Fluidic devices for analyte removal and / or capture and associated systems and methods

Abstract

Fluidic devices for analyte removal and / or capture and related systems and methods are generally described.

Description

[0001] FLUIDIC DEVICES FOR ANALYTE REMOVAL AND / OR CAPTURE AND ASSOCIATED SYSTEMS AND METHODS RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 750,950, filed January 29, 2025, and entitled “Fluidic Devices for Analyte Removal and / or Capture and Associated Systems and Methods,” which is incorporated herein by reference in its entirety for all purposes.

[0003] TECHNICAL FIELD

[0004] Fluidic devices for analyte removal and / or capture and related systems and methods are generally described.

[0005] SUMMARY

[0006] Fluidic devices for analyte removal and / or capture and related systems and methods are generally described. In some embodiments, the analytes are biological cells. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0007] In some embodiments, an article is provided. In some embodiments, the article comprises a substrate comprising an open channel configured such that when the substrate is positioned adjacent to a surface and fluid is flowed through the channel, an analyte is removed from the surface.

[0008] In some embodiments, an article is provided. In some embodiments, the article comprises a substrate comprising an outer surface; and an open channel in relief from the outer surface; wherein: the open channel comprises an inlet and an outlet; and the outer surface comprises a seal region configured to isolate the open channel from an external environment.

[0009] In some embodiments a method is provided. In some embodiments, the method comprises contacting a substrate comprising an open microfluidic channel to a surface comprising an analyte to form a closed conduit; and flowing a fluid through the closed conduit such that the analyte is removed from the surface.

[0010] #14855532vlOther advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0013] FIG. 1A shows, according to certain embodiments, a cross-sectional schematic illustration of a substrate comprising an open channel in relief from the surface in contact with a surface comprising an analyte.

[0014] FIG. IB shows, according to certain embodiments, a perspective view schematic illustration of a fluidic device for cell capture.

[0015] FIG. 1C shows, according to certain embodiments, a top-view schematic illustration of a substrate comprising an open channel in relief from the surface.

[0016] FIG. 2 shows, according to certain embodiments, a cross-sectional schematic illustration of a restricted fluid flow cell capture device where the microchannel, of characteristic width w, length I and height h. is formed when the device contacts tissue or cells, which form the microchannel base. Here, an external vacuum applies the reactionary force that prevents the breach of the microchannel due to pressure-driven flow.

[0017] FIG. 3 A shows, according to certain embodiments, a perspective view schematic illustration of a fluidic device for cell capture.

[0018] FIG. 3B shows, according to certain embodiments, an illustration of how the fluidic device of FIG. 3A can be used to capture cells from a fallopian tube.

[0019] #14855532vlFIG. 3C shows, according to certain embodiments, a cross-sectional schematic illustration of flow pathways within the fluidic device of FIG. 3A.

[0020] FIG. 3D shows, according to certain embodiments, a side-view schematic illustration of the bottom portion of the fluidic device of FIG. 3A.

[0021] FIG. 3E shows, according to certain embodiments, a first bottom perspective view schematic illustration of the bottom portion of the fluidic device of FIG. 3A.

[0022] FIG. 3F shows, according to certain embodiments, a front view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3A.

[0023] FIG. 3G shows, according to certain embodiments, a first bottom view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3A.

[0024] FIG. 3H shows, according to certain embodiments, a second bottom perspective view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3A.

[0025] FIG. 31 shows, according to certain embodiments, a second bottom view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3A.

[0026] FIG. 4 shows, according to certain embodiments, a system for performing cell capture using a fluidic device, including use of a fixture to hold the syringe housing in place, lab jack to bring the 3D printed microchannel and vacuum ring in contact with tissue (in this case, porcine oviduct), a tube connected to an empty syringe to manually pull vacuum, a linear stage with force actuator to apply a constant volumetric flow rate of working fluid (PBS or growth media) through the syringe plunge, and a collection tube passing into Eppendorf tubes to collect suspensions of cells.

[0027] FIG. 5 shows, according to some embodiments, the results of Fluorescent Activated Cell Sorting (FACS) of suspensions of cells collected from tissues of three sacrificed murine animal models. Cells were stained with a Live / Dead Staining assay, and subsequently cell count and viability were measured.

[0028] FIG. 6 shows, in accordance with certain embodiments, 2D culturing of cells (lOx zoom) collected from porcine oviduct tissue surface (a) and porcine ovary tissue surface (b) Cells were seeded on well plates coated with gelatin and collagen IV and cultured with regular DMEM / F12 media supplemented with a variety of growth factors, antibiotics, antifungals, and extra nutrients. Cells were cultured over the span of two

[0029] #14855532vlweeks. Both were able to spread and proliferate, however the ovarian cells grew significantly more.

[0030] FIG. 7 shows, in accordance with some embodiments, before and after images of Polystyrene petri dishes containing methylene blue stained MG63 cells using flow device at different PBS flow rates (2x zoom) including a) 0.0063 m / s, b) 0.21 m / s, c) 0.42 m / s, and d) 0.64 m / s. The flow rate corresponds to the average velocity at the inlet of the channel. Clearly, a larger proportion of cells are removed as higher velocities.

[0031] FIG. 8 shows, in accordance with certain embodiments, the correlation between percent of remaining cells on a surface at a given down-channel distance for different input velocities to the microfluidic channel.

[0032] DETAILED DESCRIPTION

[0033] Fluidic devices for analyte removal and / or capture and related systems and methods are generally described. The present disclosure describes, in certain embodiments, articles and methods for non-invasive cell removal and / or capture from surfaces using fluidic devices. Many traditional biopsy techniques, which are the principal source of human cells for diagnostic purposes (including for the development of patient-derived organelles and other emerging techniques in personalized medicine), have several problems which can lead to negative patient outcomes, including the inability to capture tumor homogeneity, the potential liberation of cancerous cells to spread to other parts of the body, scarring, and bleeding. As such, improved biopsy devices and techniques are needed.

