U.S. patent application number 17/345451 was filed with the patent office on 2021-12-02 for microfluidic device for cell separation and uses thereof.
The applicant listed for this patent is The General Hospital Corporation. Invention is credited to Ravi Kapur, Mehmet Toner, George Truskey.
Application Number | 20210370298 17/345451 |
Document ID | / |
Family ID | 1000005779681 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210370298 |
Kind Code |
A1 |
Toner; Mehmet ; et
al. |
December 2, 2021 |
Microfluidic Device For Cell Separation And Uses Thereof
Abstract
Methods for separating cells from a sample (e.g., separating
fetal red blood cells from maternal blood) include introducing a
sample including cells into one or more microfluidic channels. In
one embodiment, the device includes at least two processing steps.
For example, a mixture of cells is introduced into a microfluidic
channel that selectively allows the passage of a desired type of
cell, and the population of cells enriched in the desired type is
then introduced into a second microfluidic channel that allows the
passage of the desired cell to produce a population of cells
further enriched in the desired type. The selection of cells is
based on a property of the cells in the mixture, for example, size,
shape, deformability, surface characteristics (e.g., cell surface
receptors or antigens and membrane permeability), or intracellular
properties (e.g., expression of a particular enzyme).
Inventors: |
Toner; Mehmet; (Charlestown,
MA) ; Truskey; George; (Durham, NJ) ; Kapur;
Ravi; (Sharon, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation |
Boston |
MA |
US |
|
|
Family ID: |
1000005779681 |
Appl. No.: |
17/345451 |
Filed: |
June 11, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16138428 |
Sep 21, 2018 |
11052392 |
|
|
17345451 |
|
|
|
|
15356276 |
Nov 18, 2016 |
10081014 |
|
|
16138428 |
|
|
|
|
14665708 |
Mar 23, 2015 |
|
|
|
15356276 |
|
|
|
|
11726231 |
Mar 21, 2007 |
8986966 |
|
|
14665708 |
|
|
|
|
10529453 |
Dec 19, 2005 |
8895298 |
|
|
PCT/US2003/030965 |
Sep 29, 2003 |
|
|
|
11726231 |
|
|
|
|
60414258 |
Sep 27, 2002 |
|
|
|
60414065 |
Sep 27, 2002 |
|
|
|
60414102 |
Sep 27, 2002 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/70589
20130101; G01N 1/405 20130101; B01L 3/502753 20130101; B01L
2300/0877 20130101; G01N 2333/70582 20130101; B82Y 30/00 20130101;
B33Y 70/00 20141201; B01L 2300/12 20130101; B01L 2400/0487
20130101; B01L 1/52 20190801; B01L 2300/0867 20130101; B01L
2300/0883 20130101; B01L 2400/086 20130101; G01N 33/5091 20130101;
B01L 2300/0864 20130101; B01L 3/502746 20130101; G01N 1/40
20130101; C12N 5/0087 20130101; B33Y 80/00 20141201; B01L 9/54
20130101; G01N 33/54366 20130101; Y10T 29/49982 20150115; G01N
2333/70596 20130101; B01L 2200/0668 20130101; B01L 2200/0652
20130101; B01L 2200/0647 20130101; B01L 3/502761 20130101; B01L
2200/12 20130101; G01N 33/56966 20130101; Y10T 428/24744 20150115;
G01N 2015/1006 20130101; G01N 33/574 20130101; G01N 33/54386
20130101; G01N 2015/1081 20130101; B01L 2300/0681 20130101; B01L
2300/0816 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; B82Y 30/00 20060101 B82Y030/00; G01N 33/50 20060101
G01N033/50; G01N 33/543 20060101 G01N033/543; G01N 33/569 20060101
G01N033/569; G01N 33/574 20060101 G01N033/574; B01L 1/00 20060101
B01L001/00; C12N 5/00 20060101 C12N005/00; G01N 1/40 20060101
G01N001/40 |
Claims
1-34. (canceled)
35. A method of collecting fetal red blood cells from a sample
comprising maternal blood, the method comprising introducing a
sample of maternal blood into a lysis device containing a lysis
reagent to selectively lyse maternal blood cells, to provide a
sample enriched in fetal blood cells compared to maternal blood
cells; and introducing the sample enriched in fetal blood cells
into a cell binding device comprising an array of obstacles coated
with: (i) binding moieties that bind selectively to maternal blood
cells to produce a sample further enriched in fetal blood cells, or
(ii) binding moieties that bind selectively to fetal blood cells to
produce a sample depleted of fetal blood cells; and collecting
fetal cells from: (i) the sample further enriched in fetal blood
cells, or (ii) the obstacles in the cell binding device to which
the fetal blood cells have bound.
36. The method of claim 35, wherein the lysis reagent selective
lyses maternal red blood cells and comprises NH.sub.4Cl (0 to 150
mM)+NaHCO.sub.3 (0.001 to 0.3 mM)+acetazolamide (0.1 to 100
.mu.M)).
37. The method of claim 35, wherein the cell binding device
comprises an array of obstacles coated with binding moieties that
bind selectively to maternal blood cells to produce a sample
further enriched in fetal blood cells.
38. The method of claim 37, wherein the binding moieties are
antibodies.
39. The method of claim 37, wherein the binding moieties comprise
anti-CD45 antibodies that selectively bind to adult white blood
cells.
40. The method of claim 35, wherein the cell binding device
comprises an array of obstacles coated with binding moieties that
bind selectively to fetal blood cells to produce a sample depleted
of fetal blood cells.
41. The method of claim 40, wherein the binding moieties are
antibodies.
42. The method of claim 40, wherein the binding moieties bind to
fetal hemoglobin transferring receptor (CD71), thrombospondin
receptor (CD36), or glycophorin A (GPA), which selectively bind to
fetal blood cells.
43. The method of claim 42, wherein the binding moieties comprise
anti-CD71 antibodies that selectively bind to fetal nucleated red
blood cells.
44. The method of claim 35, wherein the fetal cells are collected
from the sample further enriched in fetal blood cells.
45. The method of claim 44, further comprising analyzing the fetal
cells.
46. The method of claim 35, wherein the fetal cells are collected
from the obstacles in the cell binding device to which the fetal
blood cells have bound.
47. The method of claim 46, further comprising analyzing the fetal
cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/529,453, having a .sctn. 371 date of Dec. 19, 2005, which is
the National Stage of PCT/US03/30965, filed Sep. 29, 2003, which
claims benefit of U.S. Provisional Application Nos. 60/414,065,
60/414,258, and 60/414,102, filed on Sep. 27, 2002, each of which
is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to the fields of medical diagnostics
and microfluidics.
[0003] There are several approaches devised to separate a
population of homogeneous cells from blood. These cell separation
techniques may be grouped into two broad categories: (1) invasive
methods based on the selection of cells fixed and stained using
various cell-specific markers; and (2) noninvasive methods for the
isolation of living cells using a biophysical parameter specific to
a population of cells of interest.
[0004] Invasive techniques include fluorescence activated cell
sorting (FACS), magnetic activated cell sorting (MACS), and
immunomagnetic colloid sorting. FACS is usually a positive
selection technique that uses a fluorescently labeled marker to
bind to cells expressing a specific cell surface marker. FACS can
also be used to permeabilize and stain cells for intracellular
markers that can constitute the basis for sorting. It is fast,
typically running at a rate of 1,000 to 1,500 Hz, and well
established in laboratory medicine. High false positive rates are
associated with FACS because of the low number of photons obtained
during extremely short dwell times at high speeds. Complicated
multiparameter classification approaches can be used to enhance the
specificity of FACS, but multianalyte-based FACS may be impractical
for routine clinical testing because of the high cost associated
with it. The clinical application of FACS is further limited
because it requires considerable operator expertise, is laborious,
results in cell loss due to multiple manipulations, and the cost of
the equipment is prohibitive.
[0005] MACS is used as a cell separation technique in which cells
that express a specific surface marker are isolated from a mixture
of cells using magnetic beads coated with an antibody against the
surface marker. MACS has the advantage of being cheaper, easier,
and faster to perform as compared with FACS. It suffers from cell
loss due to multiple manipulations and handling. Moreover, magnetic
beads often autofluoresce and are not easily separated from cells.
As a result, many of the immunofluorescence techniques used to
probe into cellular function and structure are not compatible with
this approach.
