U.S. patent application number 14/969933 was filed with the patent office on 2016-04-07 for method of growing cells on a droplet actuator.
This patent application is currently assigned to Advanced Liquid Logic, Inc.. The applicant listed for this patent is Advanced Liquid Logic, Inc. Invention is credited to Allen E. Eckhardt, Vamsee K. Pamula, Michael G. Pollack.
Application Number | 20160097047 14/969933 |
Document ID | / |
Family ID | 40753787 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160097047 |
Kind Code |
A1 |
Pollack; Michael G. ; et
al. |
April 7, 2016 |
Method of Growing Cells on a Droplet Actuator
Abstract
A method of growing cells on a droplet actuator is provided. The
method may include providing a droplet actuator including a cell
culture droplet including a cell culture medium and one or more
cells; and maintaining the droplet at a temperature suitable for
causing the cells to grow in the cell culture medium on the droplet
actuator. Related methods, droplet actuators, and systems are also
provided.
Inventors: |
Pollack; Michael G.; (San
Diego, CA) ; Pamula; Vamsee K.; (Cary, NC) ;
Eckhardt; Allen E.; (San DIego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Advanced Liquid Logic, Inc |
San Diego |
CA |
US |
|
|
Assignee: |
Advanced Liquid Logic, Inc.
San Diego
CA
|
Family ID: |
40753787 |
Appl. No.: |
14/969933 |
Filed: |
December 15, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14225879 |
Mar 26, 2014 |
9267131 |
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14969933 |
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12334575 |
Dec 15, 2008 |
8716015 |
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14225879 |
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11639566 |
Dec 15, 2006 |
7901947 |
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12334575 |
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61013535 |
Dec 13, 2007 |
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61091637 |
Aug 25, 2008 |
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60745058 |
Apr 18, 2006 |
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60745039 |
Apr 18, 2006 |
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60745043 |
Apr 18, 2006 |
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60745059 |
Apr 18, 2006 |
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60745914 |
Apr 28, 2006 |
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60745950 |
Apr 28, 2006 |
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60746797 |
May 9, 2006 |
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60746801 |
May 9, 2006 |
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60806412 |
Jun 30, 2006 |
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60807104 |
Jul 12, 2006 |
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Current U.S.
Class: |
435/29 ;
435/173.1 |
Current CPC
Class: |
G01N 2035/1034 20130101;
B01J 2219/00653 20130101; G01N 35/10 20130101; G01N 15/10 20130101;
G01N 2015/1081 20130101; Y10T 436/25 20150115; Y10T 436/11
20150115; G01N 2035/00564 20130101; G01N 2015/0088 20130101; Y10T
436/2575 20150115; B01J 2219/00527 20130101; B01J 2219/00743
20130101; C12N 13/00 20130101 |
International
Class: |
C12N 13/00 20060101
C12N013/00; G01N 15/10 20060101 G01N015/10 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made with government support under
DK066956-02 awarded by the National Institutes of Health of the
United States. The government has certain rights in the invention.
Claims
1-4. (canceled)
5. A method of growing cells on a droplet actuator, the method
comprising the steps: a. providing a droplet actuator comprising a
sample droplet loaded thereon and a cell culture medium, wherein
the sample droplet comprises one or more cells and is in an oil
filler fluid; b. merging the sample droplet and the cell culture
medium by electrowetting to form a cell culture droplet in the oil
filler fluid and comprising the one or more cells and cell culture
medium; and c. maintaining the cell culture droplet in the oil
filler fluid at a temperature suitable for causing the cells to
grow in the cell culture medium on the droplet actuator.
6. The method of claim 1 wherein the cell culture droplet is
situated between droplet actuator substrates in proximity to a
droplet operations surface.
7. The method of claim 1 wherein the one or more cells are bound to
beads.
8. The method of claim 1, wherein the sample droplet and cell
culture droplet are substantially surrounded by the oil filler
fluid.
9. The method of claim 1, wherein the oil comprises silicone
oil.
10. The method of claim 1, wherein the one or more cells of the
cell culture droplet comprises only one cell.
11. The method of claim 1, wherein the droplet actuator comprises a
heating element configured to heat the cell culture medium to an
appropriate temperature for incubation.
12. The method of claim 1, wherein the droplet actuator is
associated with a heating element configured to heat the cell
culture medium to an appropriate temperature for incubation.
13. The method of claim 1, further comprising a step prior to step
(b) wherein, a cell sorting process is conducted on the droplet
actuator to produce the droplet comprising one or more cells, and
wherein the droplet comprising one or more cells comprises one or
more cells of the same cell type.
14. The method of claim 1, wherein the cell culture medium is in a
cell culture reservoir on the droplet actuator.
15. The method of claim 1, wherein the cell culture medium is in
contact with the atmosphere or a sub-atmosphere on the droplet
actuator.
16. The method of claim 1, wherein electrowetting electrodes
fluidically connect the cell culture droplet with a fluid
reservoir.
17. The method of claim 16, wherein the fluid reservoir comprises a
volume of reagent fluid.
18. The method of claim 17, wherein after step 1(b), droplet
operations are conducted to merge the cell culture droplet with a
droplet comprising the reagent fluid from the fluid reservoir,
wherein the reagent fluid is exchanged with the cell culture medium
in the cell culture droplet.
19. The method of claim 1, wherein droplet operations are conducted
to merge the cell culture droplet with a droplet comprising
beads.
20. The method of claim 19, wherein the beads have an affinity for
one or more cells in the cell culture droplet.
21. The method of claim 20, wherein the one or more cells are bound
to the beads in the cell culture droplet.
22. The method of claim 21, wherein the beads are immobilized
within the cell culture droplet.
23. The method of claim 22, wherein the beads are magnetically
responsive beads, and wherein the magnetically responsive beads are
magnetically immobilized within the cell culture droplet.
24. The method of claim 22, wherein the beads are immobilized
within the cell culture droplet by one or more physical
barriers.
25. The method of claim 22, wherein droplet operations are
conducted to split the cell culture droplet to form one or more
droplets substantially lacking beads, wherein the one or more
droplets substantially lacking beads comprise reagent fluid and
cell culture medium, and wherein the one or more droplets
substantially lacking beads are transported away from the cell
culture droplet.
