C12Q 1/6848 20130101; G01N 2001/2886 20130101;
C12Q 1/6848 20130101; B01L 3/0293 20130101; C12Q 2547/101 20130101;
B01L 3/502761 20130101; C12Q 1/6841 20130101; B01L 2300/0864
20130101; C12Q 1/6806 20130101; B01L 2300/0816 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; G01N 1/28 20060101 G01N001/28; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under R21
CA174412 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of lysing individual cells in situ, comprising: lysing
a selected area of a tissue sample being subjected to a steam of
continuous fluid flow with a laser, thereby releasing at least a
portion of a cellular content from the tissue sample.
2. The method of claim 1, wherein the tissue sample is a live
tissue, and wherein the selected area is a single cell of the live
tissue.
3. The method of claim 1, further comprising recovering at least
one target molecule from cellular content in the stream.
4. The method of claim 3, further comprising processing the at
least one target molecule.
5. The method of claim 4, wherein processing comprises single-cell
quantitative in situ RT-PCR.
6. The method of claim 1, wherein the tissue sample has a maximum
dimension of about 200 .mu.m to about 300 .mu.m.
7. The method of claim 1, wherein the laser is a two-photon
laser.
8. The method of claim 1, wherein the step of capturing further
includes collecting the tissue sample with a tissue collection
device.
9. The method of claim 8, further including aspirating the tissue
sample with the tissue collection device, and automatically
depositing the tissue sample on an analysis platform.
10. The method of claim 9, wherein the analysis platform is
removably coupled to the tissue collection device.
11. The method of claim 9, wherein the analysis platform is a
microfluidic chip comprising a cage configured to retain the tissue
sample.
12. The method of claim 11, wherein the cage comprises a fluid
channel and a plurality of posts spaced apart within the fluid
channel.
13. A system for lysing cells, comprising: a microfluidic chip
having a fluid channel and a cage disposed within the fluid
channel, the cage sized to capture a tissue sample; a microscope
for observing the tissue sample; a laser for irradiating a selected
area of the tissue sample; and a downstream module coupled to the
microfluidic chip for processing a target molecule collected from
the tissue sample; wherein irradiating the tissue sample with the
laser lyses the selected area of the tissue sample, thereby
releasing the target molecule from the tissue sample into the fluid
channel.
14. The system of claim 13, wherein the laser is a two-photon
laser.
15. The system of claim 13, wherein the module is a quantitative
RT-PCR module.
16. A device, comprising: an apparatus including a body defining a
passage, a capillary in fluid communication with an inlet of the
passage, and an output port in fluid communication with an outlet
of the passage; and a microfluidic chip removably coupled to the
body, the microfluidic chip including a first fluid channel having
an inlet and an outlet in communication with the passage, and a
cage positioned between the inlet and the outlet, the cage
comprising a plurality of structures sized to retain a tissue
sample in the first fluid channel.
17. The device of claim 16, further comprising an RT-PCR module in
communication with the first fluid channel.
18. The device of claim 16, wherein the microfluidic chip further
comprises a second fluid channel in communication with the first
fluid channel downstream of the cage, and a third fluid channel in
communication with the first fluid channel downstream of the second
fluid channel, wherein the second fluid channel is in communication
with a source of RT-PCR reagents, and wherein the third fluid
channel is in communication with a source of material for
encapsulating the contents of the first fluid stream and the second
fluid stream.
19. The device of claim 18, further comprising a motorized platform
for dispensing liquid from the microfluidic chip into a
container.
20. The device of claim 19, further comprising a sensor for
monitoring at least one of the motorized platform and the
container, wherein the sensor is configured to reduce carryover
contamination.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. national stage application under
35 U.S.C. .sctn.371 of PCT Application No. PCT/US2014/056960, filed
Sep. 23, 2014, published as WO2015/048009, which claims priority
under 35 U.S.C. .sctn.119 from U.S. Provisional Application No.
61/883,739, filed Sep. 27, 2013, each of which is incorporated
herein by reference in its entirety.
BACKGROUND
[0003] The present disclosure relates to cellular analysis
techniques, and more particularly, to a system and method for in
situ laser lysis for analysis of live tissue at the single cell
resolution.
[0004] Cells in a live tissue have heterogeneous responses to
environmental cues because of their differences in cell types,
locations, exposure to blood supply, malignancy, and/or infection.
For instance, cell-cell communication between cancer cells and
their environments at the primary tumor and distant metastasis
sites have been shown to be important for cancer development (Zhang
W, et al. 2011, Cancer biology & therapy 11: 150-156; Calorini
L, et al. 2010, Cell communication and signaling: CCS 8: 24).
Recent technological advances in assessing gene expression at the
single cell level have enabled advancements in the way in which
investigators study diseases.
[0005] The introduction of commercially available microfluidic
high-throughput systems further enables researchers to investigate
problems at a larger scale (Citri A, et al. 2012, Nature Protocols
7: 118-127; Guo G, et al. 2010, Developmental Cell 18: 675-685).
However, such methodologies may require cells to be dissociated
from their native environments and therefore, may obscure valuable
biological states that are influenced by multicellular complexity
in situ. Current tools are not able to capture lysate from
individual cells in situ, making it difficult to analyze the
individual cells given the short-lived nature of RNA, which can
degrade on time scales of seconds to minutes, as well as the fast
response time of cellular gene-expression on the order of
minutes.
[0006] One single-cell lysate harvesting approach includes a
vacuum-like mechanical probe to continuously release lysis buffer
through a microchannel and draw in liquid surrounding target cells
(Sarkar A, et al. 2014, Nature Communications 5). However, the size
of the probe head resulted in a physical limitation with respect to
accessing the target cells. Moreover, the time required for cell
lysis is relatively lengthy at around 1 minute. Finally, harvesting
a quantity of cells to acquire statistically significant results
can be in excess of one hour, which may trigger cellular stress
responses to the surrounding cells. Accordingly, it would be useful
to provide a system and method for single cell analysis which can
be accomplished under biologically relevant conditions and on
biologically relevant time-scales.
