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

Abstract

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

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 131,430, filed on December 29, 2020, the entire content of which is incorporated herein by reference.

[0002] Field The present disclosure relates to the delivery of payloads to cells, and more specifically, to microfluidic constriction channels that can disrupt cell membranes and allow payloads to pass through perturbed membranes.

Background Art

[0003] Background The controlled delivery of various materials to cells is important in the developing medical field of cell therapy. For example, various research and therapeutic applications can involve the delivery of peptides, nucleic acids, proteins, small molecules, and nanomaterials into cells through the cell membrane. As discussed in International Publication No. 2013059343, International Publication No. 2015023982, PCT / US2015 / 058489, PCT / US2015 / 060689, and PCT / US2016 / 13113, constricted microfluidic channels can be used to deliver compounds and other payloads to cells. As disclosed in PCT / US2018 / 66295, benchtop laboratory and / or clinical systems can be configured to force a cell suspension to flow through one or more constrictions of a microfluidic chip having constricted channels or constricted pores to cause perturbations to the membranes of cells in the cell suspension.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

[0005] Summary of the Invention As described above, systems for intracellular payload delivery include systems configured to force cells through one or more constriction channels or constriction pores to induce perturbations in the cell membranes of cells in the cell suspension as the cell suspension flows through a constriction cartridge. However, known systems for intracellular payload delivery are prone to constriction clogging, have insufficient throughput rates, and are not simple enough and efficient to manufacture. Therefore, there is a need for improved systems, methods, and techniques for intracellular payload delivery, including the need for improved microfluidic cell constriction chips having high throughput speeds and low clogging speeds. There is a need for microfluidic cell constriction chips having improved geometric configurations, improved throughput, improved resistance to clogging or other failures, and / or improved ease and efficiency of manufacture. The systems, methods, and techniques disclosed herein can address one or more of these needs to improve geometric configurations, throughput, resistance to clogging or other failures, ease and efficiency of manufacture, as well as the capacity, usage, and / or manufacturing methods of microfluidic cell constriction chips and / or systems.

[0006] This specification discloses a high-throughput microfluidic chip for use in a system for delivering a payload to cells in a cell suspension. The chip comprises multiple parallelized constrictions (e.g., constriction channels) through which the cell suspension can be extruded under pressure. Cells in the cell suspension deform to pass through the cell deformation constrictions of the microfluidic chip, and their membranes may be perturbed. As described herein, the throughput and clogging characteristics of the chip can be improved due to several characteristics of the chip disclosed herein.

[0007] Firstly, the chips disclosed herein may include a number of parallelized constrictions that divide a first fluid flow region from a second fluid flow region. For example, a chip such as those disclosed herein may have hundreds or thousands of parallelized microfluidic constrictions. When a cell suspension in the first fluid flow region is pressurized, it may flow through any one of the constrictions into the second fluid flow region, thereby increasing throughput compared to chips with fewer constrictions.

[0008] Secondly, the chips disclosed herein may have constrictions where the quotient of the cross-sectional area to the perimeter of the cross-section is 1 or greater than that of previously known designs. In some embodiments, this increase in quotient can be achieved by etching deep, narrow rectangular constrictions (e.g., slit-shaped constrictions) into a substrate (e.g., a silicon substrate). The constrictions may be formed by etching sufficiently deep while maintaining the constriction sidewalls within a sufficient angular threshold parallel to one another. Furthermore, the constrictions may be formed by etching sufficiently deep while maintaining the sidewalls within a predetermined envelope (e.g., tolerance) of distance from one another, so that cells extruded through the constrictions can be deformed by the sidewalls regardless of the position (e.g., etching depth) at which the cells pass through the constrictions. In this way, each deep, narrow rectangular constriction may increase individual throughput due to a larger cross-sectional constriction area compared to a constriction with the same width configured for cells of the same size but not deeply etched into the substrate. Furthermore, since clogging of constrictions tends to be caused by the edges and corners of the constriction, a deep, narrow rectangular constriction may clog at a reduced rate compared to a constriction that has the same width for cells of the same size but is not deeply etched into the substrate. Therefore, in some embodiments, the throughput per constriction can be increased while minimizing the possibility and / or degree of constriction clogging by increasing the constriction area while minimizing the perimeter length of the cross-sectional constriction.

[0009] Thirdly, the tips disclosed herein may have a geometric shape configured to ensure a sufficiently uniform flow velocity and flow pattern as the cell suspension approaches and flows through a plurality of constrictions. In some embodiments, the plurality of constrictions may be arranged in a line that forms a barrier or wall between a first flow region upstream of the parallel plurality of constrictions and a second flow region downstream of the parallel plurality of constrictions. The barrier formed by the plurality of constrictions may be located at a sufficient distance from the tip's inlet port, and the length of the barrier may be sufficiently short relative to that distance, so that the flow velocity and flow pattern through the plurality of constrictions are sufficiently uniform to maintain high throughput and low clogging.

[0010] Fourth, the constrictions disclosed herein may have a geometric shape configured to ensure a sufficiently uniform flow pattern within and around the constriction, and to ensure sufficiently high flow velocity and throughput through the constriction. In some embodiments, the constriction has an approach region through which the fluid flows as it approaches the narrowest point of the constriction. The approach region may be defined by one or more tapered walls, such as tapered sidewalls that taper at a predetermined angle toward the narrowest point of the constriction. The predetermined angle may be set in conjunction with the width of the narrowest point of the constriction and other characteristics of the tip and cell suspension to ensure a uniform flow pattern and sufficient flow velocity and throughput.

[0011] In some embodiments, a first microfluidic chip is provided for delivering a payload to cells, the first chip comprising: a fluid inlet configured to receive a flow of cell suspension and deliver the cell suspension to a first fluid flow region within the microfluidic chip; and 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 constrictions, each of which has a cross-sectional width smaller than the diameter of a cell in the cell suspension and is perturbed as the cell membrane passes through the constriction so that the payload can pass through the perturbed cell membrane, and the quotient of the cross-sectional area to the cross-sectional perimeter of each of the plurality of constrictions is greater than or equal to 0.5 μm.

[0012] In some embodiments, a first method is provided for delivering a payload to cells, the first method comprising receiving a flow of a cell suspension into a first fluid flow region of a microfluidic chip, wherein the cell suspension comprises a plurality of cells, and causing the cell suspension to flow from the first fluid flow region through a plurality of constrictions of the microfluidic chip, wherein the cross-sectional width of each of the plurality of constrictions is smaller than the diameter of a cell in the cell suspension, thereby causing the cell membrane to deform as it passes through the constrictions so that the payload can pass through the deformed cell membrane, and the quotient of the cross-sectional area to 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 second method is provided for manufacturing a microfluidic chip for delivering a payload to cells, the second method comprising etching a substrate to form a first fluid flow region configured to allow a cell suspension to flow from an inlet port through a first fluid flow region, and etching a plurality of constrictions configured to allow a cell suspension to flow from the first fluid flow region through constrictions, wherein the cross-sectional width of each of the plurality of constrictions is smaller than the diameter of a cell in the cell suspension, thereby causing the cell membrane to deform as it passes through the constrictions, so that the payload can pass through the deformed cell membrane, and the quotient of the cross-sectional area to 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 microfluidic chip is provided for delivering a payload to cells, the second chip comprising: a fluid inlet configured to receive a flow of cell suspension and deliver the cell suspension to 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 first constrictions; and one or more of the cell suspension to flow from the second fluid flow region to a third fluid flow region within the microfluidic chip. The first and second plurality of constrictions are fluidly connected to a second fluid flow region to enable flow through a second plurality of constrictions beyond the first plurality of constrictions, wherein the cross-sectional width of each of the constrictions of the first plurality of constrictions and the second plurality of constrictions is smaller than the diameter of a cell in the cell suspension, thereby perturbing the cell membrane as it passes through the constrictions so that the payload can pass through the perturbed cell membrane, and the quotient of the cross-sectional area of ​​each of the constrictions of the first plurality of constrictions and the second plurality of constrictions to the cross-sectional perimeter is greater than or equal to 0.5 μm.