26. The method of claim 25, wherein the merging and splitting
droplet operations deliver a metabolically useful substance, drug,
or chemical to the cell culture medium of the cell culture
droplet.
27. The method of claim 25, wherein the merging and splitting
droplet operations change the pH concentration of the cell culture
medium of the cell culture droplet.
28. The method of claim 18, wherein droplet operations are
conducted to split the cell culture droplet to form one or more
droplets, wherein the merging and splitting droplet operations
change the concentration of cells within the cell culture medium of
the cell culture droplet.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. patent application Ser. No. 14/225,879, entitled "Method of
Growing Cells on a Droplet Actuator," filed on Mar. 26, 2014, the
application of which is a divisional of and claims priority to U.S.
patent application Ser. No. 12/334,575, entitled "Manipulation of
Cells on a Droplet Actuator," filed on Dec. 15, 2008, now U.S. Pat.
No. 8,716,015, the application of which claims priority to U.S.
Patent Application Nos. 61/013,535, entitled "Manipulating cells in
a droplet actuator," filed on Dec. 13, 2007; 61/091,637, entitled
"Manipulating cells in a droplet actuator," filed on Aug. 25, 2008.
U.S. patent application Ser. No. 12/334,575 is also a
continuation-in-part of and claims priority to U.S. patent
application Ser. No. 11/639,566, entitled "Droplet-Based Particle
Sorting," filed on Dec. 15, 2006, now U.S. Pat. No. 7,901,947,
which in turn claims priority to U.S. Patent Application Nos.
60/745,058, entitled "Filler Fluids for Droplet-Based
Microfluidics," filed on Apr. 18, 2006; 60/745,039, entitled
"Apparatus and Methods for Droplet-Based Blood Chemistry," filed on
Apr. 18, 2006; 60/745,043, entitled "Apparatus and Methods for
Droplet-Based PCR," filed on Apr. 18, 2006; 60/745,059, entitled
"Apparatus and Methods for Droplet-Based Immunoassay," filed on
Apr. 18, 2006; 60/745,914, entitled "Apparatus and Method for
Manipulating Droplets with a Predetermined Number of Cells" filed
on Apr. 28, 2006; 60/745,950, entitled "Apparatus and Methods of
Sample Preparation for a Droplet Microactuator," filed on Apr. 28,
2006; 60/746,797 entitled "Portable Analyzer Using Droplet-Based
Microfluidics," filed on May 9, 2006; 60/746,801, entitled
"Apparatus and Methods for Droplet-Based Immuno-PCR," filed on May
9, 2006; 60/806,412, entitled "Systems and Methods for Droplet
Microactuator Operations," filed on Jun. 30, 2006; and 60/807,104,
entitled "Method and Apparatus for Droplet-Based Nucleic Acid
Amplification," filed on Jul. 12, 2006; the entire disclosures of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The inventions relates to methods, devices and systems for
sorting cells, inoculating culture media, replenishing culture
media, growing cells, and testing cell cultures.
BACKGROUND OF THE INVENTION
[0004] Droplet actuators are used to conduct a wide variety of
droplet operations, such as droplet transport and droplet
dispensing. A droplet actuator typically includes two surfaces
separated by a gap. One or both surfaces include electrodes for
conducting droplet operations. The gap typically includes one or
more filler fluids that are relatively immiscible with the
droplets. Droplets may, for example, be reagents and/or droplet
fluids for conducting assays. In wide variety of applications, such
as the production of antibodies and assaying stem cells, samples
within droplet actuators may include cells to be manipulated and,
therefore, there is a need for new approaches to manipulating cells
within a droplet actuator.
BRIEF DESCRIPTION OF THE INVENTION
[0005] The invention provides a method of inoculating a culture
medium. The method may include providing a droplet including a
single cell type on a droplet actuator and inoculating a culture
medium with the droplet. The inoculating step may involve
conducting one or more droplet operations to bring the droplet into
contact with the culture medium. The droplet including a single
cell type may be provided by (a) providing a droplet actuator
including a sample droplet loaded thereon, the sample droplet
including cells of multiple cell types; (b) dispensing a
sub-droplet from the sample droplet; (c) analyzing the sub-droplet
to determine whether the sub-droplet includes a single cell type;
(d) repeating steps (b) and (c) until one or more droplets each
including a single cell or a single cell type is identified.
[0006] The invention also provides a method of providing a droplet
including a single cell type, the method including: providing a
droplet actuator including: a sample droplet loaded thereon, the
sample droplet including cells of multiple cell types; a bead
droplet including one or more beads having affinity for a specific
one of the cell types; conducting one or more droplet operations to
combine the bead droplet with the sample droplet, thereby
permitting cells of the specific one of the cell types to bind to
the beads; conducting a droplet based washing protocol to separate
the beads bound to cells of the specific one of the cell types from
cells of other cell types.
[0007] Further, the invention provides a method of providing a
droplet having a modified distribution of cell types, the method
including: providing a droplet actuator including a sample droplet
loaded thereon, the sample droplet including a first distribution
of cells of multiple cell types; activating a series of electrodes
to elongate the droplet, thereby providing an elongated droplet;
applying a dielectrophoretic gradient along the elongated droplet;
deactivating an intermediate one of the series of electrodes to
divide the droplet into two or more sub-droplets, each such
sub-droplet having a distribution of cells that differs from the
first distribution of cells of multiple cell types. At least one of
the sub-droplets provided may include a cell type that is enriched
relative to the sample droplet. The cell culture droplet and the
second droplet may be situated between droplet actuator substrates
in proximity to a droplet operations surface. The cell culture
droplet may be substantially surrounded by a filler fluid.
[0008] In another embodiment, the invention provides a method of
providing a metabolically useful substance to a cell culture. A
droplet actuator may be provided, including: and a cell culture
droplet loaded thereon, the sample droplet including cells and a
cell culture medium; a second droplet including a metabolically
useful substance. The method may include conducting one or more
droplet operations to combine the cell culture droplet with the
second droplet on the droplet actuator. The cell culture droplet
may be situated between droplet actuator substrates in proximity to
a droplet operations surface. The cell culture droplet and the
second droplet may be situated between droplet actuator substrates
in proximity to a droplet operations surface. The cell culture
droplet may be substantially surrounded by a filler fluid. The cell
culture droplet and the second droplet may be substantially
surrounded by a filler fluid.