[0034] Certain of the systems, devices, and methods described in this disclosure can be used for the collection of cells from tissue surfaces. In some embodiments, the systems, devices, and methods facilitate the collection of cells by contacting a substrate comprising an open microchannel in relief from a surface of the substrate to the tissue surface to form a closed microchannel and flowing a fluid through the closed microchannel. In some embodiments, flowing the liquid through the closed microchannel liberates cells from the tissue surface, allowing those cells to be collected for further analysis, culture, and / or other use. Certain of the systems, devices, and methods described in this disclosure allow for the collection of cells from tissue surfaces in a manner that is less invasive and less destructive than existing techniques. For

[0035] #14855532vlexample, the flowing of the liquid through the closed microchannel to liberate the cells from the tissue surface described above may be a less invasive and / or less destructive procedure than existing biopsy techniques. As such, the use of certain of the systems, devices, and methods described in this disclosure can provide cells for diagnostic purposes, including the development of patient-derived organelles, without causing scarring, bleeding, or the liberation of cancerous cells to spread to other parts of the body.

[0036] As described in greater detail below, certain of the systems, devices, and methods described herein may have other advantages and / or uses, such as allowing for the efficient sampling of tumor homogeneity and the development of assays based on cell adhesion strength. Assays based on cell adhesion strength are important in a variety of applications. Cells adhere to each other and the extracellular matrix (ECM), an intricate array of macromolecules including collagens, proteoglycans, elastins, and glycoproteins, which provide structural and biochemical support to cells. The adhesion strength of cells to other cells, cells to the ECM, and cells to other surfaces (e.g., plates or dishes used for cell culture, matrices used for 3D cell culture, etc.) can vary greatly based on the characteristics of the surface, the type of cells, and / or the disease state of the cells. Due to this variation, information about cell adhesion strength can be used to infer a variety of characteristics of the cells. Assays based on cell adhesion strength have the ability to distinguish cell types, disease states, and other cell features. Certain of the systems, devices, and methods described in this disclosure allow for the development and improvement of such assays.

[0037] The systems, devices, and methods described herein may be used for a variety of suitable purposes in addition to collecting cells from tissue surfaces. For example, certain of the systems, devices, and methods could be used for capturing cells from any surface, including a cell culture surface (e.g., a Petri dish, a cell culture well). In some embodiments, the systems, devices, and methods as described herein may be particularly advantageous for removing cells from such surfaces for transport to a different surface (e.g., for re-culturing). Many existing methods for detaching cells from surfaces used for cell culture have a high risk of damaging the cells, which may render the cells difficult or impossible to re-culture, or may reduce the yield of re-cultured cells. In contrast, certain of the devices, methods, and systems described herein may facilitate the removal of cells

[0038] #14855532vlfrom the surface used for cell culture without causing damage to the cells, thereby facilitating more effective and higher-yield re-culturing. In some embodiments, the systems, devices, and methods as described herein may be used to achieve highly localized and / or targeted detachment of cells from a specific region of a surface (e.g., a cell culture surface). This may allow, for example, the re-culturing of cells from a specific region of a cell culture removed from the cell culture surface.

[0039] A perspective view schematic illustration of an example article is shown in FIG. IB. In some embodiments, the article 100 comprises a substrate 101 comprising an outer surface 102. In some embodiments, the substrate comprises an open channel 103 in relief from the outer surface of the substrate. In some embodiments, as shown in FIG. IC, an open channel comprises an inlet 105 and an outlet 106. In certain embodiments, the open channel comprises three orthogonal dimensions. In some embodiments, the open channel has a length, a width, and a height. For example, in FIG. 1A, the open channel 103 has a length 401, a width 402 (running into and out of the page in FIG. 1A), and a height 403. In some embodiments, the open channel is configured such that a liquid may enter the open channel via the inlet, flow through the open channel, and exit the open channel via the outlet.

[0040] In some embodiments, the substrate may be positioned adjacent a surface. In some embodiments, an analyte is disposed on the surface. For example, an analyte may comprise one or more biological cells (e.g., a plurality of biological cells). In some embodiments, the surface comprises a tissue surface. In some embodiments, the surface comprises a cell culture surface (e.g., the surface of a cell culture plate). In some embodiments in which the surface is a tissue surface or a cell culture surface, the analyte may comprise one or more biological cells disposed thereon. The analyte may comprise any other suitable compound and / or composition that it may be desirable to remove from the surface.

[0041] It should be understood that the term “analyte” is used herein to refer to any material that one desires to remove from a surface, and subsequent identification, measurement, or other analysis of the analyte after removing from the surface is not required for a material to qualify as an analyte. In some embodiments, the analyte is a cell. Generally, unless the context clearly indicates otherwise, the term “cell” is used herein to refer to a biological cell. In some embodiments, the analyte is an animal cell.

[0042] #14855532vl-1- In certain embodiments, the analyte is a human cell. In some embodiments, the analyte is a cancer cell. In some embodiments, the analyte is a molecule (e.g., a peptide, a protein, and / or a nucleotide). In some embodiments, the analyte is a fragment of a cell. In some embodiments, the analyte is a biofouler (i.e., a material that is present due to biofouling). In some embodiments, a biofouler may be a mineralized compound, a solid particle, and / or a biological cell. In some embodiments, the analyte is a non-living colloidal solid.

[0043] In some embodiments, as described in greater detail below, the substrate may be configured such that when the outer surface of the substrate is positioned adjacent a surface (e.g., a surface comprising an analyte disposed thereon, as described above), the surface may enclose the open channel to form a closed conduit. In some such embodiments, a liquid may enter the closed conduit via the inlet, flow through the closed conduit, and exit the closed conduit via the outlet. As the liquid is flowed through the closed conduit, it may come into contact with the surface. In some such embodiments, flowing the liquid through the closed conduit may cause the analyte to be removed from the surface. In some embodiments in which the surface comprises one or more biological cells disposed thereon (e.g., in embodiments in which the surface is a tissue surface or a surface used for cell culture), flowing the liquid through the closed conduit may cause one or more of the biological cells to be removed from the surface. In some embodiments, flowing the liquid through the closed conduit and thereby causing one or more biological cells to be removed from the tissue surface or cell culture surface may be an advantageously gentle method of removing the biological cells from the surface, and may thereby result in a comparatively lower risk of damage to the biological cells than other methods for removing cells from surfaces.

