U.S. patent application number 16/338306 was filed with the patent office on 2020-01-16 for system for identifying and targeting individual cells within a heterogeneous population for selective extraction of cellular con.
The applicant listed for this patent is THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO, SINAI HEALTH SYSTEM. Invention is credited to Michael Dean CHAMBERLAIN, David CHITAYAT, Michael David Murdoch DRYDEN, Elena KOLOMIETZ, Julian Lucas LAMANNA, Aaron Ray WHEELER.
Application Number | 20200016594 16/338306 |
Document ID | / |
Family ID | 61763256 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200016594 |
Kind Code |
A1 |
WHEELER; Aaron Ray ; et
al. |
January 16, 2020 |
SYSTEM FOR IDENTIFYING AND TARGETING INDIVIDUAL CELLS WITHIN A
HETEROGENEOUS POPULATION FOR SELECTIVE EXTRACTION OF CELLULAR
CONTENT
Abstract
The present disclosure provides a system and method for
identifying and targeting individual cells within a cell population
for selective extraction of cellular content and a digital
microfluidic device having at least one hydrophilic site for
receiving cells, an imaging system including a stage for receiving
the digital microfluidic device and an imaging module for
identifying at least one targeted cell among the cells at the at
least one hydrophilic site. The system includes a pulsed laser
source for laser lysing the targeted cell thereby releasing the
cell content to produce a lysate. A control system controls the
pulsed laser source, the imaging system and the digital
microfluidic device and is programmed for coordinating steps of i)
movement of droplets on the digital microfluidic device, ii)
selection of the at least one targeted cell to be lysed located at
the at least one hydrophilic site, iii) illumination of the at
least one selected targeted cell by the pulsed laser source to lyse
the at least one selected targeted cell to produce lysate, and iv)
collection of the lysate.
Inventors: |
WHEELER; Aaron Ray;
(Toronto, CA) ; CHAMBERLAIN; Michael Dean;
(Toronto, CA) ; LAMANNA; Julian Lucas; (Toronto,
CA) ; DRYDEN; Michael David Murdoch; (Toronto,
CA) ; KOLOMIETZ; Elena; (Thornhill, CA) ;
CHITAYAT; David; (Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO
SINAI HEALTH SYSTEM |
Toronto
Toronto |
|
CA
CA |
|
|
Family ID: |
61763256 |
Appl. No.: |
16/338306 |
Filed: |
September 29, 2017 |
PCT Filed: |
September 29, 2017 |
PCT NO: |
PCT/CA2017/051158 |
371 Date: |
March 29, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62402208 |
Sep 30, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 1/33 20130101; C12Q
2565/629 20130101; B01L 7/52 20130101; B01L 2300/089 20130101; G01N
15/00 20130101; B01L 3/502753 20130101; G01N 15/1468 20130101; B01L
2400/0421 20130101; G01N 15/10 20130101; C12Q 1/6806 20130101; G01N
1/28 20130101; B01L 2200/0647 20130101; B01L 3/502715 20130101;
C12M 1/34 20130101; G01N 2015/1006 20130101; B01L 3/502792
20130101; B01L 2200/0668 20130101; C12Q 1/6881 20130101; G01N
2015/149 20130101; G01N 2015/1081 20130101; B01L 2300/0867
20130101; C12N 1/066 20130101; B01L 2400/0442 20130101; G01N 1/40
20130101; G01N 1/286 20130101; G01N 35/00 20130101; B01L 3/502761
20130101; C12M 1/42 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12N 1/06 20060101 C12N001/06; C12Q 1/6806 20060101
C12Q001/6806; G01N 1/28 20060101 G01N001/28; G01N 15/14 20060101
G01N015/14 |
Claims
1-14. (canceled)
15. A method for detection and/or isolation of fetal cells and/or
fetal analytes, the method comprising: loading a sample containing
cells on at least one site of a digital microfluidic device thereby
forming a virtual microwell at each of the at least one site;
Immobilizing the cells on the at least one site; selecting at least
one immobilized cell; lysing the at least one selected cell using a
pulsed laser source to produce lysate within its corresponding
virtual microwell; displacing a droplet of liquid to the
corresponding virtual microwell for collecting the lysate; and
moving the droplet containing the lysate from the corresponding
virtual microwell to a designated site, and optionally detecting
and/or isolating fetal analytes in the lysate.
16. The method according to claim 15 wherein the at least one site
is hydrophilic, partially hydrophilic or become hydrophilic after
protein fouling/adsorption from the sample.
17. The method according to claim 15 further including a step of
generating a map of locations of the immobilized cells, and wherein
the step of selecting the at least one immobilized cell includes
selecting the at least one cell from the map.
18. The method of claim 15 further comprising a step of labelling
the immobilized cells.
19. The method of claim 18 wherein the step of labelling the
immobilized cells further comprises fixing the cells.
20. The method of claim 15 further comprising steps of: moving the
digital microfluidic device along horizontal axes and a vertical
axis for positioning the digital microfluidic device for lysing
another at least one selected cell from immobilized cells; lysing
the other at least one selected cell using the pulsed laser source
to produce another lysate within its corresponding virtual
microwell; displacing another droplet of liquid to the
corresponding virtual microwell for collecting the other lysate;
and moving the other droplet containing the other lysate from the
corresponding virtual microwell to a designated site.
21. The method of claim 15 further comprising a step of introducing
the sample containing the cells at an initial site and displacing
the sample to the at least one site.
22. The method of claim 15, wherein said at least one site is a
plurality of sites, and including steps of moving of droplets to
said plurality of sites, selecting of a cells to be lysed at each
of said plurality of sites, selecting a first site to illuminate
the selected cell at that site, moving of the stage to move the
digital microfluidic device sequentially to bring each of the sites
into a field of view of the pulsed laser source to lyse the
selected cell to produce lysate at each site, and collecting the
lysate at each site.
23. The method according to claim 22 including calculating a
shortest distance travelled by the stage to bring each of the
plurality of sites into the field of view of the pulsed laser
source sequentially.
24. The method according to claim 22 wherein the at least one
selected targeted cell is a plurality of selected targeted cells,
including identifying a sequence of selected targeted cells to be
lysed to minimize a time to perform the lysing on all selected
targeted cells, and wherein the plurality of selected targeted
cells is within one field of view, or a plurality of field of
views, or within a plurality of sites.
25. The method of claim 15 wherein the pulsed laser source is a
nanosecond-pulsed laser.
26. The method of claim 15 wherein the pulsed laser source is a
nanosecond-pulsed laser delivering pulses of at least 1 .mu.J.
27. The method according to claim 25, wherein the nanosecond-pulsed
laser is a Nd-based laser.
28. The method according to claim 25, wherein the nanosecond-pulsed
laser produces a pulsed-laser beam within the visible spectrum.
29. The method of claim 15, wherein the pulsed laser source is a
Q-switched laser.
30. The method of claim 15, further comprising the step of
performing on chip analysis of the lysate at the designated
site.
31. The method of claim 30, further comprising the step of
collecting the droplet containing the lysate from the designated
site for off-chip analysis.
32. (canceled)
33. The method according to claim 15 for prenatal genetic testing
or screening, or detection or diagnosis of a prenatal
condition.
34. (canceled)
Description
FIELD
[0001] Devices for use in identifying and targeting individual
cells for lysis in a heterogeneous cell environment, said cell
lysates being subject to further downstream analysis. Also provided
are methods of using the devices as well as systems and kits that
comprise the devices. The devices, systems, methods and kits find
use in a variety of different applications, including testing and
diagnostics.
BACKGROUND
[0002] In the last decade it has become clear that cell populations
that were thought to be the same can actually be very heterogeneous
in nature. Two examples of this are (a) the population of cancer
cells that make up a tumour, and (b) stem cells and their progeny.
There is great interest in having methods to identify and study
individual cells or small subpopulations of cells in complex
mixtures. In addition, with the discovery of the existence of rare
cell populations (e.g., cancer cells or immune cells in blood,
fetal cells in maternal samples, etc.), there is great interest in
the development of methods to isolate and characterize individual
cells.
[0003] Single-cell genomic analysis relying on flow cytometry or
microchannel-based sorting has been previously investigated [3-10]
and the Polaris.TM. system was commercially released for this
purpose. The use of flow cytometry-based or microchannel-based
sorting for single cell genomic analysis is quite useful for
various applications, however, the use of these technologies is
limited to the analysis of suspensions of cells in liquid media.
Furthermore devices using these technologies are limited in the
number of cells that can be evaluated at the same time.
[0004] Microfluidics regards miniaturized fluid-handling
technologies for processing or manipulating extremely small volumes
of fluids. Such technologies include digital microfluidics (DMF)
(sometimes also known as "electrowetting" or
"electrowetting-on-dielectric") which relies on fluid manipulated
as droplets on an open array (with no channels), and integrated
fluidic circuits (ITC) which relies on fluid pumped through
enclosed micron-dimension channels. The integration of laser
microbeam cell lysis with ITCs (in enclosed micron dimension
channels) has been previously reported. Quinto-Su described a
number of challenges using such approach, including the tendency of
plasma-induced bubbles to clog, deform, or damage the
microchannels, making the method impractical for regular use [17].
Lai reported that some of these problems may be corrected by
focusing the laser microbeam to a smaller spot [16]. However,
downstream analysis was limited perhaps because of cell lysate
dilution in the microchannel.
SUMMARY
[0005] Disclosed herein is a system for identifying and targeting
individual cells within a cell population for selective extraction
of cellular content, comprising:
[0006] a digital microfluidic device having at least one site for
receiving cells;
[0007] an imaging system including a stage for receiving the
digital microfluidic device, the imaging system including an
imaging module for identifying at least one targeted cell among the
cells at the at least one site and a pulsed laser source for laser
lysing the at least one targeted cell thereby releasing the cell
content to produce a lysate; and
[0008] a control system for controlling the pulsed laser source,
the imaging system and the digital microfluidic device, the control
system being programmed with instructions for coordinating steps of
[0009] moving of droplets on the digital microfluidic device,
[0010] selecting of the at least one targeted cell to be lysed
located at the at least one site, [0011] illuminating of the at
least one selected targeted cell by the pulsed laser source to lyse
the at least one selected targeted cell to produce lysate, and
[0012] collecting of the lysate.
[0013] The at least one site may be hydrophilic, partially
hydrophilic or become hydrophilic after protein fouling/adsorption
from the sample.
[0014] The digital microfluidic device may include a top plate and
a bottom plate defining a space there between, and wherein each of
the at least one site has an external perimeter, each of the at
least one site being defined on a surface of at least one of the
plates for forming a corresponding virtual microwell, each
corresponding virtual microwell having a virtual wall extending
from the external perimeter of the site between the top and bottom
plates, and upon illumination of the at least one selected targeted
cell by the pulsed laser source, a plasma bubble is formed in the
virtual microwell, and upon formation of the plasma bubble, the
virtual wall deforms thereby absorbing energy released by the
expanding plasma bubble.
[0015] The at least one site may be a plurality of sites, and
wherein the control system is programmed with instructions for
coordinating [0016] moving of droplets to the plurality of sites,
[0017] selecting at least one cell to be lysed at each of the
plurality of sites, [0018] selecting a first site to illuminate
with the pulsed laser source the at least one selected cell at that
site, [0019] moving of the stage to move the digital microfluidic
device sequentially from the first site to another site to bring
each of the sites into a field of view of the pulsed laser source
to lyse the at least one selected cell to produce lysate at each
site, and [0020] collecting the lysate at each site.
[0021] In this aspect the control system may be programmed for
calculating a shortest distance travelled by the stage to bring
sequentially each of the plurality of sites into the field of view
of the pulsed laser.
[0022] The at least one selected targeted cell may be a plurality
of selected targeted cells with the control system being programmed
for [0023] identifying a sequence of selected targeted cells to be
lysed to minimize a time to perform the coordinating steps, and
[0024] wherein the plurality of selected targeted cells is within
one field of view, or a plurality of field of views, or within a
plurality of sites.
[0025] In this aspect the sequence of selected targeted cells is
based on a shortest path between the plurality of selected targeted
cells.
[0026] The imaging system may include a translation mechanism for
displacement of the stage, the translation mechanism being
controlled by the control system.
[0027] The control system may include a droplet control mechanism
or system for controlling displacement of droplets of fluid from a
fluid reservoir towards the at least one site.
[0028] The pulsed laser source may be a nanosecond-pulsed
laser.
[0029] The pulsed laser source may be a nanosecond-pulsed laser
delivering pulses of at least 1 .mu.J.
[0030] The pulsed laser source may be a Q-switched laser.
[0031] The nanosecond-pulsed laser may be a Nd-based laser.
[0032] The nanosecond-pulsed laser may be selected to produce a
pulsed-laser beam within the visible spectrum.
[0033] The present disclosure also provides a method for
identifying and targeting individual cells within a cell population
for selective extraction of cellular content, comprising:
[0034] loading a sample containing cells on at least one site of a
digital microfluidic device thereby forming a virtual microwell at
each of the at least one site;
[0035] Immobilizing the cells on the at least one site;
[0036] selecting at least one immobilized cell;
[0037] lysing the at least one selected cell using a pulsed laser
source to produce lysate within its corresponding virtual
microwell;
[0038] displacing a droplet of liquid to the corresponding virtual
microwell for collecting the lysate; and
[0039] moving the droplet containing the lysate from the
corresponding virtual microwell to a designated site.
