High-throughput microfluidic chip with parallel constrictions for perturbing cell membranes

Abstract

A microfluidic chip for delivering a payload to a cell includes a plurality of constrictions configured to allow a cell suspension to flow from a first fluid flow region within the microfluidic chip through one or more of the plurality of constrictions to a second fluid flow region, wherein a cross-sectional width of each of the plurality of constrictions is less than a diameter of a cell in the cell suspension such that a membrane of the cell is perturbed when passing through the constriction such that a payload is able to pass through the perturbed cell membrane, and wherein a quotient of a cross-sectional area and a cross-sectional perimeter of each of the plurality of constrictions is greater than or equal to 0.5 pm.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 131,430, filed December 29, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to delivering payloads into cells, and more specifically to perturbing the cell membrane to allow the payload to pass through microfluidic contractile channels of the perturbed membrane. Background Technology

[0004] In the development of cell therapy, the controlled delivery of various materials into cells is crucial in the medical field. For example, various research and therapeutic applications may involve delivering peptides, nucleic acids, proteins, small molecules, and nanomaterials through cell membranes. As discussed in WO 2013059343, WO 2015023982, PCT / US2015 / 058489, PCT / US2015 / 060689, and PCT / US2016 / 13113, constrictive microfluidic channels can be used to deliver compounds and other payloads into cells. As disclosed in PCT / US2018 / 66295, benchtop laboratory and / or clinical systems can be configured to force cell suspensions through one or more constrictions of a microfluidic chip with constrictive channels or orifices to induce disturbances in the cell membrane within the cell suspension. Summary of the Invention

[0005] As explained above, systems for intracellular payload delivery include those configured to force cells through one or more contractile channels or orifices to cause disturbance of the cell membrane in the cell suspension as the cell suspension flows through the constriction box. However, known systems for intracellular payload delivery are prone to clogging of the constriction, have insufficient throughput, and are not simple or efficient to manufacture.

[0006] Therefore, there is a need for improved systems, methods, and techniques for intracellular payload delivery, including improved microfluidic cell contraction chips with high throughput and low clogging rates. Microfluidic cell contraction chips with improved geometry, improved throughput, improved resistance to clogging or other failures, and / or improved ease of fabrication and efficiency are required. The systems, methods, and techniques disclosed herein address one or more of these needs to improve the geometry, throughput, resistance to clogging or other failures, ease of fabrication and efficiency, and capacity of microfluidic cell contraction chips and / or systems, thereby improving methods of use and / or manufacturing.

[0007] This document discloses a high-throughput microfluidic chip for use in a system for delivering payloads to cells in a cell suspension. The chip includes multiple parallel constrictions (e.g., constriction channels) through which the cell suspension can be forced under pressure. The cells in the cell suspension can deform due to the cell deformation constrictions of the microfluidic chip, and their membranes are perturbed. As explained herein, the throughput and clogging properties of the chip can be improved due to several characteristics of the chip disclosed herein.

[0008] First, the chips disclosed herein may include a large number of parallel constrictions separating a first fluid flow region from a second fluid flow region. For example, chips such as those disclosed herein may have hundreds or thousands of parallel microfluidic constrictions. When the cell suspension fluid in the first fluid flow region is pressurized, it may be able to flow through any one of the constrictions into the second fluid flow region, thereby increasing throughput compared to chips with fewer constrictions.

[0009] Second, the chip disclosed herein may have one or more narrow sections, for which the quotient of cross-sectional area to cross-sectional perimeter is higher than in previously known designs. In some embodiments, this quotient can be increased by etching deep, narrow rectangular narrow sections (e.g., slit-shaped narrow sections) into a substrate (e.g., a silicon substrate). The narrow sections can be formed by etching deep enough while maintaining the sidewalls of the narrow sections within a sufficient angular threshold that are parallel to each other. Furthermore, the narrow sections can be formed by etching deep enough while maintaining the sidewalls within a predetermined envelope (e.g., tolerance) of distance from each other, such that cells forced through the narrow sections can deform due to the sidewalls, regardless of the location of the cell through the narrow section (e.g., the etching depth). In this way, each deep, narrow rectangular narrow section can have increased individual throughput due to the larger cross-sectional area of ​​the narrow sections, compared to narrow sections with a width for a cell configuration of the same size but not etched as deep into the substrate. Furthermore, since clogging of the narrow section can be easily caused by its edges and corners, a deep, narrow rectangular narrow section may clog at a reduced rate compared to a narrow section with a width for a cell configuration of the same size but not etched as deep into the substrate. In some embodiments, therefore, increasing the cross-sectional area of ​​the narrow section while minimizing its perimeter can increase the throughput per narrow section while minimizing the likelihood and / or extent of clogging.

[0010] Third, the chip disclosed herein can have a geometry configured to ensure sufficiently uniform flow rate and flow pattern as the cell suspension approaches and flows through the plurality of constrictions. In some embodiments, the plurality of constrictions can be arranged in a line, forming a barrier or wall between a first flow region upstream of the plurality of parallel constrictions and a second flow region downstream of the plurality of parallel constrictions. The barrier formed by the plurality of constrictions can be located at a sufficiently large distance from the chip's inlet port, and the length of the barrier can be sufficiently short relative to the distance such that the flow rate and flow pattern through the plurality of constrictions are sufficiently uniform to maintain high throughput and low clogging.

[0011] Fourth, the narrowing section disclosed herein can have a geometry configured to ensure a sufficiently uniform flow pattern within and around the narrowing section and to ensure sufficiently high flow velocities and throughput through the narrowing section. In some embodiments, the narrowing section has an approach region through which fluid flows as it approaches the narrowest point of the narrowing section. The approach region can be defined by one or more tapered walls (such as tapered sidewalls) that taper gradually toward the narrowest point of the narrowing section at a predetermined angle. The predetermined angle can be set in combination with the width of the narrowest point of the narrowing section and other properties of the chip and cell suspension to ensure a uniform flow pattern and sufficient flow velocities and throughput.

[0012] In some embodiments, a first microfluidic chip is provided for delivering a payload to a cell, the first chip comprising: a fluid inlet configured to receive a flow of cell suspension and allow the cell suspension to pass through a first fluid flow region within the microfluidic chip; a plurality of constrictions fluidly connected to the first fluid flow region to allow the cell suspension to flow from the first fluid flow region to a second fluid flow region within the microfluidic chip through one or more of the plurality of constrictions, wherein: the cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cell in the cell suspension, such that the cell membrane is disturbed when passing through the constriction, allowing the payload to pass through the disturbed cell membrane; and; and the quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of constrictions is greater than or equal to 0.5 μm.

[0013] In some embodiments, a first method for delivering a payload to a cell is provided, the first method comprising: receiving a flow of a cell suspension comprising a plurality of cells into a first fluid flow region of a microfluidic chip; and allowing the cell suspension to flow from the first fluid flow region through a plurality of constrictions in the microfluidic chip, wherein: the cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, such that the cell membrane is deformed when passing through the constriction, allowing the payload to pass through the deformed cell membrane; and the quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of constrictions is greater than or equal to 0.5 μm.

[0014] In some embodiments, a second method is provided for manufacturing a microfluidic chip for delivering a payload to cells, the second method comprising: etching into a substrate to form a first fluid flow region configured to allow a cell suspension to flow through the first fluid flow region from an inlet port; and etching into the substrate to form a plurality of constrictions configured to allow the cell suspension to flow through the constrictions from the first fluid flow region, wherein: the cross-sectional width of each of the plurality of constrictions is smaller than the diameter of a cell in the cell suspension, such that the cell membrane is deformed when passing through the constriction, allowing the payload to pass through the deformed cell membrane; and the quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of constrictions is greater than or equal to 0.5 μm.

[0015] In some embodiments, a second microfluidic chip is provided for delivering a payload to cells. The second chip includes: a fluid inlet configured to receive a flow of cell suspension and allow the cell suspension to pass through a first fluid flow region within the microfluidic chip; a first plurality of constrictions fluidly connected to the first fluid flow region to allow the cell suspension to flow from the first fluid flow region to a second fluid flow region within the microfluidic chip through one or more of the first plurality of constrictions; and a second plurality of constrictions fluidly connected to the second fluid flow region to allow the cell suspension to flow from the second fluid flow region to a third fluid flow region within the microfluidic chip through one or more of the second plurality of constrictions, wherein: the cross-sectional width of each of the first plurality of constrictions and the second plurality of constrictions is smaller than the diameter of the cells in the cell suspension, such that the cell membrane is disturbed when passing through the constriction, allowing the payload to pass through the disturbed cell membrane; and the quotient of the cross-sectional area and the cross-sectional perimeter of each of the first plurality of constrictions and the second plurality of constrictions is greater than or equal to 0.5 μm.

[0016] In some embodiments, any one or more of the features, characteristics, or elements discussed above with respect to any embodiment may be incorporated into any other embodiment mentioned above or described elsewhere herein. In some embodiments, any one or more features, characteristics, or elements discussed elsewhere in this disclosure may be incorporated into any one or more of the embodiments mentioned above. Attached Figure Description

[0017] Figures 1A-1C illustrate various views of a chip for delivering payloads to cells according to some embodiments. Figure 1A shows a top view of the chip according to some embodiments and an enlarged highlight of the chip's constriction; Figure 1B shows a top view of the chip's constriction and guide channel according to some embodiments; Figure 1C shows a cross-sectional view of the chip's constriction according to some embodiments.

[0018] Figures 2A and 2B illustrate the fabrication steps of a chip for delivering payloads to cells according to some embodiments. Figure 2A shows the narrowing after the etching step according to some embodiments; Figure 2B shows the narrowing after the deposition step according to some embodiments.

[0019] Figures 3A-3C show various views of the flow lines at different magnifications according to some embodiments, illustrating the flow patterns of cell suspensions passing through a chip to deliver payloads to cells, wherein the chip does not have angled proximity areas.

[0020] Figure 4 illustrates a flow pattern of a cell suspension passing through a chip to deliver a payload to the cell, according to some embodiments, wherein the chip has an angled proximity region.

[0021] Figures 5A-5C illustrate streamlines showing flow patterns of various cell suspensions through different embodiments of a chip for delivering payloads to cells, wherein the embodiments have access regions defined by different corresponding angles (or, in the case of Figure 5C, the embodiments do not have angled access regions adjacent to the constriction).

[0022] Figures 6A-6C illustrate the flow rates of various cell suspensions through different embodiments of a chip for delivering payloads to cells, wherein the embodiments have access regions defined by different corresponding angles (or, in the case of Figure 5I, the embodiment does not have an angled access region adjacent to the narrowing).

[0023] Figure 7 illustrates a chip for delivering a payload to a cell according to some embodiments, wherein the chip has multiple sets of parallel narrow sections, and wherein the sets are connected in series with each other.

[0024] Figure 8 illustrates methods for delivering payloads to cells according to some implementation schemes.

[0025] Figure 9 shows a holding chip for delivering payloads to cells according to some embodiments.

[0026] Figures 10A-10C illustrate various chips for delivering payloads to cells, wherein the chips have fluid flow regions with various geometries.

[0027] Figure 11 shows experimental delivery profile data for a chip used to deliver payloads to cells according to some implementation schemes.

[0028] Figure 12 shows experimental delivery profile data for a chip used to deliver payloads to cells according to some implementation schemes.