SUMMARY OF THE DISCLOSURE
[0007] The present invention overcomes the aforementioned drawbacks
by providing a system and method for in situ laser lysis for
analysis of live tissue at the single cell resolution.
[0008] In accordance with one aspect of the present disclosure, a
method for lysing individual cells in situ includes the steps of
capturing a tissue sample comprising a cellular content, subjecting
the tissue sample to a stream of continuous fluid flow, lysing a
selected area of the tissue sample with a laser, thereby releasing
at least a portion of the cellular content from the tissue sample,
recovering at least one target molecule from the cellular content
in the stream, and processing the at least one target molecule.
[0009] In accordance with another aspect of the present disclosure,
a system for lysing cells, includes a microfluidic chip having a
fluid channel and a cage disposed within the fluid channel, the
cage sized to capture a tissue sample, a microscope for observing
the tissue sample, a laser for irradiating a selected area of the
tissue sample, and a downstream module coupled to the microfluidic
chip for processing a target molecule collected from the tissue
sample. Irradiating the tissue sample with the laser lyses the
selected area of the tissue sample, thereby releasing the target
molecule from the tissue sample into the fluid channel.
[0010] In accordance with a further aspect of the present
disclosure, a device includes an apparatus including a body
defining a passage, a capillary in fluid communication with an
inlet of the passage, and an output port in fluid communication
with an outlet of the passage. The device further includes a
microfluidic chip removably coupled to the body, the microfluidic
chip including a first fluid channel having an inlet and an outlet
in communication with the passage, and a cage positioned between
the inlet and the outlet, the cage comprising a plurality of
structures sized to retain a tissue sample in the first fluid
channel.
[0011] The foregoing and other aspects and advantages of the
invention will appear from the following description. In the
description, reference is made to the accompanying drawings which
form a part hereof, and in which there is shown by way of
illustration a preferred embodiment of the invention. Such
embodiment does not necessarily represent the full scope of the
invention, however, and reference is made therefore to the claims
and herein for interpreting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is an illustration of one example of a tissue
collection device and microfluidic chip according to the present
disclosure.
[0013] FIG. 2 is a schematic illustration of a tissue capture cage
within a microfluidic channel of the microfluidic chip of FIG.
1.
[0014] FIG. 3A is a schematic illustration of a laser lysis process
performed on a captured tissue sample. The tissue sample may be
irradiated with a two-photon laser and the lysate collected and
combined with one or more components prior to encapsulation.
[0015] FIG. 3B is an enlarged view of an actual tissue sample
schematically shown in FIG. 3A.
[0016] FIG. 4A is an illustration of single-photon (1P) laser
excitation intensity profile.
[0017] FIG. 4B is an illustration of two-photon (2P) laser
excitation intensity profile.
[0018] FIG. 5 is a schematic illustration of an RT-qPCR module.
[0019] FIG. 6A is a schematic illustration of a multi-channel
microfluidic device for the preparation of sample lysates with
different compositions of reagents (e.g., PCR reagents) for
downstream analysis, and the corresponding expression level
profiles for each prepared droplet.
[0020] FIG. 6B is a bright field image of an example microfluidic
device according to FIG. 6A.
[0021] FIG. 7 is a workflow example of a method for performing
SQUIRT-PCR according to the present disclosure.
[0022] FIG. 8 is a schematic illustration of the principle of the
SQUIRT-PCR system. An ultrafast (pulsed) laser lyses a cell of
interest in a living tissue via a two-photon (2P) process. The
system collects cellular contents, performs highly multiplexed
RT-qPCR, and sequentially (cell-by-cell) reconstructs a 3D spatial
map of mRNA expression with a large number of genes.
[0023] FIG. 9 is a schematic illustration of a microfabricated cage
in a microfluidic cassette for holding a tissue sample during
lysis. The `plume` of cellular contents may be collected downstream
of the channel, and the microfluidic cassette may be implemented on
a fluorescence microscope with a motorized X-Y-Z stage. A tube
connects the outlet of the microfluidic cassette with, and
dispenses the cell lysate into a well of a 96-well plate.
Dispensing may be controlled by a drop sensor at the tubing end.
After lysis, a plurality of analyses, such as reverse transcription
and pre-amplification of the collected RNA samples may be
performed.
[0024] FIG. 10 is a photographic image of an example device for
performing qPCR with an integrated analysis platform. The device
includes an integrated fluidics circuit which has 96.times.96 PCR
micro-chambers capable of analyzing 96 genes in each of the 96
single-cell samples.
[0025] FIG. 11A is a fluorescence image of a stained nuclei
recorded during lysis. The crosshair at the center indicates the
position of the focused 2P laser beam. The insert shows the four
lysis locations and the sequential lysis pattern on one of the
nuclei.
[0026] FIG. 11B is an enlarged partial view of FIG. 11A.
Irradiation with a 100 ms long pulse train resulted in significant
rupture of the nucleus.
[0027] FIG. 11C is a fluorescence image illustrating the nucleus
after irradiation with four pulse trains targeted at locations
shown in FIG. 11A.
[0028] FIGS. 12A-12C are combined fluorescence and brightfield
micrographs showing two-photon laser lysis (2PLL) of individual
cells in an intact live 3D cell cluster. FIG. 12A is an image of
the cell cluster immobilized in the microfabricated cage. FIGS. 12B
and 12C show sequential application of the 2PLL to two green
fluorescent protein (GFP) positive cells demonstrating accurate
lysis events that are highly confined to the separate target cells
with no effect on adjacent cells.