[0040] The at least one site is hydrophilic, partially hydrophilic
or become hydrophilic after protein fouling/adsorption from the
sample.
[0041] The method may further include a step of generating a map of
locations of the immobilized cells, and wherein the step of
selecting the at least one immobillized cell includes selecting the
at least one cell from the map.
[0042] The method may further comprise a step of labelling the
immobilized cells. This step of labelling the immobilized cells may
further comprise fixing the cells.
[0043] The method may further comprise steps of:
[0044] moving the digital microfluidic device along horizontal axes
and a vertical axis for positioning the digital microfluidic device
for lysing another at least one selected cell from immobilized
cells;
[0045] lysing the other at least one selected cell using the pulsed
laser source to produce another lysate within its corresponding
virtual microwell;
[0046] displacing another droplet of liquid to the corresponding
virtual microwell for collecting the other lysate; and
[0047] moving the other droplet containing the other lysate from
the corresponding virtual microwell to a designated site.
[0048] The method further comprise a step of introducing the sample
containing the cells at an initial site and displacing the sample
to the at least one site.
[0049] The at least one site is a plurality of sites, and including
steps of
[0050] moving of droplets to said plurality of sites,
[0051] selecting of a cells to be lysed at each of said plurality
of sites,
[0052] selecting a first site to illuminate the selected cell at
that site,
[0053] moving of the stage to move the digital microfluidic device
sequentially to bring each of the sites into a field of view of the
pulsed laser source to lyse the selected cell to produce lysate at
each site, and
[0054] collecting the lysate at each site.
[0055] In this aspect the method may include calculating a shortest
distance travelled by the stage to bring each of the plurality of
sites into the field of view of the pulsed laser source
sequentially.
[0056] The at least one selected targeted cell may a plurality of
selected targeted cells, and the method may further include
[0057] identifying a sequence of selected targeted cells to be
lysed to minimize a time to perform the lysing on all selected
targeted cells, and wherein the plurality of selected targeted
cells is within one field of view, or a plurality of field of
views, or within a plurality of sites.
[0058] In the method the pulsed laser source may be a
nanosecond-pulsed laser.
[0059] In the method the pulsed laser source may be a
nanosecond-pulsed laser delivering pulses of at least 1 .mu.J.
[0060] In the method the nanosecond-pulsed laser may be a Nd-based
laser.
[0061] The nanosecond-pulsed laser may be selected to produce a
pulsed-laser beam within the visible spectrum.
[0062] The pulsed laser source may be a Q-switched laser.
[0063] The method may further comprise the step of performing on
chip analysis of the lysate at the designated site, and may further
comprise the step of collecting the droplet containing the lysate
from the designated site for off-chip analysis.
[0064] The method may be used in the case there the individual
cells are fetal cells, and may be configured for prenatal genetic
testing or screening, or detection or diagnosis of a prenatal
condition.
[0065] Also disclosed herein is method for detection and/or
isolation of fetal cells and/or fetal analytes, the method
comprising:
[0066] loading a sample containing cells on at least one site of a
digital microfluidic device thereby forming a virtual microwell at
each of the at least one site;
[0067] immobilizing the cells on the at least one site;
[0068] selecting at least one immobilized fetal cell;
[0069] lysing the at least one selected cell using a pulsed laser
source to produce lysate within its corresponding virtual
microwell;
[0070] displacing a droplet of liquid to the corresponding virtual
microwell for collecting the lysate; and
[0071] moving the droplet containing the lysate from the
corresponding virtual microwell to a designated site, and
optionally detecting and/or isolating fetal analytes in the
lysate.
BRIEF DESCRIPTION OF DRAWINGS
[0072] Embodiments will now be described, by way of example only,
with reference to the drawings, in which:
[0073] FIG. 1 is a schematic diagram of a system for identifying
and targeting individual cells within a cell population for
selective extraction of cellular content according to an embodiment
of the present disclosure;
[0074] FIG. 2 shows A) a schematic of an assembled DMF device for a
cell culture, B) an enlarged portion of the assemble DMF device
showing an array of electrodes and hydrophilic sites for passive
and active dispensing, and C) a side view of the enlarged portion
of the assembled DMF device according to an embodiment of the
present disclosure;
[0075] FIGS. 3A and 3B are schematics depicting the use of a system
for identifying and targeting individual cells within a cell
population for selective extraction of cellular content according
to an embodiment of the present disclosure;
[0076] FIG. 4A shows a schematic depicting a cell mapping step
according to an embodiment of the present disclosure;
[0077] FIG. 4B shows a schematic depicting a laser microbeam lysis
of a selected cell according to an embodiment of the present
disclosure;
[0078] FIG. 5 shows captured images of passive dispensing of a
droplet for the removal of cell lysis lysate from the virtual
microwell according to an embodiment of the present disclosure;
[0079] FIG. 6 shows brightfield images of trophoblast cells A)
before laser microbeam lysis and B) after laser microbeam lysis
according to an embodiment of the present disclosure;
[0080] FIG. 7 shows immunofluorescent images of GFP-expressing U87
cells A) before laser microbeam lysis and B) after laser microbeam
lysis according to an embodiment of the present disclosure;
[0081] FIG. 8 shows PCR analysis of a single laser lysed cell. A)
The detection of the GFP gene by whole genome amplification and PCR
from the lysis of single GFP-expressing U87 cells. B) The detection
of short tandem repeats of four chromosomes from 1, 3 or 5 laser
lysed cells.
[0082] FIG. 9 shows that only the specifically targeted cells are
lysed. Red (B16-tdTomato) and green (U87-GFP) cells where mixed
together and 5 green (GFP) cells where lysed. PCR was then used to
detect the presence of either the GFP or the tdTomato gene in the
cell lysate. Only the GFP gene was detected in the lysate
indicating that the red cells surrounding the green cells where not
lysed.
[0083] FIG. 10 shows A) an electropherogram of on-chip laser lysed
cells showing size of DNA and B) QF-PCR of the laser lysed cells
according to an embodiment of the present invention C) Next
generation sequencing of the laser lysed cells according to an
embodiment of the present disclosure;
[0084] FIG. 11 is a flowchart showing the high level architecture
of the interaction of the different modules for automated control
cell loading, staining, map generation and cell lysis according to
an embodiment of the present disclosure;
[0085] FIG. 12 is a flowchart showing the steps required for
automation of cell loading, culture and treatment according to an
embodiment of the present disclosure;
[0086] FIG. 13 is a flowchart showing the steps required for
automation of cell staining according to an embodiment of the
present disclosure;
[0087] FIG. 14 is a flowchart showing the steps required for
automation of cell map generation according to an embodiment of the
present disclosure; and
[0088] FIG. 15 is a flowchart showing the steps required for
automation of cell lysis according to an embodiment of the present
disclosure.
DETAILED DESCRIPTION
[0089] Various embodiments and aspects of the disclosure will be
described with reference to details discussed below. The following
description and drawings are illustrative of the disclosure and are
not to be construed as limiting the disclosure. The figures are not
to scale. Numerous specific details are described to provide a
thorough understanding of various embodiments of the present
disclosure. However, in certain instances, well-known or
conventional details are not described in order to provide a
concise discussion of embodiments of the present disclosure.
[0090] As used herein, the terms, "comprises" and "comprising" are
to be construed as being inclusive and open ended, and not
exclusive. Specifically, when used in the specification and claims,
the terms "comprises" and "comprising" and variations thereof mean
the specified features, steps or components are included. These
terms are not to be interpreted to exclude the presence of other
features, steps or components.
[0091] As used herein, the term "exemplary", "illustrative" and
"for example" mean "serving as an example, instance, or
illustration," and should not be construed as preferred or
advantageous over other configurations disclosed herein.
[0092] As used herein, the terms "about" and "approximately" are
meant to cover variations that may exist in the upper and lower
limits of the ranges of values, such as variations in properties,
parameters, and dimensions. In one non-limiting example, the terms
"about" and "approximately" mean plus or minus 10 percent or
less.
[0093] Unless defined otherwise, all technical and scientific terms
used herein are intended to have the same meaning as commonly
understood to one of ordinary skill in the art.
[0094] According to an embodiment, the present invention provides a
system for identifying and targeting individual cells within a cell
population for selective extraction of cellular content. According
to another embodiment, the present invention provides a system for
identifying and targeting individual adherent cells within a
heterogeneous cell population for selective extraction of cellular
content. The system may comprise a DMF device and an imaging system
having a lysis module and an imaging module.
[0095] According to an embodiment, the present invention provides a
method for identifying and targeting individual cells within a cell
population for selective extraction of cellular content using a
system comprising a DMF device and an imaging system having a lysis
module and an imaging module. According to another embodiment, a
method for identifying and targeting individual adherent cells
within a heterogeneous cell population for selective extraction of
cellular content is also provided. The method may use a system
comprising a DMF device and an imaging system having a lysis module
and an imaging module. The adherent cell samples may be introduced
onto a DMF device, where they may be cultured and interrogated
using microfluidic immunocytochemistry. Cells of interest may be
identified and selected and their content extracted selectively by
laser microbeam lysis. The selected cell lysate contents may be
collected into droplets which may be analyzed on-chip or
off-chip.
[0096] In an embodiment there is provided a system for identifying
and targeting individual cells within a cell population for
selective extraction of cellular content. The system includes a
digital microfluidic device having at least one site for receiving
cells and an imaging system including a stage for receiving the
digital microfluidic device, the imaging system including an
imaging module for identifying at least one targeted cell among the
cells at the at least one site and a pulsed laser source for laser
lysing the at least one targeted cell thereby releasing the cell
content to produce a lysate. The system includes a control system
for controlling the pulsed laser source, the imaging system and the
digital microfluidic device. The control system is programmed with
instructions for coordinating steps of [0097] moving of droplets on
the digital microfluidic device, [0098] selecting of the at least
one targeted cell to be lysed located at the at least one site,
[0099] illuminating of the at least one selected targeted cell by
the pulsed laser source to lyse the at least one selected targeted
cell to produce lysate, and [0100] collecting of the lysate.
[0101] In some embodiments the at least one site for receiving
cells may be hydrophilic, partially hydrophilic or become
hydrophilic after protein fouling/adsorption from the sample.
[0102] In an embodiment the digital microfluidic device may include
a top plate and a bottom plate defining a space there between, and
wherein each of the at least one site has an external perimeter,
each of the at least one site being defined on a surface of at
least one of the plates for forming a corresponding virtual
microwell, each corresponding virtual microwell having a virtual
wall extending from the external perimeter of the site between the
top and bottom plates. Upon illumination of the at least one
selected targeted cell by the pulsed laser source, a plasma bubble
is formed in the virtual microwell, and upon formation of the
plasma bubble, the virtual wall deforms thereby absorbing energy
released by the expanding plasma bubble.
[0103] In an embodiment the at least one sites is a plurality of
sites, and wherein the control system is programmed with
instructions for coordinating [0104] moving of droplets to said
plurality of sites, [0105] selecting at least one cell to be lysed
at each of said plurality of sites, [0106] selecting a first site
to illuminate with the pulsed laser source the at least one
selected cell at that site, [0107] moving of the stage to move the
digital microfluidic device sequentially from the first site to
another site to bring each of the sites into a field of view of the
pulsed laser source to lyse the at least one selected cell to
produce lysate at each site, and [0108] collecting the lysate at
each site.
[0109] In the embodiment having a plurality of sites, the control
system may be programmed for calculating a shortest distance
travelled by the stage to bring sequentially each of the plurality
of sites into the field of view of the pulsed laser.
[0110] In an embodiment the at least one selected targeted cell is
a plurality of selected targeted cells, and the control system may
be programmed for [0111] identifying a sequence of selected
targeted cells to be lysed to minimize a time to perform the
coordinating steps, and [0112] wherein the plurality of selected
targeted cells is within one field of view, or a plurality of field
of views, or within a plurality of sites.
[0113] The sequence of selected targeted cells may be based on a
shortest path between the plurality of selected targeted cells.
[0114] In an embodiment the imaging system may include a
translation mechanism for displacement of the stage, the
translation mechanism being controlled by the control system.
[0115] In an embodiment the control system may include a droplet
control means for controlling displacement of droplets of fluid
from a fluid reservoir towards the at least one site.
[0116] In an embodiment the pulsed laser source is a
nanosecond-pulsed laser.
[0117] In an embodiment the pulsed laser source may be a
nanosecond-pulsed laser delivering pulses of at least 1 .mu.J.
[0118] In an embodiment the nanosecond-pulsed laser may be a
Nd-based laser.
[0119] In an embodiment the nanosecond-pulsed laser produces a
pulsed-laser beam within the visible spectrum.
[0120] In an embodiment the pulsed laser source may be a Q-switched
laser.
[0121] According to an embodiment, referring to FIG. 1, a system
for identifying and targeting individual cells within a cell
population for selective extraction of cellular contents 1 may
comprise a DMF device 40 and an imaging system 30.
[0122] The imaging system 30 may be equipped with a lysis module 10
and an imaging module 20. The imaging system 30 may be any suitable
optical imaging system known in the art that can capture
brightfield and fluorescent images and that may direct a laser beam
into a focused laser beam for forming a plasma bubble at the same
plane of imaging. According to an embodiment, the imaging system 30
may be a microscope such as an inverted microscope. The imaging
system 30 may have a motorized stage 32 for cell localization,
image tilting and autofocusing and an optical unit 31 configured
for both brightfield and fluorescent imaging. One possible but not
limiting example of the inverted microscope is an Olympus IX-71
series model. According to another embodiment, the imaging system,
instead of being equipped with the motorized stage 32, may have a
fixed stage and motorized lens and mirrors for cell localization,
image tiling and autofocusing.