[0044] The substrate may comprise any of a variety of suitable materials. For example, in some embodiments, the substrate may comprise a polymer, a thermoplastic, a thermoset, an elastomer, a polymer blend, and / or a polymer composite. In some embodiments, the substrate comprises a polymer which is suitable for use in additive manufacturing (AM). In certain embodiments, the substrate may comprise a polycarbonate, a polyamide, a polystyrene, polylactic acid, and / or polydimethyl siloxane (PDMS). In some embodiments, the substrate may comprise a non-porous material. In some embodiments, the non-porous material may be a metal. In certain embodiments,

[0045] #14855532vlthe substrate may comprise a cellulosic material. In certain embodiments, the substrate may comprise wood.

[0046] The open channel in relief from the outer surface of the substrate may have any of a variety of suitable heights. In some embodiments, the open channel has a height which is relatively small. For example, an open channel in relief from the outer surface of the substrate may have a height of less than or equal to 5 mm, less than or equal to 1 mm, less than or equal to 500 micrometers, less than or equal to 100 micrometers, less than or equal to 50 micrometers, less than or equal to 10 micrometers, less than or equal to 5 micrometers, or less than or equal to 1 micrometer (and / or, in some embodiments, as little as 0.5 micrometers, as little as 0.1 micrometers, or less). In some embodiments, the open channel in relief from the outer surface of the substrate has a height of greater than or equal to 0.1 micrometers, greater than or equal to 0.5 micrometers, greater than or equal to 1 micrometer, greater than or equal to 5 micrometers, greater than or equal to 10 micrometers, greater than or equal to 50 micrometers, greater than or equal to 100 micrometers, greater than or equal to 500 micrometers, greater than or equal to 1 mm, or greater. Combinations of these ranges are also possible (e.g., in some embodiments, the open channel in relief from the outer surface of the substrate may have a height of less than or equal to 500 micrometers and greater than or equal to 5 micrometers).

[0047] The open channel in relief from the outer surface of the substrate may have any of a variety of suitable widths. For example, an open channel in relief from the outer surface of the substrate may have a width of less than or equal to 5 mm, less than or equal to 1 mm, less than or equal to 500 micrometers, less than or equal to 100 micrometers, less than or equal to 50 micrometers, less than or equal to 10 micrometers, less than or equal to 5 micrometers, or less than or equal to 1 micrometer (and / or, in some embodiments, as little as 0.5 micrometers, as little as 0.1 micrometers, or less). In some embodiments, the open channel in relief from the outer surface of the substrate has a width of greater than or equal to 0.1 micrometers, greater than or equal to 0.5 micrometers, greater than or equal to 1 micrometer, greater than or equal to 5 micrometers, greater than or equal to 10 micrometers, greater than or equal to 50 micrometers, greater than or equal to 100 micrometers, greater than or equal to 500 micrometers, greater than or equal to 1 mm, or greater. Combinations of these ranges are also possible (e.g., in some embodiments, the open channel in relief from the outer

[0048] #14855532vlsurface of the substrate may have a width of less than or equal to 500 micrometers and greater than or equal to 5 micrometers).

[0049] The open channel in relief from the outer surface of the substrate may have any of a variety of suitable lengths. For example, an open channel in relief from the outer surface of the substrate may have a length of less than or equal to 50 cm, less than or equal to 10 cm, less than or equal to 5 cm, less than or equal to 1 cm, less than or equal to 5 mm, less than or equal to 1 mm, less than or equal to 500 micrometers, less than or equal to 100 micrometers, less than or equal to 50 micrometers, or less than or equal to 10 micrometers, or less. In some embodiments, an open channel in relief from the outer surface of the substrate may have a length of greater than or equal to 10 micrometers, greater than or equal to 50 micrometers, greater than or equal to 100 micrometers, greater than or equal to 500 micrometers, greater than or equal to 1 mm, greater than or equal to 5 mm, greater than or equal to 10 mm, or greater than or equal to 1 cm. Combinations of these ranges are also possible (e.g., in some embodiments, the open channel in relief from the outer surface of the substrate may have a length of less than or equal to 50 cm and greater than or equal to 1 cm).

[0050] In some embodiments, the substrate comprises a seal region. For example, in FIG. IB, the substrate 101 comprises a seal region 104. In certain embodiments, the seal region may be configured such that when the substrate comprising an open channel in relief from the surface of the substrate is in contact with a surface (e.g., a surface comprising an analyte disposed thereon, as discussed above) to form a closed conduit, the seal region couples and / or seals the surface to the substrate. In some embodiments, the seal region may be configured to couple and / or seal the surface to the substrate such that the surface and the substrate will remain coupled under the influence of an opposing force. In some embodiments, the seal region may be configured to couple and / or seal the surface to the substrate such that the surface and the substrate will remain coupled under the influence of an opposing force of at least 1 N, at least 2.5 N, at least 5 N, at least 10 N, or at least 25 N. In some embodiments, the seal region may be configured to couple and / or seal the surface to the substrate such that the surface and the substrate will remain coupled under the influence of an opposing force of less than or equal to 50 N, less than or equal to 25 N, less than or equal to 10 N, less than or equal to 5 N, or less than or equal to 2.5 N. Combinations of these ranges are also possible (e.g. the seal region may

[0051] #14855532vlbe configured to couple and / or seal the surface to the substrate such that the surface and the substrate will remain coupled under the influence of an opposing force of at least 1 N and less than or equal to 50 N).

[0052] The seal region may comprise any of a variety of suitable sealing mechanisms. In some embodiments, the seal region may comprise a vacuum seal. For example, in some embodiments, the system can include a source of vacuum (e.g., a vacuum pump, a fluid flow pump, a vacuum-creating piston (such as a syringe) or another suitable vacuum source), and the source of the vacuum can be in fluidic communication with a channel that surrounds the open channel formed in relief from the outer surface of the substrate, such that when the substrate is placed in contact with a surface to transform the open channel formed in relief from the outer surface of the substrate into a closed channel or conduit, a vacuum is created around the closed channel or conduit, sealing the closed channel or conduit from an external environment (e.g., as shown in FIG. 2).

[0053] The present disclosure is not limited to the creation of seal regions using vacuum channels, and other types of seal regions may be used. In certain embodiments, the seal region may comprise an adhesive and / or a reversible adhesive. In some embodiments, the seal region may comprise a region configured to cause temporary modifications to a surface when the substrate is contacted with the surface. In some embodiments, the seal region may comprise a hydrogel, a charged hydrogel, and / or a treated polymer (e.g. treated PDMS). In certain embodiments, the seal region comprises a double-sided adhesive (e.g., double-sided tape).

