(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2015/0260711 A1
`(43) Pub. Date:
`Sep. 17, 2015
`Toner et al.
`
`US 2015026O711A1
`
`(54)
`
`(71)
`
`(72)
`
`(21)
`(22)
`
`(60)
`
`(60)
`
`MCROFLUDIC DEVICE FORCELL
`SEPARATION AND USES THEREOF
`
`Applicant: The General Hospital Corporation,
`Boston, MA (US)
`Inventors: Mehmet Toner, Charlestown, MA (US);
`George Truskey, Durnham, NC (US);
`Ravi Kapur, Sharon, MA (US)
`Appl. No.: 14/665,708
`Filed:
`Mar. 23, 2015
`Related U.S. Application Data
`Continuation of application No. 1 1/726.231, filed on
`Mar. 21, 2007, now Pat. No. 8,986,966, which is a
`division of application No. 10/529,453, filed on Dec.
`19, 2005, now Pat. No. 8,895,298, filed as application
`No. PCT/US2003/030965 on Sep. 29, 2003.
`Provisional application No. 60/414,065, filed on Sep.
`27, 2002, provisional application No. 60/414,102,
`filed on Sep. 27, 2002, provisional application No.
`60/414.258, filed on Sep. 27, 2002.
`Publication Classification
`
`(51)
`
`Int. C.
`GOIN33/543
`GOIN L/40
`
`(2006.01)
`(2006.01)
`
`(2006.01)
`(2006.01)
`
`BOIL 3/00
`GOIN33/569
`(52) U.S. Cl.
`CPC. G0IN33/54366 (2013.01); G0IN33/56966
`(2013.01); G0IN 1/405 (2013.01); BOIL
`3/502746 (2013.01); B0IL3/502761 (2013.01);
`BOIL 2.200/12 (2013.01); B01 L 2200/0668
`(2013.01); B01 L 2400/086 (2013.01); BOIL
`2300/12 (2013.01)
`
`(57)
`
`ABSTRACT
`
`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).
`
`SLT NLE
`
`
`
`TOPLAYER (GLASS)
`
`2
`
`
`
`
`
`
`
`
`
`BOTTOM LAYER
`(SILICON)
`
`MICROPOST
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 1
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 1 of 25
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`US 2015/0260711 A1
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`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 2
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 2 of 25
`
`US 2015/0260711 A1
`
`
`
`W
`
`w
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`- - - - - - a n e a na - - - - - - - -
`()
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`a
`
`a
`as a
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 3
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 3 of 25
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`US 2015/0260711 A1
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`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 4
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 4 of 25
`
`US 2015/0260711 A1
`
`uuuu 9
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 5
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 5 of 25
`
`US 2015/0260711 A1
`
`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 6
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 6 of 25
`
`US 2015/0260711 A1
`
`FIG. 6A
`Compression Molding
`
`
`
`Spincoating of
`thermoplast on
`hard substrate
`
`Stamp with
`nanorelief
`
`Hot embossing
`and demolding
`
`
`
`4 mm
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 7
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 7 of 25
`
`US 2015/0260711 A1
`
`El 100
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`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 8
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 8 of 25
`
`US 2015/0260711 A1
`
`
`
`(SSV19) HBXVI JOI
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`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 9
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 9 of 25
`
`US 2015/0260711 A1
`
`
`
`50 um
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`50 uma-ae).......
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`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 10
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 10 of 25
`
`US 2015/0260711 A1
`
`(
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`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 11
`
`

