U.S. patent application number 13/566930 was filed with the patent office on 2012-11-29 for sheath flow devices and methods.
This patent application is currently assigned to CYNVENIO BIOSYSTEMS, INC.. Invention is credited to Andre' De Fusco, Paul W. Dempsey, Paul Pagano, Jiangrong Karen Qian, Hyongsok Tom Soh, Yanting Zhang.
Application Number | 20120301883 13/566930 |
Document ID | / |
Family ID | 43309472 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301883 |
Kind Code |
A1 |
Pagano; Paul ; et
al. |
November 29, 2012 |
SHEATH FLOW DEVICES AND METHODS
Abstract
The invention relates generally to fluid processing and, in
particular aspects, processing fluids for detection, selection,
trapping and/or sorting of particulate moieties. Sheath flow
devices described allow isolation of target species from fluid
samples while avoiding non-specific binding of unwanted species to
the surfaces of the separation device. Biological fluid processing,
detection, sorting or selection of cells, proteins, and nucleic
acids is described. The invention finds particular use in
diagnostic settings, analyzing a patient's medical condition,
monitoring and/or adjusting a therapeutic regimen and producing
cell based products.
Inventors: |
Pagano; Paul; (Moorpark,
CA) ; Zhang; Yanting; (Santa Barbara, CA) ;
Qian; Jiangrong Karen; (Thousand Oaks, CA) ; Soh;
Hyongsok Tom; (Santa Barbara, CA) ; Dempsey; Paul
W.; (Studio City, CA) ; De Fusco; Andre';
(Westlake Village, CA) |
Assignee: |
CYNVENIO BIOSYSTEMS, INC.
Westlake Village
CA
|
Family ID: |
43309472 |
Appl. No.: |
13/566930 |
Filed: |
August 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12813285 |
Jun 10, 2010 |
8263387 |
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13566930 |
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61185919 |
Jun 10, 2009 |
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61313625 |
Mar 12, 2010 |
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Current U.S.
Class: |
435/6.11 ;
204/450; 435/173.9; 435/29; 435/325; 435/377; 435/39; 435/6.1 |
Current CPC
Class: |
B03C 2201/18 20130101;
B03C 2201/22 20130101; B01L 2300/0816 20130101; G01N 35/0098
20130101; G01N 33/54366 20130101; B01L 2400/043 20130101; B03C 1/01
20130101; B03C 2201/26 20130101; B01L 2400/0487 20130101; B01L
3/502761 20130101; G01N 2800/52 20130101; B01L 3/502776 20130101;
B01L 2400/086 20130101; G01N 35/1095 20130101; G01N 1/405 20130101;
G01N 33/57407 20130101; G01N 33/585 20130101; B03C 1/0332 20130101;
B01L 2200/0652 20130101; B03C 1/288 20130101 |
Class at
Publication: |
435/6.11 ;
435/173.9; 435/29; 435/39; 435/325; 435/6.1; 435/377; 204/450 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C25B 7/00 20060101 C25B007/00; C12Q 1/68 20060101
C12Q001/68; C12N 5/071 20100101 C12N005/071; C12Q 1/02 20060101
C12Q001/02; C12Q 1/06 20060101 C12Q001/06 |
Claims
1. A method of separating a target species from a fluid sample in a
sheath flow device, comprising: (a) establishing a laminar sheath
of buffer flow along a planar surface of the sheath flow device;
(b) establishing a laminar flow of the fluid sample adjacent to the
laminar sheath of buffer flow; and (c) deflecting the target
species from the laminar flow of the fluid sample and into the
laminar sheath of buffer flow; wherein the laminar sheath of buffer
flow and the laminar flow of the fluid sample are adjacent.
2. The method of claim 1, further comprising establishing a second
laminar sheath of buffer flow, adjacent to the laminar flow of the
fluid sample, wherein (b) comprises establishing the laminar flow
of the fluid sample between the first and second laminar buffer
flows.
3. The method of claim 2, wherein establishing the first and second
laminar sheaths of buffer flow and the laminar flow of the fluid
sample are sufficient to maintain separate flow planes
substantially free of turbulent flow.
4. The method of claim 3, wherein deflecting the target species
from the laminar flow of the fluid sample and into the laminar
sheath of buffer flow comprises using at least one of a magnetic
force, an acoustic force, an electrophoretic force and an optical
force.
5. The method of claim 4, wherein the target species comprises at
least one of a cell, a bacterium, a virus, a protein and a nucleic
acid.
6. The method of claim 5, wherein the targets species is isolated
in a trapping station over which the laminar sheath of buffer flow
passes.
7. The method of claim 6, wherein the trapping station employs
magnetic force in order to trap the targets species selectively
labeled with magnetic particles.
8. The method of claim 7, wherein the target species is a
circulating tumor cell and the fluid sample is whole blood.
9. A method of analyzing a patient the method comprising: (a)
receiving a sample comprising tumor cells from a patient; (b)
separating said tumor cells from the sample by a separation method
comprising: (i) labeling the sample with magnetic particles having
a specific affinity for said tumor cells, thereby producing a
labeled sample, (ii) passing the labeled sample through a fluidic
device comprising a sorting region having a magnetic field gradient
effective to deflect and/or trap the magnetic particles from the
labeled sample and thereby separate the tumor cells from the
sample, wherein the sample flows in a sheath of buffer solution to
reduce nonspecific binding of the sample to the fluidic device; and
(c) characterizing the tumor cells separated from the sample in
(b).
10. The method of claim 9, wherein the characterizing in (c)
provides information for diagnosing a condition, screening for a
clinical trial, assessing the effectiveness of a therapeutic
treatment, and measuring the effectiveness of surgery.
11. The method of claim 9, wherein the sample is a fluid sample
taken from the patient.
12. The method of claim 9, wherein the sample is not taken from a
biopsy of the patient.
13. The method of claim 9, wherein the sample is a blood
sample.
14. The method of claim 13, wherein the tumor cells are circulating
tumor cells from a non-haematologic cancer.
15. The method of claim 9, wherein the characterizing is a count of
said tumor cells.
16. The method of claim 9, wherein the characterizing is a
molecular characterization of said tumor cells.
17. The method of claim 16, wherein said molecular characterization
is a genetic mutation in said tumor cells.
18. A method of monitoring and, if appropriate, adjusting a
patient's treatment regimen, the method comprising: (a) receiving a
sample comprising tumor cells from a patient undergoing a first
treatment regimen; (b) separating said tumor cells from the sample
by a separation method comprising: (i) labeling the sample with
magnetic particles having a specific affinity for said tumor cells,
thereby producing a labeled sample, (ii) passing the labeled sample
through a fluidic device comprising a sorting region having a
magnetic field gradient effective to deflect and/or trap the
magnetic particles from the labeled sample and thereby separate the
tumor cells from the sample, wherein the sample flows in a sheath
of buffer solution to reduce nonspecific binding of the sample to
the fluidic device; and (c) characterizing the tumor cells
separated from the sample in (b) to suggest a future treatment for
the patient.
19. The method of claim 18, wherein the first treatment regimen is
a chemotherapy regimen.
20. The method of claim 19, wherein the future treatment is a
different chemotherapy regimen.
21. The method of claim 18, wherein suggesting the future treatment
for the patient comprises predicting a future effectiveness of the
first treatment regimen.
22. The method of claim 18, wherein suggesting a future treatment
for the patient comprises identifying a second treatment regimen
that is different than the first treatment regimen and accounts for
a characteristic of the tumor cells not previously observed for the
patient.
23. The method of claim 22, further comprising: (d) receiving a
sample comprising tumor cells from the patient after the patient
has undergone the second treatment regimen; and (e) thereafter
performing (b) and (c) on the sample and tumor cells received in
(d).
24. A method of providing cell based products, the method
comprising: (a) receiving a sample comprising target cells; (b)
separating said target cells from the sample by a separation method
comprising: (i) labeling the target cells with magnetic particles
having a specific affinity for said target cells, thereby producing
a population of labeled cells in the sample, (ii) passing the
sample through a fluidic device comprising a sorting region having
a magnetic field gradient effective to deflect and/or trap at least
a portion of the population of labeled cells from the sample and
thereby separate the target cells from the sample, wherein the
sample flows in a sheath of buffer solution to reduce nonspecific
binding of the sample to the fluidic device; and (c) deriving a
cell based product from the target cells separated from the sample
in (b).
25. The method of claim 24, further comprising treating the target
cells separated from the sample in (b) to produce the cell based
product.
26. The method of claim 25, wherein the target cells are stem cells
and treating the target cells comprising treating the stem cells to
become an effective therapeutic agent.
27. The method of claim 26, wherein treating the stem cells to
become an effective therapeutic agent comprises differentiating the
stem cells to produce a more specific cell type.
28. The method of claim 24, wherein the sample is a fluid sample
taken from a patient.
29. The method of claim 28, wherein the fluid sample is a blood
sample.
30. The method of claim 24, further comprising characterizing the
target cells separated from the sample in (b).
31. The method of claim 30, wherein the characterizing is a count
of said target cells.
32. The method of claim 30, wherein the characterizing is a
molecular characterization of said target cells.
33. The method of claim 32, wherein said molecular characterization
is a genetic sequence of said target cells.
34. The method of any of claims 9, 18, and 24, wherein the sorting
region has a substantially rectangular interior space for bounding
the sample flow and the sheath of buffer solution, wherein the
interior space has first and second lateral dimensions transverse
to the sample flow, wherein the second lateral dimension is at
least about 2 times larger than the first lateral dimension such
that nonspecific binding is reduced along the larger of the first
and second lateral dimensions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims priority to
co-pending U.S. patent application Ser. No. 12/813,285, filed Jun.
10, 2010, entitled "SHEATH FLOW DEVICES AND METHODS," which claims
benefit of and priority to U.S. Provisional Application No.
61/185,919 filed Jun. 10, 2009, and U.S. Provisional Application
No. 61/313,625 filed Mar. 12, 2010, the contents of which are
incorporated by reference herein in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates generally to fluid processing and, in
particular aspects, processing fluids for detection, selection or
sorting of particulate moieties. In other aspects the present
invention relates to biological fluid processing, detection,
sorting or selection of cells, proteins, and nucleic acids. Other
aspects are disclosed herein.
BACKGROUND
[0003] Particle sorting technologies are widely used for targeting
moieties in suspension, but undue contamination can be a technical
hurdle to overcome. For example, one may seek to sort relatively
rare cells from a complex whole blood sample. Contamination by
other cell types may require the need for pre-treatment to enrich
for target cells, or post-treatment to remove unwanted non-target
cells.
[0004] Moreover, contaminating moieties may localize to container
sidewalls or other surfaces. Although one may minimize non-specific
binding with surface coatings, such as silicone-based products,
this may be insufficient. Where there is a horizontal fluid flow
plane, and the particles flow in suspension along that plane, they
may sink to the bottom (depending on density, viscosity, and other
characteristics). Particles may then form a barrier, clogging up
the flow path. Plus, contaminating particles may be co-localized in
(for example) microfluidic devices configured with a trapping
structure. Particularly where target particles are extremely rare,
non-specific binding can confound sorting or detection of the
target species.
[0005] There are ways to enhance particle sorting specificity. In a
microfluidic volume, hydrodynamic focusing may be used to transport
target moieties. Conventional approaches to hydrodynamic focusing
involve using two outer fluidic flows on each side of a central
sample flow to laterally constrain the sample flow. See generally,
P. Crosland-Taylor, "A device for counting small particles
suspended in fluid through a tube," Nature 171:37-38 (1953) doi:
10.1038/171037b0 for a seminal paper on the subject of flow
cytometry, and the use of fluidic sheaths.
[0006] In general, sheath flow is a particular type of laminar
flow. Although sheath flow may be configured as an outer flow
"tube" surrounding a fluid stream, or other fluidic path for total
or partial surrounding of a fluid stream, sheath flow herein also
includes a fluidic flow path in laminar flow with respect to an
adjacent, parallel fluid flow path. Thus, what is a laminar flow
plane on a solid surface is considered a sheath flow plane when on
a fluid "surface" (e.g., the adjacent, parallel fluid flow path).
Sheath flow implies substantially no turbulent flow, as undue
turbulence would result in intermixing of fluidic flow paths (and
the fluidic laminar flow plane layer would no longer function as a
"sheath"). As such, laminar flow, rather than turbulent flow, is
necessary to create sheath flow.
[0007] Depending on the architecture and fluid characteristics,
sheath flow may function to hydrodynamically focus a fluid sample
(by surrounding a sample flow path without intermixing), or, where
there is a layer (or sheath incompletely surrounding a fluid flow),
the fluid in a laminar flow plane may act as a fluidic extension of
a device wall--essentially acting as a fluidic barrier between a
fluidic sample and surrounding solid surfaces.
[0008] Sheath flow is particularly useful in a microfluidic
context, where particles in suspension may disrupt or block
microfluidic circuitry. Thus, hydrodynamic focusing or sheath flow
allows for faster sample flow velocity, and higher throughput.
[0009] Nevertheless, creating laminar sheath flow useful for
microfluidic devices (or larger devices) is problematic.
Conventional devices, such as conventional flow cytometers require
complex instrumentation with specialized components, and
fabrication is particularly detailed. Although there may be
particular geometries and architectures reportedly creating
microfluidic sheath flow, there exists a need for easy to
manufacture, predictable sheath flow devices useful at the macro-
and micro-fluidic scales, particularly for improved sensitivity in
target moiety sorting, and reducing non-specific binding to
surfaces.
SUMMARY
[0010] The present invention provides sheath flow based devices,
manufacturing and instrumentation systems, methods of use, methods
of manufacture, and related aspects.
[0011] The present invention stems from the observation that sheath
flow may be obtained downstream of a laminar flow established on a
solid surface. Where the solid surface is discontinued, the laminar
flow essentially continues as established, except, where configured
such that the flow plane abuts a fluid stream in an adjacent,
parallel fluid flow plane, it functions as "sheath flow." This
observation gave rise to a number of aspects and applications of
the present invention. Thus, sheath flow, advantageous in many
fluidics applications, is easily established without the need for
complex instrumentation.
[0012] In particular aspects, the present invention relates
processing biological fluids and particles, including, but not
limited to blood and fractions thereof, cells, nucleic acids and
proteins, and other analytes found in or relating to biological
functions. Other aspects will be apparent one of ordinary skill in
the art in view of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic illustration of a sheath flow device
where four component plates are used to establish a laminar buffer
flow above and below a sample laminar flow.
[0014] FIGS. 2A and 2B are illustrations presenting a
cross-sectional side view and a top view, respectively, of a device
configured for magnetophoretic particle separation.
[0015] FIG. 2C shows various structures of magnetic traps suitable
for sheath flow devices described herein.
[0016] FIGS. 3A and 3B are illustrations presenting a
cross-sectional side view and a top view, respectively, showing a
removable magnetic trapping station.
[0017] FIG. 4 is a reproduction of photographs illustrating the
non-specific binding of blood cells on a solid microfluidic channel
surface in a device without sheath flow (left panel) and then using
sheath flow (right panel).
[0018] FIGS. 5A and 5B are graphs illustrating rare cell purity and
rare cell recovery, respectively, from whole blood using sheath
flow devices as described herein.
[0019] FIG. 6A is an exploded perspective illustrating components
of a sheath flow device.
[0020] FIG. 6B is a photograph of the assembled sheath flow device
of FIG. 6A.
[0021] FIG. 6C is a perspective cross section illustration showing
details of the sheath flow device described in FIGS. 6A and 6B.
[0022] FIG. 7A is an exploded perspective illustrating components
of a sheath flow device.
[0023] FIG. 7B is a perspective of the assembled sheath flow device
depicted in FIG. 7A.
