Devices and methods for isolation of biological entities in suspension

Abstract

The present disclosure is generally directed to various methods and apparatuses for isolation of biological entities in suspension utilizing various techniques. One example application is the extraction and denudation of oocytes from a fluid. Oocytes in biological fluids may need to be isolated and / or denuded before they can be effectively used in various applications, such as in vivo fertilization (IVF) applications. Certain aspects are thus directed to systems and / or methods that allow for more efficient cell isolation and separation. For example, the device may comprise various regions, such as a filter region, a denuder region, and / or a capture region, each comprising posts which optionally have teeth, with multiple pressure sources to control fluid flow, or the like. In some cases, regions such as these can be used as individually, or combined, or can all be in a single microfluidic device.

Description

[0001] DEVICES AND METHODS FOR ISOLATION OF BIOLOGICAL ENTITIES IN SUSPENSION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 518,322, filed August 8, 2023, entitled “Microfluidic Methods and Apparatuses for Denudation of Oocytes Utilizing Oscillatory Flow,” and of U.S. Provisional Patent Application Serial No. 63 / 518,323, filed August 8, 2023, entitled “Devices and Methods for Isolation of Biological Entities in Suspension.” Each of the above is incorporated herein by reference in its entirety.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant. No. HD 105529 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] Certain aspects of the present disclosure are generally directed to microfluidic methods and apparatuses for isolation of biological entities in suspension.

[0008] BACKGROUND

[0009] Biological fluids are often complex in nature and contain both wanted and unwanted components (e.g., biological debris, contaminants, etc.). As such, these fluids often need to be pre-processed and / or purified before they can be used in various applications. For example, biological fluids containing reproductive materials such as oocytes often require complicated and multi-step pre-processing before purified and denuded oocytes can be extracted for subsequent use.

[0010] Several systems and / or methods, predominantly manual, for extracting oocytes are available. However, the systems and / or methods can be complicated and inefficient, and thus, more effective systems and / or method for extracting oocytes are still needed.

[0011] SUMMARY

[0012] Certain aspects of the present disclosure are generally directed to devices and methods for isolation of biological entities in suspension. For example, some embodiments are generally directed to the isolation, denudation, and / or concentration of oocytes, or other cells such as those described herein. In some cases, oocytes and / or other cells may be concentrated, e.g., into a relatively small volume. This may be useful, for example, for fertilization or banking applications. 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.

[0013] One aspect is generally drawn to a microfluidic device. According to one set of embodiments, the device comprises a fluidic channel comprising a filter region, a denuder region, a first pressure source upstream of the filter region, and a second pressure source downstream of the denuder region. In some cases, the filter region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some the first sub-channels have a minimum cross-sectional dimension of between 100 micrometers and 3000 micrometers. In certain cases, the denuder region comprises a second plurality of posts defining second sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross-sectional dimensions of between about 50 micrometers and about 350 micrometers.

[0014] In another set of embodiments, the device comprises a fluidic channel comprising a denuder region, a capture region, a first pressure source upstream of the denuder region, and a second pressure source downstream of the capture region. In some embodiments, the denuder region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some of the first sub-channels have a minimum cross-sectional dimension of between about 80 micrometers and about 350 micrometers. In certain cases, the capture region comprises a second plurality of posts defining second subchannels between neighboring pairs of posts, wherein at least some of the second subchannels have a minimum cross-sectional dimension of less than 150 micrometers.

[0015] Still another set of embodiments is generally drawn to a microfluidic device, comprising a fluidic channel comprising a denuder region and a capture region, wherein the denuder region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some of the first sub-channels have a minimum cross-sectional dimension of between about 80 micrometers and about 350 micrometers, and the capture region comprises a second plurality of posts defining second sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross-sectional dimension of less than 120 micrometers; a first port upstream of the denuder region; a second port downstream of the denuder region and upstream of the capture region; and a third port downstream of the capture region.

[0016] In yet another set of embodiments, the device comprises a fluidic channel comprising a filter region, a denuder region downstream the filter region, and a capture region downstream the denuder region; a first port upstream of the filter region; a second port downstream of the denuder region and upstream of the capture region; a third port downstream of the capture region; and an additional port downstream of the filter region and upstream of the denuder region. In some cases, the filter region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some the first sub-channels have a minimum cross-sectional dimension of between 100 micrometers and 3000 micrometers. In certain embodiments, the denuder region comprises a second plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross-sectional dimension of between 80 micrometers and 350 micrometers. In one embodiment, the capture region comprises a second plurality of posts defining second sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross- sectional dimensions of less than 120 micrometers.

[0017] Another aspect is generally drawn to a method. In one set of embodiments, the method is a method of separating oocyte complex from a fluid and denuding the oocyte complex. In some embodiments, the method comprises flowing the fluid containing the oocyte complex through a microfluidic device, wherein the microfluidic device comprises a fluidic channel comprising a filter region comprising a first plurality of posts, a denuder region comprising a second plurality of posts, a first pressure source upstream of the filter region, and a second pressure source downstream of the denuder region.

[0018] In another set of embodiments, the method is a method of denuding an oocyte complex comprising an oocyte and bounded non-oocyte cells to separate the oocyte from the non-oocyte cells. In some embodiments, the method comprises providing a fluidic channel comprising a denuder region, a capture region, a first port upstream of the denuder region, a second port downstream of the denuder region and upstream of the capture region, and a third port downstream of the capture region; at a first point in time, causing fluid to flow from the first port, through the denuder region and the capture region, to the third port, such that the complex passes through the denuder region and is trapped by the capture region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; at a second point in time, causing the fluid to flow from the third port, through the capture region and the denuder region, to the first port, such that the complex passes through the denuder region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; and at a third point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the oocyte exits through the second port. The method, in accordance with yet another set of embodiments, is a method of controlling an oocyte complex comprising an oocyte and bounded non-oocyte cells. In some embodiments, the method comprises causing a first flow of a fluid containing the complex in a first direction through a fluidic channel; removing at least some of the non-oocyte cells from the complex as the oocyte flows through the fluidic channel in the first direction; trapping the complex in an exit region of the fluidic channel; and flowing the fluid containing the trapped complex in a second direction opposed to the first direction in the fluidic channel.

[0019] The method, in still another set of embodiments, is a method of processing a fluid containing one or more non-target entities and an oocyte complex comprising an oocyte and bounded non-oocyte cells. In certain embodiments, the method comprises providing a fluidic channel comprising a filter region, a denuder region, a capture region, a first port upstream of the filter region, a second port downstream of the denuder region and upstream of the capture region, a third port downstream of the capture region, and an additional port downstream of the filter region and upstream of the denuder region; at a first point in time, causing fluid to flow from the first port, through the filter region, to the additional port, such that at least a portion of the non-target entities passes through the filter region, wherein some of the nontarget entities undergo a reduction in size as the non-target entities pass through the filter region; at a second point in time, causing fluid to flow from the additional port, through the filter region, to the first port, such that at least a portion of the non-target entities passes through filter region, wherein some of the non-target entities undergo a reduction in size as the non-target entities pass through the filter region; at a third point in time, causing fluid to flow from the first port, through the filter region to the denuder region, such that the oocyte complex passes from the filter region to the denuder region; at a fourth point in time, causing fluid to flow from the additional port, through the denuder region and the capture region, to the third port, such that the oocyte complex passes through the denuder region and is trapped by the capture region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; at a fifth point in time, causing fluid to flow from the third port, through the capture region and the denuder region, to the additional port, such that the complex passes through the denuder region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; and at a sixth point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the oocyte exits through the second port. In another set of embodiments, the method is directed to processing a fluid containing one or more non-target entities and an oocyte complex comprising an oocyte and non-oocyte cells. In certain cases, the method comprises providing a fluidic channel comprising a filter region, a denuder region, and a capture region; causing repeated flow of the fluid through the filter region, such that at least a portion of the non-target entities undergoes a reduction in size as the non-target entities flows within the filter region; and causing repeated flow of the fluid through the denuder region, such that at least a portion of the non-oocyte cells is removed from the oocyte complex as the complex flows through the denuder region.

[0020] The method, in still another set of embodiments, is a method of controlling an oocyte complex comprising an oocyte and bounded non-oocyte cells. In one embodiment, the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts; trapping the complex in an exit region of the fluidic channel; releasing the complex from the exit region; and passing the released complex through the plurality of posts.

[0021] The method, in yet another set of embodiments, is a method of controlling an oocyte complex comprising an oocyte and bounded non-oocyte cells. In one embodiment, the method comprises causing a flow of a fluid containing the complex through a fluidic channel; and repeatedly trapping and releasing the complex in an exit region of the fluidic channel.

[0022] In one set of embodiments, the method is a method of controlling an oocyte complex comprising an oocyte and bounded non-oocyte cells. In certain cases, the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining subchannels between neighboring pairs of posts, at least one of the sub-channels having a minimum cross-sectional dimension of between 120 micrometers and 180 micrometers; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

[0023] In another set of embodiments, the method is a method of controlling an oocyte complex comprising an oocyte and bounded non-oocyte cells. In one embodiment, the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the subchannels having a minimum cross-sectional dimension of 1.0 to 1.4 times an average diameter of the oocyte; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

[0024] The method, in yet another set of embodiments, is a method of denuding an target entity-secondary entity comprising an target entity and one or more secondary entities to separate the target entity from the secondary entities. In one embodiment, the method comprises providing a fluidic channel comprising a denuder region, a capture region, a first port upstream of the denuder region, a second port downstream of the denuder region and upstream of the capture region, and a third port downstream of the capture region; at a first point in time, causing fluid to flow from the first port, through the denuder region and the capture region, to the third port, such that the complex passes through the denuder region and is trapped by the capture region, wherein at least some of the secondary entities are removed from the complex as the complex flows through the denuder region; at a second point in time, causing the fluid to flow from the third port, through the capture region and the denuder region, to the first port, such that the complex passes through the denuder region, wherein at least some of the secondary entities are removed from the complex as the complex flows through the denuder region; and at a third point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the target entity exits through the second port.

[0025] In another set of embodiments, the method is a method of controlling an target entitysecondary entity comprising an target entity and one or more secondary entities. In one embodiment, the method comprises causing a first flow of a fluid containing the complex in a first direction through a fluidic channel; removing at least some of the secondary entities from the complex as the target entity flows through the fluidic channel in the first direction; trapping the complex in an exit region of the fluidic channel; and flowing the fluid containing the trapped complex in a second direction opposed to the first direction in the fluidic channel.

[0026] In yet another set of embodiments, the method is a method of controlling an target entity-secondary entity comprising an target entity and one or more secondary entities. In certain embodiments, the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts; trapping the complex in an exit region of the fluidic channel; releasing the complex from the exit region; and passing the released complex through the plurality of posts.

[0027] The method, in still another set of embodiments, is a method of controlling an target entity-secondary entity comprising an target entity and one or more secondary entities. In certain cases, the method comprises causing a flow of a fluid containing the complex through a fluidic channel; and repeatedly trapping and releasing the complex in an exit region of the fluidic channel.

[0028] In one set of embodiments, the method is a method of controlling an target entitysecondary entity comprising an target entity and one or more secondary entities. In certain cases, the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the sub-channels having a minimum cross-sectional dimension of between 120 micrometers and 180 micrometers; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

[0029] The method, in another set of embodiments, is a method of controlling an target entity-secondary entity comprising an target entity and one or more secondary entities. In one embodiment, the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the sub-channels having a minimum cross-sectional dimension of 1.0 to 1.4 times an average diameter of the target entity; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

[0030] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Non-limiting embodiments of the present disclosure 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 disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0033] FIG. 1A is a schematic illustration showing one embodiment of a microfluidic device, in accordance with some embodiments;

[0034] FIG. IB is a schematic illustration showing a fluid flow in a first direction in a microfluidic device at a first point in time, in accordance with some embodiments;

[0035] FIG. 1C is a schematic illustration showing a fluid flow in a second direction in a microfluidic device at a second point in time, in accordance with some embodiments;

[0036] FIG. ID is a schematic illustration showing a fluid flow in microfluidic device at a third time point, in accordance with some embodiments;

[0037] FIG. 2 is a schematic illustration showing a filter region, in accordance with some embodiments; FIG. 3 is a schematic illustration showing a denuder region, in accordance with some embodiments;

[0038] FIG. 4 is a schematic illustration showing a concentrator region, in accordance with some embodiments;

[0039] FIG. 5 is a schematic illustration showing a capture region, in accordance with some embodiments;

[0040] FIGS. 6A-6B are schematic illustrations showing velocity profiles within a denuder region (FIG. 6A) versus a capture region (FIG. 6B), in accordance with some embodiments;

[0041] FIGS. 7-12 are schematic illustrations showing various steps of inducing oscillatory flow within a microfluidic device for extracting and denuding oocytes, in accordance with some embodiments;

[0042] FIGS. 13A-13B are schematic illustrations showing fluidic constriction channels with bi-directional extruded features (e.g., teeth) having a fluid flow in a first direction (FIG. 13 A) versus fluid flow in a second direction (FIG. 13B), in accordance with some embodiments;

[0043] FIGS. 14A-14C are schematic illustrations showing fluid flow in a fluidic channel, including in a first direction (FIG. 14A) and in a second direction (FIG. 14B), in accordance with some embodiments;

[0044] FIGS. 15A-15D are schematic illustrations showing various example of extruded features, in accordance with some embodiments;

[0045] FIG. 16 is a schematic illustration showing an embodiment of a microfluidic device for repeated (non-oscillatory) fluid flow, in accordance with some embodiments;

[0046] FIGS. 17A-17F are schematic illustrations showing another embodiment of a microfluidic device during operation, in accordance with some embodiments;

[0047] FIG. 18 is a schematic illustration showing yet another embodiment of a microfluidic device, in accordance with some embodiments;

[0048] FIGS. 19A-19F are schematic illustrations showing specific designs of a microfluidic device, in accordance with some embodiments;

[0049] FIG. 20 is a schematic illustration showing a specific design of a microfluidic device having a circular form factor, in accordance with some embodiments;

[0050] FIGS. 21A-21C are schematic illustrations showing microfluidic devices comprising angled walls, in certain embodiments;

[0051] FIG. 22 shows a photomicrograph of denuded oocytes following passage through the denudation region shown in FIG. 21C, in accordance with one embodiment; FIG. 23 shows a schematic illustration of a first concentration region of an exemplary microfluidic device, in accordance with some embodiments;

[0052] FIGS. 24A-24C show illustrations and uses of a second concentration region of an exemplary micro fluidic device, in accordance with some embodiments;

[0053] FIGS. 25A-25B show illustrations of exemplary inertial focusing regions, in accordance with some embodiments; and

[0054] FIGS. 26A-26D illustrate flow speeds to focus oocytes using an inertial focusing region, according to some embodiments.

[0055] DETAILED DESCRIPTION

[0056] The present disclosure is generally directed to various methods and apparatuses for isolation of biological entities in suspension utilizing various techniques. One example application is the extracting and denudation of oocytes from a fluid (e.g., a biological fluid or non biological fluids such as culture media). Oocytes in biological fluids may need to be isolated and / or denuded before they can be effectively used in various applications, such as in vivo fertilization (IVF) applications and oocyte banking. Certain aspects are thus directed to systems and / or methods that allow for more efficient cell isolation and separation. For example, the device may comprise various regions, such as a filter region, a denuder region, and / or a capture region, each comprising posts which optionally have teeth, with multiple pressure sources to control fluid flow, or the like. In some cases, regions such as these can be used as individually, or combined, or can all be in a single microfluidic device.

[0057] In addition, in some embodiments, the devices further comprise an inertial focusing region capable of focusing the cells within the biological fluid to specific locations within a fluid channel. For example, some inertial focusing regions may focus target cells to a middle of a channel width. In other embodiments, an inertial focusing region may focus target cells to the middle of a channel width and along an axis perpendicular to the direction of fluid flow (e.g., top or bottom of the channel).

[0058] Certain aspects as discussed herein are generally directed to microfluidic methods and apparatuses for isolation of biological entities in suspension. Biological fluids are often complex in nature and contain unwanted components (e.g., biological debris, contaminants, etc.) that often need to be pre-processed and / or purified before the fluids can be used in various applications. For example, biological fluids containing oocytes may need to be pre- processed before purified and denuded oocytes can be extracted for subsequent use. Accordingly, certain aspects of the disclosure are directed to systems, devices and / or methods that collectively allow for a highly efficient isolation of biological entities in suspension, such as oocyte extraction, isolation, and denudation process.

[0059] Certain devices and methods described herein may have various advantages compared to conventional devices and methods for isolating biological entities in suspension. In some embodiments, a microfluidic device is disclosed herein. Referring now to FIG. 1A, in one set of embodiments, an example microfluidic device 10 comprises a first port 20, a third port 40, and various regions (e.g., filter region 100, denuder region 200, concentrator region 300, capture region 400) in the fluidic channel disposed in between the two ports. During operation, a biological fluid containing a wanted biological entity, such as an oocyte complex, and any unwanted impurities and / or debris (if present) may be fed into the device via the first port 20, optionally followed by repeated flows across the fluidic channel from the first port to the third port, via the intervening regions, and vice versa. In some embodiments, an oocyte complex may include an oocyte and one or more types of secondary cells (i.e., nonoocyte cell(s)) bounded to (e.g., surrounding or attached to) the oocyte, including, but not limited to, cumulus cells, granulosa cells, mural cells, thecal cells, red blood cells, or combinations thereof. For example, the oocyte complex may be flowed back-and-forth, e.g., in an oscillatory fashion through the channel, and / or the oocyte may be flowed through the same channel in a certain direction a plurality of times. As an example, in these figures, e.g., such as an oocyte may flow from the first port 20 to the third port 40 in a first direction 13 (e.g., from left to right) as shown in FIG. IB, and then from the third port 40 to the first port 20 in a second direction 15 (e.g., from right to left) as shown in FIG. 1C. Such an oscillatory flow across the fluidic channel may be repeated multiple times within the fluidic channel and may allow for effective isolation and denudation of oocytes. In other embodiments, however, the oocyte may be caused to flow in a single direction multiple times repeatedly through the microfluidic channel (e.g., in the direction shown in FIG. 16). Additionally, the repeated back-and-forth flow described herein may facilitate removal and extraction of any trapped impurities and / or debris from the device, such that the device is prevented from being clogged during use.

[0060] In some embodiments, the device described herein may allow for selective and / or simultaneous actuation of a plurality of ports and / or pressure sources in the device during operation, as shown in FIGS. IB- ID. Such selective actuation may allow for precise and timed control of fluid flow within the device, including the directionality of fluid flow within the device, the type of fluid flow (e.g., oscillatory fluid flow), and / or the timing of various fluid flows into and / or out of the device, as desired. In some cases, the plurality of ports may be simultaneously actuated (by a controller) to transition from one configuration to another, such as from a first configuration to a second configuration, a second configuration to a third configuration, and vice versa.

[0061] For example, the microfluidic device may further comprise a plurality of ports, such as port 20, 30, 40, 50, 60, and / or 70, at least some of which are coupled to pressure sources, such as pressure sources 12 and 14, e.g., as shown in FIGS. IB- ID. In some cases, the plurality of ports may be simultaneously actuated to have a first configuration at a first point in time. For example, when in a first configuration, a first set of ports may be selectively actuated to open (e.g., port 20, 50, 60, and 70) while a second set of ports may be actuated to close (e.g., port 30 and 40), such that a fluid containing a target entity (e.g., oocyte) may be caused to flow (by a pressure source 12) from a reservoir into an inlet region of the device via one or more ports (e.g., port 20), through various intermediate regions (e.g., regions 100, 200, 300, and / or 400) of the device in a first direction (e.g., a forward direction), to a sealed exit region (e.g., region sealed by port 40), e.g., as shown in FIG. IB. As described in more detail below, one or more ports, e.g., ports 50, 60, and / or 70, may be actuated to open to allow excess fluid and / or waste to flow therethrough, while one or more ports, e.g., ports 30 and 40, may be selectively closed to retain target entities (e.g., oocyte complex) within the device for further processing and / or prevent target entities from exiting the device.

[0062] In some embodiments, the plurality of ports may be actuated to have a second configuration at a second point in time. For example, when in a second configuration, a third set of ports may be selectively actuated to open (e.g., port 20, 40, and 50) while a fourth set of ports may be actuated to close (e.g., port 30, 60, 70), such that a fluid may be caused to flow (by a pressure source 14) into an exit region of the device via one or more ports (e.g., port 40), through the various intermediate regions (e.g., regions 400, 300, 200, and / or 100) of the device in a second direction (e.g., backward direction), and exit the inlet region of the device via one or more ports (e.g., port 20) into the reservoir (via arrow 22), e.g., as shown in FIG. 1C. As described in more detail below, one or more ports, e.g., port 50, may be actuated to open to allow excess fluid and / or waste to flow therethrough (via arrow 51), while one or more ports, e.g., ports 30, 60, and / or 70, may be actuated to close to prevent target entities from exiting the device and / or to facilitate and direct fluid flow in the second direction.

[0063] The plurality of ports, according to some embodiments, may be actuated repeatedly to transition from the first configuration to the second configuration, and vice versa, for any number of times as described elsewhere herein, e.g., at least 1 time, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, and / or up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above-referenced ranges are possible (e.g., at least 1 time and up to 1000 times, at least 5 times and up to 1000 times). Other ranges are also possible.

[0064] In some embodiments, the plurality of ports may be actuated to have a third configuration at a third point in time. For example, when in a third configuration, a fifth set of ports may be selectively actuated to open (e.g., ports 30, 40, and 50) while a sixth set of ports may be actuated to close (e.g., port 20, 60, 70), such that a fluid may be caused to flow (by a pressure source 14) into an exit region of the device via one or more ports (e.g., port 40), through the various intermediate regions (e.g., region 400) in the second direction (e.g., a backward direction), and exit via one or more ports (e.g., ports 30 and 50), e.g., as shown in FIG. ID. As described in more detail below, one or more ports, e.g., port 30, may be actuated to open such that target entities (e.g., denuded oocyte) may be released from the device, while one or more ports, e.g., port 50, may be actuated to open to allow excess fluid and / or waste to flow therethrough. It should be understood that FIGS. 1A-1D are used to illustrate one embodiment of a microfluidic device in the present disclosure and that other embodiments are possible, as discussed in more detail below and shown in FIGS. 17A-18.

[0065] In some embodiments, a microfluidic device is provided. For example, the microfluidic device may comprise a plurality of regions connected in series, such as a filter region, a denuder region downstream the filter region, a concentrator region downstream the denuder region, and / or a capture region downstream of the concentrator region. According to some embodiments, the microfluidic device may further comprise a plurality of ports disposed adjacent one or more of the above-reference regions configured to control fluid flow into and / or out of the regions.

[0066] FIG. 1 shows one non-limiting embodiment of a microfluidic device 10. As shown in FIG. 1, the microfluidic device 10 may comprise a fluid channel comprising a filter region 100 and a denuder region 200 downstream of the filter region 100. As shown, the fluidic channel may further comprise a capture region 400 positioned downstream the denuder region 200. In some embodiments, the fluidic channel may optionally comprise a concentrator region 300 positioned between the denuder region 100 and the capture region 400. The device 10 may further comprise two or more pressure sources, such as a first pressure source 12 (e.g., a pump) upstream of the filter region 100 and / or the denuder region 200, and a second pressure source 14 (e.g., a pump) downstream of one or more regions, such as the denuder region 200, the concentrator region 300, and / or the capture region 400, according to some embodiments. In some embodiments, the microfluidic device further comprises a plurality of ports configured for receiving and / or outputting a fluid. For example, referring to FIG. 1, the microfluidic device 10 may further comprise a plurality of ports, such as port 20, 30, 40, 50, 60, and / or 70.

[0067] It should be understood that while FIG. 1 illustrates an embodiment in which the device comprises regions 100-400, in other embodiments, one or more regions may not be present in the device. For example, in one set of embodiments, the device 10 may comprise the filter region 100, the denuder region 200, with the first pressure source 12 positioned upstream of the filter region 100, and the second pressure source 14 positioned downstream of the denuder region 200. In another set of embodiments, the device 10 may comprise the denuder region 200 and the capture region 400 downstream of the denuder region 200, with the first pressure source 12 positioned upstream of the denuder region 200 and the second pressure source 14 positioned downstream the capture region 400.

[0068] Specifics of each of the above-reference regions and / or ports are described in more detail below.