[0029] FIG. 13 is a plot demonstrating the ability of SQUIRT-PCR to
lyse and collect single cell contents with minimal carryover
contamination from two sequential lysis events and negligible
cellular stress. The data was obtained with three individual cells
where each cell was lysed and cell contents were collected in three
sequential 60 .mu.l fractions. The solid curve is a best-fit line
to the .beta.-actin mRNA levels across the fractions from all three
cells. The .beta.-actin level was the highest in the first two
fractions and subsequently returned to the background level after
the 3rd collection. This finding clearly suggests that SQUIRT-PCR
efficiently collects RNA samples from single cells and the fluid
flow conditions effectively eliminate carryover contamination
between cell lysing events. The dashed curve represents a fit to
the HSP70 mRNA which was at low levels in the three cells. Notably,
the initial lysing event of the first cell did not induce HSP70
gene expression in the second two cells, which indicates that laser
lysis does not induce cellular stress during cell lysis.
[0030] FIG. 14 is a schematic illustration of an example
microfluidic chip with a tissue capture cage according to the
present disclosure.
[0031] FIGS. 15A-15D are optical images showing four different
embodiments of the microfabricated cage for immobilization of
tissue clusters.
[0032] FIG. 16 is an optical image of a cell cluster trapped in an
example cage according to FIG. 14.
[0033] FIG. 17A is a 3D model of an example platform for loading a
single tissue sample into the microfluidic cassette. The model
includes the loading head including a glass capillary, the
microfluidic cassette on its top, and a miniature camera on the
bottom.
[0034] FIG. 17B is a picture showing a possible embodiment of the
loading head in FIG. 17A mounted on a motorized microscope
stage.
[0035] FIG. 17C is a micrograph of a target cell cluster with the
tip of a glass capillary on the right.
[0036] FIGS. 18A and 18B are schematic illustrations of an
experimental design for evaluating a PEG-treated PDMS chip which
included one syringe filled with cell medium and another syringe
filled with cell medium+RBCL mRNA that passed through a valve, the
microfluidic chip, before being deposited into a 96-well plate.
After flushing the entire system with water, the system was primed
with a solution of medium and RNA up until the valve. The valve was
then switched to allow only RNA-free medium to flood the entire
system up until reaching the microtiter plate. At this point, the
valve was switched to allow the RBCL RNA-rich medium to flow
through the chip before the collection started into the 96-well
plate.
[0037] FIG. 18C is a plot of RT-qPCR analysis showing RBCL levels
in the flow-through samples collected from untreated (solid line)
and PEG-treated (dashed line) samples as a function of collected
volume represented in well numbers. Data was collected with the
experimental setup shown in FIGS. 18A and 18B. The volume collected
per well as about 40 (about 3 drops).
[0038] Like numbers will be used to describe like parts from Figure
to Figure throughout the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention is presented in several varying
embodiments in the following description with reference to the
Figures, in which like numbers represent the same or similar
elements. Reference throughout this specification to "one
embodiment," "an embodiment," or similar language means that a
particular feature, structure, or characteristic described in
connection with the embodiment is included in at least one
embodiment of the present invention. Thus, appearances of the
phrases "in one embodiment," "in an embodiment," and similar
language throughout this specification may, but do not necessarily,
all refer to the same embodiment.
[0040] The described features, structures, or characteristics of
the invention may be combined in any suitable manner in one or more
embodiments. In the following description, numerous specific
details are recited to provide a thorough understanding of
embodiments of the system. One skilled in the relevant art will
recognize, however, that the system and method may both be
practiced without one or more of the specific details, or with
other methods, components, materials, and so forth. In other
instances, well-known structures, materials, or operations are not
shown or described in detail to avoid obscuring aspects of the
invention.
[0041] In general, one aspect of the present disclosure includes a
system and method for laser lysis of individual cells in situ
without the requirement of disaggregating live tissues a priori.
The proposed laser lysis method may be used in any setting for any
kind of single-cell analysis that requires harvesting of single
cell contents and, in addition to DNA and RNA, may be utilized for
collecting proteins or other molecules of interest. The in situ
laser lysis device may be used in connection with any downstream
module capable of collecting single cell contents. In one aspect,
cells in live tissue may respond to environmental insults
differently because of their inherent differences in cell types,
locations, exposure to blood supply, intrinsic heterogeneity,
infection, or a combination thereof. Hence, the disclosed in situ
laser lysis system may provide single cell contents from live
tissue at known locations. The system and method may allow for
analysis of detailed responses at the genomic, transcriptional, and
protein expression levels in healthy tissues, diseased cells (e.g.,
cancer), their neighboring cells in a diseased (e.g., cancer)
tissue, and the like. Accordingly, the present disclosure may
provide a better understanding of how cell-cell communication takes
place in diseased tissue in situ.
[0042] In one embodiment of the present invention, the in situ
laser lysis device may allow a user to perform single cell in situ
quantitative reverse transcription polymerase chain reaction
(SQUIRT-PCR) for in situ gene expression heterogeneity analysis
using mRNA collected from individual cells. The device may include
a two-photon laser in order to separately lyse a sequence of
individual cells, one cell at a time, located at known coordinates
within a three-dimensional (3D) tissue or cell cluster. Further,
this system may allow for rapid release of cellular contents from
different individual cells for a plurality of downstream analyses,
including quantitative reverse transcription polymerase chain
reaction (RT-qPCR) profiling. In one aspect, cellular contents may
include DNA (chromosomal, plasmid, and the like), RNA (mRNA,
non-coding RNA, ribosomal RNA, and the like), protein, small
molecules, membrane components, and the like. In one embodiment,
the single-cell lysate may be immediately transported to an
emulsion-based (oil-droplet) RT-qPCR module to profile mRNA
expression. Accordingly, the present disclosure may provide for a
highly multiplexed platform capable of detecting dozens of mRNA
sequences from each droplet of the single cell lysate.