[0009] The invention provides a method of growing cells on a
droplet actuator. The method includes providing a droplet actuator
including a cell culture droplet including a cell culture medium
and one or more cells; and maintaining the droplet at a temperature
suitable for causing the cells to grow in the cell culture medium
on the droplet actuator. The cell culture droplet may be situated
between droplet actuator substrates in proximity to a droplet
operations surface. The cell culture droplet may be surrounded by a
filler fluid. The cells may include cells bound to beads.
[0010] The invention also provides a method of providing a
hybridoma. A droplet actuator may be provided including: a B-cell
droplet including a B-cell; and a myeloma cell droplet including a
myeloma cell. The method may involve conducting droplet operations
to combine the B-cell droplet with the myeloma cell droplet under
conditions suitable to cause the fusion of the B-cell with the
myeloma cell to produce a hybridoma. The B-cell droplet is situated
between droplet actuator substrates in proximity to a droplet
operations surface. The myeloma cell droplet is situated between
droplet actuator substrates in proximity to a droplet operations
surface. The hybridoma may be grown and tested on the droplet
actuator. The B-cell droplet may be surrounded by a filler fluid.
The myeloma cell droplet may be surrounded by a filler fluid.
[0011] In a further embodiment, the invention provides a method of
monitoring a cell culture. The method may include providing droplet
actuator including a cell culture droplet including a cell culture
medium and one or more cells; conducting one or more droplet
operations to dispense a sample droplet from the cell culture
medium; and testing the sample droplet for one or more target
substances. The cell culture droplet may be situated between
droplet actuator substrates in proximity to a droplet operations
surface. The sample droplet may be dispensed using
electrode-mediated droplet operations between droplet actuator
substrates in proximity to a droplet operations surface. Testing
may be effected using steps including electrode-mediated droplet
operations between droplet actuator substrates. The cell culture
droplet may be substantially surrounded by a filler fluid. The
sample droplet may be substantially surrounded by a filler
fluid.
[0012] Testing may be effected while the sample droplet is
substantially surrounded by a filler fluid. Testing may involve
conducting one or more electrode-mediated, droplet-based assays on
the droplet actuator. The target substances may include
metabolically useful substances.
[0013] One or more droplet operations may be used to replace the
sample droplet from the cell culture droplet with a replacement
droplet added to the cell culture droplet, the replacement droplet
including one or more metabolically useful substances. The
replacement droplet is dispensed and transported from a droplet
actuator reservoir by electrode mediated droplet operations into
contact with the cell culture droplet. The replacement droplet may
be selected to replace one or more specific substances identified
as deficient in the testing step. The testing and replacement of
one or more target substances may be automated. The testing may
include quantifying one or more metabolic substances.
[0014] The invention also provides a method of monitoring a cell
culture including: providing cell culture including a cell culture
medium and one or more cells and a fluid path to a droplet
actuator; providing a cell culture droplet from the cell culture to
the droplet actuator via the fluid path; testing the sample droplet
for one or more metabolically useful substances. The method may
also include replacing one or more metabolically useful substances
identified as deficient by the testing step.
DEFINITIONS
[0015] As used herein, the following terms have the meanings
indicated.
[0016] "Activate" with reference to one or more electrodes means
effecting a change in the electrical state of the one or more
electrodes which, in the presence of a droplet, results in a
droplet operation.
[0017] "Bead," with respect to beads on a droplet actuator, means
any bead or particle that is capable of interacting with a droplet
on or in proximity with a droplet actuator. Beads may be any of a
wide variety of shapes, such as spherical, generally spherical, egg
shaped, disc shaped, cubical and other three dimensional shapes.
The bead may, for example, be capable of being transported in a
droplet on a droplet actuator or otherwise configured with respect
to a droplet actuator in a manner which permits a droplet on the
droplet actuator to be brought into contact with the bead, on the
droplet actuator and/or off the droplet actuator. Beads may be
manufactured using a wide variety of materials, including for
example, resins, and polymers. The beads may be any suitable size,
including for example, microbeads, microparticles, nanobeads and
nanoparticles. In some cases, beads are magnetically responsive; in
other cases beads are not significantly magnetically responsive.
For magnetically responsive beads, the magnetically responsive
material may constitute substantially all of a bead or one
component only of a bead. The remainder of the bead may include,
among other things, polymeric material, coatings, and moieties
which permit attachment of an assay reagent. Examples of suitable
magnetically responsive beads are described in U.S. Patent
Publication No. 2005-0260686, entitled, "Multiplex flow assays
preferably with magnetic particles as solid phase," published on
Nov. 24, 2005, the entire disclosure of which is incorporated
herein by reference for its teaching concerning magnetically
responsive materials and beads. The fluids may include one or more
magnetically responsive and/or non-magnetically responsive beads.
Examples of droplet actuator techniques for immobilizing
magnetically responsive beads and/or non-magnetically responsive
beads and/or conducting droplet operations protocols using beads
are described in U.S. patent application Ser. No. 11/639,566,
entitled "Droplet-Based Particle Sorting," filed on Dec. 15, 2006;
U.S. Patent Application No. 61/039,183, entitled "Multiplexing Bead
Detection in a Single Droplet," filed on Mar. 25, 2008; U.S. Patent
Application No. 61/047,789, entitled "Droplet Actuator Devices and
Droplet Operations Using Beads," filed on Apr. 25, 2008; U.S.
Patent Application No. 61/086,183, entitled "Droplet Actuator
Devices and Methods for Manipulating Beads," filed on Aug. 5, 2008;
International Patent Application No. PCT/US2008/053545, entitled
"Droplet Actuator Devices and Methods Employing Magnetic Beads,"
filed on Feb. 11, 2008; International Patent Application No.
PCT/US2008/058018, entitled "Bead-based Multiplexed Analytical
Methods and Instrumentation," filed on Mar. 24, 2008; International
Patent Application No. PCT/US2008/058047, "Bead Sorting on a
Droplet Actuator," filed on Mar. 23, 2008; and International Patent
Application No. PCT/US2006/047486, entitled "Droplet-based
Biochemistry," filed on Dec. 11, 2006; the entire disclosures of
which are incorporated herein by reference.