[0024] FIG. 8 is a process flow diagram of a method of sorting a
sample in accordance with various embodiments.
[0025] FIGS. 9A and 9B show purity and recovery of hematopoietic
cells isolated from cord blood using a sheath flow device as
described herein.
DETAILED DESCRIPTION
[0026] As noted above, the present invention stems from the
observation that once a laminar flow path is established on a solid
surface, it can continue as a sheath flow abutting an adjacent,
parallel laminar flow path plane. Thus, even without the solid
surface used to originally establish laminar flow, the fluid will
continue in the flow path and flow plane as established when
adjacent to one or more parallel fluid laminar flow planes. In some
embodiments, a laminar sample flow path is established adjacent to
one or two buffer flow paths. In this way, a sheath flow is
established, whereby the sample flow path is prevented from
contacting interior surfaces of a sheath flow device.
[0027] FIG. 1 depicts an exploded perspective of a sheath flow
device, 100, of the invention. Sheath flow device 100 includes four
parallel plates, 105, 110, 115 and 120, that, when adjoined, form
sheath flow device 100. A sample fluid is introduced via an access
port in plate 105, in the "z" axis, as indicated by dashed arrow
125. The fluid sample strikes, for example, a milled surface on
plate 115 and is deflected and, by virtue of the chambering, the
sample fluid is directed along the solid surface thereby
establishing a laminar flow path. The solid surface is discontinued
(e.g. as the fluid passes the end of the solid surface or otherwise
discontinues contact with the solid surface) in the laminar flow
path plane, and the fluid continues as a laminar flow. Buffer fluid
is introduced via inlets in plates 120 and 105, as indicated by
dashed arrows 130 and 135. Each of the buffer streams strikes a
similar surface, on plates 115 and 110, respectively, an also
establish laminar flows adjacent to the sample laminar flow. These
laminar flows continue as separate layers of laminar flow, thereby
creating a sheath flow. That is, the sample laminar flow is
sheathed by the buffer laminar flows, for example, preventing the
sample flow from touching certain interior surfaces of the sheath
flow device.
[0028] Thus, the present invention, in one aspect, provides a
sheath flow device including: (a) a first laminar flow establishing
solid surface upstream of a sheath flow plane area; (b) a sample
laminar flow area in parallel with the sheath flow plane area; and
optionally, (c) a second laminar flow establishing solid surface
upstream of a second sheath flow plane area.
[0029] As with the configuration illustrated in FIG. 1, one may
establish sheath flow using a device of the present invention
configured to establish one or more parallel laminar flow planes on
corresponding solid surfaces upstream of corresponding sheath flow
planes. Embodiments described herein include sheath flow devices
with magnetic separation stations, which deflect and optionally
trap magnetically labeled sample components. Exemplary magnetic
trapping stations are described in, e.g., U.S. Patent Publication
20090053799A1, "Trapping Magnetic Sorting System for Target
Species," herein incorporated by reference for all purposes. In
certain embodiments, the sheath flow devices employ non-magnetic
forces to drive separation. Examples of these forces include
acoustic forces, optical forces, and dielectric forces.
[0030] FIGS. 2A and 2B are illustrations presenting a
cross-sectional side view and a top view, respectively, of a sheath
flow device, 200, of the present invention configured for
magnetophoretic particle separation. Device 200 includes a top
plate, 205, and a bottom plate, 210. A complex sample containing a
target moiety (e.g., cells 220 as illustrated) is in a laminar flow
layer between a top fluidic layer and a bottom fluidic layer. A
structure for particle selection, here, a nickel ferromagnetic
structure, 230, is located on the inside surface of top plate 205,
such that the sample in laminar flow is prevented from direct
contact by the top buffer sheath flow. In one embodiment, the
nickel structure is isolated, for example coated with a material,
so that the collected targets species, such as live cells, do not
directly contact the (potentially toxic) nickel. External
controllable force (here, magnetic) is applied to
magnetophoretically sort selective particles (as illustrated, cells
selectively labeled with magnetic particles 215, and thereby be
responsive to magnetic selection). FIG. 2B illustrates a top-down
view, showing that waste is not deflected or trapped by the
magnetophoretic structure, and passes in the sample fluid flow.
Further aspects of this embodiment are described below.
[0031] FIG. 2C shows nine variations of magnetic trapping grids or
patterns that can be used in sheath flow devices described herein.
These serve as a magnetic field gradient generating (MFG)
structures. In one embodiment, the magnetic trapping grids or
patterns are made of magnetic materials. In another embodiment, the
magnetic trapping grids or patterns are formed, for example
micromachined or screen printed, from materials which are used in
conjunction with external magnets that induce a highly localized
strong magnetic field in the magnetic trapping grid or pattern in
order to pull the magnetic-particle-labeled target species from the
sample flow, through the buffer sheath flow and onto the magnetic
trapping grid or pattern.
[0032] In general, devices and related methods and systems of the
present invention establish discrete fluidic laminar flow layers
that are substantially free or essentially free of turbulent flow,
such that the fluidic layers remain discrete. As used herein with
reference to the laminar sheath flow, the term "substantially free
of turbulent flow" with reference to laminar sheath flow denotes
that there may be some turbulent flow, although the sheath flow
result is still achieved. The term "essentially free" as used in
this context denotes that there may be some unavoidable turbulent
flow, but, as indicated above, the desired laminar sheath flow
layers are achieved.
[0033] Sheath flow, in some aspects, provides a fluidic barrier
between a sample fluid (from which one seeks a separation or
detection of a target moiety) and a solid surface, thereby
functioning as an isolation barrier to solid surfaces. For example,
sheath flow is used to reduce non-specific binding of any
non-target moieties to the solid surfaces. The present invention
thus provides means to prevent unwanted interaction of a sample
fluid's components with solid surfaces of the device. Desirable
interactions, for example selective collection of fluid components,
is achieved with undesirable interactions between the sample fluid
and solid surfaces. For example, devices of the invention can be
used to prevent undue non-specific binding of non-target moieties
within a sample fluid laminar flow plane, by providing a sheath
flow barrier substantially preventing sample fluid contact with
solid surfaces--like sidewalls, or top or bottom solid surfaces,
trapping stations (as described above) and the like.
[0034] Additionally, sheath flow may be employed to "pinch" or
otherwise laterally narrow the flow of sample to thereby provide a
narrow flow of sample that can be used for purposes such as
counting cells in the sample. In certain embodiments, the laminar
sheath flow bounds a laminar sample flow having a width comparable
in dimension to that of the cells or other target species being
detected. Other aspects will be apparent to one of ordinary skill
in the art in view of the present disclosure.
[0035] Terminology:
[0036] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by one of ordinary skill in
the art.
[0037] General terminology: In this application, the use of the
singular includes the plural unless specifically stated otherwise.
In this application, the word "a" or "an" means "at least one"
unless specifically stated otherwise. The phrase "at least one of"
followed by a list means any one or more members of the list and
does not mean that all listed members must be present. The use of
"or" means "and/or" unless specifically stated otherwise. In the
context of a multiple dependent claim, the use of "or" refers back
to more than one preceding independent or dependent claim in the
alternative only. Furthermore, the use of the term "comprising," as
well as other forms, such as "comprises" and "comprised," is not
limiting. Also, terms such as "element" or "component" encompass
both elements and components including one unit and elements or
components that include more than one unit unless specifically
stated otherwise. Where a "skilled practitioner" is referenced,
this refers to an ordinary skilled practitioner in the art to which
the subject matter pertains, in context, unless otherwise
noted.
[0038] Fluid mechanics/fluid dynamics terminology: The various
terms describing fluid mechanics (including microfluidics), are
used in their conventional technical meanings
[0039] The term "fluid" and the term "liquid" are used synonymously
herein to refer to substances that flow and optionally take the
shape of a container. Under some circumstances, there may be
gaseous or solid substances that flow. For example, finely granular
materials may flow.
[0040] The term, "fluidic circuit" refers to a configuration of
fluidically interconnected functional areas located in the present
sheath flow devices. As described in more detail herein, functional
areas include reservoirs or compartments and channels through which
fluids may flow. The channels may be optional, for example, where
two compartments are directly fluidically connected as by an
adjoining wall. Two reservoirs or chambers may be reversibly
fluidically connected, such as via adjoining wall that may be
sealed and opened, or may be porous, allowing only certain size
particles to flow through. A skilled practitioner will appreciate
the numerous configurations possible for the present fluidic
circuitry. Apart from configuration, there are similarly wide
varieties of choices to integrate fluidic circuits, such as the
flow control structural elements described herein.
[0041] Particle sorting terminology: The term "moiety" as used from
time to time herein denotes a "portion" and includes reference to a
particle. A "particle" refers to a small object that behaves as a
whole unit in terms of its transport and properties. The term
"analyte" can be a "moiety" or a "particle" and is used in its
ordinary meaning as a substance the presence of which is detected,
or a characteristic of which is measured, in an analytical
procedure.
[0042] Biological and biochemical terminology: Where specific
categories of molecules are discussed, such as nucleic acids or
proteins, synthetic forms are included, such as mimetic or isomeric
forms of naturally occurring molecules. Unless otherwise indicated,
modified versions are similarly encompassed, so long as the desired
functional property is maintained. For example, an aptamer
selective for a CD34 cell surface protein includes chemical
derivatives (e.g., pegylated, creation of a pro-form, derivatized
with additional active moieties, such as enzymes, ribozymes,
etc.)
[0043] The term "biological fluid" denotes the source of the fluid,
and includes (but is not limited to) amniotic fluid, aqueous humor,
blood and blood plasma (and herein blood refers to the plasma
component, unless otherwise expressly stated or indicated in
context), cerumen (ear wax), Cowper's fluid, chime, interstitial
fluid, lymph fluids, mammalian milk, mucus, pleural fluid, pus,
saliva, sebum, semen, serum, sweat tears, urine, vaginal secretion,
vomit and exudates (from wounds or lesions).
[0044] The term "selective binding molecule" denotes a molecule
that selectively, but not necessarily specifically, binds to a
particular target moiety. The binding is not random. Selective
binding molecules may be selected from among various antibodies or
permutations (poly- or monoclonal, peptibodies, humanized,
foreshortened, mimetics, and others available in the art), aptamers
(which may be DNA, RNA, or various protein forms, and may be
further modified with additional functional moieties, such as
enzymatic or colorimetric moieties), or may be particular to a
particular biological system. Proteins may be expressed with
particular "tags" such as a "His-tag", and a skilled practitioner
will determine appropriate kinds of selective binding molecules or
detectable labels are suitable. The list is not exhaustive.
[0045] It should be understood that embodiments of the invention
are not limited to biological or even organic samples, but extend
to non-biological and inorganic materials. Thus, the apparatus and
methods described herein can be used to screen, analyze, modify, or
otherwise process a wide range of biological and non-biological
substances in liquids. The target and/or non-target species may
include small or large chemical entities of natural or synthetic
origin such as chemical compounds, supermolecular assemblies,
proteins, organelles, fragments, glasses, ceramics, etc. In certain
embodiments, they are monomers, oligomers, and/or polymers having
any degree of branching. They may be expressed on a cell or virus
or they may be independent entities. They may also be complete
cells or viruses themselves.
[0046] General Considerations:
[0047] General considerations for making and using the present
invention include the overall device configuration, materials,
manufacturing systems, instrumentation systems, and applications.
Particular embodiments including working examples are also
presented. Prophetic examples are also included below.
[0048] In making or using the present invention, one will generally
consider fluid dynamics principles including: compressible versus
incompressible flow, viscous versus inviscid flow, steady versus
unsteady flow, laminar versus turbulent flow, Newtonian versus
non-Newtonian fluids, subsonic versus transonic, supersonic and
hypersonic flows, non-relativistic versus relativistic flows,
magnetohydrodynamics (or other considerations of particle sorting).
The list is incomplete, but a practitioner will recognize that
fluid, particle, device and sorting methods must all integrate.
[0049] Thus, among other things, the present invention may be used
for selecting rare cells, such as circulating tumor cells, from a
blood sample, while minimizing non-selective binding that could
confound results. For particles of cellular dimensions, physical
constraints imply that the hydrodynamically focused stream requires
a velocity of several meters per second. At this speed a typical
cell would traverse its own diameter in a few microseconds.
[0050] The present devices may be configured for additional
functions. For instance, one may seek to first isolate cells, and
then culture the cells in situ; thus the present devices may be
additionally configured for use as a bioreactor. Moreover, one may
first culture cells, or use pre-grown cells, to obtain protein (or
other target moiety) on the present sheath flow device. The target
protein (or moiety) may then be detectably labeled and suspended in
fluid, to be sorted in the present sheath flow device. The present
invention may be used for sorting or screening libraries of
chemicals, i.e., by selecting for protein to which an aptamer from
an aptamer library.
[0051] The present sheath flow device may be used in any way that
current fluid handling devices are used. General considerations
include the desired manufacturing method systems, the desired use,
and the desired related instrumentation (if any). One will consider
device geometries in conjunction with sample size and application
requirements, means for controlling fluid flow direction, path, and
rate; means for selecting or sorting particulate matter (if
desired), as well as adaptation with instrumentation required.
[0052] The present sheath flow devices, related methods and
systems, have industrial application in both macro-fluidic as well
as microfluidic samples, and, as more fully described below, may be
configured and adapted for a wide variety of purposes. Microfluidic
devices are available for such purposes as on--protein
purification, rare cell separation, and screening for rare
molecules (such as proteins or aptamers) in a sample. These may be
configured with the present laminar flow establishing surface, to
establish sheath flow in fluidic layers as described herein. See,
e.g., J. Qian, X. Lou, Y. Zhang, Y. Xiao, H. T. Soh, "Rapid
Generation of Highly Specific Aptamers via Micromagnetic Selection"
Analytical Chemistry (2009); U. Kim and H. T. Soh, "Simultaneous
Sorting of Multiple Bacterial Targets Using Integrated
Dielectrophoretic-Magnetic Activated Cell Sorter" Lab on a Chip
(2009); Y. Liu, J. D. Adams, K. Turner, F. V. Cochran, S. Gambhir,
and H. T. Soh, Controlling the Selection Stringency of Phage
Display Using a Microfluidic. Lab on a Chip (2009); X. Lou, J.
Qian, Y. Xiao, L. Viel, A. E. Gerdon, E. T. Lagally, P. Atzberger,
T. M. Tarasow, A. J. Heeger, and H. T. Soh, "Micromagnetic
Selection of Aptamers in Microfluidic Channels," Proceedings of the
National Academy of Sciences, USA, 106 (9) 2989-2994 (2009), all of
which are herein incorporated by reference.
[0053] General Configuration of Sheath Flow Devices:
[0054] The present sheath flow devices will be configured in
accordance with the principle of establishing a laminar flow on a
solid surface that continues as sheath flow once the fluid layer
comes in contact with another fluid layer (such as upon
discontinuation of contact with the solid surface, as described
herein). The present sheath flow devices can have any number of
configurations and architectures so long as a solid surface is
available to establish a laminar flow upstream of fluidic contact
with an adjacent fluid in a parallel laminar flow path plane.
[0055] Generally, the present invention provides devices configured
for sheath flow acting as a barrier between a fluid sample flow
path and a device surface. This barrier inhibits or prevents
interaction of the sample fluid with device surfaces. In one
example, this barrier substantially prevents non-specific binding
of moieties in the subject fluid sample to the solid surface. By
substantially preventing non-specific binding, deleterious effects,
such as disruption of path flow by accumulation of moieties, or
contamination of selected rare moieties, can be avoided.