[0054] In certain embodiments in which the seal region comprises a vacuum ring, the vacuum ring may be connected to an external vacuum source. For example, as shown in FIG. IB, the seal region 104 is configured as a vacuum ring that is fluidically connected to vacuum source 203. In some embodiments (e.g., vacuum source 203 in FIG. IB), the external vacuum source is a syringe. In some embodiments, the syringe comprises a plunger and a barrel, such that when the substrate is contacted to a surface and the plunger of the syringe is pulled back, vacuum is generated to seal the substrate to the surface. In certain embodiments, the generation of vacuum in the vacuum ring is automatically actuated.

[0055] In some embodiments, the inlet of an open channel in relief from the outer surface of a substrate may be fluidically connected to a reservoir. For example, in FIG.

[0056] #14855532vlIB, the inlet of the open channel 103 is fluidically connected to reservoir 201. In certain embodiments, the reservoir is configured such that the flow rate of a fluid from the reservoir to the inlet of the channel is controllable. In some embodiments (e.g., reservoir 201 in FIG. IB), the reservoir comprises a syringe. In certain embodiments, the syringe is configured such that it is automatically actuated (e.g., via a syringe pump or other actuation device). In some embodiments, the syringe is sterile.

[0057] In some embodiments, the reservoir may contain a fluid. The reservoir may contain a fluid that is suitable for transferring biological cells from one location to another. For example, the fluid may comprise a buffer solution, a saline solution, a growth medium, or another aqueous solution configured to stably transfer the biological cells (e.g., without causing damage to the biological cells). This may be advantageous when the device is used to remove one or more biological cells from a surface. For example, in some embodiments, the substrate may be positioned adjacent a surface comprising one or more biological cells disposed thereon (e.g., a tissue surface, a cell culture surface) to form a closed conduit, and a fluid may be flowed into the closed conduit by flowing the fluid from the reservoir, to the inlet of the closed conduit, and through the closed conduit, thereby causing one or more biological cells to be removed from the surface. In some such embodiments, as described below, the one or more biological cells removed from the surface may be flowed out of the closed conduit through the outlet thereof to a different location and / or for further testing, culture, and / or processing.

[0058] In some embodiments, the outlet of an open channel in relief from the outer surface of a substrate may be fluidically connected to an outflow conduit. For example, in FIG. IB, the outlet of the open channel 103 is fluidically connected to the outflow conduit 202. In certain embodiments, the outflow conduit comprises tubing. In some embodiments, the tubing is sterile. In certain embodiments, the outflow conduit comprises a collection vessel. In some embodiments, the collection vessel is sterile. It may be advantageous for the collection vessel to be sterile in embodiments in which the substrate is positioned adjacent a surface comprising one or more biological cells disposed thereon (e.g., a tissue surface, a cell culture surface) to form a closed conduit, and a fluid is flowed into the closed conduit (e.g., from a reservoir via the inlet of the closed conduit, as described above) and through the closed conduit, thereby causing one

[0059] #14855532vlor more biological cells to be removed from the surface, the one or more biological cells may be flowed into the collection vessel via the outlet of the closed conduit. In some such embodiments, it is advantageous for the collection vessel to be sterile in order to limit or avoid damage to and / or contamination of the biological cells, which may be subjected to further testing, culture, and / or processing.

[0060] The systems and articles described herein can be manufactured using a variety of suitable techniques. For example, in some embodiments, the article can be manufactured using additive manufacturing, micromachining, or a combination of both. Using additive manufacturing or micromachining may in some embodiments allow for the achievement of small feature size in the article, for example an open microchannel in relief from the surface of a substrate having a small height. In some embodiments, the article described herein may be reusable.

[0061] According to certain embodiments, methods of removing an analyte from a surface are described. In certain embodiments, the method comprises contacting a substrate comprising an open microfluidic channel (e.g., an open channel in relief from the outer surface of the substrate, as described above) to a surface comprising an analyte to form a closed conduit. For example, in FIG. 1A, closed conduit 301 comprises surface 150 comprising analyte 140. In certain embodiments, a fluid 120 may be flowed through the closed conduit 301. In some embodiments, flowing a fluid through the closed conduit may cause the analyte 140 to be removed from the surface, becoming free analyte 141 in the fluid. In certain embodiments, the substrate comprising an open microfluidic channel may have any of the characteristics of a substrate described above. In some embodiments, the open microfluidic channel may have any of the characteristics of the open channel described above.

[0062] In certain embodiments, after the analyte has been removed from a surface, the analyte may be identified. In certain embodiments, after the analyte has been removed from a surface, one or more properties of the analyte may be measured (e.g., a viability of the analyte, a genome of the analyte, or one or more other properties).

[0063] In certain embodiments, the surface may comprise tissue. The tissue may comprise, for example, animal tissue such as human tissue. In some embodiments, the removal of the analyte may be performed in vivo. For example, removal of one or more biological cells from tissue may be performed in vivo, in some embodiments. In certain

[0064] #14855532vlembodiments, the tissue may be biopsied tissue. In certain embodiments, the tissue may comprise cancer cells.

[0065] In certain embodiments, flowing a fluid through a closed conduit may comprise flowing a fluid into an inlet of the conduit. In some embodiments, a fluid is flowed from a first location that is fluidically connected to the conduit to the inlet of the conduit. In some embodiments, flowing a fluid through a closed conduit may comprise flowing the fluid out of an outlet of the conduit. In some embodiments, the fluid is flowed to a second location from the outlet of the conduit.

[0066] In some embodiments, flowing a fluid through the closed conduit comprises applying a positive pressure to the fluid upstream of the inlet of the conduit. In some embodiments, the first location comprises a syringe. In certain embodiments, applying a positive pressure to the fluid upstream of the inlet of the conduit comprises depressing the plunger of the syringe. In some embodiments, flowing a fluid through the closed conduit comprises applying a negative pressure to the fluid downstream of the outlet of the conduit. In some embodiments, the second location comprises a syringe. In some embodiments, applying a negative pressure to the fluid downstream of the outlet of the conduit comprises pulling back the plunger of the syringe.