[0029] Figures 13A-13O show schematic information of chips according to some embodiments and experimental data from test chips with multiple sets of parallel narrow sections, wherein the multiple sets are connected in series with each other. Detailed Implementation

[0030] The following discloses a high-throughput microfluidic chip for use in a system for delivering payloads to cells in a cell suspension. The chip includes multiple parallel constrictions (e.g., constriction channels) through which the cell suspension can be forced under pressure. The cells in the cell suspension can deform due to the cell deformation constrictions of the microfluidic chip, and their membranes are perturbed. As explained herein, the throughput and clogging properties of the chip can be improved due to several characteristics of the chip disclosed herein.

[0031] The following descriptions of Figures 1A-1C illustrate exemplary embodiments of a microfluidic chip having parallel constrictions for intracellular payload delivery. Figures 2A-2B then describe the fabrication of the constrictions in the microfluidic chip for intracellular payload delivery. Figures 3A-3C, 4, 5A-5C, and 6A-6C then describe various geometries of the proximity region adjacent to the constrictions of the chip for intracellular payload delivery. Figure 7 then describes an exemplary embodiment of a microfluidic chip having multiple sets of parallel constrictions, wherein said multiple sets are connected in series. Figure 8 then describes methods of using chips such as those disclosed herein. As described below, in some embodiments, the chip and / or chip geometry described with reference to Figures 1-7 can be used in method 800. Figure 9 then describes a holding chip for delivering payloads to cells according to some embodiments. Figures 10A-10C then describe chips with fluid flow regions having various geometries. The descriptions of Figures 11, 12, and 13A-13O then refer to experimental data from several examples described herein.

[0032] The following description illustrates exemplary systems, methods, techniques, parameters, etc. However, it should be understood that such description is not intended to be a limitation on the scope of this disclosure, but is intended to be provided as a description of exemplary embodiments.

[0033] definition

[0034] For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any definition listed below conflicts with any document incorporated herein by reference, the listed definition shall prevail.

[0035] As used herein, unless otherwise indicated, the singular forms “a / an” and “the” include plural indicators.

[0036] It should be understood that the aspects and embodiments of the invention described herein include “comprising aspects and embodiments,” “consisting of aspects and embodiments,” and / or “substantially consisting of aspects and embodiments.”

[0037] To be further understood, the terms “includes” and / or “comprises” specify the presence of the said feature, integer, step, operation, element and / or component, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0038] Depending on the context, the term "if" can be interpreted as "when," "based on," "in response to determination," or "in response to detection." Similarly, depending on the context, the phrase "if determined" or "if [the condition or event of the statement] is interpreted as "based on determination," "in response to determination," "based on the detection of [the condition or event of the statement]," or "in response to the detection of [the condition or event of the statement]."

[0039] As used herein, the term "about" refers to a commonly known range of error for a corresponding value that is readily known to those skilled in the art. When this document refers to a value or parameter as "about," it includes (and describes) the implementation scheme involving that value or parameter itself.

[0040] Although the terms “first,” “second,” etc., are used in this description to describe various elements, these elements should not be limited by the terms. These terms are only used to distinguish one element from another.

[0041] For any structural and functional features described herein, the methods for determining these features are known in the art.

[0042] All references cited in this article (including patent applications and publications) are incorporated in their full text by way of citation.

[0043] Microfluidic chip geometry for intracellular payload delivery

[0044] The following describes a microfluidic chip used to increase throughput and reduce cell blockage.

[0045] Figures 1A-1C illustrate various views of a chip 100 for delivering payloads to cells according to some embodiments. Figure 1A shows a top view of the chip 100 according to some embodiments and an enlarged protrusion of the chip's constriction; Figure 1B shows a top view of the chip 100's constriction and guide channel according to some embodiments; Figure 1C shows a cross-sectional view of the chip 100's constriction according to some embodiments.

[0046] The microfluidic chip 100 is configured to receive a fluid flow at a fluid inlet 110. The fluid may include a cell suspension. In some embodiments, the fluid may include a payload for delivery into the cell, wherein the payload may include any suitable payload for delivery into the cell.

[0047] For the purposes of this description, the orientation and dimension of chip 100 can be referred to by the following conventions: the x-direction or x-dimension can refer to the horizontally extending dimension in Figure 1A (the x-direction is the overall flow direction of fluid through chip 100 from the inlet to the outlet); the y-direction or y-dimension can refer to the vertically extending dimension in Figure 1A; and the z-direction or z-dimension can refer to the dimension extending into and out of the page in Figure 1A.

[0048] In some embodiments, chip 100 may be a microfluidic chip configured to guide fluid flow through one or more constrictions (e.g., constriction channels) that are narrow enough in at least one dimension to deform cells forced to pass through the constrictions under pressure. The deformation of the cells as they pass through the constrictions under pressure can cause perturbation of the cell membrane, allowing payloads suspended in the cell suspension (either suspended in the suspension before passing through the constrictions or added to the suspension afterward) to enter the cell through the perturbated cell membrane.

[0049] In the example of chip 100, fluid flowing through chip 100 (e.g., cell suspension) can flow into chip 100 via inlet port 102 and can flow from inlet port 102 into first fluid flow region 104. Fluid can flow from first fluid flow region 104 (e.g., by being forced under pressure) through multiple parallel constrictions 106 and into second fluid flow region 108. Fluid can then flow out of chip 100 from second fluid flow region 108 via outlet port 110. The distance between fluid inlet port 102 and fluid outlet port 110 can extend in a direction perpendicular to the planar surface (e.g., inner surface) of the substrate of chip 100. The distance between fluid inlet port 102 and fluid outlet port 110 can be 5 mm to 30 mm, 8 mm to 25 mm, or 10 mm to 20 mm. In some embodiments, the distance between the fluid inlet port 102 and the fluid outlet port 110 can be less than or equal to 450 mm, 250 mm, 100 mm, 50 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, or 8 mm. In some embodiments, the distance between the fluid inlet port 102 and the fluid outlet port 110 can be greater than or equal to 5 mm, 8 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 50 mm, 100 mm, 250 mm, or 450 mm. In some embodiments, the fluid inlet port 102 and the fluid outlet port 110 of the microfluidic chip 100 can be interchanged.

[0050] In some embodiments, the first fluid flow region 104, the narrowing portion 106, and the second fluid flow region 108 can all be formed as recessed spaces within a substrate, such as a silicon substrate. In some embodiments, etching can be performed in the z-direction by etching down into the top surface of the substrate. In some embodiments, wet or dry etching can be used. In some embodiments, deep reactive ion etching (DRIE) can be used. After etching into the substrate to define the recessed spaces forming the first fluid flow region 104, the narrowing portion 106, and / or the second fluid flow region 108, a top layer can be attached to the substrate layer to surround regions 104 and 108 and the narrowing portion 106 in the z-direction.

[0051] In some embodiments, the inlet port 102 and / or the outlet port 110 may be formed as an opening in the top layer; in some embodiments, the inlet port 102 and / or the outlet port 110 may be formed as an opening in an etched substrate layer, such as an opening through the bottom of the substrate layer in the z direction, or an opening through the side of the substrate in the x and / or y directions.

[0052] In some embodiments, chip 100 may include pillars 120 that may extend through a first fluid flow region 104 and / or a second fluid flow region 108. Pillars 120 may serve as a support structure supporting the top layer of chip 100 and / or a support structure to which the top layer of chip 100 may be joined. In some embodiments, the shape and / or placement of pillars 120 may be selected to minimize disruption to the uniform flow pattern and velocity of all portions of the boundary formed by the narrowing 106 between the first fluid flow region 104 and the second fluid flow region 108; for example, the y-direction width of pillar 120 may be less than its x-direction length to provide sufficient xy-surface area for support and / or joining without excessively increasing the y-direction width and obstructing fluid flow in the x-direction.

[0053] In some implementations, one or more additional and / or alternative support mechanisms may be used, such as rods or pillars formed separately from the substrate and / or external support mechanisms, such as one or more backsheets that may cover the front and / or rear sides of the chip 100.

[0054] The enlarged circular view in Figure 1A shows an enlarged view of a subset of the plurality of narrow sections 106 of the chip 100. As shown, the narrow sections 106 can be arranged parallel to each other such that they collectively form the boundary between the first fluid flow region 104 and the second fluid flow region 108. In the example of the chip 100, the narrow sections 106 are arranged side by side in a straight line (extending in the y-direction). In some embodiments, a group of narrow sections in a parallel flow arrangement can form a boundary with a curved shape and / or a linear shape. In some embodiments, the shape of the boundary can extend diagonally through the chip 100 in the xy-plane, thereby increasing its total length. In some embodiments, the shape of the boundary can extend through the chip 100 in the xy-plane at a serpentine or stepped angle, thereby increasing its total length.

[0055] Figure 1B shows an enlarged partial top view of chip 100, illustrating four constrictions 106 of chip 100. As shown in the enlarged circular view in Figure 1A, the constrictions 106 in Figure 1B are arranged side-by-side in a straight line (extending in the y-direction), providing corresponding flow paths from a first fluid flow region 104 on the left to a second fluid flow region 108 on the right. In the illustration of Figure 1B, the uppermost (in the y-direction) and surrounding areas of the four constrictions are annotated to show the shape and extent of the various regions of chip 100. The annotated areas include the constrictions 106, the upstream proximity region 112a, the downstream proximity region 112b, the upstream guide channel 114a, and the downstream guide channel 114b.

[0056] Figure 1C shows an enlarged partial cross-sectional view of chip 100 (showing the cross-sectional zy plane), illustrating three narrow sections 106 of chip 100. As shown in Figures 1A and 1B, they are arranged side-by-side in a straight line (extending in the y-direction), providing corresponding flow paths from the first fluid flow region 104 to the second fluid flow region 108. As shown in Figure 1C, the narrow sections 106 may have a height (e.g., etch depth) extending in the z-direction, for example, extending downward into the substrate from the top side in the z-direction. In some embodiments, the height of the narrow sections 106 may be the same as the height of one or more of the corresponding upstream access region 112a, the corresponding downstream access region 112b, the corresponding upstream guide channel 114a, and / or the corresponding downstream guide channel 114b.

[0057] In some implementations, the dimensions of any one or more components shown in Figure 1B and / or Figure 1C may be selected to increase throughput, reduce congestion, and maintain the effectiveness of payload delivery and cell viability after delivery.

[0058] In some embodiments, the narrowing portion 106 may have a cross-sectional narrowing width (in the y-direction as shown in FIG. 1B) greater than or equal to 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. In some embodiments, the cross-sectional narrowing width (in the y-direction as shown in FIG. 1B) may be less than or equal to 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. In some embodiments, the cross-sectional narrowing width may have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm. In some embodiments, the cross-sectional narrowing width may be set and / or selected based on the diameter of the cell (e.g., the diameter in the suspension) that will be disturbed by passing through the narrowing portion. For example, the range described herein can be configured to process cells with diameters between approximately 1.4 μm and approximately 10 μm when in suspension. Given the disclosure herein, those skilled in the art will understand that different cross-sectional narrowing widths (and corresponding other chip sizes) can be used when processing cells of other diameters. For example, the processing of mononuclear cells can be accomplished with a cross-sectional narrowing width greater than 10 μm; even larger cells can be processed with a cross-sectional narrowing width up to approximately 0.1 mm.

[0059] In some embodiments, the narrowed portion 106 may have a narrowed portion length (in the x direction shown in FIG1B) greater than or equal to 2.5 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 500 μm, 1 mm, 5 mm, 1 cm, or 2 cm. In some embodiments, the narrowed portion length (in the x direction shown in FIG1B) may be less than or equal to 2.5 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 500 μm, 1 mm, 5 mm, 1 cm, or 2 cm. In some implementations, the length of the narrow section can have a tolerance of ±2μm, ±1.5μm, ±1μm, or ±0.5μm.