[0123] The lysis module 10 may comprise a pulsed laser source 11
which emits a laser microbeam 120. The pulsed laser source 11 is
aligned using a dichroic mirror 21 into the magnification objective
lens 24 and the opening 321 of the motorized stage 32. The pulsed
laser source 11 may be a laser source capable of producing a
high-energy pulsed laser beam. According to an embodiment, the
pulsed laser source 11 may be a nanosecond-pulsed laser delivering
pulses of at least 1 .mu.J. According to another embodiment, the
pulsed laser source 11 may be a nanosecond-pulsed laser delivering
pulses of >4-6 .mu.J and able to produce pulses of approximately
0.5 to 50 ns or a faster laser such as an ultrafast laser
(femtosecond-pulsed laser or picosecond-pulsed laser). The pulsed
laser source 11 may be a Q-switched laser, gain-switched laser,
mode-locked laser or a pulsed-pumped laser. According to another
embodiment, the pulsed laser source 11 may be able to emit a pulsed
laser beam within the visible spectrum. More particularly, the
pulsed laser source 11 may be a Nd-based laser, e.g., a 532 nm
Q-switched Nd:YAG laser. According to an embodiment, the
nanosecond-pulsed laser may produce a pulsed-laser beam within the
visible spectrum.
[0124] The imaging module 20 may comprise a light source 22 which
emits a light beam 220. The light source 22 may be aligned using a
dichroic mirror 21 into the magnification objective lens 24 and the
opening 321 of the motorized stage 32. The imaging module may
further comprise an excitation filter 25 and an emission filter
26.
[0125] The system 1 may further comprise a control system 3 which
may allow the coordinated management of the droplet manipulation
and lysis of the targeted cells. The motorized stage 32 may be
configured to be controlled by the control system 3 to program and
manage droplet movement on the DMF device 40. For example the
motorized stage 32 may be modified with an array of pogo pins 401
that allow the control system 3 to control the droplet movement as
shown in FIG. 2.
[0126] According to another embodiment, the control system 3 may
comprise a droplet control system or mechanism, a computer and a
control board. An example of such a system is the open source
"DropBot" system (described at
http://microfluidics.utoronto.ca/dropbot/) which may be controlled
centrally via a computer and control board, allowing for the
coordinated management of droplet manipulation and lysis of
targeted cells. The motorized stage 32 may be configured to be
controlled by the droplet control system to program and manage
droplet movement on the DMF device 40. For example the motorized
stage 32 may be modified with an array of pogo pins 401 that allow
the control board through the interface of the droplet control
system to control the droplet movement.
[0127] The DMF device 40 may rest upon the motorized stage 32 which
is designed to move laterally to reposition the targeted cells and
vertically to focus on a selected targeted cell.
[0128] DMF is a fluid handling technology in which sample and
reagents are manipulated as discrete droplets on a hydrophobic
surface. DMF systems actuate droplets through the application of
electrical potentials on a generic array of insulated electrodes.
This format enables software-reconfigurable, concurrent droplet
operations including merging, mixing, splitting and metering from
reservoirs. Complex multi-step procedures can be performed on DMF
devices such as DMF device 40, e.g., integrated multiplexed ELISA,
long-term multigenerational cell culture and immunocytochemistry. A
DMF-based immunocytochemistry method has the advantage of providing
the possibility of evaluating adherent cells in situ.
[0129] As shown in FIG. 2, the DMF device 40 may be a two-plate DMF
device comprising a bottom plate 41 and a top plate 42. The bottom
plate 41 may comprise a glass or plastic substrate 46 patterned
with electrodes 43 which are coated with a dielectric material 431
and a hydrophobic material 45. The top plate 42 may comprise a
glass or plastic substrate 46 coated with a conductive layer, i.e.,
electrodes 43, which is pattern-coated with the hydrophobic
material 45. The patterned coating may be such that the top plate
42 includes patterned hydrophilic sites 44. Both dielectric
material 431 and hydrophobic material 45 may be any suitable
dielectric material and hydrophobic material known to those skilled
in the art respectively, e.g., Parylene.TM. C for dielectric
material 431 and Teflon.TM. AF for the hydrophobic material 45.
Reservoirs 48 are located at both extremities of the bottom plate
41. Droplets 49, which may contain reagents or samples, may be
displaced on the DMF device 40 from or to the reservoirs 48 either
through active dispensing or passive dispensing. Active dispensing
is achieved by actuating electrodes 43 to electrostatically
stretch, neck, and pinch a droplet 49. This active dispensing
renders possible the reliable and precisely on-demand dispensing at
the microliter scale of cell samples and reagents. Passive
dispensing is implemented by taking advantage of variations in
surface energy on the surface of the DMF device 40 that is
primarily hydrophobic but patterned with hydrophilic regions.
Passive dispensing occurs spontaneously as a droplet 49 is
translated across the hydrophilic site 44. When the droplet 49 is
translated across the hydrophilic site 44, surface tension effects
result in spontaneous formation of a sub-droplet on the site.
Passive dispensing is particularly useful for adherent mammalian
cell cultures, allowing for cell 100 seeding onto dry hydrophilic
sites 44, as well as for subsequent media and/or reagent exchange
on droplet-bearing sites 44. One advantage of passive dispensing is
the formation of virtual micro-wells 47 at the hydrophilic sites
44. Virtual microwells 47 are not confined on the sides like
traditional wells, but are defined by the surface properties of the
bottom and top and plates 41 and 42 and virtual vertical wall 471,
which is simply defined by the air-liquid interface of the droplet
49 that is held in place at the hydrophilic site 44 by surface
tension of the liquid (see [19] Eydelnant et al. (2012) Lab Chip
12(4):750-757). The volume of the virtual micro-wells 47 is
dictated by the diameter of the hydrophilic sites 44 and the
distance between bottom 41 and top plates 42. According to an
embodiment, the site 44 may be hydrophilic, partially hydrophilic
or become hydrophilic after protein fouling/adsorption from the
sample.
[0130] As shown in FIG. 2, cells 100 from a sample to be analyzed
reside between the bottom plate 41 and top plate 42 of the DMF
device 40. As the cells 100 cannot grow on a hydrophobic material,
the cells 100 are cultured on the hydrophilic sites 44 of the top
plate 42 and in the virtual microwell 47.
[0131] According to an embodiment, as shown in FIG. 3A, a sample
containing heterogeneous cells 100 may be introduced on the DMF
device 40. The sample may be introduced into reservoir 48 under the
form of a droplet 49a. The droplet 49a containing the cells 100 is
then displaced on the DMF device 40 from the reservoir 48 towards
to the hydrophilic site 44 through active dispensing. Once the
cells 100 have reached the hydrophilic site 44, the virtual
microwell 47 is formed by passive dispensing and the cells 100 may
be cultured for a sufficient amount of time to achieve cellular
adhesion on the hydrophilic site 44. Alternatively, prior assembly
of the DMF device 40 by assembling the top plate 42 onto the bottom
plate 41, the sampled cells 100 may be introduced onto the
hydrophilic site 44 of the top plate 42, and optionally cultured,
and the DMF device 40 is then assembled and mounted onto the
motorized stage 32. In this embodiment, the sampled cells 100 may
be a cell suspension introduced as a droplet, part of a biopsied
tissue sample or a smear (e.g. cells are contained within a sample
having high extracellular matrix content, highly viscous in nature)
onto the hydrophilic site 44.
[0132] As shown in FIG. 3A, cells 100 present within the virtual
microwell 47 are then labelled with imaging reagents that interact
with or bind to cell-specific markers, surface and/or
intracellular, to identify targeted cells 110 using techniques well
known in the literature. The imaging reagents may include but are
not limited to cell stains such as DAPI and eosin and/or
fluorescent dyes such as fluorescein derivatives, BODIPY
derivatives, cyanine derivatives, etc. The imaging reagents may
include binding agents that bind to cell specific markers,
including but are limited to antibodies conjugated to a detectable
label such as the fluorescent dyes listed above. Immunofluorescence
staining protocols for DMF applications known to be suitable by the
skilled person may be used. For example, the protocol described by
Ng et al. may be used for immunocytochemical labelling the cells
for the purpose of the present invention [15].
[0133] It will be appreciated by one skilled in the art that any
label that can be detected by physical, chemical, spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical,
electromagnetic and other related analytical methods may be
conjugated to an imaging reagent, in particular a binding agent. A
detectable label that may be utilized includes, without limitation,
a radioisotope, fluorophore, chromophore, mass label, electron
dense particle, magnetic particle, spin label, chemi-luminescent
molecule, electro-chemically active molecule, enzyme, cofactor, and
enzyme substrates. In aspects of the disclosure, the label is a
binding agent disclosed herein or known in the art. In aspects of
the disclosure, the label is an immunocytochemical label, in
particular an antibody.
[0134] The immunocytochemical labelling reagents or stains and dyes
or similar imaging reagents 105 may be introduced into reservoir 48
under the form of a droplet 49b. The droplet 49b containing the
reagents 105 may be then displaced on the DMF device 40 from the
reservoir 48 towards the virtual microwell 47 through active
dispensing. The droplet 49b may then be actuated through the
virtual microwell 47 for immunocytochemical labelling. Once the
labelling has been done, the fluid containing the excess of
immunocytochemical labelling reagents 105 is carried to the
opposite reservoir 48 as droplet 49b'. According to an embodiment,
the step of immunocytochemical labelling the cells may comprise
additional steps such as introduction of additional reagents,
washes, etc.
[0135] According to an embodiment, the step of immunocytochemical
labelling the cells 100 may comprise the step of fixing the cells
prior to labelling. The fixing step may comprise the use of any
acceptable fixative agent known in the art. The fixing step may
comprise the use of denaturing fixative or crosslinking fixative.
Non-limiting examples include paraformaldehyde, methanol, ethanol,
acetone, and glutaraldehyde. The system 1 may perform the lysis of
cells that are fixed or live. Live cells have limited means for
selection (e.g., external cell markers and morphology) but may be
compatible with a broad range of analysis, such as genomic,
proteomic and metabolomics profiling. Fixed cells are more amenable
for selection (intracellular markers can be probed by
immunocytochemistry) and may be stable over long time periods;
however, the analytical techniques amenable for probing their
contents may be limited.
[0136] According to an embodiment, an adherent cancer cell line,
U87, was stably transduced with green fluorescent protein (GFP) and
then cultured on a DMF device for two days. As shown in FIGS. 7A
and 7B, a particular cell was targeted with a 3-nanosecond laser
pulse and its lysate content was collected immediately after lysis,
showing that the cell has been lysed and is no longer visible.
[0137] FIGS. 6A, 6B and 7A, 7B exemplify the flexibility of the
system 1 to lyse both fixed and live cells on DMF devices.
According to an embodiment, FIGS. 6A and 6B display lysis of
paraformaldehyde-fixed trophoblast cells from the CVS isolation (6A
prior to lysis and 6B after lysis). For the laser lysis, the media
was replaced with PBS and cells were lysed into the PBS droplet as
shown in FIGS. 6A and 6B. The lysate was then used for
genotyping.
[0138] According to another embodiment, FIGS. 7A and 7B display
lysis of live cells, using the U87 cell line (7A prior to lysis and
7B after lysis). U87 cells stably transduced with green fluorescent
protein (GFP) were cultured on a DMF device for two days. As shown
in FIGS. 7A and 7B, a particular cell was targeted with a
3-nanosecond laser pulse and its lysate content was collected
immediately after lysis, showing that the cell has been lysed and
is no longer visible. The cell lysate generated from a single U87
cell was collected and the whole genome was amplified by MDA and
the resulting sample was analyzed by PCR for the GFP gene to show
that it is possible to obtain results from a single cell as shown
in FIG. 8A. FIG. 8B shows the PCR analysis for short tandem repeats
on four chromosomes indicating that there is a high level of genome
coverage generated from the laser lysis of cells. As shown in FIG.
9, only the cells targeted for lysis are lysed. Red (B16-tdTomato)
and green (U87-GFP) cells where mixed together and 5 green (GFP)
cells where lysed via the laser microbeam. Whole genome
amplification and PCR were then used to detect the presence of
either the GFP or the tdTomato gene in the cell lysate. Only the
GFP gene was detected in the lysate indicating that the red cells
surrounding the green cells where not lysed.
[0139] Once the cells 100 have been immunocytochemically labelled,
the imaging system 30 may use the imaging module 20 to generate
images of the virtual microwell 47 in brightfield imaging for size
and shape discrimination and fluorescence imaging for detection of
the nucleus and labelled cells 110 tagged with the cell-specific
biomarkers. FIGS. 6A and 6B show the lysis of trophoblast cells in
brightfield imaging and FIGS. 7A and 7B show the lysis of U87 cells
in fluorescence imaging respectively. Image analysis software, such
as CellProfiler.TM. (http://cellprofiler.org/), may be used to
determine the cells of interest based on the labeling pattern of
the cell markers and cell size/shape. The captured images may be
processed by machine learning algorithms to identify the targeted
cells 110 and plot their locations in the virtual microwell 47 to
generate a map of the locations of the targeted cells 110.
Algorithms relying on biomarkers and cell size/shape may be
implemented to identify the targeted cells to be lysed, e.g., cells
111, 112 and 113 (FIG. 3A).