[0015] In some embodiments, any one or more features, characteristics, or elements described above with respect to any of the embodiments may be incorporated into any of the other embodiments described above or elsewhere in this specification. In some embodiments, any one or more features, characteristics, or elements described elsewhere in this disclosure may be incorporated into any of the one or more above-described embodiments. In embodiments of the present invention, for example, the following items are provided. (Item 1) A microfluidic chip for delivering a payload to cells, wherein the chip is A fluid inlet configured to receive the flow of a cell suspension and deliver the cell suspension to a first fluid flow region within the microfluidic chip, A plurality of constrictions fluidly connected to the first fluid flow region, to enable the cell suspension to flow from the first fluid flow region to the second fluid flow region within the microfluidic chip through one or more constrictions, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, and the cell membrane is perturbed as it passes through the constrictions so that the payload can pass through the perturbed cell membrane. The quotient of the cross-sectional area of ​​each of the aforementioned constricted portions with respect to the perimeter of the cross-section is greater than or equal to 0.5 μm, and the plurality of constricted portions, A microfluidic chip comprising a fluid outlet configured to allow the cell suspension to flow out of the microfluidic chip from the second fluid flow region. (Item 2) The microfluidic chip according to item 1, wherein the cross-sectional width of each of the plurality of constrictions is less than or equal to one or greater than 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. (Item 3) A microfluidic chip according to any one of items 1 to 2, wherein the cross-sectional height of each of the plurality of constricted portions is greater than or equal to 20 μm. (Item 4) The microfluidic chip according to item 3, wherein each of the constricted portions is formed by etching the substrate of the microfluidic chip, and the cross-sectional height of each of the plurality of constricted portions is defined by the etching depth of the etching. (Item 5) The microfluidic chip described in item 4, wherein the substrate contains silicon. (Item 6) A microfluidic chip according to any one of items 4 to 5, wherein the etching includes deep reactive ion etching. (Item 7) The microfluidic chip according to any of items 1 to 6, wherein the plurality of constrictions include more than 1,000 constrictions. (Item 8) The microfluidic chip according to any one of items 1 to 7, wherein the plurality of constrictions are arranged in parallel with each other so as to form a boundary between the first fluid flow region and the second fluid flow region. (Item 9) The microfluidic chip according to item 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. (Item 10) A microfluidic chip according to any of items 8 to 9, wherein the ratio of the distance from the inlet port to the furthest 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. (Item 11) A microfluidic chip according to any of items 8 to 10, wherein the aforementioned boundary includes a portion that extends in a straight line. (Item 12) A microfluidic chip as described in any of items 8-11, wherein the length of the aforementioned boundary is greater than or equal to 4 mm. (Item 13) The microfluidic chip described in any of items 1 to 12, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions. (Item 14) The microfluidic chip described in any of items 1 to 13 is configured to operate at a pressure greater than or equal to 10 psi. (Item 15) A microfluidic chip according to any one of items 1 to 14, wherein each of the plurality of constrictions is positioned adjacent to an approach region which includes a tapered wall that narrows toward the constriction. (Item 16) The microfluidic tip according to item 15, wherein the tapered wall tapers toward the constriction at an angle greater than or equal to 10 degrees from the side wall of the constriction and less than or equal to 80 degrees. (Item 17) The microfluidic chip according to any one of items 1 to 16, wherein the microfluidic chip is configured such that the average volume flow rate per constriction of the plurality of constrictions is greater than or equal to 1 μL / min. (Item 18) A microfluidic chip according to any one of items 1 to 17, comprising one or more pillars that intersect the first fluid flow region and connect a first inner surface plane of the first fluid flow region to a second inner surface plane of the first fluid flow region. (Item 19) A microfluidic chip according to any one of items 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. (Item 20) A method for delivering a payload to a cell, wherein the method is Receiving a flow of a cell suspension into a first fluid flow region of a microfluidic chip, wherein the cell suspension contains a plurality of cells, This includes causing the cell suspension to flow from the first fluid flow region through a plurality of constrictions of the microfluidic chip, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, so that the cell membrane is deformed when passing through the constrictions, allowing the payload to pass through the deformed cell membrane. A method wherein the quotient of the cross-sectional area of ​​each of the plurality of constricted portions with respect to the cross-sectional perimeter is greater than or equal to 0.5 μm. (Item 21) The method according to item 20, wherein the cell suspension comprises the payload. (Item 22) The method according to any one of items 20 to 21, comprising bringing the payload into contact with the cell suspension after perturbation of the cell membrane. (Item 23) The method according to item 22, wherein the percentage of cells to which the payload is delivered after the payload is brought into contact with the cell suspension is greater than or equal to 30%. (Item 24) The method according to any one of items 20 to 23, wherein the percentage of live cells in the cell suspension after the cell suspension has passed through the plurality of constrictions of the microfluidic chip is greater than or equal to 30%. (Item 25) The method according to any one of items 20 to 24, wherein the cross-sectional width of each of the plurality of constrictions is less than or equal to one or greater than 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. (Item 26) The method according to any one of items 20 to 25, wherein the cross-sectional height of each of the plurality of constricted portions is greater than or equal to 20 μm. (Item 27) The method according to any one of items 20 to 26, wherein the plurality of constrictions include more than 1,000 constrictions. (Item 28) The method according to any one of items 20 to 27, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions. (Item 29) The method according to any one of items 20 to 28, wherein the flow of the cell suspension from the first fluid flow region through the plurality of constrictions is forced to a pressure greater than or equal to 10 psi. (Item 30) The method according to any one of items 20 to 29, wherein the flow of the cell suspension from the first fluid flow region through the plurality of constrictions is performed at an average volume flow rate per constriction of the plurality of constrictions greater than or equal to 1 μL / min. (Item 31) A method for manufacturing a microfluidic chip for delivering a payload to cells, wherein the method is Etching the substrate to form a first fluid flow region configured to allow the cell suspension to flow from the inlet port through the first fluid flow region, This includes etching the substrate to form a plurality of constrictions configured to allow the cell suspension to flow from the first fluid flow region through the constrictions, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, so that the cell membrane is deformed when passing through the constrictions, allowing the payload to pass through the deformed cell membrane. A method wherein the quotient of the cross-sectional area of ​​each of the plurality of constricted portions with respect to the cross-sectional perimeter is greater than or equal to 0.5 μm. (Item 32) The method according to item 31, comprising fixing a cover layer to the etched substrate to surround the first fluid flow region and the plurality of constrictions. (Item 33) The method according to any one of items 31 to 32, wherein the etching of the substrate for forming the plurality of constrictions includes deep reactive ion etching. (Item 34) The method according to any one of items 31 to 33, wherein etching the substrate to form the plurality of constrictions includes etching to a depth greater than or equal to 50 μm. (Item 35) The method according to any one of items 31 to 34, comprising depositing a layer of material on the substrate after etching the substrate, wherein the width of the plurality of constrictions is reduced by depositing the layer. (Item 36) A microfluidic chip for delivering a payload to cells, wherein the chip is A fluid inlet configured to receive the flow of a cell suspension and deliver the cell suspension to a first fluid flow region within the microfluidic chip, A plurality of first constrictions fluidly connected to the first fluid flow region to enable the cell suspension to flow from the first fluid flow region to the second fluid flow region within the microfluidic chip through one or more first constrictions, A plurality of second constrictions, fluidly connected to the second fluid flow region, to enable the cell suspension to flow from the second fluid flow region to the third fluid flow region within the microfluidic chip through one or more second constrictions, The cross-sectional width of each of the first and second plurality of constrictions is smaller than the diameter of the cells in the cell suspension, thereby perturbing the cell membrane as it passes through the constrictions, so that the payload can pass through the perturbed cell membrane. A microfluidic chip comprising: a first plurality of constrictions and a second plurality of constrictions, wherein the quotient of the cross-sectional area of ​​each of the first plurality of constrictions with respect to the cross-sectional perimeter of each of the constrictions is greater than or equal to 0.5 μm. (Item 37) The microfluidic chip according to item 36, wherein the cross-sectional width of each of the first and second plurality of constrictions is less than or equal to one or greater than 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. (Item 38) A microfluidic chip according to any one of items 36 to 37, wherein the cross-sectional height of each of the first plurality of constrictions and the second plurality of constrictions is greater than or equal to 20 μm. (Item 39) The microfluidic chip according to item 38, wherein each of the first plurality of constrictions and the second plurality of constrictions is formed by etching the substrate of the microfluidic chip, and the cross-sectional height of each of the plurality of constrictions is defined by the etching depth of the etching. (Item 40) A microfluidic chip according to any one of items 36 to 39, wherein each of the first plurality of constrictions and the second plurality of constrictions includes more than 1,000 constrictions. (Item 41) A microfluidic chip according to any of items 36 to 40, wherein the first plurality of constrictions and the second plurality of constrictions are spaced apart from each other by the nearest spacing distance greater than or equal to 25 μm. (Item 42) The microfluidic chip according to any one of items 36 to 41, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions. (Item 43) A microfluidic chip as described in any of items 36-42, configured to operate at a pressure greater than or equal to 10 psi. (Item 44) The microfluidic chip according to any one of items 36 to 43, wherein the microfluidic chip is configured such that the average volume flow rate per constriction of the first plurality of constrictions and the second plurality of constrictions is greater than or equal to 1 μL / min. [Brief explanation of the drawing]

[0016] [Figure 1] Figures 1A to 1C show various diagrams of a chip for delivering a payload to cells according to several embodiments. Figure 1A shows an overhead view of the chip and a magnified highlight of the narrowed portion of the chip according to several embodiments. Figure 1B shows an overhead view of the narrowed portion and guide channel of the chip according to several embodiments. Figure 1C shows a cross-sectional view of the narrowed portion of the chip according to several embodiments.

[0017] [Figure 2] Figures 2A and 2B show the manufacturing process of a chip for delivering a payload to cells according to several embodiments. Figure 2A shows the constricted area after the etching process according to several embodiments. Figure 2B shows the constricted area after the deposition process according to several embodiments.

[0018] [Figure 3] Figures 3A–3C are various diagrams at different magnifications of the flowline showing the flow pattern of a cell suspension through a tip for delivering a payload to cells, according to several embodiments, where the tip does not have an inclined approach region.

[0019] [Figure 4] Figure 4 shows flow lines illustrating the flow pattern of a cell suspension through a chip for delivering a payload to cells, according to several embodiments, where the chip has an inclined approach region.

[0020] [Figure 5] Figures 5A to 5C show flow lines illustrating various cell suspension flow patterns through different embodiments of the chip for delivering a payload to cells, where the embodiments have approach regions defined by different angles (or, in the case of Figure 5C, the embodiment does not have an inclined approach region adjacent to a constriction).

[0021] [Figure 6-1]Figures 6A–6C show the flow rates of various cell suspensions through different embodiments of the chip for delivering the payload to cells, where the embodiments have approach regions defined by different angles (or, in the case of Figure 5I, the embodiments do not have inclined approach regions adjacent to the constriction). [Figure 6-2] Figures 6A–6C show the flow rates of various cell suspensions through different embodiments of the chip for delivering the payload to cells, where the embodiments have approach regions defined by different angles (or, in the case of Figure 5I, the embodiments do not have inclined approach regions adjacent to the constriction). [Figure 6-3] Figures 6A–6C show the flow rates of various cell suspensions through different embodiments of the chip for delivering the payload to cells, where the embodiments have approach regions defined by different angles (or, in the case of Figure 5I, the embodiments do not have inclined approach regions adjacent to the constriction).

[0022] [Figure 7] Figure 7 shows a chip for delivering a payload to cells according to several embodiments, the chip having multiple sets of parallelized constrictions, the sets being in series with each other.

[0023] [Figure 8] Figure 8 shows several embodiments of methods for delivering a payload to cells.

[0024] [Figure 9] Figure 9 shows a cartridge holding a chip for delivering a payload to cells, according to several embodiments.

[0025] [Figure 10] Figures 10A to 10C show various chips for delivering payloads to cells, each chip having a fluid flow region with various geometric shapes.

[0026] [Figure 11] Figure 11 shows experimental delivery profile data of chips for delivering payloads to cells in several embodiments.

[0027] [Figure 12] Figure 12 shows experimental delivery profile data of chips for delivering payloads to cells in several embodiments.

[0028] [Figure 13-1] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-2] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-3] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-4] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-5] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-6] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-7] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-8] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-9] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-10] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-11] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-12] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-13] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-14] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Figure 13-15] Figures 13A to 13O show schematic chip information and experimental data from test chips having a set of parallelized constrictions according to several embodiments, where the sets are in series with each other. [Modes for carrying out the invention]

[0029] Detailed description of the invention A high-throughput microfluidic chip for use in a system for delivering a payload to cells in a cell suspension is disclosed below. The chip comprises multiple parallelized constrictions (e.g., constriction channels) through which the cell suspension can be extruded under pressure. Cells in the cell suspension deform to pass through the cell deformation constrictions of the microfluidic chip, and their membranes may be perturbed. As described herein, the throughput and clogging characteristics of the chip can be improved due to several characteristics of the chip disclosed herein.

[0030] The following description of Figures 1A to 1C illustrates exemplary embodiments of a microfluidic chip having parallelized constrictions for intracellular payload delivery. Next, the description of Figures 2A to 2B illustrates the fabrication of constrictions within a microfluidic chip for intracellular payload delivery. Next, the description of Figures 3A to 3C, 4, 5A to 5C, and 6A to 6C illustrates various geometric shapes of approach regions adjacent to the constrictions of the chip for intracellular payload delivery. Next, the description of Figure 7 illustrates exemplary embodiments of a microfluidic chip having multiple sets of parallelized constrictions, the sets being in series with respect to each other. Next, the description of Figure 8 illustrates a method of using the chip as disclosed herein. As described below, the geometric shapes of the chip and / or chip described with reference to Figures 1 to 7 can be used in some embodiments of Method 800. Next, the description of Figure 9 illustrates a cartridge-holding chip for delivering a payload to cells, according to some embodiments. Next, the description of Figures 10A to 10C illustrates a chip having fluid flow regions with various geometric shapes. Next, the descriptions of Figures 11, 12, and 13A-13O relate to experimental data from several examples described herein.

[0031] The following descriptions include exemplary systems, methods, techniques, parameters, etc. However, it should be noted that such descriptions are not intended to limit the scope of this disclosure and are instead provided as descriptions of exemplary embodiments. definition

[0032] For the purposes of interpreting this Specified, the following definitions apply, and wherever used in the singular, the plural form is also included, and vice versa. In the event of any conflict between the following definitions and any documents incorporated herein by reference, the following definitions shall prevail.

[0033] As used herein, the singular “a,” “an,” and “the” include plural references unless otherwise indicated.

[0034] The aspects and embodiments of the present invention described herein are understood to include the terms "comprising," "consisting," and "consisting essentially of."