[0018] "Droplet" means a volume of liquid on a droplet actuator
that is at least partially bounded by filler fluid. For example, a
droplet may be completely surrounded by filler fluid or may be
bounded by filler fluid and one or more surfaces of the droplet
actuator. Droplets may, for example, be aqueous or non-aqueous or
may be mixtures or emulsions including aqueous and non-aqueous
components. Droplets may take a wide variety of shapes; nonlimiting
examples include generally disc shaped, slug shaped, truncated
sphere, ellipsoid, spherical, partially compressed sphere,
hemispherical, ovoid, cylindrical, and various shapes formed during
droplet operations, such as merging or splitting or formed as a
result of contact of such shapes with one or more surfaces of a
droplet actuator. For examples of droplet fluids that may be
subjected to droplet operations using the approach of the
invention, see International Patent Application No. PCT/US
06/47486, entitled, "Droplet-Based Biochemistry," filed on Dec. 11,
2006. In various embodiments, a droplet may include a biological
sample, such as whole blood, lymphatic fluid, serum, plasma, sweat,
tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal
fluid, vaginal excretion, serous fluid, synovial fluid, pericardial
fluid, peritoneal fluid, pleural fluid, transudates, exudates,
cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal
samples, liquids containing single or multiple cells, liquids
containing organelles, fluidized tissues, fluidized organisms,
liquids containing multi-celled organisms, biological swabs and
biological washes. Moreover, a droplet may include a reagent, such
as water, deionized water, saline solutions, acidic solutions,
basic solutions, detergent solutions and/or buffers. Other examples
of droplet contents include reagents, such as a reagent for a
biochemical protocol, such as a nucleic acid amplification
protocol, an affinity-based assay protocol, an enzymatic assay
protocol, a sequencing protocol, and/or a protocol for analyses of
biological fluids.
[0019] "Droplet Actuator" means a device for manipulating droplets.
For examples of droplet actuators, see U.S. Pat. No. 6,911,132,
entitled "Apparatus for Manipulating Droplets by
Electrowetting-Based Techniques," issued on Jun. 28, 2005 to Pamula
et al.; U.S. patent application Ser. No. 11/343,284, entitled
"Apparatuses and Methods for Manipulating Droplets on a Printed
Circuit Board," filed on filed on Jan. 30, 2006; U.S. Pat. No.
6,773,566, entitled "Electrostatic Actuators for Microfluidics and
Methods for Using Same," issued on Aug. 10, 2004 and U.S. Pat. No.
6,565,727, entitled "Actuators for Microfluidics Without Moving
Parts," issued on Jan. 24, 2000, both to Shenderov et al.; Pollack
et al., International Patent Application No. PCT/US2006/047486,
entitled "Droplet-Based Biochemistry," filed on Dec. 11, 2006, the
disclosures of which are incorporated herein by reference. Methods
of the invention may be executed using droplet actuator systems,
e.g., as described in International Patent Application No.
PCT/US2007/009379, entitled "Droplet manipulation systems," filed
on May 9, 2007. In various embodiments, the manipulation of
droplets by a droplet actuator may be electrode mediated, e.g.,
electrowetting mediated or dielectrophoresis mediated. Examples of
other methods of controlling fluid flow that may be used in the
droplet actuators of the invention include devices that induce
hydrodynamic fluidic pressure, such as those that operate on the
basis of mechanical principles (e.g. external syringe pumps,
pneumatic membrane pumps, vibrating membrane pumps, vacuum devices,
centrifugal forces, and capillary action); electrical or magnetic
principles (e.g. electroosmotic flow, electrokinetic pumps
piezoelectric/ultrasonic pumps, ferrofluidic plugs,
electrohydrodynamic pumps, and magnetohydrodynamic pumps);
thermodynamic principles (e.g. gas bubble
generation/phase-change-induced volume expansion); other kinds of
surface-wetting principles (e.g. electrowetting, and
optoelectrowetting, as well as chemically, thermally, and
radioactively induced surface-tension gradient); gravity; surface
tension (e.g., capillary action); electrostatic forces (e.g.,
electroosmotic flow); centrifugal flow (substrate disposed on a
compact disc and rotated); magnetic forces (e.g., oscillating ions
causes flow); magnetohydrodynamic forces; and vacuum or pressure
differential. In certain embodiments, combinations of two or more
of the foregoing techniques may be employed in droplet actuators of
the invention.
[0020] "Droplet operation" means any manipulation of a droplet on a
droplet actuator. A droplet operation may, for example, include:
loading a droplet into the droplet actuator; dispensing one or more
droplets from a source droplet; splitting, separating or dividing a
droplet into two or more droplets; transporting a droplet from one
location to another in any direction; merging or combining two or
more droplets into a single droplet; diluting a droplet; mixing a
droplet; agitating a droplet; deforming a droplet; retaining a
droplet in position; incubating a droplet; heating a droplet;
vaporizing a droplet; cooling a droplet; disposing of a droplet;
transporting a droplet out of a droplet actuator; other droplet
operations described herein; and/or any combination of the
foregoing. The terms "merge," "merging," "combine," "combining" and
the like are used to describe the creation of one droplet from two
or more droplets. It should be understood that when such a term is
used in reference to two or more droplets, any combination of
droplet operations that are sufficient to result in the combination
of the two or more droplets into one droplet may be used. For
example, "merging droplet A with droplet B," can be achieved by
transporting droplet A into contact with a stationary droplet B,
transporting droplet B into contact with a stationary droplet A, or
transporting droplets A and B into contact with each other. The
terms "splitting," "separating" and "dividing" are not intended to
imply any particular outcome with respect to volume of the
resulting droplets (i.e., the volume of the resulting droplets can
be the same or different) or number of resulting droplets (the
number of resulting droplets may be 2, 3, 4, 5 or more). The term
"mixing" refers to droplet operations which result in more
homogenous distribution of one or more components within a droplet.
Examples of "loading" droplet operations include microdialysis
loading, pressure assisted loading, robotic loading, passive
loading, and pipette loading. Droplet operations may be
electrode-mediated. In some cases, droplet operations are further
facilitated by the use of hydrophilic and/or hydrophobic regions on
surfaces and/or by physical obstacles. Droplet operations may be
discrete flow operations, in which each overall operation involves
discrete steps, and each discrete step is mediated by the one or
more electrodes upon which the droplets reside and/or adjacent
electrodes. In certain cases, discrete flow droplet operations may
involve movement of droplets through a surrounding filler fluid, as
compared to movement of filler fluid to cause droplet
movements.