[0140] Furthermore, the cells may be targeted based on cell
morphology and phenotype, including but not limited to cell size,
shape, surface marker, intracellular marker, dye/drug uptake,
etc.
[0141] According to an embodiment, the cells may be targeted based
on positive and/or negative selection.
[0142] FIG. 4A illustrates an example of the mapping process where
cultured cells are labelled with biomarkers such as Fetal Cell
Marker 1 and Fetal Cell Marker 2. The cells are then imaged and
image analysis and cell mapping are performed to identify and
select targeted cells, such as cells 111, 112 and 113. During the
cell mapping, algorithms may be applied to determine an optimal
displacement path for the motorized stage 32 between cells 111, 112
and 113.
[0143] Once the mapping has been performed and the targeted cells
have been selected, the system 1 may adjust its operation mode from
imaging mode to cell lysis mode. The targeting map is then used to
move the DMF device 40 so that a selected targeted cell, such as
cell 111, 112 or 113, is brought into the laser microbeam 120 path
for lysis. After the selected targeted cell is lysed, one or more
droplets 49 may be passed over the virtual microwell 47 to collect
the cellular contents and the automated targeting system moves the
DMF device to the next cell.
[0144] Now referring to FIG. 4B, once the adhered cells 100 have
been cultured and immunocytochemically labelled, a labelled cell
110 is selected. A high-energy nanosecond-pulsed laser beam 120 may
be focused through the front and rear optical lenses of the imaging
device 30, producing a microscopic plasma bubble 121 in proximity
to the targeted selected cell 110. The plasma bubble 121 expands
rapidly and with enough force to disrupt the cell membrane 110b and
lyse the selected labelled cell 110 releasing the cell content 110c
as cell lysate 114 into the surrounding solution of the virtual
microwell 47. According to an embodiment, the pulsed laser source
11 may fire the laser beam 120 for a duration of 3 nanoseconds
leading in the formation of the plasma bubble 121 and the collapse
of the targeted labelled cell 110.
[0145] According to another embodiment, the lysis may be done in a
very short period of time such as 0.5 seconds or less per cell. The
"wall" of the virtual microwell 47 or virtual vertical wall 471 may
be simply defined by the air-liquid interface of the droplet that
is held in place at the hydrophilic site 44 by surface tension of
the liquid. This means that the virtual vertical wall 471 is
flexible and deformable which allows the absorption of the energy
of the expanding plasma bubble by expanding/deforming as the bubble
grows and then snapping back into the original shape afterwards
based on the lowest energy state of the air/liquid interface
without damaging the DMF device 40.
[0146] Alternatively, the plasma bubble 121 may lyse the selected
labelled cell 110 and other cells that are within a few micrometers
(.mu.m) of the focal spot releasing the contents of a plurality of
cells as cell lysate into the surrounding solution of the virtual
microwell 47.
[0147] In one particular embodiment, the technique may be carried
out using substrates formed from optically transparent materials
(e.g., glass microscope slides similar to those used to form DMF
devices) for sampling of adherent cell contents by capillary
electrophoresis [16-18] as shown in U.S. Pat. No. 6,156,576, which
is incorporated herein by reference in its entirety.
[0148] According to an embodiment, the repetition rate (or pulse
repetition frequency) and the pulse energy of the pulsed laser
source 11 may be adjusted according to the type of cells, state of
the cells and the material of interest to be collected. According
to an embodiment, one or more laser pulses may be needed to lyse
the cell 110.
[0149] As the laser microbeam lysis process is dependent on
achieving very high optical power in a confined volume, if the
focal point of the objective 24 is not correctly positioned at the
same distance as the targeted cell 110, the defocused portion of
the laser beam 120 may not attain high enough power to cause cell
lysis. For this reason, it is preferable (but not essential) to
maintain correct focus for the lysis of each selected labelled cell
110. As factors such as changes in temperature and deviations from
planarity of the DMF top plate 42 may influence the focal point of
the objective 24 or the distance between the selected labelled cell
110 and the objective 24, the focus must be adjusted for each
selected cell 110 using an autofocus system.
[0150] According to an embodiment, the autofocus system (not shown)
may use a simple software feature that adjusts the vertical
position of the motorized stage 32 to maximize the contrast
detected by the coupled imaging module 20. Alternatively the
autofocus system may comprise an active sensor to measure the
distance between the objective 24 and the inner surface of the DMF
top plate 42 improving the speed of focusing. In addition, slight
changes to the lysis module 10 conveying the laser microbeam 120 to
the objective 24, for instance due to temperature or vibration, may
change the angle at which the laser beam 120 enters the objective
24, altering the focal point of the laser microbeam 120. Because
the plasma bubble 121 produced affects only a small area, a small
change in the point of impact may impair proper cell lysis. To
prevent this, the focal point of the laser beam 120 may be measured
by placing a test substrate with a fluorescent coating on the
motorized stage and low power laser pulses applied to image the
actual focal point. With this information updated as needed, the
placement of the stage 32 may be corrected by the control system 3
to position the laser focal point in the intended location.
[0151] To efficiently lyse a multitude of cells in sequence, it may
be desirable to minimize the distance the motorized stage 32 has be
moved to reach all the cells, reducing the time required per
sample. Using the data acquired during the mapping process,
algorithms may be applied to determine an optimal displacement path
for the motorized stage 32 to move from one to another selected
targeted cell 110.
[0152] As shown in FIG. 3B, after cells 100 were cultured on the
hydrophilic site 44, a selected cell or plurality of selected cells
from the labelled cells 110 is lysed by laser microbeam lysis. As
shown in FIG. 3B and FIG. 5, once the selected labelled cell or
cells 110 is/are lysed within the virtual microwell 47, the droplet
of liquid covering the cells is replaced with a new droplet. A
fresh droplet 49 of reagent may be dispensed from the reservoir 48
and may then be driven through the virtual microwell 47, displacing
the original fluid droplet 49 that contains the cell lysate. The
original fluid is carried to a reservoir 48 on the opposite side of
the device 40.
[0153] As shown in the still images of FIG. 5, a fresh droplet 49
is dispensed from the reservoir 48 on one side of the device 40 as
shown and then driven through the virtual microwell 47, displacing
the original fluid in the virtual microwell 47 that contains the
cell lysates. The original fluid is carried to a reservoir 48 on
the other side of the device 40. The observation that droplets 49
are moveable before and after lysis demonstrates that the laser
microbeam plasma bubble 121 does not damage either surface of the
DMF device 40 and that droplet 49 movement is unhindered after
lysis. This is an indication that the use of lysis module to
extract cellular content of a targeted cell cultured within a DMF
device may be compatible with DMF technology.
[0154] Prior to reaching the reservoir 48 on the other side of the
device 40, the droplet 49 containing the cell lysate 112 may be
further manipulated on the DMF device 40. Once the droplet 49
reaches the other side reservoir 48, the droplet 49 may be then
collected for downstream analysis off-chip or used for on-chip
analysis. By carefully controlling the timing of the laser
illumination in synchronicity with the droplet movement through the
use of the control system 3, one may generate arrays of droplets
containing the contents of single cells (or the contents of
multiple cells, if desired) within a short period of time.
According to an embodiment, the DMF device 40 may have a multitude
of reservoirs 48 on both side of the device (as shown in FIG. 2).
Once the droplet 49 has collected the cell lysate from the virtual
microwell 47, the droplet may be divided and the resulting droplets
may be displaced to the same reservoir 48 or displaced to different
reservoirs 48.
[0155] According to an embodiment, when successively lysing a
multitude of cells 110, such as cells 111, 112 and 113, the
droplets 49c', 49d' and 49e' may be carried to the same reservoir
48. Under such set-up, cleaning droplets may be introduced on the
device 40 to clean the droplet path and the reservoir 48 before the
displacement of droplets 49c', 49d' and 49e' to reservoir 48.
Alternatively other cleaning means may be used to prevent
contamination. According to another embodiment, when the DMF device
40 has a several of reservoirs 48 on both sides of the device (as
shown in FIG. 2), each droplets 49c', 49d' and 49e' may be
displaced to a different reservoir 48 therefore minimizing
contamination.
[0156] According to an embodiment of the present disclosure, an
advantage of combining the lysis module 10 with the DMF device 40
is the precise control of the illumination timing of the laser
microbeam 120 from the pulsed laser source 11, performed by the
control system 3 in coordination with the displacement of droplets
49 over the adherent cells 100, performed by the control system
3.
[0157] Such coordinated control over the laser
microbeam-illumination timing and the droplet displacement enables
the user to rapidly generate an array of droplets, each of which
may contain the content of an individual cell while minimizing
contamination and degradation. Alternatively the contents of
multiple cells may be contained within a droplet.
[0158] In an embodiment, the DMF device 40, and the imaging system
30 comprising the lysis module 10 and the imaging module 20 may all
be controlled (or at least coordinated) by the control system 3
programmed with control software, where steps that do not interfere
can be run concurrently (e.g., droplets can move while the stage is
repositioning), but checkpoints where certain conditions should be
met to proceed will ensure the system is in the correct state at
critical points. e.g. the laser source 11 will not fire until the
stage 32, DMF device 40 and droplet 49 are in position. Also, the
droplet 49 will not be moved away until lysis is confirmed by
comparing the camera images before and after; etc. Automatic error
handling could also be performed: droplet movement or laser lysis
could be retried if it is detected that the system is not in the
correct state, only notifying the operator if the error cannot be
corrected.
[0159] According to an embodiment, FIG. 3B illustrates the
coordination between droplet movement and laser lysis. The droplet
49c is displaced from the reservoir 48 toward the virtual microwell
47 as the laser beam 120 lyses the targeted cell 111 releasing the
cell content of cell 111c. Droplet 49c is then driven through the
virtual microwell 47 displacing the original fluid in the virtual
microwell 47 that contains the cell lysate 111c. The original fluid
is carried to the opposite reservoir 48 as droplet 49c'. As droplet
49c' reaches the reservoir 48 and is collected for analysis, a
second droplet 49d is displaced from the reservoir 48 toward the
virtual microwell 47 as the laser beam 120 lyses the targeted cell
112 releasing the contents of cell 112c.
[0160] Droplet 49d is then driven through the virtual microwell 47
displacing the previous fluid in the virtual microwell 47 that
contains the cell lysate 112c. The fluid containing the lysate 112c
is carried to the opposite reservoir 48 as droplet 49d'. As droplet
49d' reaches the reservoir 48 and is collected for analysis, a
second droplet 49e is displaced from the reservoir 48 toward the
virtual microwell 47 as the laser beam 120 lyses the targeted cell
113 releasing the contents of cell 113c. Droplet 49e is then driven
through the virtual microwell 47 displacing the previous fluid in
the virtual microwell 47 that contains the cell lysate 113c. The
fluid containing the lysate 113c is carried to the opposite
reservoir 48 as droplet 49e'. This process may be repeated until
all of the targeted cells 110 are lysed and their contents
collected. The lysate for different lysis may be brought to
different reservoirs or alternatively collected at the same
reservoir.
[0161] It will be understood that droplets 49c, 49d and 49e are
non-limiting examples and that one or more droplets may be passed
through the microwell to collect the lysate content 111c, 112c, and
113c of cells 111, 112, and 113 respectively. As the droplets 49c,
49d and 49e are passed through the virtual microwell 47 under the
control of control system 3, the control system 3 displaces the
motorized stage 32 to adjust the DMF device 40 to position and
focus on the next targeted cell for lysis. To minimize
contamination and degradation of the lysate content, the control
system 3 in communication with the lysis module 10 timely
coordinates the dispensing of the droplets 49, the adjustment of
vertical and horizontal positions of the motorized stage 32 and the
firing of the pulsed laser source 11.
[0162] According to an embodiment, under the control of the control
system 3, the lysis-stage displacement-lysis-stage
displacement-etc. may be performed on the time scale of about 5
seconds or less per step for movement within a virtual microwell
and 15 seconds or less per step when moving between adjacent
microwells. Once the lysates 111c, 112c and 113c of selected cells
111, 112 and 113 are collected for off-chip analysis, genomic,
metabolomic, transcriptomics and/or proteomic analysis of the lysed
cell's contents may be performed and the obtained information may
be subsequently correlated to the cell morphology and phenotype
information catalogued prior to analysis. These "omic" based
analyses are of great importance for many research and clinical
applications from the study of stem cells and their differentiation
to understand circulating tumour cells and their role in metastatic
disease. According to another embodiment, the lysates 111c, 112c
and 113c may be collected for on-chip analysis such as ELISA and
miRNA detection by electrochemistry or other detection modalities
that can interface with DMF devices. The system for identifying and
targeting individual cells within a cell population for selective
extraction of cellular content 1 may also be configured in a mode
to lyse multiple cells before the lysate is collected.
[0163] According to an embodiment, to ensure that targeting maps
may be imported from external sources or exported to other
instruments and that the motorized stage 32 may be moved to
position the DMF device 40 accurately, the coordinate space of the
imaging system 30 must be aligned with that of the DMF device 40.
Because there are always slight changes in position and rotation of
the DMF device 40 during fabrication, two or more alignment marks
(not shown) may be added to the DMF top plate 42 to be located by
the imaging system 30 and may be used as reference points. To
locate the marks, the imaging system 30 scans the areas where the
marks should be found and their locations can be detected by one of
several methods including finding the autocorrelation maximum with
a template image of the alignment mark or methods based on feature
recognition [20]. Since the positional relationship within the
alignment marks and between the marks and the virtual microwells 47
may be defined by the design of the DMF device 40, assuming the
device 40 is coplanar with the motorized stage 32, the translation
and rotation needed to convert between the coordinates of the DMF
device 40 and coordinates of the imaging system 30 may be
calculated from the locations of the marks.