[0060] In some embodiments, the narrowed portion 106 may have a narrowed portion height (in the z-direction as shown in FIG1C) greater than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the narrowed portion height may be less than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the narrowed portion height may have a tolerance of ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0061] In some embodiments, the narrowing portion 106 may have a sidewall draft angle, wherein the sidewall refers to one of the walls that extend vertically in the z-direction and define the width of the narrowing portion, as shown in FIG1C. In some embodiments, the sidewall draft angle may be greater than or equal to 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, or 0.06°. In some embodiments, the sidewall draft angle may be less than or equal to 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, or 0.06°. In some embodiments, the sidewall draft angle may have a tolerance of ±0.02°, ±0.01°, or ±0.005°. In some embodiments, the sidewall draft angle may be greater than or equal to 1°, 2°, 5°, or 10°; in some embodiments, the sidewall draft angle may be less than or equal to 1°, 2°, 5°, or 10°. The permissible range of sidewall draft angles can depend on the height of the z-dimensional narrowing, because for a given draft angle, a higher (deeper) narrowing may exhibit a greater variation in the width of the cross-section narrowing than a shorter (shallower) narrowing. (If the variation in narrowing width is too significant, fluid flow will pass through the wider portion of the narrowing rather than the narrower portion, thus avoiding the narrower part of the channel and potentially allowing fluid flow through the narrowing without disturbing the cell membrane.) Therefore, a chip with a shorter (shallower) z-dimensional narrowing height can have a larger sidewall draft angle than a chip with a higher (deeper) z-dimensional narrowing height.

[0062] In some embodiments, the narrowed portion 106 may have a sidewall roughness. In some embodiments, the sidewall roughness may be greater than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, 0.05 μm, 0.01 μm, or 0.001 μm. In some embodiments, the sidewall roughness may be less than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, 0.05 μm, 0.01 μm, or 0.001 μm.

[0063] In some embodiments, the access region 112a or 112b may include a narrow end and a wide end, wherein the narrow end is adjacent to a corresponding constriction 106, and the wide end is adjacent to a corresponding guide channel 114a or 114b. The access region may have sidewalls comprising linear walls that form an angle between the opening at the narrow end and the opening at the wide end, defining an angle greater than 0° and less than 90° with respect to a straight line in the x-direction. The angle formed between the sidewalls of the access regions may be greater than 0° and less than 180°. In some embodiments, the sidewalls of the access region may include one or more curved portions (e.g., defining a serpentine shape when viewed overhead in the z-direction) and / or corners (e.g., defining a stepped shape when viewed overhead in the z-direction) between the narrow and wide ends.

[0064] In some embodiments, the cross-sectional approach width (in the y-direction as shown in Figure 1B) at the narrow end can be greater than or equal to 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. In some embodiments, the cross-sectional approach width (in the y-direction as shown in Figure 1B) at the narrow end can be less than or equal to 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. In some embodiments, the cross-sectional approach width at the narrow end can have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm.

[0065] In some embodiments, the cross-sectional area width at the wide end (in the y-direction as shown in Figure 1B) can be greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, the cross-sectional area width at the wide end (in the y-direction as shown in Figure 1B) can be less than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, the cross-sectional area width at the wide end can have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm.

[0066] In some embodiments, the approach region length (in the x-direction as shown in Figure 1B) can be greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, the narrowing length can have a tolerance of ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0067] In some embodiments, the proximity region height (in the z-direction as shown in Figure 1C) can be greater than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the proximity region height (in the z-direction as shown in Figure 1C) can be less than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the proximity region height can have a tolerance of ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm. In some embodiments, the proximity region height can be any suitable height in the z-direction that is less than the height of the substrate.

[0068] In some embodiments, the approach regions 112a or 112b may have sidewall draft angles, wherein the sidewall refers to one of the walls extending vertically in the z-direction and defining the width of the approach region. In some embodiments, the sidewall draft angle of the approach region may be greater than or equal to 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, or 0.06°. In some embodiments, the sidewall draft angle of the approach region may be less than or equal to 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, or 0.06°. In some embodiments, the sidewall draft angle of the approach region may have a tolerance of ±0.02°, ±0.01°, or ±0.005°. In some embodiments, the sidewall draft angle may be greater than or equal to 1°, 2°, 5°, or 10°; in some embodiments, the sidewall draft angle may be less than or equal to 1°, 2°, 5°, or 10°. The allowable range of the sidewall draft angle can depend on the height of the approach region in the z-dimension, in a manner similar to that discussed above regarding the sidewall draft angle and height of the narrow section.

[0069] In some embodiments, the proximity regions 112a or 112b may have sidewall roughness. In some embodiments, the sidewall roughness of the proximity regions may be greater than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, or 0.05 μm. In some embodiments, the sidewall roughness of the proximity regions may be less than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, or 0.05 μm.

[0070] In some embodiments, the approach area may have sidewalls extending from the wide end of the approach area to the narrow end of the approach area. In some embodiments, the opposing sidewalls of the approach area may form an angle greater than or equal to 160°, 140°, 120°, 100°, 80°, 60°, 40°, or 20° with each other. In some embodiments, the opposing sidewalls of the approach area may form an angle less than or equal to 160°, 140°, 120°, 100°, 80°, 60°, 40°, or 20° with each other. In some embodiments, the sidewall of the approach area may form an angle less than or equal to 80°, 70°, 60°, 50°, 40°, 30°, 20°, or 10° with the sidewall of the adjacent narrowing portion. In some embodiments, the sidewall of the approach area may form an angle greater than or equal to 80°, 70°, 60°, 50°, 40°, 30°, 20°, or 10° with the sidewall of the adjacent narrowing portion.

[0071] In some implementations, the guide channel 114a or 114b may extend between the end near the corresponding access area (112a or 112b) and the opposite end away from the corresponding access area.

[0072] In some embodiments, the cross-sectional guide channel width (in the y-direction as shown in Figure 1B) can be greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, the cross-sectional guide channel width (in the y-direction as shown in Figure 1B) can be less than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, the cross-sectional guide channel width can have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm.

[0073] In some embodiments, the guide channel length (in the x-direction shown in Figure 1B) can be greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, the guide channel length can have a tolerance of ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0074] In some embodiments, the guide channel height (in the z-direction as shown in Figure 1C) can be greater than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the guide channel height (in the z-direction as shown in Figure 1C) can be less than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the guide channel height can have a tolerance of ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm. In some embodiments, the guide channel height can be any suitable height in the z-direction that is less than the height of the substrate.

[0075] In some embodiments, guide channels 114a or 114b may have sidewall draft angles, wherein the sidewall refers to one of the walls that extends vertically in the z-direction and defines the width of the guide channel. In some embodiments, the sidewall draft angle of the guide channel may be greater than or equal to 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, or 0.06°. In some embodiments, the sidewall draft angle of the proximity area may be less than or equal to 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, or 0.06°. In some embodiments, the sidewall draft angle of the guide channel may have a tolerance of ±0.02°, ±0.01°, or ±0.005°.

[0076] In some embodiments, guide channels 114a or 114b may have sidewall roughness. In some embodiments, the sidewall roughness of the guide channels may be greater than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, or 0.05 μm. In some embodiments, the sidewall roughness of the guide channels may be less than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, or 0.05 μm.

[0077] In some embodiments, adjacent parallel narrowing portions (and / or corresponding proximity regions or guide channels) may be offset from each other in the y-direction by a spacing 116 less than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm.

[0078] In some embodiments, adjacent parallel narrowed portions may be separated from each other in the y-direction by a narrowed portion sidewall thickness 118 less than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, adjacent parallel narrowed portions may be separated from each other in the y-direction by a narrowed portion sidewall thickness 118 greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm.

[0079] In some embodiments, adjacent parallel guide channels may be separated from each other in the y-direction by a guide channel sidewall thickness 122 of less than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, or 250 μm. In some embodiments, adjacent parallel guide channels may be separated from each other in the y-direction by a guide channel sidewall thickness 122 of greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, or 250 μm.

[0080] In some embodiments, the substrate forming the first flow region 104, the narrowing portion 106, and / or the second flow region 108 may have a thickness (in the z-direction) of less than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 5 mm. In some embodiments, the substrate forming the first flow region 104, the narrowing portion 106, and / or the second flow region 108 may have a thickness (in the z-direction) greater than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 5 mm. A substrate thickness greater than or equal to about 400 μm can help reduce the risk of substrate breakage.

[0081] In some embodiments, the top layer of chip 100 (e.g., a layer placed on top of the substrate after etching the substrate layer) may have a thickness (in the z-direction) of less than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 5 mm. In other embodiments, the top layer of chip 100 (e.g., a layer placed on top of the substrate after etching the substrate layer) may have a thickness (in the z-direction) greater than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 5 mm.

[0082] In some embodiments, inlet port 102 and / or outlet port 110 may be formed as openings with a diameter less than or equal to 200 μm, 400 μm, 500 μm, 1 mm, or 5 mm. In some embodiments, inlet port 102 and / or outlet port 110 may be formed as openings with a diameter greater than or equal to 200 μm, 400 μm, 500 μm, 1 mm, or 5 mm. In some embodiments, the opening may be circular. In some embodiments, the opening may have any shape, and its dimension in the y-direction, x-direction, or another direction in the xy-plane may be less than or equal to any of the aforementioned diameters; in some embodiments, the opening may have any shape, and its dimension in the y-direction, x-direction, or another direction in the xy-plane may be greater than or equal to any of the aforementioned diameters.

[0083] In some embodiments, all or part of the first fluid region 104 and / or all or part of the second fluid region 108 may have a height in the z-direction greater than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm (e.g., the etch depth into the substrate of the chip 100). In some embodiments, the height (in the z-direction) of all or part of the first fluid region 104 and / or all or part of the second fluid region 108 may be less than or equal to 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, or 120 μm. In some embodiments, the height of the first fluid region 104 and / or the height of the second fluid region 108 may have tolerances of ±3μm, ±2μm, ±1.5μm, ±1μm or ±0.5μm.

[0084] In some embodiments, any one or more narrow sections 106 can be characterized by the quotient of the area of ​​the narrow section and the perimeter of the narrow section. In some embodiments, the quotient of the area of ​​the narrow section and the perimeter of the narrow section can be greater than or equal to 0.5 μm, 0.75 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.25 μm, 1.5 μm, 2 μm, 3 μm, or 5 μm. In some embodiments, the quotient of the area of ​​the narrow section and the perimeter of the narrow section can be less than or equal to 0.5 μm, 0.75 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.25 μm, 1.5 μm, 2 μm, 3 μm, or 5 μm. In some embodiments, narrow sections with a higher quotient are less prone to clogging than narrow sections with a lower quotient. In some embodiments, for a given narrowing width (which may be determined by the cell diameter (e.g., the diameter of a cell in a suspension) of the cells (multiple) disturbed by passing through the narrowing), a narrowing with a higher quotient is less prone to clogging than a narrowing with a lower quotient. In some embodiments, increasing the quotient can be achieved by etching a deep, narrow rectangular narrowing (e.g., a slit-shaped narrowing) into a substrate (e.g., a silicon substrate) as described herein. The narrowing can be formed by etching sufficiently deep and / or depositing a narrowing sidewall material while maintaining the narrowing sidewalls within a sufficient angular threshold of parallelness as described herein. Furthermore, the narrowing can be formed by etching sufficiently deep and / or depositing a narrowing sidewall material while maintaining the sidewalls within a predetermined envelope (e.g., a tolerance) of distance from each other, such that cells forced through the narrowing can deform due to the sidewalls, regardless of the location of the cell passing through the narrowing (e.g., the etching depth). In this way, each deep, narrow rectangular narrow section can have increased individual flux due to its larger cross-sectional area, compared to a narrow section with the width of a cell configured to perturb the same diameter but not etched as deep into the substrate. Furthermore, since clogging of the narrow section can be caused by its edges and corners, a deep, narrow rectangular narrow section may clog at a reduced rate compared to a narrow section with the width of a cell configured to perturb the same diameter but not etched as deep into the substrate. In some embodiments, therefore, increasing the cross-sectional area of ​​the narrow section while minimizing its perimeter can increase the flux per narrow section while minimizing the likelihood and / or extent of clogging.