[0021] "Filler fluid" means a fluid associated with a droplet
operations substrate of a droplet actuator, which fluid is
sufficiently immiscible with a droplet phase to render the droplet
phase subject to electrode-mediated droplet operations. The filler
fluid may, for example, be a low-viscosity oil, such as silicone
oil. Other examples of filler fluids are provided in International
Patent Application No. PCT/US2006/047486, entitled, "Droplet-Based
Biochemistry," filed on Dec. 11, 2006; and in International Patent
Application No. PCT/US2008/072604, entitled "Use of additives for
enhancing droplet actuation," filed on Aug. 8, 2008.
[0022] "Immobilize" with respect to magnetically responsive beads,
means that the beads are substantially restrained in position in a
droplet or in filler fluid on a droplet actuator. For example, in
one embodiment, immobilized beads are sufficiently restrained in
position to permit execution of a splitting operation on a droplet,
yielding one droplet with substantially all of the beads and one
droplet substantially lacking in the beads.
[0023] "Magnetically responsive" means responsive to a magnetic
field. "Magnetically responsive beads" include or are composed of
magnetically responsive materials. Examples of magnetically
responsive materials include paramagnetic materials, ferromagnetic
materials, ferrimagnetic materials, and metamagnetic materials.
Examples of suitable paramagnetic materials include iron, nickel,
and cobalt, as well as metal oxides, such as Fe.sub.3O.sub.4,
BaFe.sub.12O.sub.19, CoO, NiO, Mn.sub.2O.sub.3, Cr.sub.2O.sub.3,
and CoMnP.
[0024] "Washing" with respect to washing a magnetically responsive
bead means reducing the amount and/or concentration of one or more
substances in contact with the magnetically responsive bead or
exposed to the magnetically responsive bead from a droplet in
contact with the magnetically responsive bead. The reduction in the
amount and/or concentration of the substance may be partial,
substantially complete, or even complete. The substance may be any
of a wide variety of substances; examples include target substances
for further analysis, and unwanted substances, such as components
of a sample, contaminants, and/or excess reagent. In some
embodiments, a washing operation begins with a starting droplet in
contact with a magnetically responsive bead, where the droplet
includes an initial amount and initial concentration of a
substance. The washing operation may proceed using a variety of
droplet operations. The washing operation may yield a droplet
including the magnetically responsive bead, where the droplet has a
total amount and/or concentration of the substance which is less
than the initial amount and/or concentration of the substance.
Other embodiments are described elsewhere herein, and still others
will be immediately apparent in view of the present disclosure.
[0025] The terms "top" and "bottom" are used throughout the
description with reference to the top and bottom substrates of the
droplet actuator for convenience only, since the droplet actuator
is functional regardless of its position in space.
[0026] When a liquid in any form (e.g., a droplet or a continuous
body, whether moving or stationary) is described as being "on",
"at", or "over" an electrode, array, matrix or surface, such liquid
could be either in direct contact with the
electrode/array/matrix/surface, or could be in contact with one or
more layers or films that are interposed between the liquid and the
electrode/array/matrix/surface.
[0027] When a droplet is described as being "on" or "loaded on" a
droplet actuator, it should be understood that the droplet is
arranged on the droplet actuator in a manner which facilitates
using the droplet actuator to conduct one or more droplet
operations on the droplet, the droplet is arranged on the droplet
actuator in a manner which facilitates sensing of a property of or
a signal from the droplet, and/or the droplet has been subjected to
a droplet operation on the droplet actuator.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 illustrates a cell sorting process conducted in a
droplet actuator;
[0029] FIG. 2 illustrates a process of sorting droplets in a
droplet actuator by the types of cells contained therein;
[0030] FIGS. 3A and 3B illustrate side views of a first and second
step, respectively, of a method of using a droplet actuator for
separating different types of cells;
[0031] FIG. 4 illustrates a process for merging a droplet
containing one or more cells with a droplet of, for example, a
reagent;
[0032] FIG. 5 illustrates a cell incubation process for growing
cells in a droplet actuator, e.g., growing cells from a single
cell;
[0033] FIG. 6 illustrates a cell fusing process of merging droplets
that contain different types of cells;
[0034] FIG. 7 illustrates a process of separating different cell
types by use of beads in a droplet actuator;
[0035] FIG. 8 illustrates a cell incubation process of growing
cells on beads in a droplet actuator; and
[0036] FIG. 9 illustrates a liquid exchange process in a cell
culture reservoir.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The invention provides methods of manipulating cells within
a droplet actuator. For example, by use of operations, such as,
dispensing droplets from a cell suspension, analyzing the number of
droplets in the dispensed droplet, merging the droplet with other
droplets containing either specific reagents or other cells,
detecting a property of the droplet, and incubating the droplet at
a particular temperature. Embodiments of the invention provide a
wide variety of techniques, of which the following are examples:
(1) sorting droplets by the number of cells in a droplet, (2)
sorting droplets by the types of cells in a droplet, (3) merging
cell-containing droplets with reagent droplets, (4), incubating
cell-containing droplets in order to grow more cells, (5) fusing
droplets with different types of cells in a single droplet, (6)
separating a single droplet with different types of cells into
multiple droplets, each with a reduced number of cell types, (7)
growing cells on beads via incubation, (8) culturing cells in a
culture reservoir, and (9) performing liquid exchange in a
cell-containing culture reservoir.
8.1 Sorting Cell-Containing Droplets
[0038] FIG. 1 illustrates a cell sorting process 100 conducted in a
droplet actuator.
[0039] Droplets are dispensed from a parent droplet or reservoir
containing a suspension of cells and dispensed droplets are sorted
by the number of cells contained therein. FIG. 1 shows an
arrangement of electrodes 110, e.g., electrowetting electrodes, in
the droplet actuator. A sensor 114 is provided for detecting the
number of cells in a droplet. Sensor 114 may be any suitable
detection mechanism for detecting the number of cells in a droplet.
Examples include optical detection mechanisms, electrical detection
mechanisms, and florescence-based detection mechanisms. Cells may
be labeled to facilitate detection. A sample reservoir contains a
volume of sample liquid 118 that contains a quantity of cells 122.
Droplet operations are used to dispense and transport droplets from
the sample, such as a droplet 126. Each dispensed droplet may
include a random number of cells. Dispensed droplets are
transported along electrodes 110 and into sensing proximity with
sensor 114.