[0035] It should be further understood that the terms “includes,” “including,” “comprises,” and / or “comprising” specify the presence of the described features, integers, processes, actions, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, processes, actions, elements, components, and / or groups thereof.

[0036] The term "if" can be interpreted, depending on the context, as meaning "when," "upon," "in response to determining," or "in response to detecting." Similarly, the phrases "if it is determined" or "[the described condition or event] is detected" can be interpreted, depending on the context, as meaning "at the time of determination," "in response to determination," "[the described condition or event] is detected," or "[the described condition or event] is detected."

[0037] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. References to values ​​or parameters “about” herein include (and are described) embodiments that apply to the value or parameter itself.

[0038] In this specification, terms such as "first," "second," etc., are used to describe various elements, but these elements should not be limited by these terms. These terms are used solely to distinguish one element from another.

[0039] For any structural or functional properties described herein, methods for determining these properties are known in the art.

[0040] All references cited herein, including patent applications and publications, are incorporated in their entirety by reference. Geometric shape of microfluidic chip for intracellular payload delivery

[0041] The following describes a microfluidic chip designed to improve throughput and reduce cell clogging.

[0042] Figures 1A to 1C show various diagrams of the chip 100 for delivering a payload to cells according to several embodiments. Figure 1A shows an overhead view of the chip 100 and a magnified highlight of the constricted portion of the chip according to several embodiments. Figure 1B shows an overhead view of the constricted portion and guide channel of the chip 100 according to several embodiments. Figure 1C shows a cross-sectional view of the constricted portion of the chip 100 according to several embodiments.

[0043] The microfluidic chip 100 is configured to receive a fluid flow at the fluid inlet 110. The fluid may include a cell suspension. In some embodiments, the fluid may include a payload for delivery to cells, and the payload may include any suitable cargo for delivery to cells.

[0044] For the purposes of this specification, the orientation and dimensions of tip 100 may be referred to by the following conventions: the x-direction or x-dimension may refer to the dimension extending horizontally in Figure 1A (the x-direction is the overall direction of fluid flow through tip 100 from inlet to outlet); the y-direction or y-dimension may refer to the dimension extending vertically in Figure 1A; and the z-direction or z-dimension may refer to the dimension passing in and out of the plane of paper in Figure 1A.

[0045] In some embodiments, the tip 100 may be a microfluidic tip configured to guide a fluid flow through a constriction (e.g., a constriction channel) that is sufficiently narrow in at least one dimension to deform cells as they are pushed through the constriction under pressure. The deformation of the cells as they pass through the constriction under pressure may cause perturbation of the cell membrane so that a payload suspended in the cell suspension (either suspended in the suspension before passing through the constriction or added to the suspension afterward) can pass through the perturbed cell membrane and enter the cell.

[0046] In the example of the chip 100, the fluid (e.g., cell suspension) flowing through the chip 100 can enter the chip 100 through the inlet port 102 and from the inlet 102 into a first fluid flow region 104. From the first fluid flow region 104, the fluid can flow (e.g., by being forced under pressure) through a plurality of parallel constrictions 106 into a second fluid flow region 108. The fluid can then flow out of the chip 100 from the second fluid flow region 108 through the outlet port 110. The distance between the fluid inlet port 102 and the fluid outlet port 110 can extend in a direction perpendicular to the plane (e.g., inner surface) of the substrate of the chip 100. The distance between the fluid inlet port 102 and the fluid outlet port 110 may be 5-30 mm, 8-25 mm, or 10-20 mm. In some embodiments, the distance between the fluid inlet port 102 and the fluid outlet port 110 may 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 may 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 may be interchangeable.

[0047] In some embodiments, the first fluid flow region 104, the constriction 106, and the second fluid flow region 108 may all be formed as recessed spaces within a substrate such as a silicon substrate. In some embodiments, etching may be performed in the z direction by etching the upper surface of the substrate. In some embodiments, wet etching or dry etching may be used. In some embodiments, deep reactive ion etching (DRIE) may be used. After etching the substrate to define the recessed spaces forming the first fluid flow region 104, the constriction 106, and / or the second fluid flow region 108, the uppermost layer may be fixed on the substrate layer so as to surround regions 104 and 108 and the constriction 106 in the z direction.

[0048] In some embodiments, the inlet port 102 and / or outlet port 110 may be formed as openings in the uppermost layer. In some embodiments, the inlet port 102 and / or outlet port 110 may be formed as openings in the etched substrate layer, such as openings passing through the bottom of the substrate layer in the z direction, or openings passing through the sides of the substrate in the x and / or y directions.

[0049] In some embodiments, the tip 100 may include pillars 120 that may extend through a first fluid flow region 104 and / or a second fluid flow region 108. The pillars 120 may function as support structures that hold the uppermost layer of the tip 100 and / or can connect the uppermost layer of the tip 100. In some embodiments, the shape and / or arrangement of the pillars 120 may be selected to ensure a uniform flow pattern and minimize disturbance of the flow velocity to all parts of the boundary formed by the constriction 106 between the first fluid flow region 104 and the second fluid flow region 108. For example, the pillars 120 may have a width in the y-direction that is shorter than the length in the x-direction, so as to provide a sufficient xy surface area of ​​the pillar for support and / or connection without excessively increasing the width in the y-direction and obstructing the fluid flow in the x-direction.

[0050] In some embodiments, one or more additional and / or alternative support mechanisms, such as poles or pillars formed separately from the substrate, may be used, and / or one or more external support mechanisms, such as backings, that can cover the front and / or rear surfaces of the chip 100 may be used.

[0051] The enlarged circular view in Figure 1A shows an enlarged view of a subset of the constrictions 106 of the tip 100. As shown, the constrictions 106 may be arranged in parallel to one another so as to collectively form a boundary between a first fluid flow region 104 and a second fluid flow region 108. In the example of tip 100, the constrictions 106 are arranged linearly (extending in the y-direction) side by side. In some embodiments, a set of constrictions in a flow arrangement arranged in parallel to one another can form a curved and / or linear boundary. In some embodiments, the shape of the boundary may extend diagonally across the tip 100 in the xy-plane, thereby increasing its overall length. In some embodiments, the shape of the boundary may extend across the tip 100 in the xy-plane at a meandering or stepped angle, thereby increasing its overall length.

[0052] Figure 1B shows an overhead view of a magnified portion of the tip 100, showing the four constrictions 106 of the tip 100. As in the circular magnified view of Figure 1A, the constrictions 106 in Figure 1B are arranged linearly (extending in the y-direction) and adjacent to each other, providing respective flow paths from the first fluid flow region 104 on the left to the second fluid flow region 108 on the right. In the diagram of Figure 1B, the top (y-direction) and surrounding regions of the four constrictions are annotated to show the shape and extent of various regions of the tip 100. The annotated regions include the constrictions 106, the upstream approach region 112a, the downstream approach region 112b, the upstream guide channel 114a, and the downstream guide channel 114b.

[0053] Figure 1C shows an enlarged partial cross-sectional view of the chip 100 (showing the cross-sectional plane zy), showing three constrictions 106 of the chip 100. As shown in Figures 1A and 1B, they are arranged linearly (extending in the y-direction) side by side and provide respective flow paths from the first fluid flow region 104 to the second fluid flow region 108. As shown in Figure 1C, the constrictions 106 may have a height (e.g., etching depth) that extends in the z-direction, for example, downward in the substrate in the z-direction from the top surface of the substrate. In some embodiments, the height of the constrictions 106 may be the same as or greater than the height of one of the corresponding upstream approach region 112a, the corresponding downstream approach region 112b, the corresponding upstream guide channel 114a, and / or the corresponding downstream guide channel 114b.

[0054] In some embodiments, one or more dimensions of the components shown in Figure 1B and / or Figure 1C may be selected to increase throughput, reduce clogging, and maintain the effectiveness of payload delivery and cell viability after delivery.

[0055] In some embodiments, the constriction 106 may have a cross-sectional constriction width (in the y-direction as shown in Figure 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 constriction width (in the y-direction as shown in Figure 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 constriction 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 constriction width may be set and / or selected according to the diameter of the cells perturbed by passing through the constriction (e.g., the diameter in suspension). For example, the ranges enumerated herein may be configured to process cells with a diameter of approximately 1.4 μm to approximately 10 μm while in suspension. In light of the disclosure herein, those skilled in the art will understand that different cross-sectional constriction widths (and corresponding other dimensions of the tip) may be used in use cases where cells of other diameters are processed. For example, processing of monocytes may be achieved with a cross-sectional constriction width greater than 10 μm. Processing of even larger cells may be achieved with a cross-sectional constriction width of up to approximately 0.1 mm.

[0056] In some embodiments, the constriction 106 may have a constriction length (in the x-direction shown in Figure 1B) that is 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 constriction length (in the x-direction shown in Figure 1B) 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 embodiments, the constriction length may have a tolerance of ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0057] In some embodiments, the constriction 106 may have a constriction height 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 the z direction as shown in Figure 1C). In some embodiments, the constriction 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 constriction height may have a tolerance of ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0058] In some embodiments, the constriction 106 may have a sidewall draft, where a sidewall refers to one of the walls that extend perpendicularly in the z-direction and define the width of the constriction, as shown in Figure 1C. In some embodiments, the sidewall draft 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 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 may have a tolerance of ±0.02°, ±0.01°, or ±0.005°. In some embodiments, the sidewall draft may be greater than or equal to 1°, 2°, 5°, or 10°. In some embodiments, the sidewall draft may be less than or equal to 1°, 2°, 5°, or 10°. Since a higher (deeper) constriction can exhibit a greater variation in cross-sectional constriction width than a shorter (shallower) constriction for a given draft angle, the acceptable range of sidewall draft may depend on the z-dimensional constriction height (if the variation in constriction width is too large, the fluid flow will flow through the wider portion of the constriction rather than the narrower portion, bypassing the narrower portion of the channel and allowing the fluid to flow through the constriction without perturbing the cell membrane). Therefore, a tip with a shorter (shallower) z-dimensional constriction height may have a higher sidewall draft angle than a tip with a higher (deeper) z-dimensional constriction height.

[0059] In some embodiments, the constricted portion 106 may have 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, or 0.05 μm, 0.01 μm, or 0.001 μm.

[0060] In some embodiments, the approach area 112a or 112b may include a narrow end and a wide end, the narrow end being proximal to the corresponding constriction 106 and the wide end being proximal to the corresponding guide channel 114a or 114b. The approach area may have side walls including a straight wall that slopes between the opening of the narrow end and the opening of the wide end, defining an angle greater than 0° and less than 90° from a straight line in the x-direction. The angle between the side walls of the approach area may be greater than 0° and less than 180°. In some embodiments, the side walls of the approach area may include one or more curved portions (e.g., defining a meandering shape when viewed from above in the z-direction) and / or angles (e.g., defining a step-like shape when viewed from above in the z-direction) between the narrow end and the wide end.

[0061] In some embodiments, the width of the cross-sectional approach area at the narrow end (in the y-direction as shown in Figure 1B) may 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 width of the cross-sectional approach area at the narrow end (in the y-direction as shown in Figure 1B) may be less than or equal to the 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm approach area. In some embodiments, the width of the cross-sectional approach area at the narrow end may have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm.

[0062] In some embodiments, the width of the cross-sectional approach area at the wide end (in the y-direction as shown in Figure 1B) may 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 width of the cross-sectional approach area at the wide end (in the y-direction as shown in Figure 1B) may 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 width of the cross-sectional approach area at the wide end may have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm.