[0006] A magnetic colloid system has been used in the isolation of
cells from blood. This colloid system uses ferromagnetic
nanoparticles that are coated with goat anti-mouse IgG that can be
easily attached to cell surface antigen-specific monoclonal
antibodies. Cells that are labeled with ferromagnetic nanoparticles
align in a magnetic field along ferromagnetic Ni lines deposited by
lithographic techniques on an optically transparent surface. This
approach also requires multiple cell handling steps including
mixing of cells with magnetic beads and separation on the surfaces.
It is also not possible to sort out the individual cells from the
sample for further analysis.
[0007] Noninvasive techniques include charge flow separation, which
employs a horizontal crossflow fluid gradient opposing an electric
field in order to separate cells based on their characteristic
surface charge densities. Although this approach can separate cells
purely on biophysical differences, it is not specific enough. There
have been attempts to modify the device characteristics (e.g.,
separator screens, buffer counterflow conditions, etc.) to address
this major shortcoming of the technique. None of these
modifications of device characteristics has provided a practical
solution given the expected individual variability in different
samples.
[0008] Since the prior art methods suffer from high cost, low
yield, and lack of specificity, there is a need for a method for
depleting a particular type of cell from a mixture that overcomes
these limitations.
SUMMARY OF THE INVENTION
[0009] The invention features methods for separating cells from a
sample (e.g., separating fetal red blood cells from maternal
blood). The method begins with the introduction of a sample
including cells into one or more microfluidic channels. In one
embodiment, the device includes at least two processing steps. For
example, a mixture of cells is introduced into a microfluidic
channel that selectively allows the passage of a desired type of
cell, and the population of cells enriched in the desired type is
then introduced into a second microfluidic channel that allows the
passage of the desired cell to produce a population of cells
further enriched in the desired type. The selection of cells is
based on a property of the cells in the mixture, for example, size,
shape, deformability, surface characteristics (e.g., cell surface
receptors or antigens and membrane permeability), or intracellular
properties (e.g., expression of a particular enzyme).
[0010] In practice, the method may then proceed through a variety
of processing steps employing various devices. In one step, the
sample is combined with a solution in the microfluidic channels
that preferentially lyses one type of cell compared to another
type. In another step, cells are contacted with a device containing
obstacles in a microfluidic channel. The obstacles preferentially
bind one type of cell compared to another type. Alternatively,
cells are arrayed individually for identification of the cells of
interest. Cells may also be subjected to size, deformability, or
shape based separations. Methods of the invention may employ only
one of the above steps or any combination of the steps, in any
order, to separate cells. The methods of the invention desirably
recover at least 75%, 80%, 90%, 95%, 98%, or 99% of the desired
cells in the sample.
[0011] The invention further features a microfluidic system for the
separation of a desired cell from a sample. This system may include
devices for carrying out one or any combination of the steps of the
above-described methods. One of these devices is a lysis device
that includes at least two input channels; a reaction chamber
(e.g., a serpentine channel); and an outlet channel. The device may
additional include another input and a dilution chamber (e.g., a
serpentine channel). The lysis device is arranged such that at
least two input channels are connected to the outlet through the
reaction chamber. When a dilution chamber is present, it is
disposed between the reaction chamber and the outlet, and another
inlet is disposed between the reaction and dilution chambers. The
system may also include a cell depletion device that contains
obstacles that preferentially bind one type of cell when compared
to another type, e.g., they are coated with anti-CD45, anti-CD36,
anti-GPA, or anti-CD71 antibodies. The system may also include an
arraying device that contains a two-dimensional array of locations
for the containment of individual cells. The arraying device may
also contain actuators for the selective manipulation (e.g.,
release) of individual cells in the array. Finally, the system may
include a device for size based separation of cells. This device
includes sieves that only allow passage of cells below a desired
size. The sieves are located with a microfluidic channel through
which a suspension of cells passes, as described herein. When used
in combination, the devices in the system may be in liquid
communication with one another. Alternatively, samples that pass
through a device may be collected and transferred to another
device.
[0012] By "a depleted cell population" is meant a population of
cells that has been processed to decrease the relative population
of a specified cell type in a mixture of cells. Subsequently
collecting those cells depleted from the mixture also leads to a
sample enriched in the cells depleted.
[0013] By an "enriched cell population" is meant a population of
cells that has been processed to increase the relative population
of a specified cell type in a mixture of cells.
[0014] By "lysis buffer" is meant a buffer that, when contacted
with a population of cells, will cause at least one type of cell to
lyse.
[0015] By "to cause lysis" is meant to lyse at least 90% of cells
of a particular type.
[0016] By "not lysed" is meant less than 10% of cells of a
particular type are lysed. Desirably, less that 5%, 2%, or 1% of
these cells are lysed.
[0017] By "type" of cell is meant a population of cells having a
common property, e.g., the presence of a particular surface
antigen. A single cell may belong to several different types of
cells.
[0018] By "serpentine channel" is meant a channel that has a total
length that is greater than the linear distance between the end
points of the channel. A serpentine channel may be oriented
entirely vertically or horizontally. Alternatively, a serpentine
channel may be "3D," e.g., portions of the channel are oriented
vertically and portions are oriented horizontally.
[0019] By "microfluidic" is meant having one or more dimensions of
less than 1 mm.
[0020] By "binding moiety" is meant a chemical species to which a
cell binds. A binding moiety may be a compound coupled to a surface
or the material making up the surface. Exemplary binding moieties
include antibodies, oligo- or polypeptides, nucleic acids, other
proteins, synthetic polymers, and carbohydrates.
[0021] By "obstacle" is meant an impediment to flow in a channel,
e.g., a protrusion from one surface.
[0022] By "specifically binding" a type of cell is meant binding
cells of that type by a specified mechanism, e.g., antibody-antigen
interaction. The strength of the bond is generally enough to
prevent detachment by the flow of fluid present when cells are
bound, although individual cells may occasionally detach under
normal operating conditions.
[0023] By "rows of obstacles" is meant is meant a series of
obstacles arranged such that the centers of the obstacles are
arranged substantially linearly. The distance between rows is the
distance between the lines of two adjacent rows on which the
centers are located.
[0024] By "columns of obstacles" is meant a series of obstacles
arranged perpendicular to a row such that the centers of the
obstacles are arranged substantially linearly. The distance between
columns is the distance between the lines of two adjacent columns
on which the centers are located.
[0025] The methods of the invention are able to separate specific
populations of cells from a complex mixture without fixing and/or
staining. As a result of obtaining living homogeneous population of
cells, one can perform many functional assays on the cells. The
microfluidic devices described herein provide a simple, selective
approach for processing of cells.
[0026] Other features and advantages of the invention will be
apparent from the following description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a schematic layout of a microfluidic device that
enables selective lysis of cells.
[0028] FIG. 2 is an illustration of the channel layout for the
introduction of three fluids to the device, e.g., blood sample,
lysis buffer, and diluent.
[0029] FIG. 3 is an illustration of a repeating unit of the
reaction chamber of the device where a sample of cells is passively
mixed with a lysis buffer. In one example, 133 units are connected
to form the reaction chamber.
[0030] FIG. 4 is an illustration of the outlet channels of the
device.
[0031] FIG. 5 is an illustration of a device for cell lysis.
[0032] FIGS. 6A and 6B are illustrations of a method for the
fabrication of a device of the invention.
[0033] FIG. 7 is a schematic diagram of a cell binding device.
[0034] FIG. 8 is an exploded view of a cell binding device.
[0035] FIG. 9 is an illustration of obstacles in a cell binding
device.
[0036] FIG. 10 is an illustration of types of obstacles.
[0037] FIG. 11A is a schematic representation of a square array of
obstacles. The square array has a capture efficiency of 40%. FIG.
11B is a schematic representation of an equilateral triangle array
of obstacles. The equilateral triangle array has a capture
efficiency of 56%.
[0038] FIG. 12A is a schematic representation of the calculation of
the hydrodynamic efficiency for a square array. FIG. 12B is a
schematic representation of the calculation of the hydrodynamic
efficiency for a diagonal array
[0039] FIGS. 13A-13B are graphs of the hydrodynamic (13A) and
overall efficiency (13B) for square array and triangular array for
a pressure drop of 150 Pa/m. This pressure drop corresponds to a
flow rate of 0.75 mL/hr in the planar geometry.
[0040] FIG. 14A is a graph of the overall efficiency as a function
of pressure drop. FIG. 14B is a graph of the effect of the obstacle
separation on the average velocity.
[0041] FIG. 15 is a schematic representation of the arrangement of
obstacles for higher efficiency capture for an equilateral
triangular array of obstacles in a staggered array. The capture
radius r.sub.cap.sub.2=0.3391. The obstacles are numbered such that
the first number refers to the triangle number and the second
number refers to the triangle vertex. The staggered array has a
capture efficiency of 98%.