[0085] In some embodiments, the total surface area of ​​the column 120 surrounded by the first fluid flow region 104 in the xy plane can be 5-10% of the area of ​​the first fluid flow region 104 itself in the xy plane. In some embodiments, the total surface area of ​​the column 120 surrounded by the first fluid flow region 104 in the xy plane can be less than or equal to 1%, 5%, 10%, 15%, 20%, 25%, or 50% of the area of ​​the first fluid flow region 104 itself in the xy plane. In some embodiments, the total surface area of ​​the column 120 surrounded by the first fluid flow region 104 in the xy plane can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, or 50% of the area of ​​the first fluid flow region 104 itself in the xy plane.

[0086] In some embodiments, the total surface area of ​​the column 120 surrounded by the second fluid flow region 108 in the xy plane can be 5-10% of the area of ​​the second fluid flow region 108 itself in the xy plane. In some embodiments, the total surface area of ​​the column 120 surrounded by the second fluid flow region 108 in the xy plane can be less than or equal to 1%, 5%, 10%, 15%, 20%, 25%, or 50% of the area of ​​the second fluid flow region 108 itself in the xy plane. In some embodiments, the total surface area of ​​the column 120 surrounded by the first fluid flow region 108 in the xy plane can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, or 50% of the area of ​​the first fluid flow region 108 itself in the xy plane.

[0087] In some embodiments, the total chip length of chip 100 in the x-direction can be greater than or equal to 2mm, 4mm, 10mm, 20mm, 30mm, 40mm, 50mm, 100mm, 250mm, 450mm, or 500mm. In other embodiments, the total chip length of chip 100 in the x-direction can be less than or equal to 2mm, 4mm, 10mm, 20mm, 30mm, 40mm, 50mm, 100mm, 250mm, 450mm, or 500mm.

[0088] In some embodiments, the total chip width of chip 100 in the y-direction can be greater than or equal to 2mm, 4mm, 10mm, 20mm, 30mm, 40mm, 50mm, 100mm, 250mm, 450mm, or 500mm. In other embodiments, the total chip width of chip 100 in the y-direction can be less than or equal to 2mm, 4mm, 10mm, 20mm, 30mm, 40mm, 50mm, 100mm, 250mm, 450mm, or 500mm.

[0089] In some embodiments, the total chip height (e.g., thickness) of chip 100 in the z-direction can be less than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, 3 mm, or 5 mm. In some embodiments, the total chip height (e.g., thickness) of chip 100 in the z-direction can be greater than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, 3 mm, or 5 mm. In some embodiments, the total chip height can be between 400 μm and 3 mm.

[0090] In some embodiments, the etched regions forming the first fluid flow region 104 and the second fluid flow region 108 may have a combined surface area (e.g., in the xy plane) that is less than or equal to 75%, 50%, 40%, 30%, 20%, or 10% of the surface area of ​​the substrate into which the microfluidic chip and / or the flow regions are etched. In some embodiments, the etched regions may have a combined surface area greater than or equal to 75%, 50%, 40%, 30%, 20%, or 10% of the surface area of ​​the substrate into which the microfluidic chip and / or the flow regions are etched.

[0091] In some embodiments, chip 100 may have a substrate (e.g., a silicon substrate) to which a narrow portion is etched, the substrate having a substrate thickness in the z-direction of less than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 3 mm. In some embodiments, chip 100 may have a substrate (e.g., a silicon substrate) to which a narrow portion is etched, the substrate having a substrate thickness in the z-direction of greater than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 3 mm.

[0092] In some embodiments, the set of parallel narrowing portions may include 1001 to 1500 narrowing portions, 1501 to 2000 narrowing portions, 2001 to 2500 narrowing portions, 2501 to 5000 narrowing portions, or 5001 to 10000 narrowing portions. In some embodiments, the set of parallel narrowing portions 106 in chip 100 may include more than or equal to 1, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, or 5000 parallel narrowing portions. In some embodiments, the set of parallel narrowing portions 106 in chip 100 may include fewer than or equal to 1, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, or 5000 parallel narrowing portions. As disclosed herein, chips with a relatively large number of parallel constrictions can achieve high volumetric flow rates, high cell throughput, and reduced clogging of cell suspensions.

[0093] In some embodiments, chip 100 may include one or more components made of metal, plastic, polymer, and / or glass. In some embodiments, the substrate of chip 100 may include silicon. In some embodiments, the top layer of chip 100 (e.g., a layer bonded or attached to the substrate) may include glass and / or quartz.

[0094] In the example shown in Figure 1B, from the top view depicted in Figure 1B, the narrowing portion 106 has a rectangular cross-sectional shape. Therefore, the narrowing portion 106 shown in Figure 1B has a uniform width of cross-section at all points along its length (in the x-direction) (in the y-direction, as shown in Figure 1B). In some other or alternative embodiments, the narrowing portion (such as narrowing portion 106) may have a trapezoidal, curved, stepped, or other irregular cross-sectional shape, as seen from the top view depicted in Figure 1B.

[0095] For example, in some embodiments, the narrowing may have a tapered shape, rather than a uniform cross-sectional narrowing width (in the y-direction as shown in FIG. 1B), such that its cross-sectional width in the y-direction increases or decreases (linearly or otherwise) along the length of the narrowing (in the x-direction). In some embodiments, the cross-sectional width of the tapered narrowing may increase or decrease by about 1%, 2%, 3%, 5%, 10%, 25%, 50%, 100%, 200%, or 500% along the entire length of the narrowing (from any cross-sectional narrowing width given herein).

[0096] In some embodiments, the narrowing may have multiple segments of narrowing width, rather than a uniform cross-sectional narrowing width (in the y-direction as shown in FIG. 1B), wherein the narrowing has a stepped shape, and wherein the multiple segments of the narrowing (in the x-direction as shown in FIG. 1B) have cross-sectional widths different from one or more of the other segments. For example, in some embodiments, the narrowing may have two segments, wherein the first segment is narrower than the second segment, or wherein the second segment is narrower than the first segment. The cross-sectional width of the narrowing segments may monotonically increase in the flow direction of the narrowing, may monotonically decrease in the flow direction of the narrowing, or may increase and decrease in the flow direction of the narrowing (e.g., in the case of a narrowing having three or more narrowing segments). The segments may be distinguished from each other by steps (in the y-direction) formed in one or two narrowing walls. The steps between the narrowing segments may be formed as right-angled steps and / or may be formed by tapered transition regions between the steps.

[0097] In some embodiments, the cross-sectional narrowing width of any one or more narrowing segments may be equal to any cross-sectional narrowing width disclosed herein. In some embodiments, adjacent narrowing segments may have cross-sectional narrowing widths that differ from each other by about 1%, 2%, 3%, 5%, 10%, 25%, 50%, 100%, 200%, or 500%.

[0098] In some embodiments, the length of any one or more narrowing segments may be equal to any narrowing length disclosed herein. Alternatively, a set of narrowing segments forming the entire narrowing may have a length equal to the sum of any narrowing lengths disclosed herein.

[0099] In some embodiments, adjacent narrowed segments are separated by a tapered narrowed transition region, the length of which may be equal to about 0.1%, 0.5%, 1%, 2%, 3%, 5%, 10%, 25%, 50%, or 100% of any narrowed length disclosed herein.

[0100] In some embodiments, a narrowing with a non-uniform (e.g., tapered) cross-sectional width and / or a narrowing consisting of multiple segments with different cross-sectional widths can allow the chip to extend the amount of time that cells passing through the narrowing are subjected to pressure from the narrowing wall, thereby extending the amount of time that pores in the cell wall are open and improving payload delivery efficiency and effectiveness. In some embodiments, a narrowing with an initial narrower portion followed by a wider portion can allow for rapid and efficient pore formation in the initial portion and allow the pores to remain open as cells pass through the wider portion. Compared to implementations where the entire narrowing has a narrower width, using a wider narrowing followed by a narrower narrowing can achieve efficient payload delivery while increasing cell viability.

[0101] Figures 2A and 2B illustrate the manufacturing steps of a chip for delivering a payload to a cell according to some embodiments. Specifically, Figure 2A shows a partial cross-sectional view of a narrowed portion of the chip (e.g., narrowed portion 106 of chip 100) after an etching step according to some embodiments; Figure 2B shows a partial cross-sectional view of a narrowed portion of the chip after a deposition step performed after the etching step according to some embodiments.

[0102] As shown in Figure 2A, the etching step for forming a narrow portion in a chip may include etching downwards (e.g., downwards in the z-direction) into the substrate of the chip to remove material from the substrate to form a channel therein. In some embodiments, the etching step may include dry etching and / or wet etching. In some embodiments, the etching step may include deep reactive ion etching.

[0103] In some implementations, the etched channel may have one or more dimensions (e.g., height in the z-direction and / or width in the y-direction) that are larger than the target size of the narrowing at the end of the manufacturing process (e.g., after deposition).

[0104] As shown in Figure 2B, after the etching step, a deposition step can be performed, in which a material layer is deposited onto the etched substrate. In some embodiments, the deposited material may include SiO, plastic, glass, silicon-derived materials, plastic-derived materials, glass-derived materials, metals (e.g., gold, silver, stainless steel, and / or aluminum), and / or metal-derived materials. In some embodiments, the deposition of this material layer may cause the etched channel to narrow due to the material deposited on the vertical (z-direction) sidewalls of the channel, and the channel may be narrowed by this deposition process to form a narrowed portion with a desired narrowed portion size.

[0105] In some embodiments, the deposited layer may have a thickness greater than or equal to 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, or 2 μm. In some embodiments, the deposited layer may have a thickness less than or equal to 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, or 2 μm.

[0106] In some implementations, the narrowing after the deposition step may have one or more dimensions that are the same as any of the dimensions of the narrowing 106 described with reference to Figures 1A-1C.

[0107] The flow properties of microfluidic chips disclosed in this article

[0108] The following describes various fluid flow characteristics of the fluid flowing through the microfluidic chip described herein, as well as various chip properties that may affect these fluid flow characteristics. Specifically, pressure, shear stress, shear rate, cell flow rate, cell volumetric flow rate, cell blockage rate, and cell flux are provided below.

[0109] As explained above, the microfluidic chip provided herein is designed to operate under pressure to ensure that cells in a cell suspension are forced from an upstream fluid flow region through a constriction to a downstream fluid flow region, causing disturbance of the cell membrane as it passes through the constriction. In some embodiments, the microfluidic chip 100 may be configured to operate at pressures of 1 PSI to 200 PSI, 10 PSI to 150 PSI, or 25 PSI to 100 PSI. In some embodiments, the microfluidic chip 100 may be configured to operate at pressures less than or equal to 1 PSI, 5 PSI, 10 PSI, 25 PSI, 50 PSI, 75 PSI, 100 PSI, 125 PSI, 150 PSI, or 200 PSI. In some implementations, the microfluidic chip 100 can be configured to operate at pressures greater than or equal to 1 PSI, 5 PSI, 10 PSI, 25 PSI, 50 PSI, 75 PSI, 100 PSI, 125 PSI, 150 PSI, or 200 PSI.