[0069] In some embodiments, the fluidic channel comprises a filter region. In some cases, the filter region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, e.g., as shown in FIG. 1A. As described in more detail below, the filter region may be configured to filter out one or more components (e.g., oocyte complex, debris) from a fluid introduced (e.g., follicular fluid) into the first sub-channels, according to some embodiments. Debris may include any organic (e.g., granulose cells, cell clumps, tissue, red blood cell clusters, blood clots, etc.) or inorganic material other than the targeted cells or cell complexes (e.g., cumulus-oocyte-complex, oocyte, etc.). In some embodiments, components having sizes larger than the minimum cross-sectional dimensions of the subchannels may be trapped by the sub-channels, while components having sizes smaller than the minimum cross-sectional dimension of the sub-channels may be flowed through the subchannels.

[0070] FIG. 2 illustrates one non-limiting embodiment of the filter region 100 shown in FIG. 1 A. As shown, the filter region 100 comprises a first plurality of posts 110 defining first subchannels 120 between neighboring pairs of posts 110. Some of the first sub-channels 120 may have a minimum cross-sectional dimension a. As shown, the inlet of the filter region may be fluidically connected to the first pressure source 12 via the first port 20 (as shown in FIG. 1A), and the outlet of the filter region may be fluidically connected to the denuder region (as shown in FIG. 1A). As described in more detail below, the filter region may allow for size-based selective filtering of target entities (e.g., oocyte complex) and / or non-target entities (e.g., debris) from a fluid flowing through the filter region. In some embodiments, the first plurality of posts may be arranged in any of a variety of appropriate patterns and may have any of a variety of appropriate shapes.

[0071] The first plurality of posts within the filter region of the fluidic channel may be arranged in any appropriate fashion. For instance, in one embodiment, the first plurality of posts are arranged in an array across the fluidic channel. The array of posts, in some cases, may comprise one or more rows of posts, where each row comprises one or more posts. The posts may be arranged in any suitable arrangement, for example, a rectangular grid or in a staggered fashion, e.g., as shown in FIG. 1A and / or in FIG. 2. However, it should be understood that the plurality of posts is not limited to being arranged in an ordered fashion, and that in some cases, at least one of the plurality of posts may be arranged randomly across the fluidic channel.

[0072] In some embodiments, the array of posts in the filter region may comprise n number of rows of post(s) and / or sub-channels, where n is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and / or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000, or at least 1 and no more than 100). Other ranges are also possible. In one set of embodiments, the array of posts comprises a single row of post and / or sub-channels. Referring to FIG. 1A and FIG. 2 as a non-limiting example, the array of posts 110 comprises multiple rows of posts and sub-channels.

[0073] In some embodiments, the array of posts in the filter region may comprise m number of post(s) and / or sub-channels per row, where m is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and / or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000, or at least 1 and no more than 100). Other ranges are also possible. In one set of embodiments, the array of posts comprises a single post per row.

[0074] In some embodiments, some (or all) of the sub-channel(s) within the filter region of the fluidic channel may have a minimum cross-sectional dimension a of between of greater than or equal to 5 micrometers, greater than or equal to 7 micrometers, greater than or equal to 10 micrometers, greater than or equal to 20 micrometers, greater than or equal to 30 micrometers, greater than or equal to 50 micrometers, greater than or equal to 70 micrometers, greater than or equal to 80 micrometers, greater than or equal to 100 micrometers, greater than or equal to 125 micrometers, greater than or equal to 150 micrometers, greater than or equal to 200 micrometers, greater than or equal to 300 micrometers, greater than or equal to 400 micrometers, greater than or equal to 500 micrometers, greater than or equal to 600 micrometers, greater than or equal to 700 micrometers, greater than or equal to 800 micrometers, greater than or equal to 1000 micrometers, greater than or equal to 1500 micrometers, greater than or equal to 2000 micrometers, greater than or equal to 2500 micrometers, and / or less than or equal to 3000 micrometers, less than or equal to 2500 micrometers, less than or equal to 2000 micrometers, less than or equal to 1500 micrometers, less than or equal to 1000 micrometers, less than or equal to 800 micrometers, less than or equal to 700 micrometers, less than or equal to 600 micrometers, less than or equal to 500 micrometers, less than or equal to 400 micrometers, less than or equal to 300 micrometers, less than or equal to 200 micrometers, less than or equal to 150 micrometers, less than or equal to 125 micrometers, less than or equal to 100 micrometers, less than or equal to 80 micrometers, less than or equal to 70 micrometers, less than or equal to 50 micrometers, less than or equal to 30 micrometers, less than or equal to 20 micrometers, less than or equal to 10 micrometers, less than or equal to 7 micrometers, less than or equal to 5 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 70 micrometers and less than or equal to 3000 micrometers, greater than or equal to 100 micrometers and less than or equal to 3000 micrometers, greater than or equal to 150 micrometers and less than or equal to 300 micrometers, greater than or equal to 300 micrometers and less than or equal to 1000 micrometers, or greater than or equal to 1000 and less than or equal to 3000 micrometers).

[0075] Other ranges are also possible. In some embodiments, the above-referenced ranges may be particularly advantageous for certain type of applications, e.g., denudation of oocyte complexes.

[0076] In some embodiments, the minimum cross-sectional dimension of the channel(s) in the filter region may vary depending on the application and / or the target entity of interest. For example, some or all of the channel(s) may have minimum cross-sectional dimensions (in one or more ranges described above) that are larger than the cross-sectional dimension of a target entity of interest. As an example, in embodiments in which the target entity of interest is the oocyte, some or all of the channel(s) may have minimum cross-sectional dimensions (in one or more ranges described above) that are larger than the cross-sectional dimension of an oocyte, e.g., between 70 micrometers and 3000 micrometers.

[0077] In some embodiments, some of the sub-channels in the filter region may have dimensions that differ from the rest of the sub-channels. For example, in certain embodiments, the sub-channels may be arranged such that the sub-channel(s) adjacent an inlet of the filter region may have minimum cross-sectional dimension(s) larger than the minimum cross-sectional dimension(s) of sub-channel(s) further away from the inlet of the filter region, such as the outlet of the filter region. For example, referring to FIG. 1A and FIG. 2, the sub-channels 130 adjacent an inlet 101 of the filter region 100 (such as those in the first two rows) may have sub-channels with minimum cross-sectional dimension(s) larger than cross-sectional dimension(s) of sub-channel(s) further away from the inlet of the filter region, e.g., adjacent an outlet 102 of the filter region.

[0078] In some embodiments, the plurality of sub-channels may be divided into one or more stages, with some or all of the stages containing sub-channels having different minimum cross-sectional dimensions from the those in the other stage(s). For example, in some embodiments, the sub-channels may comprise a first stage of sub-channels having a minimum cross-sectional dimension of greater than or equal 3000 micrometers and less than or equal to 5000 micrometers. In some cases, the sub-channels may comprise a second stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 1000 micrometers and less than 3000 micrometers. In some cases, the sub-channels may comprise a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 300 micrometers and less than 1000 micrometers. In some cases, the sub-channels may comprise a fourth stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to greater than or equal to 150 micrometers and less than 300 micrometers, etc. FIG. 2 can be used to illustrate one such embodiment. As shown, the subchannels may be divided into a plurality of stages, e.g., stage 1 (si), stage 2 (s2), stage 3 (s3), stage 4 (s4). According to some embodiments, a stage of sub-channels adjacent an inlet 201 of the filter region may have a minimum cross-sectional dimension larger than the minimum cross-sectional dimension of a subsequent stage, such as a stage of sub-channels adjacent an outlet 102 of the filter region.

[0079] The plurality of posts within the filter region may have any of a variety of appropriate shapes, including but not limited to, triangles, polygons, circles,, and / or any other shapes described elsewhere herein with respect to the posts in the denuder region. In some cases, the plurality of posts may have smooth surfaces. In some cases, the plurality of posts may have surfaces comprising teeth, as described elsewhere with respect to the posts in the denuder region, such as the teeth shown in FIGS. 15A-15D. The presence of teeth or jagged edging on the post(s) in the filter region may, according to some embodiments, facilitate initial dissociation of one or more non-target entities (e.g., debris, tissues, etc.) from the target entities and aid subsequent denudation of secondary entities from the target entities downstream the filter region.

[0080] In some embodiments, the fluidic channel comprises a denuder region downstream of the filter region. In some cases, the denuder region comprises a second plurality of posts defining first sub-channels between neighboring pairs of posts, e.g., as shown in FIG. 1A and FIG. 3.

[0081] FIG. 3 illustrates one non-limiting embodiment of the denuder region 200 shown in FIG. 1A. As shown, the denuder region 200 comprises a second plurality of posts 210 defining second sub-channels 220 between neighboring pairs of posts 210. In some cases, sub-channels may be defined by post 210 and wall 207 in the denuder region 200. The use of parallel sub-channels may advantageously provide a plurality of paths for a target entity to pass through, which may reduce the risk of chip clogging and increase denudation efficiency.

[0082] As shown, the inlet of the denuder region 200 may be fluidically connected to the outlet of the filter region 100 (as shown in FIG. 1A), and the outlet of the denuder region 200 may be fluidically connected to the second pressure source 14, via one or more intermediate regions (such as the concentrator region 300 and / or the capture region 400 shown in FIG. 1A). As described in more detail below, the second plurality of posts may be arranged in any of a variety of appropriate patterns and may have any of a variety of appropriate shapes.

[0083] In some embodiments, the sub-channel(s) between the posts in the denuder region may have a minimum cross-sectional dimension that is less than the minimum cross-sectional dimension of the sub-channel(s) within the filter region of the fluidic channel, according to some embodiments. For example, the sub-channel(s) between the posts in the denuder region may have minimum cross-sectional dimensions that are at least 10 micrometers (e.g., at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, and / or up to 50 micrometers, up to 70 micrometers, up to 100 micrometers) less than the minimum cross-sectional dimension of the sub-channel(s) in the filter region.

[0084] The plurality of posts within the denuder region may be arranged in any appropriate fashion. For instance, in one embodiment, the plurality of posts are arranged in an array across the fluidic channel. The array of posts, in some cases, may comprise one or more rows of posts, where each row comprises one or more posts. The posts may be arranged in any suitable arrangement, for example, a rectangular grid or in a staggered fashion, e.g., as shown in FIG. 3. However, it should be understood that the plurality of posts is not limited to being arranged in an ordered fashion, and that in some cases, at least one of the plurality of posts may be arranged randomly across the fluidic channel.

[0085] In some embodiments, the array of posts may comprise n number of rows of post(s) and / or sub-channels, where n is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and / or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000). Other ranges are also possible. In one set of embodiments, the array of posts comprises a single row of post and / or sub-channels. Referring to FIG. 3 as a non-limiting example, the array of posts 210 comprises multiple rows of posts and sub-channels.

[0086] In some embodiments, the array of posts may comprise m number of post(s) and / or sub-channels per row, where m is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and / or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000). Other ranges are also possible. In one set of embodiments, the array of posts comprises a single post per row.

[0087] While FIG. 3 illustrates an embodiment in which the denuder region 200 comprises one or more posts, it should be understood that the disclosure is not so limited, and that in certain embodiments, the denuder region does not necessarily include a post therein. FIG. 14A can be used to illustrate one such embodiment. As shown, denuder region 200a does not contain any posts therein. As shown, in accordance with certain embodiments, the denuder region 200a comprise a single sub-channel 230a therein formed between extruded wall portions of the fluidic channel. The sub-channel 230a may have any properties (e.g., minimum cross-sectional dimension) described respective to the sub-channels shown in FIG. 3.

[0088] The one or more posts within the denuder region may individually have any of a variety of appropriate shapes and forms. If more than one post is present, the posts may have the same or different shapes and / or sizes. In some embodiments, at least one of the plurality of posts comprises a shape such as kite-shaped, a hexagon, rectangular, circular, triangular, polygonal, etc. For example, FIG. 1A can be used to illustrate a plurality of posts within the fluidic channel 110 having a hexagonal shaped structure. As another example, FIG. 14C can be used to illustrate a plurality of posts having kite shapes. Other shapes are also possible.

[0089] In some embodiments, at least some of the sub-channel(s) (for example, defined between two neighboring posts) in the denuder region may have an inlet portion, an outlet portion, and a constriction portion between the inlet portion and outlet portion. In some cases, the constriction portion of the sub-channel(s) may have a cross-sectional dimension that is less than those of the inlet portion and outlet portion. FIG. 3 can be used to illustrate one such embodiment. For example, as shown in FIG. 3, a sub-channel defined by neighboring posts may include an inlet portion a, an outlet portion c, and a constriction portion b therebetween, with the constriction portion b having a cross-sectional dimension less than the inlet and outlet portions. In some embodiments, at least one or more (e.g., some) of the sub-channel(s) have a minimum cross-sectional dimension w in the constriction portion of the sub-channel(s), e.g., as shown in FIG. 3. In some embodiments, the minimum cross-sectional dimension of the sub-channel(s) may be sized so as to allow denudation of a target entity of interest (e.g., an oocyte), e.g., such as having size(s) comparable to (or slightly larger) than the size of the target entity (e.g., oocyte).

[0090] In some embodiments, some (or all) of the sub-channel(s) within the denuder region may have a minimum cross-sectional dimension w of greater than or equal to 20 micrometers, greater than or equal to 50 micrometers, greater than or equal to 70 micrometers, greater than or equal to 80 micrometers, greater than or equal to 100 micrometers, greater than or equal to 120 micrometers, greater than or equal to 125 micrometers, greater than or equal to 130 micrometers, greater than or equal to 140 micrometers, greater than or equal to 150 micrometers, greater than or equal to 160 micrometers, greater than or equal to 170 micrometers, greater than or equal to 180 micrometers, greater than or equal to 200 micrometers, greater than or equal to 250 micrometers, greater than or equal to 300 micrometers, and / or less than or equal to 350 micrometers, less than or equal to 300 micrometers, less than or equal to 250 micrometers, less than or equal to 200 micrometers, less than or equal to 180 micrometers, less than or equal to 170 micrometers, less than or equal to 160 micrometers, less than or equal to 150 micrometers, less than or equal to 140 micrometers, less than or equal to 130 micrometers, less than or equal to 125 micrometers, less than or equal to 100 micrometers, less than or equal to 80 micrometers, less than or equal to 70 micrometers, less than or equal to 50 micrometers, or less. Combinations of the above- referenced ranges are possible (e.g., between 80 micrometers and 350 micrometers, between 70 micrometers and 350 micrometers, between 120 micrometers and 180 micrometers, between 150 micrometers and 180 micrometers, between 120 micrometers and 160 micrometers, or between 120 micrometers and 150 micrometers). Other ranges are also possible.

[0091] In some embodiments, the minimum cross-sectional dimension of the sub-channels in the denuder region may depend, at least in part, on the type of target entity that is being denuded in the denuder region. As described elsewhere herein, the target entity may be an oocyte. Depending on the particular type or species of oocyte (e.g., human oocyte, bovine oocyte, mouse oocyte), the size of the oocyte may also vary. In some embodiments, at least some of the sub-channels in the denuder region may have a minimum cross-sectional dimension that is at least 1.01 times (at least 1.1 times, at least 1.15 times, at least 1.2 times, at least 1.25 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, or more, and / or up to 3 times, up to 2.5 times, up to 2 times, up to 1.5 times, up to 1.4 times, up to 1.3 times, up to 1.25 times, up to 1.2 times, up to 1.15 times, up to 1.1 times, etc.) the size (e.g., diameter) of the target entity (e.g., a bare oocyte).

[0092] However, in some cases, the minimum cross-sectional dimension of the sub-channels may also be slightly smaller than the target entity (e.g. a bare oocyte). For example, at least some of the sub-channels in the denuder region may have a minimum cross-sectional dimension that is at least 0.8 times, at least 0.82 times, at least 0.85 times, at least 0.87 times, at least 0.9 times, at least 0.92 times, at least 0.94 times, at least 0.95 times, at least 0.96 times, at least 0.97 times, at least 0.98 times, at least 0.99 times, etc. and / or up to 0.99 times, up to 0.98 times, up to 0.97 times, up to 0.96 times, up to 0.95 times, up to 0.94 times, up to 0.92 times, up to 0.9 times, up to 0.87 times, up to 0.85 times, up to 0.82 times, up to 0.8 times the size (e.g., diameter) of the target entity (e.g., a bare oocyte). Without wishing to be bound by any theory, it is believed that slightly smaller dimensions may artificially activate oocytes, and / or induce proliferation towards embryo growth.

[0093] In some embodiments, the dimensions referenced above may be particularly advantageous for application related to oocyte denudation. However, it should be understood that the disclosure is not so limited, and that in some embodiments, the minimum cross- sectional dimension of the sub-channels in the denuder region may be scaled relative to the size of the target entity (e.g., a cell, a tissue, etc.). For example, in some embodiments in which the target entity is a particular type of cell (e.g., a tumor cell), the minimum cross- sectional dimension of the sub-channels be in the size range of micrometers, such as less than 100 microns, less than 50 microns, less than 25 microns, less than 20 microns, less than 15 microns, or less and / or down to 10 microns, down to 5 microns, or less. For example, in some embodiments in which the target entity is a particular type of tissue, the minimum cross-sectional dimension of the sub-channels be in the size range of millimeter, such as at least 0.5 mm, at least 1 mm, at least 2 mm, at least 10 mm, or more.

[0094] In some embodiments, some of the sub-channels in the denuder region may have dimensions that differ from the rest of the sub-channels. For example, in certain embodiments, the sub-channels may be arranged such that the sub-channel(s) adjacent an inlet of the microfluidic device may have minimum cross-sectional dimension(s) larger than the minimum cross-sectional dimension(s) of sub-channel(s) further away from the inlet of the device. For example, referring to FIG. 3, the sub-channels 200 adjacent an inlet 201 of the denuder region 200 may have minimum cross-sectional dimension(s) larger than cross- sectional dimension(s) of sub-channel(s) further away from the inlet 201 of the denuder region 200, e.g., adjacent an outlet 202. In some embodiments, the minimum cross-sectional dimension of the sub-channels furthest away from the inlet 201 (adjacent outlet 202) may have the smallest minimum cross-sectional dimension amongst the other sub-channels in the denuder region, such as having a minimum cross-section dimension that is comparable to the size of the target entity.

[0095] In some embodiments, starting from a first row or set of sub-channels (e.g., si) adjacent the inlet of the denuder region, each subsequent row or set of sub-channels (e.g., s2, s3) may have minimum cross-sectional dimensions that decrease by an increment of at least 5 micrometers (e.g., at least 10 micrometers, at least 15 micrometers, or more, and / or up to 20 micrometers, up to 25 micrometers) from the minimum cross-sectional dimensions of the preceding row or set of sub-channels. In some embodiments, such an arrangement in the denuder region may allow for gradual removal (e.g., denudation) of secondary entities from the target entities, which would lead to an overall more efficient denudation process. As an exemplary example, in embodiments in which the target entity is an oocyte, the incremental decrease in sub-channel dimensions may allow for gradual removal of non-oocyte cells from the oocyte as the oocyte complex passes through each row or set of sub-channels. As described in more detail below, non-oocyte cell(s)) may include, but not limited to, cumulus cells, granulosa cells, mural cells, thecal cells, red blood cells, or combinations thereof.

[0096] Depending on the particular application, the sub-channels in the denuder region may be sized that a desired level of denudation can be achieved, in some embodiments. For example, in applications in which complete denudation of secondary entities (e.g., non- oocyte cells) from the target entity (e.g., oocyte) is desired, at least some of the sub-channels (e.g., sub-channels in stage ,s3) may have minimum cross-sectional dimensions comparable or approximately equal to the size of the target entity. For another example, in applications in which partial denudation of secondary entities (e.g., non-oocyte cells) from the target entity (e.g., oocyte) is desired, at least some of the sub-channels (e.g., sub-channels in stage ,s3) may have minimum cross-sectional dimensions that are slightly larger than the size of the target entity.

[0097] In some embodiments, the plurality of sub-channels in the denuder region may be divided into one or more stages, with some or all of the stages containing sub-channels having different minimum cross-sectional dimensions from the those in the other stage(s). For example, in some embodiments, the sub-channels may comprise a first stage of subchannels having a minimum cross-sectional dimension of greater than or about equal to 180 micrometers and less than or equal to 200 micrometers, a second stage of sub-channels having a minimum cross-sectional dimension of greater than 150 micrometers and less than or equal to 200 micrometers, a third stage of sub-channels having a minimum cross-sectional dimension of greater than 120 micrometers and less than or equal to 150 micrometers, etc.

[0098] In some embodiments, the plurality of posts and / or sub-channels in the denuder region may be arranged such that a row of posts and / or sub-channels is offset from a subsequent row of posts and / or sub-channels, e.g., as shown in FIG. 3. For example, in one set of embodiments, the sub-channel may be arranged such that the longitudinal axis of subchannels) in one row of the fluidic channel passes through (or coincides with) the longitudinal axis of post(s) in a subsequent row. However, it should be understood the disclosure is not so limited, and that any appropriate arrangement of the posts and / sub- channels are possible.

[0099] In some embodiments, at least a portion of the walls forming the sub-channels in the denuder region (and / or filter region) may comprises a plurality of extruded features (e.g., teeth). In some embodiments, the minimum cross-sectional dimension w of a sub-channel may correspond to a distance between the outermost edges (e.g., tips) of extruded features (e.g., teeth) on opposing walls of the sub-channel(s). For example, as shown in FIG. 3, at least a portion of the walls on the plurality of posts 210 defining the sub-channel(s) 220 comprise extruded features (e.g., teeth) 240. As shown in FIG. 3, the minimum cross- sectional dimension w of the sub-channel may correspond to the distance between the outermost edges of extruded features 240 on respective walls of neighboring posts 210. Similarly, as shown in FIGS. 14A-14B, at least a portion of the extruded wall portions of the denuder region 200a may comprise teeth, and the minimum cross-sectional dimension w may correspond to the distance between the tips of teeth on opposing walls of the fluidic channel.

[0100] While FIG. 3 shows one set of embodiments in which the walls forming the subchannels comprise a plurality of extruded features (e.g., teeth), it should be understood that the disclosure is not so limited, and that in other embodiments, at least one (or both) of the walls forming the sub-channels may be substantially smooth (e.g., lack of teeth thereon).

[0101] The denuder region, according to some embodiments, may advantageously have a relatively small length. According to some embodiments, the length of denuder region may be measured horizontally across from the first (row of) sub-channel(s) or post(s) in the denuder region to the last (row of) sub-channel(s) or post(s) in the denuder region. FIG. 3 can be used to illustrate this. For example, as shown in FIG. 3, the length L of the denuder region can be measured from the first row post(s) or sub-channel(s) to the last row of post(s) or sub-channel(s).

[0102] In some embodiments, the length of denuder region within the microfluidic device may be relatively short, such as at least 50 micrometers, at least 100 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 8 mm, at least 10 mm, at least 20 mm, at least 40 mm, at least 60 mm, at least 80 mm, at least 100 mm, at least 150 mm, and / or no more than 200 mm, no more than 150 mm, no more than 100 mm, no more than 80 mm, no more than 60 mm, no more than 40 mm, no more than 20 mm, no more than 10 mm, no more than 8 mm, no more than 5 mm, no more than 3 mm, no more than 2 mm, no more than 1 mm, no more than 500 micrometers, no more than 100 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., at least 10 mm and no more than 200 mm, at least 10 mm and no more than 100 mm, at least 20 micrometers and no more than 200 mm, at least 20 micrometers and no more than 200 mm, at least 50 micrometers and no more than 200 mm, or at least 50 micrometers and no more than 10 mm). Other ranges are also possible. Without wishing to be bound by any theory, it is believed that due to the ability to flow entities (such as oocytes) repeatedly through a denuder region, the length of the denuder region can be relatively short, as the effective length of the denuder region that the entities (e.g., cells) actually experience is the product of the length of the denuder region and the number of times that the entities pass therethrough (e.g., in one direction, or in more than one direction). In contrast, in many other systems, only a single pass of the entities through the denuder region is contemplated. However, it should be understood that the length of the denuder region is not limited to the above-referenced ranges, and that in some cases, the length of the denuder region may be longer, e.g., such as 10 cm or longer.

[0103] In some embodiments, each of the sub-channel(s) in the denuder region described above may have a length of at least 50 micrometers, at least 100 micrometers, 200 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 4 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, or more, and / or no more than 200 mm, no more than 100 mm, no more than 50 mm, no more than 20 mm, no more than 10 mm, no more than 5 mm, no more than 2 mm, no more than 1 mm, no more than 500 micrometers, no more than 200 micrometers, no more than 100 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., at least 200 micrometers and no more than 1 mm, at least 200 micrometers and no more than 10 mm, at least 200 micrometers and no more than 100 mm). Other ranges are also possible.