`

`Sep. 17, 2015 Sheet 11 of 25
`
`
`
`S/0260711 A1 US 201
`
`FIG. 1 1A
`
`FIG. 11B
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`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 12
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 12 of 25
`
`US 2015/0260711 A1
`
`FIG. 12A
`
`
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`w w
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`it wox it
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`it we la P w a
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`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 13
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 13 of 25
`
`US 2015/0260711 A1
`
`FIG. 13A 100
`
`90
`
`8 O
`
`5 O
`
`14O 150 16O 17O 18O 190 200
`Minimal distance between cylinders (micron)
`
`F G. 1 3B 100
`90
`Adhesion
`is included
`
`
`
`as at a is Square array
`
`Triangular array
`
`Pressure gradient = 150 Palm
`Viscosity = 1.2 cF
`
`160
`150
`140
`Minimal distance between cylinders (micron)
`
`170
`
`-r n x xy sea Square array
`
`Triangular array
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 14
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 14 of 25
`
`US 2015/0260711 A1
`
`FIG. 14A 60
`59
`
`Triangular array
`1 s 150 microns
`Viscosity = 1.2cP
`
`SO
`
`15O
`1OO
`Pressure gradient (Palm)
`
`2OO
`
`FIG. 14B
`Adhesion
`is included
`S C
`
`5E-05
`4.5E-05
`
`
`
`Pressure gradient = 150 Palm
`Wiscositv = 1.
`iscosity = 1.2cP
`
`4E-05
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`3.5E-05
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`
`2.5E-05
`
`2E-05
`
`1.5E-05
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`17O
`16O
`15O
`140
`Minimal distance between cylinders (micron)
`
`as as as a
`
`Square array - Triangular array
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 15
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 15 of 25
`
`US 2015/0260711 A1
`
`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 16
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 16 of 25
`
`US 2015/0260711 A1
`
`F.G. 16A
`
`
`
`7.5
`5
`O.6 1.25 2.5
`Flow rate (mL/h)
`
`O
`
`s
`
`F.G. 16B
`
`60
`
`50
`
`40
`
`O
`
`10-5 10-6
`10-3
`10-1
`Ratio of target cells to WBC
`10 target cells)
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 17
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 17 of 25
`
`US 2015/0260711 A1
`
`QNV LETNI)NN
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`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 18
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 18 of 25
`
`US 2015/0260711 A1
`
`FIG. 18
`
`SiO2
`
`YYZZZZZZYZZZZZZYZZZZZZZZY
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`Thermal band top glass layer
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`| Deposit anti-CD45
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`Anti- CD45
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`
`
`
`
`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 19
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 19 of 25
`
`US 2015/0260711 A1
`
`2
`2
`S
`9 : C
`3 5s.
`2
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`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 20
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 20 of 25
`
`US 2015/0260711 A1
`
`
`
`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 21
`
`

`

`Patent Application Publication
`
`Sep. 17,2015 Sheet 21 of 25
`
`US 2015/0260711 Al
`
`OUTLET
`
`FIG.21
`
`INLET
`
`° o oO ° °
`
`COLLECTION
`
`PORTORRESERVOIR
`
`FLOW INLET
`
`Yita v. MacNeil IP, IPR2020-01139, Page 22
`
`MacNeil Exhibit 2173
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 22
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 22 of 25
`
`US 2015/0260711 A1
`
`FIG.22A
`
`FIG.22B
`
`FIG. 22C
`
`
`
`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 23
`
`

`

`Patent Application Publication
`
`US 2015/0260711 A1
`
`
`
`
`
`HTTIGOW S?SAT ___
`
`
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 24
`
`

`

`Patent Application Publication
`
`Sep. 17, 2015 Sheet 24 of 25
`
`US 2015/0260711 A1
`
`FIG. 24A
`
`
`
`Sieve Filters
`
`
`
`Smaller
`RBC/platelets
`OOOOOOOOOOOOOOOOC
`OOOOOOOOOOOOOOOOOOOOO
`
`net
`
`
`
`FIG. 24B
`
`Outlet
`High pressure
`(Suction)
`
`Outlet
`Low preSSure
`(Suction)
`
`Outlet
`High pressure
`(Suction)
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 25
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`

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`Patent Application Publication
`
`Sep. 17, 2015 Sheet 25 of 25
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`US 2015/0260711 A1
`
`??SEM || 30
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`Squè6e38 HSH
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`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 26
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`