[0042] FIG. 16A is a graph of the percent capture of cells as a
function of the flow rate for a 100 .mu.m diameter obstacle
geometry with a 50 .mu.m edge-to-edge spacing. The operating flow
regime was established across multiple cell types: cancer cells,
normal connective tissue cells, and maternal and fetal samples. An
optimal working flow regime is at 2.5 ml/hr. FIG. 16B is a graph of
the percent capture of cells as a function of the ratio of targets
cells to white blood cells. The model system was generated by
spiking defined number of either cancer cells, normal connective
tissue cells, or cells from cord blood into defined number of cells
from buffy coat of adult blood. The ratio of the contaminating
cells to target cells was incrementally increased 5 log with as few
as 10 target cells in the mixture. Yield was computed as the
difference between number of spiked target cells captured on posts
and number of cells spiked into the sample.
[0043] FIG. 17 is an illustration of various views of the inlet and
outlets of a cell binding device.
[0044] FIG. 18 is an illustration of a method of fabricating a cell
binding device.
[0045] FIG. 19 is an illustration of a mixture of cells flowing
through a cell binding device.
[0046] FIG. 20A is an illustration of a cell binding device for
trapping different types of cells in series. FIG. 20B is an
illustration of a cell binding device for trapping different types
of cells in parallel.
[0047] FIG. 21 is an illustration of a cell binding device that
enables recovery of bound cells.
[0048] FIG. 22A is an optical micrograph of fetal red blood cells
adhered to an obstacle of the invention. FIG. 22B is a fluorescent
micrograph showing the results of a FISH analysis of a fetal red
blood cell attached to an obstacle of the invention. FIG. 22C is a
close up micrograph of FIG. 22B showing the individual
hybridization results for the fetal red blood cell.
[0049] FIG. 23 is an illustration of a cell binding device in which
beads trapped in a hydrogel are used to capture cells.
[0050] FIG. 24A is an illustration of a device for size based
separation. FIG. 24B is an electron micrograph of a device for size
based separation.
[0051] FIG. 25 is a schematic representation of a device of the
invention for isolating and analyzing fetal red blood cells.
[0052] Figures are not necessarily to scale.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The invention features methods for separating a desired cell
from a mixture or enriching the population of a desired cell in a
mixture. The methods are generally based on sequential processing
steps, each of which reduces the number of undesired cells in the
mixture, but one processing step may be used in the methods of the
invention. Devices for carrying out various processing steps may be
separate or integrated into one microfluidic system. The devices of
the invention are a device for cell lysis, a device for cell
binding, a device for arraying cells, and a device for size, shape,
or deformability based separation. In one embodiment, processing
steps are used to reduce the number of cells prior to arraying.
Desirably, the methods of the invention retain at least 75%, 80%,
90%, 95%, 98%, or 99% of the desired cells compared to the initial
mixture, while potentially enriching the population of desired
cells by a factor of at least 100, 1000, 10,000, 100,000, or even
1,000,000 relative to one or more non-desired cell types. The
methods of the invention may be used to separate or enrich cells
circulating in the blood (Table 1).
TABLE-US-00001 TABLE 1 Types, concentrations, and sizes of blood
cells. Cell Type Concentration (cells/.mu.l) Size (.mu.m) Red blood
cells (RBC) 4.2-6.1 10 .sup.6 4-6 Segmented Neutrophils 3600 >10
(WBC) Band Neutrophils (WBC) 120 >10 Lymphocytes (WBC) 1500
>10 Monocytes (WBC) 480 >10 Eosinophils (WBC) 180 >10
Basophils (WBC) 120 >10 Platelets 500 10 .sup.3 1-2 Fetal
Nucleated Red Blood 2-50 10 .sup.-3 8-12 Cells
Devices
A. Cell Lysis
[0054] One device of the invention is employed to lysis of a
population of cells selectively, e.g., maternal red blood cells, in
a mixture of cells, e.g., maternal blood. This device allows for
the processing of large numbers of cells under nearly identical
conditions. The lysis device desirably removes a large number of
cells prior to further processing. The debris, e.g., cell membranes
and proteins, may be trapped, e.g., by filtration or precipitation,
prior to any further processing.
[0055] Device. A design for a lysis device of the invention is
shown in FIG. 1. The overall branched architecture of the channels
in the device permits equivalent pressure drops across each of the
parallel processing networks. The device can be functionally
separated into four distinct sections: 1) distributed input
channels carrying fluids, e.g., blood, lysis reagent, and wash
buffer, to junctions 1 and 2 (FIG. 2); 2) a serpentine reaction
chamber for the cell lysis reaction residing between the two
junctions (FIG. 3); 3) a dilution chamber downstream of Junction 2
for dilution of the lysis reagent (FIG. 3); and 4) distributed
output channels carrying the lysed sample to a collection vial or
to another microfluidic device (FIG. 4).
[0056] Input/Output Channels. The branched input and output
networks of channels enable even distribution of the reagents into
each of the channels (8, as depicted in FIG. 1). The three ports
for interfacing the macro world with the device typically range in
diameter from 1 mm-10 mm, e.g., 2, 5, 6, or 8 mm. Air tight seals
may be formed with ports 1, 2, and 3, e.g., through an external
manifold integrated with the device (FIG. 1). The three solution
vials, e.g., blood, lysing reagent, and diluent, may interface with
such a manifold. The input channels from ports 1, 2, and 3 to the
reaction and mixing chambers, for the three solutions shown in FIG.
1, may be separated either in the z-plane of the device (three
layers, each with one set of distribution channels, see FIG. 2) or
reside in the external manifold. If residing in the external
manifold, the distribution channels are, for example, CNC (computer
numerically controlled) machined in stainless steel and may have
dimensions of 500 .mu.m diameter. The manifold may hermetically
interface with the device at ports that are etched into locations
1', 2', and 3' shown in FIG. 1. Locating the distribution channels
in a manifold reduces the complexity and cost of the device.
Retaining the distribution channels on the device will allow
greater flexibility in selecting smaller channel size, while
avoiding any issues of carry-over contamination between samples.
Each sample input channel may have a separate output, or as
depicted in FIG. 4, the output channels for each sample input are
combined. As an alternative to a manifold, tubing for each fluid
input or output may be attached to the device, e.g., by compression
fitting to gaskets or nipples or use of watertight connections such
as a luer lock. The channels on the device transporting the fluids
to the mixing junctions and chambers beyond, can range from 10
.mu.m-500 .mu.m in width and depth, e.g., at most 10 .mu.m, 25
.mu.m, 50 .mu.m, 75 .mu.m, 100 .mu.m, 150 .mu.m, 200 .mu.m, 250
.mu.m, 350 .mu.m, or 450 .mu.m width and depth. The channel
architecture is desirably rectangular but may also be circular,
semi-circular, V-shaped, or any other appropriate shape. In one
embodiment, the output channel (or channels) has a cross-sectional
area equal to the sum of the cross-sectional areas of the input
channels.
[0057] Reaction and Dilution Chambers. For lysis and dilution, two
fluid streams are combined and allowed to pass through the
chambers. Chambers may be linear or serpentine channels. In the
device depicted in FIG. 1, the sample and lysis buffer are combined
at junction 1, and the lysed sample and the diluent are combined at
junction 2. Serpentine architecture of the reaction chamber and
dilution chamber enables sufficient resident time of the two
reacting solutions for proper mixing by diffusion or other passive
mechanisms, while preserving a reasonable overall footprint for the
device (FIG. 3). The serpentine channels may be constructed in 2D
or in 3D, e.g., to reduce the total length of the device or to
introduce chaotic advection for enhanced mixing. For short
residence times, a linear chamber may be desired. Exemplary
resident times include at least 1 second, 5 seconds, 10 seconds, 30
seconds, 60 second, 90 seconds, 2 minutes, 5 minutes, 30 minutes, 1
hour, or greater that 1 hour. The flow rate of fluids in the
reaction/dilution chambers can be accurately controlled by
controlling the width, depth, and effective length of the channels
to enable sufficient mixing of the two reagents while enabling
optimal processing throughput. In one embodiment, the serpentine
mixing chambers for cell lysis (reaction chamber) and for dilution
of the lysed sample (dilution chamber) have a fluid volume each of
.about.26 .mu.l. Other examples of reaction/dilution chamber
volumes range from 10-200 .mu.l, e.g., at most 20, 50, 100, or 150
.mu.l. In some embodiments, the width and depth of the reaction and
dilution chambers have the same range as the input and output
channels, i.e., 10 to 500 .mu.m. Alternatively, the chambers may
have a cross-sectional area equal to the combined areas of any
input (or output channels) in order to ensure a uniform velocity of
flow through the device. In one example, the chambers are 100 .mu.m
100 .mu.m channels. The total length of the chambers may be at
least 1 cm, 5 cm, 10 cm, 20 cm, 30 cm, 40 cm, or 50 cm.