[0056] Spatially, where a sheath flow device is positioned
horizontally (with respect to a horizon), the flow paths are
vertically aligned (e.g., layered one atop the other) with respect
to each other. This is illustrated in FIGS. 1 and 6A-C, where the
top sheath and the bottom sheath flows are parallel with and
adjacent to either side of the sample flow plane. The top sheath is
on "top", the bottom sheath is on the "bottom" and therefore
stacked in a vertical orientation, although the flow plane is
horizontal.
[0057] One may similarly configure the present devices in a
vertical orientation (perpendicular to a horizon). With respect to
a vertical orientation, the flow paths are layered horizontally,
(e.g., left to right with respect to each other).
[0058] Depending on the fluid circuitry, the flow paths may or may
not be in a flat plane. Where the flow paths are configured in the
shape of a semi-circular channel, circular tube, or similar
channeled configuration, the flow paths will be substantially
parallel (non-intersecting, in accordance with Euclidian geometry
for a curved planar three dimensional shape).
[0059] In some circumstances, one may have a substantially
stationary fluid "pool" acting as a fluid sheath. Although strictly
speaking a pool of fluid is not flowing, particularly viscous
liquids (e.g., substances taking the shape of a container in which
they are in) may display fluid movement characteristics but
extremely slowly. As such, one may configure a device of the
present invention to have such a "pool" area, such as a bottom
fluid layer (see below) upon which a less viscous fluid sample
flows.
[0060] Size: The present device can be scaled to any size, with
particular consideration of fluid mechanic/dynamics, and fluid
characteristics. In one embodiment, the devices of the invention
are scaled for microfluidic circuitry. In other embodiments,
devices of the invention are configured for liter or multi-liter
scale use.
[0061] With respect to the laminar flow establishing area,
considerations include the surface area necessary to establish a
laminar flow in view of the fluid characteristics.
[0062] As one of ordinary skill in the art would appreciate, fluid
characteristics include viscosity, particulate matter
characteristics (such as hydrodynamics), particulate matter
concentration, and the degree to which the fluid is miscible (or
otherwise reacts with) fluid in an adjoining layer, for
example.
[0063] With regard to size, the amount of pressure for a desired
amount/rate of flow may be calculable based on fluid dynamics
considerations, including: compressible versus incompressible flow,
viscous versus inviscid flow, steady versus unsteady flow, laminar
versus turbulent flow, Newtonian versus non-Newtonian fluids,
subsonic versus transonic, supersonic and hypersonic flows,
non-relativistic versus relativistic flows, magnetohydrodynamics,
and other approximations according to methods known in the art. One
of ordinary skill in the art would consider fluid dynamics in view
of the overall system, including the device materials.
[0064] The sheath flow device will have a direction over which a
separation force acts to deflect target species from a sample flow
plane into a sheath flow plane. Generally, this direction will be
perpendicular to the direction of flow and the sheath and sample
flows will form parallel adjacent layers, sitting on top of one
another along this direction. The device dimensions in this
direction (the device's "height") will be relatively small, often
the smallest in the device, and on the order of the distance over
which the separation force acts. As these forces are often short
range forces, the device dimension in this direction often will be
on the order of one millimeter or less. In contrast, the transverse
device dimension, the dimension in a direction perpendicular to
both the direction of flow and the direction of the separation
force, can be quite large. This allows the devices to scale to
relatively large sizes and allow corresponding increases in
throughput, in some cases on the order of hundreds of milliliters
per hour in a microfluidic format. In some cases, this transverse
device dimension is at least about 10 centimeters or even at least
about 20 centimeters. In a specific embodiment, the device's height
is about 700 micrometers and its transverse dimension is about 20
centimeters. The dimensions presented here are well suited for use
with magnetophoretic separation mechanisms including any of those
described elsewhere herein.
[0065] Currently, the working examples relate to microfluidic sizes
(generally having fluidic channel diameters of 1 mm or less, see
the Examples, below). Apart from limitations on practicable
application, the lower limit of size is constrained predominantly
by manufacturing methods. For example, if one desires
nano-electronic components, one may have such components integrated
using micro-lithographic techniques. For microfluidic applications,
one may seek to proportions limiting turbulence in fluid flow and
optimizing laminar flow in a desired path. The present sheath flow
devices may be configured for use with sample volumes of 1-10
.mu.L, 10-100 .mu.L, 100-1000 .mu.L, 1000 .mu.L-100 ml, for
example.
[0066] The present sheath flow devices may be scalable to virtually
any size, again with practical considerations such as fluid
mechanic and fluid dynamics principles and the desired application.
The general principle of establishing a laminar flow upstream of a
sheath flow is broadly applicable to devices larger than those for
microfluidic applications, and one of ordinary skill in the art
will recognize the general fluid dynamic principles allowing
scale-up. For example, in some embodiments the present devices can
be configured for use with larger sample volumes from between about
100 ml to 1 liter, and in some cases multi-liter scale.
[0067] Number of and characteristics of fluid layers: The term
"fluid layer" is used herein to denote a discrete fluidic flow path
plane, substantially free of turbulent flow. The "fluid layer" may
be a laminar flow on a solid surface, or, a sheath flow with
respect to an adjacent, abutting, fluid layer in a parallel
plane.
[0068] While not in a two-dimensional planar orientation, the
present invention also applies to devices having a total sheath
surrounding sample fluid. A combination of these laminar flow
establishing surfaces may be used to create fluid layers
surrounding a sample, thereby permitting 3D hydrodynamic focusing
to be readily achieved. In this way, a sample flow is isolated from
a channel wall (in device fluidic circuitry) both horizontally and
vertically.
[0069] Number of Fluid Layers: In a general aspect, the present
invention provides devices (and related methods and systems)
including at least three discrete, serially adjoining, fluid
laminar flow layers. The laminar flow paths are positioned adjacent
to each other, and parallel with each other, in series. Each
laminar flow path is parallel with respect to the others, and
layered perpendicularly with respect to their flow paths.
[0070] Three layers may be used (as in the working example below),
such that a sample laminar flow plane is sandwiched between an
upper and lower layer (for example). One may add additional fluid
layers, although the extent to which a laminar (sheath) flow plane
is continued in the absence of solid support will largely depend on
fluid mechanic considerations. One may consider the characteristics
of the fluidic circuitry, such as the geometric configuration,
channel wall composition and frictional force. Other considerations
relate to the flow velocity and force, the flow plane dimensions,
the fluidic volume in each flow plane layer, and other fluid
mechanic considerations appreciated by a skilled practitioner.
[0071] Fluid Layer Thickness: Related to the number of fluid
layers, the thickness (or depth) of each fluid layer is important.
A practitioner will appreciate that device configuration and
application, as well as fluid characteristics will determine fluid
layer thickness.
[0072] For example, where a sheath flow device is configured for
magnetophoretic, the magnetic force should be greater than the
resistance provided by a fluid layer providing sheath flow. That
is, the magnetic force applied to separate magnetically labeled
target particles from solution should be sufficient to pull the
particles to the desired region (say, a trapping station, see
above), through the fluid layer.
[0073] Thus, for example, where one seeks a magnetophoretic device
having a trapping station on an upper surface, such that the
magnetic force is applied to the top of the device in a horizontal
plane, the present sheath flow device may include a relatively
viscous (with respect to sample viscosity), slow moving (or
stationary) first (e.g., bottom) fluid layer, a relatively
non-viscous (with respect to the bottom layer) sample layer, and
top layer essentially free of viscosity. In that way, the sheath
flow of the top layer substantially prevents non specific binding
in the magnetic trapping station (located on the top surface), yet
doesn't prevent magnetically labeled target particles from
traveling through the layer to be trapped.
[0074] Structural Elements:
[0075] In general, the present sheath flow devices function to
provide fluidic movement to effectuate a particular application,
beyond simple fluid containment or storage.
[0076] Structural features provide for this fluid circuitry. The
present sheath flow devices include one or more access ports, one
or more reservoirs, and one or more channels providing fluidic
communication between or among access ports and reservoirs. Other
structural components are also described.
[0077] Access ports: In general, depending on the overall
configuration and materials, the present sheath flow devices will
have at least one access port where materials are admitted or
exited, and at least one reservoir for containing a fluid. Access
ports, used for fluid (or other material) entry or exit, may be
configured to operate with other apparatus or instrumentation as
part of an overall system.
[0078] Access ports may connect with external environment, such as
by providing a way for fluid fill or fluid exit. For fluid fill,
the access port may be configured for fill via syringe, pipette, or
by automated filling instrumentation. This is illustrated in FIG.
6B, where an access port is fitted with an adapter suitable for a
syringe. Fluid exit may be, for example, waste disposal. Or, exit
may be part of a positive selection scheme, whereby particulate
matter in a suspension is selectively captured. Various types of
external interconnects for access ports may be used, such as tubing
studs, hose barb connections, O-ring connections, or other external
types of interconnections.
[0079] Access ports, rather than being an external interconnection,
may alternatively be in fluid communication with another portion of
the device, such as a separately enclosed reservoir. Functionally,
the same purpose is served, e.g., fluid fill or fluid partitioning
(exit).
[0080] Devices of the present invention can have one or more than
one access port for a variety of functions, such as for introducing
a number of different fluids or for exiting separate moieties, and
each may optionally be connected with the external environment or
with another portion of the device.
[0081] Reservoirs: The present device also includes at least one
reservoir for containing a fluid, and performing any functions on
the fluid. Reservoirs may serve a functional purpose, and include
additional structural elements to effect such purpose. For example,
where cultured cells are desired, one may have suitable cell
culture apparatus integrated with the reservoir, such as (but not
limited to) aeration devices, mixers, or temperature controls.
These devices may form a part of the present sheath flow device, or
may be part of related processing instrumentation where their
device integration is temporary (when the sheath flow is operably
connected to the instrumentation). Reservoirs may be adapted to
operate within a system for fulfilling a function.
[0082] Device Functional Components: One may configure or adapt the
present devices for filling and measuring fixed volumes, or for
continuous flow. The present device may be configured for
multiplexed functions (such as cell lysis and protein isolation).
The present device may be configured for a single or multi-step
process or assay, and may be configured for reagent storage. The
present device may include filtration configurations or
adaptations.
[0083] For example, the present sheath flow device may further
include a structure for trapping target moieties, as a form of
particle sorting, for example. For example, as explained below, one
may include a ferromagnetic grid for magnetophoretic particle
sorting, to act as a "trapping station" for magnetically bound
target species. Separating particles from a suspension may be
particularly advantageously performed in the present sheath flow
device. For example, where magnetophoretic separation of a
particular moiety from a complex mix is desired, one may have
suitable magnetophoretic trapping structures in place, such as a
ferromagnetic structures as described in U.S. Patent Publication
20090053799A1 cited above. This is illustrated in FIG. 6B, where a
ferromagnetic trapping station is included on an upper layer.
[0084] A variety of particle sorting/detecting/analytical
functionalities may be included, as indicated in this
specification, passim.
[0085] Examples of operational modules that may be integrated with
magnetic trapping sorters in sheath flow fluidics devices include
(a) additional enrichment modules such as fluorescence activated
cell sorters and washing modules, (b) reaction modules such as
sample amplification (e.g., PCR) modules, restriction enzyme
reaction modules, nucleic acid sequencing modules, target labeling
modules, chromatin immunoprecipitation modules, crosslinking
modules, and even cell culture modules, (c) detection modules such
as microarrays of nucleic acids, antibodies or other highly
specific binding agents, and fluorescent molecular recognition
modules, and (d) lysis modules for lysing cells, disrupting viral
protein coats, or otherwise releasing components of small living
systems. Each of these modules may be provided before or after the
magnetic sorter. There may be multiple identical or different types
of operational modules integrated with a magnetic sorter in a
single fluidics system. Further, one or more magnetic sorters may
be arranged in parallel or series with respect to various other
operational modules. Some of these operational modules may be
designed or configured as traps in which target species in a sample
are held stationary or generally constrained in particular
volume.
[0086] As should be apparent from the above examples of modules,
operations that may be performed on target and/or non-target
species in modules of integrated fluidics devices include sorting,
coupling to magnetic particles (sometimes referred to herein as
"labeling"), binding, washing, trapping, amplifying, removing
unwanted species, precipitating, cleaving, diluting, ligating,
sequencing, synthesis, labeling (e.g., staining cells),
cross-linking, culturing, detecting, imaging, quantifying, lysing,
etc.
[0087] Specific examples of biochemical operations that may be
performed in the magnetic sorting modules of integrated fluidic
devices include synthesis, purification, and/or screening of
plasmids, aptamers, proteins, and peptides; evaluating enzyme
activity; and derivatizing proteins and carbohydrates. A broad
spectrum of biochemical and electrophysiological assays may also be
performed, including: (1) genomic analysis (sequencing,
hybridization), PCR and/or other detection and amplification
schemes for DNA, and RNA oligomers; (2) gene expression; (3)
enzymatic activity assays; (4) receptor binding assays; and (5)
ELISA assays. The foregoing assays may be performed in a variety of
formats, such as: homogeneous, bead-based, and surface bound
formats. Furthermore, devices as described herein may be utilized
to perform continuous production of biomolecules using specified
enzymes or catalysts, and production and delivery of biomolecules
or molecules active in biological systems such as therapeutic
agents. Fluidic devices as described herein may also be used to
perform combinatorial syntheses of peptides, proteins, and DNA and
RNA oligomers.
[0088] Integration and Fluidic Circuitry: Integration of functional
elements may be accomplished any number of ways. In general, one
may fluidically connect access ports and reservoirs in all
combinations via flow channels. Flow channels may be adapted for
the kind of flow so desired, and may be of any dimensions that
permit desired fluidic movement.
[0089] Flow control structural elements may be selected from a wide
variety. These can be valves, porous membranes, mixers, pumps, and
other traditional flow control structural elements.
Non-conventional flow controls may be used, such as adaptation of
material that solidifies in situ to block flow.
[0090] One should consider the laminar flow establishing surface in
conjunction with integration of fluidic circuitry. For example, if
there is a laminar flow established upstream of a sheath flow, the
sheath flow should be non-interfering in a backward-incompatible
(upstream) way.
[0091] All or part of the present device may be biodegradable, such
as using polymeric material that degrades to non-toxic constituent
moieties in the presence of heat, sunlight, water, etc.
[0092] Inventory considerations further include product
identification, such as bar coding, RFID, or other means to
identify devices. The present devices may be further packaged with
related reagents. For example, for use with biological reagents,
one package the present device with suitable buffers, media,
detectable labeling moieties, apparatus (such as syringes for fluid
fill), or other items.
[0093] Wash or Carrier Fluids: The present devices may be
configured or adapted for use with, for example, aqueous buffers
(with or without detergents), alcohols (methanol, ethanol,
isopropanol for example), organic solvents (hexane, fluorocarbons,
aromatic for example), or a combination of any of the above. These
fluids may, in certain embodiments, serve as the sheath flow
fluid.
[0094] Electrochemical/electro-active: The present devices may
include one or more printed circuit boards, interdigitated
electrodes, sputter or screen printed electrodes, or capacitance
arrays. For example, one may pre-prepare circuit boards on
polymeric material, and use that to manufacture the present sheath
flow devices. These elements may be controlled by associated logic
which directs operation of trapping stations and/or various pre-
and post-processing modules or stations.
[0095] Operability with forces used for particle trapping or
sorting: One of the most promising applications for the present
device is particle sorting, and there are number of ways this can
be done. A controllable force, such as magnetic, acoustic,
electrophoretic, or optical is used to move a responsive particle
suspended in a fluid.
[0096] One may, for example, contemplate use of magnetic activated
particle sorting (such as cell sorting). Practicably, this involves
using magnetic beads to which a selective binding molecule is
attached. When the selective binding molecule binds to the desired
target, the magnet is thus so attached. The desired target can then
be trapped or sorted using magnetic force, and optionally a
ferromagnetic trapping station. Thus, one may embed ferromagnetic
material in the sheath flow device material, such as a printed
magnetic area or by incorporating ferromagnetic dust particles into
a manufacturing material (for application in a particular area, for
example). One may similarly use other controllable forces as are
available in the art such as acoustic, electrophoretic or other
forces. A skilled practitioner will appreciate the appropriate
device configuration to accommodate the present system.