[0164] According to an embodiment, the control system 3 may be
programmed with modules of instructions that define the operating
parameters of the system 1. The modules may be independent of each
other and may be assembled in different configurations as required.
There are a multitude of modules that may be generated depending on
the experiments being performed.
[0165] According to an embodiment, as shown in FIG. 11, the control
system 3 may be programmed with the following modules of
instructions fitting together to identify and target individual
cells within a population for the selective extraction of cellular
content: 1) cell loading and treatment, 2) cell staining, 3) map
generation and 4) laser cell lysis.
[0166] An iteration of the automated cell loading and treatment
module is shown in FIG. 12. In FIG. 12 and also FIGS. 13, 14 and 15
that are described below, the circles represent inputs to the
system (e.g., cells, drugs, etc.). The rectangles are the action
and triangles are times. The module, as shown in FIG. 12, may load
the cells into the virtual microwells 47 by loading a single cell
suspension into the DMF device 40 and the program moving the
droplet 49 over the hydrophilic sites 44 to form the virtual
microwells 47. If the stage 32 is outfitted with temperature and
CO.sub.2 controls so that the temperature and CO.sub.2 levels of
the DMF device 40 are regulated, then the control system 3 may
monitor the cell density via the imaging module 20. For live cell
imaging and analysis 37.degree. C. and 5% CO.sub.2 are the optimal
conditions of operation. Note that for fixed cells room temperature
and no CO.sub.2 can be used for imaging and analysis. Once the
desired cell density is reached the control system 3 will
automatically treat the cells with different reagent (drugs,
ligands, etc.) in various patterns (changes in concentration,
duration, etc.).
[0167] An example of a possible cell treatment is stimulation with
PDGF as demonstrated in Ng et al. (ref. 15). An iteration of the
cell staining module (FIG. 13) shows the automated steps required
for performing immunocytochemistry on the DMF device 40 and/or
treating the cells 100 with a dye or stain (e.g., DAPI). An
iteration of the map generation module (FIG. 14) describes the
steps require for the control system 3 to identify cells of
interest and generate a map of their positions. To generate the
map, images of the cells 100 are taken at the lowest resolution
required to identify the parameters of interest. For example, if
cell size and shape are the parameters then 5.times. or 10.times.
magnification would be used. However, if the parameters were in
subcellular localization of a protein then higher magnification
would be required. The images are tiled together to map the entire
virtual micro well 47 and this may be used to locate the cells of
interest 110. The parameters used to identify the cells of interest
110 may be determined beforehand and inputted into the program. In
its simplest form, the parameter may be a cut-off threshold based
on cell size. Alternatively the parameter may be a multi-parameter
threshold based on several factors. One possible example of this
may be machine learning where a training set may be used to develop
rules for identification of the cells of interest.
[0168] FIG. 15 shows an iteration of the cell lysis module which
controls the steps of the laser lysis. After the cells of interest
110 are identified, the user may have the options to tell the
control system 3 which cells 110 are to be collected (all or a
subset of the cells of interest and/or control cells), the number
of cells lysed per droplet, etc. From there, the control system 3
may generate a target list and cell lysis pattern (or route). This
lysis pattern may minimize the distance traveled between lysis
events to minimize the operational time required to lyse all of the
targeted cells 110 [21, 22]. If there is any simple on-device
sample processing and analysis, this may also be performed
automatically by the program and then the droplet 49 may be stored
for collect off the device for further analysis. Complex on-device
sample processing or analysis may have their own program modules
that would run after the cell lysis module. These example modules
show the major high level steps and decision points of the workflow
therefore some steps may not be shown and each box of the flowchart
may have several substeps that are not shown.
[0169] According to an embodiment, the systems, methods and kits of
the present invention may be used for identification, isolation
and/or characterisation of cellular material/analytes as
non-invasive testing. In an aspect, the systems, methods and kits
may be used as non-invasive diagnostics or screenings for
pathological conditions. In another aspect, the systems, methods
and kits may be used for non-invasive genetic, genomic, metabolic,
transcriptomics and/or proteomic diagnostics and/or screenings. In
an aspect, the analytes may include without limitation, nucleic
acids, proteins (for example, amino acids, peptides, enzymes,
antigens, antibodies, cytokines, lipoproteins, glycoproteins,
growth factors or hormones), lipids, carbohydrates, metabolites or
combinations thereof. Examples of pathological conditions which may
be detected, diagnosed or screened using the systems, methods and
kits of the disclosure, include without limitation a cell
proliferation condition. Examples of cell proliferation conditions
include without limitation, cancers of the colorectum, breast,
lung, liver, pancreas, lymph node, colon, prostate, brain, head and
neck, skin, liver, kidney, and heart; inflammatory conditions, such
as inflammation conditions of the skin; conditions related to
obesity; such as proliferation of adipocytes; viral conditions, and
cardiac conditions.
[0170] In an aspect, the analysis of analytes (e.g., detection
and/or levels of proteins and/or characterization) may be
determined using a variety of methods known to a person skilled in
the art such as immunoassays in various formats such as
radioimmunoassay (RIA), chemiluminescence- and
fluorescence-immunoassays, enzyme immunoassay (EIA), enzyme-linked
immunoassays (ELISA), luminex-based bead arrays, protein microarray
assays, and rapid test formats such as immunochromatographic strip
tests, and Selected/Multiple reaction monitoring (SRM/MRM). The
immunoassay may be a homogenous or heterogeneous assay, competitive
or non-competitive. Examples of other methods for analysis of
proteins include spectrometry, mass spectrometry, Matrix Assisted
Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass
Spectrometry, microscopy, northern blot, isoelectric focussing,
SDS-PAGE, PCR, quantitative RT-PCR, gel electrophoresis, DNA
microarray, protein sequencing, proteome analysis, and antibody
microarray, or combinations thereof.
[0171] In an aspect, the cellular analyte is nucleic acid. The
nucleic acid may be DNA (for example, genomic, mitochondrial, and
complementary cDNA from RNA), or RNA (for example, rRNA, mRNA and
miRNA). In an aspect, the nucleic acids are nucleotides,
oligonucleotides, DNAs, RNAs, or DNA-RNA hybrids. In an embodiment,
the nucleic acids are DNAs, in particular, double-stranded DNAs,
single-stranded DNAs, multi-stranded DNAs, complementary DNAs,
genomic DNAs or non-coding DNAs. In an embodiment, the nucleic
acids are RNAs, in particular, messenger RNAs (mRNAs), microRNAs
(miRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs),
transfer RNAs (tRNAs), small interfering RNAs (siRNAs),
heterogeneous nuclear RNAs (hnRNAs), or small hairpin RNAs
(shRNAs).
[0172] Below is a general procedure for processing cells using a
system or method of the invention: [0173] Obtaining a cell sample;
[0174] Generating a single cell suspension from sample [0175]
Loading cell suspension into a DMF device [0176] Moving the cell
suspension as a droplet on at least one site of the DMF device to
form a virtual microwell at each of the at least one site [0177]
Immobilizing the cells from cell suspension droplet at the desired
site [0178] Selecting at least one immobilized cell [0179] Lysing
at least one selected cell using a pulsed laser source to produce a
lysate within its corresponding virtual microwell [0180] Displacing
a droplet of liquid to the corresponding virtual microwell for
collecting the lysate [0181] Moving the droplet containing the
lysate from the corresponding virtual microwell to a designated
site.
[0182] In an aspect, a method is provided for detecting the
presence of an abnormality using rare cells and/or analytes in a
sample of a mixed cell population comprising loading the sample on
a digital microfluidic system of the disclosure to enrich for the
rare cells and/or analytes, and determining the presence or absence
of the abnormality by analyzing the enriched rare cells and/or
analytes.
[0183] In aspects, the systems, methods and kits of the disclosure
may be used for detecting rare cells that are in a sample at a
concentration of less than 1:2, 1:4. 1:5, 1:10, 1:20, 1:50, 1:100,
1:200, 1:300, 1:400, 1:500, 1:1000, 1:1500, 1:2000, 1:5000,
1:10,000, 1:20,000, 1:50,000, 1:100,000, 1:200,000, 1:500,000,
1:1,000,000, 1:2,000,000, 1:5,000,000, 1:10,000,000, 1:20,000,000,
1:50,000,000, 1:100,000,000 of all cells in the sample. In some
embodiments, the sample comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90 or 100 rare cells.
[0184] According to an embodiment, the cell sample may be used
directly as obtained from the source or following a pretreatment to
modify the character of the sample. The sample can be derived from
any biological source, such as tissues, extracts, cell cultures,
and physiological fluids, such as, for example, whole blood, plasma
and serum, and can be obtained from any animal subject. Animals can
be human or a domesticated animal such as a cow, pig, horse,
rabbit, dogs, cat, or goat.
[0185] According to an embodiment, the systems, methods and kits of
the disclosure may be used with a cell sample that has been
processed to be enriched with rare cells.
[0186] In an embodiment the present disclosure provides a method
for identifying and targeting individual cells within a cell
population for selective extraction of cellular content. The method
includes loading a sample containing cells on at least one site of
a digital microfluidic device thereby forming a virtual microwell
at each of the at least one site; Immobilizing the cells on the at
least one site; selecting at least one immobilized cell; lysing the
at least one selected cell using a pulsed laser source to produce
lysate within its corresponding virtual microwell; displacing a
droplet of liquid to the corresponding virtual microwell for
collecting the lysate; and
moving the droplet containing the lysate from the corresponding
virtual microwell to a designated site.
[0187] The meaning of the terms "immobilizing" and "immobilized" is
not limited to "inducing cell adhesion" and "adhered/adherent".
[0188] In some embodiments the at least one site for receiving
cells may be hydrophilic, partially hydrophilic or become
hydrophilic after protein fouling/adsorption from the sample.
[0189] In an embodiment the method may include a step of generating
a map of locations of the immobilized cells, and wherein the step
of selecting the at least one immobilized cell includes selecting
the at least one cell from the map.
[0190] In an embodiment the method may further comprise a step of
labelling the immobilized cells, and this may also comprise fixing
the cells.
[0191] In an embodiment the method may further comprise the steps
of:
[0192] moving the digital microfluidic device along horizontal axes
and a vertical axis for positioning the digital microfluidic device
for lysing another at least one selected cell from immobilized
cells;
[0193] lysing the other at least one selected cell using the pulsed
laser source to produce another lysate within its corresponding
virtual microwell;
[0194] displacing another droplet of liquid to the corresponding
virtual microwell for collecting the other lysate; and
[0195] moving the other droplet containing the other lysate from
the corresponding virtual microwell to a designated site.
[0196] In an embodiment the method may further comprise a step of
introducing the sample containing the cells at an initial site and
displacing the sample to the at least one site.
[0197] In an embodiment the at least one site is a plurality of
sites, and including steps of
[0198] moving of droplets to said plurality of sites,
[0199] selecting of a cells to be lysed at each of said plurality
of sites,
[0200] selecting a first site to illuminate the selected cell at
that site,
[0201] moving of the stage to move the digital microfluidic device
sequentially to bring each of the sites into a field of view of the
pulsed laser source to lyse the selected cell to produce lysate at
each site, and
[0202] collecting the lysate at each site.
[0203] In this embodiment where the at least one site is a
plurality of sites, the method may include calculating a shortest
distance travelled by the stage to bring each of the plurality of
sites into the field of view of the pulsed laser source
sequentially.
[0204] In another embodiment where the at least one site is a
plurality of sites, the
at least one selected targeted cell is a plurality of selected
targeted cells, and the method may further include
[0205] identifying a sequence of selected targeted cells to be
lysed to minimize a time to perform the lysing on all selected
targeted cells, and wherein the plurality of selected targeted
cells is within one field of view, or a plurality of field of
views, or within a plurality of sites.
[0206] In an embodiment of the method the pulsed laser source may
be a nanosecond-pulsed laser.
[0207] In an embodiment of the method the pulsed laser source may
be a nanosecond-pulsed laser delivering pulses of at least 1
.mu.J.
[0208] In an embodiment of the method the nanosecond-pulsed laser
may be a Nd-based laser.
[0209] In an embodiment of the method the nanosecond-pulsed laser
may produce a pulsed-laser beam within the visible spectrum.
[0210] In an embodiment of the method the pulsed laser source may
be a Q-switched laser.
[0211] In an embodiment the method may further comprise the step of
performing on chip analysis of the lysate at the designated site
and in this embodiment the method may further comprise the step of
collecting the droplet containing the lysate from the designated
site for off-chip analysis.
Prenatal and Neonatal Diagnostics
[0212] The system and method of the present invention may be used
for a wide range of applications including, but not limited to:
identification of rare fetal cells for genomic analysis for
non-invasive prenatal genetic diagnostic screening, identification
of circulating tumour cells for cancer diagnostics, differentiation
between cells that respond (or not) to a drug, cell analysis based
on changes of protein localization, evaluation of stem cells at
various stages of differentiation and the identification of
congenital anomalies. In an aspect, the system and method of the
invention are used for non-invasive prenatal genetic diagnosis of
chromosome abnormalities and single gene disorders.