[0058] For lysis of maternal RBCs, device output flow rates may
range from processing 5-16 .mu.l of blood per second resulting in a
20-60 minute processing time for 20 ml sample, or 10-30 min
processing time for 10 ml sample. It is expected that the sample
volume required for capturing sufficient number of fetal cells will
be lower than 10 ml because of the efficiency of the process. As
such, it is expected that the device throughput per sample will be
less than 10 minutes. A residence time of >30 seconds from the
time of convergence of the two solutions, maternal blood and lysis
reagent, within the passive mixer is deemed sufficient to obtain
effective hemolysis (T. Maren, Mol. Pharmacol. 1970, 6:430).
Alternatively, the concentration of the lysis reagent can be
adjusted to compensate for residence time in the reaction chamber.
The flow rates and residence times for other cell types may be
determined by theory or experimentation. In one embodiment, the
flow rates in each channel are limited to <20 .mu.l/sec to
ensure that wall shear stress on cells is less than 1 dyne/cm.sup.2
(cells are known to be affected functionally by shear stress >1
dyne/cm.sup.2 though deleterious effects are not seen in most cells
until after 10 dynes/cm.sup.2). In one embodiment, the flow rate in
each channel is at most 1, 2, 5, 10, 15 .mu.l/sec. Referring to
FIG. 1, the effective length of the diluent input channel leading
to junction 2 may be shorter than the effective length of the
reaction chamber. This feature enables the diluent to flow into and
prime the channels downstream of junction 2, prior to arrival of
the lysed sample at junction 2. The overflow buffer pre-collected
in the output vial may act as a secondary diluent of the lysed
sample when collected, e.g., for further processing or analysis.
Additionally, the diluent primes the channels downstream of
junction 2 to enable smoother flow and merging of the lysed sample
with the buffer in the diluting chamber, and this priming
eliminates any deleterious surface tension effects from dry
channels on the lysed sample. The diameter of the channels carrying
the diluent may be adjusted to enable the diluent to reach junction
2 at the same time as the lysed blood to prevent any problems
associated with air forced from the reaction chamber as the sample
and lysis buffers are introduced.
[0059] Although the above description focuses on a device with
eight parallel processing channels, any number of channels, e.g.,
1, 2, 4, 16, or 32, may be employed depending on the size of the
device. The device is described in terms of combining two fluids
for lysis and dilution, but three or more fluids may be combined
for lysis or dilution. The combination may be at one junction or a
series of junctions, e.g., to control the timing of the sequential
addition of reactants. Additional fluid inputs may be added, e.g.,
to functionalize the remaining cells, alter the pH, or cause
undesirable components to precipitate. In addition, the exact
geometry and dimensions of the channels may be altered (exemplary
dimensions are shown in FIG. 5). Devices of the invention may be
disposable or reusable. Disposable devices reduce the risk of
contamination between samples. Reusable devices may be desirable in
certain instances, and the device may be cleaned, e.g., with
various detergents and enzymes, e.g., proteases or nucleases, to
prevent contamination.
[0060] Pumping. In one embodiment, the device employs negative
pressure pumping, e.g., using syringe pumps, peristaltic pumps,
aspirators, or vacuum pumps. The negative pressure allows for
processing of the complete volume of a clinical blood sample,
without leaving unprocessed sample in the channels. Positive
pressure, e.g., from a syringe pump, peristaltic pump, displacement
pump, column of fluid, or other fluid pump, may also be used to
pump samples through a device. The loss of sample due to dead
volume issues related to positive pressure pumping may be overcome
by chasing the residual sample with buffer. Pumps are typically
interfaced to the device via hermetic seals, e.g., using silicone
gaskets.
[0061] The flow rates of fluids in parallel channels in the device
may be controlled in unison or separately. Variable and
differential control of the flow rates in each of channels may be
achieved, for example, by employing, a multi-channel individually
controllable syringe manifold. In this embodiment, the input
channel distribution will be modified to decouple all of the
parallel networks. The output may collect the output from all
channels via a single manifold connected to a suction (no
requirements for an airtight seal) outputting to a collection vial
or to another microfluidic device. Alternately, the output from
each network can be collected separately for downstream processing.
Separate inputs and outputs allow for parallel processing of
multiple samples from one or more individuals.
[0062] Fabrication. A variety of techniques can be employed to
fabricate a device of the invention, and the technique employed
will be selected based in part on the material of choice. Exemplary
materials for fabricating the devices of the invention include
glass, silicon, steel, nickel, poly(methylmethacrylate) (PMMA),
polycarbonate, polystyrene, polyethylene, polyolefins, silicones
(e.g., poly(dimethylsiloxane)), and combinations thereof. Other
materials are known in the art. Methods for fabricating channels in
these materials are known in the art. These methods include,
photolithography (e.g., stereolithography or x-ray
photolithography), molding, embossing, silicon micromachining, wet
or dry chemical etching, milling, diamond cutting, Lithographie
Galvanoformung and Abformung (LIGA), and electroplating. For
example, for glass, traditional silicon fabrication techniques of
photolithography followed by wet (KOH) or dry etching (reactive ion
etching with fluorine or other reactive gas) can be employed.
Techniques such as laser micromachining can be adopted for plastic
materials with high photon absorption efficiency. This technique is
suitable for lower throughput fabrication because of the serial
nature of the process. For mass-produced plastic devices,
thermoplastic injection molding, and compression molding is
suitable. Conventional thermoplastic injection molding used for
mass-fabrication of compact discs (which preserves fidelity of
features in sub-microns) may also be employed to fabricate the
devices of the invention. For example, the device features are
replicated on a glass master by conventional photolithography. The
glass master is electroformed to yield a tough, thermal shock
resistant, thermally conductive, hard mold. This mold serves as the
master template for injection molding or compression molding the
features into a plastic device. Depending on the plastic material
used to fabricate the devices and the requirements on optical
quality and throughput of the finished product, compression molding
or injection molding may be chosen as the method of manufacture.
Compression molding (also called hot embossing or relief
imprinting) has the advantages of being compatible with
high-molecular weight polymers, which are excellent for small
structures, but is difficult to use in replicating high aspect
ratio structures and has longer cycle times. Injection molding
works well for high-aspect ratio structures but is most suitable
for low molecular weight polymers.
[0063] A device may be fabricated in one or more pieces that are
then assembled. In one embodiment, separate layers of the device
contain channels for a single fluid, as in FIG. 1. Layers of a
device may be bonded together by clamps, adhesives, heat, anodic
bonding, or reactions between surface groups (e.g., wafer bonding).
Alternatively, a device with channels in more than one plane may be
fabricated as a single piece, e.g., using stereolithography or
other three-dimensional fabrication techniques.
[0064] In one embodiment, the device is made of PMMA. The features,
for example those shown in FIG. 1, are transferred onto an
electroformed mold using standard photolithography followed by
electroplating. The mold is used to hot emboss the features into
the PMMA at a temperature near its glass transition temperature
(105.degree. C.) under pressure (5 to 20 tons) (pressure and
temperature will be adjusted to account for high-fidelity
replication of the deepest feature in the device) as shown
schematically in FIG. 6A. The mold is then cooled to enable removal
of the PMMA device. A second piece used to seal the device,
composed of similar or dissimilar material, may be bonded onto the
first piece using vacuum-assisted thermal bonding. The vacuum
prevents formation of air-gaps in the bonding regions. FIG. 6B
shows a cross-section of the two-piece device assembly at the
junction of Port 1 (source for blood sample) and feed channel.
[0065] Chemical Derivitization. To reduce non-specific adsorption
of cells or compounds released by lysed cells onto the channel
walls, one or more channel walls may be chemically modified to be
non-adherent or repulsive. The walls may be coated with a thin film
coating (e.g., a monolayer) of commercial non-stick reagents, such
as those used to form hydrogels. Additional examples chemical
species that may be used to modify the channel walls include
oligoethylene glycols, fluorinated polymers, organosilanes, thiols,
poly-ethylene glycol, hyaluronic acid, bovine serum albumin,
poly-vinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and
agarose. Charged polymers may also be employed to repel oppositely
charged species. The type of chemical species used for repulsion
and the method of attachment to the channel walls will depend on
the nature of the species being repelled and the nature of the
walls and the species being attached. Such surface modification
techniques are well known in the art. The walls may be
functionalized before or after the device is assembled.