[0067] In certain embodiments, the fluid that is flowed through the conduit comprises a liquid. In some embodiments, the fluid that is flowed through the conduit comprises an aqueous solution. In some embodiments, the aqueous solution comprises a buffer solution and / or a saline solution. In certain embodiments, the fluid that is flowed through the conduit comprises a growth medium.

[0068] In some embodiments, the fluid is flowed through the closed conduit at a controllable volumetric flow rate. In certain embodiments, the volumetric flow rate of the fluid may be changed in discrete increments over a period of time. In certain embodiments, changing the volumetric flow rate of the fluid in discrete increments over a period of time may result in analytes having different masses, densities, and / or adhesive strengths being removed from the surface at different points in time. For example, during a first period of time, fluid may flow through the conduit at a first volumetric flow rate, and a first type of analyte having a first adhesion strength may be removed from the surface. In some such embodiments, during a second period of time, fluid may be flowed through the conduit at a second volumetric flow rate, and a second

[0069] #14855532vltype of analyte having a second adhesion strength different from the first adhesion strength may be removed from the surface. In some such embodiments, during a third period of time, fluid may be flowed through the conduit at a third volumetric flow rate, and a third type of analyte having a third adhesion strength different from the first adhesion strength and the second adhesion strength may be removed from the surface.

[0070] In some embodiments, the flow rate at which an analyte is removed from the surface may be used to determine the mass, density, adhesive strength and / or another property of the analyte. In some embodiments, flowing a fluid through the conduit at at least two different flow rates over at least two different periods of time may be performed as part of a process during which multiple different types of analytes are selectively removed based on the mass, density, adhesive strength and / or other properties of the analytes. This may, for example, facilitate the isolation of different cell types having different adhesion strengths. In some embodiments, fluid can be flowed through the enclosed channel or conduit such that a first type of analyte (e.g., a first type of cells) can be removed from the surface such that at least 80 wt%, at least 90 wt%, at least 95 wt%, at least 99 wt%, or 100 wt% of the analytes present within a first exit stream are analytes of the first type. In some embodiments, subsequently, fluid can be flowed through the enclosed channel or conduit such that a second type of analyte (e.g., a second type of cells) can be removed from the surface such that at least 80 wt%, at least 90 wt%, at least 95 wt%, at least 99 wt%, or 100 wt% of the analytes present within a second exit stream are analytes of the second type. In certain embodiments, subsequently, fluid can be flowed through the enclosed channel or conduit such that a third type of analyte (e.g., a third type of cells) can be removed from the surface such that at least 80 wt%, at least 90 wt%, at least 95 wt%, at least 99 wt%, or 100 wt% of the analytes present within a third exit stream are analytes of the third type.

[0071] This may be advantageous for removing unwanted cells from a tissue, as the adhesion strength of cancer cells can act as a useful physical biomarker indicating their invasiveness or metastatic potential.

[0072] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0073] EXAMPLE

[0074] #14855532vlLeveraging cell-adhesion mechanics is important for developing alternative mechanisms of live cell capture for cancer diagnosis and development of patient-derived organelles which avoid the pitfalls of patient pain, scarring, tumor seeding and epithelial overgrowth associated with traditional live cell capture techniques, while allowing for multiple sampling points to apprehend tumor heterogeneity.

[0075] This Example describes, in accordance with certain embodiments, a device which takes advantage of the increased shear stresses of restricted fluid flow to detach and capture viable cells from tissue with minimal damage. The device, illustrated in FIG. 3A, comprises a microfluidic channel in relief from the surface of a substrate. When contacted to a tissue surface and adhered to that surface via the application of vacuum pressure through a vacuum ring within the substrate, this device allows for the formation of a closed conduit with tissue or cells forming the base of said closed conduit. The establishment of this closed conduit, and subsequent application of force to drive fluid flow, enables a restricted flow which can de-adhere whole cells from the matrix or substrate to which they are bound when the shear stresses induced by the working fluid acting on the cells exceed their adhesion strength. This device can be configured to apply distinct and increasing levels of shear stress to a tissue site, to collect and assess cells removed at different shear stresses, a diagnostic method of “shear spectroscopy.” The device comprised a vacuum ring, as shown in FIG. 3A, which was used to maintain the integrity of the closed conduit formed by the microchannel and the tissue surface. Other mechanisms for maintaining the integrity of the closed conduit are also possible, including temporary surface energy modifications, suction cup-like elastomers, or mechanical grasping. The inclusion of such a mechanism was useful at least because, to drive flow at a constant speed against viscous stresses, such as those encountered in a restricted channel, establishing a pressure gradient throughout the fluid is helpful (and, under some conditions, necessary). The lowest pressure existed at the outlet of the channel, which, in the case of this device, was exposed to air and was therefore at atmospheric pressure. Thus, the inlet to the conduit experienced the highest pressure, while the pressures throughout the conduit were at intermediate values. Under many conditions, without any opposing forces, the pressures in the conduit pushed the tissue or substrate away, breaching the conduit, undesirably reducing the fluidic shear stresses and allowing the spread of cells away from the biopsy site. In order to counteract this, the

[0076] #14855532vldevice used in this Example employed a vacuum seal surrounding the microchannel to maintain the integrity of the connection between the microchannel and the tissue surface. Other mechanisms for ensuring connection integrity are possible, such as temporary surface energy modifications, suction cup-like elastomers, or even mechanical grasping.

[0077] One additional consideration in the design of this device is that, when detaching cells through the use of physical mechanisms such as restricted fluid flow, it can be important to ensure that the physical or mechanical forces or stresses do not exceed those required to rupture the cell membranes. Cells have numerous cellular mechanisms and signaling pathways to facilitate membrane repair. However, the size of the injury to the membrane is an important factor determining whether these pathways will be successful, or the cell will lyse and die, particularly in the case of eukaryotic cells (which include human cells). Here, an injury on the scale of nanometers may be enough to prevent spontaneous recovery of the cell membrane. However, the mechanical stresses required to rupture the cell membrane of eukaryotic cells can be on the order of several thousand pascals (the kPa range) for both compressive and tensile stress application. Thus, since the adhesion strength of cells lies an order of magnitude or less below this range, it should be possible to detach the cell while avoiding membrane compromise and cell death. It can be confirmed that the microchannels used in this Example meet this criterion using a simple mathematical test.