[0213] In an aspect, a method is provided for detecting the
presence of an abnormality using rare cells and/or analytes in a
sample of a mixed cell population comprising loading the sample on
a digital microfluidic system of the disclosure to enrich for the
rare cells and/or analytes, and determining the presence or absence
of the abnormality by analyzing the enriched rare cells and/or
analytes.
[0214] In aspects, the systems, methods and kits of the disclosure
may be used for detecting rare cells that are in a sample at a
concentration of less than 1:2, 1:4. 1:5, 1:10, 1:20, 1:50, 1:100,
1:200, 1:300, 1:400, 1:500, 1:1000, 1:1500, 1:2000, 1:5000,
1:10,000, 1:20,000, 1:50,000, 1:100,000, 1:200,000, 1:500,000,
1:1,000,000, 1:2,000,000, 1:5,000,000, 1:10,000,000, 1:20,000,000,
1:50,000,000, 1:100,000,000 of all cells in the sample. In some
embodiments, the sample comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90 or 100 rare cells. In embodiments of the
disclosure, the rare cells are fetal cells, in particular fetal
cells in a sample of fetal cells and maternal cells.
[0215] The systems, methods and kits of the disclosure may be used
in the detection of fetal cells or fetal analytes, and in
particular they may be used to detect, diagnose or aid in the
diagnosis of prenatal or neonatal conditions. In an aspect, the
systems, methods and kits of the disclosure are used to diagnose or
aid in the prenatal diagnosis of genetic conditions. Analytes
include without limitation, nucleic acids, proteins, lipids,
carbohydrates, metabolites, or combinations thereof.
[0216] Examples of conditions which may be detected or diagnosed
using the systems, methods and kits of the disclosure, include
without limitation, autosomal dominant conditions such as
achondroplasia, osteogenesis imperfecta, Marfan syndrome,
polycystic kidney disease, Waardenburg syndrome, and the like,
autosomal recessive conditions such as Cockayne syndrome, cystic
fibrosis, Tay-Sachs disease, sickle cell anemia, congenital adrenal
hyperplasia, alpha- and beta-thalassemia; X-linked disorders such
as fragile X syndrome, Duchenne's muscular dystrophy, hemophilia A
and B; imprinting disorders such as Angelman syndrome/Prader Willi
syndrome and microscopic and submicroscopic chromosome
abnormalities, and the like. (See also, for example, Milunsky A,
and Milunsky, J M, editors. Genetic Disorders and the Fetus:
Diagnosis, Prevention and Treatment, 7th ed. John Wiley & Sons,
2015 relating to prenatal genetic conditions.)
[0217] In embodiments of the disclosure, the prenatal condition is
a chromosome abnormality. Chromosome abnormalities include, without
limitation, a gain or loss of an entire chromosome or a region of a
chromosome comprising one or more genes or an abnormal number of a
chromosome.
[0218] Examples of conditions involving chromosome abnormalities
include without limitation, Down syndrome and DiGeorge syndrome,
trisomy 13, trisomy 18, trisomy 21 (Down Syndrome), monosomy,
triploidy, tetraploidy, Klinefelter's Syndrome (XXY), and other sex
chromosome aneuploidies or autosomal chromosome abnormalities and
combinations thereof.
[0219] In an embodiment, the prenatal condition is a single gene
disorder.
[0220] In an embodiment, the prenatal condition comprises, is
chosen from or is selected from the group consisting of DiGeorge
syndrome, trisomy 13, trisomy 18, trisomy 21 (Down Syndrome),
Klinefelter's Syndrome (XXY), monosomy, triploidy, tetraploidy and
other sex chromosome or autosomal chromosome aneuploidies, and
combinations thereof.
[0221] In an embodiment, the prenatal condition is a trisomy, more
particularly, trisomy 21, trisomy 18, trisomy 13 or a combination
thereof.
[0222] Examples of other conditions which may be detected or
diagnosed using the systems, methods and kits of the disclosure,
include without limitation a cell proliferation condition. Examples
of cell proliferation conditions include without limitation,
cancers of the colorectum, breast, lung, liver, pancreas, lymph
node, colon, prostate, brain, head and neck, skin, liver, kidney,
and heart; inflammatory conditions, such as inflammation conditions
of the skin; conditions related to obesity; such as proliferation
of adipocytes; viral conditions, and cardiac conditions.
[0223] In an aspect, a method is provided for enriching fetal cells
and/or fetal analytes in a sample containing a mixed population of
cells (e.g. fetal and maternal cells) comprising subjecting the
sample to a microfluidic system of the disclosure having elements
for separating and isolating the fetal cells and/or analytes.
[0224] In an aspect, a method is provided for obtaining and using
fetal cells and/or fetal analytes from a sample to perform prenatal
diagnosis the method comprising a microfluidic device of the
disclosure having elements for separating and isolating the fetal
cells and/or analytes from the sample.
[0225] Positive and/or negative selection may be used in the
methods of the disclosure. In a particular aspect, the method
includes a diagnostic device comprising a microfluidic device of
the disclosure having elements to separate and isolate fetal cells
and/or fetal analytes from a heterogenous sample, wherein the
elements comprise binding agents that bind fetal cell-specific
markers and/or maternal cell-specific markers, including surface or
intracellular markers. In a particular aspect, the method includes
a diagnostic device comprising a microfluidic device of the
disclosure having elements to separate and isolate fetal cells
and/or fetal analytes from a heterogenous sample, wherein the
elements comprise binding agents that bind fetal cell-specific
markers, including surface or intracellular markers. In a
particular aspect, the method includes a diagnostic device
comprising a microfluidic device of the disclosure having elements
to separate and isolate fetal cells and maternal cells from a
heterogenous sample. In an embodiment, the fetal cell-specific
markers may comprise, be chosen from, or be selected from the group
consisting of Fetal Cell Marker 1, Fetal Cell Marker 2, 5T4, HLA-G,
CD227, .beta. subunit of chorionic gonadotropin (.beta.-CG),
placental lactogen, cytokeratin 7, placental alkaline phosphatase,
NDOG1, PSG1, PSG9, MMP14, MCAM, KCNQ4, CLDN6, F3, PEG10, FLT1, CBG,
GCM1, GPA, CD45, EGFR, APOB, CD71, CD36, CD34, HbF, FB3-2, H3-3,
HAE 9, FB3-2, HBE, APOC3, AMBP, CPB2, ITIH1, APOH, HPX, AHSG, APOB,
BPG, carbonic anhydrase (CA), and thymidine kinase. In an
embodiment, the maternal cell-specific markers comprise, are chosen
from, are selected from, or selected from the group consisting of
CD90, CD73, CD44, CD105, and CD29.
[0226] Methods of the disclosure may also select fetal cells based
on morphological characteristics such as size and shape (see for
example, James et al, Human Reproduction 22(8): 2111-2119, 2007;
Bulmer et al, Prenatal Diagnosis 23:34-39, 2003; Moser et al,
Placenta 32: 197-199, 2011; and Caruso et al, Int J Mol Cell Med.
1(2): 64-74, 2012, for morphological characteristics of fetal
cells).
[0227] The methods enable non-invasive acquisition of fetal cells
and fetal analytes from a pregnant subject which can be used for a
variety of purposes, including but not limited to, the
identification of fetal cells in cervical samples, determination of
fetal cell density to predict high risk pregnancies, genetic
analysis of fetal nucleic acids, determination of the fetal
karyotype, genetic analysis of fetal DNA from fetal cells,
detection of abnormalities in the fetus, detection of possible
complications of pregnancy, and determination of biomarkers or
growth factors to predict prenatal and postnatal conditions.
[0228] A sample can be used directly as obtained from the source or
following a pretreatment to modify the character of the sample. The
sample can be derived from any biological source, such as tissues,
extracts, cell cultures, and physiological fluids, such as, for
example, whole blood, plasma and serum, and can be obtained from
any animal subject. Animals can be human or a domesticated animal
such as a cow, pig, horse, rabbit, dogs, cat, or goat. In an
embodiment, a sample can be obtained from an animal suspected of
being pregnant, pregnant, or that has been pregnant, to detect the
presence of a prenatal condition.
[0229] The system, kits and methods of the disclosure are
particularly useful for obtaining fetal cells and fetal analytes
from an endocervical sample. Endocervical samples or cervical
mucosal samples can be obtained using standard non-invasive
procedures known to a person skilled in the art, including but not
limited to intrauterine lavage, aspiration of cervical mucus, or
removal of surface tissue from the cervical or endocervical canal.
In an embodiment, a cervical mucosal sample is obtained from the
endocervical canal using a cytobrush. An endocervical sample may be
obtained with a commercially available kit such as a ThinPrep.RTM.
kit (Hologic Corporation, Marlborough, Mass.) which comprises a
cytological brush and a fixative solution (which may or may not be
used). A sample collection can be incorporated as a sample
collection device in a kit disclosed herein.
[0230] In an embodiment, the endocervical sample is obtained from
the subject during the first trimester of pregnancy. In an
embodiment, the endocervical sample is obtained from the subject
during the early second trimester of pregnancy.
[0231] Samples may be collected from subjects repeatedly at
different times, for example, over the term of a pregnancy, and can
be used to verify results from earlier detections and/or to
identify an alteration as a result of, for example, treatment.
[0232] In an aspect, the method includes a diagnostic device
comprising a microfluidic device of the disclosure having elements
to separate and isolate fetal cells or fetal analytes from maternal
cells in an endocervical sample, wherein the elements comprise
binding agents that bind fetal cell-specific markers and/or
maternal markers, including surface or intracellular markers. In an
embodiment, the fetal cell-specific markers comprise, may be chosen
from, or be selected from the group consisting of Fetal Cell Marker
1, Fetal Cell Marker 2, 5T4, HLA-G, CD227, .beta. subunit of
chorionic gonadotropin (.beta.-CG), placental lactogen, cytokeratin
7, placental alkaline phosphatase, NDOG1, PSG1, PSG9, MMP14, MCAM,
KCNQ4, CLDN6, F3, PEG10, FLT1, CBG, GCM1, GPA, CD45, EGFR, APOB,
CD71, CD36, CD34, HbF, FB3-2, H3-3, HAE 9, FB3-2, HBE, APOC3, AMBP,
CPB2, ITIH1, APOH, HPX, AHSG, APOB, BPG, carbonic anhydrase (CA),
and thymidine kinase. In an embodiment, the maternal cell-specific
markers comprise, are chosen from, are selected from, or selected
from the group consisting of CD90, CD73, CD44, CD105, and CD29.
[0233] Binding agents that bind fetal cell-specific markers or
maternal cell-specific markers include substances such as
polypeptides or antibodies that specifically bind to one or more
cell-specific marker, or in some cases an intracellular organelle
(e.g., ribosomes, endoplasmic reticulum) or cell nucleic acid. A
substance "specifically binds" if it reacts at a detectable level
with one or more cell-specific marker, and does not react
detectably with polypeptides or peptides containing an unrelated or
different sequence. Binding properties may be assessed using
methods knows in the art such as an ELISA, which may be readily
performed by those skilled in the art (see for example, Newton et
al, Develop. Dynamics 197: 1-13, 1993). Examples of binding agents
include without limitation ribosomes, with or without a peptide
component, aptamers, RNA molecules, and polypeptides, and in
particular antibodies. Antibodies may be synthetic antibodies,
monoclonal antibodies, polyclonal antibodies, recombinant
antibodies, antibody fragments (such as Fab, Fab', F(ab')2), dAb
(domain antibody; see Ward, et al, 1989, Nature, 341:544-546),
antibody heavy chains, intrabodies, humanized antibodies, human
antibodies, antibody light chains, single chain F.sub.vs (scFv)
(e.g., including monospecific, bispecific etc.), anti-idiotypic
(ant-Id) antibodies, proteins comprising an antibody portion,
chimeric antibodies (for example, antibodies which contain the
binding specificity of murine antibodies, but in which the
remaining portions are of human origin), derivatives, such as
enzyme conjugates or labeled derivatives, diabodies, linear
antibodies, disulfide-linked Fvs (sdFv), multispecific antibodies
(e.g., bispecific antibodies), epitope-binding fragments of any of
the above, and any other modified configuration of an
immunoglobulin molecule that comprises an antigen recognition site
of the required specificity. An antibody includes an antibody of
any type (e.g. IgA, IgD, IgE, IgG, IgM and IgY), any class (e.g.
IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g. IgG2a
and IgG2b), and the antibody need not be of any particular type,
class or subclass. An antibody may be from any animal origin
including birds and mammals (e.g. human, murine, donkey, sheep,
rabbit, goat, guinea pig, horse, or chicken). A binding agent may
be labelled with a detectable label as disclosed herein. A binding
agent labelled with a detectable label is sometimes referred to
herein as an imaging reagent.
[0234] In an aspect the disclosure provides a method for retrieving
fetal analytes from a sample, in particular an endocervical sample,
comprising (i) obtaining the sample, in particular an endocervical
sample; (ii) introducing the sample into a microfluidic system of
the disclosure that allows separation and isolation of fetal cells,
and (iii) producing from the fetal cells a lysate comprising fetal
analytes.