[0066] The channel walls may also be coated in order to capture
materials in the sample, e.g., membrane fragments or proteins.
[0067] Methods. In the present invention, a sample of cells, e.g.,
maternal blood, is introduced into one or more microfluidic
channels. A lysis buffer containing reagents for the selective
lysis for a population of cells in the sample is then mixed with
the blood sample. Desirably, the mixing occurs by passive means,
e.g., diffusion or chaotic advection, but active means may be
employed. Additional passive and active mixers are known in the
art. The lysis reaction is allowed to continue for a desired length
of time. This length of time may be controlled, for example, by the
length of the channels or by the rate of flow of the fluids. In
addition, it is possible to control the volumes of solutions mixed
in the channels by altering the relative volumetric flow rates of
the solutions, e.g., by altering the channel size or velocity of
flow. The flow may be slowed down, increased, or stopped for any
desired period of time. After lysis has occurred, a diluent may be
introduced into the channel in order to reduce the concentration of
the lysis reagents and any potentially harmful species (e.g.,
endosomal enzymes) released by the lysed cells. The diluent may
contain species that neutralize the lysis reagents or otherwise
alter the fluid environment, e.g., pH or viscosity, or it may
contain reagents for surface or intracellular labeling of cells.
The diluent may also reduce the optical density of the solution,
which may be important for certain detection schemes, e.g.,
absorbance measurements.
[0068] Exemplary cell types that may be lysed using the methods
described herein include adult red blood cells, white blood cells
(such as T cells, B cells, and helper T cells), infected white
blood cells, tumor cells, and infectious organisms (e.g., bacteria,
protozoa, and fungi). Lysis buffers for these cells may include
cell specific IgM molecules and proteins in the complement cascade
to initiate complement mediated lysis. Another kind of lysis buffer
may include viruses that infect a specific cell type and cause
lysis as a result of replication (see, e.g., Pawlik et al. Cancer
2002, 95:1171-81). Other lysis buffers are known in the art.
[0069] A device of the invention may be used for the selective
lysis of maternal red blood cells (RBCs) in order to enrich a blood
sample in fetal cells. In this example, a maternal blood sample,
10-20 ml, is processed within the first one to three hours after
sample collection. If the processing is delayed beyond three hours,
the sample may be stored at 4.degree. C. until it is processed. The
lysis device of the invention allows mixing of the lysis reagent
(NH.sub.4Cl (0 to 150 mM)+NaHCO.sub.3 (0.001 to 0.3
mM)+acetazolamide (0.1 to 100 .mu.M)) with the maternal blood to
enable selective lysis of the maternal red blood cells by the
underlying principle of the Orskov-Jacobs-Stewart reaction (see,
for example, Boyer et al. Blood 1976, 47:883-897). The high
selective permeability of the carbonic anhydrase inhibitor,
acetazolamide, into fetal cells enables selective hemolysis of the
maternal red blood cells. Endogenous carbonic anhydrase in the
maternal cells converts HCO.sub.3.sup.- to carbon dioxide, which
lyses the maternal red blood cells. The enzyme is inhibited in the
fetal red blood cells, and those cells are not lysed. A diluent
(e.g., phosphate buffered saline) may be added after a period of
contact between the lysis reagents and the cell sample to reduce
the risk that a portion of the fetal red bloods cells will be lysed
after prolonged exposure to the reagents.
B. Cell Binding
[0070] Another device of the invention involves depletion of whole
cells from a mixture by binding the cells to the surfaces of the
device. The surfaces of such a device contain substances, e.g.,
antibodies or ligands for cell surface receptors, that bind a
particular subpopulation of cells. This step in method may employ
positive selection, i.e., the desired cells are bound to the
device, or it may employ negative selection, i.e., the desired
cells pass through the device. In either case, the population of
cells containing the desired cells is collected for analysis or
further processing.
[0071] Device. The device is a microfluidic flow system containing
an array of obstacles of various shapes that are capable of binding
a population of cells, e.g., those expressing a specific surface
molecule, in a mixture. The bound cells may be directly analyzed on
the device or be removed from the device, e.g., for further
analysis or processing. Alternatively, cells not bound to the
obstacles may be collected, e.g., for further processing or
analysis.
[0072] An exemplary device is a flow apparatus having a flat-plate
channel through which cells flow; such a device is described in
U.S. Pat. No. 5,837,115. FIG. 7 shows an exemplary system including
an infusion pump to perfuse a mixture of cells, e.g., blood,
through the microfluidic device. Other pumping methods, as
described herein, may be employed. The device may be optically
transparent, or have transparent windows, for visualization of
cells during flow through the device. The device contains obstacles
distributed, e.g., in an ordered array or randomly, throughout the
flow chamber. The top and bottom surfaces of the device are
desirably parallel to each other. This concept is depicted in FIG.
8. The obstacles may be either part of the bottom or the top
surface and desirably define the height of the flow channel. It is
also possible for a fraction of the obstacles to be positioned on
the bottom surface, and the remainder on the top surface. The
obstacles may contact both the top and bottom of the chamber, or
there may be a gap between an obstacle and one surface. The
obstacles may be coated with a binding moiety, e.g., an antibody, a
charged polymer, a molecule that binds to a cell surface receptor,
an oligo- or polypeptide, a viral or bacterial protein, a nucleic
acid, or a carbohydrate, that binds a population of cells, e.g.,
those expressing a specific surface molecule, in a mixture. Other
binding moieties that are specific for a particular type of cell
are known in the art. In an alternative embodiment, the obstacles
are fabricated from a material to which a specific type of cell
binds. Examples of such materials include organic polymers (charged
or uncharged) and carbohydrates. Once a binding moiety is coupled
to the obstacles, a coating, as described herein, may also be
applied to any exposed surface of the obstacles to prevent
non-specific adhesion of cells to the obstacles.
[0073] A geometry of obstacles is shown in FIG. 9. In one example,
obstacles are etched on a surface area of 2 cm 7 cm on a substrate
with overall dimensions of 2.5 cm 7.5 cm. A rim of 2 mm is left
around the substrate for bonding to the top surface to create a
closed chamber. In one embodiment, obstacle diameter is 50 .mu.m
with a height of 100 .mu.m. Obstacles may be arranged in a
two-dimensional array of rows with a 100 .mu.m distance from
center-to-center. This arrangement provides 50 .mu.m openings for
cells to flow between the obstacles without being mechanically
squeezed or damaged. The obstacles in one row are desirably
shifted, e.g., 50 .mu.m with respect to the adjacent rows. This
alternating pattern may be repeated throughout the design to ensure
increased collision frequency between cells and obstacles. The
diameter, width, or length of the obstacles may be at least 5, 10,
25, 50, 75, 100, or 250 .mu.m and at most 500, 250, 100, 75, 50,
25, or 10 .mu.m. The spacing between obstacles may be at least 10,
25, 50, 75, 100, 250, 500, or 750 .mu.m and at most 1000, 750, 500,
250, 100, 75, 50, or 25 .mu.m. Table 2 lists exemplary spacings
based on the diameter of the obstacles.
TABLE-US-00002 TABLE 2 Exemplary spacings for obstacles. Obstacle
diameter Spacing between (.mu.m) obstacles (.mu.m) 100 50 100 25 50
50 50 25 10 25 10 50 10 15
[0074] The dimensions and geometry of the obstacles may vary
significantly. For example, the obstacles may have cylindrical or
square cross sections (FIG. 10). The distance between obstacles may
also vary and may be different in the flow direction compared to
the direction orthogonal to the flow. In some embodiments, the
distance between the edges of the obstacles is slightly larger than
the size of the largest cell in the mixture. This arrangement
enables flow of cells without them being mechanically squeezed
between the obstacles and thus damaged during the flow process, and
also maximizes the numbers of collisions between cells and the
obstacles in order to increase the probability of binding. The flow
direction with respect to the orientation of the obstacles may also
be altered to enhance interaction of cells with obstacles.
[0075] Exemplary arrangements of obstacles are shown in FIGS.
11A-11B. Each of these arrangements has a calculated capture
efficiency. The calculation of cell attachment considered two
different geometries: a square array (FIG. 11A), and an equilateral
triangular array (FIG. 11B). Overall, results are presented in
terms of the efficiency of adhesion. The calculations consist of
two parts, computing the hydrodynamic efficiency (.eta.) and the
probability of adhesion. The hydrodynamic efficiency was determined
as the ratio of the capture radius to the half-distance between the
cylinders (FIGS. 12A and 12B). For the square array,
.eta.=(2r.sub.cap/l)*100%, and for other arrays,
.eta.=((r.sub.cap1+r.sub.cap2)/d.sub.1)*100%, where
d.sub.1=d.sub.2=l/ 2 for a diagonal square array, and d.sub.1=l
3/2, d.sub.2=l/2 for a triangular array. The probability of
adhesion represents the fraction of cells that can resist the
applied force on the cell assuming an average of 1.5 bonds per cell
and 75 pN per bond.