[0110] In some embodiments, the microfluidic chip 100 can provide a fluid flow rate greater than or equal to 0.5 m / s, 1 m / s, 5 m / s, 10 m / s, 15 m / s, 20 m / s, 25 m / s, 30 m / s, 40 m / s, or 50 m / s through the narrowed portion of the chip. In other embodiments, the microfluidic chip 100 can provide a fluid flow rate less than or equal to 0.5 m / s, 1 m / s, 5 m / s, 10 m / s, 15 m / s, 20 m / s, 25 m / s, 30 m / s, 40 m / s, or 50 m / s through the narrowed portion of the chip.

[0111] As explained above, the microfluidic chip presented in this paper is specifically designed for high throughput. Due to the specific constriction geometry, the number of cells that can pass through the constriction (and be perturbed by it) in a given time unit is greater than that of known microfluidic chips.

[0112] In some embodiments, the microfluidic chip 100 can provide a volumetric flow rate through a single constriction of the chip greater than or equal to 0.5 μL / min, 1 μL / min, 10 μL / min, 100 μL / min, 500 μL / min, 1 mL / min, or 5 mL / min. In other embodiments, the microfluidic chip 100 can provide a volumetric flow rate through a single constriction of the chip less than or equal to 0.5 μL / min, 1 μL / min, 10 μL / min, 100 μL / min, 500 μL / min, 1 mL / min, or 5 mL / min.

[0113] In some embodiments, the microfluidic chip 100 can provide a total volumetric flow rate greater than or equal to 0.5 mL / min, 1 mL / min, 10 mL / min, 100 mL / min, 500 mL / min, 1 L / min, or 5 L / min (combined on all parallel constrictions 106).

[0114] In some embodiments, the microfluidic chip 100 can provide a cell throughput rate through the constricted portion of the chip of less than or equal to 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 30 mL / min, or 50 mL / min.

[0115] In some embodiments, the microfluidic chip 100 can provide a total cell throughput of less than or equal to 5 mL / min, 10 mL / min, 50 mL / min, 100 mL / min, 200 mL / min, or 500 mL / min.

[0116] The microfluidic chip described herein has been designed to minimize cell clogging in fluid flow regions and / or constrictions. According to the embodiments provided herein, the number of cells required to clog the microfluidic chip depends on the cross-sectional area of ​​the constriction, the particle / cell diameter, and the particle / cell flow rate. This relationship can be defined as:

[0117] A = cross-sectional area, D = particle diameter, V = particle velocity

[0118] Below, Figures 3A-3C, 4, 5A-5C and 6A-6C illustrate various implementations of chips that can have different flow properties, such as implementations with proximity regions having sidewalls at different angles.

[0119] Figures 3A-3C illustrate various views of the flow lines at different magnifications according to some embodiments, showing the flow patterns of cell suspensions passing through an exemplary microfluidic chip 300 to deliver payloads to cells, wherein the chip does not have an angled approach area.

[0120] Figure 3A shows a chip 300, which includes an inlet port 302, a first flow region 304, a narrowing 306, a second flow region 308, and an outlet port 310. Components of chip 300 may share any or more common characteristics with corresponding (e.g., similarly numbered) components of chip 100 described above with respect to Figures 1A-1C. Figure 3B shows an enlarged partial view of chip 300, including several narrowings 306. Figure 3C shows a further enlarged partial view of chip 300, including two narrowings 306.

[0121] As shown in Figures 3A-3C, chip 300 does not have a conical or angled proximity region adjacent to the narrowed portion 306. Instead, the sidewall of the narrowed portion 306 is perpendicular to the endwalls of the first flow region 304 and the second flow region 308.

[0122] In chip 300, a fluid (e.g., a cell suspension) can flow from inlet port 302 to outlet port 310 along the x-axis dimension (diagonally on the pages in Figures 3A-3C). The curved black lines running through the first flow region 304, the second flow region 308, and the constriction 306 illustrate the flow pattern of the fluid (e.g., the cell suspension) flowing through chip 300. Vortices in the pattern indicate flow interruptions, and cells in the cell suspension may aggregate at the points of flow interruption. Flat points in the flow pattern indicate areas where cells can aggregate during flow. Points at line forks in the flow pattern indicate areas where cells can aggregate.

[0123] Figure 4 illustrates a flow pattern according to some embodiments, showing a cell suspension passing through an exemplary microfluidic chip 400 to deliver a payload to the cell, wherein the chip has an angled proximity region.

[0124] Figure 4 illustrates a chip 400, which includes an inlet port 402, a first flow region 404, a narrowing section 406, a second flow region 408, and an outlet port 410. Components of chip 400 may share any or more common characteristics with the corresponding (e.g., similarly numbered) chip 100 described above with respect to Figures 1A-1C and / or chip 300 described above with respect to Figures 3A-3C.

[0125] The difference between chip 400 and chip 300 may be that the narrowing portion 406 may have a tapered approach region adjacent to each respective narrowing portion. In the example shown, chip 400 has an approach region having sidewalls that converge toward the narrowing portion 406 at a 20° angle and diverge from the narrowing portion 406 at a 20° angle (wherein the angle is defined by the angle formed by the opposite approach region sidewalls to each other).

[0126] In chip 400, fluid (e.g., cell suspension) can flow from inlet port 402 to outlet port 410 along the x-axis dimension (diagonally on the page in Figure 4). The curved black lines running through the first flow region 404, the second flow region 408, and the narrowing 406 illustrate the flow pattern of the fluid (e.g., cell suspension) flowing through chip 400.

[0127] Figure 4 uses the same conventions for representing flow patterns as Figures 3A-3C, where eddies in the pattern indicate flow interruptions, flat spots indicate areas where cells can aggregate during flow, and points at line bifurcation indicate areas where cells can aggregate. Therefore, it should be noted that the flow pattern in Figure 4 is smoother, more uniform, and less cluttered than those in Figures 3A-3C. This suggests that proximity regions with tapered sidewalls can improve flow in microfluidic chips, help prevent cell aggregation and / or chip clogging, increase chip throughput, and improve chip lifetime.

[0128] Figures 5A-5C illustrate streamlines showing flow patterns of various cell suspensions through different embodiments of the chip for delivering payloads to cells, wherein the embodiments have access regions defined by different corresponding angles (or, in the case of Figure 5I, the embodiment does not have an angled access region adjacent to the constriction).

[0129] The embodiments shown in Figures 5A-5C illustrate partial close-up views of a chip that may share some or all of the common characteristics with the chips (and their components) described above with reference to Figures 1A-1C, 3A-3C, and / or Figure 4. In the embodiments shown in Figures 5A-5C, a fluid (e.g., a cell suspension) may flow from left to right across the page in the x-axis direction through the shown constriction.

[0130] The chip embodiments in Figures 5A-5B have approach regions defined by different corresponding angles, and in the case of Figure 5C, the embodiment does not have an angled approach region adjacent to the narrowing. The embodiment in Figure 5A has an approach region whose sidewalls form a 20° angle with their corresponding opposite sidewalls; Figure 5B shows a 40° angle embodiment; and Figure 5C does not include any tapered or angled approach regions, similar to those shown in Figures 3A-3C (this can be referred to as a "180° angle embodiment").

[0131] The fluid flow patterns in the various embodiments shown in Figures 5A-5C are illustrated by arrows indicating the fluid flow regions, proximity regions, and constrictions. As shown, for embodiments with more gradually angled proximity regions, the fluid flow patterns are generally smoother, more uniform, and less cluttered. This suggests that proximity regions with more gradually angled sidewalls can improve flow in microfluidic chips, help avoid cell aggregation and / or chip clogging, increase chip throughput, and improve chip lifetime.

[0132] Figures 6A-6C illustrate the flow rates of various cell suspensions through chip embodiments similar to those shown in Figures 5A-5C. In Figures 6A-6C, as indicated by the color keys in Figure 6A, the ten shades across the gradient represent flow rates from (a) the lower end between 0 m / s and 2.327 m / s to (b) the upper end between 20.939 m / s and 23.265 m / s. As the gradient shows, the flow rates are higher in and around the constriction. In some embodiments, it is desirable to configure the chip geometry such that areas where the fluid slows down and / or stagnates are minimized (in size and / or number) and / or eliminated. The chip embodiments in Figures 6A-6B have proximity regions defined by different corresponding angles, and in the case of Figure 6C, the embodiment does not have an angled proximity region adjacent to the constriction. The embodiment in Figure 6A has an approach area, the sidewalls of which form a 40° angle with their respective opposite sidewalls; Figure 6B shows a 60° angle embodiment; and Figure 6C does not include any tapered or angled approach areas, similar to those shown in Figures 3A-3C (this may be referred to as a "180° angle embodiment").

[0133] In some embodiments, chip 100 may be configured to ensure a sufficiently uniform flow rate and flow pattern as the cell suspension approaches and flows through the multiple constrictions. In some embodiments, the arrangement of inlet port 102 relative to a set of parallel constrictions 106 may help ensure a uniform flow rate and flow pattern. Chip 100 may be configured such that the lateral (e.g., y-direction) extent of inlet port 102 relative to the barrier is sufficiently spaced (e.g., in the x-direction) from the barrier (e.g., a line or curve) formed by the set of parallel constrictions 106.

[0134] In some implementations, the plurality of narrow sections can be arranged in a line, forming a barrier or wall between a first flow region upstream of the parallel plurality of narrow sections and a second flow region downstream of the parallel plurality of narrow sections. The barrier formed by the plurality of narrow sections can be located at a sufficiently large distance from the chip's inlet port, and the length of the barrier can be sufficiently short relative to the distance, such that the flow velocity and flow pattern through the plurality of narrow sections are sufficiently uniform to maintain high throughput and low congestion.

[0135] In some embodiments, the inlet port 102 may be spaced from the set of parallel narrowing sections by a minimum spacing distance greater than or equal to 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 30 mm (e.g., the length of the shortest straight line between the inlet port 102 and any point on the set of parallel narrowing sections). In some embodiments, the inlet port 102 may be spaced from the set of parallel narrowing sections by a minimum spacing distance less than or equal to 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 30 mm.

[0136] In some embodiments, the ratio of the distance from the entry port 102 to the nearest point on the boundary to the length of the boundary is greater than or equal to 0.0001, 0.005, 0.001, 0.01, 0.5, 0.1, 1, 10, 50, 100, or 250. In some embodiments, the ratio of the distance from the entry port 102 to the nearest point on the boundary to the length of the boundary is less than or equal to 0.0001, 0.005, 0.001, 0.01, 0.5, 0.1, 1, 10, 50, 100, or 250.

[0137] In some embodiments, the ratio of the distance from the entrance port 106 to the farthest point on the boundary to the distance from the entrance port 106 to the nearest point on the boundary is less than or equal to 5, 2.5, 2.25, 2, 1.75, 1.5, 1.25, 1.1, 1.05, 1.01, or 1.001. In some embodiments, the ratio of the distance from the entrance port 106 to the farthest point on the boundary to the distance from the entrance port 106 to the nearest point on the boundary is greater than or equal to 5, 2.5, 2.25, 2, 1.75, 1.5, 1.25, 1.1, 1.05, 1.01, or 1.001. In some embodiments, the ratio of the distance from the entrance port 106 to the farthest point on the boundary to the distance from the entrance port 106 to the nearest point on the boundary is equal to 1.