[0235] In an aspect, the disclosure provides a method for detecting
and/or isolating fetal analytes from a sample, in particular an
endocervical sample, comprising a mixed population of cells, in
particular fetal cells and maternal cells, the method comprising
introducing the sample into a microfluidic system that allows (i)
separation and isolation of the fetal cells in the sample, (ii)
lysis of the isolated fetal cells, and (iv) detection and/or
isolation of fetal analytes from the lysed fetal cells. The
microfluidic system may optionally comprise culturing of cells in
the sample to enrich for fetal cells. In an embodiment, the system
comprises a digital microfluidic device of the disclosure, and
elements to separate and isolate fetal cells from other cells in an
endocervical sample such elements, chosen from, selected from, or
selected from the group consisting of binding agents that bind one,
two or more fetal cell-specific markers and/or maternal
cell-specific markers. In an embodiment, the fetal-cell specific
markers comprise, may be chosen from or be selected from the group
consisting of Fetal Cell Marker 1, Fetal Cell Marker 2, 5T4, HLA-G,
CD227, .beta. subunit of chorionic gonadotropin (.beta.-CG),
placental lactogen, cytokeratin 7, placental alkaline phosphatase,
NDOG1, PSG1, PSG9, MMP14, MCAM, KCNQ4, CLDN6, F3, PEG10, FLT1, CBG,
GCM1, GPA, CD45, EGFR, APOB, CD71, CD36, CD34, HbF, FB3-2, H3-3,
HAE 9, FB3-2, HBE, APOC3, AMBP, CPB2, ITIH1, APOH, HPX, AHSG, APOB,
BPG, carbonic anhydrase (CA), and thymidine kinase. In an
embodiment, the maternal cell-specific markers comprise, are chosen
from, are selected from, or selected from the group consisting of
CD90, CD73, CD44, CD105, and CD29. The binding agents may be
labelled with a detectable label as disclosed herein.
[0236] In an aspect, the fetal analyte is a fetal protein, (for
example, amino acids, peptides, enzymes, antigens, antibodies,
cytokines, lipoproteins, glycoproteins, growth factors or
hormones). In an embodiment, the fetal analyte is a growth
factor.
[0237] In an aspect, a method is provided for detecting or
isolating a fetal protein from a sample comprising fetal cells, the
method comprising introducing the sample into a microfluidic system
that allows (i) separation and isolation of fetal cells in the
sample; (ii) lysis of the isolated fetal cells, (iii) detection or
isolation of a fetal protein from the lysed fetal cells, and (iv)
optionally analysis of the fetal protein. The microfluidic system
may also comprise culturing of cells in the sample to enrich for
fetal cells.
[0238] The analysis of proteins (e.g., detection and/or levels of
proteins) may be determined using a variety of methods known to a
person skilled in the art such as immunoassays in various formats
such as radioimmunoassay (RIA), chemiluminescence- and
fluorescence-immunoassays, enzyme immunoassay (EIA), enzyme-linked
immunoassays (ELISA), luminex-based bead arrays, protein microarray
assays, and rapid test formats such as immunochromatographic strip
tests, and Selected/Multiple reaction monitoring (SRM/MRM). The
immunoassay may be a homogenous or heterogeneous assay, competitive
or non-competitive. Examples of other methods for analysis of
proteins include spectrometry, mass spectrometry, Matrix Assisted
Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass
Spectrometry, microscopy, northern blot, isoelectric focussing,
SDS-PAGE, PCR, quantitative RT-PCR, gel electrophoresis, DNA
microarray, protein sequencing, proteome analysis, and antibody
microarray, or combinations thereof.
[0239] In an aspect, the fetal analyte is a fetal nucleic acid. In
an aspect, a method is provided for detecting and/or isolating
fetal nucleic acids from a sample comprising fetal cells, the
method comprising introducing the sample into a microfluidic system
that allows (i) separation and isolation of fetal cells in the
sample; (ii) lysis of the isolated fetal cells, (iii) detection
and/or isolation of fetal nucleic acids from the lysed fetal cells,
and (iv) optionally analysis of the fetal nucleic acids. The
microfluidic system may also comprise culturing of cells in the
sample to enrich for fetal cells.
[0240] A fetal nucleic acid may be DNA (for example, genomic,
mitochondrial, and complementary cDNA from RNA), or RNA (for
example, rRNA, mRNA and miRNA). In an aspect, the fetal nucleic
acids are nucleotides, oligonucleotides, DNAs, RNAs, or DNA-RNA
hybrids. In an embodiment, the fetal nucleic acids are DNAs, in
particular, double-stranded DNAs, single-stranded DNAs,
multi-stranded DNAs, complementary DNAs, genomic DNAs or non-coding
DNAs. In an embodiment, the fetal nucleic acids are RNAs, in
particular, messenger RNAs (mRNAs), microRNAs (miRNAs), small
nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs
(tRNAs), small interfering RNAs (siRNAs), heterogeneous nuclear
RNAs (hnRNAs), or small hairpin RNAs (shRNAs).
[0241] The methods and systems disclosed herein may be used for
detecting the presence or absence of a target sequence, detection
of polymorphisms, single-polynucleotide polymorphism analysis,
haplotype analysis, amplification of a sequence for sequence
identification, gene expression analysis, quantification of nucleic
acids, analysis as well as other applications apparent to one
skilled in the art.
[0242] In an embodiment, the fetal analyte, in particular, nucleic
acid, fetal protein or growth factor, is associated with a prenatal
condition.
[0243] In an embodiment, a method is provided for detecting or
diagnosing a prenatal condition in a sample, in particular an
endocervical sample, comprising: (a) introducing the sample into a
microfluidic system of the disclosure to separate, isolate and lyse
fetal cells in the sample; (b) isolating a fetal protein from the
lysed fetal cells; (c) analyzing the fetal protein: and (c)
detecting or diagnosing a prenatal condition wherein the presence
or absence of a specific fetal protein is indicative of the
prenatal condition.
[0244] In an embodiment, a method is provided for detecting or
diagnosing a prenatal condition in a sample, in particular an
endocervical sample, comprising: (a) introducing the sample onto a
microfluidic system to separate, isolate and lyse fetal cells in
the sample; (b) isolating a fetal nucleic acid from the lysed fetal
cells; (c) analyzing the fetal nucleic acid: and (c) detecting or
diagnosing a prenatal condition wherein the presence or absence of
a specific fetal nucleic acid is indicative of the prenatal
condition.
[0245] In an embodiment, the disclosure provides a method for
diagnosing a prenatal condition in an endocervical sample
comprising introducing the sample onto a microfluidic system that
allows (i) culturing of cells in the sample to enrich for fetal
cells, (ii) separation and isolation of the enriched fetal cells,
(iii) lysis of the isolated fetal cells, (iv) isolation of fetal
nucleic acids from the lysed fetal cells, and (iv) analysis of the
fetal nucleic acids.
[0246] A method or system of the disclosure detecting and/or
isolating fetal nucleic acids may comprise analyzing the fetal
nucleic acids for fetal-specific nucleotide sequences. In an
embodiment, the fetal-specific nucleotide sequence is a
Y-chromosome sequence.
[0247] In aspects, the microfluidic system comprises a microfluidic
device and elements to separate and isolate fetal cells and/or
maternal cells from other cells in a sample, in particular an
endocervical sample, such elements comprising, chosen from,
selected from, or selected from the group consisting of binding
agents that bind one, two or more fetal cell-specific markers
and/or maternal cell-specific markers. In a particular embodiment,
the fetal cell-specific markers may comprise, be chosen from or be
selected from the group consisting of Fetal Cell Marker 1, Fetal
Cell Marker 2, 5T4, HLA-G, CD227, .beta. subunit of chorionic
gonadotropin (.beta.-CG), placental lactogen, cytokeratin 7,
placental alkaline phosphatase, NDOG1, PSG1, PSG9, MMP14, MCAM,
KCNQ4, CLDN6, F3, PEG10, FLT1, CBG, GCM1, GPA, CD45, EGFR, APOB,
CD71, CD36, CD34, HbF, FB3-2, H3-3, HAE 9, FB3-2, HBE, APOC3, AMBP,
CPB2, ITIH1, APOH, HPX, AHSG, APOB, BPG, carbonic anhydrase (CA),
and thymidine kinase. In an embodiment, the maternal cell-specific
markers comprise, are chosen from, are selected from, or selected
from the group consisting of CD90, CD73, CD44, CD105, and CD29. The
binding agents may be labelled with a detectable label as disclosed
herein.
[0248] A method or system disclosed herein comprising detecting or
isolating fetal nucleic acids may further comprise amplifying fetal
nucleic acids prior to, or simultaneous with the isolation and/or
analysis of the nucleic acids, or subsequent to isolation of the
nucleic acids. Amplified nucleic acids may be detected and then
pooled for analysis, pooled for subsequent detection and analysis,
or detected and not subsequently pooled.
[0249] In an embodiment, a system, method or kit disclosed herein
comprises amplification reagents necessary for amplifying desired
fetal nucleic acids. Amplification reagents may be selected based
on the amplification method but can include primers, polymerase,
reverse transcriptase, nucleotides, cofactors, metal ions, buffers
and similar reagents.
[0250] In an embodiment, amplification is carried out in a system
of the disclosure wherein the amplification reagents are provided
by prepositioning the reagents in the system, by combining the
reagents with the fetal analyte, by a combination of both, or by
any other suitable method.
[0251] Amplicons of fetal nucleic acids may be generated using
methods known in the art [see Persing, David H., "In Vitro Nucleic
Acid Amplification Techniques" in Diagnostic Medical Microbiology:
Principles and Applications (Persing et al., Eds.), pp. 51-87
(American Society for Microbiology, Washington, D.C. (1993)) for a
discussion of known amplification methods]. Illustrative
non-limiting examples of nucleic acid amplification methods
include, but are not limited to, Polymerase Chain Reaction (PCR)
[see, for example, Dieffenbach and Dvksler, 1995, PCR Primer: A
Laboratory Manuel. CSHL press. Cold Spring Harbor, USA; U.S. Pat.
Nos. 4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth.
Enzymol. 155: 335 (1987); and, Murakawa et al., DNA 7: 287 (1988)];
reverse transcription polymerase chain reaction (RT-PCR); strand
displacement amplification (SDA) [see for example, Walker, G. et
al., Proc. Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. Pat. Nos.
5,270,184 and 5,455,166; European Pat. No. 0684315]; transcription
mediated amplification (TMA) (see for example, U.S. Pat. Nos.
5,480,784, 5,399,491 and 5,824,518 and Published US Application No.
20060046265); nucleic acid sequence based amplification (NASBA)
[see for example, Sooknanan and Malek, 1995, Biotechnology 13:563;
U.S. Pat. No. 5,130,238; Lizardi et al., BioTechnol. 6: 1197
(1988); Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)];
SPIA.RTM. [Nugen Technologies, San Carlos, Calif., U.S. Pat. No.
6,251,639, WO 02/72772 and US 20030017591]; the ligase chain
reaction (LCR) [see for example, Wu and Wallace, 1989, Genomics
4:560; Weiss, R., Science 254: 1292 (1991), and Landegren et al,
1988, Science 241:1077]; and, self-sustained sequence replication
[see for example, Guatelli et al, 1990 Proc Nat Acad Sci USA
87:1874].
[0252] Amplicons and non-amplified nucleic acids may be analyzed
(i.e., detected, quantified, sequenced and the like) using routine
methods known in the art. Examples of methods include, without
limitation, mass detection of mass modified amplicons (e.g.,
matrix-assisted laser desorption ionization (MALDI) mass
spectrometry and electrospray (ES) mass spectrometry), a primer
extension method (see for example, iPLEX.TM.; Sequenom, Inc.),
microsequencing methods, ligase chain reaction, ligase sequence
determination methods (see for example, U.S. Pat. Nos. 5,679,524
and 5,952,174, and WO 01/27326), mismatch sequence determination
methods (see for example, U.S. Pat. Nos. 5,851,770; 5,958,692;
6,110,684; and 6,183,958), direct DNA sequencing, restriction
fragment length polymorphism, allele specific oligonucleotide
analysis, methylation-specific PCR, pyrosequencing analysis,
acycloprime analysis, reverse dot blot, GeneChip microarrays,
Dynamic allele-specific hybridization, Peptide nucleic acid (PNA)
and locked nucleic acids (LNA) probes, TaqMan.TM. Molecular
Beacons, intercalating dye (for example, SYBR.TM., Pico Green
(Molecular Probes, Inc., Eugene, Oreg.), ethidium bromide),
fluorescence resonance energy transfer (FRET) based systems,
AlphaScreen (Perkin Elmer), SNPstream.TM. (Beckman Coulter),
genetic bit analysis (GBA), multiplex minisequencing, SNaPshot,
GOOD assay, microarray miniseq, arrayed primer extension (APEX),
microarray primer extension (e.g., microarray sequence
determination methods), Tag arrays, Coded microspheres,
Template-directed incorporation (TDI), fluorescence polarization,
colorimetric oligonucleotide ligation assay (OLA), Sequence-coded
OLA, microarray ligation, Padlock probes, Invader assays,
hybridization methods, conventional dot blot analyses, single
strand conformational polymorphism analysis (SSCP) (see for
example, Orita et al., Proc. Natl. Acad. Sci. U.S.A. 86: 27776-2770
(1989)), denaturing gradient gel electrophoresis (DGGE),
heteroduplex analysis, mismatch cleavage detection, and cloning and
sequencing, electrophoresis, hybridization probes and quantitative
real time polymerase chain reaction (QRT-PCR), digital PCR,
nanopore sequencing, chips, closed-tube methods for detection and
quantification of alleles or paralogs (see for example, Turner et
al. BMC Medical Genetics (2015) 16:115).