[0040] In one example scenario, the droplets of interest are those
droplets that contain a single cell 122 only and any droplets that
contain no cells 122 or two or more cells 122 are discarded or
returned to the sample. In this example, when a droplet arrives at
sensor 114, the number of cells 122 that are contained therein is
determined. In this example, when single-cell droplets, such as
single-cell droplets 130, are detected, single-cell droplets 130
are transported along a certain electrode path for further
processing. In contrast, when droplets that contain no cells 122 or
two or more cells 122, such as droplets 134, are detected, droplets
134 are transported along a certain different electrode path that
returns droplets 134 back to the source volume of sample liquid 118
or, alternatively, to a waste reservoir (not shown). The parent
droplet may have a concentration of cells selected to statistically
(e.g., using Poisson distribution statistics) maximize the number
of dispensed droplets including single cells. Droplet operations
may be used to dilute excessively concentrated parent droplets in
order to improve or maximize the occurrence of dispensed droplets
with single cells.
[0041] Cell sorting process 100 of sorting droplets by the number
of cells is not limited to targeting and processing single-cell
droplets only. The target droplets of interest may contain any
desired number of cells depending on the intended purpose of the
droplet/cell operations within the droplet actuator. For example,
two-celled droplets may be targeted and all others are discarded,
one- or two-celled droplets may be targeted and all others are
discarded, and so on.
[0042] FIG. 2 illustrates a process 200 of sorting droplets in a
droplet actuator by the types of cells contained therein. FIG. 2
shows an arrangement of electrodes 210, e.g., electrowetting
electrodes, wherein the location of a sensor 214 is arranged along
a transport path for detecting the cell type in a droplet. Sensor
214 may be any suitable detection mechanism for detecting the cell
type in a droplet, such as, but not limited to, optical detection
mechanisms, electrical detection mechanisms, and florescent-based
detection mechanisms. A sample reservoir contains a volume of
sample liquid 218 that contains a quantity of various types of
cells. In one example, sample liquid 218 contains a quantity of a
first cell type 222 and a quantity of a second cell type 224.
Droplet operations are used to dispense droplets that contain a
random number and cell type and transport the dispensed droplets
into proximity with sensor 214.
[0043] In one example scenario, the droplets of interest are those
droplets that contain the first cell type 222 only and any droplets
that contain no cells at all or at least one of the second cell
type 224 are discarded. Therefore, when a droplet arrives at sensor
214, the type(s) of cells contained therein is determined. In this
example, when droplets that contain one or more of the first cell
type 222 only, such as droplets 226, are detected, droplets 226 are
transported along a certain electrode path for forming a sample
volume 230 that contains the first cell type 222 only. By contrast,
when droplets that contain no cells at all or at least one of the
second cell type 224, such as droplets 234, are detected, droplets
234 are transported along a different electrode path for forming a
waste volume 238 that may contain both the first cell type 222 and
the second cell type 224. Alternatively, an electrode path (not
shown) may be provided for forming a sample volume that contains
the second cell type 224 only.
[0044] In another example, the sorting process is used to enrich
the concentration of one cell type relative to another cell type.
For example, any droplet containing the target cell type may be
sorted to one location while any droplet not containing the target
cell type may be sorted to a second location. Thus, the first
location is enriched with the target cell type while the second
location is depleted of the target cell type. This process can be
repeated any number of times to achieve a desired level of
purification. When the target cell type is labeled, for example,
with a fluorescent tag, the sensor may simply need to detect
whether or not any signal is present in the droplet to perform this
process. For more concentrated cell suspensions the sensor may be
used to detect whether the total signal of the droplet exceeds a
certain threshold indicating whether the droplet is enriched or
depleted of the target cell type. The process can be repeated many
times over so that even a relatively small enrichment at each step
can produce a substantial amount of purification.
[0045] Cell sorting process 200 is not limited to processing two
types of cells only. Any number of types of cells may be detected
and sorted accordingly into any number of cell type-specific sample
volumes. By use of a cell sorting process, such as cell sorting
process 200, the invention provides a method of providing droplets
with enriched or pure concentrations of pre-selected cell
types.
[0046] FIGS. 3A and 3B illustrate side views of a first and second
step, respectively, of a method of using a droplet actuator 300 for
separating different types of cells. Droplet actuator 300 includes
a top plate 310 and a bottom plate 314 that are arranged with a gap
therebetween. A set of electrodes 318, e.g., electrowetting
electrodes, are associated with bottom plate 314. A quantity of
sample fluid 322 is provided in the gap of droplet actuator 300.
Additionally, sample fluid 322 contains a quantity of cells 326 of
interest that are intermixed with a quantity of other types of
cells 330. Furthermore, when the dielectric properties of the
different types of cells within sample fluid 322 are different,
certain electrodes 318 may be used to manipulate certain cells by
use of dielectrophoresis (DEP). DEP is the lateral motion imparted
on uncharged particles (e.g., cells) as a result of polarization
that is induced by non-uniform electric fields (e.g., induced via
electrodes 318). For example, FIG. 3A shows a certain electrode 318
that is near one end of the slug of sample fluid 322 is energized
in a manner that corresponds to the dielectric properties of the
cells 326 of interest. In doing so, the cells 326 of interest are
attracted and immobilized (due to DEP) near one end of the slug of
sample fluid 322, as shown in FIG. 3A, while the other types of
cells 330 that have different dielectric properties are not
attracted.
[0047] FIG. 3B shows that once the cells 326 of interest are
attracted and immobilized (due to DEP) near one end of the slug of
sample fluid 322, a droplet splitting operation may occur in order
to create a droplet 334 of sample fluid that contains substantially
the cells 326 of interest only. By use of the method shown in FIGS.
3A and 3B, cells of interest are separated from unwanted cells via
splitting. In another embodiment, DEP may be used to enrich a
droplet with cells of interest, and a cell sorting method such as
the method described with respect to FIG. 2 may be employed to
further isolate a specific cell type.