[0138] In some implementations, chips characterized by the above ratios can exhibit excellent flow uniformity, throughput, and clog resistance.

[0139] Chip implementation scheme with multiple sets of parallel narrow sections

[0140] Figure 7 illustrates a chip for delivering a payload to cells according to some embodiments, wherein the chip has multiple sets of parallel constrictions, and wherein the sets are connected in series. Figure 7 may share one or more common features with the chip 100 described above with reference to Figures 1A-1C, including that the chip 700 may be configured to guide the flow of fluid (e.g., cell suspension) from inlet port 702 to outlet port 710 through multiple cell-deformable constrictions, forming a fluid path between the ports.

[0141] However, while chip 100 includes a single set of narrowing sections 706 arranged in parallel to each other to form a fluid flow path between the first fluid flow region 104 and the second fluid flow region 108, chip 700 may include multiple sets of parallel fluid narrowing sections. As shown in FIG7, chip 700 may include a first set of narrowing sections 706a, a second set of narrowing sections 706b, a third set of narrowing sections 706c, a fourth set of narrowing sections 706d, and a fifth set of narrowing sections 706e. The narrowing sections within each of the sets 706a-706e may be arranged in parallel to each other, similar to a set of parallel narrowing sections 106 in chip 100, providing a parallel fluid flow path from the upstream fluid flow region to the downstream fluid flow region. However, the sets of parallel narrowing sections 706a-706b may be arranged in series with each other, such that the fluid flow path provided by the first set (e.g., 706a) may be upstream of the fluid flow path provided by the second flow path (e.g., 706b). In chip 700, fluid flowing from inlet port 702 to outlet port 710 can pass through first fluid flow region 730, second fluid flow region 732, third fluid flow region 734, fourth fluid flow region 736, fifth fluid flow region 738 and finally sixth fluid flow region 740, which can be arranged in series with each other and can be separated by the multiple sets of narrowing sections 706 as shown.

[0142] In some embodiments, adjacent narrowing groups (e.g., 706a and 706b) may be offset from each other in the flow direction (e.g., the x-direction in FIG7) by less than or equal to 25 μm, 50 μm, 100 μm, 500 μm, 1 mm, 1 cm, or 5 cm.

[0143] Although the example in Figure 7 shows chip 700 with five separate parallel narrowing groups, any suitable number of parallel narrowing groups can be provided to the chip. For example, the chip can have 2, 3, 4, 5 or more, 10 or more, or 20 or more parallel narrowing groups arranged in series.

[0144] Methods for delivering payloads to cells using microfluidic chips as described herein

[0145] Figure 8 illustrates a method for delivering a payload to a cell according to some embodiments. The method of Figure 8 can be implemented using one or more chips having parallel tapers as described herein, such as (but not limited to) chip 100 of Figures 1A-1C or chip 700 of Figure 7. Figure 8 is described below with reference to chip 100 as an example.

[0146] At step 802, the microfluidic chip (e.g., microfluidic chip 102 of FIG. 1) receives a fluid flow entering a first fluid flow region. The fluid (such as a cell suspension) comprises multiple cells. In some embodiments, the fluid may also include a payload to be delivered to one or more cells in the fluid. Specifically, the microfluidic chip receives the fluid flow in a first fluid flow region located upstream of a set of parallel constrictions that fluidly connect the first fluid flow region to a second fluid flow region downstream of the set of parallel constrictions.

[0147] At step 804, the microfluidic chip (e.g., microfluidic chip 102 of FIG. 1) induces a disturbance in the cell membrane of the cell by causing cells from the fluid to flow through the set of parallel constrictions in the microfluidic chip. As described herein, the set of constrictions can be formed by etching channels in the substrate of the chip and can be configured to guide the cell suspension toward and through the constrictions.

[0148] In step 806, payload delivery to the disturbed cells is initiated. Payload delivery may occur in a second fluid flow region downstream of the set of parallel constrictions after the cell suspension passes through the constrictions and the resulting cell membrane disturbance. In some embodiments, the payload is introduced into the cell suspension after the disturbance, at the second fluid flow region, and / or in a fluid container or reservoir separate from chip 100. Following payload delivery, the cell membrane may heal.

[0149] In some implementations, such as according to method 800, cells treated by one or more constrictions of the microfluidic chip described herein may exhibit a survival percentage greater than or equal to 30%, 50%, 70%, 90%, 95%, or 99% after passing through the constriction(s).

[0150] In some implementations, such as according to method 800, cells processed through one or more constrictions of the microfluidic chip described herein may exhibit a payload delivery percentage greater than or equal to 30%, 50%, 70%, 90%, 95%, or 99%.

[0151] Chips arranged in the box

[0152] Figure 9 shows a housing 900 in which two chips 902a and 902b are held. The housing 900 holds the chips inside and guides fluid in and out of the chips held therein. As shown in the cross-sectional view of Figure 9, an O-ring 904 can press against and surround the chip's inlet or outlet port, thereby holding the chip in place and forming a seal around the inlet / outlet port, and facilitating fluid inflow or outflow from the chip without leakage.

[0153] Fluid flow region geometry

[0154] Figures 10A-10C illustrate chips with various fluid flow region geometries. Specifically, while the chip 100 shown in Figures 1A-1C has a fluid flow region of an irregular pentagonal shape (in the top view of the xy plane), the boxes in Figures 10A-10C may have fluid flow regions of other shapes.

[0155] The box 1000a shown in Figure 10A has a rectangular fluid flow area; the box 1000b shown in Figure 10B has a triangular fluid flow area; and the box 1000c shown in Figure 10C has a rectangular fluid flow area, which together form a square.

[0156] Example 1

[0157] The performance of different chips was compared. The first group of two parallel chips had a narrowed section width of 4 μm, and the second group of two parallel chips had a narrowed section width of 4.5 μm. Each chip contained 75 parallel-arranged narrowed sections. The narrowed section length was 10 μm for all chips. All chips were tested at a pressure of 50 PSI and a temperature range of 2–8 °C. The concentration of the blood product Hemacare LP used was 7.20 x 10⁻⁶ for all chips. 7 Cells / mL. For all chips, RPMI delivery medium and 0.01 mg / mL 3 kDa dextran AF 680 delivery material were used.

[0158] The chip's performance is shown in Table 1 below:

[0159]

[0160] Table 1

[0161] Example 2

[0162] The performance of a chip with 75 parallel-arranged narrow sections, each 4 μm wide and etched to a depth of 66 μm, was tested. The chip was subjected to microfluidic extrusion at 40 PSI, 50 PSI, and 60 PSI. These three passes, or “extrusions,” are plotted against three control extrusions: Control 1 (chip with narrow sections of 30 μm length, 4 μm width, and 20 μm depth tested at 60 PSI); Control 2 (dextran added to cell suspension without microfluidic extrusion); and Control 3 (no dextran added and no microfluidic extrusion), to show the percentage of viable B cells, the percentage of dextran delivery, and the relative average fluorescence intensity, as shown in Figure 11.

[0163] The right-hand plot in Figure 11 is a histogram showing the fluorescence analysis of dextran uptake by cells under different conditions. The first three histograms represent compression at the following different pressures: 40 PSI, 50 PSI, and 60 PSI. “STD” indicates the compression conditions of a chip with a constriction of 30 μm in length, 4 μm in width, and 20 μm in depth, tested at 60 PSI. “SQZendo” indicates dextran delivery by simply contacting cells with dextran without compression, and shows dextran uptake into cells via endocytosis. “No contact” indicates fluorescence of cells without dextran and without compression.

[0164] The performance of a chip with 75 parallel-arranged narrow sections, each 4 μm wide and etched to a depth of 97 μm, was tested. The chip was subjected to microfluidic extrusion at pressures of 15 PSI, 30 PSI, 45 PSI, and 60 PSI. These four extrusions are plotted against three control extrusions: Control 1 (chip with narrow sections of 30 μm length, 4 μm width, and 20 μm depth tested at 60 PSI); Control 2 (dextran added to cell suspension without microfluidic extrusion); and Control 3 (no dextran added and no microfluidic extrusion), to show the percentage of viable B cells, the percentage of dextran delivery, and the relative average fluorescence intensity, as shown in Figure 12.

[0165] The right-hand plot in Figure 12 is a histogram showing the fluorescence analysis of dextran uptake by cells under different conditions. The first four histograms represent compression at the following different pressures: 15 PSI, 30 PSI, 45 PSI, and 60 PSI. “STD” indicates the compression conditions of a chip with a constriction of 30 μm in length, 4 μm in width, and 20 μm in depth, tested at 60 PSI. “SQZendo” indicates dextran delivery by simply contacting cells with dextran without compression, and shows dextran uptake into cells via endocytosis. “No contact” indicates fluorescence of cells without dextran and without compression.

[0166] Example 3

[0167] A chip with 950 parallel-arranged narrow sections was fabricated, each narrow section having a width of 4.5 μm, a length of 10 μm, and an etch depth of 80 μm. The overall dimensions of the chip are 30 mm long, 20 mm wide, and 1225 μm high (thickness). The chip includes inlet fluid ports spaced 24 mm from the outlet fluid port, each port having a diameter of 2.00 mm. The chip comprises a silicon substrate with a height (thickness) of 600 μm and a glass top layer with a thickness of 600 μm.

[0168] The target narrow section dimensions for chip manufacturing are a narrow section width of 4.50±0.20μm, a narrow section length of 10±1μm, and a narrow section height (depth) of 80±2μm.

[0169] The specifications for manufacturing the chip are shown in Table 2 below:

[0170]

[0171]

[0172] Table 2

[0173] The width of the narrow section is measured as 10% of the average width of all narrow sections on the chip, and the measurement is taken at the top of the narrow section.

[0174] The change in width of the narrowed section is measured within a given narrowed section, based on a SEM cross-section of a selected chip on the wafer.

[0175] The chip manufacturing process is configured such that 80% of the chips manufactured are expected to be within the specifications listed in Table 2.

[0176] Example 4

[0177] The performance of six chip types was tested, each of which has multiple sets of parallel-arranged narrow sections connected in series. The six chip types are schematically illustrated in Figure 13A.

[0178] The chip layout 5f has five sets of parallel narrow sections, which are connected in series and spaced apart by a first gap distance.

[0179] The chip layout 5c has five sets of parallel narrow sections, which are connected in series and spaced apart from each other by a second interval distance smaller than the first interval distance.

[0180] The chip layout 10 has ten sets of parallel narrow sections, which are connected in series and spaced apart by a second spacing distance.

[0181] Chip type 1 has a “5f” chip layout, with the upstream proximity area at a 20° angle and the downstream proximity area at a 45° angle.

[0182] Chip type 2 has a “5c” chip layout, with the upstream proximity area at a 20° angle and the downstream proximity area at a 45° angle.

[0183] Chip type 3 has a “10” chip layout, with the upstream proximity area at a 20° angle and the downstream proximity area at a 45° angle.

[0184] Chip type 4 has a “5f” chip layout, with the upstream proximity area at a 20° angle and the downstream proximity area at a 60° angle.

[0185] Chip type 5 has a “5c” chip layout, with the upstream proximity area at a 20° angle and the downstream proximity area at a 60° angle.

[0186] Chip type 6 has a “10” chip layout, with the upstream proximity area at a 20° angle and the downstream proximity area at a 60° angle.