[0043] In one aspect, any suitable method for the lysis of cell or
tissue samples may be employed. For example, the cells to be
analyzed may be individually held on a substrate, conglomerated
into a tissue (or cell cluster) sample, or the like. Embodiments of
a system may allow for indefinite sampling at the single cell
level. In one aspect, sampling may be carried out to better
understand the distribution of DNA markers or RNA markers in larger
collections of cells or tissues. In another aspect, information of
the spatial location of each cell in the tissue may be retained,
thereby enabling a better understanding of cellular heterogeneity
and the functional relevance in tissues.
[0044] In some embodiments, a system and method for in situ single
cell laser-lysis may include a microfluidic chip. In one example, a
tissue sample may be selected and then loaded into the microfluidic
chip. In some embodiments, the tissue sample may have a dimension
of about 100 .mu.m to about 300 .mu.m. In other embodiments, the
sample may include one or more individual cells, multiple cells or
tissues. The sample may be acquired from any suitable source, such
as from a batch of similar tissue samples (or individual cells)
floating in media.
[0045] In some embodiments, sample collection may be performed with
a Tissue Collection Device (TCD), which may be coupled to a
microfluidic chip according to the present disclosure. A TCD may
allow for the capture of a tissue sample (or individual cell) and
placement of the sample into a cage or other containment device
included in the design of the microfluidic chip.
[0046] Turning now to FIG. 1, and embodiment of a TCD 20 may
include an apparatus 22 and a microfluidic chip 24. The apparatus
22 may have a body 26, a clamp plate 28, a control valve 30, a
capillary 32, a camera 34 and an output port 36. The microfluidic
chip 24 may be a two part assembly including a microfabricated
polydimethylsiloxane (PDMS) component and a glass slide. The
microfluidic chip 24 may be manufactured by microfabricating a
master mold out of a silicon wafer, then pouring degassed PDMS
liquid over the mold, and after curing, removing PDMS.
[0047] With reference to FIGS. 1 and 2, the microfluidic chip 24
may include a cage 38. The cage 38 may include a plurality of posts
40 or other like baffles to prevent the passage of a cell or tissue
sample 42 flowing therethrough. Accordingly, the cage 38 may have a
specific size and geometry to allow the capture of the tissue
sample 42 while still allowing a fluid flowing through the cage 38
to wash away the lysed cell contents. Note that arrows shown in
FIG. 2 are indicative of the direction of fluid flow. Also, it will
be appreciated that a system and method may be compatible with both
tissue samples and single cell samples.
[0048] One aspect of the present system and method may include the
ability to select and collect a particular tissue sample from a
multitude of free floating tissue samples and deliver said sample
to a confined space within a microfluidic channel. Another aspect
of the present system and method may include the ability to
transfer a captured tissue sample to a downstream device. Examples
of downstream devices may include analytical devices,
instrumentation or characterization platforms for performing
tissue-level analysis.
[0049] In some embodiments, an array of tissue samples may be
maintained in a media bath. The capillary 32 of the TCD 20 may be
lowered into the bath, such as with an automated system with visual
feedback. Once a tip 44 of the capillary 32 is in proximity to the
tissue sample 42 in the media bath, the tissue sample 42 may be
drawn into the capillary 32, such as with a source of vacuum in
communication with an output line 46 of the TCD 20. As the tissue
sample 42 flows through the TCD 20 and into the microfluidic chip
24, the tissue sample 42 may eventually become caught in the cage
38 of the microfluidic chip 24 as shown in FIG. 2. At this point, a
user may observe the tissue sample 42 with the camera 34.
Thereafter, the user may turn off the control valve 30, thereby
locking the TCD 20 into a steady-state in which the microfluidic
chip 24 may be removed from the TCD 20. In some embodiments, the
tissue sample 24 retained on the microfluidic chip 24 may be
incubated for a period of time by transferring the microfluidic
chip 24 from the TCD 20 to an incubator for facilitating surface
bonding. In other embodiments, the microfluidic chip 24 may be
transferred to a microscope plate for analysis with a microscope
inspection station.
[0050] With reference to FIGS. 3A and 3B, the cell or tissue sample
42 on the microfluidic chip 24 may be subjected to a sheath of
continuous fluid (e.g., buffer) flow as indicated by the arrows. In
some embodiments, the microscope plate containing the microfluidic
chip 24 may be set up on a microscope station equipped with a laser
50. The microscope may visualize the tissue sample 24. The user may
select a target area 52 of the tissue sample 24 to be lysed. Using
the laser 50, the selected target area 52 of the tissue sample 24
may be irradiated with the laser 50, thereby releasing at least a
portion of the contents 54 of the target area 52 of the tissue
sample 24. The contents 54 may then flow downstream in the fluid
flowing through the microfluidic chip 24. In one aspect, spatial
data (e.g., positional coordinates) for each cell in the tissue
sample 42 may be collected and stored while allowing for continuous
lysis of the individual cells within the tissue sample 42 as shown
in FIGS. 3A and 3B.
[0051] In some embodiments, the laser 50 may be a two-photon laser.
With reference to FIGS. 4A and 4B, the laser profiles for a single
photon (1P) laser may differ from that of a two-photon (2P) laser.