[0076] For the triangular array, more cells adhered to the second
set of obstacles than the first set. FIGS. 13A-13B show that the
efficiency declines as the spacing between obstacles increases. As
the spacing increases there is a larger region outside the capture
radius and the cells never contact the obstacles. Further, for the
flow rates examined (0.25-1 mL/h), the overall probability of
adhesion is high because the force pr cell is less than the force
to break the bonds.
[0077] For a triangular array and a spacing of 150 microns, the
overall efficiency of capture drops 12% as the flow rate increases
from 0.25 to 1 mL/h (FIGS. 14A-14B). Adhesion is not improved by
going to lower flow rates since hydrodynamic capture is not
improved. The mean velocity increases as the spacing between
obstacles increases. The reason for this is that the calculations
used a constant pressure drop. This differs from the experiments in
which the flow rate is held fixed and the pressure drop varies. The
results may be extrapolated from one case to another by one skilled
in the art.
[0078] A repeating triangular array provides limited capture of
target cells because most of the capture occurs in the first few
rows. The reason for this is that the flow field becomes
established in these rows and repeats. The first capture radius
does not produce much capture whereas most of the capture is within
the second capture radius (FIG. 15). Once cells within the capture
radii are captured, the only way in which capture could occur is
through cell-cell collisions to shift cells off their streamlines
or secondary capture. With reference to FIG. 15, in order to
enhance capture, after the flow field is established, the rows are
shifted by a distance in the vertical direction (normal to flow) by
a distance equal to r.sub.cap.sub.2=0.3391. The first five columns
form two regular regions of equilateral triangles. This allows the
flow to be established and be consistent with the solution for an
equilateral triangular array. To promote capture of cells that fall
outside r.sub.cap.sub.2, the fourth column is shifted downward by a
distance r.sub.cap.sub.2. All columns are separated by a distance
equal to l/2. A cell which falls outside r.sub.cap.sub.2 is shown
being captured by the first obstacle in the fourth triangle
(seventh column). Triangles 4 and 5 would be equilateral. In
triangle 6, the vertex 3 is shifted downward by a distance
r.sub.cap.sub.2. This arrangement may be repeated every third
triangle, i.e., the repeat distance is 2.5l. FIGS. 16A and 16B
illustrate the efficiency of capture as a function of flow rate and
relative population of the desired cells.
[0079] The top layer is desirably made of glass and has two slits
drilled ultrasonically for inlet and outlet flows. The slit
inlet/outlet dimensions are, for example, 2 cm long and 0.5 mm
wide. FIG. 17 shows the details for the inlet/outlet geometry. A
manifold may then be incorporated onto the inlet/outlet slits. The
inlet manifold accepts blood cells from an infusion syringe pump or
any other delivery vehicle, for example, through a flexible,
biocompatible tubing. Similarly the outlet manifold is connected to
a reservoir to collect the solution and cells exiting the
device.
[0080] The inlet and outlet configuration and geometry may be
designed in various ways. For example, circular inlets and outlets
may be used. An entrance region devoid of obstacles is then
incorporated into the design to ensure that blood cells are
uniformly distributed when they reach the region where the
obstacles are located. Similarly, the outlet is designed with an
exit region devoid of obstacles to collect the exiting cells
uniformly without damage.
[0081] The overall size of an exemplary device is shown in FIG. 9
(top inset). The length is 10 cm and the width is 5 cm. The area
that is covered with obstacles is 9 cm 4.5 cm. The design is
flexible enough to accommodate larger or smaller sizes for
different applications.
[0082] The overall size of the device may be smaller or larger,
depending on the flow throughput and the number of cells to be
depleted (or captured). A larger device could include a greater
number of obstacles and a larger surface area for cell capture.
Such a device may be necessary if the amount of sample, e.g.,
blood, to be processed is large.
[0083] Fabrication. An exemplary method for fabricating a device of
the invention is summarized in FIG. 18. In this example, standard
photolithography is used to create a photoresist pattern of
obstacles on a silicon-on-insulator (SOI) wafer. A SOI wafer
consists of a 100 .mu.m thick Si(100) layer atop a 1 .mu.m thick
SiO.sub.2 layer on a 500 .mu.m thick Si(100) wafer. To optimize
photoresist adhesion, the SOI wafers may be exposed to
high-temperature vapors of hexamethyldisilazane prior to
photoresist coating. UV-sensitive photoresist is spin coated on the
wafer, baked for 30 minutes at 90.degree. C., exposed to UV light
for 300 seconds through a chrome contact mask, developed for 5
minutes in developer, and post-baked for 30 minutes at 90.degree.
C. The process parameters may be altered depending on the nature
and thickness of the photoresist. The pattern of the contact chrome
mask is transferred to the photoresist and determines the geometry
of the obstacles.
[0084] Upon the formation of the photoresist pattern that is the
same as that of the obstacles, the etching is initiated. SiO.sub.2
may serve as a stopper to the etching process. The etching may also
be controlled to stop at a given depth without the use of a stopper
layer. The photoresist pattern is transferred to the 100 .mu.m
thick Si layer in a plasma etcher. Multiplexed deep etching may be
utilized to achieve uniform obstacles. For example, the substrate
is exposed for 15 seconds to a fluorine-rich plasma flowing
SF.sub.6, and then the system is switched to a fluorocarbon-rich
plasma flowing only C.sub.4F.sub.8 for 10 seconds, which coats all
surfaces with a protective film. In the subsequent etching cycle,
the exposure to ion bombardment clears the polymer preferentially
from horizontal surfaces and the cycle is repeated multiple times
until, e.g., the SiO.sub.2 layer is reached.
[0085] To couple a binding moiety to the surfaces of the obstacles,
the substrate may be exposed to an oxygen plasma prior to surface
modification to create a silicon dioxide layer, to which binding
moieties may be attached. The substrate may then be rinsed twice in
distilled, deionized water and allowed to air dry. Silane
immobilization onto exposed glass is performed by immersing samples
for 30 seconds in freshly prepared, 2% v/v solution of
3-[(2-aminoethyl)amino] propyltrimethoxysilane in water followed by
further washing in distilled, deionized water. The substrate is
then dried in nitrogen gas and baked. Next, the substrate is
immersed in 2.5% v/v solution of glutaraldehyde in phosphate
buffered saline for 1 hour at ambient temperature. The substrate is
then rinsed again, and immersed in a solution of 0.5 mg/mL binding
moiety, e.g., anti-CD71, anti-CD36, anti-GPA, or anti-CD45, in
distilled, deionized water for 15 minutes at ambient temperature to
couple the binding agent to the obstacles. The substrate is then
rinsed twice in distilled, deionized water, and soaked overnight in
70% ethanol for sterilization.
[0086] There are multiple techniques other than the method
described above by which binding moieties may be immobilized onto
the obstacles and the surfaces of the device. Simple
physio-absorption onto the surface may be the choice for simplicity
and cost. Another approach may use self-assembled monolayers (e.g.,
thiols on gold) that are functionalized with various binding
moieties. Additional methods may be used depending on the binding
moieties being bound and the material used to fabricate the device.
Surface modification methods are known in the art. In addition,
certain cells may preferentially bind to the unaltered surface of a
material. For example, some cells may bind preferentially to
positively charged, negatively charged, or hydrophobic surfaces or
to chemical groups present in certain polymers.
[0087] The next step involves the creation of a flow device by
bonding a top layer to the microfabricated silicon containing the
obstacles. The top substrate may be glass to provide visual
observation of cells during and after capture. Thermal bonding or a
UV curable epoxy may be used to create the flow chamber. The top
and bottom may also be compression fit, for example, using a
silicone gasket. Such a compression fit may be reversible. Other
methods of bonding (e.g., wafer bonding) are known in the art. The
method employed may depend on the nature of the materials used.
[0088] The cell binding device may be made out of different
materials. Depending on the choice of the material different
fabrication techniques may also be used. The device may be made out
of plastic, such as polystyrene, using a hot embossing technique.