[0078] The shear stress induced by fluid flow next to a wall, also known as the wall shear stress, is given by the following formula (Eqn. 1):

[0079]

[0080] where u is the velocity of the fluid, at the distance y from the wall, and / z is the dynamic viscosity of the fluid. The above criterion, in conjunction with the no-slip boundary condition (u|y=0= 0) indicates that one can increase shear stress either by enhancing viscosity of the working fluid, increasing the flow rate, or by constricting the flow geometry (diminishing y). Conversely, the Navier slip condition dictates a non-zero velocity at y = 0, that is the existence of a slip velocity. Such a boundary condition, typically seen in free surface flows, has a reduced shear stress because of the diminished velocity gradient as is evident from Eqn. 1. Thus, as a design principle, a constricted flow was chosen that exploited the no-slip boundary condition at all the interfaces, as well as miniaturizing the channel dimensions to increase bulk dissipation or viscous #14855532vlstress. Additionally, this constricted flow constricted the flow, thus delimiting the spread of cancer cells to other sites in the body.

[0081] In the Poiseuille framework, the flow can be modeled as a fully developed laminar flow between infinite plates, is given by Eqn. 2:

[0082]

[0083] where uavgis the average velocity between the plates and h is the distance between plates. Consider the use of water, phosphate buffer solution (PBS), or growth media such as RPMI, all of which have a dynamic viscosity around 1 mPa s, as the working fluid. Even at the limit of the breakdown of the assumption of laminarity, for a typical Reynold’s number of 2000, a flow of 10 m / s through a constriction of h = 150 pm, which is the height of the microchannel used in this Example, yields a shear stress of the order of 400 Pa (a shear value able to remove cells on the higher end of adhesion strength). This is far below the shear stress at which there is the substantial risk of cell rupture, indicating that the microchannels formed by the device used in this Example can be safely used to remove cells from a surface without risking cell rupture.

[0084] In order to achieve shear stresses in the appropriate range for these applications, the size of the microchannel must be taken into consideration. A larger microchannel requires a larger device, which would be easier to manufacture and would have a larger area over which to capture cells. However, this results in reduced precision and requires higher flow rates to achieve the same shear stresses that a device with a smaller microchannel height could produce. This leads to more dilute cell suspensions, which potentially complicates the production of organoids or other diagnostics. Additionally, for a larger channel to achieve the same shear stresses as a smaller channel, the opposing forces required to maintain the integrity of the channel will be much higher. As an extremely rudimentary example, we can model the microchannel pictured in FIG. IB as a rectangular duct with height h. width w, and length I. With a fully developed viscous laminar flow, we can write the following equations (Eqn. 3 - Eqn. 5):

[0085] AP = 4 ^l / Dh(Eqn. 3) <

[0086]

[0087] AP

[0088] ^reactive l (Eqn. 5)

[0089] #14855532vl is the average wall shear stress, Dhis the hydraulic diameter, and Po is the Poiseuille Number (a factor accounting for the geometry of the rectangular channel). We look at the same average shear stress of 25 Pa for two cases: 1) w = I = 2500 \im, h = 500 pm and 2) w = I = 500 pm, h = 500 pm (a 5-time reduction in channel dimensions). Both cases achieve the same AP of 300 Pa. However, in 1) Q ~ 3 ml / s and ^reactive ~ 1 mN, while in 2) Q ~ 0.02 ml / s and Freactive ~ 0.04 mN (a reduction of two-orders of magnitude). Clearly, reducing the size of the device aids in operating the device with lower forces. With these design considerations accounted for, the device in FIG. 3 A was fabricated and used to remove cells from a tissue surface. An illustration of this device being used to remove cells from tissue (specifically, a fallopian tube) is pictured in FIG. 3B. In Inset 1, the device contacts the tissue at a desired location, and light manual pressure is applied. In Inset 2, a cell outflow tube is placed into clean collection container. In Inset 3, a plunger of the syringe, initially fully plunged, is pulled back until a vacuum seal is established. In Inset 4, a plunger of the syringe is pushed down until all working fluid passes through the microchannel and is collected. In Inset 5, a plunger of the syringe applying vacuum pressure is slowly released, releasing the vacuum seal. In Inset 6, the device is removed so that it is no longer contacting tissue.

[0090] FIG. 3C shows, according to certain embodiments, a cross-sectional schematic illustration of flow pathways within the fluidic device of FIG. 3 A. As shown in FIG. 3C, one syringe can be connected to the vacuum ring, and the syringe can be used to pull vacuum on the volume of the vacuum ring to establish a seal with the underlying substrate.

[0091] FIGS. 3D-3I show, according to certain embodiments, various view of the bottom portion of the device of FIG. 3A. For example, FIG. 3D is a side view schematic illustration of the bottom portion of the fluidic device of FIG. 3A. FIG. 3E shows, according to certain embodiments, a first bottom perspective view schematic illustration of the bottom portion of the fluidic device of FIG. 3 A. FIG. 3F shows, according to certain embodiments, a front view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3 A. FIG. 3G shows, according to certain embodiments, a first bottom view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3 A. FIG. 3H shows, according to certain embodiments, a second bottom perspective view schematic illustration of the fluidic

[0092] #14855532vldevice of the bottom portion of the fluidic device of FIG. 3A. FIG. 31 shows, according to certain embodiments, a second bottom view schematic illustration of the fluidic device of the bottom portion of the fluidic device of FIG. 3A.

[0093] There are several possibilities of manufacturing the device, including additive manufacturing (e.g., 3D printing), micromachining, or a combination of both. These methods are particularly useful given the small feature size (on the order of hundreds of micrometers) for the channel heights and overall complexity of the flow channel.