[0104] In some cases, some (or all) of the sub-channel(s) in the denuder region comprise inlet portions and / or outlet portions that are tapered towards the constriction portions of the subchannels). For example, as shown in FIG. 3, the inlet portion a and outlet portion b may be tapered into the constriction portion b of the sub-channel. In some cases, the presence of such tapered inlet and / or outlet portions may effectively direct bulk fluid flow into the subchannels) from the inlet and / or outlet portion into the constriction portion, thereby facilitating denudation of target entities (e.g., oocyte) as they travel through the subchannels). In embodiments in which the fluidic channels comprise posts, the post may have any of a variety of shapes (e.g., hexagonal, kite-like, etc. or other shapes such as those described herein) that can lead to formation of sub-channel(s) having tapered inlet and / or outlet portions, as described above. However, it should be understood that the present disclosure is not so limited, and that in certain embodiments, at least one or more of the inlet and / or outlet portions may not be tapered.

[0105] As noted above, the sub-channel(s) in one or more regions (e.g., denuder region and / or filter region) of the device may optionally be formed by one or more wall(s) (of neighboring posts) comprising teeth. In some cases, the presence of teeth on the wall(s) of the sub-channels may advantageously aid denudation of target entities (e.g., oocyte complexes) and they pass through the sub-channel(s). In embodiments in which a plurality of posts is present in the fluidic channel, the wall(s) of the sub-channel(s) are defined by neighboring pairs of posts, e.g., as shown in FIG. 3. In some cases, both opposing walls forming the sub-channel may comprise teeth. In other cases, only one of the opposing walls forming the sub-channel may comprise teeth. In some cases, at least a portion on the posts 210 forming the walls of inlet portion a, constriction portion b. and outlet portion c of the sub-channel(s) comprises teeth. In some cases, the constriction portion b of the subchannels) comprises teeth. In some cases, the inlet portion a and / or outlet portion c of the sub-channel(s) comprises teeth.

[0106] The teeth on the post(s) and / or wall(s) of the sub-channel(s) in one or more regions (e.g., denuder region and / or filter region) of the device may have any of a variety of appropriate shapes, including, but not limited to, round, triangular, rectangular, saw-teeth, quadrilaterals, and / or other polygonals. While FIG. 3 illustrate one embodiment of posts comprising teeth that are saw-teethed, it should be understood that the disclosure is not so limited, and that in other embodiments, the teeth may have other shapes, such as saw-teeth, rectangular, triangular, and circular, as shown in FIGS. 15A-15D. In some embodiments, in may be particularly advantageous for the teeth on the post(s) to have one or more sharp features (e.g., an edge, a corner, a point, a tip, etc.). The presence of these sharp features in the teeth may allow for more efficient denudation of secondary entities from the target entity (by inducing tear and disruption of the secondary entities) and as the target entity gains contact with these features. Non-limiting example of teeth on posts and / or walls having one or more sharp features include saw-teeth, rectangular, triangular, as shown in FIG. 3, FIGS. 15A-15C, and / or FIGS. 19A-19F.

[0107] The teeth on the post(s) and wall(s) may have any of a variety of appropriate dimensions and / or orientations. For example, as shown in FIGs. 15A-15C, the teeth may comprises a first length (e.g., LFEAT-I), a second length (e.g., LFEAT.3a first height (e.g., LFEAT-2), a second height (e.g., LFEAT-4), a first angle (O-FEAT-I), and a second angle (e.g., <FEAT- 2). The first length (e.g., / -FEAT-.?), may have any of a variety of values, such at between 5 micrometers and 30 micrometers, between 10 micrometers and 20 micrometers, or between 15 micrometers and 30 micrometers. The second length (e.g., LFEAT-3) may have any of a variety of values, such at between 0 micrometers and 30 micrometers, between 10 micrometers and 20 micrometers, or between 15 micrometers and 30 micrometers. The first height (e.g., LFEAT-2) may have any of a variety of appropriate values, such as between 30 micrometers and 80 micrometers, between 40 micrometers and 60 micrometers, or between 50 micrometers and 80 micrometers. The second height (e.g., LFEAT-4) may have any of a variety of appropriate values, such as between 10 micrometers and 50 micrometers, between 20 micrometers and 40 micrometers, or between 30 micrometers and 50 micrometers. In some embodiments, the angles aFEAT-i,2) can be between 20 degrees and 80 degrees, or 30 degrees and 60 degrees,. Other ranges are possible. For example, in some cases, the length of the feature sides (LFEAT-1,2,3,4) can be as small as 1 pm and as large as 1000 pm. In some embodiments, the angles (aFEAT-1,2) can be as small as 0.1° and as large as 179.9°.

[0108] In one example, the extruded featured may be triangular shaped and may have the following geometrical parameters: LFEAT.3= 0. In another example, the extruded features may be rectangular shaped and may have the following parameters: LFEAT-2= LFEAT-4, and -FEAT-I =aFEAT-2- In yet another example, the extruded featured may be circular shaped and the features may be defined by the circle radius (RFEAT), the offset of its center from the wall (LOFFSETani the distance between features LFEAT), as shown in FIG. 15D. The teeth on the one or more wall(s) of the sub-channel(s) in one or more regions (e.g., denuder region and / or filter region) of the device may be oriented in any of a variety of appropriate directions. In some cases, it may be particularly advantageous to have some teeth oriented towards a direction facing a bulk fluid flow across the fluidic channel, as this may lead to an increase in denudation efficiency of target entities as they enter the sub-channel(s). In some embodiments, the teeth may be oriented at any angle, such as being saw-teethed, e.g., as shown in FIG. 3. In some cases, according to some embodiments, the teeth on one or more wall(s) of the sub-channel may be unidirectional. For example, as shown in FIG. 14C, the teeth 140 on the posts 120a and / or 120b may be oriented facing a first direction 12 of bulk fluid flow through the sub-channel(s).

[0109] In some cases, the teeth on one or more wall(s) of the sub-channel in one or more regions (e.g., denuder region and / or filter region) of the device may be bi-directional, such as comprising a first set of teeth that is oriented facing a first direction of bulk fluid flow across the sub-channel, and a second set of teeth that is oriented facing a second direction of bulk fluid flow across the sub-channel, e.g., as shown in FIG. 3. As shown, in the constriction portion of the sub-channel between neighboring pair of posts, each of the opposing walls comprise a first set of teeth that is oriented facing a forward fluid flow through the subchannel and a second set of teeth that is oriented facing a backward fluid flow through the sub-channel.

[0110] The first set of teeth may be oriented at any of a variety of appropriate angles facing a first direction of bulk fluid flow across the sub-channel. For example, the first set of teeth may be oriented at an angle between 0.1° and 90°, between 5° and 80°, between 20° and 70°, or between 30° and 60° facing the bulk fluid flow across the sub-channel. In some cases, the second set of teeth may be oriented in a direction that is substantially opposite the first set of teeth. For example, the second set of teeth may have a tip that is oriented in a direction that is at least 5 degrees, at least 10 degrees, at least 20 degrees, at least 30 degrees, at least 35 degrees, at least 40 degrees, at least 45 degrees, at least 60 degrees, at least 70 degrees, at least 90 degrees, or more, and / or up to 120 degrees, up to 140 degrees, up to 160 degrees, or up to 179 degrees, or up to 179.9 degrees clockwise from the tip of the first set of teeth. Combinations of the above-referenced ranges are possible (e.g., at least 5 degrees and up to 179.9 degrees, or at least 30 degrees and up to 160 degrees). Other ranges are also possible. Without wishing to be bound by any particular theory, the presence of bi-directional teeth may advantageously enhance denudation efficiency compared to unidirectional teeth during oscillatory flow, under otherwise identical conditions. Combinations of different configurations of size, shapes and directionality may be used from the above to facilitate denudation.

[0111] In some embodiments, an average flow velocity of the fluid within the denuder region is at least 10 times (e.g., at least 15 times, at least 20 times, at least 25 times, at least 50 times, and / or up to 100 times) faster than the average flow velocity of the fluid within the capture region.

[0112] In some embodiments, the fluidic channel comprises an inertial focusing region, e.g., positioned on a microfluidic device between a denuder region and a concentrator region, or anywhere else within the device. In some embodiments, the inertial focusing region comprises at least one fluidic channel having an inlet and an outlet. Other configurations are also possible in some embodiments. For example, in some cases, the inertial focusing region comprises a fluid channel, an inlet port, an outlet port, and at least two waste ports. In some embodiments, a pumping element is used to move a fluid comprising suspended particles (e.g., oocytes and contaminant cells) along the channel in a laminar flow.

[0113] Without wishing to be bound by any particular theory, it is generally believed that the fluid, the channel (e.g., channel dimensions), and the pumping element (e.g., pumping speed) may be configured in certain embodiments to cause inertial forces to act on the particles and to focus the particles into one or more stream lines. For example, in some embodiments, fluid channels comprising a generally serpentine structure, as shown in FIG. 25A, permit focusing of a particle suspended within the fluid (e.g., an oocyte) to the middle of the fluid channel width. Likewise, curved fluid channels, such as those shown in FIG. 25B may be used to focus a particle suspended within a fluid (e.g., an oocytes) to the top / middle or bottom middle of the fluid channel width. Those of ordinary skill in the art will understand and appreciate that other configurations may permit focusing of the suspended particles to other positions within the fluid channel. For example, in some implementations, inertial focusing of a particle to a streamline can be achieved by varying a flow rate of a fluid carrying suspended particles flowed through a channel of constant cross-section. In some implementations, focusing can be achieved by a reduction in the area of a cross-section of a channel through which a flux of particles passes. Particles can be localized within an area having a width of, e.g., 1.05, 2, 3, 4, or 5 times the width of the particles. Localization can occur at any location within the channel, e.g., at an unobstructed portion of the channel. Localization can occur in a portion of the channel having less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1 %, or 0.1 % reduction in cross-sectional area. Those of ordinary skill in the art will be aware of inertial focusing techniques, e.g., as discussed in Int. Pat. Apl. Nos. WO 2008 / 130977, WO 2016 / 073486, WO 2016 / 073481, or WO 2016 / 073448, each incorporated herein by reference.

[0114] FIGS. 25A-25B show illustrations of exemplary inertial focusing regions (FIG. 25 A and 25B), in accordance with some embodiments. FIGS 25A and 25B show exemplary inertial focusing regions for use, for example, between a denudation region and a concentration region. FIG. 25A design permits focusing of the target cell to the middle of the channel width, whereas the design illustrated in FIG. 25B permits focusing of the target cell to the middle the channel width either at the top or bottom of the z-plane, according to some embodiments.

[0115] In some embodiments, a device may comprise a single inertial focusing region. In other embodiments, however, one or more inertial focusing regions may be present, e.g., linked in series (e.g., daisy-chained together such that the outlet of a first inertial focusing region is fed into the inlet of a second inertial focusing region). In some embodiments, serially-linked inertial focusing regions have the same design (e.g., A-A-A, wherein A represents an inertial focusing region having design A); however, this is not a requirement, and alternating designs may be used (e.g., A-B-A or A-B-C, etc., wherein A, B, and C represent n inertial focusing regions having designs A, B, and C, respectively), as other nonlimiting examples.

[0116] An inertial focusing region may be positioned at any suitable location on a microfluidic device. For example, in some embodiments, the inertial focusing region is positioned between a denuder region and a concentration region. In some cases, the inertial focusing region focuses a target cell to the middle of the fluid channel width prior to entering the concentrator region. In some embodiments, the inertial focusing region focuses a target cell (e.g., oocytes) to the middle of the fluid channel width and / or toward a top (e.g., middle top) or bottom (e.g., middle bottom) portion of the sub-channel prior to entering the concentrator region. In some embodiments, the fluid flow rate of a fluid through the channel enables inertial lift to focus the oocytes away from a side channel configured to siphon fluid away from a main fluid channel. In some embodiments, the fluid channel leading into the concentrator region is configured to direct a target cell (e.g., oocytes) to a plane perpendicular to the fluid flow and away from the plane of the siphoning side channels.

[0117] Without wishing to be bound by any particular theory, it is believed that such configurations may permit better separation of the target cell (e.g., oocytes) from contaminant cells by focusing the contaminant cells to enter a streamline, different than the target cell, that directs them toward a plurality of sidewall channels (e.g., waste stream) in the concentrator region. The configurations are also believed to improve the yield of the target cells in certain embodiments as they direct the target cell toward a streamline that avoids the sidewall channels in the concentration region.

[0118] As non-limiting illustrative examples, FIGS. 26A-26D illustrate flow speeds to focus oocytes using an inertial focusing region having a 1000 micrometer wide inlet channel, 2 waste channels and 1 outlet channel (FIG. 26 A). FIG. 26B shows waste removal from the inertial focusing region, according to some embodiments. FIG. 26C shows photomicrographs illustrating the fluid composition prior to and after passage through the inertial focusing region. FIG. 26D shows the oocyte position within the microfluidic channel as determined by time-lapse optical microscopy, according to some embodiments.

[0119] In some embodiments, the fluidic channel comprises a capture region downstream of the denuder region. In some cases, the capture region comprises a third plurality of posts defining third sub-channels between neighboring pairs of posts, e.g., as shown in FIG. 1A and FIG. 5. As described in more detail below, the capture region may be configured to trap or immobilize one or more target entities (e.g., denuded oocyte) in the device, according to some embodiments. In some cases, at least some of the sub-channels may have minimum cross-sectional dimensions comparable to or smaller than the sizes of target entities (e.g., denuded oocyte complex), such that the target entities may be trapped by the sub-channels in the capture region.

[0120] FIG. 5 illustrates one non-limiting embodiment of the capture region 400 shown in FIG. 1. As shown, the capture region 400 comprises a plurality of posts 410 defining second sub-channels 420 between neighboring pairs of posts 410. As shown, the inlet of the capture region 400 may be fluidically connected to the outlet of the concentrator region 300 (as shown in FIG. 1A), and the outlet of the capture region 400 may be fluidically connected to the second pressure source 14 via port 40. As described in more detail below, the presence of one or more capture regions may advantageously allow temporary entrapment of target entities (e.g., denuded oocytes) therein, thereby preventing undesired release of the target entities and allowing the target entities to be subjected to oscillatory flow within the device until reaching a desired level of denudation within the device. As described in more detail below, the plurality of posts within the capture region may be arranged in any of a variety of appropriate patterns and may have any of a variety of appropriate shapes described with respective to the denuder region.

[0121] The plurality of posts within the capture region of the fluidic channel may be arranged in any appropriate fashion. For instance, in one embodiment, the plurality of posts are arranged in an array across the capture region. The array of posts, in some cases, may comprise one or more rows of posts, where each row comprises one or more posts. The posts may be arranged in any suitable arrangement, for example, a triangular grid or in a staggered fashion, e.g., as shown in FIG. 1A and / or in FIG. 5. However, it should be understood that the plurality of posts is not limited to being arranged in an ordered fashion, and that in some cases, at least one of the plurality of posts may be arranged randomly across the capture region.

[0122] In some embodiments, the array of posts in the capture region may comprise n number of rows of post(s) and / or sub-channels, where n is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and / or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000). Other ranges are also possible. In one set of embodiments, the array of posts comprises a single row of post and / or sub-channels. Referring to FIG. 1A and FIG. 5 as a non-limiting example, the array of posts 110 comprises multiple rows of posts and sub-channels.

[0123] In some embodiments, the array of posts in the capture region may comprise m number of post(s) and / or sub-channels per row, where m is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and / or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000). Other ranges are also possible. In one set of embodiments, the array of posts comprises a single post per row.

[0124] In some embodiments, some (or all) of the sub-channel(s) within the capture region of the fluidic channel may have a minimum cross-sectional dimension d (e.g., as shown in FIG. 5) of greater than or equal to 30 micrometers, greater than or equal to 40 micrometers, greater than or equal to 50 micrometers, greater than or equal to 65 micrometers, greater than or equal to 80 micrometers, greater than or equal to 90 micrometers, greater than or equal to 100 micrometers, greater than or equal to 110 micrometers, greater than or equal to 120 micrometers, greater than or equal to 130 micrometers, greater than or equal to 140 micrometers, and / or less than or equal to 150 micrometers, less than or equal to 140 micrometers, less than or equal to 130 micrometers, less than or equal to 120 micrometers, less than or equal to 110 micrometers, less than or equal to 100 micrometers, less than or equal to 90 micrometers, less than or equal to 80 micrometers, less than or equal to 65 micrometers, less than or equal to 50 micrometers, less than or equal to 40 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., greater than or equal to 30 micrometers and less than 120 micrometers, greater than or equal to 30 micrometers and less than or equal to 100 micrometers, greater than or equal to 30 micrometers and less than 80 micrometers, greater than or equal to 120 micrometers and less than or equal to 150 micrometers, or greater than or equal to 80 micrometers and less than or equal to 120 micrometers, or between 50 micrometers and 150 micrometers). Other ranges are also possible. In one set of embodiments, some (or all) of the sub-channel(s) within the capture region of the fluidic channel may have a minimum cross-sectional dimension d of less than or equal to 120 micrometers.

[0125] In some embodiments, the above-referenced ranges may be particularly advantageous for certain type of applications, e.g., denudation of oocyte complexes. However, it should be understood that the disclosure is not so limited, and that in some embodiments, depending on the application, the minimum cross-sectional dimension of the sub-channels in the capture region may be scaled relative to the size of the target entity (e.g., a cell, a tissue, etc.). For example, in some embodiments in which the target entity is a particular type of cell (e.g., a tumor cell), the minimum cross-sectional dimension of the sub-channels be in the size range of micrometers, such as less than 100 microns, less than 50 microns, less than 25 microns, less than 20 microns, less than 15 microns, or less and / or down to 10 microns, down to 5 microns, or less. For example, in some embodiments in which the target entity is a particular type of tissue, the minimum cross-sectional dimension of the sub-channels be in the size range of millimeter, such as at least 0.5 mm, at least 1 mm, at least 2 mm, at least 10 mm, or more.

[0126] In some embodiments, at least some (or all) of the sub-channels in the capture regions have a dimension sized such that a fully denuded (or dissociated) target entity (e.g., an oocyte) cannot pass therethrough when subjected to flow. For example, according to some embodiments, at least some (or all) of the sub-channels in the capture regions may have a size that is at least 10 micrometers, at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, and / or up to 50 micrometers, at least 60 micrometers, at least 80 micrometers, or up to 100 micrometers, or more, smaller than the size (e.g., diameter) of the fully denuded (or dissociated) target entity. In some cases, according to some embodiments, at least some (or all) of the sub-channels in the capture regions may have a size that is at least 10%, at least 20%, at least 30%, at least 40%, or more, and / or up to 50%, up to 60%, up to 70%, or up to 80% less than the size of a fully denuded (or dissociated) target entity.

[0127] In some embodiments, the sub-channel(s) between the posts in the capture region may have a minimum cross-sectional dimension that is less than the minimum cross-sectional dimension of the sub-channel(s) within the denuder region of the fluidic channel, according to some embodiments. For example, the sub-channel(s) between the posts in the capture region may have minimum cross-sectional dimensions that are at least 10 micrometers (e.g., at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, and / or up to 50 micrometers, up to 70 micrometers, up to 100 micrometers, up to 120 micrometers, up to 150 micrometers, or more) less than the minimum cross-sectional dimension of the sub-channel(s) in the denuder region. In some embodiments, the plurality of sub-channels in the capture region may be sized to only allow denuded entities (e.g., denuded or bare oocytes without the bound non-oocyte cells) to flow readily therethrough while trapping larger target complexes (e.g., oocyte complex) and / or debris.

[0128] In some embodiments, some of the sub-channels in the capture region may have dimensions that differ from the rest of the sub-channels. For example, in certain embodiments, the sub-channels may be arranged such that the sub-channel(s) adjacent an inlet of the capture region may have minimum cross-sectional dimension(s) larger than the minimum cross-sectional dimension(s) of sub-channel(s) further away from the inlet of the capture region, such as the outlet of the capture region. For example, referring to FIG. 1A and FIG. 5, the sub-channels 420 adjacent an inlet 401 of the capture region 400 (such as those in the first two rows) may have sub-channels with minimum cross-sectional dimension(s) larger than cross-sectional dimension(s) of sub-channel(s) further away from the inlet of the capture region, e.g., adjacent an outlet 402 of the capture region.

[0129] In some embodiments, the plurality of sub-channels may be divided into one or more stages, with each some or all of the stages containing sub-channels having different minimum cross-sectional dimensions from the those in the other stage(s). For example, in some embodiments, the sub-channels may comprise a first stage of sub-channels having a minimum cross-sectional dimension of greater than or equal 150 micrometers and less than or equal to 200 micrometers. In some cases, the sub-channels may comprise a second stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 120 micrometers and less than 150 micrometers. In some cases, the sub-channels may comprise a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 80 micrometers and less than 120 micrometers. FIG. 5 can be used to illustrate one such embodiment. As shown, the sub-channels may be divided into a plurality of stages, e.g., stage 1 (si), stage 2 (s2), stage 3 (s3). According to some embodiments, a stage of subchannels adjacent an 401 inlet of the capture region may have a minimum cross-sectional dimension larger than the minimum cross-sectional dimension of a stage of sub-channels adjacent an outlet 402 of the capture region.

[0130] The plurality of posts within the capture region may have any of a variety of appropriate shapes, including but not limited to, triangles, polygons, and / or any other shapes described elsewhere herein with respect to the posts in the denuder region and / or filter region. In some cases, the plurality of posts may have smooth surfaces. In some cases, the plurality of posts may have surfaces comprising teeth, as described elsewhere with respect to the posts in the denuder region and / or filter region.

[0131] In some embodiments, the fluidic channel comprises a concentrator region. In some cases, the concentrator region comprises a one or more filters configured to remove at least a portion of a fluid and / or waste, as shown FIG. 1A and FIG. 4. For example, the one or more filters may comprise a plurality of sub-channels sized to permit flow of wastes and fluids, and a central channel configured to allow flow of target entities therethrough.

[0132] FIG. 4 and FIG. 23 illustrate one non-limiting embodiment of the concentrator region 300 shown in FIG. 1. As shown in FIG. 4, in one set of embodiments, the concentrator region 300 may comprise a central fluidic channel 315 surrounded by one or more filters, e.g., filters 316a and 316b. At least one (or both) of the one or more filters 316a and 316b may comprise a plurality of posts 310. The plurality of posts 310 may, in some embodiments, define a plurality of sub-channels 320 between neighboring pairs of posts 310. According to some embodiments, the sub-channels 320 may be sized to allow fluid and / or waste to flow therethrough and leave the device via channels 317 and ports 60 and 70 (as shown by arrows 61 and 71), while preventing target entities (e.g., denuded oocyte complex) from passing therethrough. The central fluidic channel 315, according to some embodiments, allows the target entities to flow therethrough from the denuder region 200 to the capture region 400 (as shown in FIG. 1A). In addition, in one embodiment, the concentrator region may be used to slow down the target entities as they enter capture region. For example, the target entities may flow through the concentrator region while slowing down substantially, allowing them to be gently captured by the capture region. In some cases, this may be because the volumetric flow rate exiting the concentrator is less than the inlet flowrate, e.g., less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, etc.

[0133] The plurality of sub-channels 320 in the concentrator region 300 may have any of a variety of appropriate minimum cross-sectional dimensions. In some embodiments, some (or all) of the sub-channel(s) may have a cross-sectional dimension of 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 20 micrometers, greater than or equal to 30 micrometers, greater than or equal to 40 micrometers, greater than or equal to 50 micrometers, greater than or equal to 60 micrometers, greater than or equal to 100 micrometers, greater than or equal to 200 micrometers, greater than or equal to 300 micrometers, or more, and / or less than or equal to 400 micrometers, less than or equal to 300 micrometers, than or equal to 200 micrometers, than or equal to 100 micrometers, less than or equal to 60 micrometers, less than or equal to 50 micrometers, less than or equal to 40 micrometers, less than or equal to 30 micrometers, less than or equal to 20 micrometers, less than or equal to 10 micrometers, less than or equal to 5 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., between 1 micrometer and 300 micrometers, between 1 micrometer and 200 micrometers, between 5 micrometers and 50 micrometers, between 1 micrometer and 20 micrometers). Other ranges are also possible. In some embodiments, at least some (or all) of the subchannels in the concentrator region have minimum cross-sectional dimensions less than the minimum cross-sectional dimensions of sub-channels in the denuder region.

[0134] FIGS. 24A-24C illustrate a non-limiting embodiment of concentrator region 300 shown in FIG. 1. As shown in FIG. 24A, in one set of embodiments, the concentrator region 300 may comprise two sidewalls 2410 that define a central fluidic channel 2405. At least one (or both) of the side walls 2410 comprise a plurality of sidewall channels 2415 oriented in a direction that is orthogonal to the central fluidic channel 2405. FIG. 24B shows a scanning electron photomicrograph of the second concentration region illustrating the difference between the main channel height and the side channel height. FIG. 24C shows fluid comprising oocytes flowing through the second concentration region, in accordance with some embodiments.