[0063] In some embodiments, the length of the approach region (in the x-direction shown in Figure 1B) may 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 length of the constriction may have a tolerance of ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0064] In some embodiments, the height of the approach region (in the z-direction as shown in Figure 1C) may 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 height of the approach region (in the z-direction as shown in Figure 1C) 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 approach region. In some embodiments, the approach region height may have a tolerance of ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm. In some embodiments, the height of the approach area may be any suitable height lower than the height of the substrate in the z-direction.

[0065] In some embodiments, the approach area 112a or 112b may have a sidewall draft, where a sidewall refers to one of the walls extending perpendicular to the z-direction and defining the width of the approach area. In some embodiments, the sidewall draft of the approach area 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 of the approach 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 of the approach area may have a tolerance of ±0.02°, ±0.01°, or ±0.005°. In some embodiments, the sidewall draft may be greater than or equal to 1°, 2°, 5°, or 10°. In some embodiments, the sidewall draft may be less than or equal to 1°, 2°, 5°, or 10°. The tolerance for the sidewall draft may depend on the z-dimensional approach region height, as with respect to the constriction sidewall draft and constriction height described above.

[0066] In some embodiments, the approach region 112a or 112b may have sidewall roughness. In some embodiments, the sidewall roughness of the approach region 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 approach region may be less than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, or 0.05 μm.

[0067] In some embodiments, the approach area may have side walls extending from the wider end of the approach area to the narrower end. In some embodiments, opposing side walls of the approach area may form angles with each other greater than or equal to 160°, 140°, 120°, 100°, 80°, 60°, 40°, or 20°. In some embodiments, opposing side walls of the approach area may form angles with each other less than or equal to 160°, 140°, 120°, 100°, 80°, 60°, 40°, or 20°. In some embodiments, the side walls of the approach area may form angles with the side walls of adjacent constrictions less than or equal to 80°, 70°, 60°, 50°, 40°, 30°, 20°, or 10°. In some embodiments, the sidewalls of the approach area may form an angle greater than or equal to 80°, 70°, 60°, 50°, 40°, 30°, 20°, or 10° with the sidewalls of the adjacent constriction.

[0068] In some embodiments, the guide channel 114a or 114b may extend between an end adjacent to the corresponding approach region (112a or 112b) and the opposite end distal to the corresponding approach region.

[0069] In some embodiments, the cross-sectional guide channel width (in the y-direction as shown in Figure 1B) may 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) may 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 may have a tolerance of ±0.3 μm, ±0.2 μm, ±0.1 μm, or ±0.05 μm.

[0070] In some embodiments, the guide channel length (in the x-direction as shown in Figure 1B) may 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 may have a tolerance of ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0071] In some embodiments, the height of the guide channel (in the z-direction as shown in Figure 1C) may 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 height of the guide channel (in the z-direction as shown in Figure 1C) 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 guide channel may have a tolerance of ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm. In some embodiments, the height of the guide channel may be any suitable height lower than the height of the substrate in the z-direction.

[0072] In some embodiments, the guide channel 114a or 114b may have a sidewall draft, where a sidewall refers to one of the walls extending perpendicular to the z-direction and defining the width of the guide channel. In some embodiments, the sidewall draft 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 of the approach 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 of the guide channel may have a tolerance of ±0.02°, ±0.01°, or ±0.005°.

[0073] In some embodiments, the guide channel 114a or 114b may have sidewall roughness. In some embodiments, the sidewall roughness of the guide channel 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 channel may be less than or equal to 0.3 μm, 0.2 μm, 0.15 μm, 0.10 μm, or 0.05 μm.

[0074] In some embodiments, adjacent parallelized constrictions (and / or corresponding approach regions or guide channels) may be offset from each other in the y direction by a pitch 116 less than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm. In some embodiments, adjacent parallelized constrictions (and / or corresponding approach regions or guide channels) may be offset from each other in the y direction by a pitch 116 greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm.

[0075] In some embodiments, adjacent parallel constrictions may be separated from each other in the y-direction by a constriction 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 constrictions may be separated from each other in the y-direction by a constriction sidewall thickness 118 greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 250 μm, or 500 μm.

[0076] In some embodiments, adjacent parallel guide channels may be separated from each other in the y direction by a guide channel sidewall thickness 122 that is 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 that is greater than or equal to 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, or 250 μm.

[0077] In some embodiments, the substrate on which the first flow region 104, the constriction 106, and / or the second flow region 108 are formed may have a thickness (in the z direction) 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 on which the first flow region 104, the constriction 106, and / or the second flow region 108 are formed 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 approximately 400 μm may help reduce the risk of substrate failure.

[0078] In some embodiments, the top layer of the chip 100 (e.g., the layer placed on top of the substrate layer after the substrate layer has been etched) may have a thickness (in the z direction) less than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 5 mm. In some embodiments, the top layer of the chip 100 (e.g., the layer placed on top of the substrate layer after the substrate layer has been etched) 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.

[0079] In some embodiments, the inlet port 102 and / or outlet port 110 may be formed as openings having a diameter less than or equal to 200 μm, 400 μm, 500 μm, 1 mm, or 5 mm. In some embodiments, the inlet port 102 and / or outlet port 11 may be formed as openings having a diameter greater than or equal to 200 μm, 400 μm, 500 μm, 1 mm, or 5 mm. In some embodiments, the openings may be circular. In some embodiments, the openings may have any shape having dimensions in the y direction, the x direction, or another direction in the xy plane that is less than or equal to any of the above diameters. In some embodiments, the openings may have any shape having dimensions in the y direction, the x direction, or another direction in the xy plane that is greater than or equal to any of the above diameters.

[0080] 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 an etching depth to the substrate of the chip 100 that is greater than or equal to the height in the z direction (e.g., 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). Alternatively, the height (in the z-direction) of 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 ±3 μm, ±2 μm, ±1.5 μm, ±1 μm, or ±0.5 μm.

[0081] In some embodiments, one or more constrictions 106 may be characterized by a quotient of the area of ​​the constriction section to the perimeter of the constriction section. In some embodiments, the quotient of the area of ​​the constriction section to the perimeter of the constriction section may 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 constriction section to the perimeter of the constriction section may 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, constrictions with a higher quotient may be less prone to clogging than constrictions with a lower quotient. In some embodiments, for a given constriction width (which may be determined by the cell diameter of the cell(s) perturbed by passing through the constriction (e.g., the diameter of a cell in suspension)), a constriction with a higher quotient may be less prone to clogging than a constriction with a lower quotient. In some embodiments, this increase in quotient can be achieved by etching a deep, narrow rectangular constriction (e.g., slit-shaped) into a substrate (e.g., a silicon substrate), as described herein. The constriction can be formed by etching and / or depositing a constriction sidewall material to a sufficient depth while maintaining the constriction sidewalls within a sufficient angular threshold parallel to one another, as described herein. Furthermore, the constriction can be formed by etching and / or depositing a constriction sidewall material to a sufficient depth while maintaining the sidewalls within a predetermined envelope (e.g., tolerance) of distance from one another, so that cells extruded through the constriction can be deformed by the sidewalls regardless of where the cells pass through the constriction (e.g., etching depth). In this way, each deep, narrow rectangular constriction is configured to perturb cells of the same diameter, but compared to constrictions with a width that is not deeply etched into the substrate, the larger cross-sectional constriction area can lead to increased individual throughput.Furthermore, since clogging of constrictions can be caused by the edges and corners of the constriction, a deep, narrow rectangular constriction may clog at a reduced rate compared to a neck with a width that is not deeply etched into the substrate, even though it is configured to perturb cells of the same diameter. Therefore, in some embodiments, the throughput per constriction can be increased while minimizing the possibility and / or degree of constriction clogging by increasing the constriction area while minimizing the perimeter length of the cross-sectional constriction.

[0082] In some embodiments, the pillar 120 enclosed by the first fluid flow region 104 may have a collective total surface area in the xy plane that is 5 to 10% of the area of ​​the first fluid flow region 104 itself in the xy plane. In some embodiments, the pillar 120 enclosed by the first fluid flow region 104 may have a collective total surface area in the xy plane that is 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 pillar 120 enclosed by the first fluid flow region 104 may have a collective total surface area in the xy plane that is 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.

[0083] In some embodiments, the pillar 120 surrounded by the second fluid flow region 108 may have a collective total surface area in the xy plane that is 5 to 10% of the area of ​​the second fluid flow region 108 itself in the xy plane. In some embodiments, the pillar 120 surrounded by the second fluid flow region 108 may have a collective total surface area in the xy plane that is 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 pillar 120 surrounded by the first fluid flow region 108 may have a collective total surface area in the xy plane that is 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.

[0084] In some embodiments, the chip 100 may have a chip length in the x-direction greater than or equal to 2 mm, 4 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 100 mm, 250 mm, 450 mm, or 500 mm. In some embodiments, the chip 100 may have a chip length in the x-direction less than or equal to 2 mm, 4 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 100 mm, 250 mm, 450 mm, or 500 mm.

[0085] In some embodiments, the chip 100 may have a y-direction chip width greater than or equal to 2 mm, 4 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 100 mm, 250 mm, 450 mm, or 500 mm. In some embodiments, the chip 100 may have a y-direction chip width less than or equal to 2 mm, 4 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 100 mm, 250 mm, 450 mm, or 500 mm.

[0086] In some embodiments, the tip 100 may have a total tip height (e.g., thickness) in the z direction that is 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 tip 100 may have a total tip height (e.g., thickness) in the z direction that is 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 tip height may be between 400 μm and 3 mm.

[0087] 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 microfluidic chip and / or substrate on which the flow regions are etched. In some embodiments, the etched regions may have a combined surface area that is greater than or equal to 75%, 50%, 40%, 30%, 20%, or 10% of the surface area of ​​the microfluidic chip and / or substrate on which the flow regions are etched.

[0088] In some embodiments, the chip 100 may have a substrate (e.g., a silicon substrate) with an etched constriction having a substrate thickness in the z direction less than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 3 mm. In some embodiments, the chip 100 may have a substrate (e.g., a silicon substrate) with an etched constriction having a substrate thickness in the z direction greater than or equal to 300 μm, 400 μm, 600 μm, 800 μm, 1 mm, 2 mm, or 3 mm.

[0089] In some embodiments, the set of parallelized constrictions may include 1001 to 1500 constrictions, 1501 to 2000 constrictions, 2001 to 2500 constrictions, 2501 to 5000 constrictions, or 5001 to 10000 constrictions. In some embodiments, the set of parallelized constrictions 106 within chip 100 may include 1, 5, 10, 25, 50, 100, 250, 500, 1000, 2,500, or parallelized constrictions greater than or equal to 5,000. In some embodiments, the set of parallelized constrictions 106 within chip 100 may include 1, 5, 10, 25, 50, 100, 250, 500, 1000, 2,500, or parallelized constrictions less than or equal to 5,000. As disclosed herein, a tip having a relatively large number of parallelized constrictions may enable high volumetric flow rates of cell suspensions, high cell throughput, and reduced clogging.

[0090] In some embodiments, the chip 100 may comprise one or more components made of metal, plastic, polymer, and / or glass. In some embodiments, the substrate of the chip 100 may comprise silicon. In some embodiments, the top layer of the chip 100 (e.g., a layer bonded or fixed on top of the substrate layer) may comprise glass and / or quartz.