Nonlinear two-photon excitation concentrates energy in a
femtoliter-scale volume, and may therefore allow precise
subcellular operations (Heisterkamp A, et al. 2005, Optics Express
13: 3690). The excitation may be rapid, highly localized, and the
process may be compatible with optical microscopes. Two-photon
laser lysis is different from the conventional single-photon-based
lysis methods that have poor longitudinal resolution for 3D
structures (Dhawan M, et al. 2002, Analytical and Bioanalytical
Chemistry 374: 421-426; Lai H-H, et al. 2008, Journal of The Royal
Society Interface 5: S113-S121; Quinto-Su P A, et al. 2008, Lab
Chip 8: 408-414; Rau K R, et al. 2006, Biophysical Journal 91:
317-329; Rau K R, et al. 2004, Applied Physics Letters 84:
2940-2942; Sims C E, et al. 1998, Anal Chem 70: 4570-4577). In one
aspect, the present system and method may combine two-photon
excitation to lyse cells in tissues and sequentially harvest their
cell contents automatically.
[0052] Tuning again to FIGS. 3A and 3B, a RT-qPCR module 70 may be
in downstream communication with the microfluidic chip 24. In this
case, the lysed single-cell contents 54 (e.g., DNA, RNA, protein,
small molecules and the like) may mix downstream with a stream 56
including, for example, components for performing RT-PCR (e.g.,
primers and RT-PCR master-mix) in a stream 58 on the microfluidic
chip 24. Primers and other components present in stream 56 may be
provided to facilitate the amplification of nucleic acid templates
(e.g., DNA, RNA) in the downstream qRT-PCR module 70. Further
downstream in the microfluidic chip 24, the stream 58 may mix with
an oil containing stream 60. Accordingly, the contents of stream 58
may be distributed into discrete droplets 62 by the perpendicular
flow of oil from stream 60.
[0053] In one aspect, the aqueous stream 58 may not mix with the
oil stream 60. In another aspect, the volume of droplets 62 may be
controlled by varying the flow rate of the stream 58 and the stream
60, the geometry of the microfluidic chip, or the like. The aqueous
phase contents of droplets 62 may continue to mix as the droplets
62 flow downstream. In one aspect, the microfluidic chip 24 may be
configured to enable the contents of the droplets 62 to achieve
homogeneity by the time the droplets 62 reach the RT-qPCR module
70.
[0054] Turning now to FIG. 5, the droplets 62 may flow from the
microfluidic chip 24 into a small diameter passage 72 of the
downstream RT-qPCR module 70. The passage 72 may be continuous and
may extend the length of the entire qRT-PCR module 70. The droplets
62 may come into contact with a first sub-system 74 for performing
reverse transcription (RT). In one aspect, the sub-system 74 may be
uniformly heated to convert RNA in the droplets 62 to cDNA. One
suitable temperature for heating the sub-system 74 to may be about
40 degrees Celsius. The droplets 62 may continue to flow downstream
through the subsystem 74 to a subsystem 76 for performing PCR. The
subsystem 76 may include a dual-zone heating block 78. Fluid tubing
80 may wrap around the periphery of the heating block 78 one or
more times. For example, the number of time the fluid tubing 80
wraps around the heating block 78 may equal the number of
amplification cycles the experiment calls for (e.g., 40 cycles=40
wraps). A first zone 82 of the heating block 78 may be maintained
at a temperature (e.g., about 95.degree. C.) to carry out a
denaturing step of a PCR protocol. A second zone 84 of the heating
block 78 may be maintained at a temperature (e.g., about 60.degree.
C.) to carry out an annealing/extension step of a PCR protocol.
[0055] The subsystem 76 may further include a fluorescence detector
86. The fluorescence detector 86 may be used to monitor each
amplification cycle of a PCR protocol. The fluorescence signal may
be obtained by exciting the droplets 62 with an LED, laser source
or other suitable excitation source. The fluorescence detector 86
may measure the fluorescence emission from the droplets 62 with any
suitable detector, such as a photodetector, photomultiplier tube,
charge coupled device or the like. The data from the fluorescence
detector 86 may be read and processed by software. After the
subsystem 76 the tubing 80 and thus droplets 62 may flow to a
downstream container (not shown).
[0056] In some embodiments, fluids (buffer, primers, master-mix,
oil) flowing through the microfluidic chip 24 or RT-qPCR module 70
may be controlled by a pressure-driven pumping system that allows
for a smooth, continuous flow. The hardware components, control
systems, data collection and processing may be carried out with
software. Data collected from the TCD 20 or RT-PCR module 70 may be
output in real-time to a monitoring system. Accordingly, a user may
store the data for later use, or adjust system parameters based on
the design of the experiment.
[0057] In some embodiments, it may be possible to apply different
perturbagens upstream of the tissue sample 42 so as to discover
their effects with real time exposure. If extended exposure is
necessary, the perturbagen may be introduced into the microfluidic
chip 24 and all flow stopped while the effect was put to the tissue
sample 42. The perturbagen may then be washed away, and,
optionally, a second perturbagen may be introduced. This may be
used to discover the effects of multiple dosing on patients organ
cells.
[0058] In some embodiments, DNA, RNA or other cellular components
inside the microfluidic chip 24 may be partitioned or encapsulated
into much smaller droplets separated by oil and directed into
different channels of a multi-channel splitter 90 as shown in FIGS.
6A and 6B. Each channel 92 may be individually addressed with a
different composition (e.g., primers, master-mix and the like) that
may be combined with the microdroplet 94 during passage through the
combination junction 96. The microdroplets 94 may be combined into
a single stream of separated microdroplets in communication with a
downstream RT-qPCR module, maintained in multiple distinct passages
that may be wound through the RT-qPCR module, or a combination
thereof. For example, the number of tubes wrapped around the PCR
module may equal forty times the number of separated microchannels
92.