The obstacles and the necessary other structures are embossed into
the plastic to create the bottom surface. A top layer may then be
bonded to the bottom layer. Injection molding is another approach
that can be used to create such a device. Soft lithography may also
be utilized to create either a whole chamber made out of
poly(dimethylsiloxane) (PDMS), or only the obstacles may be created
in PDMS and then bonded to a glass substrate to create the closed
chamber. Yet another approach involves the use of epoxy casting
techniques to create the obstacles through the use of UV or
temperature curable epoxy on a master that has the negative replica
of the intended structure. Laser or other types of micromachining
approaches may also be utilized to create the flow chamber. Other
suitable polymers that may be used in the fabrication of the device
are polycarbonate, polyethylene, and poly(methyl methacrylate). In
addition, metals like steel and nickel may also be used to
fabricate the device of the invention, e.g., by traditional metal
machining. Three-dimensional fabrication techniques (e.g.,
stereolithography) may be employed to fabricate a device in one
piece. Other methods for fabrication are known in the art.
[0089] Methods. The methods of the invention involve contacting a
mixture of cells with the surfaces of a microfluidic device. A
population of cells in a complex mixture of cells such as blood
then binds to the surfaces of the device. Desirably, at least 60%,
70%, 80%, 90%, 95%, 98%, or 99% of cells that are capable of
binding to the surfaces of the device are removed from the mixture.
The surface coating is desirably designed to minimize nonspecific
binding of cells. For example, at least 99%, 98%, 95%, 90%, 80%, or
70% of cells not capable of binding to the binding moiety are not
bound to the surfaces of the device. The selective binding in the
device results in the separation of a specific living cell
population from a mixture of cells. Obstacles are present in the
device to increase surface area for cells to interact with while in
the chamber containing the obstacles so that the likelihood of
binding is increased. The flow conditions are such that the cells
are very gently handled in the device without the need to deform
mechanically in order to go in between the obstacles. Positive
pressure or negative pressure pumping or flow from a column of
fluid may be employed to transport cells into and out of the
microfluidic devices of the invention. In an alternative
embodiment, cells are separated from non-cellular matter, such as
non-biological matter (e.g., beads), non-viable cellular debris
(e.g., membrane fragments), or molecules (e.g., proteins, nucleic
acids, or cell lysates).
[0090] FIG. 19 shows cells expressing a specific surface antigen
binding to a binding moiety coated onto obstacles, while other
cells flow through the device (small arrow on cells depict the
directionality of cells that are not bound to the surface). The top
and bottom surfaces of the flow apparatus may also be coated with
the same binding moiety, or a different binding moiety, to promote
cell binding.
[0091] Exemplary cell types that may be separated using the methods
described herein include adult red blood cells, fetal red blood
cells, trophoblasts, fetal fibroblasts, white blood cells (such as
T cells, B cells, and helper T cells), infected white blood cells,
stem cells (e.g., CD34 positive hematopoeitic stem cells),
epithelial cells, tumor cells, and infectious organisms (e.g.,
bacteria, protozoa, and fungi).
[0092] Samples may be fractionated into multiple homogeneous
components using the methods described herein. Multiple similar
devices containing different binding moieties specific for a
population of cells may be connected in series or in parallel.
Serial separation may be employed when one seeks to isolate rare
cells. On the other hand, parallel separation may be employed when
one desires to obtain differential distribution of various
populations in blood. FIGS. 20A and 20B show parallel and serial
systems for the separation of multiple populations of cells from
blood. For parallel devices, two or more sets of obstacles that
bind different types of cells may be located in distinct regions or
they may be interspersed among each other, e.g., in a checkerboard
pattern or in alternating rows. In addition, a set of obstacles may
be attached to the top of the device and another set may be
attached to the bottom of the device. Each set may then be
derivatized to bind different populations of cells. Once a sample
has passed through the device, the top and bottom may be separated
to provide isolated samples of two different types of cells.
[0093] The cell binding device may be used to deplete the outlet
flow of a certain population of cells, or to capture a specific
population of cells expressing a certain surface molecule for
further analysis. The cells bound to obstacles may be removed from
the chamber for further analysis of the homogeneous population of
cells (FIG. 21). This removal may be achieved by incorporating one
or more additional inlets and exits orthogonal to the flow
direction. Cells may be removed from the chamber by purging the
chamber at an increased flow rate, that is higher shear force, to
overcome the binding force between the cells and the obstacles.
Other approaches may involve coupling binding moieties with
reversible binding properties, e.g., that are actuated by pH,
temperature, or electrical field. The binding moiety, or the
molecule bound on the surface of the cells, may also be cleaved by
enzymatic or other chemical means.
[0094] In the example of fetal red blood cell isolation, a sample
having passed through a lysis device is passed through a cell
binding device, whose surfaces are coated with CD45. White blood
cells expressing CD45 present in the mixture bind to the walls of
the device, and the cells that pass through the device are enriched
in fetal red blood cells. Alternatively, the obstacles and device
surfaces are coated with anti-CD71 in order to bind fetal nucleated
red blood cells (which express the CD71 cell surface protein) from
a whole maternal blood sample. One percent of adult white blood
cells also express CD71. A sample of maternal blood is passed
through the device and both populations of cells that express CD71
bind to the device. This results in the depletion of fetal red
blood cells from the blood sample. The fetal cells are then
collected and analyzed. For example, cells are collected on a
planar substrate for fluorescence in situ hybridization (FISH),
followed by fixing of the cells and imaging. FIGS. 22A-22C show the
use of FISH on a cell bound to an obstacle in a binding device of
the invention. The cell, of fetal origin, is stained for X and Y
chromosomes using fluorescent probes. These data show the
feasibility of optical imaging of FISH stained cells on posts for
detection and diagnosis of chromosomal abnormalities.
[0095] Alternative Embodiments. Another embodiment of the cell
binding device utilizes chemically derivatized glass/plastic beads
entrapped in a loosely cross-linked hydrogel, such as, but not
limited to, poly(vinyl alcohol), poly(hydroxyl-ethyl methacrylate),
polyacrylamide, or polyethylene glycol (FIG. 23). The chemically
derivatized beads serve as the obstacles in this embodiment. A
mixture of cells is directed into the cell depletion device via two
diametrically opposed inputs. Positive pressure (e.g., from an
infusion pump or column of fluid) or negative pressure (e.g., from
a syringe pump in pull mode, a vacuum pump, or an aspirator) drives
the liquid through the hydrogel. The interaction of the cells in
the sample with the chemically derivatized beads dispersed in the
three-dimensional volume of the hydrogel results in either
depletion of cells, e.g., white blood cells, (negative selection)
or capture of cells, e.g., fetal red blood cells, (positive
selection). The molecular weight, cross-link density, bead density,
and distribution and flow rates can be optimized to allow for
maximal interaction and capture of relevant cells by the beads. The
high-water content hydrogel provides a structure to trap the beads
while allowing ease of flow through of the sample. The sample is
then collected through two diametrically opposed outputs. The
bifurcated input/output channel design assures maximal homogeneous
distribution of the sample through the volume of the hydrogel.
[0096] In yet another embodiment, the beads are replaced by direct
chemical derivatization of the side chains of the hydrogel polymer
with the binding moiety (e.g., synthetic ligand or monoclonal
antibody (mAb)). This approach can provide a very high density of
molecular capture sites and thereby assure higher capture
probability. An added advantage of this approach is a potential use
of the hydrogel based cell depletion device as a sensor for fetal
cell capture in the positive selection mode (select for fetal cells
with specific mAb), for example, if the polymer backbone and side
chain chemistry is designed to both capture the fetal cells and in
the process further cross-link the hydrogel. The cells bind to
numerous side chains via antigen-mAb interaction and thus serve as
a cross-linker for the polymer chains, and the reduction in flow
output over time due to increased polymer cross-link density can be
mathematically equated to the number of fetal cells captured within
the 3D matrix of the polymer. When the desired number of fetal
cells is captured (measured by reduction in output flow rate), the
device can stop further processing of the maternal sample and
proceed to analysis of the fetal cells. The captured fetal cells
can be released for analysis by use of a photoactive coupling agent
in the side chain. The photoreactive agent couples the target
ligand or mAb to the polymer backbone, and on exposure to a pulse
of UV or IR radiation, the ligands or mAbs and associated cells are
released.