[0091] In the example shown in Figure 1B, the constricted portion 106 has a rectangular cross-sectional shape when viewed from the downward angle shown in Figure 1B. Therefore, the constricted portion 106 shown in Figure 1B has a uniform (y-direction shown in Figure 1B) cross-sectional constricted portion width along its length (x-direction) at all points. In some additional or alternative embodiments, the constricted portion (e.g., constricted portion 106) may have a trapezoidal, curved, stepped, or other irregular cross-sectional shape when viewed from the downward angle shown in Figure 1B.

[0092] For example, in some embodiments, the constriction may have a tapered shape such that its cross-sectional width in the y-direction increases or decreases (linearly or otherwise) along the length of the constriction (x-direction), rather than having a uniform cross-sectional constriction width (in the y-direction as shown in Figure 1B). In some embodiments, the cross-sectional width of the tapered constriction may increase or decrease by about 1%, 2%, 3%, 5%, 10%, 25%, 50%, 100%, 200%, or 500% along the entire length of the constriction (from any of the cross-sectional constriction widths shown herein).

[0093] In some embodiments, the constriction may have a multi-stage constriction width rather than a uniform cross-sectional constriction width (in the y-direction as shown in Figure 1B), and the constriction may have a stepped shape in which multiple steps of the constriction (in the x-direction as shown in Figure 1B) have different cross-sectional widths from one or more other steps. For example, in some embodiments, the constriction may have two steps, where the first step is narrower than the second step, or the second step is narrower than the first step. The cross-sectional width of the steps in the constriction may increase monotonically in the direction of the flow in the constriction, decrease monotonically in the direction of the flow in the constriction, or both increase and decrease in the direction of the flow in the constriction (for example, in the case of a constriction having three or more steps). The steps may be distinguished from each other by steps (in the y-direction) formed on one or both constriction walls. The steps between steps in the constriction may be formed as right-angle steps and / or by tapered transition regions between steps.

[0094] In some embodiments, any one or more constriction steps may have a constriction width equal to any of the constriction widths disclosed herein. In some embodiments, adjacent constriction steps may have constriction widths that differ from each other by about 1%, 2%, 3%, 5%, 10%, 25%, 50%, 100%, 200%, or 500%.

[0095] In some embodiments, any one or more steps in the constriction may have a constriction length equal to any of the constriction lengths disclosed herein. Alternatively, a set of steps in the constriction that form the entire constriction may have a total length equal to any of the constriction lengths disclosed herein.

[0096] In some embodiments in which adjacent constricted sections are separated by a tapered constriction transition region, the length of the tapered constriction transition region may be equal to about 0.1%, 0.5%, 1%, 2%, 3%, 5%, 10%, 25%, 50%, or 100% of any of the constriction lengths disclosed herein.

[0097] In some embodiments, constrictions having non-uniform (e.g., tapering) cross-sectional widths and / or multiple sections of different cross-sectional widths may allow the tip to extend the time that cells passing through the constriction are subjected to pressure from the constriction wall, thereby extending the time that the cell wall pores remain open and increasing payload delivery efficiency and effectiveness. In some embodiments, a constriction having an initial narrower section followed by a wider section may allow pores to form rapidly and effectively in the initial section and remain open as cells pass through the wider section. By using a wider constriction following a narrower constriction, effective payload delivery may be achieved while increasing cell viability compared to embodiments where the entire constriction has a narrower constriction width.

[0098] Figures 2A and 2B illustrate the manufacturing process of a chip for delivering a payload to cells according to several embodiments. Specifically, Figure 2A shows a partial cross-sectional view of the narrowed portion of the chip after the etching process, for example, the narrowed portion 106 of chip 100, according to several embodiments. Figure 2B shows a partial cross-sectional view of the narrowed portion of the chip after the deposition process, which is carried out after the etching process, according to several embodiments.

[0099] As shown in Figure 2A, the etching process for forming a constriction within the chip may include etching down (e.g., downward in the z direction) onto the substrate of the chip to remove material from the substrate and form a channel therein. In some embodiments, the etching process may include dry etching and / or wet etching. In some embodiments, the etching process may include deep reactive ion etching.

[0100] In some embodiments, the etched channel may have dimensions (e.g., height in the z direction and / or width in the y direction) that are greater than or equal to the target dimensions of the constriction at the end of the manufacturing process (e.g., after deposition).

[0101] As shown in Figure 2B, following the etching process, a deposition process may be carried out in which a layer of material is deposited on the etched substrate. In some embodiments, the deposited material may include SiO, plastic, glass, silicon-derived material, plastic-derived material, glass-derived material, metal (e.g., gold, silver, stainless steel, and / or aluminum), and / or metal-derived material. In some embodiments, the deposition of this material layer can narrow the etched channel with the material deposited on the sidewalls perpendicular (z-direction) of the channel, and this deposition process may narrow the channel to form a constriction having a desired constriction dimension.

[0102] 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.

[0103] In some embodiments, the constricted portion after the deposition process may have a dimension equal to or greater than any of the dimensions of the constricted portion 106 described with reference to Figures 1A to 1C. Flow characteristics of microfluidic chips disclosed herein

[0104] Various fluid flow characteristics of fluids flowing through the microfluidic chips described herein, along with various chip characteristics that may affect the fluid flow characteristics, are described below. Specifically, pressure, shear stress, shear rate, cell flow rate, cell volume flow rate, cell clogging rate, and cell throughput are provided below.

[0105] As described above, the microfluidic chips provided herein are 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, thereby perturbing the cell membranes of the cells by passing through the constriction. In some embodiments, the microfluidic chip 100 may be configured to operate at pressures of 1–200 PSI, 10–150 PSI, or 25–100 PSI. In some embodiments, the microfluidic chip 100 may be configured to operate at pressures less than or equal to 1, 5, 10, 25, 50, 75, 100, 125, 150, or 200 PSI. In some embodiments, the microfluidic chip 100 may be configured to operate at pressures greater than or equal to 1, 5, 10, 25, 50, 75, 100, 125, 150, or 200 PSI.

[0106] In some embodiments, the microfluidic chip 100 may provide a fluid flow velocity through the constriction of the chip 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. In some embodiments, the microfluidic chip 100 may provide a fluid flow velocity through the constriction of the chip 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.

[0107] As described above, the microfluidic chips provided herein are specifically designed to have high throughput. Due to the geometric shape of a particular constriction, the number of cells that can pass through (and be perturbed by) the constriction is greater than that of known microfluidic chips per given unit of time.

[0108] In some embodiments, the microfluidic tip 100 may provide a volumetric flow rate through a single constriction of the tip 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 some embodiments, the microfluidic tip 100 may provide a volumetric flow rate through a single constriction of the tip 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.

[0109] In some embodiments, the microfluidic tip 100 may provide an overall volumetric flow rate (combined across all parallel constrictions 106) 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. In some embodiments, the microfluidic tip 100 may provide an overall volumetric flow rate (combined across all parallel constrictions 106) less 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.

[0110] In some embodiments, the microfluidic chip 100 may provide a cell throughput rate through a narrow section of the chip that is less than or equal to 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 30 mL / min, or 50 mL / min. In some embodiments, the microfluidic chip 100 may provide a cell throughput rate through a narrow section of the chip that is greater than or equal to 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 30 mL / min, or 50 mL / min.

[0111] In some embodiments, the microfluidic chip 100 may provide an overall cell throughput rate less than or equal to 5 mL / min, 10 mL / min, mL / min, 50 mL / min, 100 mL / min, 200 mL / min, or 500 mL / min. In some embodiments, the microfluidic chip 100 may provide an overall cell throughput rate greater than or equal to 5 mL / min, 10 mL / min, mL / min, 50 mL / min, 100 mL / min, 200 mL / min, or 500 mL / min.

[0112] The microfluidic chips described herein are designed to minimize cell clogging in the fluid flow region and / or constriction. The number of cells required to clog the microfluidic chip according to the embodiments provided herein 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 follows:

number

[0113] Figures 3A to 3C, 4, 5A to 5C, and 6A to 6C below illustrate various embodiments of the chip, which may have different flow characteristics, including embodiments having approach regions with sidewalls at different angles.

[0114] Figures 3A–3C show various diagrams at different magnifications of the flow line illustrating the flow pattern of a cell suspension through an exemplary microfluidic chip 300 for delivering a payload to cells, according to several embodiments, where the chip does not have an inclined approach region.

[0115] Figure 3A shows a tip 300 including an inlet port 302, a first flow region 304, a constriction 306, a second flow region 308, and an outlet port 310. The components of tip 300 may share any one or more characteristics in common with the corresponding (e.g., similarly numbered) components of tip 100 described above with respect to Figures 1A to 1C. Figure 3B shows an enlarged sub-view of tip 300 including some of the constrictions 306. Figure 3C shows a further enlarged sub-view of tip 300 including two of the constrictions 306.

[0116] As shown in Figures 3A to 3C, the tip 300 does not have a tapered or inclined approach region adjacent to the constriction 306. Rather, the side walls of the constriction 306 are perpendicular to the end walls of the first flow region 304 and the second flow region 308.

[0117] In tip 300, a fluid (e.g., a cell suspension) can flow from the inlet port 302 to the outlet port 310 in the x-axis dimension (diagonal lines of the paper in Figures 3A to 3C). The curved black lines across the first flow region 304, the second flow region 308, and the constriction 306 show the flow pattern of the fluid (e.g., a cell suspension) flowing throughout tip 300. The eddies in the pattern indicate interruptions in the flow, where cells in the cell suspension may aggregate at the points where the flow is interrupted. Flat points in the flow pattern indicate regions where cells may aggregate between flows. Points in the flow pattern where lines branch indicate regions where cells may aggregate.

[0118] Figure 4 shows flow lines illustrating the flow pattern of a cell suspension through an exemplary microfluidic chip 400 for delivering a payload to cells, according to several embodiments, where the chip has an inclined approach region.

[0119] Figure 4 shows a chip 400 including an inlet port 402, a first flow region 404, a constriction 406, a second flow region 408, and an outlet port 410. The components of chip 400 may share any one or more characteristics common to the corresponding (e.g., similarly numbered) components of chip 100 described with respect to Figures 1A to 1C and / or chip 300 described with respect to Figures 3A to 3C.

[0120] The tip 400 may differ from the tip 300 in that each constriction 406 may have a tapered approach region adjacent to each constriction. In the illustrated example, the tip 400 has approach regions whose side walls converge toward the constriction 406 at a 20° angle and branch off from the constriction 406 at a 20° angle (the angle is defined by the angle formed between the opposing approach region side walls).

[0121] In tip 400, a fluid (e.g., a cell suspension) can flow from the inlet port 402 to the outlet port 410 in the x-axis dimension (diagonally upward in the plane of the paper in Figure 4). The curved black lines across the first flow region 404, the second flow region 408, and the constricted section 406 illustrate the flow pattern of the fluid (e.g., a cell suspension) flowing throughout tip 400.

[0122] The same rules used in Figures 3A-3C to represent the flow patterns are used in Figure 4, where eddies in the pattern indicate flow interruptions, flat points in the flow pattern indicate regions where cells may aggregate during flow, and points in the flow pattern where lines branch indicate regions where cells may aggregate. Therefore, it should be noted that the flow patterns in Figure 4 are smoother, more uniform, and less disordered than the flow patterns in Figures 3A-3C, and the approach regions with tapered sidewalls can improve flow within the microfluidic chip, help avoid cell aggregation and / or chip clogging, improve chip throughput, and improve the usable life of the chip.

[0123] Figures 5A to 5C show flow lines illustrating various cell suspension flow patterns through different embodiments of the chip for delivering a payload to cells, where the embodiments have approach regions defined by different angles (or, in the case of Figure 5I, the embodiments do not have inclined approach regions adjacent to the constriction).