[0048] In an alternative embodiment, different types of beads that
have different affinities for different types of cells may be
provided within sample fluid 322. In one example, certain beads
within sample fluid 322 may have an affinity for the cells 326 of
interest and substantially no affinity for the other types of cells
330 and, thus, the cells 326 of interest only bind to these certain
beads. Additionally, the beads may have different magnetic
properties, for example, by having magnetically responsive beads of
different sizes, by providing a mix of magnetically responsive
beads and non-magnetically responsive beads, and any combination
thereof. As a result, a magnetic field strength that corresponds to
the beads that have an affinity for the cells 326 of interest may
be applied in order to attract and immobilize the target beads near
one end of the slug of sample fluid 322. Again, a subsequent
droplet splitting operation may occur in order to create a droplet
334 of sample fluid that is enriched for the cells 326 of interest
or contains substantially the cells 326 of interest only.
8.2 Merging Droplets Containing Cells
[0049] FIG. 4 illustrates a process 400 for merging a droplet
containing one or more cells with a droplet of, for example, a
reagent. FIG. 4 shows an arrangement of electrodes 410, e.g.,
electrowetting electrodes, along which a cell-containing droplet,
such as a cell-containing droplet 414, and a droplet of reagent,
such as reagent droplet 418, may be manipulated. In particular, a
first step of cell merging process 400 shows cell-containing
droplet 414 and reagent droplet 418 being transported toward one
another along electrodes 410 via electrowetting. A second step of
cell merging process 400 shows a merged droplet 422, which is
cell-containing droplet 414 and reagent droplet 418 that have been
combined into a single droplet. The reagent may, for example,
include a nutrient or other reagent for which the cell has a
metabolic requirement, a drug or other molecule used to perform a
treatment on the cell, such a lysis reagent, or any chemical useful
for performing an analysis on the cell.
8.3 Incubating Cells in Droplets
[0050] FIG. 5 illustrates a cell incubation process 500 for growing
cells in a droplet actuator, e.g., growing cells from a single
cell. FIG. 5 shows an arrangement of electrodes 510, e.g.,
electrowetting electrodes, along which a cell-containing droplet,
such as a cell-containing droplet 514 may be manipulated. In
particular, a first step of cell incubation process 500 shows
cell-containing droplet 514 that contains, for example, a single
cell only. A second step of cell incubation process 500 is a
temperature control step that maintains cell-containing droplet 514
at a temperature that promotes cell growth. The second step shows
an incubated droplet 518, which is a droplet that contains multiple
cells that have grown over time from the single cell. By use of a
cell incubation process, such as cell incubation process 500, cells
can proliferate within a droplet actuator. By use of a cell sorting
process as described above with respect to FIGS. 1 and 2, and an
incubation process, droplets may be obtained having a substantially
pure population of cell types.
8.4 Fusing Cells in Droplets
[0051] FIG. 6 illustrates a cell fusing process 600 of merging
droplets that contain different types of cells. FIG. 6 shows an
arrangement of electrodes 610, e.g., electrowetting electrodes,
along which a droplet that contains a first cell type, such as a
droplet 614, and a droplet that contains a second cell type, such
as droplet 618, may be manipulated. In particular, a first step of
cell fusing process 600 shows droplet 614 and droplet 618 being
transported toward one another along electrodes 610 via
electrowetting. A second step of cell fusing process 600 shows a
fused droplet 622, which is droplet 614 and droplet 618 that have
been combined into a single droplet that contains both the first
and second types of cells, e.g., fusion of a B-cell are with a
myeloma cell to produce an antibody-producing hybridoma. In anther
example, a fusing process, such as cell fusing process 600, may be
used in the in vitro fertilization (IVF) process, i.e., fusing a
sperm cell with an egg cell.
8.5 Using Beads for the Manipulation of Cells
[0052] FIG. 7 illustrates a process 700 of separating different
cell types by use of beads in a droplet actuator. FIG. 7 shows an
arrangement of electrodes 710, e.g., electrowetting electrodes,
along which a droplet that contains, for example, a first and
second cell type, such as a cell-containing droplet 714, and a
droplet that contains beads, such as bead-containing droplet 718,
may be manipulated. In particular, the beads of bead-containing
droplet 718 may be, for example, magnetically responsive beads.
Examples of suitable magnetically responsive beads are described in
U.S. Patent Publication No. 2005-0260686, entitled, "Multiplex flow
assays preferably with magnetic particles as solid phase,"
published on Nov. 24, 2005. Additionally, the beads of
bead-containing droplet 718 may have an affinity for a certain cell
type. In one example, the beads of bead-containing droplet 718 may
have an affinity for the first cell type only and substantially no
affinity for the second cell type.
[0053] A first step of cell separation process 700 shows
cell-containing droplet 714 and bead-containing droplet 718 being
merged along electrodes 710 using electrode-mediated droplet
operations. A second step of cell separation process 700 shows a
merged droplet 722, which is cell-containing droplet 714 and
bead-containing droplet 718 that have been combined into a single
droplet that contains both the first and second cell type along
with the beads. The second step of cell separation process 700 also
shows that the first cell type within merged droplet 722 bind to
the beads because the beads have an affinity for the first cell
type only. By contrast, cells of the second cell type do not bind
to the beads and, thus, remain substantially suspended within
merged droplet 722. A third step of cell separation process 700
illustrates a droplet-based wash procedure using wash buffer
droplet 724 that is used to remove the unbound second cell type
while the beads are restrained in place. The result is a
cell-containing droplet 726 that has a substantially pure cell
type. The droplet 730 of unbound cells may be subjected to further
droplet operations and/or other processing or analysis.
8.6 Growing Cells
[0054] FIG. 8 illustrates a cell incubation process 800 of growing
cells on beads in a droplet actuator. FIG. 8 shows an arrangement
of electrodes 810, e.g., electrowetting electrodes, along which a
droplet that contains a certain cell type, such as a
cell-containing droplet 814, and a droplet that contains beads,
such as bead-containing droplet 818, may be manipulated. In
particular, the beads of bead-containing droplet 818 may be, for
example, magnetically responsive beads. Additionally, the beads of
bead-containing droplet 818 may have an affinity for the particular
cell type within cell-containing droplet 814.