[0097] Optical Detection: The present devices may be configured to
permit optical detection. This has practical applicability, for
example, if colorimetric, fluorescent, or luminescent detectable
markers are desired. Other optical interfaces may include fiber
optic, surface plasmon resonance, attenuated total reflection or
other optic interfaces.
[0098] Other Features/Special requirements: The present devices may
be sterilized (such as with ethylene oxide, considering the
durability of the selected material to other sterilization
techniques, such as autoclavability). There may be surface
compatibility with cell culture requirements, and additional
surface energy in materials or configurations so selected. The
present device may be, for example, gas permeable. One may seek
internal coatings, such as silicon, to minimize non-specific
binding to a surface.
[0099] Magnetophoretic and Magnetic Trapping Embodiments: With the
above principles in mind, various designs including both sheath
flow and magnetic sorting may be envisaged. In certain embodiments
magnetic sorting involves a trapping mode where the non-target and
target species are sequentially eluted after the application of the
external magnetic field. In other words, the species attached to
magnetic particles are held in place while the unattached species
are eluted. Then, after this first elution step is completed, the
species that are attached magnetic field and were prevented from
being eluted are freed in some manner such that they can be eluted
and recovered.
[0100] In accordance with some embodiments, a trapping module of a
MACS system includes a channel through which a sample, including
species attached and not attached to magnetic particles flow. One
side of the channel includes a magnetic field gradient generating
structure that generates a magnetic field gradient with the
application of a magnetic field from an external source. This
magnetic field gradient attracts and captures magnetic particles
along with the attached species. After the sample flows through the
trapping module, the captured particles may be released by changing
the applied magnetic field or by cleaving the link between the
magnetic particles and the attached species.
[0101] For example in certain embodiments, a fluidic separating
device may be characterized by the following features: (a) at least
one sample inlet or access channel configured to provide a sample
stream in the fluidic separating device; (b) at least one sheath
flow inlet channel configured to provide one or more fluid streams
that form a sheath around the sample stream within the fluidic
separating device, and thereby reduce nonspecific binding of
components of the sample to the device; (c) a sorting station
fluidly coupled to the sample and sheath flow inlets and located
along a path of the sample stream; and (d) a magnetic field
gradient generator for interacting with an external magnetic field
to produce a change in magnetic field gradient in the sorting
station and thereby deflecting and/or trapping magnetic particles
from the sample stream.
[0102] In some cases, the sorting station has a substantially
rectangular interior space for bounding fluid flowing through the
sorting station. In such cases, the interior space has first and
second lateral dimensions transverse to (e.g., perpendicular to)
the sample stream's direction of flow. In various embodiments, the
second lateral dimension (which is often a width on the horizontal
plane) is at least about 2 times larger than the first lateral
dimension (which is often a height in the vertical direction). The
device may be configured such that the at least one sheath flow
inlet channel is configured to provide two sheath streams separated
from one another by the sample stream along the first lateral
dimension.
[0103] Further, in some embodiments employing an interior
rectangular space, the interior space is defined, in part, by two
substantially parallel and substantially planar surfaces separated
by a distance not greater than about 2 millimeters, and in other
cases not greater than about 1 millimeter. In such embodiments, the
at least one sheath flow inlet channel is configured to provide two
sheath streams flowing in contact with the two parallel planar
surfaces and separated from one another by the sample stream. A
first sheath flow inlet channel may include a substantially planar
surface located upstream of the sorting station and oriented
substantially parallel to the two substantially planar surfaces of
the interior space. Additionally, the device may include a second
sheath flow inlet channel, having its own substantially planar
surface located upstream of the sorting station and oriented
substantially parallel to the two substantially planar surfaces of
the interior space. The sample inlet channel may have its own
substantially planar surfaces parallel to the two substantially
parallel and substantially planar surfaces of the sorting station
interior space. These surfaces may be provided on planar structures
that also provide planar surfaces employed in the sheath flow inlet
channels. Exemplary structures are depicted in FIGS. 1, 2 and
6A-6C.
[0104] The magnetic field gradient generator of the fluidic
separating device may include a plurality of ferromagnetic elements
patterned on the sorting device proximate the sorting station, and
may have a permanent magnet proximate the plurality of
ferromagnetic elements. The plurality of ferromagnetic elements may
be disposed within a fluid pathway of the sorting station to allow
fluid contact between the ferromagnetic elements and the sample
stream. It may be desirable to provide a protective or passivating
layer over the ferromagnetic elements in order to prevent the
elements from coming in direct contact with the flowing sheath and
sample. Such layer is useful to prevent, e.g., poisoning or
contamination of sample components such as cells. Examples suitable
protective layers include thin (e.g., 10 nm to 1 micrometer) layers
of silicon oxide, silicon nitride, or other suitably inert barrier
material. In certain embodiments, the ferromagnetic elements
include nickel elements such as micropatterned elements. Examples
are set forth in FIGS. 8A-8H. As shown, the ferromagnetic
structures are provided in an organized or a random pattern, such
as parallel lines, an orthogonal grid, and rectangular arrays of
regular or irregular geometric shapes. The structures may be
regular or reticulated as shown. In a specific example, nickel
ferromagnetic elements are coated with a thin silica passivating
layer. In various embodiments, the magnetic field gradient
generator is configured to temporarily capture the magnetic
particles and then release the magnetic particles.
[0105] As described above in relation to FIGS. 2A and 2B,
ferromagnetic structures are formed on the inside surface of a
lower wall of a flow channel. These serve as a magnetic field
gradient generating (MFG) structures. An external magnetic field is
typically used as the driving force for trapping the magnetic
particles flowing through the fluid medium. The MFG structures may
shape the external magnetic field in order to create locally high
magnetic field gradients to assist capturing flowing magnetic
particles. In the depicted embodiment of FIG. 2A, the external
magnetic field is provided by an array of permanent magnets of
alternating polarity. More generally, the external magnetic field
may be produced by one or more permanent magnets and/or
electromagnets. In some embodiments, a collection of magnets such
as those shown in FIG. 2A are moveable, individually or as a unit,
in order to dynamically vary the magnetic field applied to the
trapping region.
[0106] In certain embodiments, the magnetic field is controlled
using an electromagnet. In other embodiments, permanent magnets may
be used, which are mechanically movable into and out of proximity
with the sorting station such that the magnetic field gradient in
the sorting region can be locally increased and decreased to
facilitate sequential capture and release of the magnetic
particles. In some cases using an electromagnet, the magnetic field
is controlled so that a strong field gradient is produced early in
the process (during capture of the magnetic particles) and then
reduced or removed later in the process (during release of the
particles).
[0107] In one embodiment, the trapping region of the device is
removable. In another embodiment, the removable trapping region is
disposable. In a specific embodiment, where the plate incorporating
the trapping region, e.g. a nickel grid, is disposable, the
trapping region is defined by an outermost perimeter that is scored
such that after the trapping operation and washing to remove any
waste, the trapping region can be "snapped" out of the plate
incorporating the trapping region. This is depicted in FIGS. 3A and
3B, a side view and top view of a plate, 300, incorporating a
nickel grid trapping region, 310, and scores, 315, in the plate
such that, for example, a suction cup device can be used to pull
the removable trapping region from the plate by applying force
pulling force sufficient to break along the scores in the plate.
The removable trapping region can be removed with or without the
magnetic field applied, depending on the need. Once removed, target
cells can be analyzed or manipulated on the grid or removed and
analyzed or manipulated off the grid.
[0108] As shown in the example of FIG. 2A, the magnetic particles
are coated with one or more molecular recognition elements (e.g.,
antibodies) specific for a marker of a target cell or other species
to be captured. Thus, one or more magnetic particles, along with a
bound cell or other target species, flow as a combined unit into
the trapping module. For large target species having many exposed
binding moieties (e.g., mammalian cells), it will be common to have
multiple magnetic particles affixed.
[0109] In some embodiments, the trapping region is relatively thin
but may be quite wide to provide relatively high throughput. In
other words, the cross-sectional area of the channel itself is
relatively large while the height or depth of the channel is quite
thin. The thinness of the channel may be defined by the effective
reach of the magnetic field which is used to attract the magnetic
particles flowing through the trapping region in the fluid medium.
In some embodiments, therefore, the cross-sectional height of the
trapping region interior is about 2 millimeters or less. In some
cases, the height is about 1.5 millimeters or less, or about 1
millimeter or less, or even about 0.8 millimeters or less. The
width of the trapping region interior space is dictated by a
combination of throughput considerations and fabrication
limitations. In some embodiments, the width is less than 1
millimeter, but can be larger than 30 cm. In a specific example,
the height is about 0.5 to 1 millimeter and the width is about 15
to 25 centimeters. Further, throughput may be improved by
connecting multiple separate sheath flow trapping devices in
parallel. In some embodiments, a single sample supply feeds each of
these parallel devices. Similarly, a single supply of buffer or
other sheath flow fluid may feed the devices. The number of such
devices operated in parallel may be 2, 3, 4, 5, or even more.
[0110] Various details of fluidics systems suitable for use with
this invention are discussed in other contexts in the description
of flow modules in U.S. patent application Ser. No. 11/583,989,
published as U.S. 2008/0124779A1, entitled, "Microfluidic
magnetophoretic device and methods for using the same," filed Oct.
18, 2006, naming Sang-Hyun Oh, et al. as inventors, which is
incorporated herein by reference for all purposes. Examples of such
details include buffer composition, magnetic particle features,
external magnet features, ferromagnetic materials for MFG's, flow
conditions, sample types, integration with other modules, control
systems for fluidics and magnetic elements, binding mechanisms
between target species and magnetic particles, etc. Generally, in a
magnetic trapping module the applied external magnetic field will
be relatively higher (considering the overall design of the module)
than that employed in a continuous flow magnetic flow sorter of the
type described in U.S. patent application Ser. No. 11/583,989
(supra). In any event, the magnetic force exerted on target species
should be sufficiently greater than the hydrodynamic drag force in
order to ensure that the target species (coupled to magnetic
particles) are captured and held in place against the flowing
fluid.
[0111] In a typical positive selection example as shown in FIG. 8,
a magnetic trapping process 800 proceeds as follows. First, a
sample such as a biological specimen potentially containing the
target cells are labeled with small magnetic particles coated with
a capture moiety (e.g., an antibody) specific for the surface
marker of the target cell in operation 801. This labeling process
may take place on or off the microfluidic sorting device. After
this labeling, the sample is flowed into the sorting station
(including a trapping region) with or without concurrently flowing
buffer solution in operation 803. Buffer may be delivered through
one or more sheath flow inlets and sample through one or more
others, see 805. In a specific embodiment, buffer is flowed first
to establish a sheath flow of one or two laminar flows of buffer.
One laminar flow of buffer is used to prevent unspecific binding to
an internal surface of the device in proximity to the sorting
station. When two laminar buffer flows are used, one is used as
described above and the other to protect another internal surface
of the device from unspecific binding, where the sample laminar
flow is between the two buffer laminar flows. The sorting station
is energized with an external magnetic field in operation 807 to
hold the magnetically labeled target cells or other species in
place against the hydrodynamic drag force exerted by the flowing
fluid in operation 809. This occurs while continuously eluting the
unlabeled non-target species in operation 811. As explained above,
the magnetic field is typically applied by an external magnet
positioned proximate the sorting station. After most, or all, of
the sample solution has flowed clear of the sorting station, the
magnetic components may be released in operation 813 by any of a
number of different mechanisms including some that involve
modifying the magnetic field gradient and/or increasing the
hydrodynamic force. For example, the magnetic field in the chamber
may be reduced, removed, or reoriented and concurrently the sample
inlet flow is replaced with release agent (for releasing the
captured species) and/or buffer flow. Ultimately the previously
immobilized magnetic components, or just their captured species
(now purified), flow out of the chamber in a buffer solution. The
purified sample component including the target species may then be
collected at an outlet of the sorting chamber in operation 815,
which, in some configurations may be located directly downstream
from the trapping chamber. In some cases, the purified target
species are delivered to one or more post-processing stations. Then
the process flow ends.
[0112] In some embodiments, the trapped target material is directly
removed from the trapping region. The material so removed can be
analyzed, used to produce cell products, manipulated for other
purposes, etc. In one case, the trapping region of the device is
designed to be peeled away, with trapped material attached, from
the remainder of the device. To this end, the device may be
designed with a thin perimeter or weakened region around the
magnetic field gradient generating region. With this configuration,
a technician can detach the trapping region to provide external
access to the trapped material after the sample has passed through
the device. To facilitate the detachment process, the device may be
designed with mechanical design provisions such as tabs, notches or
ridges.
[0113] For context an example of a trapping-type magnetic
separation system will now be described. The system employs
disposable fluidics chips or cartridges, each housing fluidics
elements that include a magnetic trapping module. In one mode of
operation (positive selection), a sample such as a small quantity
of blood is provided to a receiving port in the cartridge and then
the cartridge with sample in tow is inserted into a processing and
analysis instrument. Within the chip, the magnetic particles and
the target species (if any) from the sample are sorted and
concentrated at the magnetic trapping module. After sample has been
processed in this manner, trapped species may be released and
collected in output tubes. This may be accomplished by various
means including reducing or eliminating the external magnetic field
applied to the trapping module or applying a reagent that releases
captured species from magnetic particles. Alternatively, or in
conjunction, the hydrodynamic force exerted on the magnetic
particles may be increased. In certain embodiments, a chassis
houses the system components including a pressure system (such as a
syringe pump and a pressure controller) that provides the principal
driving force for flowing sample through the trapping module. Of
course, other designs may be employed using alternative driving
forces such as a continuous pump or a pneumatic system. Buffer from
buffer reservoirs is also provided to the cartridge under the
controlled by a buffer pump and a flow control module.
[0114] Sheath Flow Device Manufacturing Systems: In general, the
present devices and related instrumentation may be manufactured
according to available methods, so long as configuration to allow a
laminar flow establishing surface is a component.
[0115] Lithography and etching technologies may be used to
manufacture the precise design for desired microfluidic flow. At a
reduced cost, one may use injection molding for preparation of a
rigid base having a particular configuration. Preparing the base
alone, however, leaves the chambers and channels (in which the
liquid flows) open. To enclose the device, one may then seal a top
layer of similarly rigid material using laminate, heat, acoustic or
laser (to adhere a top layer for a sealed device).
[0116] Materials, such as glass, vinyls or other conventional
plastic polymers, may be used for "lab on a chip" and other
devices. The cartridge design (channels, inlets, compartments and
other fluid flow paths or containment areas) may be configured.
Interconnection elements, such as ports use for inlet or outflow,
or for pressurized flow/stoppage, may be attached, or formed as
part of the injection molded design (for example). Other elements,
such as electro-, mechanical, or various sensors may be in
similarly dedicated location.
[0117] The present sheath flow device manufacturing systems include
apparatuses for fluid fill, assembly, separating, coding (such as
bar coding), sterilizing, and packaging.
[0118] The present manufacturing systems include those in
compliance with various governmental or industry regimes, including
food and drug requirements (e.g., FDA, EMEA), quality control
organizations (e.g., International Organization for
Standardization), and other regimes set up to ensure quality for a
particular purpose.
[0119] Sheath Flow Device Instrumentation Systems: The present
devices and methods may be adapted or configured to work in
conjunction with host instruments or to meet system requirements.