[0124] The embodiments shown in Figures 5A to 5C are partial enlarged views of a chip that may share some or all of the characteristics common to the chip (and its components) described above with reference to Figures 1A to 1C, Figures 3A to 3C, and / or Figure 4. In the embodiments shown in Figures 5A to 5C, a fluid (e.g., a cell suspension) can flow through the illustrated constriction in the x-axis direction from left to right on the plane of the paper.

[0125] The tip embodiments in Figures 5A and 5B have approach areas defined by different angles, while in Figure 5C, the embodiment does not have an inclined approach area adjacent to the constriction. The embodiment in Figure 5A has an approach area with side walls that form a 20° angle with each opposite side wall. Figure 5B shows an embodiment with a 40° angle. Figure 5C does not include a tapered or inclined approach area similar to those shown in Figures 3A and 3C (this is sometimes referred to as the "180° angle embodiment").

[0126] The fluid flow patterns in various embodiments shown in Figures 5A to 5C are indicated by arrows showing the fluid flow region, approach region, and constriction. As illustrated, the fluid flow patterns are generally smoother, more uniform, and less disordered in embodiments with a more gently sloping approach region. Approach regions with more gently sloping sidewalls can improve flow within the microfluidic chip, help avoid cell aggregation and / or chip clogging, improve chip throughput, and improve chip usable life.

[0127] Figures 6A–6C show the flow velocities of various cell suspensions through tip embodiments similar to those shown in Figures 5A–5C. In Figures 6A–6C, as shown in the color key in Figure 6A, the 10 shades spanning the gradient represent the flow velocities from (a) 0 m / s–2.327 m / s at the lower end to (b) 20.939 m / s–23.265 m / s at the upper end. As indicated by the gradient, the flow velocity is higher within and around the constriction. In some embodiments, it may be desirable to configure the geometric shape of the tip so that zones where the fluid slows down and / or stagnates are minimized and / or eliminated (in size and / or volume). The tip embodiments in Figures 6A–6B have approach areas defined by different angles, respectively, while in the case of Figure 6C, the embodiment does not have an inclined approach area adjacent to the constriction. The embodiment in Figure 6A has an approach area with side walls that form a 40° angle with each opposite side wall. Figure 6B shows an embodiment with a 60° angle. Figure 6C does not include a tapered or inclined approach area similar to those shown in Figures 3A-3C (this is sometimes referred to as the "180° angle embodiment").

[0128] In some embodiments, the tip 100 may be configured to ensure a sufficiently uniform flow velocity and flow pattern as the cell suspension approaches and flows through multiple constrictions. In some embodiments, the arrangement of the inlet port 102 relative to a set of parallel constrictions 106 may help ensure a uniform flow velocity and flow pattern. The tip 100 may be configured such that the inlet port 102 is sufficiently far (e.g., in the x-direction) from the lateral (e.g., y-direction) range of the barrier (e.g., a straight line or a curve) formed by the set of parallel constrictions 106.

[0129] In some embodiments, the constrictions may be arranged in a line that forms a barrier or wall between a first flow region upstream of the parallel constrictions and a second flow region downstream of the parallel constrictions. The barrier formed by the constrictions may be located at a sufficient distance from the inlet port of the tip, and the length of the barrier may be sufficiently short relative to that distance, and the flow velocity and flow pattern through the constrictions may be sufficiently uniform to maintain high throughput and low clogging.

[0130] In some embodiments, the inlet port 102 may be spaced apart from a set of parallelized constrictions 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 (for example, the length of the shortest straight line between the inlet port 102 and any point on the set of parallelized constrictions). In some embodiments, the inlet port 102 may be spaced apart from a set of parallelized constrictions 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.

[0131] In some embodiments, the ratio of the distance from the inlet 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 inlet 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.

[0132] In some embodiments, the ratio of the distance from the inlet port 106 to the furthest point on the boundary to the distance from the inlet 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 inlet port 106 to the furthest point on the boundary to the distance from the inlet 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 inlet port 106 to the furthest point on the boundary to the distance from the inlet port 106 to the distance on the nearest point on the boundary is equal to 1.

[0133] In some embodiments, chips featuring the above ratio can exhibit excellent flow uniformity, throughput, and clogging resistance. Embodiment of a chip having multiple sets of parallelized constrictions

[0134] Figure 7 shows a chip for delivering a payload to cells according to several embodiments, the chip having multiple sets of parallelized constrictions, the sets being in series with respect to each other. Figure 7 may share one or more characteristics in common with the chip 100 described above with reference to Figures 1A-1C, and the chip 700 may be configured to guide the flow of fluid (e.g., cell suspension) from the inlet port 702 to the outlet port 710 through multiple cell deformation constrictions that form a fluid path between the ports.

[0135] However, while tip 100 includes a single set of constrictions 706 arranged in parallel to each other to form a fluid flow path between a first fluid flow region 104 and a second fluid flow region 108, tip 700 may include multiple sets of fluidically parallelized constrictions. As shown in Figure 7, tip 700 may include a first set of constrictions 706a, a second set of constrictions 706b, a third set of constrictions 706c, a fourth set of constrictions 706d, and a fifth set of constrictions 706e. The constrictions in each of the individual sets 706a to 706e may be arranged in parallel to each other, as in the set of parallelized constrictions 106 in tip 100, providing a fluidically parallelized flow path from the upstream fluid flow region to the downstream fluid flow region. However, the parallelized sets of constrictions 706a to 706b may be provided in series with respect to each other, such that the fluid flow path provided by the first set (e.g., 706a) is upstream of the fluid flow path provided by the second set (e.g., 706b). In tip 700, the fluid flowing from the inlet port 702 to the outlet port 710 can traverse the first fluid flow region 730, the second fluid flow region 732, the third fluid flow region 734, the fourth fluid flow region 736, the fifth fluid flow region 738, and finally the sixth fluid flow region 740, which may be arranged in series with respect to each other or separated by the set of constrictions 706 as shown.

[0136] In some embodiments, adjacent sets of constrictions (e.g., 706a and 706b) may be offset from each other in the flow direction (e.g., in the x-direction in Figure 7) by an amount less than or equal to 25 μm, 50 μm, 100 μm, 500 μm, 1 mm, 1 cm, or 5 cm. In some embodiments, adjacent sets of constrictions (e.g., 706a and 706b) may be offset from each other in the flow direction (e.g., in the x-direction in Figure 7) by an amount greater than or equal to 25 μm, 50 μm, 100 μm, 500 μm, 1 mm, 1 cm, or 5 cm.

[0137] The example in Figure 7 shows a chip 700 having five separate sets of parallelized constrictions, but the chip may have any appropriate number of sets of parallelized constrictions. For example, the chip may have sets of 2, 3, 4, 5 or more, 10 or more, or 20 or more parallelized constrictions arranged in series. Method for delivering a payload to cells using the microfluidic chip described herein.

[0138] Figure 8 illustrates a method for delivering a payload to cells according to several embodiments. The method in Figure 8 may be carried out using one or more chips having parallelized constrictions as described herein, such as (but not limited to) chip 100 in Figures 1A–1C or chip 700 in Figure 7. Figure 8 is described below with illustrative reference to chip 100.

[0139] In step 802, the microfluidic chip (e.g., microfluidic chip 102 in Figure 1) receives a fluid flow into a first fluid flow region. The fluid, such as a cell suspension, contains multiple cells. In some embodiments, the fluid may also contain a payload to be delivered to one or more cells in the fluid. Specifically, the microfluidic chip receives a fluid flow in the first fluid flow region located upstream of a set of parallelized constrictions, which fluidizes the first fluid flow region to a second fluid flow region downstream of the set of parallelized constrictions.

[0140] In step 804, the microfluidic chip (e.g., microfluidic chip 102 in Figure 1) perturbs the cell membranes of cells from the fluid by causing the cells to flow through a set of parallelized constrictions within the microfluidic chip. As described herein, the set of constrictions may be formed by etching channels within the substrate of the chip and may be configured to guide the cell suspension toward and through the constrictions.

[0141] In step 806, the payload is delivered to the perturbed cells. Payload delivery may occur in a second fluid flow region downstream of the set of parallel constrictions, after the cell suspension has passed through the constrictions and the resulting perturbation of the cell membrane. In some embodiments, the payload is introduced into the cell suspension in the second fluid flow region and / or in a fluid vessel or reservoir separate from tip 100 after the perturbation. After payload delivery, the cell membrane may heal.

[0142] In some embodiments, cells treated through one or more constrictions of the microfluidic chip described herein, for example according to Method 800, may exhibit viability after passing through constrictions (which may be greater than or equal to) 30%, 50%, 70%, 90%, 95%, or 99%.

[0143] In some embodiments, cells processed through one or more constrictions of the microfluidic chip described herein, for example according to Method 800, may exhibit payload delivery rates greater than or equal to 30%, 50%, 70%, 90%, 95%, or 99%. Chips placed inside the cartridge

[0144] Figure 9 shows a cartridge 900 that holds two tips 902a and 902b inside. The cartridge 900 can hold the tips inside and guide fluid flow to and from the tips held inside. As shown by the cross-sectional view in Figure 9, the O-ring 904 can press the tips, surround the inlet or outlet port of the tips, hold the tips in place, and form a seal around the inlet / outlet port, facilitating fluid flow into and out of the tips without leakage. Geometric shape of the fluid flow region

[0145] Figures 10A to 10C show tips with various fluid flow region geometric shapes. Specifically, the tip 100 shown in Figures 1A to 1C has a fluid flow region formed as an irregular pentagon (in an overhead view in the xy plane), but the cartridges in Figures 10A to 10C may have fluid flow regions of other shapes.

[0146] Cartridge 1000a, shown in Figure 10A, has a rectangular fluid flow region. Cartridge 1000b, shown in Figure 10B, has a triangular fluid flow region. Cartridge 1000c, shown in Figure 10C, both have rectangular fluid flow regions that form a square. Example 1

[0147] The performance of different chips was compared, with the first set of parallel chips having a constriction width of 4 μm and the second set of parallel chips having a constriction width of 4.5 μm. Each chip contained 75 constrictions arranged in parallel. For all chips, the constriction length was 10 μm. For all chips, a pressure of 50 PSI and a temperature of 2-8°C were used. For all chips, 7.20 × 10⁶ doses of the blood product Hemacare LP were administered. 7 The solution was used at a concentration of cells / mL. For all tips, RPMI delivery medium and 0.01 mg of ML 3kDa dextran AF680, the delivery material, were used.

[0148] The chip's performance is shown in Table 1 below. [Table 1] Example 2

[0149] The performance of a chip having 75 constrictions arranged in parallel, each constriction having a width of 4 μm and an etching depth of 66 μm, was tested. These three runs, or "squees," in which the chip was microfluidically squeezed under pressures of 40 PSI, 50 PSI, and 60 PSI, were plotted against three control squeezes: Control 1 (tested at 60 PSI, chip with constrictions 30 μm long, 4 μm wide, and 20 μm deep), Control 2 (no microfluidic squeeze, dextran added to cell suspension), and Control 3 (no dextran added, no microfluidic squeeze), showing the percentage of live B cells, the percentage of dextran delivered, and the relative mean fluorescence intensity, as shown in Figure 11.