[0135] According to some embodiments, the plurality of sidewall channels may be sized to allow fluid and / or waste to flow therethrough and leave the device (e.g., via one or more channels and / or outlet ports), while preventing target entities (e.g., denuded oocyte complex) from passing therethrough. For example, in some embodiments, the plurality of sidewall channels of at least one of the two sidewalls is not centered relative to the central fluidic channel in a direction orthogonal to fluid flow within the central fluidic channel. It is believed that such configurations allow a fluid comprising a plurality of target cells and contaminants to flow through the concentrator region such that at least a portion of contaminants in the fluid are pulled into the plurality of sidewall channels from central fluidic channel along a first plane while the target cells flow through the central fluidic channel along a second plane, different than the first plane.

[0136] In some embodiments, the target entities may flow through the concentrator region while slowing down substantially, allowing them to be captured by the capture region. In some cases, this may be because the volumetric flow rate exiting the concentrator is less than the inlet flowrate, e.g., less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, etc.

[0137] The walls of the sidewall channels, according to some embodiments, are angled, at an angle other than 0 degrees relative to perpendicular walls. As examples, the walls of the sidewalls may be less than 20 degrees, less than 15 degrees, between 2 degrees and 10 degrees, etc. In some embodiments, the wall angle of the sidewall channels is greater than or equal to 2 degrees, greater than or equal to 3 degrees, greater than or equal to 4 degrees, greater than or equal to 5 degrees, greater than or equal to 6 degrees, greater than or equal to 7 degrees, greater than or equal to 8 degrees, greater than or equal to 9 degrees, greater than or equal to 10 degrees, greater than or equal to 12 degrees, greater than or equal to 14 degrees, greater than or equal to 16 degrees, greater than or equal to 18 degrees, or greater than or equal to 20 degrees. In some embodiments, the wall angle of the sidewall channels is less than or equal to 20 degrees, less than or equal to 18 degrees, less than or equal to 16 degrees, less than or equal to 14 degrees, less than or equal to 12 degrees less than or equal to 10 degrees, less than or equal to 9 degrees, less than or equal to 8 degrees, less than or equal to 7 degrees, less than or equal to 6 degrees, less than or equal to 5 degrees, less than or equal to 4 degrees, less than or equal to 3 degrees, less than or equal to 2 degrees, or less than or equal to 1 degrees (i.e., as determined relative to perpendicular walls). Combinations of the above recited ranges are all possible in some embodiments. For example, in some embodiments, the wall angle of the sidewall channels is greater than or equal to 1 degree and less than or equal to 10 degrees.

[0138] In some embodiments, a plurality of sidewall channels has an average cross-sectional dimension of between 1 micrometer and 5 micrometers. In some embodiments, the plurality of sidewall channels have an average cross-sectional dimension of 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 20 micrometers, greater than or equal to 30 micrometers, greater than or equal to 40 micrometers, greater than or equal to 50 micrometers, greater than or equal to 60 micrometers, greater than or equal to 100 micrometers, greater than or equal to 200 micrometers, greater than or equal to 300 micrometers, or more, and / or less than or equal to 400 micrometers, less than or equal to 300 micrometers, than or equal to 200 micrometers, than or equal to 100 micrometers, less than or equal to 60 micrometers, less than or equal to 50 micrometers, less than or equal to 40 micrometers, less than or equal to 30 micrometers, less than or equal to 20 micrometers, less than or equal to 10 micrometers, less than or equal to 5 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., between 1 micrometer and 300 micrometers, between 1 micrometer and 200 micrometers, between 5 micrometers and 50 micrometers, between 1 micrometer and 20 micrometers). Other ranges are also possible. In some embodiments, at least some (or all) of the plurality of sidewall channels in the concentrator region have an average cross-sectional dimension less than the average cross-sectional dimensions of sub-channels in the denuder region.

[0139] In some embodiments, a sidewall has an average height of between 100 microns and 400 microns. In some embodiments, the average height of the sidewall is greater than or equal to 100 microns, greater than or equal to 130 microns, greater than or equal to 160 microns, greater than or equal to 190 microns, greater than or equal to 220 microns, greater than or equal to 250 microns, greater than or equal to 280 microns, greater than or equal to 310 microns, greater than or equal to 340 microns, greater than or equal to 370 microns, or greater than or equal to 400 microns. In some embodiments, the average height of a sidewall is less than or equal to 400 microns, less than or equal to 370 microns, less than or equal to 340 microns, less than or equal to 310 microns, less than or equal to 280 microns, less than or equal to 250 microns, less than or equal to 220 microns, less than or equal to 190 microns, less than or equal to 160 microns, less than or equal to 130 microns, or less than or equal to 100 microns. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the average height of a sidewall is greater than or equal to 100 micron and less than or equal to 400 microns.

[0140] In one set of embodiments, at least some (or all) of the sub-channels in the concentrator region may be sized such that a target entity (e.g., oocyte) cannot pass therethrough when subjected under an applied flow. For example, as shown in FIG. 4, while smaller entities (e.g., wastes, impurities, and / or debris) may pass through the subchannels (e.g., 320) in the concentrator region, the target entity instead passes through the main channel (e.g., 315) and does not pass through the sub-channels. In some cases, the selective removal of small entities versus the target entity via the sub-channel(s) may not be (entirely) based on the absolute cross-sectional dimension of the sub-channels, but rather based on the flow profile in the concentrator region, such as the fraction of flow separated from the main channel at the sub-channel junctions based on the downstream resistance to flow.

[0141] In some embodiments, a concentrator region may operate under flow conditions that allow inertial lift of target entities (e.g., oocytes), for example, toward a center of a central fluid channel (e.g., away from one or more side channels configured to siphon fluid away from the central fluid channel). Without wishing to be bound by any particular theory, it is believed passive inertial migration of the target entities towards the center of the fluid channel enables the side channels to siphon larger volumes of fluid than possible with random distribution of target entities across the channel cross-section. The skilled artisan will understand that the above flow regime may be characterized by Reynolds number (Rep), which is a dimensionless number that accounts for the dimensions of the channel as well as the flow properties. It is known in the art that Repmay calculated using the following equation: Rep= p x U x a2 / (p x Dh), where p is the density of the carrier fluid, U is the average flow velocity, a is the size of the target entity, p is the fluid viscosity and Dh is the hydraulic diameter of the channel.

[0142] In some embodiments, a flowrate in a fluid channel at an entrance of a concentrator region is about 5 mL / min and the flowrate at an exit of the concentrator region is about 0.5 mL / min. In some embodiments about 90% of the flow is siphoned through the sidewall channels. Those of skill in the art will understand that such conditions corresponds to a Repof about 26 at the entrance of the concentrator region and an Repof about 2.6 at the exit of the concentrator region. Again, without wishing to be bound by any particular theory, it is believed that an Rep> 1 may be sufficient to achieve good inertial focusing of a target entity. However, other values of Repat the inlet and the outlet of the concentrator region are also possible (e.g., the values will be different based on the flowrate, channel dimensions and the amount of flow percentage that is siphoned out for a particular design), and this disclosure is not so limited. For example, Repvalues of < 1 produce sufficient inertial lift forces (e.g., from the side walls) to push target entities away from the sidewalls because the inertial lift forces scale more strongly with the size of the target entity (a4-a6) near the side walls, as compared to the channel center where the correlation is weaker (e.g., large target entities, like oocytes, are still pushed away from the sidewall channels even at lower particle Reynolds numbers). In some embodiments, the lower bound for the flow regime where concentrator function is enabled via inertial lift occurs at about an Repvalue of > 0.001. In some embodiments, the upper limit of the channel flowrate is about an Re value of 2300 (e.g., the point where the flow transitions from laminar to turbulent and the focusing effects are generally lost). In still other embodiments, other ranges of Reynolds numbers may be used.

[0143] In some embodiments, a flowrate in a fluid channel at an entrance of a concentration region is between 1 mL / min and 10 mL / min. In some embodiments, the flowrate in a fluid channel at an entrance of the concentration region is greater than or equal to 1 mL / min, greater than or equal to 2 mL / min, greater than or equal to 3 mL / min, greater than or equal to 4 mL / min, greater than or equal to 5 mL / min, greater than or equal to 6 mL / min, greater than or equal to 7 mL / min, greater than or equal to 8 mL / min, greater than or equal to 9 mL / min, or greater than or equal to 10 mL / min. In some embodiments, the flowrate in a fluid channel at an entrance of the concentration region is less than or equal to 10 mL / min, less than or equal to 9 mL / min, less than or equal to 8 mL / min, less than or equal to 7 mL / min, less than or equal to 6 mL / min, less than or equal to 5 mL / min, less than or equal to 4 mL / min, less than or equal to 3 mL / min, less than or equal to 2 mL / min, or less than or equal to 1 mL / min. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the flowrate in a fluid channel at an entrance of the concentration region is greater than or equal to 1 mL / min and less than or equal to 10 mL / min. In some embodiments, the flowrate in a fluid channel at an entrance of the concentration region is about 5 mL / min.

[0144] In some embodiments, a flowrate in a fluid channel at an exit of a concentration region is between 0.1 mL / min and 1 mL / min. In some embodiments, the flowrate in a fluid channel at an entrance of the concentration region is greater than or equal to 0.1 mL / min, greater than or equal to 0.2 mL / min, greater than or equal to 0.3 mL / min, greater than or equal to 0.4 mL / min, greater than or equal to 0.5 mL / min, greater than or equal to 0.6 mL / min, greater than or equal to 0.7 mL / min, greater than or equal to 0.8 mL / min, greater than or equal to 0.9 mL / min, or greater than or equal to 1.0 mL / min. In some embodiments, the flowrate in a fluid channel at an exit of the concentration region is less than or equal to 1.0 mL / min, less than or equal to 0.9 mL / min, less than or equal to 0.8 mL / min, less than or equal to 0.7 mL / min, less than or equal to 0.6 mL / min, less than or equal to 0.5 mL / min, less than or equal to 0.4 mL / min, less than or equal to 0.3 mL / min, less than or equal to 0.2 mL / min, or less than or equal to 0.1 mL / min. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the flowrate in a fluid channel at the exit of the concentration region is greater than or equal to 0.1 mL / min and less than or equal to 1.0 mL / min. In some embodiments, the flowrate in a fluid channel at an exit of the concentration region is about 0.5 mL / min.

[0145] In some embodiments, between 60% and 95% of the flow through a central fluid channel is siphoned through the sidewall channel. In some embodiments, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, or greater than or equal to 95% of the flow through a central fluid channel is siphoned through the sidewall channel. In some embodiments, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, less than or equal to 75%, less than or equal to 70%, less than or equal to 65%, or less than or equal to 60% of the flow through a central fluid channel is siphoned through the sidewall channel. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, greater than or equal to 60% and less than or equal to 95% of the flow through a central fluid channel is siphoned through the sidewall channel. In some embodiments about 90% of the flow is siphoned through the sidewall channels.

[0146] In some embodiment, a fluid flowing through a channel (e.g., at an entrance or exit of a concentrator region on the device) has an Repvalue of between 0.001 and 2300. In some embodiments, a fluid flowing through a channel has an Re value of greater than or equal to 0.001, greater than or equal to 0.01, greater than or equal to 0.1, greater than or equal to 1, greater than or equal to 10, greater than or equal to 100, greater than or equal to 1000, greater than or equal to 1500, greater than or equal to 2000, or greater than or equal to 2300. In some embodiments, a fluid flowing through the channel has an Re value of less than or equal to 2300, less than or equal to 2000, less than or equal to 1500, less than or equal to 1000, less than or equal to 100, less than or equal to 10, less than or equal to 1, less than or equal to 0.1, less than or equal to 0.01, or less than or equal to 0.001. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the fluid flowing through a channel has an Re value of greater than or equal to 0.001 and less than or equal to 2300. In some embodiments, the fluid flowing through an entrance of the concentration region has an Repvalue of about 26. In some embodiments, the fluid flowing through an exit of the concentration region has an Repof about 2.6.

[0147] In some embodiments, the microfluidic device comprises a first pressure source upstream of the denuder region (and / or filter region) and a second pressure source downstream of capture region. In some embodiments, the device further comprises a first port adjacent and connected to the first pressure source and a third port adjacent and connected to the second pressure source. In some embodiments, the first pressure source and / or second pressure source independently comprises one or more pumps (e.g., syringe pumps, peristaltic pumps, positive displacement pumps, etc.).

[0148] According to some embodiments, the microfluidic device comprises a first port positioned upstream of and in fluidic communication with the filter region (and / or the denuder region). For example, as shown in FIG. 1 A, the microfluidic device 10 comprises a first port 20 positioned upstream of and in fluidic communication with the filter region 100 (and denuder region 200). According to some embodiments, the first port 20 may be fluidically connected to the pressure source 12. The first port 20, according to some embodiments, may be configured to transition from one position (e.g., an open position that allows for fluid flow) to another (e.g., a closed position that blocks fluid flow), or vice versa, during operation. For example, the first port 20, according to some embodiments, may be configured to receive or output a fluid from the filter region 100 when in an open position.

[0149] In some embodiments, the microfluidic device further comprises a third port downstream of the capture region, as shown in FIG. 1A and FIG. 5. For example, as shown, the microfluidic device 10 further comprises a third port 40 downstream of the capture region 400. The third port, in some embodiments, may be fluidically connected to the second pressure source 14. The third port 40, similar to the first port, may also be actuated from one position (e.g., a closed position that blocks fluid flow) to another (e.g., an open position that allows for fluid flow), or vice versa, during operation. In some embodiments, when the third port 40 is in an open position, fluidic connectivity may be established between the capture region 40 and the second pressure source 14, such that a fluid is caused to flow into the capture region 40.

[0150] As described elsewhere herein, the presence of at least two pressure sources may advantageously allow for oscillatory flow across the fluidic channel, such as causing flow across the plurality of regions (e.g., filter region, denuder region, concentrator region, and / or capture region) in a first direction and then in a second direction (opposed to the first direction). For example, the first pressure source, according to some embodiments, may be configured exert pressure in a first direction, such that a fluid within the fluidic channel may be caused or induced to flow in the first direction within the fluidic channel. Conversely, according to some embodiments, the second pressure source may be configured to exert a pressure in a second direction, such that the fluid within the fluidic channel may be caused or induced to flow in the second direction within the fluidic channel.

[0151] FIG. 1A can be used to illustrate one such embodiment. As shown, the microfluidic device 10 comprises the first port 20 and the third port 40. In some cases, the first port 20 may be positioned adjacent and connected to the first pressure source 12 and the third port 14 may be positioned adjacent and connected to the second pressure source 14. The first pressure source 12, according to some embodiments, may be configured exert pressure in a first direction 13, such that a fluid within the fluidic channel may flow in the first direction 13 (e.g., a forward direction). Conversely, according to some embodiments, the second pressure source 14 may be configured to exert a pressure in a second direction 15, such that the fluid within the fluidic channel may flow in the second direction 15 (e.g., a reverse direction).

[0152] According to some embodiments, the microfluidic device further comprises a second port positioned downstream of the denuder region and upstream of the capture region. For example, as shown in FIG. 2, the microfluidic device 10 may further comprise a second port 30 positioned downstream of the denuder region 200 and upstream of the capture region 400. The second port, according to some embodiments, may be configured to output target entities (e.g., denuded oocyte) from the fluidic channel during operation. Similar to the first port and / or the second port, the second port may also be actuated from one position (e.g., a closed position that blocks fluid flow and seals the fluidic channel from the external environment) to another (e.g., an open position that allows for fluid flow or connectivity with the external environment), or vice versa.

[0153] In some embodiments, the microfluidic device may further comprise one or more additional ports (e.g., a fourth, a fifth port, a sixth port, etc.) configured to output wastes out of the device, e.g., port 50, 60, and 70 as shown in FIG. 1A. Specifically, the device 10 may further comprise a fourth port 50 downstream of the capture region 400, and / or one or more additional ports, e.g., a fifth port 60 and a sixth port 70 downstream of the concentrator region 300.

[0154] While FIGS. 1A-1D are used to illustrate one embodiment of a microfluidic device comprising multiple ports and pressure sources, it should be understood that the disclosure is not so limited, and that in certain embodiments, additional ports and / or pressure sources may be present in the microfluidic device to control fluid flow within the device.

[0155] For example, in some embodiments, the microfluidic device may further comprise additional port(s) and / or pressure source(s) in an intermediate region of the fluidic channel, e.g., between the filter region and the denuder, e.g., as shown in FIG. 17A. As shown, the microfluidic device 10 may further comprise an additional port (e.g., a seventh port 80) positioned downstream of the filter region 100 and upstream of the denuder region 200. The microfluidic device 10 may further comprise an additional pressure source (e.g., a third pressure source 80) adjacent and coupled to the additional port 80 and configured to direct flow into and / or out of the port 80 via an application of positive or negative pressure. The additional pressure source 16 may be connected to a reservoir (not shown) external the device.

[0156] The additional port (e.g., seventh port 80), similar to other ports described elsewhere herein, may also be actuated to transition from one position to another. For example, in one set of embodiments, the additional port (e.g., seventh port 80) may be configured to transition from a first position, e.g., a closed or sealed position that prevents fluidic connectivity between regions of the fluidic channel (e.g., filter region 100 and / or denuder region 200) and the third pressure source (e.g., pressure source 80), to a second position, e.g., an open position that establishes fluid connectivity between the fluidic channel (e.g., filter region 100 and / or denuder region 200) and the third pressure source, or vice versa, during operation. In some embodiments, when in the first position, the additional port may allow for fluidic communication between region(s) (e.g., between filter region or denuder region) within the fluidic channel, e.g., as shown in FIG. 17C.

[0157] In some cases, the additional port (e.g., port 80 in FIG. 17A) between the filter region and the denuder region may comprise a three-way valve. According to some embodiments, the three-way valve may be actuated from a first configuration (e.g. where fluidic connectivity is established between the filter region 100 and the third pressure source 80 that is coupled to the port 16) as shown in FIG. 17B, to a second position (e.g., where fluidic connectivity is established between the filter region 100 and the denuder region 200) as shown in FIG. 17C, and to a third configuration (e.g., where fluidic connectivity is established between the denuder region 200 and the third pressure source 80 that is coupled to the port 16) as shown in FIG. 17D, according to some embodiments.

[0158] In some cases, the presence of the additional port between the filter region and the denuder region may advantageously allow for separate control of flow properties within each of the filter region and the denuder region, such that each region may be treated as a modular unit independent of the other regions. This may allow each of the regions to be used separately to perform different functions during operation. For example, as described in more detail below, in some embodiments, while repeated flow (e.g., oscillatory flow) of a fluid may be performed within the filter region to break down larger non-target entities (e.g., debris and / or impurities), repeated flow (e.g., oscillatory flow) of a fluid may be separately performed within the other regions of the fluidic channel (e.g., denuder region and capture region) to denude target entities (e.g., oocyte complex), as described in more detail below.

[0159] In some embodiments, the device described in FIG. 17A-17F may allow for selective and / or simultaneous actuation of a plurality of ports and / or pressure sources in the device during operation, similar to the device illustrated in FIGS. IB- ID. Such selective actuation may allow for precise and timed control of fluid flow within the device, including the directionality of fluid flow within the device, the type of fluid flow (e.g., oscillatory fluid flow), and / or the timing of various fluid flows into and / or out of the device, as desired. In some cases, the plurality of ports may be simultaneously actuated (by a controller) to transition from one configuration to another.

[0160] For example, as shown in FIGS. 17A-17F, the microfluidic device 10 may further comprise a plurality of ports, such as port 20, 30, 40, 50, 60, 70 and 80, at least some of which are coupled to pressure sources, such as pressure sources 12, 14, and 16. In some cases, the plurality of ports may be simultaneously actuated to have a first configuration at a first point in time. For example, when in a first configuration, a first set of ports may be selectively actuated to open (e.g., port 20) while a second set of ports may be actuated to close (e.g., port 30, 40, 50, 60, 70 and 80), such that a fluid containing a target entity (e.g., oocyte complex) and non-target entities (e.g., debris and / or impurities) (if present) may be caused to flow (by a pressure source 12) from a reservoir into an inlet region of the device via one or more ports (e.g., port 20), through the filter region (e.g., filter region 100) of the device in a first direction (e.g., a forward direction 13a), to a sealed region (e.g., outlet of the filter region 100 sealed by port 80), e.g., as shown in FIG. 17A. In such a configuration, port 80 is actuated to seal the outlet of the filter region to block fluidic connectivity between the filter region and the denuder region, in some embodiments.

[0161] In some embodiments, the plurality of ports may be actuated to have a second configuration at a second point in time. For example, when in a second configuration, a third set of ports may be selectively actuated to open (e.g., port 20 and 80) while a fourth set of ports may be actuated to close (e.g., port 30, 40, 50, 60 and 70), such that a fluid may be caused to flow (by a pressure source 16) into a region of the device via one or more ports (e.g., port 80), through the filter region (e.g., filter region 100) of the device in a second direction (e.g., a backward direction 15a), and exit the inlet region of the device via one or more ports (e.g., port 20) into the reservoir (not shown), e.g., as shown in FIG. 17B. At the second point in time, the additional port (e.g., port 80) may be actuated to establish fluid connectivity between the pressure source 16 and the filter region 100, and block fluid connectivity between the filter region 100 and denuder region 200.

[0162] The plurality of ports, according to some embodiments, may be actuated repeatedly to transition from the first configuration to the second configuration, and vice versa, for any number of times as described elsewhere herein, e.g., at least 1 time, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, and / or up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above-referenced ranges are possible (e.g., at least 5 times and up to 1000 times). Other ranges are also possible.

[0163] In some cases, the plurality of ports may be simultaneously actuated to have a third configuration at a third point in time. For example, when in a third configuration, a fifth set of ports may be selectively actuated to open (e.g., port 20, 50, 60 and 70) while a sixth set of ports may be actuated to close (e.g., port 30, 40 and 80), such that the fluid containing the target entity (e.g., oocyte complex) in the filter region may be caused to flow (by a pressure source 12) from filter region (e.g., filter region 100) in a first direction (e.g., a forward direction 13b), to subsequent regions (e.g., denuder region 200 and / or capture region 400), e.g., as shown in FIG. 17C. As described in more detail below, one or more ports, e.g., ports 50, 60, and / or 70, may be actuated to open to allow excess fluid and / or waste to flow therethrough, while one or more ports, e.g., ports 30 and 40, may be selectively closed to retain target entities (e.g., oocyte complex) within the device for further processing and / or prevent target entities from exiting the device. The additional port (e.g., port 80) may be actuated to allow fluid flow from the filter region to the denuder region, according to some embodiments.

[0164] In some cases, the plurality of ports may be simultaneously actuated to have a fourth configuration at a fourth point in time. For example, when in a fourth configuration, a seventh set of ports may be selectively actuated to open (e.g., port 50, 60, 70 and 80) while an eighth set of ports may be actuated to close (e.g., port 20, 30 and 40), such that fluid flow (e.g., flow 81b) may be caused by a pressure source 16 from the additional port (e.g., port 80) positioned between the filter region and the denuder region, through various intermediate regions (e.g., regions, 200, 300, and / or 400) of the device in a first direction (e.g., a forward direction 13c), to a sealed exit region (e.g., region sealed by port 40), e.g., as shown in FIG. 17D. As described in more detail below, one or more ports, e.g., ports 50, 60, and / or 70, may be actuated to open to allow excess fluid and / or waste to flow therethrough, while one or more ports, e.g., ports 30 and 40, may be selectively closed to retain target entities (e.g., oocyte complex) within the device for further processing and / or prevent target entities from exiting the device. The additional port 80 may be actuated to establish fluid connectivity between the pressure source 16 and the denuder region 200, but block fluid connectivity between the filter region 100 and denuder region 200, according to some embodiments.

[0165] In some embodiments, the plurality of ports may be actuated to have a fifth configuration at a fifth point in time. For example, when in a fifth configuration, a nineth set of ports may be selectively actuated to open (e.g., port 40, 50, 60 and 70) while a tenth set of ports may be actuated to close (e.g., port 20, 30 and 80), such that a fluid 31 may be caused to flow (by a pressure source 14) into an exit region of the device via one or more ports (e.g., port 40), through the various intermediate regions (e.g., regions 400, 300, and / or 200) of the device in a second direction (e.g., a backward direction 15b), to a seal region (e.g., inlet of denuder region 200 sealed by port 80), e.g., as shown in FIG. 17E. As described in more detail below, one or more ports, e.g., port 50, may be actuated to open to allow excess fluid and / or waste to flow therethrough (via arrows 51), while one or more ports, e.g., ports 30, 60 and / or 70, may be actuated to close to prevent target entities from exiting the device and / or to facilitate and direct fluid flow in the second direction. The additional port (e.g., port 80) may be actuated to prevent fluid flow from the denuder region to the filter region, according to some embodiments.