[0059] In some embodiments, the microfluidic chip 24 may be
maintained to provide a suitable microenvironment for the tissue
sample 42. In one aspect, the microfluidic chip 24 may be
transparent for compatibility with one or more optical microscopes,
lasers or other analytical equipment. Moreover, the structure of
the microfluidic chip 24 may be configured to reduce physical
stress that may be imparted to the tissue sample 42, for example,
to minimize artificial bias.
[0060] Turning to FIG. 7, an example method 200 for performing
SQUIRT-PCR may include a first step 202 in which a tissue sample is
captured. As described herein, a tissue sample may include a single
cell or a portion thereof as well as a plurality of cells or
tissues. The tissue sample may be captured using any suitable
device such as a TCD or other device configured to capture, retain
or otherwise isolate a tissue sample. Thereafter, in a step 204,
the tissue sample may be selectively lysed. Lysis may include the
use of a laser (e.g., a two-photon laser) to target one or more
portions of a tissue sample. Lysis may further include the use of a
microscope to visualize the tissue sample. Visualization may be
useful for guiding the laser or other lysing apparatus in order to
target a particular area or portion of the tissue sample for
lysis.
[0061] Following lysis, a step 206 may include recovery of the
lysate. In one aspect, it may be useful to retain the tissue sample
or portions thereof while recovering soluble portions of the
lysate. For example, it may be useful to recover DNA, RNA, small
molecules, proteins, or the like. The method of recovering the
lysate may include a microfluidic device for capturing the selected
portions of the tissue while enabling other portions to be
partitioned for recovery. A step 208 may include combing the
recovered lysate from the step 206 with analysis reagents. Suitable
analysis reagents may include, primers, buffer, salts, polymerase,
dyes, solvents (e.g., betain, DMSO), reverse transcriptase,
nucleotides and the like. For example, it may be useful to combine
the lysate with reagent for performing RT-qPCR. The step 208 may
further include encapsulating the combined lysate and reagents. For
example, the aqueous lysate and reagents may be combined with an
immiscible organic material such as oil.
[0062] In a step 210, the combined lysate and reagents may be
subjected to one or more amplification processes. Example
amplification processes may include thermal cycling processes or
protocols for performing reverse transcription, preamplification,
polymerase chain reaction and the like. In one aspect, DNA or RNA
templates present in the lysate may be amplified for detection in a
step 212, wherein target molecules present in the lysate may be
detected. Target molecules may include DNA, RNA, small molecules,
proteins and the like. In one example, optical detection method may
be used to qualitatively or quantitatively analyze the amplified
target molecules.
[0063] It will be appreciated that in some embodiments, one or more
steps of the method 200 may be omitted. For example, in the case
that a target molecule includes a protein that may be present in
the recovered lysate, it may be useful to omit an amplification
step. Instead (or in addition), it may be useful to recover the
protein target molecule for analysis with another technique such as
mass spectrometry or nuclear magnetic resonance. Other variations
of the method 200 may also fall within the scope of the present
disclosure.
[0064] In general, the in situ single cell laser lysis system of
the present invention may enable more accurate gene expression
profiling of cells as the single-cell contents may flow directly
from live tissue to RT-qPCR in a relatively short time as compared
with other systems. In one aspect, the small scale of the
microfluidic channels within the in situ laser lysis device may
enable the use of microliter scale volumes for sample processing.
In another aspect, the elapsed time interval between cell lysing
and lysate encapsulation may be on the order of seconds. In yet
another aspect, completion of RT-qPCR may occur on the order of
about one hour.
[0065] In some embodiments, the present system and method may be
applied to basic biomedical research, clinical applications,
assessment of population-level heterogeneity in gene expression
levels in normal or tumor cells with single-cell resolution and the
like. In other embodiments, the present system and method may
provide increased throughput with respect to the number of genes
that may be quantified simultaneously. In still other embodiments,
the present system and method may provide a highly multiplexed
platform capable of detecting dozens of mRNA sequences for each
initial droplet eluted from a given sample. This multiplexed
platform may allow end-users to select cells located on the surface
of the live tissue for downstream analysis and then lyse and
analyze those cells at particular time points while retaining
spatial information with respect to the tissue.
[0066] In some embodiments, one or more automation techniques may
be applied to a SQUIRT-PCR system and method. For example,
automation may be provided to harvest single-cell contents into
single-wells of standard microtiter plates. The microtiter plates
with single-cell lysates may be used by any downstream instrument
for analysis of DNA, RNA, protein, small molecules, or combinations
thereof. In one aspect, high-throughput quantitative mRNA profiling
may be performed by harvesting single cell mRNA and interfacing a
SQUIRT-PCR system with a high-throughput analytical platform. In
one aspect, a high-throughput analytical platform may include
thermal cycling and fluorescence detection capabilities. One
example platform includes the BIOMARK HD system from FLUIDIGM.
Accordingly, the present disclosure may provide an understanding of
cellular heterogeneity in live intact tissue.
[0067] In some embodiments, SQUIRT-PCR system 100 may be used to
construct a 3D map of mRNA expression in living tissue samples of
up to 96 genes as shown in FIG. 8. In a first step 102, an
ultrafast (pulsed) laser may lyse a cell-of-interest in a living
tissue such as with a two-photon process. In a next step 104, the
system 100 may collect the intracellular contents of the tissue
sample and in a sub-step 104a, perform highly multiplexed RT-qPCR,
while in a sub-step 104b, the system 100 may perform 3D image
segmentation to reconstruct a 3D spatial map of mRNA expression
with a large number of genes in a next step 106. The SQUIRT-PCR
system 100 may automatically dispense single cell lysates into a
standard microtiter plate format. As such, a user may use a
SQUIRT-PCR system to analyze intracellular analytes of interest
using one or more analytical platforms at the single-cell level in
situ. Example applications where SQUIRT-PCR may be applied include
epigenomics, RNA-seq, enzyme activity assays, mass spectroscopy,
and the like.