[0150] The graph on the right in Figure 11 is a histogram showing fluorescence analysis of dextran uptake by cells under different conditions. The top three histograms represent squeeze under different pressures: 40 PSI, 50 PSI, and 60 PSI. "STD" represents the squeeze condition with a tip having a constriction of 30 μm in length, 4 μm in width, and 20 μm in depth, tested at 60 PSI. "SQZendo" represents the delivery of dextran by simply bringing cells into contact with dextran without squeezing, showing dextran uptake into cells by endocytosis. "No Contact" represents the fluorescence of cells that did not contain dextran and were not squeezed.

[0151] The performance of a chip having 75 parallel-arranged constrictions, each constriction having a width of 4 μm and an etching depth of 97 μm, was tested. The chip was subjected to microfluidic squeeze under pressures of 15 PSI, 30 PSI, 45 PSI, and 60 PSI. These four squeezes were plotted against three control squeezes: Control 1 (tested at 60 PSI, chip with constrictions 30 μm long, 4 μm wide, and 20 μm deep), Control 2 (no microfluidic squeeze, dextran added to cell suspension), and Control 3 (no dextran added, no microfluidic squeeze), showing the percentage of live B cells, the percentage of dextran delivered, and the relative mean fluorescence intensity, as shown in Figure 12.

[0152] The graph on the right in Figure 12 is a histogram showing fluorescence analysis of dextran uptake by cells under different conditions. The top four histograms represent squeeze under different pressures: 15 PSI, 30 PSI, 45 PSI, and 60 PSI. "STD" represents the squeeze condition tested at 60 PSI with a tip having a constriction of 30 μm in length, 4 μm in width, and 20 μm in depth. "SQZendo" represents the delivery of dextran by simply bringing cells into contact with dextran without squeezing, showing dextran uptake into cells by endocytosis. "No Contact" represents the fluorescence of cells without dextran and without squeeze. Example 3

[0153] A chip was fabricated in which 950 constricted sections, each 4.5 μm wide, 10 μm long, and 80 μm deep, were arranged in parallel. The overall dimensions of the chip were 30 mm in length, 20 mm in width, and 1225 μm in height (thickness). The chip included an inlet fluid port 24 mm away from the outlet fluid port, with each port having a diameter of 2.00 mm. The chip consisted of a silicon substrate with a height (thickness) of 600 μm and a top layer of glass with a thickness of 600 μm.

[0154] The target dimensions for the constricted section used to manufacture the chip were a constricted section width of 4.50 ± 0.20 μm, a constricted section length of 10 ± 1 μm, and a constricted section height (depth) of 80 ± 2 μm.

[0155] The specifications for manufacturing the chip were as shown in Table 2 below: [Table 2]

[0156] The narrowing width was measured as the average narrowing width of 10% of all narrowing areas on the tip, measured at the top of each narrowing area.

[0157] The variation in the narrowing width was measured within a given narrowing, based on SEM cross-sections of selected chips across the wafer.

[0158] The chip manufacturing process was configured such that 80% of the chips produced were expected to fall within the specifications listed in Table 2. Example 4

[0159] The performance of six chip types was tested, each of which has multiple sets of constrictions arranged in parallel, with sets in series. The six chip types are schematically shown in Figure 13A.

[0160] The chip layout 5f has five sets of parallel constrictions, which are in series with respect to each other and spaced apart by a first interval.

[0161] The chip layout 5c has five sets of parallel constrictions, which are in series with respect to each other and spaced apart by a second spacing distance that is smaller than a first spacing distance.

[0162] The chip layout 10 has 10 sets of parallel constrictions, these sets are in series with each other and spaced apart by a second spacing distance.

[0163] Chip type 1 has a "5f" chip layout in which the upstream approach area is inclined at an angle of 20° and the downstream approach area is inclined at an angle of 45°.

[0164] Chip type 2 has a "5c" chip layout in which the upstream approach area is tilted at an angle of 20° and the downstream approach area is tilted at an angle of 45°.

[0165] Chip type 3 has a "10" chip layout in which the upstream approach area is tilted at an angle of 20° and the downstream approach area is tilted at an angle of 45°.

[0166] Chip type 4 has a "5f" chip layout in which the upstream approach area is tilted at an angle of 20° and the downstream approach area is tilted at an angle of 60°.

[0167] Chip type 5 has a "5c" chip layout in which the upstream approach area is tilted at an angle of 20° and the downstream approach area is tilted at an angle of 60°.

[0168] Chip type 6 has a "10" chip layout in which the upstream approach area is tilted at an angle of 20° and the downstream approach area is tilted at an angle of 60°.

[0169] For each of the six tip types, the length of the constricted area was 10 μm, the width was 3 μm, and the height (etching depth) was 70 μm.

[0170] For each of the six types of chips, 88 chips were tested. The 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 running the chip both "forward" and "backward". The results are shown in Figures 13B–13H according to the angle of the approach region that was actually on the upstream side during the run. That is, in the run labeled "45°" for chip type 1, the chip was reversed so that the 45° approach region was on the upstream side and the 20° approach region was on the downstream side. The results are shown below.

[0171] Figure 13B shows the survival and delivery rate results of a Chip Type 1 test at various pressures (30, 45, 60, 75, 90, and 105 PSI) using blood from three different donors (A, B, and C) in various orientations.

[0172] Figure 13C shows the detection of active GFP in cells from a tip-type 1 test at various pressures (30, 45, 60, 75, 90, and 105 PSI) using blood from three different donors (A, B, and C) in various orientations.

[0173] Figure 13D shows the effectiveness results of testing tip type 1 at various pressures (30, 45, 60, 75, 90, and 105 PSI) and in various orientations.

[0174] Figures 13E-13H show FACS plots for the chip type 1 test. GFP fluorescence in cells is plotted against dextran fluorescence in cells.

[0175] Figure 13I shows the survival and delivery rate results of a chip type 3 test at various pressures (45, 60, 75, 90, and 105 PSI) using blood from three different donors (A, B, and C) in various orientations.

[0176] Figure 13J shows the results of the test of chip type 3 at various pressures (45, 60, 75, 90, and 105 PSI) using blood from three different donors (A, B, and C) in various orientations.

[0177] Figure 13K shows the effectiveness results of testing tip type 3 at various pressures (45, 60, 75, 90, and 105 PSI) and in various orientations.

[0178] Figures 13L-13O show FACS plots of the chip type 3 test. GFP fluorescence in cells is plotted against dextran fluorescence in cells. Embodiment

[0179] The following is an enumerated list of specific embodiments. In some embodiments, even if the dependencies of the embodiments do not explicitly indicate that the embodiments can be combined, they can be combined with any one or more of the features of any one or more of the following embodiments or any one or more other embodiments. 1. A microfluidic chip for delivering a payload to a cell, wherein the chip is A fluid inlet configured to receive the flow of a cell suspension and deliver the cell suspension to a first fluid flow region within the microfluidic chip, A plurality of constrictions fluidly connected to the first fluid flow region, to enable the cell suspension to flow from the first fluid flow region to the second fluid flow region within the microfluidic chip through one or more constrictions, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, and the cell membrane is perturbed as it passes through the constrictions so that the payload can pass through the perturbed cell membrane. The quotient of the cross-sectional area of ​​each of the aforementioned constricted portions with respect to the perimeter of the cross-section is greater than or equal to 0.5 μm, and the plurality of constricted portions, A microfluidic chip comprising a fluid outlet configured to allow the cell suspension to flow out of the microfluidic chip from the second fluid flow region. 2. The microfluidic chip according to Embodiment 1, wherein the cross-sectional width of each of the plurality of constricted portions is less than or equal to one or greater than 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 any one of Embodiments 1 to 2, wherein the cross-sectional height of each of the plurality of constricted portions is greater than or equal to 20 μm. 4. The microfluidic chip according to Embodiment 3, wherein each of the plurality of constricted portions is formed by etching the substrate of the microfluidic chip, and the cross-sectional height of each of the plurality of constricted portions is defined by the etching depth of the etching. 5. The microfluidic chip according to Embodiment 4, wherein the substrate contains silicon. 6. A microfluidic chip according to any one of embodiments 4 to 5, wherein the etching includes deep reactive ion etching. 7. A microfluidic chip according to any one of embodiments 1 to 6, wherein the plurality of constrictions include more than 1,000 constrictions. 8. A microfluidic chip according to any one of embodiments 1 to 7, wherein the plurality of constrictions are arranged in parallel with each other so as to form a boundary between the first fluid flow region and the second fluid flow region. 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. 10. A microfluidic chip according to any one of embodiments 8 to 9, wherein the ratio of the distance from the inlet port to the furthest 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. A microfluidic chip according to any one of embodiments 8 to 10, wherein the boundary extends in a straight line. 12. A microfluidic chip according to any of embodiments 8 to 11, wherein the length of the boundary is greater than or equal to 4 mm. 13. The microfluidic chip according to any one of embodiments 1 to 12, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions. 14. The microfluidic chip according to any of embodiments 1 to 13, wherein the microfluidic chip is configured to operate at a pressure greater than or equal to 10 psi. 15. A microfluidic chip according to any one of embodiments 1 to 14, wherein each of the plurality of constrictions is positioned adjacent to an approach region which includes a tapered wall that narrows toward the constriction. 16. The microfluidic tip according to Embodiment 15, wherein the tapered wall tapers toward the constricted portion at an angle greater than or equal to 10 degrees from the side wall of the constricted portion and less than or equal to 80 degrees. 17. The microfluidic chip according to any one of embodiments 1 to 16, wherein the microfluidic chip is configured such that the average volume flow rate per constricted portion of the plurality of constricted portions is greater than or equal to 1 μL / min. 18. A microfluidic chip according to any one of embodiments 1 to 17, comprising one or more pillars that intersect the first fluid flow region and connect a first inner surface plane of the first fluid flow region to a second inner surface plane of the first fluid flow region. 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. 20. A method for delivering a payload to a cell, wherein the method is Receiving a flow of a cell suspension into a first fluid flow region of a microfluidic chip, wherein the cell suspension contains a plurality of cells, This includes causing the cell suspension to flow from the first fluid flow region through a plurality of constrictions of the microfluidic chip, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, so that the cell membrane is deformed when passing through the constrictions, allowing the payload to pass through the deformed cell membrane. A method wherein the quotient of the cross-sectional area of ​​each of the plurality of constricted portions with respect to the cross-sectional perimeter is greater than or equal to 0.5 μm. 21. The method according to Embodiment 20, wherein the cell suspension comprises the payload. 22. The method according to any one of embodiments 20 to 21, comprising bringing the payload into contact with the cell suspension after perturbation of the cell membrane. 23. The method according to Embodiment 22, wherein the percentage of cells to which the payload is delivered after the payload is brought into contact with the cell suspension is greater than or equal to 30%. 24. The method according to any one of embodiments 20 to 23, wherein the percentage of viable cells in the cell suspension after the cell suspension has passed through the plurality of constrictions of the microfluidic chip is greater than or equal to 30%. 25. The method according to any one of Embodiments 20 to 24, wherein the cross-sectional width of each of the plurality of constrictions is smaller than or equal to one or more than one of 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 Embodiments 20 to 25, wherein the cross-sectional height of each of the plurality of constrictions is greater than or equal to 20 μm. 27. The method according to any one of Embodiments 20 to 26, wherein the plurality of constrictions includes more than 1000 constrictions. 28. The method according to any one of Embodiments 20 to 27, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions. 29. The method according to any one of Embodiments 20 to 28, wherein flowing the cell suspension from the first fluid flow region through the plurality of constrictions includes forcing the fluid flow at a pressure greater than or equal to 10 psi. 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 includes flowing the cell suspension at an average volume flow rate per constriction greater than or equal to 1 μL / min. 31. A method of manufacturing a microfluidic chip for delivering a payload to a cell, the method comprising: etching a substrate to form a first fluid flow region configured to allow a cell suspension to flow from an inlet port through the first fluid flow region; and etching the substrate to form a plurality of constrictions configured to allow the cell suspension to flow from the first fluid flow region through the 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, whereby the cell membrane is deformed as it passes through the constriction so that the payload can pass through the deformed cell membrane. A method in which the quotient of the cross-sectional area with respect to the circumferential length of each of the plurality of constrictions is greater than or equal to 0.5 μm. 32. The method according to embodiment 31, comprising fixing a cover layer to the etched substrate to surround the first fluid flow region and the plurality of constrictions. 33. The method according to any one of embodiments 31 to 32, wherein the etching of the substrate to form the plurality of constrictions includes deep reactive ion etching. 34. The method according to any one of embodiments 31 to 33, wherein etching the substrate to form the plurality of constrictions includes an etching step to a depth greater than or equal to 50 μm. 35. The method according to any one of embodiments 31 to 34, comprising depositing a layer of material on the substrate after etching the substrate, and reducing the width of the plurality of constrictions by depositing the layer. 36. A microfluidic chip for delivering a payload to a cell, the chip comprising: A fluid inlet configured to receive a flow of cell suspension and send the cell suspension to 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 within the microfluidic chip through one or more than one first plurality of constrictions to a second fluid flow region; 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 within the microfluidic chip through one or more than one second plurality of constrictions to a third fluid flow region, wherein The cross-sectional width of each of the constrictions of the first plurality of constrictions and the second plurality of constrictions is smaller than the diameter of the cells in the cell suspension, whereby the cell membrane is perturbed as it passes through the constriction so that the payload can pass through the perturbed cell membrane. A microfluidic chip comprising: a first plurality of constrictions and a second plurality of constrictions, wherein the quotient of the cross-sectional area of ​​each of the first plurality of constrictions with respect to the cross-sectional perimeter of each of the constrictions is greater than or equal to 0.5 μm. 37. The microfluidic chip according to Embodiment 36, wherein the cross-sectional width of each of the first plurality of constrictions and the second plurality of constrictions is less than or equal to one or greater than 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm. 38. A microfluidic chip according to any one of embodiments 36 to 37, wherein the cross-sectional height of each of the first plurality of constrictions and the second plurality of constrictions is greater than or equal to 20 μm. 39. The microfluidic chip according to Embodiment 38, wherein each of the first plurality of constrictions and the second plurality of constrictions is formed by etching the substrate of the microfluidic chip, and the cross-sectional height of each of the plurality of constrictions is defined by the etching depth of the etching. 40. A microfluidic chip according to any one of embodiments 36 to 39, wherein each of the first plurality of constrictions and the second plurality of constrictions includes more than 1,000 constrictions. 41. A microfluidic chip according to any of embodiments 36 to 40, wherein the first plurality of constrictions and the second plurality of constrictions are spaced apart from each other by the nearest spacing distance greater than or equal to 25 μm. 42. The microfluidic chip according to any one of embodiments 36 to 41, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions. 43. A microfluidic chip according to any of embodiments 36 to 42, configured to operate at a pressure greater than or equal to 10 psi. 44. The microfluidic chip according to any one of embodiments 36 to 43, wherein the microfluidic chip is configured such that the average volume flow rate per constriction of the first plurality of constrictions and the second plurality of constrictions is greater than or equal to 1 μL / min.