[0166] The plurality of ports, according to some embodiments, may be actuated repeatedly to transition from the fourth configuration to the fifth configuration, and vice versa, for any number of times as described elsewhere herein, e.g., at least 1 time, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, and / or up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above-referenced ranges are possible (e.g., at least 5 times and up to 1000 times). Other ranges are also possible.

[0167] In some embodiments, the plurality of ports may be actuated to have a sixth configuration at a sixth point in time. For example, when in a sixth configuration, an eleventh set of ports may be selectively actuated to open (e.g., ports 30, 40, and 50) while a twelve set of ports may be actuated to close (e.g., port 20, 60, 70 and 80), such that a fluid may be caused to flow (by a pressure source 14) into an exit region of the device via one or more ports (e.g., port 40), through the various intermediate regions (e.g., region 400) in the second direction (e.g., a backward direction 15c), and exit via one or more ports (e.g., ports 30 and 50), e.g., as shown in FIG. 17F. As described in more detail below, one or more ports, e.g., the second port 30, may be actuated to open such that target entities (e.g., denuded oocyte) may be released from the device, while one or more ports, e.g., port 50, may be actuated to open to allow excess fluid and / or waste to flow therethrough.

[0168] While FIGS. 17A-17D are used to illustrate one embodiment of a microfluidic device comprising seven ports, it should be understood that the disclosure is not so limited, and that in certain embodiments, additional ports may be present in the microfluidic device to control fluid flow within the device. For example, as shown in FIG. 18, in addition to the various ports shown in FIG. 17, an eighth port (port 85) may be present in the device. Port 85, according to some embodiments, may be positioned between the concentrator region 300 and the denuder region 200 to control flow between the two regions. For example, as described in more detail elsewhere herein, oscillatory flow may be applied separately in each of the filter region and denuder regions, independent of the other regions. The eighth port (e.g., port 85), similar to the seventh port (e.g., port 80), may also be actuated to transition from one position to another. For example, in one set of embodiments, the eighth port 75 may be configured to transition from a first position, e.g., a closed or sealed position that prevents fluidic connectivity between the denuder region 200 and the concentrator region 300, to a second position, e.g., an open position that establishes fluid connectivity between the denuder region 200 and the concentrator region 300, or vice versa, during operation.

[0169] In some embodiments, by simultaneously adjusting the various ports (e.g., port 80 and / or 85 as shown in FIG. 18), flow may be independent controlled within each of the filter region and / or denuder region. For example, as shown in FIG. 18, when ports 80 and 85 are maintained in closed positions, the denuder region 200 may be isolated from other regions and flow (e.g., oscillatory flow) may be independent controlled within the denuder region, in some embodiments. In some embodiments, a method of denuding an oocyte complex comprising an oocyte and bounded secondary cells (i.e., non-oocyte cells) to separate the oocyte from the bounded secondary cells in a fluid is described herein, e.g., as shown in FIGS. 1B-1D. As described in more detail below, the method may employ an oscillatory flow of a fluid through the fluidic channel, such as a first fluid flow (e.g., a forward flow) from the first port 20, through one or more regions (e.g., regions 100, 200, 300, 400), to the third port 40, e.g., as shown in FIG. 1C, and subsequently followed by a second fluid flow (e.g. a reversed or backward flow) from the third port 40, through the one or more regions, to the first port 20, as shown in FIG. ID.

[0170] The oocyte complex may be contained within a biological fluid introduced into the device. The fluid may include any of a variety of types of fluids, including, but not limited to, follicular fluid, fluid containing ovarian tissue, fluid containing inseminated cumulus oocyte complexes, and / or any other fluid containing other tissues and / or debris in addition to the oocyte. The fluid may also include cell culture media in some embodiments. In some embodiments, an oocyte complex may include an oocyte and one or more types of secondary cells (i.e., non-oocyte cell(s)) bounded to (e.g., surrounding or attached to) the oocyte, including, but not limited to, cumulus cells, granulosa cells, mural cells, thecal cells, red blood cells, or combinations thereof. For example, in the fluid sample, the oocyte may be surrounded by or bounded to one or more layers of secondary entities, such as one or more layers of cumulus cells and / or granulosa cells, to form secondary entity bounded oocyte complex, such as cumulus oocyte complex (COC). The fluid sample may be derived from any of a variety of mammals, including, but not limited to, human, mouse, bovine, porcine, ovine, etc.. The oocyte and / or oocyte complex may have any of a variety of sizes described herein, depending on the type of mammal.

[0171] While the method described herein is predominantly directed to a method of denuding an oocyte complex to produce denuded oocytes (that are either partially or fully denuded), it should be understood that the present disclosure is not so limited, and that in some embodiments, the methods may be used to process a fluid containing already denuded oocytes. For example, in some embodiments, such a method may comprise separating already denuded oocytes from the fluid and / or contaminants (e.g., tissues, debris, etc.) contained within the fluid via oscillatory flow. For example, oscillatory flow (e.g., as shown in FIGS. 1B-1C) may be induced either in the filter region (e.g., region 100) and / or denuder region (e.g., region 200) to break down larger contaminants in the fluid to smaller sizes, as described elsewhere herein. In some embodiments, the smaller sized contaminants may be removed from the device (e.g., via flows 51, 61, 71 as shown in FIG. IB) and the already denuded oocytes in the fluid may be captured in the capture region (e.g., region 400 as shown in FIG. ID) and subsequently released from the device (e.g., via flow 41 as shown in FIG. ID) for future use. In some embodiments, the method described herein may allow for (simultaneous) separation of oocyte complex from a fluid and denuding the oocyte complex within a single device. The method, according to some embodiments, comprises flowing a fluid containing the oocyte complex and any impurities and / or debris therein through one or more microfluidic devices described herein, e.g., as shown in FIGS. 1A-1D and / or FIG. 17A- 18, such that the oocyte complex is separated from the fluid (and any impurities and / or debris) and subsequently denuded.

[0172] In some embodiments, the method comprises providing a fluidic channel comprising a denuder region, a capture region, a first port upstream of the denuder region, a second port downstream of the denuder region and upstream of the capture region, and a third port downstream of the capture region. For example, as shown in FIG. IB, the method comprises providing a fluidic channel comprising the denuder region 200, the capture region 400, the first port 20 upstream of the denuder region, the second port 30 downstream of the denuder region and upstream of the capture region, and the third port 40 downstream of the capture region 40.

[0173] In some embodiments, at a first point in time, a fluid is caused to flow from the first port, through the denuder region and the capture region, to the third port. In some embodiments, the fluid may pass via a filter region (when present) prior to being passed through the denuder region and the capture region. Referring again to FIG. IB, at a first point in time, fluid 21 is caused to flow from the first port 20, through the denuder region 200 and the capture region 400, to the third port 40. The fluid may be passed via the filter region 100 downstream of the first port 20 prior to being passed through the denuder region 200 and the capture region 400. The fluid may contain the oocyte complex and one or more nontarget entities (e.g., debris) described elsewhere herein. In some embodiments, a first pressure is exerted by the first pressure source 12 to cause the fluid to flow from the first port 20, through the denuder region 200 and the capture region 400, to the third port 40.

[0174] In some embodiments, as fluid is caused to flow from the first port, the complex may pass through the denuder region and may be trapped by the capture region. As the complex passes through the denuder region, at least some of the bounded non-oocyte cells are removed from the complex. In some cases, at least 1%, at least 5%, at least 10%, at least 20%, and / up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, or up to 90%, up to 95%, up to 99%, or up to 100% of the non-oocyte cells is removed as the oocyte complex flows through the denuder region in the first direction. For example, referring to FIG. IB, under the fluid flow 21 from the first port 12, the complex may pass through the denuder region 200 and may be trapped by the capture region 400. As shown in FIG. 13A (a zoomed view of the denuder region 200), at least a portion of bounded non-oocyte cells 18b may be removed as oocyte complex 18 flows through the denuder region 200 containing the sub-channel(s) 240 under the fluid flow.

[0175] In some embodiments, when a fluid is caused to flow as illustrated in FIG. IB, one or more ports may be actuated to open and / or close during the flow. For example, as shown, during the fluid flow from the first port 20, the second port 30 and third port 40 may be configured to remain in a closed position, such that no target entities (e.g., oocyte complex) can flow out of the fluidic channel via these ports. One or more ports, e.g., port 50, 60, and / or 70 may be actuated to open to release non-target entities (e.g., waste) and / or fluid (e.g., fluid that does not contain target entities) to flow out of the fluidic channel. As noted elsewhere herein, at the first point in time, the plurality of ports may have a first configuration.

[0176] In some embodiments, at a second point in time, the fluid is caused to flow from the third port, through the capture region and the denuder region, to the first port. In embodiments in which a concentrator region is present, the fluid is caused to flow from the third port, through the capture region and the concentrator region, prior to being flowed through the denuder region and to the first port. For example, referring to FIG. 1C, at a second point in time, the fluid is caused to flow via flow 31 from the third port 40, through the capture region 400, the concentrator region 300, and the denuder region 300, to the first port 20. In some embodiments, a second pressure is exerted by the second pressure source 14 to cause the fluid to flow (via flow 31) from the third port 40, through the capture region 400 and the denuder region 200, to the first port 20.

[0177] In some embodiments, as the fluid flows from the third port, the complex trapped in the capture region may be caused to flow out of (e.g., released from) the capture region and passes through the denuder region. In some cases, at least some of the bounded non-oocyte cells may be removed from the complex as the complex flows through the denuder region. In some cases, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, and / up to 60%, up to 70%, up to 80%, up to 90%, or up to 100% of the non-oocyte cells is removed as the oocyte complex flows through the denuder region in the second direction. For example, referring again to FIG. 1C, as the fluid flows from the third port 40 via flow 31, the complex trapped in the capture region may be released from the capture region and pass through the denuder region 200. As shown in FIG. 13B (a zoomed in view of the denuder region 200), at least some of the bounded non-oocyte cells 18b may be removed from the complex 18 as the complex 18 flows through the denuder region 200.

[0178] In some embodiments, depending on the desired application, a predetermined amount of bounded non-oocyte cells may be removed from the complex using the method described herein. For example, in some cases, at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more, and / or up to 99%, up to 99.9%, or 100% of the total amount of non-oocyte cells original present in the complex may be removed from the complex using the method described herein. For example, in embodiments in which partial or full denudation is desired, a relatively high amount of non-oocyte cells originally present in the complex may be removed from the complex, such as between 50% and 100%, between 90% and 100%, etc. In embodiments in which little to no denudation is desired, a relatively small amount of non-oocyte cells may be removed from complex, e.g., greater than 1% and less than 50%, or in some cases, as low as 0.1 % and down to 0%.

[0179] In some embodiments, the level of denudation may be controlled, at least in part, by controlling the number of interactions the complex has with the teeth on walls of opposing posts. The number of interactions may correlate with the number of teeth on each wall and the number of times the complex flows past the teeth during repeated flow (e.g., oscillatory flow).

[0180] In some embodiments, when a fluid is caused to flow as illustrated in FIG. 1C, one or more ports may be actuated to open and / or close during the flow. For example, as shown, during the fluid flow from the third port 40, the second port 30 and one or more additional ports (e.g., port 60 and / or 70) may be configured to remain in a closed position, such that no target entities (e.g., oocyte complex) can flow out of the fluidic channel via these ports. Accordingly, the fluid can flow past these ports, through the concentrator region 300, and into the denuder region 200, the filter region 200, to the first port 20. In some cases, the first port 20 may be connected to a fluid reservoir (not shown), such that the fluid is further flowed out the first port 20 and into the fluid reservoir. In some cases, the fourth port 50 downstream of the capture region 400 may be transition to an open position to allow target entities (e.g., waste) and / or fluid (e.g., fluid that does not contain target entities) to flow out of the fluidic channel. As noted elsewhere herein, at the second point in time, the plurality of ports may have a second configuration. Certain embodiments comprise, at a third point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the oocyte trapped within the capture region may exit through the second port. For example, referring to FIG. ID, at a third point in time, a fluid may be caused to flow (via flow 32) from the third port 40, through the capture region 400, to the second port 30. With flow 32, denuded oocytes trapped within the capture region 400 may be caused to flow out of the capture filter 400, and exit through the second port 30. In some embodiments, the second port 30 may be actuated to open during flow 32, such that fluid connectivity is established between the capture region and an oocyte collection reservoir (not shown) and fluid connectivity is blocked between the capture region and upstream regions (e.g., concentrator region, denuder region, etc.). One or more ports, e.g., port 50, may be actuated to open during the flow 32 such that waste (e.g., waste fluid from flow 32) may be caused to flow out of the channel. In some embodiments, the fluid flow 32 may be caused by a pressure exerted by the second pressure source 14. As noted elsewhere herein, at the third point in time, the plurality of ports may have a third configuration.

[0181] In some embodiments, the method further comprises, between the second point in time and the third point in time, repeatedly causing a fluid to flow between various ports of Fig 17A-D, therefore oscillating the fluid flow at selected regions of the device to enable efficient washing and / or denudation of the captured entities. For example, as shown in FIG. IB, such that the complex passes through the denuder region 200 and is trapped by the capture region 400, and then causing a fluid to flow from the third port 40 to the first port 20, as shown in FIG. 1C, such that the trapped complex is caused to flow and pass through the denuder region 200, in an alternating (and periodic) fashion. As noted elsewhere herein, during repeated flows, the plurality of ports may be configured to transition from one configuration to another, e.g., from a first configuration to a second configuration, and vice versa.

[0182] In some embodiments, after causing repeated fluid flows (e.g., first flow and second flows) across various regions of the device (e.g., filter and / or denuder regions), a steady flow of fluid (e.g., washing fluid) may be applied for a period of time in either the first direction or second direction through the device to purge any non-target entities, e.g., debris and / or byproducts (e.g., non-oocyte cells denuded from the complex generated during the repeated flow) out of the device, e.g., as shown in FIG. IB. For example, as shown in FIG. IB, wash solutions may be flowed into the device via flow 21 to purge non-target entities (e.g., debris) and / byproducts from the device via ports 61, 71, and / or 51. Any of a variety of buffer solutions may be used as the washing solution. By alternating between repeated flow (e.g., oscillatory flow) and steady flows of fluids, the method may allow for efficient removal of non-target entities from the chip while allowing for more efficient denudation of the target entities.

[0183] In some embodiments, the repeated flow (e.g., oscillatory flow comprising first and second flows) may be caried out a first period of time, followed by a steady flow in a first or a second direction for a second period of time. For example, the repeated flow (e.g., oscillatory flow) may be carried out for a first period of time of at least 0.01 s, at least 0.1 s, at least 10 s, at least 15 s, at least 60 s, at least 100 s, and / or up to 200 s, up to 400 s, up to 600 s, up to 1000 s, up to 1500 s, up to 2000 s, up to 2500 s, up to 3000 s, up to 3600 s, or more. The steady flow, according to some embodiments, may be carried out for a second period of time of at least 0.01 s, at least 0.1 s, at least 10 s, at least 15 s, at least 60 s, at least 100 s, and / or up to 200 s, up to 400 s, up to 600 s, up to 1000 s, up to 1500 s, up to 2000 s, up to 2500 s, up to 3000 s, up to 3600 s, or more.

[0184] The above-referenced process (e.g., first flow, second flow, optionally followed by a purging / washing step) may be repeated for any number of times, such as at least 1 round, at least 2 rounds, at least 3 rounds, at least 5 rounds, at least 10 rounds, at least 20 rounds, at least 30 rounds, at least 50 rounds, at least 100 rounds, and / or up to 200 rounds, up to 500 rounds, up to 1000 rounds, or more, of a flow from the first port to the third (i.e., a first flow) and a flow from the third port to the first port (i.e., a second flow). Combinations of the above-referenced ranges are possible (e.g., at least 1 round and up to 1000 rounds, at least 5 rounds and up to 1000 rounds). Other ranges are also possible.

[0185] The repeated causing step may be performed until a predetermined amount of denudation is achieved, according to some embodiments. For example, depending on the desired application, the predetermined denudation level may range from 0% to 100%. In some embodiments in which partial or full denudation of the oocyte complex is desired, the repeated causing step is performed until the oocyte complex is denuded to a relatively high denudation level. For example, in some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or more, and / or up to 95%, up to 97%, up to 99%, up to 99.5%, up to 99.9% of the bounded non-oocyte cells in the complex are removed during repeated flow of the complex through the fluidic channel (e.g., the denuder region) described herein. In some embodiments in which little or no denudation of the oocyte complex is desired, a relative low denudation level may be achieved using the method / device described herein, such as low as 1%, as low as 0.5%, or as low as 0%. The above-referenced process, e.g., the step of repeating a first flow (e.g., a flow 13 from the first port to the third port as shown in FIG. IB, or flow 13a, 13b, or 13c as shown in FIGS. 17A, 17C, and 17D) and a second flow (e.g., a flow 15 from the third port to the first port as shown in FIG. 1C or flow 15a, 15b, or 15c as shown in FIG. 17B, 17E, and 17E), followed optionally by a steady flow washing step, may be carried out at any of a variety of appropriate frequencies. In some embodiments, the repeating step (and optionally followed by a washing step) may be carried out at a rate of at least 0.1 rounds of flow per minute, at least 1 round of flow per minute, at least 2 rounds of flow per minute, at least 4 rounds of flow per minute, at least 6 rounds of flow per minute, at least 8 rounds of flow per minute, at least 10 rounds of flow per minute, at least 20 rounds of flow per minute, at least 40 rounds of flow per minute, at least 60 rounds of flow per minute, at least 100 rounds of flow per minute, at least 500 rounds of flow per minute, at least 1,000 rounds of flow per minute, at least 5,000 rounds of flow per minute, or more, and / or no more than 10,000 rounds of flow per minute, no more than 5,000 rounds of flow per minute, no more than 1,000 rounds of flow per minute, no more than 500 rounds of flow per minute, no more than 100 rounds of flow per minute, no more than 60 rounds of flow per minute, no more than 40 rounds of flow per minute, no more than 20 rounds of flow per minute, no more than 10 rounds of flow per minute, no more than 8 rounds of flow per minute, no more than 6 rounds of flow per minute, no more than 4 rounds of flow per minute, no more than 2 rounds of flow per minute, no more than 1 round of flow per minute, or less, where one round of flow corresponds to a first flow in the first direction followed by a second flow in the second direction. Combinations of the above-referenced ranges are possible (e.g., at least 4 rounds of flow per minute and no more than 60 rounds of flow per minute, or at least 0.1 rounds of flow per minute and no more than 10,000 rounds of flow per minute). Other ranges are also possible.

[0186] The first flow and / or second flow may have any of a variety of appropriate mean flow velocities. For example, in some embodiments, the first flow and / or second flow has a mean flow velocity of at least 0.01 cm / s, at least 0.1 cm / s, at least 1 cm / s, at least 5 cm / s, at least 10 cm / s, at least 12 cm / s, at least 15 cm / s, at least 20 cm / s, at least 24 cm / s, at least 30 cm / s, at least 50 cm / s, at least 100 cm / s, at least 500 cm / s, at least 1,000 cm / s, at least 5,000 cm / s, or more, and / or no more than 10,000 cm / s, no more than 5,000 cm / s, no more than 1,000 cm / s, no more than 500 cm / s, no more than 100 cm / s, no more than 50 cm / s, no more than 30 cm / s, no more than 24 cm / s, no more than 20 cm / s, no more than 15 cm / s, no more than 12 cm / s, no more than 10 cm / s, no more than 5 cm / s, no more than 1 cm / s, no more than 0.1 cm / s, or less. Combinations of the above-referenced ranges are possible (e.g., between 12 cm / s and 24 cm / s, between 0.01 cm / s and 50 cm / s, or between 0.01 cm / s and 10,000 cm / s). Other ranges are also possible. The first flow and the second flow may have mean flow velocities that are same or different, according to some embodiments.

[0187] The first flow and / or second flow may have any of a variety of appropriate flow rates. For example, in some embodiments, the first flow and / or second flow has a flow rate of at least 0.01 mL / min, at least 0.1 mL / min, at least 1 mL / min, at least 2 mL / min, at least 5 mL / min, at least 10 mL / min, at least 15 mL / min, at least 50 mL / min, at least 100 mL / min, at least 500 mL / min, at least 1,000 mL / min, at least 5,000 mL / min, or more, and / or no more than 10,000 mL / min, no more than 5,000 mL / min, no more than 1,000 mL / min, no more than 500 mL / min, no more than 100 mL / min, no more than 50 mL / min, no more than 15 mL / min, no more than 10 mL / min, no more than 5 mL / min, no more than 2 mL / min, no more than 1 mL / min, no more than 0.1 mL / min, or less. Combinations of the above-referenced ranges are possible (e.g., between 0.01 mL / min and 1,000 mL / min, between 1 mL / min and 100 mL / min, between 2 mL / min and 1,000 mL / min, or between 5 mL / min and 15 mL / min). Other ranges are also possible. The first flow and the second flow may have flow rates that are same or different, according to some embodiments.

[0188] The first flow and / or second flow may have any of a variety of appropriate duty cycles. The duty cycle may be measured as a percentage of time the fluid flows in one direction (e.g., first flow in a first direction or second flow in a second direction) during one round of oscillatory flow (e.g., one round of first flow and second flow). For example, a first flow in a first direction may have a duty cycle of at least 0.1%, at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 70%, at least 80%, at least 90%, and / or up to 95%, up to 97%, up to 99%, up to 99.5%, up to 99.9%. Combinations of the abovereferenced ranges are possible (e.g., at least 0.1% and up to 99.9%, or at least 70% and up to 99.9%). Other ranges are also possible. To illustrate this, as a non-limiting example, a first flow in a first direction having duty cycle of 70% may correspond to a first flow moving in a first direction for 70% of the time, and a second flow moving in a second direction for 30% of the time, during one round of oscillatory flow.

[0189] In some embodiments, prior to the first time point, the method further comprises selectively isolating one or more oocyte complex from the fluid in the filter region, prior to flowing the complex through the denuder region, the capture region, to the third port. For example, as shown in FIG. 1A or FIG. 17A, one of more complex from the fluid may be selectively retained in (e.g., extracted by) the filter region 100 by sub-channels 110 having sizes comparable and / or slightly smaller than the complex. In some cases, the isolated complex may be caused to flow through the filter region 100 via an induced oscillatory flow, e.g., as shown in FIG. 1B-1C, where a first pressure is exerted to drive flow 21 followed by a second pressure to drive flow 31. Such an oscillatory flow may exert sufficient shear stress against the complex such that the complex may be caused to flow through the sub-channels in the filter region 100, to the denuder region 100.

[0190] Alternatively or additionally, an enzymatic solution may be flowed via the flow 21 for a period of time, such that at least a portion of the non-oocyte cells around the complex is removed, thereby allowing the complex to move through the sub-channels in the filter region 100. According to some embodiments, the enzymatic solution comprises an enzyme capable of selectively dissociating and / or separating the non-oocyte cells from each other, without negatively impacting the target oocytes. In some embodiments, the use of an enzymatic solution may be localized to certain regions (e.g., filter region) of the device, such that the complex is exposed to the enzymatic solution while only residing within certain regions of the device. It should be understood that while certain embodiments comprise employing an enzymatic solution to chemically weaken and / or remove bounded non-oocyte cells, not all embodiments are so limiting, and that in some embodiments, an enzymatic solution is not employed.

[0191] In some embodiments, the fluid sample introduced into the fluidic channel may contain additional non-target entities and / or impurities suspended in the fluid, in addition to the one or more oocyte complex. The additional non-target entities and / or impurities may comprise blood clots, debris, aggregated blood cells, individual blood cells (e.g., red blood cells, platelets), and / or inorganic impurities (e.g., impurities from follicular fluid collection process). Alternatively or additionally, the non-target entities and / or impurities my include individual or aggregated non-oocyte cells (e.g., cumulus cells, granulosa cells, etc.) that are not part of the oocyte complex, e.g., that is, non-oocyte cells that are not bounded to the oocyte and are instead separately suspended in the fluid sample. These addition non-target entities and / or impurities may, in some cases, be trapped within various regions of the device, e.g., such as the filter region and / or denuder region and in turn lead to clogging of the chip. Accordingly, some methods described herein may comprise washing and / or removing the non-target entities and / or impurities from the microfluidic device via oscillatory flow.