[0187] For each of the six chip types, the narrow section has a length of 10 μm, a width of 3 μm, and a height (etch depth) of 70 μm.

[0188] For each of the six chip types, 88 chips were tested. These six chip types were tested using different pressures and / or blood from different donors. Furthermore, the chips were tested in various directions, for example, by moving the chips both "forward" and "backward". Figures 13B-13H indicate the results based on the angle of the approach area actually upstream during movement. That is, for chip type 1, marked "45°", the chip was reversed so that the 45° approach area was upstream and the 20° approach area was downstream. The results are described below.

[0189] Figure 13B shows the survival and delivery percentage results for tests on chip type 1 at different pressures (30 PSI, 45 PSI, 60 PSI, 75 PSI, 90 PSI, and 105 PSI), in different orientations, and using blood from three different donors (A, B, and C).

[0190] Figure 13C shows the detection of active GFP in cells tested for chip type 1 under different pressures (30 PSI, 45 PSI, 60 PSI, 75 PSI, 90 PSI, and 105 PSI), in different orientations, and using blood from three different donors (A, B, and C).

[0191] Figure 13D shows the power results of tests conducted on chip type 1 at different pressures (30 PSI, 45 PSI, 60 PSI, 75 PSI, 90 PSI, and 105 PSI) and in different orientations.

[0192] Figures 13E-13H show FACS plots for tests on chip type 1. The plots show GFP fluorescence in cells relative to dextran fluorescence in cells.

[0193] Figure 13I shows the survival and delivery percentage results for tests on chip type 3 under different pressures (45 PSI, 60 PSI, 75 PSI, 90 PSI, and 105 PSI), in different orientations, and using blood from three different donors (A, B, and C).

[0194] Figure 13J shows the percentage of expression results for chip type 3 under different pressures (45 PSI, 60 PSI, 75 PSI, 90 PSI and 105 PSI), in different orientations, and using blood from three different donors (A, B and C).

[0195] Figure 13K shows the power results of tests on chip type 3 under different pressures (45 PSI, 60 PSI, 75 PSI, 90 PSI and 105 PSI) and in different orientations.

[0196] Figures 13L-13O show FACS plots for tests on chip type 3. The plots show GFP fluorescence in cells relative to dextran fluorescence in cells.

[0197] Implementation Plan

[0198] The following is an enumerated list of some implementation schemes. In some implementation schemes, any one or more features of any one or more of the following implementation schemes may be combined with any one or more other implementation schemes, even if the subordination of the implementation schemes does not explicitly indicate that the implementation schemes can be combined.

[0199] 1. A microfluidic chip for delivering a payload to a cell, the chip comprising:

[0200] A fluid inlet is configured to receive a flow of cell suspension and allow the cell suspension to pass through a first fluid flow region within the microfluidic chip;

[0201] Multiple constrictions fluidly connected to the first fluid flow region allow the cell suspension to flow from the first fluid flow region to a second fluid flow region within the microfluidic chip through one or more of the multiple constrictions, wherein:

[0202] Each of the plurality of constrictions has a cross-sectional width smaller than the diameter of the cells in the cell suspension, such that the cell membrane is disturbed when passing through the constriction, allowing the payload to pass through the disturbed cell membrane; and

[0203] The quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of narrow sections is greater than or equal to 0.5 μm; and

[0204] A fluid outlet is configured to allow the cell suspension to flow out of the microfluidic chip from the second fluid flow region.

[0205] 2. The microfluidic chip according to embodiment 1, wherein the cross-sectional width of each of the plurality of narrow portions is less than or equal to one or more of the following: 1.8μm, 1.9μm, 2μm, 3μm, 4μm, 5μm, 6μm or 10μm.

[0206] 3. The microfluidic chip according to any one of embodiments 1 to 2, wherein the cross-sectional height of each of the plurality of narrow portions is greater than or equal to 20 μm.

[0207] 4. The microfluidic chip according to embodiment 3, wherein each of the narrow portions is formed by etching into the substrate of the microfluidic chip, wherein the cross-sectional height of each of the plurality of narrow portions is defined by the etching depth.

[0208] 5. The microfluidic chip according to embodiment 4, wherein the substrate comprises silicon.

[0209] 6. The microfluidic chip according to any one of embodiments 4 to 5, wherein the etching includes deep reactive ion etching.

[0210] 7. The microfluidic chip according to any one of embodiments 1 to 6, wherein the plurality of narrowing portions includes more than 1,000 narrowing portions.

[0211] 8. The microfluidic chip according to any one of embodiments 1 to 7, wherein the plurality of narrow portions are arranged in parallel to each other to form a boundary between the first fluid flow region and the second fluid flow region.

[0212] 9. The microfluidic chip according to embodiment 8, wherein the ratio of the distance from the inlet port to the nearest point on the boundary to the length of the boundary is greater than or equal to 0.5.

[0213] 10. The microfluidic chip according to any one of embodiments 8 to 9, wherein the ratio of the distance from the inlet port to the farthest point on the boundary to the distance from the inlet port to the nearest point on the boundary is less than or equal to 1.5.

[0214] 11. The microfluidic chip according to any one of embodiments 8 to 10, wherein the boundary includes a portion extending in a linear manner.

[0215] 12. The microfluidic chip according to any one of embodiments 8 to 11, wherein the length of the boundary is greater than or equal to 4 mm.

[0216] 13. The microfluidic chip according to any one of embodiments 1 to 12, wherein the microfluidic chip is configured to operate across all constrictions at a total throughput rate greater than or equal to 1 mL / min.

[0217] 14. The microfluidic chip according to any one of embodiments 1 to 13, wherein the microfluidic chip is configured to operate at a pressure greater than or equal to 10 psi.

[0218] 15. The microfluidic chip according to any one of embodiments 1 to 14, wherein each of the plurality of narrow portions is arranged adjacent to a corresponding proximity region, the proximity region comprising a tapered wall that tapers toward the narrow portion.

[0219] 16. The microfluidic chip according to embodiment 15, wherein the tapered wall gradually tapers toward the narrowed portion at an angle greater than or equal to 10 degrees and less than or equal to 80 degrees to the sidewall of the narrowed portion.

[0220] 17. The microfluidic chip according to any one of embodiments 1 to 16, wherein the microfluidic chip is configured such that the average volumetric flow rate per narrow section of the plurality of narrow sections is greater than or equal to 1 μL / min.

[0221] 18. A microfluidic chip according to any one of embodiments 1 to 17, comprising one or more pillars intersecting the first fluid flow region and connecting a plane of a first inner surface of the first fluid flow region to a plane of a second inner surface of the first fluid flow region.

[0222] 19. The microfluidic chip according to any one of embodiments 1 to 18, wherein the ratio of the total etched area forming the first fluid flow region and the second fluid flow region is less than or equal to 50% of the surface area of ​​the microfluidic chip.

[0223] 20. A method for delivering a payload to a cell, the method comprising:

[0224] Receives a flow of cell suspension, comprising a plurality of cells, into a first fluid flow region of the microfluidic chip; and

[0225] The cell suspension is allowed to flow from the first fluid flow region through multiple narrow sections of the microfluidic chip, wherein:

[0226] Each of the plurality of constrictions has a cross-sectional width smaller than the diameter of the cells in the cell suspension, such that the cell membrane is deformed when passing through the constriction, allowing the payload to pass through the deformed cell membrane; and

[0227] The quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of narrow sections is greater than or equal to 0.5 μm.

[0228] 21. The method according to embodiment 20, wherein the cell suspension includes the payload.

[0229] 22. The method according to any one of embodiments 20 to 21, comprising contacting the payload with the cell suspension after the cell membrane is disturbed.

[0230] 23. The method according to embodiment 22, wherein after the payload is brought into contact with the cell suspension, the percentage of the payload delivered to the cells is greater than or equal to 30%.

[0231] 24. The method according to any one of embodiments 20 to 23, wherein after the cell suspension passes through the plurality of constrictions of the microfluidic chip, the percentage of surviving cells in the cell suspension is greater than or equal to 30%.

[0232] 25. The method according to any one of embodiments 20 to 24, wherein the cross-sectional width of each of the plurality of narrow portions is less than or equal to one or more of the following: 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm or 10 μm.

[0233] 26. The method according to any one of embodiments 20 to 25, wherein the cross-sectional height of each of the plurality of narrow portions is greater than or equal to 20 μm.

[0234] 27. The method according to any one of embodiments 20 to 26, wherein the plurality of narrowing portions comprises more than 1,000 narrowing portions.

[0235] 28. The method according to any one of embodiments 20 to 27, wherein the microfluidic chip is configured to operate across all constrictions at a total throughput rate greater than or equal to 1 mL / min.

[0236] 29. The method according to any one of embodiments 20 to 28, wherein causing the cell suspension to flow from the first fluid flow region through the plurality of constrictions comprises forcing the fluid to flow at a pressure greater than or equal to 10 psi.

[0237] 30. The method according to any one of embodiments 20 to 29, wherein flowing the cell suspension from the first fluid flow region through the plurality of constrictions comprises flowing the cell suspension at an average volumetric flow rate per constriction greater than or equal to 1 μL / min.

[0238] 31. A method for manufacturing a microfluidic chip for delivering a payload into a cell, the method comprising:

[0239] Etching into the substrate to form a first fluid flow region, the first fluid flow region being configured to allow cell suspension to flow from the inlet port through the first fluid flow region; and

[0240] Etching is performed into the substrate to form a plurality of narrow sections, the plurality of narrow sections being configured to allow the cell suspension to flow from the first fluid flow region through the narrow sections, wherein:

[0241] Each of the plurality of constrictions has a cross-sectional width smaller than the diameter of the cells in the cell suspension, such that the cell membrane is deformed when passing through the constriction, allowing the payload to pass through the deformed cell membrane; and

[0242] The quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of narrow sections is greater than or equal to 0.5 μm.

[0243] 32. The method according to embodiment 31, comprising attaching a cover layer to the etched substrate to surround the first fluid flow region and the plurality of narrowing portions.

[0244] 33. The method according to any one of embodiments 31 to 32, wherein etching into the substrate to form the plurality of narrow portions includes deep reactive ion etching.

[0245] 34. The method according to any one of embodiments 31 to 33, wherein etching into the substrate to form the plurality of narrow portions includes etching to a depth greater than or equal to 50 μm.

[0246] 35. The method according to any one of embodiments 31 to 34, comprising depositing a material layer on the substrate after etching into the substrate, wherein depositing the layer results in a reduction in the width of the plurality of narrow portions.

[0247] 36. A microfluidic chip for delivering a payload to a cell, the chip comprising:

[0248] A fluid inlet is configured to receive a flow of cell suspension and allow the cell suspension to pass through a first fluid flow region within the microfluidic chip;

[0249] A plurality of constrictions fluidly connected to the first fluid flow region to allow the cell suspension to flow through one or more of the plurality of constrictions from the first fluid flow region to a second fluid flow region within the microfluidic chip; and

[0250] A second plurality of constrictions, fluidly connected to the second fluid flow region to allow the cell suspension to flow from the second fluid flow region to a third fluid flow region within the microfluidic chip through one or more of the second plurality of constrictions, wherein:

[0251] Each of the first plurality of constrictions and the second plurality of constrictions has a cross-sectional width smaller than the diameter of the cells in the cell suspension, such that the cell membrane is disturbed when passing through the constriction, allowing the payload to pass through the disturbed cell membrane; and

[0252] The quotient of the cross-sectional area and the cross-sectional perimeter of each of the first plurality of narrowing portions and the second plurality of narrowing portions is greater than or equal to 0.5 μm.