[0055] A first step of cell incubation process 800 shows
cell-containing droplet 814 and bead-containing droplet 818 being
transported toward one another along electrodes 810 via
electrowetting. A second step of cell incubation process 800 shows
a merged droplet 822, which is cell-containing droplet 814 and
bead-containing droplet 818 that have been combined into a single
droplet that contains both the cells and the beads. The second step
of cell incubation process 800 also shows that the cells within
merged droplet 822 bind to the beads because the beads have an
affinity for the particular cell type. A third step of cell
incubation process 800 is a temperature control step that maintains
merged droplet 822 at a temperature that promotes cell growth. The
third step of cell incubation process 800 shows an incubated
droplet 826, which is a droplet that contains multiple cells that
have grown over time upon the surface of the beads. By use of a
cell incubation process, such as cell incubation process 800, cells
can proliferate within a droplet actuator. In particular, the beads
provide a means for growing cells on surfaces other than the
droplet actuator surface so that the cells can be subsequently
manipulated in the droplet actuator.
[0056] Embodiments of the invention may be provided for culturing
cells on a droplet actuator. A cell-containing droplet, such as a
droplet that contains one or more cells and/or cell-types, may be
transported using droplet operations into contact with a cell
culture medium. The cell culture medium may be included in a cell
culture reservoir or well. When necessary, the cell culture medium
may be in contact with the atmosphere or with a sub-atmosphere on
the droplet actuator. The droplet actuator may include or be
associated with a heating element configured to heat the cell
culture medium to an appropriate temperature for incubation.
[0057] FIG. 9 illustrates a liquid exchange process 900 in a cell
culture reservoir. FIG. 9 shows an arrangement of electrodes 910,
e.g., electrowetting electrodes, which fluidically connect a fluid
reservoir 914 and a cell culture droplet 918. The arrangement is
useful, for example, for performing a liquid exchange process
supplying reagents, such as reagents metabolically useful
substances, to cell culture droplet 918. Fluid reservoir 914 may
contain, for example, a volume of reagent fluid 922. Cell culture
droplet 918 may contain, for example, a volume of cell culture
medium 926 that contains a quantity of cells 930. Cells 930 may be
immobilized within cell culture droplet 918. In one example, cells
930 may be bound to magnetically responsive beads that are within
cell culture droplet 918, whereby the magnetically responsive beads
may be magnetically immobilized. Similarly, non-magnetically
responsive beads may be physically immobilized, e.g., using one or
more physical barriers as described in International Patent
Application No. PCT/US08/74151, filed on Aug. 25, 2008, entitled
"Bead Manipulations on a Droplet Actuator," the entire disclosure
of which is incorporated herein by reference. Any mechanism for
immobilizing or retaining cells 930 within cell culture droplet 918
is suitable. Liquid may be exchanged using droplet operations for
merging nutrient-containing droplets into contact with cell culture
droplet 918. In some cases, droplet splitting operations may also
be used to remove droplets including reduced quantities of such
nutrients from the cell culture droplet 918.
[0058] In one example, by use of droplet operations, droplets of
reagent fluid 922 may be dispensed from fluid reservoir 914 and
transported along electrodes 910 and into cell culture droplet 918.
By introducing reagent fluid 922 into cell culture medium 926 of
cell culture droplet 918, reagent fluid 922 is exchanged with cell
culture medium 926. Subsequently, one or more droplets 934, which
are formed of a mixture of reagent fluid 922 and cell culture
medium 926, are transported away from cell culture droplet 918; all
the while, cells 930 are held immobilized within cell culture
droplet 918. In alternative embodiments, cells 930 are not
immobilized.
[0059] Example purposes of a liquid exchange process, such as
liquid exchange process 900, may include, but are not limited to,
delivering in a metered fashion various substances, such as
metabolically useful substances, drugs or chemicals, to cell
culture medium 926 of cell culture droplet 918, changing the PH
concentration of cell culture medium 926 of cell culture droplet
918, changing the concentration of cells 930 within cell culture
medium 926 of cell culture droplet 918, and any combinations
thereof.
8.7 Inoculation of a Cell Culture Medium
[0060] The droplet actuator of the invention may include a cell
culture medium arranged in sufficient proximity to one or more
droplet operations electrodes to permit a droplet comprising a cell
to be introduced to the culture medium. The culture medium itself
may be composed on the droplet actuator by combining various
droplets including medium components. The culture medium may or may
not be subject to droplet operations. In accordance with the
invention, a culture medium may be provided on the droplet
actuator. A droplet including one or more cells may be transported
via droplet operations into contact with the culture medium. The
inoculated culture medium may be incubated on the droplet actuator.
A droplet may be contacted with a viscous culture medium and
removed from the culture medium in order to capture one or more
cultured cells, e.g., using the techniques described in
International Patent Application No. PCT/US08/74151, filed on Aug.
25, 2008, entitled "Bead Manipulations on a Droplet Actuator," the
entire disclosure of which is incorporated herein by reference.
8.8 Testing Cells
[0061] Cells on a droplet actuator may be tested using a wide
variety of techniques. A cell may be produced on the droplet
actuator and tested on the droplet actuator. A cell may be supplied
from an external source to the droplet actuator for testing. A
reporter assay may be conducted using droplet operations on the
droplet actuator to determine whether a gene of interest is being
expressed. A RT-PCR assay may be conducted using droplet operations
on the droplet actuator using material extracted from the cells
using a droplet-based extraction protocol to determine the presence
and quantity of mRNA for the gene of interest. An immunoassay may
be conducted using droplet operations on the droplet actuator to
determine the presence and the amount of protein produced. An
enzymatic assay may be conducted using droplet operations on the
droplet actuator to determine the activity of the protein. Two or
more of these assays or assay types may be conducted on a single
droplet actuator.
[0062] The results of a combination of the foregoing assays would
show the relationship between the expression of the gene, the
amount of protein product and the activity of the protein. Cells
may be treated with pathogens, therapeutic agents or other test
substances or conditions, and the foregoing assays may be conducted
to elucidate the effect of the test substance on the cell.
CONCLUDING REMARKS
[0063] The foregoing detailed description of embodiments refers to
the accompanying drawings, which illustrate specific embodiments of
the invention. Other embodiments having different structures and
operations do not depart from the scope of the present invention.
This specification is divided into sections for the convenience of
the reader only. Headings should not be construed as limiting of
the scope of the invention. The definitions are intended as a part
of the description of the invention. It will be understood that
various details of the present invention may be changed without
departing from the scope of the present invention. Furthermore, the
foregoing description is for the purpose of illustration only, and
not for the purpose of limitation, as the present invention is
defined by the claims as set forth hereinafter.
* * * * *