[0253] A system of the disclosure may be used in combination with a
sequence analysis apparatus or sequence analysis component(s),
including without limitation, the Illumina Genomic Analyzer (or
Solexa platform), the SOLID System (Applied Biosystems), the
Helicos True Single Molecule DNA sequencing platform (Harris T D et
al. 2008 Science, 320, 106-109), the SMRT.TM. system of Pacific
Biosciences, or a nanopore sequencing platform (Soni G V and Meller
A. 2007 Clin Chem 53: 1996-2001).
[0254] The methods of the disclosure can be used as a clinical
laboratory service. In an aspect, the methods include a sample
collection device capable of obtaining a sample, for example an
endocervical sample, from a subject.
[0255] In an aspect, the disclosure provides a kit comprising a
diagnostic device comprising a microfluidic system of the
disclosure for isolating fetal cells and/or fetal analytes from a
sample, in particular an endocervical sample, wherein the system
allows separation of the fetal cells, lysis of the lysed fetal
cells, and optionally detection or isolation of fetal analytes. The
kit optionally comprises components for culturing of cells in the
sample to enrich for fetal cells. In an embodiment, the kit
comprises binding agents. In an embodiment, the kit comprises
imaging agents.
[0256] In an embodiment, the disclosure provides a diagnostic kit
comprising (a) a diagnostic device comprising a digital
microfluidic system for separating and isolating fetal nucleic
acids from an endocervical sample wherein the system allows
culturing of cells in the sample to enrich for fetal cells,
separation of the enriched fetal cells, lysis of the separated
fetal cells, and optionally isolation of fetal analytes, wherein
the system comprises binding agents that bind to one, two or more
fetal-cell specific markers and/or maternal cell-specific markers.
In a particular embodiment, the fetal cell-specific markers
comprise, are chosen from or selected from the group consisting of
Fetal Cell Marker 1, Fetal Cell Marker 2, 5T4, HLA-G, CD227, .beta.
subunit of chorionic gonadotropin (.beta.-CG), placental lactogen,
cytokeratin 7, placental alkaline phosphatase, NDOG1, PSG1, PSG9,
MMP14, MCAM, KCNQ4, CLDN6 and F3, facilitating separation of fetal
cells. In a particular embodiment, the maternal cell-specific
markers comprise, are chosen from or selected from the group
consisting of CD90, CD73, CD44, CD105, and CD29. The binding agents
may be labelled with a detectable label disclosed herein.
[0257] A diagnostic kit may additionally comprise a sample
collection device capable of obtaining an endocervical sample from
a subject. A diagnostic kit may additionally comprise reagents for
amplifying fetal nucleic acids.
[0258] A microfluidic device or system in the method and kits of
the disclosure relating to fetal cells and fetal analytes is
preferably a microfluidic device or system disclosed herein,
however, it will be appreciated by those skilled in the art that
other suitable microfluidic devices or systems can be utilized.
(See for example, Wu, J et al, Analyst. 2017 Jan. 26;
142(3):421-441; Shields C W et al, Cytometry B Clin Cytom. 2017
March; 92(2):115-125; Shields et al, Lab Chip. 2015 Mar. 7;
15(5):1230-49 for a description of microfluidic devices and
methods).
[0259] In an aspect, the disclosure relates to the use of a DMF
method for identification and isolation of fetal cells and/or fetal
analytes within a sample. In another aspect the disclosure relates
to a method for detecting and/or isolating fetal cells and/or fetal
analytes from a sample. In embodiments, the sample comprises fetal
cells and maternal cells. In embodiments, the sample is an
endocervical or cervical mucosal sample. In embodiments, the sample
is an endocervical or cervical mucosal sample from a pregnant
subject.
[0260] In embodiments, the method comprises:
[0261] (a) loading the sample on at least one hydrophilic site of a
digital microfluidic device thereby forming a virtual microwell at
each of the at least one hydrophilic site;
[0262] (b) immobilizing the cells on the at least one site;
[0263] (c) selecting at least one immobilized fetal cell;
[0264] (d) lysing the at least one selected fetal cell using a
pulsed laser source to produce lysate within its corresponding
virtual microwell, wherein the lysate comprises the analytes;
[0265] (e) displacing a droplet of liquid to the corresponding
virtual microwell for collecting the lysate; and
[0266] (f) moving the droplet containing the lysate from the
corresponding virtual microwell to a designated site, and
optionally detecting and/or isolating analytes in the lysate.
[0267] The digital microfluidic device generally comprises an
imaging system including a stage for receiving the digital
microfluidic device. The imaging system includes an imaging module
for identifying the selected fetal cell and the pulsed laser
source.
[0268] In an embodiment, cells are immobilized in (b) by culturing
the cells in the sample to thereby induce cell adhesion on the at
least one hydrophilic site to produce adhered cells, and at least
one adhered fetal cell is selected in (c).
[0269] Positive and/or negative selection or morphological
characteristics as disclosed herein or known in the art may be used
to select an immobilized fetal cell in (c).
[0270] In an embodiment, the method further comprises generating a
map of locations of the immobilized (in particular adhered cells),
and wherein the step of selecting the at least one immobilized (in
particular adhered cell) includes selecting the at least one cell
from the map.
[0271] In another embodiment, the method further comprises
immunocytochemically labelling cells in the sample or labelling
adherent cells, and optionally further comprises fixing the
cells.
[0272] In another embodiment, the method further comprises:
[0273] (i) moving the digital microfluidic device along horizontal
axes and a vertical axis for positioning the digital microfluidic
device for lysing another at least one selected cell from
immobilized cells (in particular adhered cells);
[0274] (ii) lysing the other at least one selected cell using the
pulsed laser source to produce another lysate within corresponding
virtual microwell;
[0275] (iii) displacing another droplet of liquid to the
corresponding virtual microwell for collecting the other lysate;
and
[0276] (iv) moving the other droplet containing the other lysate
from the corresponding virtual microwell to a designated site.
[0277] In another embodiment, the method further comprises
introducing the sample containing the cells at an initial site and
displacing the sample to the at least one hydrophilic site.
[0278] In an embodiment, the at least one hydrophilic site is a
plurality of hydrophilic sites, and the method comprises: [0279]
(i) moving droplets to said plurality of hydrophilic sites, [0280]
(ii) selecting of cells to be lysed at each of said plurality of
hydrophilic sites, [0281] (iii) selecting a first hydrophilic site
to illuminate the selected cell at that hydrophilic site, [0282]
(iv) moving the stage to move the digital microfluidic device
sequentially to bring each of the hydrophilic sites into a field of
view of the pulsed laser source to lyse the selected cell to
produce lysate at each hydrophilic site, and [0283] (v) collecting
the lysate at each hydrophilic site, and optionally calculating a
shortest distance travelled by the stage to bring each of the
plurality of hydrophilic sites into the field of view of the pulsed
laser source sequentially.
[0284] In an embodiment, the selected fetal cell is a plurality of
fetal cells and the method comprises identifying a sequence of
selected fetal cells to be lysed to minimize a time to perform the
lysing on all selected fetal cells, and wherein the plurality of
selected fetal cells is within one field of view, or a plurality of
field of views, or within a plurality of hydrophilic sites.
[0285] In embodiments, the pulsed laser source is a
nanosecond-pulsed laser. In a particular embodiment, the pulsed
laser source is a nanosecond-pulsed laser delivering pulses of at
least 1 .mu.J, more particularly an Nd-based laser. In embodiments,
the nanosecond-pulsed laser produces a pulsed-laser beam within the
visible spectrum. In embodiments, the pulsed laser source is a
Q-switching laser.
[0286] In an embodiment, the method further comprises performing on
chip analysis of the lysate at the designated site.
[0287] In an embodiment, the method further comprises collecting
the droplet containing the lysate from the designated site for
off-chip analysis. Analysis of the lysate, in particular fetal
analytes, may be performed using the methods disclosed herein or
known in the art.
[0288] The following non-limiting example is only an example of the
use of the system and methods of the present invention.
Example
Prenatal Genetic Diagnostic Testing Using DMF/Laser-Microbeam
Lysis
[0289] The system and method of the present disclosure were used
with trophoblast cells from a cervical mucosa sample (CMS). The
trophoblast cells, which were adherent and embedded within a
sticky, viscous mucous, were not suitable for flow cytometry or
microchannels.
[0290] CMS samples were loaded onto a DMF device, where they were
seeded and cultured. The samples were stained with markers to
identify the fetal cells. After incubation, the media was
exchanged, and cells were laser lysed and the lysate was collected
for off-chip whole-genome amplification and genotyping. As shown in
FIG. 10B the CVS control sample (upper panel) and the matched laser
lysed fetal cell sample (lower panel) show the same pattern of
heterozygotic alleles by QF-PCR. As show by the circled alleles
this same is trisomy 21 positive. FIG. 10C shows genetic analysis
of the same laser lysed fetal cell sample using next generation
sequencing for analysis and it shows the same trisomy 21 result.
The performed steps were as follows:
Cervical Mucousal Sample Preparation (Example of a Specific Method
to Generate Single Cell Solution)
[0291] 1) A cervical mucosal sample is collected by cytobrush; 2)
Sample is stored up to 48 hours at 4.degree. C. in 40 ml PBS plus
10 ml AmnioMax media and 0.1 to 5 mM (final) sodium bicarbonate; 3)
Sample is warmed to RT and n-acetylcysteine is added to sample to
reach between 25 uM and 500 mM. 4) Sample is incubated at
37.degree. C. for 30 to 60 minutes with agitation. 5) Sample is
centrifuged for 10 minutes at 300.times.g at RT. 6) Supernatant is
taken down to 10 ml. 7) 15 ml of 0.25% trypsin-EDTA is added. 8)
Sample is incubated at 37.degree. C. for 10 to 30 min with
agitation. 9) Sample is centrifuged for 10 min at 300.times.g at
RT. 10) Supernatant is removed and sample is resuspended in 500 uL
of AmnioMax with 0.05% pluronic f-68. 11) Sampled is transferred to
1.5 ml tube and centrifuge 300.times.g for 10 min at RT. 12)
Sampled is resuspended in 200 uL of AmnioMax with 0.05% pluronic
f-68.
Sample Manipulation
[0292] 13) A DMF device with a top plate containing hydrophilic
sites for cell cultures is assembled. 14) Cytobrush cell sample
mixture is loaded onto the DMF device and into virtual microwells.
15) The loaded cell sample is incubated overnight at 37 C and 5%
CO.sub.2 in a humidified flask within the incubator. 16) Cells are
fixed using a crosslinking or denaturing fixative such as 4%
paraformaldehyde, Clarke's solution, HOPE, ethanol, etc. 17)
Immunocytochemical labeling using standard staining and labelling
methods is performed using two or more fetal cell markers (markers
including but are not limited to 5T4, HLA-G, CD227, cytokeratin 7,
NDOG1, PSG1, MMP14, MCAM, KCNQ4, CLDN6, and F3). 18) The DMF device
is imaged by tilting microscopy or array scanner. For tilting
microscopy the fully assembled device is imaged directly. For the
array scanner the device is disassembled and the top plate is
washed with water twice and air dried for imaging. 19) Labelled
cells are visualized using Cellprofiler to determine fetal cells
based on staining pattern of the two fetal cell markers as well as
cell size and shape. 20) A map of locations of the fetal cells in
the virtual microwells is generated 21) Reassemble the DMF device
for DMF-LCL if read on the array scanner. 22) Lyse the fetal cells
into PBS (200 .mu.s laser pump pulse, 0-20 .mu.s Q-switch delay)
23) Move PBS droplet to lysis location to collect fetal cell
lysate. 24) Move droplet with fetal cell lysate to reservoir. 25)
Collect fetal cell lysate from reservoir to use for whole genome
amplification (WGA). 26) Run WGA on fetal cell lysate. 27) Run
standard prenatal genetic test on the cDNA from the WGA. These
tests can be QF-PCR, aCGH (array comparative genomic
hybridization), NGS, etc. Specific Methods of Cell Loading and
Analysis with Laser Cell Lysis U87-GFP and B16-tdTomato Cell
Protocol [0293] 1) Assemble top and bottom plate of DMF device
[0294] 2) Generate single cell suspension of U87-GFP and/or
B16-tdTomato cells in DMEM plus 10% FBS, 1% P/S and 0.05% pluronic
F-68 [0295] 3) Load U87-GFP and/or B16-tdTomato cells into DMF
device [0296] 4) Culture U87-GFP and/or B16-tdTomato cells on
device at 37.degree. C. and 5% CO.sub.2 in a humidified flask
within the incubator. [0297] 5) Replace media with PBS [0298] 6)
Laser lyse cells [0299] 7) Collect cell contents
CVS Samples
[0299] [0300] 1) Assemble top and bottom plate of DMF device [0301]
2) Replace media on the CVS sample with AmnioMax plus 0.05%
pluronic F-68 [0302] 3) Load CVS sample into DMF device [0303] 4)
Culture CVS sample on device at 37.degree. C. and 5% CO.sub.2 in a
humidified flask within the incubator. [0304] 5) Replace media with
PBS [0305] 6) Run whole genome amplification (WGA) on fetal cell
lysate. [0306] 7) Run standard prenatal genetic test on the cDNA
from the WGA. These tests can be QF-PCR, aCGH (array comparative
genomic hybridization), NGS, etc.
[0307] The present invention is not to be limited in scope by the
specific embodiments described herein, since such embodiments are
intended as but single illustrations of one aspect of the invention
and any functionally equivalent embodiments are within the scope of
this invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0308] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety. All publications,
patents and patent applications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the methodologies, reagents, etc. which are reported therein which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
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