Adaptations or configurations include (but are not limited to) one
or more for vacuum filling, automated control of pumps and valves,
pressure flow, injection loop for sample loading, temperature
control, electro-osmotic flow, positive displacement pumping,
expected volumetric flow rate, centrifugal force processes,
humidity control, and gas exchange control, for example.
[0120] Fluid flow may be controlled by automated instrumentation,
although depending on the device, one may use manual control.
Devices of the present invention, for example, can use pneumatic or
air pressure, to establish flow via integrated ports operably
linked with automated instrumentation.
[0121] Processing Prior To Trapping:
[0122] Various processes may be performed "upstream" from or prior
to sorting in a sheath flow device. Examples of these processes
include target labeling, cell lysis, and depletion. Labeling as
explained in the following discussion may involve coupling magnetic
particles having specific binding moieties to target or non-target
components of a sample. Lysis may involve breaking cell membranes
or cell walls to release cell components (organelles, biomolecules,
etc.) into the sample. Depletion involves removing a particular
component or components in a sample prior to separation. An example
of depletion is removal of erythrocytes from a sample by acoustic
or other means. Other pre-processing operations that may be
performed on- or off-chip include a variety chemical means for
staining, fixing or introducing exogenous materials into the
cells.
[0123] In many implementations, it is necessary to insure that the
target or non-target components of a sample become "labeled" with
magnetic beads as appropriate. This labeling operation is performed
upstream (prior to) the trapping/separating stage in which the
magnetic particles are captured and held stationary in a flowing
fluid medium.
[0124] The magnetic particles will have a surface functional group
that has a specific affinity for either the target or non-target
species. Thus, when the magnetic particles come in contact with the
relevant species, they bind with those species to form conjugates.
An inventive operation pertains to a mechanism for facilitating the
binding or conjunction of the magnetic particles with the
appropriate species or component from the sample.
[0125] Typically, though not necessarily, this pre-sorting
treatment is performed in one or more separate chambers or
reservoirs located in fluid communication with the trapping region.
Such chambers or reservoirs may be located on the same device
(chip) as the trapping region or in a separate device or chip. They
may have micro fluidic dimensions or even slightly larger
dimensions if appropriate. In one example, each of one or more
pre-treatment reservoirs has a volume of approximately five
milliliters. In some cases, the reservoirs may be between 0.5 ml to
10 ml.
[0126] The magnetic beads, as well as the sample, and other
reagents to facilitate binding are each provided to the reservoir
or reservoirs. Note that the magnetic particles may be provided in
a functionalized form, in which case it will be unnecessary to
provide the other reagents. The magnetic particles are moved with
respect to the other components in the reservoir(s) to facilitate
labeling. This movement is induced by successive application of
pneumatic pressure two separate chambers in accordance with certain
embodiments. In some embodiments, this movement is induced by a
magnetic mixing mechanism of the type described for magnetic
particle release as described in a later section. The same
mechanisms for facilitating mixing may be employed; e.g., a moving
a magnetic field as by, for example, oscillating the field. Other
examples of mixing mechanisms include ultrasonic agitation or
stirring. Examples of systems and methods that provide fluidic
mixing of magnetic particles and allow for labeling and/or release
of sample species are described in detail in PCT Patent Application
No. PCT/US2009/040866, filed Apr. 16, 2009 (PCT Publication No. WO
2009/129415), incorporated herein by reference for all
purposes.
[0127] Processing After Trapping:
[0128] Various processes may be performed after sorting in a sheath
flow device. These processes may be performed in the sorting
station and/or downstream from the station. Generally, the
processes may involve quantifying target species (e.g., counting
cells), extracting molecular information about the target species
(e.g., whether a particular SNP is present), and/or extacting cell
based products (e.g., a differentiated version of trapped stem
cell). Examples of suitable processes include direct detection of
target species as by optical techniques, assaying, growth of
trapped cells or viruses, transformation of the target species
(e.g., differentiating trapped stem cells), profiling expression
patterns, and genetic characterization. Specific tools that may be
employed to characterize expression profiles and/or genetic
sequences include microarrays such as mRNA arrays and high
throughput sequencing tools. Further discussion may be found in PCT
Patent Application No. PCT/US2009/040866 (supra).
[0129] Often post separation operations involve methods for
releasing target species from magnetic particles that have been
trapped in a trapping station or otherwise separated in a sorting
station. In a typical scenario, at the end of a trapping operation,
the only sample species that remain in the trapping region are
bound to magnetic particles. For many applications, it is important
to separate the captured species from the magnetic particles prior
to further processing.
[0130] In the post separation operations described here, some
mechanism for releasing the bound species from the magnetic
particle is employed. Various binding and release systems are
available. These include, for example, release reagents that (1)
digest a linkage chemically coupling the magnetic bead to the
captured species, (2) compete with chemical or biochemical linkage
mechanisms for binding with the captured species, and (3) cleaving
the linkage with a secondary antibody. Trapped target species may
be simply concentrated, purified and/or released as described.
Alternatively they can be further analyzed and/or treated.
[0131] In some embodiments, the particles that have been captured
and washed and optionally released in the trap as described above
are exposed to one or more markers (e.g., labeled antibodies) for
target species in the sample. Certain tumor cells to be detected,
for example, express two or more specific surface antigens. To
detect these tumors, more than one marker may be used. This
combination of antigens occurs only in certain unique tumors. After
one or more labels flow through the trap for a sufficient length of
time, the captured particles/cells may be washed. Thereafter, the
particles/cells can be removed from trap for further analysis or
they may be analyzed in situ. For example, the contents of trap may
be scanned with probe beams at excitation for the first and second
labels if such labels or fluorophores for example. Emitted light is
then detected at frequencies characteristic of the first and second
labels. In certain embodiments, individual cells or particles are
imaged to characterize the contents of trap 301 and thereby
determine the presence (or quantity) of the target tumor cells. Of
course various target components other than tumor cells may be
detected. Examples include pathogens such as certain bacteria or
viruses.
[0132] In another embodiment, nucleic acid from a sample enters is
captured by an appropriate mechanism. These nucleic acids can be
detected and profiled directly without any amplification, for
example using microarrays. Alternatively, PCR reagents
(nucleotides, polymerase, and primers in appropriate buffers) enter
the trap and an appropriate PCR thermal cycling program is
performed. The thermal cycling continues until an appropriate level
of amplification is achieved. Subsequently in situ detection of
amplified target nucleic acid can be performed for, e.g.,
genotyping or detection of a particular mutation. Alternatively,
the detection can be accomplished downstream of the trap in, e.g.,
a separate chamber which might contain a nucleic acid microarray or
an electrophoresis medium. In another embodiment, real time PCR can
be conducted in trap by introducing, e.g., an appropriately labeled
intercalation probe or donor-quencher probe for the target
sequence. The probe could be introduced with the other PCR reagents
(primers, polymerase, and nucleotides for example). In situ real
time PCR is appropriate for analyses in which expression levels are
being analyzed. In either real time PCR or end point PCR, detection
of amplified sequences can, in some embodiments, be performed in
the trap by using appropriate detection apparatus such as a
fluorescent microscope focused on regions of the trap.
[0133] In some embodiments, capture elements capture and confine
cells from sample to reaction chamber in situ. Thereafter, a lysing
agent (e.g., a salt or detergent) is delivered to the chamber. The
lysing agent may be delivered in a plug of solution and allowed to
diffuse throughout the chamber, where it lyses the immobilized
cells in due course. This allows the cellular genetic material to
be extracted for subsequent amplification. In certain embodiments,
the lysing agent may be delivered together with PCR reagents so
that after a sufficient period of time has elapsed to allow the
lying agent to lyse the cells and remove the nucleic acid, a
thermal cycling program can be initiated and the target nucleic
acid detected.
[0134] In other embodiments, sample nucleic acid is provided in a
raw sample and coupled to magnetic particles containing appropriate
hybridization sequences. The magnetic particles are then sorted and
immobilized in the trap. After PCR reagents are delivered to the
chamber and all valves are closed, PCR can proceed via thermal
cycling. During the initial temperature excursion, the captured
sample nucleic acid is released from the magnetic particles.
[0135] The nucleic acid amplification technique described here is a
polymerase chain reaction (PCR). However, in certain embodiments,
non-PCR amplification techniques may be employed such as various
isothermal nucleic acid amplification techniques; e.g., real-time
strand displacement amplification (SDA), rolling-circle
amplification (RCA) and multiple-displacement amplification (MDA).
Each of these can be performed in a trap such as a chamber in the
device containing appropriate valving and flow lines.
[0136] Besides the extraction and analysis of the nucleic acids,
the captured cells themselves may be used directly as the product
of the process, or they can be manipulated to produce the desired
product. For example, the device may be used to isolate stem cells
from blood or tissue as the cell-based product. Alternatively, the
captured cells may be manipulated with reagents such as growth
factors, chemokines, and antibodies to produce the desired cell- or
molecule-based products. Multiple processes of purification and
manipulation may be performed to obtain the desired product within
the device.
[0137] One example operation employing the apparatuses and methods
of the present invention is automated protein purification,
particularly as protein is expressed in cell culture. Protein
purification may be performed manually. However, the apparatuses
and methods of the present invention provide a time and labor
saving automation that delivers a high purity product with low
cost.
[0138] In one example, desired proteins are expressed in organisms
such as virus, bacteria, insect or mammalian cells. The expressed
protein may be designed such that it may be selectively isolated
from background materials. This may be accomplished via adding one
or more selectable amino acid tags that add a stretch of amino acid
to the protein. The tag may be a His tag, FLAG tag or other
epitope-based tags (E-tags). The cells (for example) are introduced
to one of the sample reservoirs described herein, with magnetic
particles and lyses reagents in the same or one or more reservoirs.
The magnetic particles may be magnetic beads coated with a high
affinity media such as NTA-agarose or other resin containing to
nickel. Mixing between the various sample reservoirs is promoted
via one or more of the techniques described above, e.g., pneumatic,
hydraulic, or magnetic mixing. The cells are disrupted by the
lysing reagent and, under suitable conditions, the magnetic
particles bind with the target protein in the lysate. The raw
lysate is then flowed into the magnetic separation chamber where
the beads become trapped on the surface of the channel. Wash buffer
is added to elute the untagged and unbound protein and other cell
fragments. According to various embodiments, the magnetic
separation chamber may be agitated magnetically or through other
means to further remove any unbound protein stuck between trapped
particles. A highly stringent wash buffer may be used to further
elute unwanted particles. At this point, only the target protein
and bound magnetic particles remain in the chamber with very high
selectivity. The target protein may be released by using a bead
release agent into a small volume, optionally for further
processing. Lastly, the magnetic particles may be released. Because
these various operations occur on a unitary or disposable cartridge
in a machine, the procedure may be preprogrammed and automated to
save time and cost. This configuration may be used to selectively
trap other nucleic acid related products, such as RNA, which may be
so labeled so as to be similarly selectable.
[0139] General Applications:
[0140] As explained, the sheath flow devices of this invention are
widely applicable. They may be used to capture and separate many
different types of target species tagged with magnetic particles.
Thus the sheath flow fluidic devices of this invention have many
applications including, for example, biological fluid sample
preparation and analysis, separation of rare molecules or cells,
chemical library screening, point of care diagnostic in a clinical
laboratory setting, environmental testing or monitoring, consumer
products and food quality control aspects. The invention finds
particular application in academic, industrial and clinical
settings. In academic, industrial and clinical settings, sheath
flow devices of the invention find particular use in isolating cell
lines, proteins, genetic material and the like for testing,
characterization and further manipulation, for example
amplification of cell lines, proteins, mRNA, genetic code,
molecular signature proteonomics and the like. Sheath flow devices
and methods can be used in early detection and diagnosis of
disease, for example cancers via isolation of CTC's from blood
samples. In clinical settings in particular, embodiments include
screening potential patient pools for clinical trial candidacy
and/or testing biological fluids of patients already enrolled in
clinical trials for information on therapy efficacy and/or future
course of treatment regimens as well as measuring the efficacy of
adjunct therapy or neo-adjunct therapy. More specific examples are
described below.
[0141] Sheath Flow Research Tools:
[0142] The present sheath flow devices have broad use in scientific
research, including but not limited to screening molecular
libraries. For example, one can use the present devices for
screening aptamer libraries by preparing a purified and isolated
protein on the present device, and then exposing the protein to an
aptamer library, within a single device. The term "aptamer" as used
herein is meant in its broadest sense to denote oligonucleic acid
or peptide molecules which bind to a specific target molecule, and
related synthetic molecules, such as mimetics. Sheath flow devices
of the invention can be used for biomarker/drug discovery platforms
that utilize molecular or cellular library sorting such as aptamer
library, ribosome library, phage display, bacterial peptide
display, yeast display systems and the like. In another example,
sheath flow devices of the invention can be used to isolate target
cells for a variety of endpoints including cell count, typing,
amplification, differentiation and the like.
[0143] Sheath Flow Bio/Chemical Monitoring, Synthesis or Analysis:
The present invention may be configured or adapted for a variety of
biological or chemical monitoring, synthesis or analysis purposes,
such as, for example, chemical threat monitoring, nucleic acid
analysis (and amplification using, for example, polymerase chain
reaction), continuous monitoring of particular conditions, such as
closed environmental monitoring, personalized genomics and
diagnosis, chemical synthesis, such as synthesis of aptameric
therapeutics contained within viral coats or other nanocages
suitable for delivery into a physiologic environment, or other
chemical syntheses. For home use, for example, in monitoring
swimming pool or drinking water quality, one may include pH
indicator, metal indicators, or other indicators of water quality.
The present devices can be configured or adapted for production
process control, such as bioreactor monitoring in biopharmaceutical
production processes, or for the food industry. The present devices
can be adapted or configured for fluid control and analysis, gas
control and analysis. For example, by adapting the present devices
for continuous flow, one can monitor the rate at which cells are
sorted. The present devices can be configured for production
quality control, such as for supply chain monitoring. Since sheath
flow apparatus of the invention can capture target species from
liquid samples with high selectivity, this represents a strong
advantage over many conventional systems where, for example in
therapeutic settings, a target species must be isolated in high
purity from a fluid biological sample having a highly complex
mixture of species.
[0144] Configurations:
[0145] Prefilled "Kit on a Chip" Because of the ease in manufacture
and use, it is contemplated that one aspect of the present
invention is a sheath flow device prefilled with reagents useful
for a particular purpose. For example, devices may be prefilled
with reagents useful for biological sample preparation. This "kit
on a chip" sheath flow device aspect can be adapted for a variety
of applications as described above.
[0146] Such "kit on a chip" embodiments may include a variety of
reagents and may be adapted for a variety of fields, such as
biological fluid sample preparation and analysis, separation of
rare molecules or cells, chemical library screening, point of care
diagnostic in a clinical laboratory setting, environmental testing
or monitoring, consumer products and food quality control aspects,
for example. The present invention includes single or a plurality
of prefilled devices suitable for such uses. Configurations are
non-limiting, but should be considered along with related
instrumentation and methods.
[0147] Reagents can be disposed within the sheath flow device for
ease of use, for example in reservoirs in predetermined amounts.
For example, a substantially purified protein preparation may be
obtained by culturing cells so expressing the desired protein. The
subject reservoir may be so adapted to culturing the cells, and
have access ports with appropriate reagents in fluidic
communication under controlled conditions. Reagents include buffers
for lysing cells, washing cells, and removing beads selectively
bound to a moiety. Additional reagents include selective binding
molecules, such as antibodies, aptamers, and other molecules that
selectively (although not necessarily specifically) bind a target
molecule. Further reagents include various moieties allowing
capture of the selected molecule, such as magnetic beads, acoustic
beads and other beads providing that function. Reagents further
include nucleic acids such as primers suitable for selecting
particular nucleic acids from a complex mix. For example, the
present device may be used to screen genomic DNA, and amplify
selected sequences using polymerase chain reaction, within the
device itself.