[0192] In one set of embodiments, the method further comprises isolating one or more non- target entities in the filter region and / or causing oscillatory flow within the filter region such that the non-target entities dissociate into smaller sizes. For example, one or more non-target entities may be isolated by filter region 100 due to their large sizes, as shown in FIG. 1A. In some cases, under an applied oscillatory flow, e.g., as shown in FIGS. 1B-1C, the isolated one or more non-target entities may be caused to shear against the plurality of posts and / or sub-channels within the filter region 100 such that the non-target entities dissociate into smaller sizes. The dissociated non-target entities may be readily removed / washed from the filter region via one or more additional ports in the device, e.g., ports 50, 60, and / or 70. The use of an applied oscillatory flow, according to some embodiments, may advantageously facilitate re-orientation of the non-target entities (e.g., debris) by causing flow of the non- target entities in different directions. This may prevent the non-target entities from being stuck or clogged in the device in a particular orientation and thus lead to more efficient non- target entity removal (e.g., debris removal).

[0193] In some embodiments, during the repeated flows described herein, e.g., FIGS. IB- ID, one or more fluids may be employed to assist denudation of the oocyte complex and / or dissociation of larger impurities and / or debris within the fluidic channel. Such fluids may include one or more enzymatic solutions and / or buffer solutions. In some embodiments, one or more chemicals and / or enzymatic solutions may be flowed through the fluidic channel to facilitate denudation of the oocytes. In some cases, the one or more chemical and / or biological solutions may include an enzyme capable of removing one or more non-oocyte cells bounded to an oocyte. Non-limiting examples of chemicals and / or biological include hyaluronidase, cumulase, and / or pronase. The one or more chemical and / or enzymatic solutions may be flowed into the device via the one or more inlets before, during, and / or after the oscillatory flow steps, according to some embodiments.

[0194] In some embodiments, the method further comprises reducing a volumetric flow rate of the fluid from the denuder region by passing the fluid into a concentrator region adjacent the denuder region, e.g., as shown in FIG. IB. For example, as shown, as the flow 21 passes from the denuder region 200 to the concentrator region 300, a portion of the fluid is removed via the sub-channels 320 via ports 60 and 70, thereby resulting in a reduction in the volumetric flow rate of the flow passing through the concentrator 300 into the capture region 400. The removed fluid contains debris and / or removed non-oocyte cells, according to some embodiments.

[0195] In some embodiments, methods of controlling an oocyte complex comprising an oocyte and non-oocyte cells are provided.

[0196] In some embodiments, the methods comprise causing a flow of a fluid containing the complex through a fluidic channel. For example, certain embodiments comprise causing a first flow of a fluid containing the complex in a first direction through a fluidic channel. For example, as shown in FIG. 1A, the method comprises causing a first flow of a fluid 21 containing the complex in a first direction 13 through the fluidic channel (e.g., filter region 100, denuder region 200, concentrator region 300, etc.). The complex may be passed through regions of the fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, such as the denuder region 200. The sub-channels may have any of a variety of minimum cross-sectional dimensions described herein, such as at least one of the sub-channels having a minimum cross-sectional dimension of between 120 micrometers and 180 micrometers.

[0197] Certain embodiments further comprise removing at least some of the non-oocyte cells from the complex as the oocyte flows through the fluidic channel in the first direction. As the fluid containing the oocyte complex is caused to flow through the fluidic channel in the first direction, according to some embodiments, at least a portion of the bounded non-oocyte cells is removed or separated from the complex as the complex flows through the fluidic channel in the first direction. In some cases, at least 1%, at least 5%, at least 10%, at least 20%, and / up to 30%, up to 40%, or up to 50% of the bounded non-oocyte cells is removed as the oocyte complex flows through the fluidic channel in the first direction. For example, referring to FIG. 13 A, at least a portion of the bounded non-oocyte cells 18b is removed as the oocyte complex 18 flows through the fluidic channel (e.g., denuder region 200) containing the sub-channel(s) (e.g., 230) in the first direction 13.

[0198] In some embodiments, with the first flow is caused through the fluidic channel, the complex may be trapped or immobilized in an exit region of the fluidic channel, e.g., by the sub-channels in the capture region 400 shown in FIG. IB. In some embodiments, the method comprises trapping the oocyte complex (or other entities) subjected to the first flow and / or second flow in a capture region comprising a plurality of posts defining a plurality of subchannels between neighboring pairs of posts. For example, in one set of embodiments, when the oocyte complex is caused to flow through the denuder region containing the subchannels) via a first flow in a first direction, the oocyte complex may become partially denuded and the partially denuded oocyte complex may be subsequently trapped by the subchannels in the capture region.

[0199] In some embodiments, the method further comprises flowing the fluid containing the trapped complex in a second direction opposed to the first direction in the fluidic channel. Upon trapping the partially denuded oocyte complexes, the trapped oocyte complexes may be caused to flow via a second flow in a second direction, such that the trapped oocyte complexes flow through the denuder region containing the sub-channel(s). The trapped oocyte complex, according to some embodiments, may be released from the exit region via a pressure applied by a pressure source, and may be passed through a plurality of posts in the denuder region.

[0200] For example, as shown in FIG. 1C, a fluid containing the trapped complex may be caused to flow in a second direction 15, via the application of a pressure exerted by the pressure source 14. The second direction 15 (as shown in FIG. 1C) may be opposed to the first direction 13 (as shown in FIG. IB). While the first direction may correspond to a flow direction from the first port 20 to the third port 40, via one or more intervening regions (e.g., regions 100-400), the second direction may correspond to an opposed flow direction, such as from the third port 40 to the first port 20, via the one or more intervening regions (e.g., regions 400-100). The trapped oocyte complex, according to some embodiments, may be released from the capture filter 400 in the exit region via a pressure applied by the second pressure source 14, and may be passed through a plurality of posts in the denuder region 200.

[0201] In some embodiments, the second direction may be substantially opposite the first direction. For example, a flow having the second direction may be oriented in a direction that is greater than 90 degrees, greater than or equal to 120 degrees, greater than or equal to 140 degrees, greater than or equal to 160 degrees, greater than or equal to 180 degrees, greater than or equal to 200 degrees, greater than or equal to 220 degrees, greater than or equal to 240 degrees, and / or less than 270 degrees, less than or equal to 240 degrees, less than or equal to 220 degrees, less than or equal to 200 degrees, less than or equal to 180 degrees, less than or equal to 160 degrees, less than or equal to 140 degrees, or less than or equal to 120 degrees from the first direction of the first fluid in the clockwise direction. Combinations of the above-referenced ranges are possible (e.g., greater than 90 degrees and less than 270 degrees). In some embodiments, the second direction may be directly opposite the first direction, e.g., oriented about 180 degrees clockwise from the first direction.

[0202] The first pressure and / or second pressure exerted by the pressure sources may have of a variety of values. For example, in some embodiments, the first pressure and / or second pressure may have a value of at least at least 0.01 psi, at least 0.05 psi, at least 0.1 psi, at least 0.5 psi, at least 1 psi, at least 2 psi, at least 5 psi, at least 10 psi, at least 20 psi, at least 50 psi and / or up to 100 psi, up to 200 psi, up to 300 psi, up to 400 psi, up to 500 psi, or more. Combinations of the above-referenced ranges are possible (e.g., at least 0.5 psi and up to 500 psi, at least 1 psi and up to 300 psi). Other ranges are also possible.

[0203] Certain embodiments comprises repeatedly causing flow through the various regions of fluidic channel, e.g., such as repeatedly causing the oscillatory flow described herein (as shown in FIGS. 1B-1C). The oscillatory flow described herein may facilitate denudation of a target complex (e.g., oocyte complex) and / or dissociation of impurities and / or non-target entities within the fluid. Upon repeated flows, the complex may be repeatedly trapped in an exit region, e.g., the capture region 400 as shown in FIG. IB. In some embodiments, the trapped complex may be repeatedly released from the exit region of the fluidic channel, such as from the capture region 400 via second port 30 via flow 41 shown in FIG. ID.

[0204] The complex may be repeatedly passed through one or more regions of the fluidic channel and trapped for any of a variety of appropriate times, such as at least 1 time, at least 2 times, at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, and / or up to 200 times, up to 500 times, up to 1000 times, or more, of a first and second flow. Combinations of the above-referenced ranges are possible (e.g., at least 1 time and up to 1000 times, at least 5 times and up to 1000 times). Other ranges are also possible.

[0205] In some embodiments, the method further comprises releasing the denuded oocyte when a measured parameter within the device reaches a threshold value. For example, the measured parameter may be a denudation level of the oocyte within the device, and the denuded oocyte may be released from the device when the denudation level reaches a predetermined threshold value. In some embodiments, when the measured denudation level is less than the predetermined threshold value, a closed-loop controller described elsewhere herein may be employed to subject the oocyte complexes within the fluid channel to additional rounds of repeated flow (e.g., oscillatory flow) for additional denudation. The controller may be operationally linked to one or more components described here (e.g., one or more pressure sources, inlets and / or outlets, fluid controls) to control the flow of fluid and oocyte complexes within the device.

[0206] In some embodiments, upon reaching a predetermined denudation level, the controller may be operated to stop the repeated flow (e.g., oscillatory flow) through the fluidic channel. In some cases, the denuded oocytes may be released from device via one or more outlets. For example, in some embodiments in which the device comprises a first outlet downstream of the capture filter, the denuded oocytes may be caused to flow in the first direction through the plurality of sub-channels in the capture region via an application of a pressure by the first pressure source. In embodiments in which the sub-channels in the capture region are sized to allow denuded oocytes (e.g., bare oocytes without the bound non-oocyte cells) to flow readily through, the denuded oocytes may flow through the capture region and flow out of the first outlet. In some embodiments in which the sub-channels in the capture region are sized slightly smaller than the denuded oocyte, the denuded oocyte may be trapped at the capture filter. In such cases, a pressure may be applied to cause slight deformation in shape and / or size of the denuded oocyte, such that the oocyte may be forced through sub-channels in the capture region. The first outlet may be actuated to open by the controller and / or fluid controls to release the oocyte.

[0207] The repeated flows, according to some embodiments, may be oscillatory or non- oscillatory in nature. As noted elsewhere herein, in some embodiments, the repeated flows may be oscillatory in nature, e.g., such as capable of oscillating between a first flow in a first direction and a second flow in the second direction (e.g., as shown in FIGS. 1B-1D).

[0208] While FIGS. IB- ID are used to illustrate one embodiment of a method described herein, it should be understood that the disclosure is not so limited, and that in certain embodiments, other embodiments may be possible, e.g., as shown in FIGS. 17A-17F. In some cases, FIGS. 17A-17F may be employed for modular fluid control within separate regions of the fluidic channel for processing a fluid containing one or more non-target entities (e.g., debris) and oocyte complex comprising an oocyte and bounded non-oocyte cells, as described in more detail below.

[0209] For example, the method may cause repeated flow of the fluid through the filter region, such that at least a portion of the non-target entities undergoes a reduction in size as the non-target entities flows within the filter region, and separately cause repeated flow of the fluid through the denuder region, such that at least a portion of the non-oocyte cells is removed from the oocyte complex as the complex flows through the denuder region.

[0210] As shown in FIG. 17A, the method comprises providing a fluidic channel 10 comprising a filter region 100, a denuder region 200, a capture region 400, an optional concentration region 300, a first port 20 upstream of the filter region 100, a second port 30 downstream of the denuder region 100 and upstream of the capture region 400, a third port 40 downstream of the capture region 400, and an additional port (e.g., port 80) downstream of the filter region 100 and upstream of the denuder region 100. Various other ports (e.g., port 50, 60, and / or 70) and various pressure sources (e.g., source 12, 14, and 16) may be also present, as described elsewhere herein.

[0211] In some embodiments, at a first point in time, a fluid 21a containing one or more non- target entities (e.g., debris and / or impurities) and oocyte complex is caused to flow from the first port 20, through the filter region 100, to the additional port 80, such that at least a portion of the non-target entities passes through sub-channels within the filter region 100 to the port 80, as shown in FIG. 17A. In some cases, the flow 13a may cause some of the non- target entities to shear against the plurality of posts forming the sub-channels within the filter region 100, such that the non-target entities undergo a reduction in size as the non-target entities pass through (or are forced through) the sub-channels in the filter region. In some cases, as noted elsewhere herein, some of the sub-channels within the filter region may have sizes smaller than the size of the non-target entities. The flow described herein may be caused by a pressure exerted by a first pressure source (e.g., pressure source 12), and the pressure may be sufficient to cause some of the non-target entities to shear through the subchannels, thereby causing a reduction in size. In some embodiments, the plurality of ports may have a first configuration described elsewhere herein.

[0212] In some embodiments, at a second point in time, a fluid 81a may be caused to flow from the additional port 80, through the filter region 100, to the first port 20, such that at least a portion of the non-target entities passes through (or are forced through) the sub-channels within the filter region 100 in a second direction 15a, as shown in FIG. 17B. In some cases, the flow 15a may cause some of the non-target entities to shear against the plurality of posts forming the sub-channels within the filter region 100, such that the non-target entities undergo a reduction in size as the non-target entities pass through (are forced through) the sub-channels in the filter region. The flow 15a described herein may be caused by a pressure exerted by an additional pressure source (e.g., third pressure source 16), and the pressure may be sufficient to cause some of the non-target entities to shear through the sub-channels, thereby experiencing a reduction in size. The fluid 81a may include any of a variety of fluid flows, e.g., a buffer solution and / or an enzymatic solution, as described elsewhere herein. In some embodiments, the plurality of ports may resume a second configuration described elsewhere herein.

[0213] In some embodiments, the above-referenced causing steps at the first point in time and second point in time may be repeated. The repeating step may comprise repeatedly causing a fluid to flow from the first port (e.g., port 20) to the additional port (e.g., port 80), e.g., as shown in FIG. 17A, and from the additional port (e.g., port 80) to the first port (e.g., port 20), e.g., as shown in FIG. 17B, in an alternating fashion, such that the non-target entities are repeatedly caused to undergo a reduction in size as they pass through the filter region. The repeating step may be performed for any appropriate number of times described elsewhere herein. The oscillatory flow (e.g., flow 13a and 15a) may be conducted for any appropriate number of times, durations, duty cycles, ratio of duty cycles, flowrates, described elsewhere herein. Any of a variety of buffer solution and / or enzymatic solution described elsewhere herein may be employed as the fluid described herein. In some embodiments, at a third point in time, a fluid 21c may be caused to flow (by a first pressure source 12) from the first port 20, through the filter region 100 to the denuder region 200, such that the oocyte complex within the fluid passes from the filter region 100 to the denuder region 200, e.g., as shown in FIG. 17C. In some embodiments, the fluid is caused to flow until substantially all (e.g., at least 90%, at least 95%, at least 100%) of the complex has been passed from the filter region to the denuder region. In some embodiments, the plurality of ports may have a third configuration described elsewhere herein. In some embodiments, the flow may cause the reduced non-target entities to flow out of the fluidic channel via one or more ports, e.g., port 50, 60 and / or 70.

[0214] In some embodiments, at a fourth point in time, a fluid 81b is caused to flow (e.g., by the third pressure source 16) from the additional port 80, through the denuder region 200 and the capture region 400, to the third port 40, such that the oocyte complex passes through the denuder region 200 and is trapped by the capture region 400, e.g., as shown in FIG. 17D. In some such cases, at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region, as described elsewhere herein. In some embodiments, the plurality of ports may have a fourth configuration described elsewhere herein.

[0215] In some embodiments, at a fifth point in time, a fluid 31a may be caused (e.g., by a second pressure source 14) to flow from the third port 40, through the capture region 400 and the denuder region 200, to the additional port 80, such that the complex passes through the denuder region 200, e.g., as shown in FIG. 17E. In some cases, at least some of the non- oocyte cells are removed from the complex as the complex flows through the denuder region, as described elsewhere herein. In some embodiments, the plurality of ports may have a fifth configuration described elsewhere herein.

[0216] In some embodiments, the above-referenced causing steps at the fourth point in time and fifth point in time may be repeated. For example, the method may comprise repeatedly causing a fluid to flow from the additional port (e.g., port 80) to the third port (e.g., port 40), e.g., as shown in FIG. 17D, and from the third port (e.g., port 40) to the additional port (e.g., port 80), e.g., as shown in FIG. 17E, in an alternating fashion, such that the complex passes through the denuder region repeatedly. The repeating step may be performed for any appropriate number of times described elsewhere herein. The oscillatory flow (e.g., flow 13c and 15b) may be conducted for any appropriate number of times, durations, duty cycles, ratio of duty cycles, flowrates, described elsewhere herein. Any of a variety of buffer solution and / or enzymatic solution described elsewhere herein may be employed as the fluid described herein.

[0217] In some embodiments, at a sixth point in time, a fluid 31b is caused (e.g., by a second pressure source 14) to flow from the third port 40, through the capture region 400, to the second port 30, such that the oocyte exits through the second port (via arrow 41), e.g., as shown in FIG. 17C.

[0218] In some embodiments, repeated flows (e.g., oscillatory flows) may be separately controlled and induced in each of the filter region and denuder region of the device, e.g., in a modular fashion. FIG. 18 can be used to illustrate a non-limiting example of one such embodiment. As shown, the device in FIG. 18 comprises an addition port (e.g., port 85) compared to the device shown in FIGS. 17A-17F.

[0219] For example, as shown in FIG. 18, at a first point in time, a fluid 21a containing an oocyte complex and optionally one or more non-target entities (e.g., debris and / or impurities) may be caused to flow from the first port 20 and into the filter region 100.

[0220] At a second point in time, a repeated flow (e.g., oscillatory flow) may be induced within the filter region 100 between port 20 and 80, e.g., in a similar fashion as illustrated in FIGS. 17A-17B. During the repeated flow within the filter region, at least some of the nontarget entities may be repeatedly sheared against the plurality of posts forming the subchannels within the filter region 100, such that the non-target entities undergo a reduction in size as the non-target entities pass through (or are forced through) the sub-channels in the filter region. The oscillatory flow may be conducted for any appropriate number of times, durations, duty cycles, ratio of duty cycles, flowrates, described elsewhere herein.

[0221] In some embodiments, at a third point in time, the non-target entities and oocyte complex in the filter region 100 may be caused to flow (by a first pressure source 12) from the first port 20, through the filter region 100 to the denuder region 200, such that the oocyte complex within the fluid passes from the filter region 100 to the denuder region 200 until reaching port 85, e.g., as shown in FIG. 18. In some embodiments, the fluid is caused to flow until substantially all (e.g., at least 90%, at least 95%, at least 100%) of the complex has been passed from the filter region to the denuder region.

[0222] In some embodiments, at a fourth point in time, a repeated flow (e.g., oscillatory flow) may be induced within the denuder region 200 (e.g., between port 80 and port 85) with the application of pressure from one or more pressure sources (e.g., pressure sources 16 and 17), e.g., as shown in FIG. 18. At least some of the non-oocyte cells are removed from the complex as the complex repeatedly flows through the denuder region 200 between port 80 and 85, according to some embodiments. During the repeated flow within the denuder region, both port 80 and port 85 may be in positions that prevent communication with the other regions (e.g., filter region, concentrator region, capture regions) of the device. The oscillatory flow within the denuder region may be conducted for any appropriate number of times, durations, duty cycles, ratio of duty cycles, flowrates, described elsewhere herein.

[0223] In some embodiments, at a fifth point in time, a flow may be induced to flow denuded complex out of the denuder region 200 and into capture region 400, e.g., in a similar fashion as shown in FIG. 17D. During this period of time, the port 85 (as shown in FIG. 18) between the denuder region 200 and the concentrator region 300 may be in an open position to allow the denuded oocyte to exit denuder region. As the denuded oocyte enters into the concentrator region, the denuded complex is trapped in the capture region, according to some embodiments.

[0224] In some embodiments, at a sixth point in time, the trapped oocyte in the capture region 400 may be released from the capture region via port 30 and subsequently collected, e.g., in a similar fashion in as illustrated in FIG. 17F.

[0225] In some embodiments, the steps recited at the first point in time, second point in time, third point in time, fourth point in time, fifth point in time, and / or sixth point in time are performed simultaneously.

[0226] In some embodiments, fluid flow through the filter region, denuder region, inertial focusing region, concentrator region, and / or capture region is differentially regulated. In some embodiments, flow can be incoming and outgoing from any one of the ports shown in FIG. 18. For example, by adjusting the positions of the ports, flow can be alternated or oscillated between any of the following combinations of ports, such as between port 20 and 80, between port 20 and 85, between port 20 and 40, between port 80 and 85, and / or between port 80 and 40, in some embodiments.

[0227] In some embodiments, the repeated flows through the fluidic channel may be non- oscillatory in nature. For example, in some cases, at least some of the repeated flows through the channel may be in the same direction, e.g., either in a first direction or a second direction. In some embodiments, the first direction may be a clockwise direction and the second direction may be counterclockwise direction.

[0228] FIG. 16 can be used to illustrate one such embodiment. For example, as shown in FIG. 16, a microfluidic device 500 may comprise a fluidic channel 600 fluidically connecting the first port 20 with a region upstream the capture filter 400. The fluidic channel 600 in FIG. 16 may allow for repeated flow of the oocyte in the first direction 13 through the device. For example, a fluid containing an oocyte complex comprising an oocyte and non-oocyte cells may be repeatedly flowed in the same direction (e.g., clockwise or counterclockwise) through the filter region, the denuder region, the concentrator region, such that the complex is denuded and the oocyte separates from the non-oocyte cells.

[0229] In addition, in accordance with certain embodiments, it has been found that more efficient enrichment of oocytes can be achieved, compared to manual techniques. In some cases, a variety of oocytes have been determined, e.g., in various stages of maturation (e.g., in the GV stage (Germinal Vesicle), and / or the Ml (Metaphase I) and M2 (Metaphase II) stages). In contrast, manual techniques are usually unable to find and isolate immature oocytes, as they usually rely on optical visibility of COCs in the follicular fluid, and thus COCs that are not large or clearly visible may be missed during manual inspection. Human error or variability can thus be a source of errors in such manual techniques. However, in accordance with certain embodiments, cells such as oocytes can be determined based on dimensions of the oocyte, which are relatively uniform, rather than on human perception or abilities.

[0230] While the above-referenced embodiments are generally directed to methods of controlling and / or denuding an oocyte complex comprising an oocyte and non-oocyte cells, it should be understood that the disclosure is not so limited, and that in other embodiments, the methods may be generalized to controlling and / or denuding any target entity- secondary entity complex other than the oocyte complex. For example, any of the above-referenced methods and / or devices may be employed to control and / or denude target entity-secondary entity complex comprising a target entity and one or more secondary entities to separate the target entity from the secondary entities. Alternatively The target entity may include any of a variety of cell types, including, but not limited to, tumor cells, bacterial cells, etc.

[0231] In some embodiments, the above-referenced methods or devices may be employed to dissociate conglomerates or aggregates of target cells into individual cells for use in different applications, such as in cell sorting, single-cell sequencing, etc. For example, in some embodiments, the devices and / or methods described herein may be used to process a fluid containing tumor tissue or circulating tumor cell clusters (CTCCs). The fluid may be passed into a filter region (e.g., region 100 in device 10 as shown in FIGS. 1A, 17A, or 18), where the CTCCs or tissue would be captured in microfilter by the virtue of its size being larger than individual cells (or by targeting cancer-specific antibodies). In some embodiments, under an application of a repeated or oscillatory flow (e.g., as shown in FIGS. 1B-1C, FIGS. 17A-17E) and / or enzyme treatment, cell-cell interactions may be weakened and the tissue or the CTCC cluster may dissociate into individual cells in the filter region and / or denuder region. The individual cells may be in turn captured by the capture region and released from the device for further use, according to some embodiments.

[0232] In some embodiments, one or more of the devices described herein (e.g., FIGS. 1A- 18) may be coupled to and / or integrated to devices for vitrifying and freezing oocytes. For example, the denuded oocytes released from one or more of the devices may be either manually vitrified / frozen or flowed to devices for automatically vitrifying and freezing oocytes. The vitrified oocytes may be stored for future applications.

[0233] In some embodiments, the one or more devices described herein may be defined using any suitable form factor. In some embodiments, the one or more devices is defined by a rectangular form factor, for example, as shown in FIG. 19. In other embodiments, however, the one or more devices is defined by a circular form factor, for example, as shown in FIG. 20. In some embodiments, the circular form factor comprise one or more holes. In some embodiments, the one or more holes are concentric about the center of the device. Other form factors are also possible in other embodiments.