[0094] The device was used to capture cells from various tissues acquired from porcine and murine animal models. For these experiments, either a pig or mouse was sacrificed, and within 24 to 48 hours, with tissue covered in growth media and on ice in downtime, a sanitized cell capture device (similar to picture in FIG. 3A) was used to capture cells as a suspension from tissues excised from the animals. In each instance, the device was contacted with the tissue such that the open microfluidic channel and the tissue formed a closed conduit. Once the closed conduit was established, a fluid was flowed at a constant speed through the conduit. In order connect the conduit to a source of working fluid and an external vacuum, this device employed a 3D printed flexible resin Luer lock attachment secured to two syringes. A linear stage with force transducer was used apply the force to drive fluid flow, while vacuum was applied manually (see FIG. 4). The use of a linear stage with a force transducer allows for more controlled volumetric flow rates through the conduit. The collection time was inversely related to the volumetric flow rates, as the Eppendorf tubes used for cell collection were the same size for each biopsy site / test.

[0095] Once cells were collected, they were processed. In some instances, the suspension of captured cells was spun down and either plated on well plates coated with 2% gelatin (1-hour incubation) followed collagen IV solution (0.006 % w / v) (> 4-hour incubation) for 2D cell culture or seeded in Matrigel (which provides 3D structure for cells to grow on) in well plates for 3D cell culture. The cells were cultured over the course of days to weeks using a culture medium cocktail containing regular DMEM / F12 plus epidermal growth factor, insulin, antibiotics and antifungal additives, and some other nutrients. In the other instances, the suspension of captured cells was spun down, resuspended in a smaller volume of liquid, stained with a viability dye and counted with Fluorescence Activated Cell Sorting (FACS) or a benchtop fluorescent cell counter.

[0096] #14855532vlAdditionally, any marks due to vacuum at the biopsy site were shown to diminish over the course of several minutes, indicating that this device would cause minimal damage in vivo. In the murine animal model experiments, three mice were given cancer and were later sacrificed. Tumors growing on their legs were surgically removed, as well as non-cancerous tissue from the leg, liver, and skin. These tissue samples were stored in separate containers with growth media while awaiting testing. The tissue was tested using the apparatus depicted in FIG. 4 with a volumetric flow rate of 3 ml / s and a channel footprint of ~ 6 mm2, with individual suspensions of cells captured in fresh 1.5 mL Eppendorf tubes. One sample of leg tissue was not tested. The cell suspensions were individually spun down, resuspended and stained with a Live / Dead Assay. The cell suspensions were then filtered into falcon tubes, and fluorescence activated cell sorting (FACS) was performed on each sample after calibrating with a live and dead control. Significant intratissue variability can be observed in FIG. 5, which shows the cell counts and viabilities observed in these experiments. However, significant differences were observed between both the cell count and the viability between certain tissue types, like liver and normal leg tissue. Thus, these results lend credence that this device has selectivity for tissue type based on the applied flow rate. Additionally, these results demonstrate that one can capture viable cells from tissue, even if a few hours have passed since the organism was sacrificed. Also, on average there are many more cells captured from tumor compared to normal leg tissue, which is an interesting finding regarding diagnostic implications of this device. In the porcine animal model experiments, porcine reproductive system tissues were sampled one day after the animal was sacrificed. Using a sterile device, the surface of both oviduct tissue and ovary tissue were tested with a flow rate of roughly 3 mL / s and channel footprint of ~ 6 mm2. The 2D culture results of a single test for oviduct and ovary are pictured in FIG. 6. For both tests, large amounts of cells were removed from the tissue. However, after the initial media change, many cells were removed, as they did not adhere to the 2D substrate. This may be a result of the initial viability of the tissue, as it was received a full day after the animal was sacrificed, and transported tissues for transplantation may only be viable up to 12 hours for static cold storage without special media. Regardless, cell spreading and proliferation of the cells collected in both tissues were observed (though more so from the ovarian cells). Overall, this is a highly promising result that this device can capture

[0097] #14855532vllive cells that are culturable, showing its potential for the development of PDOs. The fluidic device was also used to measure the adhesion of MG63 cells (ATCC CRL-1427), a human bone osteosarcoma cell line. In this experiment, petri dishes with MG63 cultures were prepared, and microscopic images of the petri dish were taken before starting the experiment. The fluidic device was then applied normally to the petri dish with a force of around 1 to 2 N to seal the channel and prevent leakage. A given flow velocity was then applied for 5 seconds, collecting all fluid (PBS and cells) that went through the fluidic device. An image was then taken of the collection area on the petri dish. The proportion of cells remaining after flow (discretized along the length of the channel) was correlated with simulated applied shear stresses. This test was repeated several times for volumetric flow rates ranging from 0.01 mL / s to 1 mL / s (corresponding to a lOmL syringe of 8mm radius with a plunger descent speed of 3 mm / min to 300 mm / min). The speed of the plunger was set using a motorized linear stage. The correlation between the syringe plunger's descent velocity (vx) and the volumetric flow rate (Q) within the fluidic device is given by the conservation of mass of an incompressible (constant density fluid) in the syringe and in the channel (PBS). Let r be the syringe radius, S2be the channel inlet area, and v2be the average velocity inside the channel. With S2~ (2.425 mm)(0.635 mni) ~ 1.54 mm2and r = 8 mm, we have in Eqn. 6:

[0098]

[0099] > , 0.65 m / s) (Eqn. 6) Images of the petri dish were taken with an inverted microscope before and after the test at 2x magnification (FIG. 7). The footprint of the device channel was clearly visible in these images. Cells that had been in contact with the device had been detached by direct contact (given the noncompliant nature of the petri dish substrate) and were ignored in analysis of cell removal rate. Inside the square corresponding to the channel, it was seen that increasing flow speed increased the number of cells removed. Moreover, the distribution of the remaining cells was inhomogeneous, suggesting the presence of edge effects and developing flow. These images were then analyzed with Fiji (an open-source image processing toolbox built on top of ImageJ2) to determine the proportion of cells remaining after removal. The number of cells remaining on the surface after testing was

[0100] #14855532vlcompared with the number of cells on the surface before testing. The proportion of cells remaining is given in FIG. 8 as a function of x-position along the length of the channel.