[0148] Integrated Systems: A kit on a chip configuration can stand
alone, be part of or serve as an integrated system that contains
sheath flow devices of the invention. Additional processing modules
or chambers can be added "upstream" or "downstream" of the sheath
flow component of apparatus of the invention. For example, one can
prepare suitable post-expression modification fluidic circuitry,
such as providing reservoirs with, for example a desired polymeric
or other substance for derivatization and or other reaction. In a
specific example, the present sheath flow device is configured with
fluidic circuitry for protein expression from cells in culture, and
optionally additional modules for derivatizing the protein so
expressed, such as a pegylation module in which one may derivatize
the subject protein.
[0149] Examples of operational modules that may be integrated with
magnetic trapping sorters in fluidics devices include (a)
additional enrichment modules such as fluorescence activated cell
sorters and washing modules, (b) reaction modules such as sample
amplification (e.g., PCR) modules, restriction enzyme reaction
modules, nucleic acid sequencing modules, target labeling modules,
chromatin immunoprecipitation modules, crosslinking modules, and
even cell culture modules, (c) detection modules such as
microarrays of nucleic acids, antibodies or other highly specific
binding agents, and fluorescent molecular recognition modules, and
(d) lysis modules for lysing cells, disrupting viral protein coats,
or otherwise releasing components of small living systems. Each of
these modules may be provided before or after the magnetic sorter.
There may be multiple identical or different types of operational
modules integrated with a magnetic sorter in a single fluidics
system. Further, one or more magnetic sorters may be arranged in
parallel or series with respect to various other operational
modules. Some of these operational modules may be designed or
configured as traps in which target species in a sample are held
stationary or generally constrained in a particular volume.
Features of operational modules depend on the type of reaction
desired and may include a thermal management system, micromixer,
catalyst structure and sensing system. A thermal management system
may include heaters, temperature sensors, and micro heat
exchangers. All these components may be integrated to precisely
control temperatures. Such temperature control is crucial for
example when using PCR for DNA amplification.
[0150] As should be apparent from the above examples of modules,
operations that may be performed on target and/or non-target
species in modules of integrated fluidics devices include sorting,
coupling to magnetic particles (sometimes referred to herein as
"labeling"), binding, washing, trapping, amplifying, removing
unwanted species, precipitating, cleaving, diluting, ligating,
sequencing, synthesis, labeling (e.g., staining cells),
cross-linking, culturing, detecting, imaging, quantifying, lysing,
etc. The present devices may be configured to perform one of more
functions within the same device.
[0151] Examples of biochemical operations that may be performed in
the magnetic sorting modules of integrated sheath flow fluidic
devices include synthesis and/or screening of plasmids, aptamers,
proteins, and peptides; evaluating enzyme activity; and
derivatizing proteins and carbohydrates. A broad spectrum of
biochemical and electrophysiological assays may also be performed,
including: (1) genomic analysis (sequencing, hybridization), PCR
and/or other detection and amplification schemes for DNA, and RNA
oligomers; (2) gene expression; (3) enzymatic activity assays; (4)
receptor binding assays; and (5) ELISA assays. The foregoing assays
may be performed in a variety of formats, such as: homogeneous,
bead-based, and surface bound formats. Furthermore, devices as
described herein may be utilized to perform continuous production
of biomolecules using specified enzymes or catalysts, and
production and delivery of biomolecules or molecules active in
biological systems such as a therapeutic agents. Sheath flow
devices as described herein may also be used to perform
combinatorial syntheses of peptides, proteins, and DNA and RNA
oligomers as conventionally performed in macrofluidic volumes.
[0152] Specific Applications:
[0153] Cell Trapping and Manipulation: As indicated above,
exploitation of the present sheath flow devices is particularly
useful when configured or adapted for culturing, purifying or
isolating components of, or analyzing a variety of biological
materials. In particular aspects, the present devices may be
configured or adapted for cell lysis, bead-based displacement
assays, perfusion, filtration, sample preparation, chemotaxis,
whole blood separation, and a variety of other processes.
[0154] Isolation of particular cell types from a complex biological
sample is particularly useful. For example, a biological sample may
contain one or more stem cells, bacteria, human cells, bio-film
materials, mammalian cells, yeasts, algae, primary tumor cells,
immortalized cell lines, tissue or organ cultures, unicellular or
multi-cellular organisms, molds and other organisms that can be
isolated from the sample, for example a culture. Conventionally,
isolating cell types from these complex mixtures is labor intensive
and often unsuccessful due to low yield, low purity, nonviability
of isolated cells due to excessive manipulation, and the like. As
mentioned, sheath flow devices of the present invention may not
only have fluidic circuitry adapted for cell sorting but also
isolation of different cell types which can subsequently be
expanded or differentiated for use in characterization, therapy and
the like. Sheath flow apparatus of the invention allow
post-isolation processes such as expansion and differentiation ex
situ and/or in situ.
[0155] Target cell types are isolated by, for example, tagging the
cells with magnetic particles that bear a binding agent, for
example an antibody or chemically reactive group, selective for the
target cell. The sample is exposed to the magnetic particles and
thus the target cells are selectively bound to the magnetic
particle via the selective binding agent bound to the magnetic
particle. Two exemplary cell types where the sheath flow devices of
the invention find particular use are stem cells and circulating
tumor cells (CTC's). A more detailed description of applications of
sheath flow devices and methods for stem cells and CTC's
follows.
[0156] Stem Cells: Medical researchers believe that stem cell
treatments have the potential to change the face of human disease
and alleviate suffering. The propensity of stem cells to self-renew
and generate new stem cell populations offers an enormous potential
for repairing and/or replacing (via differentiation to the target
cell population) diseased and damaged tissues in the body, without
the risk of rejection and side effects. Medical researchers
anticipate using adult and embryonic stem cell technology to treat
cancer, metabolic disorders, cardiac failure, muscle damage and
neurological disorders. More specific disorders for which stem cell
therapies show promise are brain damage, cancer, spinal cord
injury, heart damage, haematopoiesis, baldness, missing teeth,
deafness, blindness and vision impairment, amyotrophic lateral
sclerosis, graft v. host disease, Crohn's disease, neural and
behavioral birth defects, diabetes, orthopedic disorders, wound
healing and infertility. See: The Leading Edge of Stem Cell
Therapeutics, by Singec I., et al., Annu Rev. Med. 58: 313-328,
2007.
[0157] A key to stem cell research and/or treatments is the
isolation of highly pure viable stem cells. As described above,
sheath flow apparatus and methods of the invention allow a user to
select and isolate particular stem cells, for example, from blood,
marrow, umbilical cord or other sources, and optionally, carry out
further manipulation of the isolated cells, for example, culturing
the cells isolated by the sheath flow device in situ and/or ex
situ.
[0158] Thus one particular aspect of the invention are sheath flow
devices and methods configured for isolating stem cells from a
biological sample. Such selective binding molecules can be
connected to a variety of suitable vehicles for selection within
the sheath flow device, such as a magnetophoretic bead, an acoustic
bead, or other moiety capable of capturing the molecule (and cell)
so selected. In one embodiment, sheath flow devices use magnetic
particles bearing moieties selective for stem cells, such as CD34+
selective binding molecules. In a particular embodiment, the
magnetic particles are used to trap and isolate the stem cells by
binding to the target stem cells, and while traveling in the sample
stream surrounded by the buffer sheath flow of the device, are
selectively pulled or deflected from the sample stream through the
sheath flow to a collection grid.
[0159] The isolated stem cells can be characterized and/or
manipulated on or off the collection grid. As mentioned, sheath
flow devices of the invention may have additional post-isolation
modules or features for amplifying stem cell populations and/or
differentiating the stem cells. Post-isolation modifications can
take place, for example depending upon the binding moiety and local
environment, before or after separation from the magnetic particles
used to trap the cells. Such post-isolation modules or features
will have, as understood by one of ordinary skill in the art,
additional features of a bioreactor for stem cell growth, such as
media, oxygen, temperature, media replacement, and the like.
[0160] In a particular example, sheath flow devices of the
invention can be configured for sorting suitable stem cells from
blood, and further culturing the stem cells for expansion. One may
optionally include selected media, growth factors, and other
materials to be pre-filled on a present sheath flow device. For
example, one may use hematopoietic stem cell selective reagents,
such as antibodies or aptamers. Such reagents may selectively bind
to, for example, CD34+ stem cells. In one example, the magnetic
particles, bound and unbound to CD34+ cells, are then held in
place, and other material is washed away. A bead release reagent,
for example commercially available, is applied and the bound cells
are released. While the beads are captured by the magnetic force,
the stem cells may be separated in a fluidic supernatant. Other
types of stem cells may include embryonic, fetal, amneonic, adult,
or induced pluoripotent stem cells.
[0161] As described above, isolated cells, in this example stem
cells, can be used to derive a cell based product from the stem
cells separated from the liquid sample in the sheath flow device.
Cell based products can include expanded cell populations,
differentiated cells and products derived from analysis of
molecular components of cells, for example, genetic coding
information, RNA, DNA, oncogenic information and the like. In the
context of stem cells, modules of the sheath flow devices of the
invention can be used to culture stem cells, expanding and/or
differentiating stem cell populations, either in situ, or removing
them for expansion and/or differentiation in a different device.
Expanded stem cell populations can be used for injection into a
patient, for example at the site of a tumor or tissue damage, for
example nerve or muscle tissue. Also stem cells can repair tissue
or aid in repair of tissue. In oncology, stem cells can be used to
aid in destruction of tumors. Additionally, cancer stem cells
(CSC's) are thought to be a component of relapse of cancerous
growths due to chemotherapy's ineffectiveness to kill CSC's while
destroying tumorous growths. Sheath flow devices of the invention
can be used to isolate, characterize and manipulate CSC's just as
other stem cells described herein.
[0162] Sheath flow devices of the invention can also be used for
tissue regeneration. For example, where the sheath flow device is
used for stem cell expansion as describe above, reagents may be
used to differentiate isolated stem cells into different types of
tissue-related cells, another example of cell based product. Such
sheath flow devices of the invention can be configured with
biocompatible scaffolding and suitable reagents for growing tissues
ex vivo. In one embodiment, a sheath flow device is configured to
culture liver or other organ tissues, for transplant, based on
cells originally isolated and grown in situ in the sheath flow
device. As the present device may be configured for stem cell
selection and in situ expansion, one may further configure the
device, including pre-filled reagents, for various applications
involving stem cell differentiation to a desired target tissue. The
sheath flow device itself may be made of biocompatible material so
that the stem cell-grown tissue (or population of cells on a
scaffold) may be applied or implanted directly into a patient. In
another example, the present sheath flow device can be configured
to isolate stem cells and generate corneal tissue suitable for a
corneal transplant.
[0163] Circulating Tumor Cells: A majority of cancer deaths are
caused by haematogenous metastatic spread and proliferation of
tumor cells in tissues throughout the body. Viable tumor derived
epithelial cells, circulating tumor cells or CTC's, have been
identified in peripheral blood from cancer patients and are likely
the origin of intractable metastatic disease (see: Nagrath, S., et
al., Isolation of Rare Circulating Tumour Cells in Cancer Patients
by Microchip Technology, Nature, Vol 450: 20/27, 1235-1240, 2007).
The study of CTC's is therefore highly relevant to the biology of
early metastatic spread and provide a powerful diagnostic source in
patients with overt metastases (see: Patna, K., et al., Detection,
Clinical Relevance and Specific Biological Properties of
Disseminating Tumour Cells, Nature Reviews, Vol. 8: 329-40, 2008).
Moreover, molecular analysis of CTC's from the blood of patients
with cancer offers the possibility of monitoring changes in tumor
genotypes during the course of treatment (see: Detection of
Mutations in EGFR in Circulating Lung-Cancer Cells, Maheswaran, S.,
et al., N Engl J Med, 359:366-77, 2008). Therefore, utilizing
liquid samples, for example from a simple blood draw, offers a huge
advantage over invasive biopsies as a source of tumor tissue for
the detection, characterization and monitoring of non-haematologic
cancers.
[0164] A key to CTC research and/or treatments is the isolation of
highly pure CTC's. Sheath flow apparatus of the invention allow a
user to select and isolate CTC's from a liquid sample, for example
from a blood draw, without having to biopsy solid tumors. Using
apparatus and methods of the invention, one may select particular
circulating tumor cells for analysis, for example from blood of a
patient being diagnosed and/or monitored for a cell proliferation
disorder such as cancer. Point of care diagnostics using sheath
flow devices of the invention are a powerful tool for early and
accurate diagnosis of, for example, cancer types and/or
progression. Also, patients with existing cancers will benefit from
a quick turnaround analysis of CTC's in their system, for example
from a simple blood draw, for formulating current and future
treatment regimens as well as measuring the efficacy of adjunct
therapies such as surgery to remove a tumorous growth.
[0165] Thus one aspect of the invention is a method of monitoring
and, if appropriate, adjusting a patient's treatment regimen, the
method including: (a) receiving a sample including tumor cells from
a patient undergoing a first treatment regimen; (b) separating the
tumor cells from the sample by a separation method including: (i)
labeling the sample with magnetic particles having a specific
affinity for the tumor cells, thereby producing a labeled sample,
(ii) passing the labeled sample through a fluidic device including
a sorting region having a magnetic field gradient effective to
deflect and/or trap the magnetic particles from the labeled sample
and thereby separate the tumor cells from the sample, where the
sample flows in a sheath of buffer solution to reduce nonspecific
binding of the sample to the fluidic device; and (c) characterizing
the tumor cells separated from the sample in (b) to suggest a
future treatment for the patient and/or to assess the efficacy of
an existing treatment. In one embodiment, the sample is a fluid
sample taken from the patient. In a specific embodiment, the sample
is a blood sample. In another embodiment, the tumor cells are
CTC's. In one embodiment, the first treatment regimen is a
chemotherapy regimen, and in a more specific embodiment, the future
treatment is a different chemotherapy regimen. In one embodiment,
characterization of the tumor cells is a count, in another
embodiment characterization of the tumor cells is a molecular
characterization. In a more specific embodiment, the molecular
characterization is a genetic mutation in the tumor cells.
[0166] In one embodiment, suggesting the future treatment for the
patient includes predicting a future effectiveness of the first
treatment regimen. In another embodiment, suggesting a future
treatment for a patient includes identifying a second treatment
regimen that is different than the first treatment regimen and
accounts for a characteristic of the tumor cells not previously
observed for the patient. In another embodiment, the method further
includes: (d) receiving a sample including tumor cells from the
patient after the patient has undergone the second treatment
regimen; and (e) thereafter performing (b) and (c) (as in the
previous paragraph) on the sample and tumor cells received in
(d).
[0167] Thus, using methods and apparatus of the invention,
clinicians can, for example, screen a potential patient pool for
candidates suitable for the clinical trials. In this way, patients,
for example, having CTC's with oncogenic coding that indicates
refractory response to intended treatment regimens can be steered
towards other possible treatments. Or, in some cases refractory
patients are specifically targeted for a particular new treatment
regimen. In another example, a patient's CTC's are analyzed for a
count and/or molecular components thereof, and from this
information it is determined whether or not a current treatment is
effective, should be continued, or another or additional treatments
should be considered. In another embodiment, a patient's CTC's are
analyzed by count and/or additional characterization to assess
whether an adjunct therapy was efficacious. In a specific example,
the patient's CTC count suggests that the adjunct therapy was
successful and that no further treatment is currently necessary. In
another specific example, the patient's CTC count suggests that the
adjunct therapy was successful, but that another therapy is
necessary based on a mutation in the CTC as compared to a previous
CTC characterization from the patient. In another example, the
patient's CTC count suggests that the adjunct therapy was
unsuccessful, and that another adjunct therapy is indicated.