C. Cell Arraying
[0097] In this device, a mixture of cells that has typically been
depleted of unwanted cells is arrayed in a microfluidic device. An
exemplary device for this step is described in International
Publication No. WO 01/35071. The cells in the array are then
assayed, e.g., by microscopy or colorimetric assay, to locate
desired cells. The desired cells may then be analyzed on the array,
e.g., by lysis followed by PCR, or the cells may be collected from
the array by a variety of mechanisms, e.g., optical tweezers. In
the exemplary device described in WO 01/35071, the cells are
introduced into the arraying device and may passively settle into
holes machined in the device. Alternatively, positive or negative
pressure may be employed to direct the cells to the holes in the
array. Once the cells have been deposited in the holes, selected
cells may be individually released from the array by actuators,
e.g., bubble actuated pumps. Other methods for immobilizing and
releasing cells, e.g., dielectrophoretic trapping, may also be used
in an arraying device. Once released from the array, cells may be
collected and subjected to analysis. For example, a fetal red blood
cell is identified in the array and then analyzed for genetic
abnormalities. Fetal red blood cells may be identified
morphologically or by a specific molecular marker (e.g., fetal
hemoglobin, transferring receptor (CD71), thrombospondin receptor
(CD36), or glycophorin A (GPA)).
D. Size-Based Separation
[0098] Another device is a device for the separation of particles
based on the use of sieves that selectively allow passage of
particles based on their size, shape, or deformability. The size,
shape, or deformability of the pores in the sieve determines the
types of cells that can pass through the sieve. Two or more sieves
can be arranged in series or parallel, e.g., to remove cells of
increasing size successively.
[0099] Device. In one embodiment, the sieve includes a series of
obstacles that are spaced apart. A variety of obstacle sizes,
geometries, and arrangements can be used in devices of the
invention. Different shapes of obstacles, e.g., those with
circular, square, rectangular, oval, or triangular cross sections,
can be used in a sieve. The gap size between the obstacles and the
shape of the obstacles may be optimized to ensure fast and
efficient filtration. For example, the size range of the RBCs is on
the order of 5-8 .mu.m, and the size range of platelets is on the
order of 1-3 .mu.m. The size of all WBCs is greater than 10 .mu.m.
Large gaps between obstacles increase the rate at which the RBCs
and the platelets pass through the sieve, but increased gap size
also increases the risk of losing WBCs. Smaller gap sizes ensure
more efficient capture of WBCs but also a slower rate of passage
for the RBCs and platelets. Depending on the type of application
different geometries can be used.
[0100] In addition to obstacles, sieves may be manufactured by
other methods. For example, a sieve could be formed by molding,
electroforming, etching, drilling, or otherwise creating holes in a
sheet of material, e.g., silicon, nickel, or PDMS. Alternatively, a
polymer matrix or inorganic matrix (e.g., zeolite or ceramic)
having appropriate pore size could be employed as a sieve in the
devices described herein.
[0101] One problem associated with devices of the invention is
clogging of the sieves. This problem can be reduced by appropriate
sieve shapes and designs and also by treating the sieves with
non-stick coatings such as bovine serum albumin (BSA) or
polyethylene glycol (PEG), as described herein. One method of
preventing clogging is to minimize the area of contact between the
sieve and the particles.
[0102] The schematic of a low shear stress filtration device is
shown in FIG. 24. The device has one inlet channel which leads into
a diffuser, which is a widened portion of the channel. Typically,
the channel widens in a V-shaped pattern. The diffuser contains two
sieves having pores shaped to filter, for example, smaller RBCs and
platelets from blood, while enriching the population of WBCs and
fetal RBCs. The diffuser geometry widens the laminar flow
streamlines forcing more cells to come in contact with the sieves
while moving through the device. The device contains 3 outlets, two
outlets collect cells that pass through the sieves, e.g., the RBCs
and platelets, and one outlet collects the enriched WBCs and fetal
RBCs.
[0103] The diffuser device typically does not ensure 100% depletion
of RBCs and platelets. Initial RBC:WBC ratios of 600:1 can,
however, be improved to ratios around 1:1. Advantages of this
device are that the flow rates are low enough that shear stress on
the cells does not affect the phenotype or viability of the cells
and that the filters ensure that all the large cells (i.e., those
unable to pass through the sieves) are retained such that the loss
of large cells is minimized or eliminated. This property also
ensures that the population of cells that pass through sieve do not
contain large cells, even though some smaller cells may be lost.
Widening the diffuser angle will result in a larger enrichment
factor. Greater enrichment can also be obtained by the serial
arrangement of more than one diffuser where the outlet from one
diffuser feeds into the inlet of a second diffuser. Widening the
gaps between the obstacles might expedite the depletion process at
the risk of losing large cells through the larger pores in the
sieves. For separating maternal red blood cells from fetal
nucleated red blood cells, an exemplary spacing is 2-4 .mu.m.
[0104] Method. The device of the invention is a continuous flow
cell sorter, e.g., that filters larger WBCs and fetal RBCs from
blood. The location of the sieves in the device is chosen to ensure
that the maximum number of particles come into contact with the
sieves, while at the same time avoiding clogging and allowing for
retrieval of the particles after separation. In general, particles
are moved across their laminar flow lines which are maintained
because of extremely low Reynolds number in the channels in the
device, which are typically micrometer sized.
[0105] Fabrication. Simple microfabrication techniques like
poly(dimethylsiloxane) (PDMS) soft lithography, polymer casting
(e.g., using epoxies, acrylics, or urethanes), injection molding,
polymer hot embossing, laser micromachining, thin film surface
micromachining, deep etching of both glass and silicon,
electroforming, and 3-D fabrication techniques such as
stereolithography can be used for the fabrication of the channels
and sieves of devices of the invention. Most of the above listed
processes use photomasks for replication of micro-features. For
feature sizes of greater than 5 .mu.m, transparency based emulsion
masks can be used. Feature sizes between 2 and 5 .mu.m may require
glass based chrome photomasks. For smaller features, a glass based
E-beam direct write mask can be used. The masks are then used to
either define a pattern of photoresist for etching in the case of
silicon or glass or define negative replicas, e.g., using SU-8
photoresist, which can then be used as a master for replica molding
of polymeric materials like PDMS, epoxies, and acrylics. The
fabricated channels and may then be bonded onto a rigid substrate
like glass to complete the device. Other methods for fabrication
are known in the art. A device of the invention may be fabricated
from a single material or a combination of materials.
Example. In one example, a device for size based separation of
smaller RBCs and platelets from the larger WBCs was fabricated
using simple soft lithography techniques. A chrome photomask having
the features and geometry of the device was fabricated and used to
pattern a silicon wafer with a negative replica of the device in
SU-8 photoresist. This master was then used to fabricate PDMS
channel and sieve structures using standard replica molding
techniques. The PDMS device was bonded to a glass slide after
treatment with O.sub.2 plasma. The diffuser geometry is used to
widen the laminar flow streamlines to ensure that the majority of
the particles or cells flowing through the device will interact
with the sieves. The smaller RBC and platelets pass through the
sieves, and the larger WBCs are confined to the central
channel.
Combination of Devices
[0106] The devices of the invention may be used alone or in any
combination. In addition, the steps of the methods described herein
may be employed in any order. A schematic representation of a
combination device for detecting and isolating fetal red blood
cells is shown in FIG. 25. In one example, a sample may be
processed using the cell lysis step, and then desired cells may be
trapped in a cell binding device. If the cells trapped are
sufficiently pure, no further processing step is needed.
Alternatively, only one of the lysis or binding steps may be
employed prior to arraying. In another example, a mixture of cells
may be subjected to lysis, size based separation, binding, and
arraying.
[0107] The methods of the invention may be carried out on one
integrated device containing regions for cell lysis, cell binding,
arraying, and size based separation. Alternatively, the devices may
be separate, and the populations of cells obtained from each step
may be collected and manually transferred to devices for subsequent
processing steps.
[0108] Positive or negative pressure pumping may be used to
transport cells through the microfluidic devices of the
invention.
Analysis
[0109] After being enriched by one or more of the devices of the
invention, cells may be collected and analyzed by various methods,
e.g., nucleic acid analysis. The sample may also be further
processed prior to analysis. In one example, cells may be collected
on a planar substrate for fluorescence in situ hybridization
(FISH), followed by fixing of the cells and imaging. Such analysis
may be used to detect fetal abnormalities such as Down syndrome,
Edwards' syndrome, Patau's syndrome, Klinefelter syndrome, Turner
syndrome, sickle cell anemia, Duchenne muscular dystrophy, and
cystic fibrosis. The analysis may also be performed to determine a
particular trait of a fetus, e.g., sex.
OTHER EMBODIMENTS
[0110] All publications, patents, and patent applications mentioned
in the above specification are hereby incorporated by reference.
Various modifications and variations of the described method and
system of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying
out the invention that are obvious to those skilled in the art are
intended to be within the scope of the invention.
[0111] Other embodiments are in the claims.
* * * * *