[0234] In some embodiments, the one or more devices described herein comprise a wall angle of between 2 degrees and 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device. In some embodiments, the wall angle is greater than or equal to 1 degree, greater than or equal to 2 degrees, greater than or equal to 3 degrees, greater than or equal to 4 degrees, greater than or equal to 5 degrees, greater than or equal to 6 degrees, greater than or equal to 7 degrees, greater than or equal to 8 degrees, greater than or equal to 9 degrees, or greater than or equal to 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device. In some embodiments, the wall angle is less than or equal to 10 degrees, less than or equal to 9 degrees, less than or equal to 8 degrees, less than or equal to 7 degrees, less than or equal to 6 degrees, less than or equal to 5 degrees, less than or equal to 4 degrees, less than or equal to 3 degrees, less than or equal to 2 degrees, or less than or equal to 1 degree, relative to an axis that is perpendicular to a bottom surface of the microfluidic device. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the wall angle is greater than or equal to 1 degree and less than or equal to 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device. In some embodiments, the wall angle is 3 degrees relative to an axis that is perpendicular to a bottom surface of the microfluidic device. In other embodiments, the wall angle is 5 degrees relative to an axis that is perpendicular to a bottom surface of the microfluidic device. In some embodiments, a device may comprise one or more regions (e.g., filter regions, denuder regions, inertial focusing regions, concentrator regions, and / or capture regions, etc.) with one or more wall angles, according to some embodiments. In some embodiments, the one or more wall angles of the one or more regions is the same. In other embodiments, the one or more wall angles of the one or more regions is different. In some embodiments, the one or more wall angles of the one or more regions may be selected from any one of the above recited ranges and / or combinations thereof. As a non-limiting example, in some cases, a filter region has a 3 degree wall angle and a denuder region has a 5 degree wall angle. In another non-limiting example, the filter region and denuder region both have a 3 degree wall angle or a 3 degree wall angle.

[0235] The skilled artisan will understand that devices comprising adjacent sidewalls having non-zero wall angles may define a channel with a trapezoidal-like shape, wherein a first portion of the trapezoidal-like channel will have a larger cross-sectional dimension than the second portion of the trapezoidal-like channel. Other shapes are also possible in some embodiments, e.g., parallelogram-like shapes. Similarly, devices comprising a plurality of posts (e.g., filter region, denuder region, and capture region) having non-zero wall angles may in some embodiments define a plurality of trapezoidal-like sub-channels between neighboring pairs of posts (see, as examples, FIG. 21A and 21B). In some embodiments, a cross-sectional dimension of a first portion of a trapezoidal-like sub-channel is larger than the cross-sectional dimension of a second portion of the trapezoidal-like sub-channel. In some embodiments, the cross-sectional dimension of the first portion of the trapezoidal-like subchannel is smaller than the cross-sectional dimension of the second portion of the trapezoidal- like sub-channel.

[0236] FIG. 21A illustrates exemplary wall dimensions for a 3-degree wall angle and FIG. 2 IB illustrates exemplary wall dimensions for a 5 -degree wall angle, as non-limiting examples. However, wall angles may vary in other embodiments, e.g., from about 1 degree to about 10 degrees, etc. FIG. 21C shows a scanning electron micrograph of a denudation region with a X degree wall angle.

[0237] In some embodiments, a plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) has an average cross-sectional dimension of between 10 microns and 200 microns. In some embodiments, the average cross-sectional dimension of the plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) 10 microns and 200 microns. In some embodiments, the average cross-sectional dimension of the plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) is greater than or equal to 10 microns, greater than or equal to 25 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns, greater than or equal to 125 microns, greater than or equal to 150 microns, greater than or equal to 175 microns, or greater than or equal to 200 microns,. In some embodiments, the average cross-sectional dimension of the trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) is less than or equal to 200 microns, less than or equal to 175 microns, less than or equal to 150 microns, less than or equal to 125 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 25 microns, or less than or equal to 10 microns. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the average cross-sectional dimension of the trapezoidal-like sub-channels is greater than or equal to 10 micron and less than or equal to 200 microns.

[0238] In some embodiments, a plurality of trapezoidal-like sub-channels in a filter region, a denuder region, and / or a capture region (e.g., formed by neighboring pairs of posts having a non-zero wall angle) has an average cross-sectional dimension of between 10 microns and 200 microns. In some embodiments, the average cross-sectional dimension of the plurality of trapezoidal-like sub-channels in the filter region, a denuder region, and / or a capture region (e.g., formed by neighboring pairs of posts having a non-zero wall angle) is greater than or equal to 10 microns, greater than or equal to 25 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns, greater than or equal to 125 microns, greater than or equal to 150 microns, greater than or equal to 175 microns, or greater than or equal to 200 microns,. In some embodiments, the average cross- sectional dimension of the trapezoidal-like sub-channels in the filter region, a denuder region, and / or a capture region (e.g., formed by neighboring pairs of posts having a non-zero wall angle) is less than or equal to 200 microns, less than or equal to 175 microns, less than or equal to 150 microns, less than or equal to 125 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 25 microns, or less than or equal to 10 microns. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the average cross- sectional dimension of the trapezoidal-like sub-channels in the filter region, denuder region, and / or a capture region is greater than or equal to 10 microns and less than or equal to 200 microns. In some embodiments, an average cross-sectional dimension of a plurality of subchannels in a filter region is between 10 microns and 200 microns and the average cross- sectional dimension of a plurality of sub-channels in a denuder region is between 10 microns and 200 microns.

[0239] In some embodiments, an average cross-sectional dimension of a plurality of subchannels in a filter region is between 10 microns and 200 microns and the average cross- sectional dimension of a plurality of sub-channels in a capture region is between 10 microns and 200 microns.

[0240] In some embodiments, an average cross-sectional dimension of a plurality of subchannels in a denuder region is between 10 microns and 200 microns and the average cross- sectional dimension of the sub-channels in a capture region is between 10 microns and 200 microns.

[0241] In some embodiments, an average cross-sectional dimension of a plurality of subchannels in a filter region is between 10 microns and 200 microns, the average cross- sectional dimension of a plurality of sub-channels in a denuder region is between 10 microns and 200 microns, and the average cross-sectional dimension of a plurality of sub-channels in the capture region is between 10 microns and 200 microns.

[0242] In some embodiments, a plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) has an average height of between 100 microns and 400 microns. In some embodiments, the average height of the plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) is greater than or equal to 100 microns, greater than or equal to 130 microns, greater than or equal to 160 microns, greater than or equal to 190 microns, greater than or equal to 220 microns, greater than or equal to 250 microns, greater than or equal to 280 microns, greater than or equal to 310 microns, greater than or equal to 340 microns, greater than or equal to 370 microns, or greater than or equal to 400 microns. In some embodiments, the average height of the plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) is less than or equal to 400 microns, less than or equal to 370 microns, less than or equal to 340 microns, less than or equal to 310 microns, less than or equal to 280 microns, less than or equal to 250 microns, less than or equal to 220 microns, less than or equal to 190 microns, less than or equal to 160 microns, less than or equal to 130 microns, or less than or equal to 100 microns. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the average cross-sectional dimension of the trapezoidal-like sub-channel is greater than or equal to 100 micron and less than or equal to 400 microns.

[0243] In some embodiments, a plurality of trapezoidal-like sub-channels in a filter region, a denuder region, and / or a capture region (e.g., formed by neighboring pairs of posts having a non-zero wall angle) has an average height of between 100 microns and 400 microns. In some embodiments, the average height of the plurality of trapezoidal-like sub-channels in the filter region, denuder region, and / or capture region is greater than or equal to 100 microns, greater than or equal to 130 microns, greater than or equal to 160 microns, greater than or equal to 190 microns, greater than or equal to 220 microns, greater than or equal to 250 microns, greater than or equal to 280 microns, greater than or equal to 310 microns, greater than or equal to 340 microns, greater than or equal to 370 microns, or greater than or equal to 400 microns. In some embodiments, the average height of the plurality of trapezoidal-like sub-channels in the filter region, denuder region, and / or capture region is less than or equal to 400 microns, less than or equal to 370 microns, less than or equal to 340 microns, less than or equal to 310 microns, less than or equal to 280 microns, less than or equal to 250 microns, less than or equal to 220 microns, less than or equal to 190 microns, less than or equal to 160 microns, less than or equal to 130 microns, or less than or equal to 100 microns. Combinations of the above recited ranges are also possible in some embodiments. For example, in some embodiments, the average cross-sectional dimension of the plurality of trapezoidal-like sub-channels in the filter region, denuder region, and / or capture region is greater than or equal to 100 micron and less than or equal to 400 microns.

[0244] In some embodiments, an average height of a plurality of sub-channels in a filter region is between 100 microns and 400 microns and the average height of a plurality of subchannels in a denuder region is between 100 microns and 400 microns.

[0245] In some embodiments, an average height of a plurality of sub-channels in a filter region is between 100 microns and 400 microns and the average height of a plurality of subchannels in a capture region is between 100 microns and 400 microns.

[0246] In some embodiments, an average height of a plurality of sub-channels in a denuder region is between 100 microns and 400 microns and the average height of the sub-channels in a capture region is between 100 microns and 400 microns.

[0247] In some embodiments, an average height of a plurality of sub-channels in a filter region is between 100 microns and 400 microns, the average height of a plurality of subchannels in a denuder region is between 100 microns and 400 microns, and the average height of a plurality of sub-channels in the capture region is between 100 microns and 400 microns.

[0248] Without wishing to be bound by any particular theory, it is generally believed that devices comprising non-zero wall angles in the denuder region may permit sequential shear induced break up of oocyte complexes, that may be advantageous for denuding oocytes in certain cases. As described above, and elsewhere herein, posts within the denuder region may in some cases comprise teeth that serve as strippers to denude the oocytes (see FIG. 13). In devices comprising non-zero wall angles, these strippers may become tapered in certain embodiments, for example, as illustrated in FIG. 21. Neighboring posts may be used in some instances to create trapezoidal-like sub-channels with a series of tapered strippers. Without wishing to be bound by any theory, it is generally believed that initially large oocyte complexes can be denuded at the widest part of the tapered strippers to yield partially denuded oocytes, which may further be denuded, if desired, via repeated passage through a more narrow portion of the tapered strippers. Accordingly, the skilled artisan will understand that denudation process can be controlled by varying one or more of the aforementioned parameters and may be tailored to met the needs of any specific target cell (e.g., oocytes).

[0249] U.S. Provisional Patent Application Serial No. 63 / 518,322, filed August 8, 2023, entitled “Microfluidic Methods and Apparatuses for Denudation of Oocytes Utilizing Oscillatory Flow,” and U.S. Provisional Patent Application Serial No. 63 / 518,323, filed August 8, 2023, entitled “Devices and Methods for Isolation of Biological Entities in Suspension” are each incorporated herein by reference in its entirety.

[0250] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0251] EXAMPLE 1

[0252] This example is related to microfluidic designs and methods that allow for specific isolation and preparation of biological entities from suspensions that contain significant debris in the background.

[0253] Biological fluids can be used to serve many purposes from basic research to therapeutics. However, they often need to be pre-processed to prior to utilization of the specific entities of interest. The entities may be rare, requiring the extraction of excess volume and other unwanted entities. In some cases, the fluid may contain biological debris, and in other cases target entity may need additional treatments. This example illustrates an effective approach for processing fluids using a microfluidic device having multiple stages. In this example, the device described herein can be used to process follicular fluid, or a biological fluid or a man-made fluid (e.g., a culture media) that contain dissected ovarian samples. For example, during In-vitro Fertilization (IVF) therapy, the first step is to collect the follicular fluid from women’s ovaries to look for the oocytes. At this stage, the oocytes are oftentimes surrounded within or attached to a tissue of cumulus cells and / or various other cell types from the ovary (e.g., granulosa cells, mural cells, thecal cells, blood cells) and this tissue-oocyte complex is called Cumulus-Oocyte-Complex (COC) (e.g., cumulus enclosed oocytes). In the embryology lab, follicular fluid aspirate (FFA), which has a significant amount of unwanted entities such as red blood cells, cumulus tissue debris, and blood clots, is often inspected under a microscope by an expert embryologist for COCs. After COCs are collected and isolated, they are cultured until fertilization. In most cases, fertilization is conducted through injection of the sperm manually into the oocyte, a procedure called Intra-Cytoplasmic-Sperm- Injection (ICSI). In order to execute ICSI effectively, the oocytes need to be separated off from the surrounding cumulus tissue, or denuded. Thus, the steps needed to effectively extract / produce denuded oocytes for effective ICSI include: i) finding and isolating COCs from the follicular fluid, and 2) denuding COCs to separate the oocytes. These same steps (z.e. isolation and denudation) are also required for long-term egg freezing (egg banking) procedure a process also known as fertility preservation used for example in oncology patients or women storing oocytes for future use.

[0254] In some applications of this invention, the working fluid that contains the oocytes is not follicular fluid, but a biological fluid containing dissected ovarian samples. Dissected ovarian samples are collected in cases where a patient is going through a medical procedure that requires the removal of their reproductive organs (e.g. ovarian cancer patients), but they wish to maintain future fertility potential via banking of immature and mature oocytes or follicles. There are also animal applications (e.g. mice, bovine, sheep, etc.) where some labs collect oocytes from sacrificed animals, where the oocytes would be found in dissected ovarian sample fluid.

[0255] This example may allow for processing of untouched follicular fluid or dissected ovarian sample, captures COCs, applies a washing protocol to reduce the debris load without losing oocytes, separates COCs from majority of the debris, and denudes COCs during the isolation process. At the end, denuded oocytes in purified (i.e. orders of magnitude higher oocyte to debris ratio) suspension are extracted.

[0256] A. Details of microfluidic device design To serve this purpose, multiple microfluidic innovations may be integrated in a single design (Figure- 1).

[0257] Components of integrated design here and their specific functions are as follows:

[0258] Filter- 1 “Microfilter” (Figure 2): This is a first filter that may be used to capture COCs and large chunks of debris. It consists of 4 stages of size -based filtration, with specific gap sizes of (from inlet to next stage): 2 rows of 3000 pm, 2 rows of 1000 pm, 2 rows of 300 pm, and 2 rows of 150 pm.

[0259] Filter-2 “Denuder” (Figure 3): This is the denuder region that allows small debris to pass through and denudes COCs. Filter posts can have saw-teeth like structures to enable oocyte denudation. Flow velocity may be also drastically elevated (Fig 6) to achieve high shear stresses and facilitate denudation. It can consist of 3 stages of denuding filters, with specific gap sizes of (from microfilter to next stage): 2 rows of 180 pm, 2 rows of 150 pm, and 2 rows of 120 pm.

[0260] Filter-3 “Concentrator” (Figure 4): The concentrator region can be used to extract large amount of fluid and small unwanted entities from the suspension, and slow the fluid flow velocity that carries target entities into Filter-4. The size-based separation of small entities to the waste outlet may not be based on the absolute gap size between the square posts, but based on the fraction of the flow separated from the main channel based on the downstream resistance to flow.

[0261] Filter-4 “Capture filter” (Figure 5): Two of these filters may be utilized in parallel on the chip. This capture filter may capture the target entities (oocytes) and allow smaller debris to pass through. Flow velocity may be drastically reduced (Fig 6) here to prevent target oocytes from squeezing through the filters and escape. It can consist of 3 stages of size-based filtration, with specific gap sizes of (from concentrator to waste): 3 rows of 150 pm, 3 rows of 120 pm, and 2 rows of 80 pm.

[0262] B. Processes of Extracting / Denuded Oocytes

[0263] Current process may be carried out by the following steps:

[0264] Step 1: As shown in Fig. 7, follicular fluid aspirate (FFA) (or other biological fluid with COCs) may steadily flow in through the main inlet. Most of excess volume in the FFA and red blood cells (along with other small untargeted particles) may flow out of the Filter-3 (concentrator) waste outlets or from Filter-4 (Capture filter) waste outlet.

[0265] Step 2: As shown in Fig. 8, wash buffer solution may then steadily flow in through the main inlet. Anything not captured by filters and small enough may get removed from the chip via the waste outlets on Filter-3 and Filter-4 stages, similar to Step 1. Step 3: As shown in Figs. 9-10, hyaluronidase or other solutions such as any of those described herein may flow in and fill the device chambers from the main inlet. The chip may be incubated with this solution for a time period without flow. After a preset time period, flow oscillation may be started. These flow oscillations may have a net positive direction towards main outlet (e.g. 5 seconds in the forward flow phase shown in Fig 9, 2 seconds in backward flow phase shown in Fig 10). At this time, tissue debris may start to loosen and COCs that were captured in Filter- 1 may start transitioning to Filter-2. Debris particles that are small enough may move through Filter-2 pass through Filter-3. If the debris particles are small enough, they may be extract from Filter-3, otherwise they may either get temporarily captured in Filter-4 or get extracted from main outlet.

[0266] Hyaluronidase flow may then be replaced by washing buffer after a preset incubation time with hyaluronidase. Oscillatory wash may continue with washing buffer. In time, large majority of debris may dissociate into smaller segments and may be pushed from Filter- 1 to Filter-4 and then get extracted from the concentrator waste or main waste outlets. Oocytes may initially transition to Filter-2; however, as they get denuded, they may be able to move through the gaps of Filter-2 and subsequently be captured in Filter-4. When flow direction changes, the oocyte may flow / pass through denudation segment again. A net positive direction of the flow may be applied to the denuded oocyte such that the oocytes can be captured by Filter-4 at the end of the whole process.

[0267] Step 4: As shown in Fig. 11, Step 2 may be repeated to send the wash buffer solution may then steadily flow in through the main inlet.

[0268] Step 5: As shown in Fig. 12, denuded oocytes may be released from the device for further processing.

[0269] As illustrated above, this example involves a method of oscillatory washing in a microfluidic device described herein. For example, as the follicular fluid flows through the device, COCs as well as a substantial amount of tissue debris may be caught within the device. In order to reduce the debris load, washing may be conducted while the direction of flow is alternated, with net flow occurring in one direction (e.g. 5 sec forward, 2 sec backward flow). Since tissue debris is flexible and does not have a uniform hydrodynamic shape / radius, this oscillatory flow pattern may allow for multiple repetitions of repeated attempts to push tissue debris through filter openings. Therefore, using the above-referenced process, unwanted entities may have higher likelihood of being extracted at the end of the process.

[0270] Furthermore, this example involves washing of captured contents with various enzymes and buffers that can help improve the filtration specificity. For example, enzyme may be used to weaken the intracellular bonds that holds the tissue together. In this application, enzymes may break apart the large tissue debris and generate smaller components while having minimal impact the target entity size / shape. Thus, this innovation selectively digests unwanted entities to smaller size so they can be extracted more easily. In the current utilization of oocyte isolation, this enzyme is hyaluronidase, and the tissue is cumulus and granulosa cells.

[0271] This example may further utilize oscillatory flow during enzyme treatment. This approach may allow more efficient interaction of the enzyme with target tissue. Without alternating or somehow perturbing the flow within the device, enzyme (or other washing solutions) may be limited to flow lines of laminar flow conditions. When portions of the device are clogged by debris, for example, variable flow conditions may improve the mixing and improve interaction rate of the enzyme with debris. Oscillatory, or otherwise perturbed, flow conditions can help increasing the efficiency of debris removal from the device. Consistent mixing during alternating flow conditions may also disturb concentration boundary layers, thereby improving enzyme interactions with the target tissues and cells.

[0272] This example may further incorporate multiple microfluidic modules where the local flow velocity in some modules (denuder) may be an order of magnitude greater than some other modules (capture) of the chip. This may be achieved by different module widths (e.g., such as the width of the denuder vs capture filter), parallelized modules (2 x capture filter), and the volumetric flowrate reduction in the concentrator module. The high flow velocity in the denuder section may also introduce localized vortices, which facilitates oocyte denudation by increased shear force and changing the orientation of the oocytes as they travel through the module.

[0273] As noted, the microfluidic described herein may incorporate a denudation segment, Filter-2, to a filtration device. Utilization of oscillatory washing and enzyme treatment can allow for denudation of oocytes as they move through Filter-2 (e.g., the denuder region). This segment, with the sawtooth features pointing towards both flow directions, can be used to works to strip-off the cell tissue surrounding the target entities, as the target entity (e.g. oocytes) move in either direction. The denudation process can start as soon as COCs get through the filtration elements of Filter- 1 and get captured in Filter-2. Therefore, filtration process and denudation may occur simultaneously. If needed, oscillation process can continue after enzyme treatment with standard buffers and debris cleaning is completed.

[0274] In this example, in-device mixing may be executed through modulation of the flow direction. Alternatively, other methods for mixing of flow may be achieved through microvortex generating herringbone-structures, mixing through ultrasonic pulses applied to the flow channels, or any other method that can be employed to disturb the flow pattern.

[0275] In this example, during oscillatory flow, a size based capture filter may be utilized to hold the target entities to avoid them from being discarded with the other debris. This may be necessary since cells needed to circulate in the device through the same functional sections multiple times. In order to facilitate the cell immobilization for oscillation, one can utilize different holding mechanisms, for example, an electrokinetic immobilization method to capture the target cells through dielectrophoretic force.

[0276] In this example, oocytes may be captured by the size based filter at the final step of the process. At the stage, majority of the debris may be extracted from the device through one of the outlets. This enriched suspension of oocytes may then extracted from the device. In some embodiments, alternative purification methods can be used for the final stage. For example, during the forward flow, denuded oocytes may be discernable from rest of the debris, and automated image recognition can be employed to identify these oocytes as they pass through. Similarly, electronic sensors can be used to identify an incoming oocyte. Upon identification of the denuded oocyte, flow direction can be instantaneously altered in order to direct the oocyte towards a different path for isolation.

[0277] Furthermore, in this example, flow may be oscillated through all the modules of the chip as previously described. Alternatively, flow can be oscillated through individual modules of the chip. For example, initial oscillatory hyaluronidase washing step could be confined to the Filter- 1 module, and the denudation oscillation step could be confined to the denudation module. In order for this approach to work effectively, one may need to ensure that all oocytes are transferred to the next module in between the oscillation steps.

[0278] EXAMPLE 2

[0279] This example is related to one embodiment of microfluidic designs and methods that allow for specific isolation and preparation of biological entities from suspensions that contain significant debris in the background.

[0280] A microfluidic device with four regions was fabricated using the geometry shown in FIGS. 19A-19F. As shown, the microfluidic device includes a microfilter region, a denudation fine filter region, a concentrating sword region, and a capturing fine filter region. The microfilter region can have post having a polygonal geometry with a smooth interface and a width Wi of about 150 micrometers between two adjacent posts, e.g., as shown in FIG. 19B. The denudation fine filter region can have posts having polygonal shapes (e.g., a rhombus) and teeth extending out from walls of the posts with width W2 and W3 of about 120 and 170 micrometers between adjacent posts, e.g., as shown in FIG. 19C. The teeth may either have a sharp edge (e.g., as shown in FIG. 19C(ii)) or a rounded edge. FIG. 19C(iii) is a photo of a manufactured denudation region having posts with teeth having rounded tips. The concentrating sword region can have square posts with a width W4 of about 40 micrometers between the posts, e.g., as shown in FIG. 19D. The capturing fine filter region can have triangular posts with a width W5 of between the posts, e.g., as shown in FIG. 19D. The width W5 between the posts may differ by increments between the different stages of posts (stage A, stage B, stage C), For examples, the stages may have variable width, such about 150 micrometers in stage A, about 120 micrometers in stage B, and about 80 micrometers in stage C. The microfluidic chip can have eight ports (e.g., ports A-G), as shown in FIG. 19F. Flow can be incoming and outgoing from any one of these ports. Flow can be alternated between any of the combinations (i.e. between A-B, between A-C, between A-F, between B-C, between B-F, etc.). The device can be operated using any appropriate pressure, such as about 20 psi.

[0281] While several embodiments of the present disclosure 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 disclosure. 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 disclosure 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 disclosure 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 disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0282] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0283] 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.”

[0284] 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. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. 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. 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 only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0285] 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.”

[0286] 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 necessarily 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.

[0287] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0288] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0289] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” 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.

Claims

CLAIMSWhat is claimed is:

1. A method of separating oocyte complex from a fluid and denuding the oocyte complex, comprising: flowing the fluid containing the oocyte complex through a microfluidic device, wherein the microfluidic device comprises a fluidic channel comprising a filter region comprising a first plurality of posts, a denuder region comprising a second plurality of posts, a first pressure source upstream of the filter region, and a second pressure source downstream of the denuder region.

2. The method of claim 1, further comprising a capture region comprising a third plurality of posts positioned downstream the denuder region.

3. The method of any one of claims 1-2, further comprising a concentrator region positioned between the filter region and the denuder region.

4. The method of any one of claims 1-3, wherein the microfluidic device is one of a plurality of microfluidic devices.

5. The method of any one of claims 1-4, wherein the first pressure source comprises a syringe pump.

6. The method of any one of claims 1-5, wherein the first pressure source comprises a peristaltic pump.

7. The method of any one of claims 1-6, wherein the first pressure source comprises a positive displacement pump.

8. The method of any one of claims 1-7, wherein the second pressure source comprises a syringe pump.

9. The method of any one of claims 1-8, wherein the second pressure source comprises a peristaltic pump.

10. The method of any one of claims 1-9, wherein the second pressure source comprises a positive displacement pump.

11. The method of any one of claims 1-10, wherein the first plurality of posts, the denuder region comprising the second plurality of posts, the first pressure source upstream of the filter region, and the second pressure source downstream of the denuder region are present in the order recited.