[0101] The device described in this Example has the demonstrated capability to detach numerous cells (on the order of 10000) from a small area of tissue (~ 6 mm2). Many of these attached cells were viable, and successful culturing of captured cells has been demonstrated. Clearly, restricted fluid flow is a viable mechanism to extract cells from tissue. In the testing involving the use of a suction mechanism to provide the opposing forces required to maintain the integrity of the closed conduit, any marks left over from the device on the tissue faded over the course of minutes, with only a light imprint remaining. Also, no blood was produced in these tests. This suggests that this device does not cause scarring or bleeding, which immediately gives it an advantage over existing tissue biopsy methods for skin and other organs. In addition, the invention exhibited minimal leaking, which will minimize risk of spreading cancerous cells throughout the body if used. If accessing internal regions of the body, the invention could likely fit the required footprint of laparoscopic tools giving the added quality of minimal invasiveness. The low scarring, bleeding, and risk of spread along with minimal invasiveness would allow for easy and safe biopsies of multiple locations to either 1) capture the heterogeneity of a tumor or 2) assess the pathology of a more representative sampling of tissue. With an automated actuation mechanism, varying and controlled flow profiles can be applied, which may allow for extraction of different cell types or chunks of tissues versus individual cells. This provides a versatility not available in other devices. If one wants to capture a suspension of individual cells to avoid the enzymatic or mechanical tissue dissociation steps common to organoid development, one can apply lower, more controlled flow rates to avoid breaking off small clumps of tissue. On the other hand, if one wants to extract small clumps of tissue among individual cells to perform cytopathological analysis that captures the underlying tissue architecture, one can apply higher flow rates. Still yet, if one wants to separately perform cytopathological analysis on cells extracted from a lesion and histopathological analysis on the lesion itself acquired in a typical tissue biopsy, one can apply lower flow rates to extract small numbers of potentially cancerous or pre-cancerous cells. Lastly, one could increase the flow rate by discrete intervals to remove and collect different cell types at different shear stresses based on their adhesion strength. This would aid in separating epithelial cells

[0102] #14855532vlfrom other cells of interest to avoid the issue of epithelial overgrowth. This would also allow one to use shear stress as a potential biophysical marker for invasiveness or metastatic potential. The device described in this Example has also demonstrated the ability to detach MG63 cells. It has been determined that cell detachment takes place beginning at a particular shear stress (about 22.5 Pa) corresponding to a flow velocity of 0.42 m / s. The device has also been tested with the OVCAR8 cells, a non-ovarian carcinoma cell line , showing similar results: the adhesion of OVCAR8 being close to that of MG63. For future applications involving the detachment of cells with different adhesion characteristics, a higher or lower flow rate than that currently used will be considered. This may involve reducing or increasing the size of the syringe. The modification of the dimensions of the device will be considered to modify the flow velocity while limiting the force required to initiate flow. The current design allows localized cell removal and detachment over an area ~6 mm2. Future modifications to the design are envisaged. For example, to improve precision by enabling more cell removal over a larger channel footprint. Shape modifications may also be considered to improve device efficiency. For example, a fluid inlet in the center of a circular channel will be tested. This would enable cells to be collected from a circular rather than a rectangular area, enabling radial symmetry.

[0103] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically

[0104] #14855532vldescribed and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0105] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0106] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0107] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0108] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not

[0109] #14855532vlnecessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0110] As used herein, “wt%” is an abbreviation of weight percentage. As used herein, “at%” is an abbreviation of atomic percentage.

[0111] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0112] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0113] When a portion (e.g., a layer, a structure, a region) is “on”, “adjacent”, “above”, “over”, “overlying”, or “supported by” another portion, it can be directly on the portion, or an intervening portion (e.g., layer, structure, region) may also be present. Similarly, when a portion is “below” or “underneath” another portion, it can be directly below the

[0114] #14855532vlportion, or an intervening portion (e.g., layer, structure, region) may also be present. A portion that is “directly adjacent”, “directly on”, “immediately adjacent”, “in contact with”, or “directly supported by” another portion means that no intervening portion is present. It should also be understood that when a portion is referred to as being “on”, “above”, “adjacent”, “over”, “overlying”, “in contact with”, “below”, or “supported by” another portion, it may cover the entire portion or a part of the portion.

[0115] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0116] #14855532vl

Claims

CLAIMS1. An article, comprising:a substrate comprising an open channel configured such that when the substrate is positioned adjacent to a surface and fluid is flowed through the channel, an analyte is removed from the surface.

2. The article of claim 1, wherein the open channel comprises an inlet and an outlet.

3. The article of any one of claims 1-2, wherein the open channel is in relief from an outer surface of the substrate.

4. The article of any one of claims 1-3, further comprising a seal region configured to isolate the open channel from an external environment.

5. An article, comprising:a substrate comprising an outer surface; andan open channel in relief from the outer surface;wherein:the open channel comprises an inlet and an outlet; andthe outer surface comprises a seal region configured to isolate the open channel from an external environment.

6. The article of claim 5, further comprising a reservoir fluidically connected to the inlet.

7. The article of any one of claims 5-6, further comprising an outflow conduit fluidically connected to the outlet.

8. The article of any one of claims 5-7, wherein the seal region comprises a vacuum seal.#14855532vl9. The article of any one of claims 5-8, wherein the seal region comprises an adhesive.

10. The article of claim 9, wherein the adhesive is a reversible adhesive.

11. The article of any one of claims 5-10, wherein the seal region is configured to couple and / or seal the outer surface of the substrate to a surface with which the seal region is contacted such that the surface and the outer surface of the substrate will remain coupled under the influence of an opposing force of at least 1 N.

12. The article of any one of claims 1-11, wherein the open channel comprises an open microfluidic channel.

13. the article of any one of claims 1-12, wherein the substrate is formed from a polymer.

14. A method, comprising:contacting a substrate comprising an open microfluidic channel to a surface comprising an analyte to form a closed conduit; andflowing a fluid through the closed conduit such that the analyte is removed from the surface.

15. The method of claim 14, wherein the analyte comprises biological cells.

16. The method of any one of claims 14-15, wherein the surface comprises tissue.

17. The method of any one of claims 14-16, wherein the fluid comprises a buffer solution.

18. The method of any one of claims 14-17, wherein the fluid comprises a growth medium.#14855532vl19. The method of any one of claims 14-18, further comprising, after the removal of the analyte from the surface, subjecting the analyte to testing, culture, and / or processing.

20. The method of any one of claims 14-19, wherein the fluid is flowed through the closed conduit at a first volumetric flow rate during a first period of time and at a second volumetric flow rate during a second period of time.#14855532vl

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