EMBODIMENTS
[0168] In accord with the description herein, one embodiment is a
fluidic separating device including: (a) at least one sample inlet
channel configured to provide a sample stream in the fluidic
separating device; (b) at least one sheath flow inlet channel
configured to provide one or more fluid streams that form a sheath
around the sample stream within the fluidic separating device, and
thereby reduce nonspecific binding of components of the sample to
the device; (c) a sorting station fluidly coupled to the sample and
sheath flow inlets and located along a path of the sample stream;
and (d) a magnetic field gradient generator for interacting with an
external magnetic field to produce a change in magnetic field
gradient in the sorting station and thereby deflecting and/or
trapping magnetic particles from the sample stream. In one
embodiment, the magnetic particles are trapped by the sorting
station.
[0169] In one embodiment, the sorting station has a substantially
rectangular interior space for bounding fluid flowing through the
sorting station, the interior space has first and second lateral
dimensions transverse to the sample stream's direction of flow, the
second lateral dimension is at least about 2 times larger than the
first lateral dimension, and the at least one sheath flow inlet
channel is configured to provide two sheath streams separated from
one another by the sample stream along the first lateral dimension.
In another embodiment, the sorting station has a substantially
rectangular interior space for bounding fluid flowing through the
sorting station, the interior space is defined, in part, by two
substantially parallel and substantially planar surfaces separated
by a distance not greater than about 2 millimeters, and the at
least one sheath flow inlet channel is configured to provide two
sheath streams flowing in contact with the two parallel planar
surfaces and separated from one another by the sample stream. In
one embodiment, the two substantially parallel and substantially
planar surfaces are separated by a distance not greater than about
1 millimeter. In one embodiment, the at least one sheath flow inlet
channel includes a first sheath flow inlet channel, which includes
a substantially planar surface located upstream of the sorting
station and oriented substantially parallel to the two
substantially planar surfaces of the interior space.
[0170] In one embodiment, the fluidic separating device further
includes a second sheath flow inlet channel, which includes its own
substantially planar surface located upstream of the sorting
station and oriented substantially parallel to the two
substantially planar surfaces of the interior space. In one
embodiment, the at least one sample inlet channel includes its own
substantially planar surfaces oriented substantially parallel to
the two substantially planar surfaces of the interior space.
[0171] In one embodiment, the magnetic field gradient generator
includes a plurality of ferromagnetic elements patterned on the
sorting device proximate the sorting station. In another
embodiment, the magnetic field gradient generator includes a
permanent magnet proximate the plurality of ferromagnetic elements.
In yet another embodiment, the plurality of ferromagnetic elements
are disposed within a fluid pathway of the sorting station to allow
fluid contact between the ferromagnetic elements and the sample
stream. The ferromagnetic elements can include micropatterned
nickel elements. In one embodiment, the magnetic field gradient
generator is configured to temporarily capture the magnetic
particles and then release the magnetic particles.
[0172] Another embodiment is a method of capturing a target species
in a sample, the method including: (a) providing the sample to at
least a first inlet channel of a fluidic sorting device, where the
sample includes magnetic particles having a specific affinity for
the target species; (b) providing a sheath flow stream to the
fluidic sorting device to produce one or more sheath streams that
form a sheath around a sample stream within the fluidic separating
device, and thereby reduce nonspecific binding of sample to the
device; (c) magnetizing a magnetic field gradient generator to trap
at least some of the magnetic particles and thereby separate the
trapped magnetic particles from the sample; and (d) analyzing
target species that are or were bound to the trapped the magnetic
particles. In one embodiment, the magnetic field gradient generator
includes a plurality of ferromagnetic elements. The ferromagnetic
elements can include micropatterned ferromagnetic material on the
fluidic device. In one embodiment, magnetizing the magnetic field
gradient generator includes applying an external magnetic field
from a permanent magnet or an electromagnet to the magnetic field
gradient generator.
[0173] Methods can further include any combination of: 1) detecting
the purified target species bound to the trapped magnetic
particles, 2) amplifying a nucleic acid of the target species in
the fluidic sorting device, 3) lysing cells in the fluidic sorting
device, where at least some of the cells include the target
species, 4) separating genetic material from cells or viruses in
the fluidic sorting device, where at least some of the cells or
viruses include the target species, and 5) recovering purified
target species including at least 50% of the target species in the
sample.
[0174] Another embodiment is a method of purifying a target species
in a sample including magnetic particles having an affinity for the
target species, the method including: (a) flowing the sample to
into a fluidic sorting device having a magnetic field gradient
generator to thereby capture at least some of the magnetic
particles, where the flowing sample is contained with or sandwiched
between a sheath of liquid that is substantially free of the
sample; (b) removing or reducing a magnetic field applied to the
magnetic field generator to thereby release captured magnetic
particles; and (c) collecting or processing purified target species
with at least some of the magnetic particles downstream from the
magnetic field generator.
[0175] Another embodiment is a sheath flow device including: (a) a
first laminar flow establishing surface upstream of a sheath flow
plane area; (b) a sample laminar flow establishing surface parallel
to the sheath flow plane area and upstream of a sample sheath flow
plane area; and optionally, (c) a second laminar flow establishing
surface upstream of a second sheath flow plane area. In one
embodiment, the sheath flow device further includes a particle
trapping station. This trapping station can be, for example, a
magnetic trapping station. The sheath flow devices described herein
can be part of a larger fluidic circuit.
[0176] Another embodiment is a sheath flow device including: (a) a
first laminar flow establishing surface upstream of a sheath flow
plane; (b) a sample laminar flow establishing surface parallel to
the sheath flow plane area and upstream of a sample sheath flow
plane area; and (c) a second laminar flow establishing surface
upstream of a second sheath flow plane area. In one embodiment, the
laminar flow establishing surfaces in (a) (b) and (c) are
sufficient to maintain separate sheath flow planes substantially
free of turbulent flow. In one embodiment, the sample laminar flow
establishing plane of subpart (b) has a top and a bottom surface,
and the second laminar flow surface of subpart (c) is a located on
the surface opposite that used for sample laminar flow. The sheath
flow device can be oriented substantially orthogonally with respect
to a horizon. The sheath flow device can further include a particle
trapping station. In one embodiment, the particle trapping station
includes a ferromagnetic component. The sheath flow device can be
adapted for magnetophoretic particle separation.
[0177] The sheath flow devices described herein can: further
include at least one reservoir containing a reagent, be adapted for
stem cell isolation from whole blood and/or be configured for
microfluidic operation. In one embodiment, the sheath flow device
includes a reservoir containing a reagent, the reagent including at
least one of a buffer, a quantity of magnetic beads, a stem cell
expansion agent, an aptamer, a composition including a protein, a
bacterial cell culture, and a composition including a bacteriophage
population.
[0178] Another embodiment is a method of separating a target
species from a fluid sample in a sheath flow device, including: (a)
pre-labeling the target species in the fluid sample with magnetic
particles using a reagent bound to the magnetic particles that
selectively binds the target species; (b) establishing a laminar
buffer flow along a planar surface of the sheath flow device; (c)
establishing a laminar flow of the fluid sample adjacent to the
laminar buffer flow; (d) pulling the target species from the
laminar flow of the fluid sample through the laminar buffer flow;
and (e) trapping the target species on a magnetic trapping station.
In one embodiment, the laminar flow is on the order of microns in
height and on the order of millimeters in width. The buffer and
sample laminar flows are adjacent by virture of their area along
the length and width dimensions being coincident, that is, they are
stacked in the height dimension. One way to increase throughput
when isolating target species using such methods is to increase the
width of the laminar flows, while keeping the height of the flows
in the micron (sub-millimeter) regime. This can be accomplished by
running multiple sheath flow devices in parallel and/or by
increasing the width of the laminar flow in the sheath device. In
one embodiment, the method further includes establishing a second
laminar buffer flow, adjacent to the first laminar buffer flow,
before establishing the fluid sample laminar flow, where the
laminar flow of the fluid sample is established between the first
and second laminar buffer flows. In one embodiment, the first and
second buffer laminar flows and the laminar flow of the fluid
sample are sufficient to maintain separate flow planes
substantially free of turbulent flow. In another embodiment, the
magnetic trapping station comprises a ferromagnetic component. In
one embodiment the target species comprises at least one of a cell,
a bacterium, a virus, a protein and a nucleic acid. In one
embodiment, the target species is a circulating tumor cell and the
fluid sample is whole blood.
[0179] Another embodiment is a method of analyzing a patient the
method including: (a) receiving a sample including tumor cells from
a patient; (b) separating the tumor cells from the sample by a
separation method including: (i) labeling the sample with magnetic
particles having a specific affinity for the tumor cells, thereby
producing a labeled sample, (ii) passing the labeled sample through
a fluidic device including a sorting region having a magnetic field
gradient effective to deflect and/or trap the magnetic particles
from the labeled sample and thereby separate the tumor cells from
the sample, where the sample flows in a sheath of buffer solution
to reduce nonspecific binding of the sample to the fluidic device;
and (c) characterizing the tumor cells separated from the sample in
(b). In one embodiment, the characterizing in (c) provides
information for diagnosing a condition, screening for a clinical
trial, assessing the effectiveness of a therapeutic treatment, and
measuring the effectiveness of surgery. In another embodiment, the
sample is a fluid sample taken from the patient. In one embodiment,
the sample is not taken from a biopsy of the patient, for example,
the sample is a blood sample. In one embodiment, the tumor cells
are circulating tumor cells from a non-haematologic cancer. In
another embodiment, the characterizing is a count of the tumor
cells and/or a molecular characterization of the tumor cells.
Molecular characterization, for example, is a genetic mutation in
the tumor cells.
[0180] Another embodiment is a method of monitoring and, if
appropriate, adjusting a patient's treatment regimen, the method
including: (a) receiving a sample including tumor cells from a
patient undergoing a first treatment regimen; (b) separating the
tumor cells from the sample by a separation method including: (i)
labeling the sample with magnetic particles having a specific
affinity for the tumor cells, thereby producing a labeled sample,
(ii) passing the labeled sample through a fluidic device including
a sorting region having a magnetic field gradient effective to
deflect and/or trap the magnetic particles from the labeled sample
and thereby separate the tumor cells from the sample, where the
sample flows in a sheath of buffer solution to reduce nonspecific
binding of the sample to the fluidic device; and (c) characterizing
the tumor cells separated from the sample in (b) to suggest a
future treatment for the patient. In one embodiment, the first
treatment regimen is a chemotherapy regimen. In one embodiment, the
future treatment is a different chemotherapy regimen. In one
embodiment, suggesting the future treatment for the patient
includes predicting a future effectiveness of the first treatment
regimen, in another embodiment, suggesting a future treatment for
the patient includes identifying a second treatment regimen that is
different than the first treatment regimen and accounts for a
characteristic of the tumor cells not previously observed for the
patient.
[0181] The method of monitoring and, if appropriate, adjusting a
patient's treatment regimen, can further include: (d) receiving a
sample including tumor cells from the patient after the patient has
undergone the second treatment regimen; and (e) thereafter
performing (b) and (c) on the sample and tumor cells received in
(d).
[0182] Another embodiment is a method of providing cell based
products, the method including: (a) receiving a sample including
target cells; (b) separating the target cells from the sample by a
separation method including: (i) labeling the target cells with
magnetic particles having a specific affinity for the target cells,
thereby producing a population of labeled cells in the sample, (ii)
passing the sample through a fluidic device including a sorting
region having a magnetic field gradient effective to deflect and/or
trap at least a portion of the population of labeled cells from the
sample and thereby separate the target cells from the sample, where
the sample flows in a sheath of buffer solution to reduce
nonspecific binding of the sample to the fluidic device; and (c)
deriving a cell based product from the target cells separated from
the sample in (b). In one embodiment, the method further includes
treating the target cells separated from the sample in (b) to
produce the cell based product. In one embodiment, the target cells
are stem cells and treating the target cells including treating the
stem cells to become an effective therapeutic agent. In one
embodiment, treating the stem cells to become an effective
therapeutic agent includes differentiating the stem cells to
produce a more specific cell type. As with other methods described
herein, the sample can be a fluid sample taken from a patient, for
example, a blood sample.
[0183] In one embodiment, the method further includes
characterizing the target cells separated from the sample in (b).
The characterization can be a count of the target cells and/or a
molecular characterization of the target cells. In one embodiment,
the molecular characterization is a genetic sequence of the target
cells.
EXAMPLES
[0184] Presented below are working and prophetic examples. Example
1 demonstrates the present device in magnetophoretically sorting
rare cells from complex blood samples. Example 2 describes kit on
chip embodiments of the invention. Examples 3-7 describe
embodiments where biologics or chemicals are part of the sample
fluids. Examples 8 and 9 describe embodiments where the devices of
the invention are used for monitoring, for example monitoring of
environmental processes and biopharmaceutical manufacturing,
respectively. Example 10 describes using devices of the invention
for point of care diagnostics. Example 11 demonstrates comparative
enrichment of hematopoietic progenitor cells (HPC's) from human
cord blood samples.
Example 1
Isolation of Rare Cells From Whole Blood, and Comparison to Device
Without Sheath Flow
[0185] This working example demonstrates that the present sheath
flow devices establish a fluid layer substantially preventing
non-specific binding of non-target blood cells to a magnetophoretic
trapping station.
[0186] We have demonstrated the microfluidic separation to enrich
extremely rare target cells in 10.sup.6 MNC's (mononuclear cells)
and whole blood based on magnetic force, which is termed as
Multi-stream Micro Magnetic Separator (M-MMS or MMS).
[0187] The device illustrated in FIG. 6A and photographed in FIG.
6B was used. FIG. 6A is an exploded perspective illustrating one
embodiment of sheath flow components of an MMS device, 600, of the
present invention. The diagram is exploded for purposes of
illustrating how individual components of the device, when
registered with one another, create sheath flow. In this example,
there are seven plates, 615, 620, 625, 630, 635, 640 and 645, each
configured with access ports and/or channels, configured to create
sheath flow when registered and buffer and sample flow are
introduced to the device.
[0188] Sample inlet port, 601, is where liquid sample is
introduced, as indicated by dashed arrow 602, showing the sample
flow path. Sample flows through analogous ports in plates 620 and
625, and an end portion of a channel in plate 630, prior to
striking area 606 (a sample laminar flow establishing surface) on
the top surface (as illustrated) of plate 635. For convenience,
sample flow arrow 602 is illustrated as if the flow is diverted by
striking area 606 when registered, thus establishing a laminar flow
in a direction parallel to and in the cavity in plate 630.
Similarly, buffer inlet port 604 is where buffer solution is
introduced, as indicated by dashed arrow 603, showing one buffer
flow path. Buffer flows through an analogous end portion of a
channel in plate 620, prior to striking area 605 (a buffer laminar
flow establishing surface) on the top surface (as illustrated) of
plate 625. For convenience, buffer flow arrow 603 is illustrated as
if the flow is diverted by striking area 605, thus establishing a
laminar flow in a direction parallel to and in the cavity in plate
625. Also similarly, another buffer inlet port 608 is where buffer
is introduced, as indicated by dashed arrow 609, showing another
buffer flow path. Buffer flows through an analogous end portion of
a channel in plate 640, prior to striking area 607 (a buffer
laminar flow establishing surface) on the bottom surface (as
illustrated) of plate 635. For convenience, buffer flow arrow 609
is illustrated as if the flow is diverted by striking area 607,
thus establishing a laminar flow in a direction parallel to and in
the cavity in plate 635.
[0189] Arrow 611 depicts a common waste flow path as flows 602, 603
and 609 form a confluence at the distal end of the channels in
plates 620, 625, 630, 635 and 640 (when registered) and exit the
device via exit port 610 as indicated by dashed arrow 611.