Claims

1. A microfluidic chip for delivering a payload to cells, wherein the chip is A fluid inlet configured to receive the flow of a cell suspension and send the cell suspension to a first fluid flow region within the microfluidic chip, A plurality of constrictions fluidly connected to the first fluid flow region, enabling the cell suspension to flow from the first fluid flow region to the second fluid flow region within the microfluidic chip through one or more of the plurality of constrictions. The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, thereby perturbing the cell membrane as it passes through the constriction, and thereby allowing the payload to pass through the perturbed cell membrane. The quotient of the cross-sectional area of ​​each of the aforementioned constricted portions with respect to the perimeter of the cross-section is greater than or equal to 0.5 μm, and the plurality of constricted portions, A fluid outlet configured to allow the cell suspension to flow out of the microfluidic chip from the second fluid flow region, One or more pillars extending through the first fluid flow region and / or the second fluid flow region, The uppermost layer, wherein the pillar 1 or beyond functions as a support structure for the uppermost layer and Microfluidic chips, including...

2. The microfluidic chip according to claim 1, wherein the cross-sectional width of each of the plurality of constricted portions is less than or equal to one or more of 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 constricted portions is greater than or equal to 20 μm.

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

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

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

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

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

9. The microfluidic chip according to 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 according to claim 8, wherein the ratio of the distance from the inlet port to the furthest 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 according to claim 8, wherein the boundary includes a portion that extends in a straight line.

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 each of the plurality of constricted portions is located adjacent to an approach region which includes a tapered wall that narrows toward the constricted portion.

14. The microfluidic tip according to claim 13, wherein the tapered wall tapers toward the constricted portion at an angle greater than or equal to 10 degrees from the side wall of the constricted portion and less than or equal to 80 degrees.

15. The microfluidic chip according to any one of claims 1 to 6, wherein the 1 or more pillars intersect with the first fluid flow region and connect the plane of the first inner surface of the first fluid flow region to the plane of the second inner surface of the first fluid flow region.

16. The microfluidic chip according to any one of claims 1 to 6, wherein the first fluid flow region, the plurality of constrictions, and the second fluid flow region are formed by etching the substrate, and 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.

17. A method for delivering a payload to a cell, wherein the method is The process involves receiving a flow of a cell suspension into a first fluid flow region of a microfluidic chip, wherein the cell suspension contains a plurality of cells. This includes causing the cell suspension to flow from the first fluid flow region through a plurality of constricted portions of the microfluidic chip, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, thereby the cell membrane is deformed when passing through the constriction, and thereby the payload can pass through the deformed cell membrane. The quotient of the cross-sectional area of ​​each of the aforementioned multiple constricted portions with respect to the perimeter of the cross-section is greater than or equal to 0.5 μm. The microfluidic chip includes one or more pillars extending through the first fluid flow region and / or the second fluid flow region, the one or more pillars functioning as support structures for the uppermost layer of the microfluidic chip. method.

18. The method according to claim 17, wherein the cell suspension comprises the payload.

19. The method according to claim 17, comprising bringing the payload into contact with the cell suspension after perturbation of the cell membrane.

20. The method according to claim 19, wherein the percentage of cells to which the payload is delivered after the payload is brought into contact with the cell suspension is greater than or equal to 30%.

21. The method according to any one of claims 17 to 20, wherein the percentage of live cells in the cell suspension after the cell suspension has passed through the plurality of constrictions of the microfluidic chip is greater than or equal to 30%.

22. The method according to any one of claims 17 to 20, wherein the cross-sectional width of each of the plurality of constricted portions is less than or equal to one or more than 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm.

23. The method according to any one of claims 17 to 22, wherein the cross-sectional height of each of the plurality of constricted portions is greater than or equal to 20 μm.

24. The method according to any one of claims 17 to 23, wherein the plurality of constricted portions include more than 1,000 constricted portions.

25. The method according to any one of claims 17 to 24, wherein the microfluidic chip is configured to operate at an overall throughput rate greater than or equal to 1 mL / min across all constrictions.

26. The method according to any one of claims 17 to 25, wherein the flow of the cell suspension from the first fluid flow region through the plurality of constrictions is forced to a pressure greater than or equal to 10 psi.

27. The method according to any one of claims 17 to 26, wherein the flow of the cell suspension from the first fluid flow region through the plurality of constrictions is performed by flowing the cell suspension at an average volume flow rate per constriction of the plurality of constrictions greater than or equal to 1 μL / min.

28. A method for manufacturing a microfluidic chip for delivering a payload to cells, wherein the method is Etching a substrate to form a first fluid flow region, which is configured to allow a cell suspension to flow from an inlet port through the first fluid flow region, wherein one or more pillars extend through the first fluid flow region and / or a second fluid flow region, and the one or more pillars function as a support structure for the uppermost layer of the microfluidic chip. The process includes etching the substrate to form a plurality of constrictions, which are configured to allow the cell suspension to flow from the first fluid flow region through the constrictions, The cross-sectional width of each of the plurality of constrictions is smaller than the diameter of the cells in the cell suspension, thereby the cell membrane is deformed when passing through the constriction, and thereby the payload can pass through the deformed cell membrane. A method wherein the quotient of the cross-sectional area of ​​each of the plurality of constricted portions with respect to the cross-sectional perimeter is greater than or equal to 0.5 μm.

29. The method according to claim 28, comprising fixing a cover layer to the etched substrate to surround the first fluid flow region and the plurality of constricted portions.

30. The method according to any one of claims 28 to 29, wherein the etching of the substrate for forming the plurality of constricted portions includes deep reactive ion etching.

31. The method according to any one of claims 28 to 29, wherein etching the substrate to form the plurality of constrictions includes etching to a depth greater than or equal to 50 μm.

32. The method according to any one of claims 28 to 29, comprising depositing a layer of material on the substrate after etching the substrate, wherein the width of the plurality of constricted portions is reduced by depositing the layer.

33. A microfluidic chip for delivering a payload to cells, wherein the chip is A fluid inlet configured to receive the flow of a cell suspension and send the cell suspension to a first fluid flow region within the microfluidic chip, A first plurality of constrictions fluidly connected to the first fluid flow region, the first plurality of constrictions enabling the cell suspension to flow from the first fluid flow region to the second fluid flow region within the microfluidic chip through one or more of the first plurality of constrictions, A second plurality of constrictions fluidly connected to the second fluid flow region, enabling the cell suspension to flow from the second fluid flow region to the third fluid flow region within the microfluidic chip through one or more of the second plurality of constrictions, The cross-sectional width of each of the first and second plurality of constrictions is smaller than the diameter of the cells in the cell suspension, thereby perturbing the cell membrane as it passes through the constriction, thereby allowing the payload to pass through the perturbed cell membrane. The second plurality of constricted portions, wherein the quotient of the cross-sectional area of ​​each of the first plurality of constricted portions with respect to the perimeter of the cross-section of each of the constricted portions is greater than or equal to 0.5 μm, One or more pillars extending through the first fluid flow region and / or the second fluid flow region, The uppermost layer, wherein the pillar 1 or beyond functions as a support structure for the uppermost layer and Microfluidic chips, including...

34. The microfluidic chip according to claim 33, wherein the cross-sectional width of each of the first plurality of constrictions and the second plurality of constrictions is less than or equal to one or more than 1.8 μm, 1.9 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, or 10 μm.

35. The microfluidic chip according to claim 33, wherein the cross-sectional height of each of the first plurality of constrictions and the second plurality of constrictions is greater than or equal to 20 μm.

36. The microfluidic chip according to claim 35, wherein each of the first plurality of constrictions and the second plurality of constrictions is formed by etching the substrate of the microfluidic chip, and the cross-sectional height of each of the plurality of constrictions is defined by the etching depth of the etching.

37. The microfluidic chip according to any one of claims 33 to 36, wherein each of the first plurality of constrictions and the second plurality of constrictions includes more than 1,000 constrictions.

38. The microfluidic chip according to claim 37, wherein the first plurality of constrictions and the second plurality of constrictions are spaced apart from each other by the nearest spacing distance greater than or equal to 25 μm.

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