[0068] In some embodiments, a system 108 may include an ultrafast
laser system with a motorized fluorescence microscope as shown in
FIG. 9. A plastic microfluidic cassette 110 may include a
microfabricated cage 112 to retain a selected tissue sample 114
introduced into a straight microfluidic channel 116. During lysis,
the system 108 may collect and automatically dispense single cell
lysates 118 into individual wells 120 of a 96-well plate 122. After
the reverse transcription reaction that converts mRNA to cDNA, and
cDNA pre-amplification using a standard protocol, an integrated
analytical platform may be used to analyze expression levels of 96
genes per cell for all lysed cells (i.e., 96 genes.times.96 cells).
In one example, the integrated platform may integrate with a
multi-well plate 124 including an integrated fluidics circuit 126
as shown in FIG. 10. Further, 3D image processing and visualization
software (3D-SC) may be developed and used to correlate single-cell
gene expression profiles with the 3D spatial locations of the
corresponding cells within the cluster, as shown in FIG. 8. In
another example, the device shown in FIG. 10 may be substituted for
(or augmented with) one or more devices for epigenomic analysis,
proteomic analysis, and the like.
[0069] In one aspect, the inventors have discovered that the cell
disruption with a two-photon laser may be achieved with sub-micron
resolution. Cell lysis may be well localized to the single-cell
level. The cells next to the targeted cell may not be damaged in
the lysis process. Combined with the image of the clusters, the
acquired mRNA expression result may be tagged with spatial
information of each lysed cells, thus generating a 3D map of mRNA
expression.
[0070] In another aspect, the inventors have discovered that
SQUIRT-PCR may be used to collect the contents of a single cell.
The extracted mRNA may be preamplified, and then analyzed using
qPCR. The sensitivity may be comparable with conventional single
cell RT-qPCR where the housekeeping genes are obviously measurable.
As expected, genes having lower relative expression levels are
observed to have larger variations in expression level
measurements.
[0071] In yet another aspect, the inventors have discovered that a
SQUIRT-PCR system may be used to collect the lysate of 10 cells in
about 200-cell cluster having a diameter of about 100 microns to
about 150 microns without apparent carryover contamination. A
FLUIDIGM BioMark HD FLEXsix platform (12.times.12 qPCR array,
capable of analyzing 12 genes for 12 individual single cells)
yielded a sparse 3D map of mRNA expression of 12 genes. The entire
process may be semi-automated and may be accomplished in about 30
minutes.
[0072] The schematic flow chart shown in FIG. 7 is generally set
forth as a logical flow chart diagram. As such, the depicted order
and labeled steps are indicative of one embodiment of the presented
method. Other steps and methods may be conceived that are
equivalent in function, logic, or effect to one or more steps, or
portions thereof, of the illustrated method. Additionally, the
format and symbols employed in FIG. 7 are provided to explain the
logical steps of the method and are understood not to limit the
scope of the method. Although various arrow types and line types
may be employed, they are understood not to limit the scope of the
corresponding method. Indeed, some arrows or other connectors may
be used to indicate only the logical flow of the method. For
instance, an arrow may indicate a waiting or monitoring period of
unspecified duration between enumerated steps of the depicted
method. Additionally, the order in which a particular method occurs
may or may not strictly adhere to the order of the corresponding
steps shown.
Examples
[0073] For optimization of SQUIRT-PCR, two-photon (2P) laser lysis
was demonstrated to effectively lyse individual cells with
sub-cellular resolution in a cluster without incurring visible
damage to the neighboring cells. Various laser settings were tested
to achieve sufficient lysis performance and mRNA harvest
efficiency. Laser settings and optical configurations found to
yield efficient lysis included 100.times.1.3 NA oil-immersion
objective lens, fundamental laser wavelength of 800 nm with a pulse
duration of 150-200 fs, 250 kHz repetition rate, 0.4 .mu.J pulse
energy, and .about.1 s total exposure time. Moreover, targeting the
nuclear membrane was discovered to easily generate micrometer-scale
intracellular cavitation bubbles (Quinto-Su P A, et al. 2008, Lab
Chip 8: 408-414; Li H, Sims C E, et al. 2001, Anal Chem 73:
4625-4631), which cause ruptures in the cell plasma membrane and
the release of cell contents from the cell. Cavitation bubbles only
existed for .about.100 ms then dissipated. The 2P excitation was
confirmed to induce the most efficient lysis when focused on the
nucleus rather than anywhere else in the cell. Using a 100 ms-long
series of pulses (25,000 pulses per series) targeted at the nuclear
envelope, the lysis of the target cells and the release of their
contents was accurately controlled. A study was performed with a 3D
esophageal cell model using a laboratory (CBDA) established
protocol. To visualize individual nuclei in cell clusters, cells
were stained with the nuclear stain, Hoechst prior to 2PLL. FIGS.
11A-11C illustrate four points at the nuclear envelope region
targeted by the focused laser beam and the resulting release of
cellular contents visualized by the disappearance of fluorescence
signal from the entire cell. FIGS. 12A-12C show a 3D cell cluster
containing two cell populations including GFP-positive and
GFP-negative cells (FIG. 12A). The cell cluster was injected into
the microfluidic chip and was effectively retained in a
microfabricated cage. GFP-positive cells were sequentially lysed
and the release of the cell contents was monitored by the decrease
in the GFP fluorescence signal (FIGS. 12B and 12C). The results
showed that although the two GFP-positive cells were next to each
other, the 2P laser precisely targeted and lysed each cell without
affecting the other cell, as indicated by the release of the
cytosolic green fluorescence protein from the individual cells. In
addition, the Hoechst-labeled nuclei facilitate identifying the
locations and areas of cell nuclei in 3D clusters. Microscopy
analysis showed that 40 minute staining duration prior to lysis was
sufficient for Hoechst to efficiently stain clusters with a
diameter of 100-150 p.m. The photobleaching of Hoechst stain does
not pose a problem for cell visualization during the projected
15-minute process duration.