[0190] Referring to FIG. 6B, a thin layer of nickel grid is
patterned on the top surface of the channel (e.g., as described in
relation to FIGS. 2A and 2B; seen in FIG. 6B as the grid pattern in
shades of gray). The nickel grid is configured to generate a strong
magnetic field gradient with the presence of external magnets and
thus permit the capture of magnetic beads labeled target analytes.
The unlabeled, background cells are not affected by the magnetic
field gradient and are continuously eluted as waste. Since
initially only pure sheath flow contact the top surface, only
magnetically trapped target analytes will be trapped on the top
surface, with minimum nonspecific binding to the surface, resulting
in extremely high purity samples. FIG. 6B shows sample inlet
fitting, 650, for example a leurlock, upper and lower buffer inlet
fittings, 655 and 660, respectively, and waste outlet fitting 665.
These fittings allow, for example, sample to be introduced via
syringe, while buffer is continually pumped into device 600 via
dedicated lines. Waste is removed, for example, to a central
reservoir via a dedicated waste line. Devices can be switched out
by simply disconnecting the lines and reconnecting to a new
device.
[0191] FIG. 6C shows a cross section, A, of a portion (upper left
quadrant) of device 600. The bottom portion of FIG. 6C, the
expanded cross section, shows sample and buffer flow paths, how
they are established adjacent to each other, and how the sample
flow is prevented from touching the top and bottom inner surfaces
of the sheath flow device 600. On the right-most portion of the
cross section the sheath of laminar buffer flow above and below the
laminar sample flow is illustrated (dashed arrows indicate flow
direction for each of top buffer, sample and bottom buffer.
[0192] FIG. 7A is an exploded perspective illustrating components
of a similar sheath flow device, 700. Sheath flow device 700 has
layers 705, 710, 715, 720, 725, 730 and 735, which when registered
adjacent to each other in a stack as depicted, form device 700.
Device 700 differs from device 600 because device 700 has all the
inlets (lower buffer inlet, 740, upper buffer inlet, 745, and
sample inlet, 755) and outlet (waste outlet 750) on top plate 705
(recall device 600 had a lower buffer inlet on the bottom plate).
This allows for convenient handling, since device 700 can be
positioned on a flat surface during operation. In this example, top
plate 705 is glass with a nickel trapping grid, layers 710, 720 and
730 are adhesive layers, for example, about 0.2 mm thick, layers
715 and 725 are polycarbonate, for example, about 0.125 mm thick,
and layer 735 is glass. FIG. 7B is a perspective of the assembled
sheath flow device 700 depicted in FIG. 7A.
[0193] The present sheath flow device was configured such that the
bottom fluid layer was matched the viscosity and density of blood,
with 50% (w/w) sucrose.
[0194] Sheath flow efficacy was first demonstrated by injecting
whole blood (in the absence of magnetic particles), at equal flow
rates, in the chip with and without sheath flow for comparison. As
shown in FIG. 4 (left panel), without sheath flow RBC's and other
cells nonspecifically attached to the surface. In FIG. 4 (right
panel) the sheath flow device demonstrated efficacy because the
surface is substantially free of non-target cells.
[0195] To quantify M-MMS ability to recover and retrieve rare
cells, MCF-7 breast cancer cells were pre-labeled with commercially
available nano-sized magnetic particles (anti-PE magnetic
particles, BD Biosciences). Pre-labeled MCF-7 tumor cells were
spiked into whole blood at 50 cells/ml, 30 cells/ml or 10 cells/ml.
The results are presented in the graphs in FIGS. 5A and 5B. The
average purity is 69%, 58% and 20% (for 50, 30, and 10 cells/ml
respectively (5A)). This is around 1.times.10.sup.7 fold enrichment
over the initial sample. The average cell recovery is 86%, 94% and
92% (for 50, 30 and 10 cells/ml respectively (5B)).
[0196] Using in-situ labeling, about 90% recovery of target cells
was achieved. The cell purity is about 13%, which is
2.times.10.sup.6 fold enrichment of the initial sample. Presumably
this was not as efficient as pre-labeled sorting because of the
inefficiencies related to in situ magnetic labeling.
[0197] As such, the present invention provides fluidic sheath flow
devices including a first fluid layer of laminar sheath flow in a
plane adjacent to and parallel with a sample fluid layer in a
laminar flow plane, and, optionally, a second fluid layer of
laminar sheath flow adjacent to a second side of the sample fluid
in a laminar flow plane, where sheath flow is established first on
a solid support.
Example 2
"Kit on a Chip"
[0198] This is a prophetic example. A sheath flow device of the
present invention is manufactured, according to a predefined
fluidic circuitry. The device is manufactured using fluid
dispensing automation instrumentation to pre-fill selected
reservoirs with desired fluids. The reservoirs are sealed, with a
portion of the pre-filled reservoir having a seal that will burst
with predetermined tensile force, such that the fluid is in fluid
communication with a different reservoir. There are several
reservoirs prefilled for a particular purpose, and fluid circuitry
allows the fluids to flow to a predetermined area upon application
of suitable force, such as pneumatic force. Thus, sheath flows of
the instant invention are achieved via fluid leaving reservoirs and
entering devices of the instant invention, e.g. as described in
FIGS. 1 and 2, which are part of the predefined fluidic circuitry.
In one example, automated instrumentation applies force with
pneumatic pistons in a predetermined temporal pattern coordinating
with the fluidic circuitry of the device to create sheath
flows.
Example 3
Biomarker Detection
[0199] This is a prophetic example. A sheath flow device of the
present invention is configured with fluidic circuitry for sorting
a biomarker from a biological fluid obtained from an individual.
The biomarker presence indicates a particular disease state. The
biomarker is selected from among a cell, a protein, a nucleic acid,
or a degradation product of any of the above. The disease state is
selected from among a cancer, a neurological disease, and an
infection. The cancer biomarker is selected from among a
circulating tumor cell, a protein, and a nucleic acid. The
neurological disease biomarker is selected from among a cell, a
protein including but not limited to an abeta 1-42 protein or
fragment or oligomer thereof, or other biomarker for a neurological
disease selected from among Alzheimer's disease, Huntington's
disease, Amylateral sclerosis ("ALS" or Lou Gehrig's disease), a
dementia, multiple sclerosis, and a disease caused by a prion. The
biomarker for infection is selected from an infectious agent and a
secondary pathogen or detectable marker of deleterious effect, and
includes, but is not limited to, a virus, bacteria, a fungus, a
prion and any other type of infectious agent. The virus may be an
HIV virus, a hepatitis virus (of any type), a flu virus (of any
type), a papilloma virus (HPV) of any type, a rabies virus, or any
other viral infectious agent. The biomarker may be a portion of the
organism or infectious agent so listed. For example, the biomarker
may be a protein associated with a viral coat.
Example 4
Aptamer Screening
[0200] This is a prophetic example. A sheath flow device of the
present invention is configured with fluidic circuitry for use of
an aptamer for detection of a rare molecule in a fluid sample. The
aptamer is optionally associated with a detectable marker. The
aptamer is exposed to a fluidic suspension under conditions for it
to bind to its target. The aptamer and target are captured in a
trapping station located within a reservoir in the present sheath
flow device. Non-target material is washed away with fluid (e.g.,
buffer) applied using pneumatic (or other force) force insufficient
to dislodge the aptamer/target from the trapping station. This
prophetic example may be used, for instance, to detect substances
in urine, blood, or other bodily fluid. For example, one may detect
trace amounts of cocaine or other illicit ingested pharmacological
agents in urine. See, e.g., Swensen, J. S. et al. Continuous,
real-time monitoring of cocaine in undiluted blood serum via a
microfluidic, electrochemical aptamer-based sensor. J. Am. Chem.
Soc. doi:10.1021/ja806531z (2009), herein incorporated by reference
for all purposes.
Example 5
Testing for Analytes in Body Fluid
[0201] This is a prophetic example. A sheath flow device of the
present invention is configured for fluidic circuitry so that an
individual (such as a human or animal) may be monitored for drug,
illicit or not, presence or dosages. The present devices may be
configured to detect or monitor medically prescribed dosages,
pharmacokinetic, body or brain performance enhancing, illicit
(methamphetamine, cocaine, marijuana (cannabinoids)) or endocrine
related, such as glucose (insulin). For example, a sheath flow
device of the present invention is configured to provide prefilled
reservoirs (or chambers) with reagents suitable for detecting
pharmaceutical or pharmaceutical degradation or downstream
metabolic agents, in a bodily fluid, such as blood or urine. A
sheath flow device is configured so that a blood (for example)
sample is dispensed into a reservoir, and reservoirs pre-filled
with suitable reagents are then permitted to open with applied
force, such as manual tensile pressure. The reagents when so
combined with the bodily fluid provide a visible detection of
whether the patient is properly dosed.
Example 6
Chemical Library Screening, Including Aptamer
[0202] This is a prophetic example. A sheath flow device of the
present invention is configured with fluidic circuitry for
screening a library of chemicals for a particular purpose. For
example, a library of aptamers may be screened against a protein
target, such as by using a phage display. The aptamer/protein
complexes may be analyzed to identify the aptamers so binding, and
any binding characteristics, and the enriched aptamers may be
subjected again to library screening. This may be performed in an
iterative process to select aptameric moieties with particular
characteristics, such as binding affinities or binding to
particular epitopes on a protein moiety for example. A sheath flow
device of this example will have a reservoir for holding, and
optionally culturing a phage display population of a predetermined
protein, and inlet port or a prefilled chamber with the subject
aptameric library to be so screened. Alternatively, one may have a
reservoir holding an aptameric library to which is dispensed a
desired protein (or other substrate for selection). The binding
reaction may be aided with force applied to a reservoir to admix
the aptamer library and protein (or other source).
Example 7
Genome Screening; DNA Analysis
[0203] This is a prophetic example. A sheath flow device of the
present invention is configured with microfluidic circuitry and
used in nucleic acid sorting. A sample of DNA is either placed
within the device, or cells containing DNA are placed within the
device, in fluid communication with reservoirs and channels for
delivering reagents suitable to bind to particular DNA sequences
(and optionally lyse cells to expose internal DNA if so desired or
required). For example, DNA primers are used to bind to specific
corresponding DNA sequences. The primers are applied to the
reservoir containing the subject DNA (such as a genome or forensic
sample). A wash fluid is added to the chamber to wash away unbound
moieties. The primer/DNA is then exposed to several rounds of
polymerase chain reaction, including applying reagent. The reagents
are suitably mixed using automated instrumentation for applying
tensile strength.
Example 8
Environmental Monitoring
[0204] This example is a prophetic example. A sheath flow device of
the present invention is configured with suitable materials and
fluidic circuitry for environmental monitoring or analysis. While
environmental fluid sample processing has much in common with
aqueous fluid processing from biological fluids (above),
modifications for field use include rugged material (e.g., made to
withstand extremes in temperature, sunlight, salinity, or other
environmental conditions), and use in the absence of reliable
electricity. For example, a homeowner may wish to monitor drinking
water, but collecting drinking water in a subject over a period of
time, and analyzing once. Or, the present sheath flow devices can
be used for monitoring microbial species indicators for oil and gas
drilling, where certain species are known to be associated with
particular oil or gas containing geologic formations. Thus, one of
ordinary skill in the art selects materials able to withstand
sample application under these conditions. Sheath flow devices of
the invention can be further configured so that manual (hand or
hand-held tool) applied pressure is sufficient for fluidic flow in
the sheath flow path. Drinking or environmental water (such as
saline or fresh water sources), soil (such as soil remediation),
PCB or superfund site clean-up monitoring, environmental radiation
monitoring, repopulation (such as algae or krill) or other
ecological purposes, as well as residential environmental
monitoring (such as water, air or soil sample monitoring or
analysis, including drinking or swimming pool water). One may use a
prefilled device containing aptamers (for example, or other
selective binding molecules) that selectively bind to heavy metals,
such as mercury, lead, iron, or even gold or silver (for
prospecting).One may monitor environmental toxins, such as arsenic,
undue pharmaceutical environmental contamination, MBE's or other
organic solvents. Acidification of oceanic areas, such as the
continental shelf areas, may be performed with the inclusion of
acidification indicators (e.g., colorimetric strips) for
example.
Example 9
Monitoring Biopharmaceutical Manufacturing
[0205] This is a prophetic example. The present sheath flow devices
may be used in the manufacture of biologicals for monitoring during
the biological process. For example, one may collect protein from a
separate bioreactor at various stages to monitor protein production
for lot-to-lot variation. Vaccine manufacturing may also be
monitored in this way. A variety of biologicals and
biopharmaceutics can be monitored for quality assurance purposes
using the present sheath flow devices.
Example 10
Point of Care, Diagnostic
[0206] This is a prophetic example. The present sheath flow devices
are configured suitably for various point of care blood panel
analyses typically performed in a clinical laboratory. The present
sheath flow devices are configured so that a patient's blood is
first deposited into a reservoir, and then, using tensile pressure,
directed to flow to be partitioned in separate reservoirs. The
blood sample so partitioned into individual reservoirs is then
separately exposed to moieties used in such clinical laboratory
practice, such as stains or dyes, or antibodies. Alternatively or
additionally, the blood so partitioned may be exposed to
alternative reagents better suited for the intended purpose, such
as liver enzyme, blood sugar, thyroid, protein C or other blood
moieties.
Example 11
Comparative Enrichment of Hematopoietic Progenitor Cells (HPC's)
from Human Cord Blood Samples
[0207] The example device of the invention, as described in Example
1 above employing sheath flow and magnetic trapping, was compared
on a side-by-side basis with a commercially available MACS.RTM.
Cell Separation Column (available from Miltenyi Biotec of Bergisch
Gladbach, Germany), a device for cell separation employing magnetic
trapping of cells labeled with magnetic particles, but without
sheath flow capability.
[0208] Frozen or 2-day old fresh cord blood multinucleate cells
(MNC's) were first filtered to remove any dead cells. The sample
was then labeled with Miltenyi CD34 Microbeads (available from
Miltenyi Biotec of Bergisch Gladbach, Germany. The labeled sample
was divided and one portion was run through the Miltenyi separation
column described above and the other portion was run through the
sheath flow magnetic separation device of the invention as
described, for example, in relation to Example 1. The a portion of
the sample run through the Miltenyi column was run again through
another Miltenyi column so that a comparison of one pass (MACS 1x)
and two passes (MACS 2x) through the Miltenyi system could be
compared to a single pass through the sheath flow device (MMS) of
the invention. Each of the three purified samples was analyzed
using standard immuno-fluorescence staining and FACS analysis as
one of ordinary skill in the art would appreciate.
[0209] FIGS. 9A and 9B show the results of the FACS analysis of
HPC's isolated using the above described methods, MACS 1x, MACs 2x
and MMS, respectively. The data is compiled for N=6 runs as
described above, with error bars included to show standard
deviation. FIG. 9A shows that the HPC purity from the MMS
separation is much better than MACS 1x and perhaps better but at
least comparable to MACS 2x. This demonstrates the improved
capabilities of the MMS technology over conventional, non-sheath
flow, devices. FIG. 9B shows that HPC recovery based on each of
MACS 1x, MACS 2x and MMS, respectively. Although the MACS 1x showed
higher recovery, the samples were of lower purity (see FIG. 9A)
than that of MACS 2x or MMS. The results also show that recovery
(and purity) with MMS is comparable to MACS 2x, but with only a
single pass through the MMS device as compared to double elution
with the MACS conventional device to achieve the equivalent purity
and recovery.
[0210] There are a wide variety of configurations and applications,
and one of ordinary skill in the art will ascertain these in view
of the present disclosure. The present invention is not limited by
the specific examples described herein.
* * * * *