[0253] 37. The microfluidic chip according to embodiment 36, wherein the cross-sectional width of each of the first plurality of narrow portions and the second plurality of narrow portions is less than or equal to one or more of the following: 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm or 10 μm.

[0254] 38. The microfluidic chip according to any one of embodiments 36 to 37, wherein the cross-sectional height of each of the first plurality of narrow portions and the second plurality of narrow portions is greater than or equal to 20 μm.

[0255] 39. The microfluidic chip according to embodiment 38, wherein each of the first plurality of narrow portions and the second plurality of narrow portions is formed by etching into the substrate of the microfluidic chip, wherein the cross-sectional height of each of the plurality of narrow portions is defined by the etching depth.

[0256] 40. The microfluidic chip according to any one of embodiments 36 to 39, wherein the first plurality of narrowing portions and the second plurality of narrowing portions each comprise more than 1,000 narrowing portions.

[0257] 41. The microfluidic chip according to any one of embodiments 36 to 40, wherein the first plurality of narrow portions and the second plurality of narrow portions are separated from each other by a nearest interval distance greater than or equal to 25 μm.

[0258] 42. The microfluidic chip according to any one of embodiments 36 to 41, wherein the microfluidic chip is configured to operate across all constrictions at a total throughput rate greater than or equal to 1 mL / min.

[0259] 43. The microfluidic chip according to any one of embodiments 36 to 42, wherein the microfluidic chip is configured to operate at a pressure greater than or equal to 10 psi.

[0260] 44. The microfluidic chip according to any one of embodiments 36 to 43, wherein the microfluidic chip is configured such that the average volumetric flow rate per narrowing of the first plurality of narrowings and the second plurality of narrowings is greater than or equal to 1 μL / min.

Claims

1. A microfluidic chip for delivering a payload to a cell, the chip comprising: A fluid inlet is configured to receive a flow of cell suspension and allow the cell suspension to pass through a first fluid flow region within the microfluidic chip; Multiple constrictions fluidly connected to the first fluid flow region allow the cell suspension to flow from the first fluid flow region to a second fluid flow region within the microfluidic chip through one or more of the multiple constrictions, wherein: The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, such that the cell membrane is disturbed when passing through the constriction, allowing the payload to pass through the disturbed cell membrane. Each of the plurality of narrow sections constitutes a slit shape; and The quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of narrow sections is greater than or equal to 0.5 μm; A fluid outlet configured to allow the cell suspension to flow out of the microfluidic chip from the second fluid flow region; and Top floor.

2. The microfluidic chip according to claim 1, wherein the cross-sectional width of each of the plurality of narrow portions is less than or equal to one or more of the following: 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm or 10 μm.

3. The microfluidic chip according to claim 1, wherein the cross-sectional height of each of the plurality of narrow portions is greater than or equal to 20 μm.

4. The microfluidic chip of claim 3, wherein each of the narrow portions is formed by etching into the substrate of the microfluidic chip, wherein the cross-sectional height of each of the plurality of narrow portions is defined by the etching depth.

5. The microfluidic chip according to claim 4, wherein the substrate comprises silicon.

6. The microfluidic chip according to claim 4, wherein the etching comprises deep reactive ion etching.

7. The microfluidic chip according to any one of claims 1 to 6, wherein the plurality of narrowing portions comprises more than 1,000 narrowing portions.

8. The microfluidic chip according to any one of claims 1 to 6, wherein the plurality of narrow portions are arranged in parallel to each other to form a boundary between the first fluid flow region and the second fluid flow region.

9. The microfluidic chip of claim 8, wherein the ratio of the distance from the inlet port to the nearest point on the boundary to the length of the boundary is greater than or equal to 0.

5.

10. The microfluidic chip of claim 9, wherein the ratio of the distance from the inlet port to the farthest point on the boundary to the distance from the inlet port to the nearest point on the boundary is less than or equal to 1.

5.

11. The microfluidic chip of claim 8, wherein the boundary includes a portion that extends in a linear manner.

12. The microfluidic chip according to claim 8, wherein the length of the boundary is greater than or equal to 4 mm.

13. The microfluidic chip according to any one of claims 1 to 6, wherein the microfluidic chip is configured to operate across all constrictions at a total throughput rate greater than or equal to 1 mL / min.

14. The microfluidic chip according to any one of claims 1 to 6, wherein the microfluidic chip is configured to operate at a pressure greater than or equal to 10 psi.

15. The microfluidic chip according to any one of claims 1 to 6, wherein each of the plurality of narrow portions is arranged adjacent to a corresponding proximity region, the proximity region comprising a tapered wall that tapers toward the narrow portion.

16. The microfluidic chip of claim 15, wherein the tapered wall tapers towards the narrowing portion at an angle greater than or equal to 10 degrees and less than or equal to 80 degrees to the sidewall of the narrowing portion.

17. The microfluidic chip according to any one of claims 1 to 6, wherein the microfluidic chip is configured such that the average volumetric flow rate per narrow section of the plurality of narrow sections is greater than or equal to 1 μL / min.

18. The microfluidic chip according to any one of claims 1 to 6, comprising one or more pillars intersecting the first fluid flow region and connecting a plane of a first inner surface of the first fluid flow region to a plane of a second inner surface of the first fluid flow region.

19. The microfluidic chip according to any one of claims 1 to 6, wherein the ratio of the total etched area forming the first fluid flow region and the second fluid flow region is less than or equal to 50% of the surface area of ​​the microfluidic chip.

20. A method for delivering a payload to a cell, the method comprising: Receives a flow of cell suspension into a first fluid flow region of a microfluidic chip, the cell suspension comprising a plurality of cells; as well as The cell suspension is allowed to flow from the first fluid flow region through multiple narrow sections of the microfluidic chip, wherein: The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, such that the cell membrane is deformed when passing through the constriction, allowing the payload to pass through the deformed cell membrane. Each of the plurality of narrow sections constitutes a slit shape; and The quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of narrow sections is greater than or equal to 0.5 μm; and the microfluidic chip includes a top layer.

21. The method of claim 20, wherein the cell suspension comprises the payload.

22. The method of claim 20, further comprising bringing the payload into contact with the cell suspension after the cell membrane has been deformed.

23. The method of claim 22, wherein after the payload is brought into contact with the cell suspension, the percentage of the payload delivered to the cells is greater than or equal to 30%.

24. The method according to any one of claims 20 to 23, wherein after the cell suspension passes through the plurality of constrictions of the microfluidic chip, the percentage of surviving cells in the cell suspension is greater than or equal to 30%.

25. The method according to any one of claims 20 to 23, wherein the cross-sectional width of each of the plurality of narrow portions is less than or equal to one or more of the following: 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm or 10 μm.

26. The method according to any one of claims 20 to 23, wherein the cross-sectional height of each of the plurality of narrow portions is greater than or equal to 20 μm.

27. The method according to any one of claims 20 to 23, wherein the plurality of narrowing portions comprises more than 1,000 narrowing portions.

28. The method according to any one of claims 20 to 23, wherein the microfluidic chip is configured to operate across all constrictions at a total throughput rate greater than or equal to 1 mL / min.

29. The method of any one of claims 20 to 23, wherein causing the cell suspension to flow from the first fluid flow region through the plurality of constrictions comprises forcing the fluid to flow at a pressure greater than or equal to 10 psi.

30. The method according to any one of claims 20 to 23, wherein flowing the cell suspension from the first fluid flow region through the plurality of constrictions comprises flowing the cell suspension at an average volumetric flow rate per constriction greater than or equal to 1 μL / min.

31. A method for manufacturing a microfluidic chip for delivering a payload into a cell, the method comprising: Etching is performed into the substrate to form a first fluid flow region, the first fluid flow region being configured to allow cell suspension to flow from the inlet port through the first fluid flow region; as well as Etching is performed into the substrate to form a plurality of narrow sections, the plurality of narrow sections being configured to allow the cell suspension to flow from the first fluid flow region through the narrow sections, wherein: The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, such that the cell membrane is deformed when passing through the constriction, allowing the payload to pass through the deformed cell membrane. Each of the plurality of narrow sections constitutes a slit shape; and The quotient of the cross-sectional area and the cross-sectional perimeter of each of the plurality of narrow sections is greater than or equal to 0.5 μm; and The microfluidic chip includes a top layer.

32. The method of claim 31, further comprising attaching a cover layer to the etched substrate to surround the first fluid flow region and the plurality of narrowing portions.

33. The method according to any one of claims 31 to 32, wherein etching into the substrate to form the plurality of narrow portions comprises deep reactive ion etching.

34. The method according to any one of claims 31 to 32, wherein etching into the substrate to form the plurality of narrow portions comprises etching to a depth greater than or equal to 50 μm.

35. The method according to any one of claims 31 to 32, comprising depositing a material layer on the substrate after etching into the substrate, wherein depositing the layer results in a reduction in the width of the plurality of narrow portions.

36. A microfluidic chip for delivering a payload to a cell, the chip comprising: A fluid inlet is configured to receive a flow of cell suspension and allow the cell suspension to pass through a first fluid flow region within the microfluidic chip; A plurality of constrictions fluidly connected to the first fluid flow region to allow the cell suspension to flow from the first fluid flow region to a second fluid flow region within the microfluidic chip through one or more of the plurality of constrictions; as well as A second plurality of constrictions, fluidly connected to the second fluid flow region to allow the cell suspension to flow from the second fluid flow region to a third fluid flow region within the microfluidic chip through one or more of the second plurality of constrictions, wherein: The cross-sectional width of each of the first plurality of narrow sections and the second plurality of narrow sections is smaller than the diameter of the cells in the cell suspension, such that the cell membrane is disturbed when passing through the narrow section, allowing the payload to pass through the disturbed cell membrane; Each of the plurality of narrow sections constitutes a slit shape; and The quotient of the cross-sectional area and the cross-sectional perimeter of each of the first plurality of narrowing portions and the second plurality of narrowing portions is greater than or equal to 0.5 μm; as well as Top floor.

37. The microfluidic chip of claim 36, wherein the cross-sectional width of each of the first plurality of narrowed portions and the second plurality of narrowed portions is less than or equal to one or more of the following: 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm or 10 μm.

38. The microfluidic chip of claim 36, wherein the cross-sectional height of each of the first plurality of narrowed portions and the second plurality of narrowed portions is greater than or equal to 20 μm.

39. The microfluidic chip of claim 38, wherein each of the first plurality of narrow portions and the second plurality of narrow portions is formed by etching into the substrate of the microfluidic chip, wherein the cross-sectional height of each of the plurality of narrow portions is defined by the etching depth.

40. The microfluidic chip according to any one of claims 36 to 39, wherein the first plurality of narrowing portions and the second plurality of narrowing portions each comprise more than 1,000 narrowing portions.

41. The microfluidic chip according to any one of claims 36 to 39, wherein the first plurality of narrow portions and the second plurality of narrow portions are separated from each other by a nearest interval distance greater than or equal to 25 μm.

42. The microfluidic chip according to any one of claims 36 to 39, wherein the microfluidic chip is configured to operate across all constrictions at a total throughput rate greater than or equal to 1 mL / min.

43. The microfluidic chip according to any one of claims 36 to 39, wherein the microfluidic chip is configured to operate at a pressure greater than or equal to 10 psi.

44. The microfluidic chip according to any one of claims 36 to 39, wherein the microfluidic chip is configured such that the average volumetric flow rate per narrowing of the first plurality of narrowings and the second plurality of narrowings is greater than or equal to 1 μL / min.

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