[0074] For mRNA expression analysis, 2PLL was demonstrated to
serially lyse individual cells in tissue samples in a single
microfluidic device. Conditions were identified for minimizing
cell-cell contamination by extensively washing tissue samples
between cell lysis cycles (FIG. 13). Single-cell mRNA yield from
2PLL was sufficient for detection with the FLUIDIGM BioMark
platform. In addition, tests were performed to determine whether
2PLL induces cellular stress by monitoring heat shock protein 70
(HSP70) mRNA levels following multiple cell lysis cycles. Results
indicated that while the HSP70 transcript level was detectable in
the cell lysates, it was consistently low in all the cells (FIG.
13), which indicated that 2PLL does not induce detectable cellular
stress during the 15-minute collection time.
[0075] A microfluidic cassette 128 capable of immobilizing cell
clusters was constructed as shown in FIG. 14. Polydimethylsiloxane
(PDMS), a biocompatible material widely used for lab-on-a-chip
applications, was selected for the fabrication of the 1''.times.1''
cassette 128. The width and height of the channel 132 are 500 .mu.m
and 100 .mu.m, respectively. When a tissue sample (e.g., a cell
cluster) is introduced into the channel 132, it is trapped in a
microfabricated "cage" 130 in the middle of the channel 132 (FIGS.
15A-15D) by fluid flow as shown in FIG. 16. Various designs for
cage 130 were tested with different post geometries and the gaps
between posts varying from 15 .mu.m to 30 .mu.m. The bottom of the
cassette 128 was a standard 170 .mu.m-thick cover glass, so it is
compatible with imaging using high numerical aperture (NA)
objective lenses. Testing determined that a loaded cell cluster can
be held still in the cage 130 with a small flow rate (<1
.mu.L/min), whereas it remains viable and intact with only
insignificant deformation when the flow rate exceeds 300 .mu.L/min
as shown in FIG. 16. The surface of the channel 132 was coated with
polyethylene glycol (PEG) to avoid surface adsorption of the
molecules in the lysate. In general, it may be useful to load only
one tissue structure into the cassette 128 at a time.
[0076] A robotic tissue loading platform with visual feedback was
implemented to pick an individual cell cluster from a large
population of clusters, and to transfer it to the microfluidic
cassette for 2PLL. This platform is based on a single-cell loading
platform (Anis Y H, et al. 2010, IEEE Transactions on Automation
Science and Engineering 7: 598-606; Anis Y, et al. 2011, Biomedical
Microdevices 13: 651-659). Before tissue loading, the user may
insert the microfluidic cassette into the loading head. The user
may use a microscope on the platform to identify a tissue sample of
interest, then use the loading head on a robotic arm to
automatically aspirate it into the microfluidic cassette and load
it into the microfabricated cage as shown in FIGS. 17A-17C. A
camera on the loading head allows the user to visually confirm that
the tissue is trapped in the cage. According to one method, the
loading process requires relatively minimal effort and takes less
than about 5 minutes. Once loading is completed, the user may
transfer the cassette from the loading head to the 2PLL station for
laser lysis.
[0077] In one aspect, adhesion of RNA to PDMS may be a concern
during the collection of lysate due to the small expected
concentrations in single cells. To test the ability of a PEG
(polyethylene glycol) surface treatment to mitigate adhesion, a PEG
coated microfluidic chip was compared experimentally to an uncoated
one. Both chips were injected with the control mRNA, RBCL
(ribulose-bisphosphate carboxylase), the RNA sample was allowed to
flow through the chip, and the flow through was collected onto the
96-well plate. The amount of RBCL absorbed by the microfluidic
channels was tested by determining RBCL RNA levels collected from
the outlet before and after the introduction of the RNA sample into
the microfluidic channels as shown in FIGS. 18A-18C. Since PDMS has
the propensity to bind RNA, the untreated microfluidic chip was
predicted to absorb significant amount of RBCL. In contrast,
minimal binding of RBCL to the PEG treated channel was anticipated
since PEG has previously been shown to block RNA from binding PDMS
(Yamanaka K, et al. 2011, The Analyst 136: 2064; Zhou J, et al.
2010, ELECTROPHORESIS 31: 2-16). Results showed that the PEG
treated channel had significantly higher RBCL RNA levels during the
entire time when RBCL passed through the channel as shown in FIG.
18C, confirming the notion that PEG coated PDMS channels can
minimize RNA absorption.
[0078] The present invention has been described in terms of one or
more preferred embodiments, and it should be appreciated that many
equivalents, alternatives, variations, and modifications, aside
from those expressly stated, are possible and within the scope of
the invention.
[0079] Each reference identified in the present application is
herein incorporated by reference in its entirety.
[0080] While present inventive concepts have been described with
reference to particular embodiments, those of ordinary skill in the
art will appreciate that various substitutions and/or other
alterations may be made to the embodiments without departing from
the spirit of present inventive concepts. Accordingly, the
foregoing description is meant to be exemplary, and does not limit
the scope of present inventive concepts.
[0081] A number of examples have been described herein.
Nevertheless, it should be understood that various modifications
may be made. For example, suitable results may be achieved if the
described techniques are performed in a different order and/or if
components in a described system, architecture, device, or circuit
are combined in a different manner and/or replaced or supplemented
by other components or their equivalents. Accordingly, other
implementations are within the scope of the present inventive
concepts.
* * * * *