12. A microfluidic device, comprising: a fluidic channel comprising a filter region, a denuder region, a first pressure source upstream of the filter region, and a second pressure source downstream of the denuder region, wherein the filter region comprises a first plurality of posts defining first subchannels between neighboring pairs of posts, wherein at least some the first subchannels have a minimum cross-sectional dimension of between 100 micrometers and 3000 micrometers; and wherein the denuder region comprises a second plurality of posts defining second sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross-sectional dimensions of between about 50 micrometers and about 350 micrometers.

13. The device of claim 12, wherein at least some of the second sub-channels have a minimum cross-sectional dimensions of between about 50 micrometers and about 100 micrometers.

14. The device of any one of claims 12 or 13, wherein at least some of the second subchannels have a minimum cross-sectional dimensions of between about 80 micrometers and about 350 micrometers.

15. The device of any one of claims 12-14, wherein the filter regions comprises multiple stages of sub-channels.

16. The device of any one of claims 12-15, wherein in the filter region, a stage of subchannels adjacent an inlet of the filter region has a minimum cross-sectional dimension larger than the minimum cross-sectional dimension of a stage sub-channels adjacent an outlet of the filter region.

17. The device of any one of claims 12-16, wherein the filter region comprises a first stage of sub-channels having a minimum cross-sectional dimension of about or equal to 3000 micrometers.

18. The device of any one of claims 12-17, wherein the filter region comprises a second stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 1000 micrometers and less than 3000 micrometers.

19. The device of any one of claims 12-18, wherein the filter region comprises a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 300 micrometers and less than 1000 micrometers.

20. The device of any one of claims 12-19, wherein the filter region comprises a fourth stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 100 micrometers and less than 300 micrometers.

21. The device of any one of claims 12-20, wherein the denuder region is downstream of the filter region.

22. The device of any one of claims 12-21, wherein the denuder region comprises multiple stages of sub-channels.

23. The device of any one of claims 12-22, Wherein in the denuder region, a stage of subchannels adjacent an inlet of the denuder region has a minimum cross-sectional dimension larger than the minimum cross-sectional dimension of a stage sub-channels adjacent an outlet of the denuder region.

24. The device of any one of claims 12-23, wherein the denuder region comprises a first stage of sub-channels having a minimum cross-sectional dimension of about or equal to 180 micrometers.

25. The device of any one of claims 12-24, wherein the denuder region comprises a first stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 180 micrometers and less than 500 micrometers.

26. The device of any one of claims 12-25, wherein the denuder region comprises a second stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 150 micrometers and less than 200 micrometers.

27. The device of any one of claims 12-26, wherein the denuder region comprises a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 80 micrometers and less than 150 micrometers.

28. The device of any one of claims 12-27, wherein the denuder region comprises a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 50 micrometers and less than 150 micrometers.

29. The device of any one of claims 12-28, wherein the denuder region comprises multiple stages of sub-channels.

30. The device of any one of claims 12-29, wherein in the denuder region, a stage of subchannels adjacent an inlet of the denuder region has a minimum cross-sectional dimension larger than the minimum cross-sectional dimension of a stage sub-channels adjacent an outlet of the denuder region.

31. The device of any one of claims 12-30, wherein the denuder region comprises a first stage of sub-channels having a minimum cross-sectional dimension of greater than 140 micrometers and less than or equal to 160 micrometers.

32. The device of any one of claims 12-31, wherein the denuder region comprises a second stage of sub-channels having a minimum cross-sectional dimension of greater than 120 micrometers and less than or equal to 140 micrometers.

33. The device of any one of claims 12-32, wherein the denuder region comprises a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 50 micrometers and less than or equal to 120 micrometers.

34. The device of any one of claims 12-33, further comprising a concentrator region downstream of the denuder region.

35. The device of any one of claims 12-34, Wherein the concentrator region comprises one or more filters sized to remove at least a portion of a fluid and / or non-target entities from the microfluidic device.

36. The device of any one of claims 12-35, Where the concentrator region comprises a central fluidic channel defined by a third plurality of posts on at least one or more side of the central fluidic channel.

37. The device of any one of claims 12-36, wherein the third plurality of posts defines a plurality of sub-channels between neighboring pairs of posts.

38. The device of any one of claims 12-37, further comprising a first port positioned upstream of the filter region, wherein the first port is configured to receive a fluid into and / or output a fluid from the filter region.

39. The device of any one of claims 12-38, further comprising a second port positioned between the denuder region and a capture region, wherein the second port is configured to output denuded oocytes.

40. The device of any one of claims 12-39, further comprising a third port downstream of the capture region, wherein the third port is configured to receive a fluid into the capture region.

41. The device of any one of claims 12-40, further comprising one or more additional ports configured to output wastes out of the device.

42. The device of any one of claims 12-41, further comprising a fifth port downstream of the capture region, wherein the fifth port is configured to output a waste from the capture region.

43. The device of any one of claims 12-42, further comprising one or more additional ports downstream of the concentrator region, wherein the one or more additional ports are configured to output a waste from the device.

44. The device of any one of claims 12-43, further comprising an additional port positioned between the filter region and the denuder region.

45. The device of any one of claims 12-44, further comprising an additional pressure source adjacent the additional port.

46. The device of any one of claims 12-45, wherein the first pressure source is positioned upstream of the filter region.

47. The device of any one of claims 12-46, wherein the second pressure source is positioned downstream of the capture region.

48. The device of any one of claims 12-47, wherein the capture region has a width that is at least 10 times the width of the denuder region.

49. The device of any one of claims 12-48, Wherein an average flow velocity of the fluid within the denuder region is at least 10 times faster than the average flow velocity of the fluid within the capture region.

50. A microfluidic device, comprising: a fluidic channel comprising a denuder region, a capture region, a first pressure source upstream of the denuder region, and a second pressure source downstream of the capture region,wherein the denuder region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some of the first sub-channels have a minimum cross-sectional dimension of between about 80 micrometers and about 350 micrometers; and wherein the capture region comprises a second plurality of posts defining second sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross-sectional dimensions of less than 150 micrometers.

51. The device of claim 50, wherein at least some of the second sub-channels have a minimum cross-sectional dimensions of less than 120 micrometers.

52. The device of any one of claims 50-51, wherein the capture region is downstream of the denuder region.

53. The device of any one of claims 50-52, wherein at least some of the sub-channels in the capture region have minimum cross-sectional dimensions less than the minimum cross-sectional dimensions of sub-channels in the denuder region.

54. The device of any one of claims 50-53, wherein all of the sub-channels in the capture region have minimum cross-sectional dimensions less than the minimum cross- sectional dimensions of sub-channels in the denuder region.

55. The device of any one of claims 50-54, wherein the capture region comprises multiple stages of sub-channels.

56. The device of any one of claims 50-55, wherein in the capture region, a stage of subchannels adjacent an inlet of the capture region has a minimum cross-sectional dimension larger than the minimum cross-sectional dimension of a stage of subchannels adjacent an outlet of the capture region.

57. The device of any one of claims 50-56, wherein the capture region comprises a first stage of sub-channels having a minimum cross-sectional dimension of about or equal to 150 micrometers.

58. The device of any one of claims 50-57, wherein the capture region comprises a second stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 120 micrometers and less than 150 micrometers.

59. The device of any one of claims 50-58, wherein the capture region comprises a third stage of sub-channels having a minimum cross-sectional dimension of greater than or equal to 80 micrometers and less than 120 micrometers.

60. The device of any one of claims 50-59, wherein the capture region comprises two or more sets of posts arrange in parallel.

61. A microfluidic device, comprising: a fluidic channel comprising a denuder region and a capture region, wherein the denuder region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some of the first sub-channels have a minimum cross-sectional dimension of between about 80 micrometers and about 350 micrometers, and the capture region comprises a second plurality of posts defining second sub-channels between neighboring pairs of posts, wherein at least some of the second sub-channels have a minimum cross-sectional dimension of less than 120 micrometers; a first port upstream of the denuder region; a second port downstream of the denuder region and upstream of the capture region; and a third port downstream of the capture region.

62. The device of claim 61, wherein at least some of the second sub-channels have a minimum cross-sectional dimensions of less than 150 micrometers.

63. The device of any one of claims 61 or 62, wherein at least some of the second subchannels have a minimum cross-sectional dimension smaller than a smallest crosssection in the denuder region.

64. The device of any one of claims 61-63, further comprising a first pressure source upstream of the filter region.

65. The device of any one of claims 61-64, further comprising a second pressure source downstream of the capture region.

66. A microfluidic device, comprising: a fluidic channel comprising a filter region, a denuder region downstream the filter region, and a capture region downstream the denuder region; a first port upstream of the filter region; a second port downstream of the denuder region and upstream of the capture region; a third port downstream of the capture region; and an additional port downstream of the filter region and upstream of the denuder region; wherein: the filter region comprises a first plurality of posts defining first sub-channels between neighboring pairs of posts, wherein at least some the first sub-channels have a minimum cross-sectional dimension of between 100 micrometers and 3000 micrometers; the denuder region comprises a second plurality of posts defining second subchannels between neighboring pairs of posts, wherein at least some of the second subchannels have a minimum cross-sectional dimension of between 80 micrometers and 350 micrometers; and the capture region comprises a third plurality of posts defining third subchannels between neighboring pairs of posts, wherein at least some of the third subchannels have a minimum cross-sectional dimensions of less than 120 micrometers.

67. The device of claim 66, further comprising a first pressure source upstream of the filter region.

68. The device of any one of claims 66-67, further comprising a second pressure source downstream of the capture region.

69. The device of any one of claims 66-68, further comprising a third pressure source adjacent the additional port.

70. The device of any one of claims 66-69, further comprising a port between the denuder region and the capture region.

71. A method of denuding an oocyte complex comprising an oocyte and bounded nonoocyte cells to separate the oocyte from the non-oocyte cells, the method comprising: providing a fluidic channel comprising a denuder region, a capture region, a first port upstream of the denuder region, a second port downstream of the denuder region and upstream of the capture region, and a third port downstream of the capture region; at a first point in time, causing fluid to flow from the first port, through the denuder region and the capture region, to the third port, such that the complex passes through the denuder region and is trapped by the capture region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; at a second point in time, causing the fluid to flow from the third port, through the capture region and the denuder region, to the first port, such that the complex passes through the denuder region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; and at a third point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the oocyte exits through the second port.

72. The method of claim 71, further comprising, between the second point in time and the third point in time, repeatedly causing the fluid to flow from the first port to the third port, such that the complex passes through the denuder region and is trapped by the capture region, and causing the fluid to flow from the third port to the first port, such that the complex passes through the denuder region, in an alternating fashion.

73. The method of any one of claims 71 or 72, wherein the repeatedly causing step is performed until at least 90% of the non-oocyte cells are removed from the complex.

74. The method of any one of claims 71-73, wherein the repeatedly causing step further causes debris associated with the complex to break down and / or dissociate into smaller entities.

75. The method of claim 74, further comprising removing the dissociated debris from the filter region via one or more additional ports in the device.

76. The method of any one of claims 71-75, wherein the fluid caused to flow from one port to another comprises a buffer solution and / or an enzymatic solution.

77. The method of claim 76, wherein the enzymatic solution comprises hyaluronidase.

78. The method of any one of claims 71-77, wherein a first pressure is exerted by the first pressure source to cause the fluid to flow from the first port, through the denuder region and the capture region, to the third port.

79. The method of any one of claims 71-78, wherein a second pressure is exerted by the second pressure source to cause the fluid to flow from the third port, through the capture region and the denuder region, to the first port.

80. The method of any one of claims 71-79, further comprising selectively isolating one or more oocyte complex from the fluid in a filter region upstream the denuder region.

81. The method of any one of claims 71-80, wherein the causing step comprises applying an oscillatory fluid flow across the filter region and / or flowing an enzyme capable of dissociating the non-oocyte cells from each other through the filter region.

82. The method of any one of claims 71-81, wherein the fluid contains one or more oocyte complex and more additional non-target entities suspended in the fluid.

83. The method of any one of claims 71-82, wherein the additional non-target entities comprises blood clots, debris, aggregated blood cells, blood clots, debris, aggregated blood cells, individual blood cells, tissue, non-blood cells, and / or inorganic impurities.

84. The method of any one of claims 71-83, further comprising isolating one or more additional non-target entities in the filter region.

85. The method of any one of claims 71-84, further comprising causing oscillatory flow within the filter region and / or the denuder region such that the non-target entities dissociate into smaller sizes.

86. The method of any one of claims 71-85, further comprising flowing the dissociated non-target entities out of the device via one or more ports.

87. The method of any one of claims 71-86, further comprising causing repeated flow of a fluid containing the non-target entities and the oocyte complex through a filter region and / or a denuder region, such that at least a portion of the non-target entities dissociates into smaller sizes, and such that at least some of the initially bounded nonoocyte cells is removed from the complex.

88. The method of any one of claims 71-87, further comprising reducing a volumetric flow rate of the fluid from the denuder region by passing the fluid into a concentrator region adjacent the denuder region.

89. The method of any one of claims 71-88, further comprising removing a fluid containing debris and / or removed non-oocyte cells via one or more ports downstream the concentrator region.

90. A method of controlling an oocyte complex comprising an oocyte and bounded non- oocyte cells, comprising: causing a first flow of a fluid containing the complex in a first direction through a fluidic channel; removing at least some of the non-oocyte cells from the complex as the oocyte flows through the fluidic channel in the first direction; trapping the complex in an exit region of the fluidic channel; and flowing the fluid containing the trapped complex in a second direction opposed to the first direction in the fluidic channel.

1. A method of processing a fluid containing one or more non-target entities and an oocyte complex comprising an oocyte and bounded non-oocyte cells, the method comprising: providing a fluidic channel comprising a filter region, a denuder region, a capture region, a first port upstream of the filter region, a second port downstream of the denuder region and upstream of the capture region, a third port downstream of the capture region, and an additional port downstream of the filter region and upstream of the denuder region; at a first point in time, causing fluid to flow from the first port, through the filter region, to the additional port, such that at least a portion of the non-target entities passes through the filter region, wherein some of the non-target entities undergo a reduction in size as the non-target entities pass through the filter region; at a second point in time, causing fluid to flow from the additional port, through the filter region, to the first port, such that at least a portion of the non-target entities passes through filter region, wherein some of the non-target entities undergo a reduction in size as the non-target entities pass through the filter region; at a third point in time, causing fluid to flow from the first port, through the filter region to the denuder region, such that the oocyte complex passes from the filter region to the denuder region; at a fourth point in time, causing fluid to flow from the additional port, through the denuder region and the capture region, to the third port, such that the oocyte complex passes through the denuder region and is trapped by the capture region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; at a fifth point in time, causing fluid to flow from the third port, through the capture region and the denuder region, to the additional port, such that the complex passes through the denuder region, wherein at least some of the non-oocyte cells are removed from the complex as the complex flows through the denuder region; and at a sixth point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the oocyte exits through the second port.

92. The method of claim 91, wherein the additional non-target entities comprise blood clots, debris, aggregated blood cells, individual blood cells, and / or inorganic impurities.

93. The method of any one of claims 91-92, further comprising causing the reduced nontarget entities to flow out of the fluidic channel.

94. The method of any one of claims 91-93, further comprising, between the second point in time and the third point in time, repeatedly causing a fluid to flow from the first port to the additional port and from the additional port to the first port in an alternating fashion, such that the non-target entities are repeatedly caused to undergo a reduction in size.

95. The method of any one of claims 91-94, further comprising, between the fifth point in time and the sixth point in time, repeatedly causing a fluid to flow from the additional port to the third port and from the third port to the first port in an alternating fashion, such that the complex passes through the denuder region repeatedly.

96. The method of any one of claims 91-95, wherein the repeatedly causing step is performed until at least 90% of the non-oocyte cells are removed from the complex.

97. The method of any one of claims 91-96, wherein the fluid caused to flow from one port to another comprises a buffer solution and / or an enzymatic solution.

98. A method of processing a fluid containing one or more non-target entities and an oocyte complex comprising an oocyte and non-oocyte cells, the method comprising: providing a fluidic channel comprising a filter region, a denuder region, and a capture region; causing repeated flow of the fluid through the filter region, such that at least a portion of the non-target entities undergoes a reduction in size as the non-target entities flows within the filter region; and causing repeated flow of the fluid through the denuder region, such that at least a portion of the non-oocyte cells is removed from the oocyte complex as the complex flows through the denuder region.

99. A method of controlling an oocyte complex comprising an oocyte and bounded nonoocyte cells, the method comprising: passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts; trapping the complex in an exit region of the fluidic channel; releasing the complex from the exit region; and passing the released complex through the plurality of posts.

100. A method of controlling an oocyte complex comprising an oocyte and bounded nonoocyte cells, the method comprising: causing a flow of a fluid containing the complex through a fluidic channel; and repeatedly trapping and releasing the complex in an exit region of the fluidic channel.

101. A method of controlling an oocyte complex comprising an oocyte and bounded nonoocyte cells, the method comprising: passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the subchannels having a minimum cross-sectional dimension of between 120 micrometers and 180 micrometers; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

102. A method of controlling an oocyte complex comprising an oocyte and bounded nonoocyte cells, the method comprising: passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the subchannels having a minimum cross-sectional dimension of 1.0 to 1.4 times an average diameter of the oocyte; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times. es.

103. A method of denuding a target entity- secondary entity comprising a target entity and one or more secondary entities to separate the target entity from the secondary entities, the method comprising: providing a fluidic channel comprising a denuder region, a capture region, a first port upstream of the denuder region, a second port downstream of the denuder region and upstream of the capture region, and a third port downstream of the capture region; at a first point in time, causing fluid to flow from the first port, through the denuder region and the capture region, to the third port, such that the complex passes through the denuder region and is trapped by the capture region, wherein at least some of the secondary entities are removed from the complex as the complex flows through the denuder region; at a second point in time, causing the fluid to flow from the third port, through the capture region and the denuder region, to the first port, such that the complex passes through the denuder region, wherein at least some of the secondary entities are removed from the complex as the complex flows through the denuder region; and at a third point in time, causing the fluid to flow from the third port, through the capture region, to the second port, such that the target entity exits through the second port.

104. A method of controlling a target entity- secondary entity comprising a target entity and one or more secondary entities, comprising: causing a first flow of a fluid containing the complex in a first direction through a fluidic channel; removing at least some of the secondary entities from the complex as the target entity flows through the fluidic channel in the first direction; trapping the complex in an exit region of the fluidic channel; and flowing the fluid containing the trapped complex in a second direction opposed to the first direction in the fluidic channel.

105. A method of controlling a target entity- secondary entity comprising a target entity and one or more secondary entities, the method comprising: passing the complex through a fluidic channel comprising a plurality of postsdefining sub-channels between neighboring pairs of posts; trapping the complex in an exit region of the fluidic channel; releasing the complex from the exit region; and passing the released complex through the plurality of posts.

106. A method of controlling a target entity- secondary entity comprising a target entity and one or more secondary entities, the method comprising: causing a flow of a fluid containing the complex through a fluidic channel; and repeatedly trapping and releasing the complex in an exit region of the fluidic channel.

107. A method of controlling a target entity- secondary entity comprising a target entity and one or more secondary entities, the method comprising: passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the subchannels having a minimum cross-sectional dimension of between 120 micrometers and 180 micrometers; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

108. A method of controlling a target entity- secondary entity comprising a target entity and one or more secondary entities, the method comprising: passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the subchannels having a minimum cross-sectional dimension of 1.0 to 1.4 times an average diameter of the target entity; trapping the complex in an exit region of the fluidic channel; and repeating the passing and trapping steps at least 10 times.

109. The device and / or method of any one of the preceding claims, wherein the non-oocyte cells comprise one or more of cumulus cells, granulosa cells, mural cells, thecal cells, red blood cells, or combinations thereof.

110. The device and / or method of any one of the preceding claims, wherein the microfluidic device is defined by a circular form factor.

111. The device and / or method of any one of the preceding claims, wherein the plurality of posts has a wall angle of between 2 degrees and 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device.

112. The device and / or method of any one of the preceding claims, wherein a cross- sectional dimension of a first portion of a trapezoidal-like sub-channel is larger than the cross-sectional dimension of a second portion of the trapezoidal-like sub-channel.

113. The device and / or method of any one of the preceding claims, wherein a cross- sectional dimension of the first portion of the trapezoidal-like sub-channel is smaller than the cross-sectional dimension of the second portion of the trapezoidal-like subchannel114. The device and / or method of any one of the preceding claims, wherein the trapezoidal-like sub-channels have an average cross-sectional dimension of between 10 microns and 200 microns.

115. The device and / or method of any one of the preceding claims, wherein the average cross-sectional dimension of the sub-channels in the filter region is between 10 microns and 200 microns and the average cross-sectional dimension of the subchannels in the denuder region is between 10 microns and 200 microns.

116. The device and / or method of any one of the preceding claims, wherein the average cross-sectional dimension of the sub-channels in the filter region is between 10 microns and 200 microns and the average cross-sectional dimension of the subchannels in the capture region is between 10 microns and 200 microns.

117. The device and / or method of any one of the preceding claims, wherein the average cross-sectional dimension of the sub-channels in the denuder region is between 10 microns and 200 microns and the average cross-sectional dimension of the subchannels in the capture region is between 10 microns and 200 microns.

118. The device and / or method of any one of the preceding claims, wherein the average cross-sectional dimension of the sub-channels in the filter region is between 10 microns and 200 microns, the average cross-sectional dimension of the sub-channels in the denuder region is between 10 microns and 200 microns, and the average cross- sectional dimension of the sub-channels in the capture region is between 10 microns and 200 microns119. The device and / or method of any one of the preceding claims, wherein the subchannels have an average height of between 100 microns and 400 microns.

120. The device and / or method of any one of the preceding claims, wherein the average height of the sub-channels in the filter region is between 100 microns and 400 microns and the average height of the sub-channels in the denuder region is between 100 microns and 400 microns.

121. The device and / or method of any one of the preceding claims, wherein the average height of the sub-channels in the filter region 100 microns and 400 microns and the average height of the sub-channels in the capture region is between 100 microns and 400 microns.

122. The device and / or method of any one of the preceding claims, wherein the average height of the sub-channels in the denuder region is between 100 microns and 400 microns and the average height of the sub-channels in the capture region is between 100 microns and 400 microns.

123. The device and / or method of any one of the preceding claims, wherein the average height of the sub-channels in the filter region is between 100 microns and 400 microns, the average height of the sub-channels in the denuder region is between 100 microns and 400 microns, and the average height of the sub-channels in the capture region is between 100 microns and 400 microns.

124. The device and / or method of any one of the preceding claims, wherein the concentrator region comprises a first sidewall and a second sidewall, the first sidewall and second side wall defining a central fluidic channel, and the first side wall and second side wall comprising a plurality of sidewall channels.

125. The device and / or method of the claim 124, wherein the plurality of sidewall channels of at least one of the two sidewalls is not centered relative to the central fluidic channel in a direction orthogonal to fluid flow within the central fluidic channel.

126. The device and / or method of any one of the preceding claims, wherein the plurality of channels have an average height of between 100 and 400 microns.

127. The device and / or method of any one of the preceding claims, wherein the plurality of channels have an average cross-sectional dimension of between 1 and 400 microns.

128. The device and / or method of any one of the preceding claims, wherein fluid is pulled into the plurality of channels from central fluidic channel along a first plane.

129. The device and / or method of any one of the preceding claims, wherein passing a fluid comprising a plurality of target cells and contaminants through the concentrator region results in at least a portion of the contaminants flowing into the plurality of channels along the first plane and the target cell flowing through the central fluidic channel along a second plane, different than the first plane.

130. The device and / or method of any one of the preceding claims, further comprising a inertial focusing region positioned on a microfluidic device between a denudation region and a concentrator region.

131. The device and / or method of any one of the preceding claims, wherein the inertial focusing region comprises a fluid channel, an inlet port, an outlet port, and at least two waste ports.

132. The device and / or method of any one of the preceding claims, wherein the inertial focusing region focuses a target cell to the middle of the fluid channel width prior to entering the concentrator region.

133. The device and / or method of any one of the preceding claims, wherein the inertial focusing region focuses a target cell to the middle of the fluid channel width and / or toward a top or bottom portion of the fluid channel prior to entering the concentrator region.

134. The method of any one of the preceding claims, wherein the steps recited at the first point in time, second point in time, third point in time, fourth point in time, fifth point in time, and / or sixth point in time are performed simultaneously.

135. The device and / or method of any one of the preceding claims, wherein fluid flow through the filter region, denuder region, inertial focusing region, concentrator region, and / or capture region is differentially regulated.

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