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`US 2015/02607 11 A1
`
`Sep. 17, 2015
`
`MCROFLUDIC DEVICE FORCELL
`SEPARATION AND USES THEREOF
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001. This application is a divisional of U.S. application
`Ser. No. 10/529,453, having a $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 Applica
`tion Nos. 60/414,065, 60/414,258, and 60/414,102, filed on
`Sep. 27, 2002, each of which is hereby incorporated by ref
`CCC.
`
`BACKGROUND OF THE INVENTION
`0002 The invention relates to the fields of medical diag
`nostics 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 catego
`ries: (1) invasive methods based on the selection of cells fixed
`and stained using various cell-specific markers; and (2) non
`invasive 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 posi
`tive 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 sort
`ing. It is fast, typically running at a rate of 1,000 to 1,500 Hz,
`and well established in laboratory medicine. High false posi
`tive rates are associated with FACS because of the low num
`ber 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 advan
`tage of being cheaper, easier, and faster to perform as com
`pared 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 ferro
`magnetic 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 ferromag
`netic nanoparticles align in a magnetic field along ferromag
`netic 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 separa
`tion, 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 differ
`ences, 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 chan
`nels. In one embodiment, the device includes at least two
`processing steps. For example, a mixture of cells is intro
`duced 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, deformabil
`ity, Surface characteristics (e.g., cell Surface receptors or anti
`gens 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 microflu
`idic channels that preferentially lyses one type of cell com
`pared 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 sub
`jected 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 chan
`nels; 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
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 27
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`US 2015/02607 11 A1
`
`Sep. 17, 2015
`
`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-dimen
`sional 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 popula
`tion 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 popu
`lation 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 con
`tacted 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 poly
`mers, 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 gen
`erally 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 cen
`ters 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 homo
`geneous 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 alysis 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 cal
`culation 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 tri
`angular 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 arrange
`ment of obstacles for higher efficiency capture for an equilat
`eral triangular array of obstacles in a staggered array. The
`capture radius r,
`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 um diameter obstacle
`geometry with a 50 um edge-to-edge spacing. The operating
`flow regime was established across multiple cell types: cancer
`cells, normal connective tissue cells, and maternal and fetal
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 28
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`Sep. 17, 2015
`
`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.
`004.5
`FIG. 19 is an illustration of a mixture of cells flow
`ing 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 offetal red blood
`cells adhered to an obstacle of the invention. FIG. 22B is a
`fluorescent micrograph showing the results of a FISH analy
`sis 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 num
`ber 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, process
`ing 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 1
`
`Types, concentrations, and sizes of blood cells.
`
`Cell Type
`Red blood cells (RBC)
`Segmented Neutrophils (WBC)
`Band Neutrophils (WBC)
`Lymphocytes (WBC)
`Monocytes (WBC)
`Eosinophils (WBC)
`Basophils (WBC)
`Platelets
`Fetal Nucleated Red Blood
`Cells
`
`Concentration (cellsill)
`4.2-6.1 10
`36OO
`120
`1SOO
`480
`18O
`120
`500 103
`2-5010 -3
`
`Size (Lm)
`4-6
`>10
`>10
`>10
`>10
`>10
`>10
`1-2
`8-12
`
`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.
`0056. 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 function
`ally 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).
`0057. Input/Output Channels.
`0058. The branched input and output networks of channels
`enable even distribution of the reagents into each of the chan
`nels (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 solu
`tion vials, e.g., blood, lysing reagent, and diluent, may inter
`face 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 con
`trolled) machined in stainless steel and may have dimensions
`of 500 um 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
`
`MacNeil Exhibit 2173
`Yita v. MacNeil IP, IPR2020-01139, Page 29
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`Sep. 17, 2015
`
`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 chan
`nels on the device transporting the fluids to the mixing junc
`tions and chambers beyond, can range from 10um-500 um in
`width and depth, e.g., at most 10 um, 25um, 50 um, 75um,
`100 m, 150 um, 200 m, 250 um, 350 um, or 450 Lim 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.
`0059
`Reaction and Dilution Chambers.
`0060 For lysis and dilution, two fluid streams are com
`bined 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 through
`put. 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 -26 ul.
`Other examples of reaction/dilution chamber volumes range
`from 10-200 ul, e.g., at most 20, 50, 100, or 150 ul. In some
`embodiments, the width and depth of the reaction and dilution
`chambers have the same range as the input and output chan
`nels, i.e., 10 to 500 um. Alternatively, the chambers may have
`a cross-se