Apparatus and method for recovering organelle vesicles from cells using a microfluidic extraction unit

Abstract

A device for recovering organelle vesicles from cells, comprising a microfluidic chip for extracting organelle vesicles from cells, the microfluidic chip having one or more microchannels (14) each having one or more contractile portions (27). A method for carrying out the device and extracellular organelle vesicles obtained by the method.

Description

[Technical Field]

[0001] The present invention generally relates to an apparatus and method for recovering intracellular components from cells.

[0002] More specifically, the present invention relates to a microfluidic extraction unit. [Background technology]

[0003] To recover intracellular components from cells, it is generally necessary to lyse the cells, that is, to break down the outer cell membrane and release the intracellular components, and manual protocols are used for this purpose.

[0004] Conventional cell lysis techniques include chemical, mechanical, electrical, thermal, and laser lysis processes.

[0005] Known technologies are costly in terms of human time and effort, complex, non-automatable, non-reproducible, and produce very little biological material, which hinders industrialization and data robustness. [Overview of the Initiative]

[0006] From an industrialization perspective, the present invention provides an apparatus for recovering organelle vesicles from cells, and the apparatus is Organelle swelling units configured to produce swollen intracellular organelle vesicles; and An extraction unit configured to extract swollen organelle vesicles from cells; The extraction unit comprises a microfluidic chip having one or more microchannels configured to receive a fluid stream containing the cells, the microchannels comprising one or more contraction sections configured to mechanically restrain the cells as they pass through it.

[0007] Such microfluidic chips enable high-frequency extraction of organelle vesicles from cells, control of lysis parameters to minimize organelle damage for the recovery of highly functional organelle vesicles, and a reproducible extraction process in which each cell experiences the same stress. This meets the industrial requirements of mass production, organelle viability, and reproducibility.

[0008] In one embodiment, at least one of the shrinking portions is connected to the upstream and / or downstream portions of the same microchannel.

[0009] The upstream portion preferably has a cross-section that decreases, preferably monotonically, from the upstream portion of the microchannel toward the contraction portion.

[0010] The downstream portion preferably has a cross-section that increases monotonically from the contracted portion toward the downstream portion of the microchannel.

[0011] In one embodiment, the upstream portion is formed by the following: At least one first wall member defining an angle with the transverse plane between 91° and 179°, preferably between 99° and 170°, for example, 105° or 130°; and / or At least one second wall member defining an angle with the transverse plane in the range of 90° to 170°, preferably 90° to 160°, for example, 95° or 120°.

[0012] In one embodiment, the downstream portion is formed by the following: At least one first wall member defining an angle with the transverse plane between 91° and 180°, preferably between 120° and 179°, for example, 150° or 170°; and / or At least one second wall member defining an angle with the transverse plane in the range of 90° to 170°, preferably 90° to 160°, for example, 95° or 120°.

[0013] When the constriction is connected to both the upstream portion and the downstream portion, it is preferable that the upstream portion is longer than the downstream portion with respect to the direction of fluid flow.

[0014] In one embodiment, the constriction has the following. 100 μm 2 to 2000 μm 2 , preferably 170 μm 2 to 650 μm 2 a cross-section in the range of, for example, 225 μm 2 or 330 μm 2 of the cross-section, and / or a width in the range of 0.5 μm to 15 μm, preferably 2 μm to 15 μm, preferably 4 μm to 15 μm, preferably 7 μm to 13 μm, for example, a width of 8 μm, 9 μm, 10 μm, 11 μm or 12 μm, and / or a height in the range of 0.5 μm to 15 μm, preferably 2 μm to 100 μm, preferably 5 μm to 100 μm, preferably 10 μm to 30 μm, for example, a height of 25 μm, 30 μm, 35 μm or 40 μm, and / or a length in the range of 1 μm to 200 μm, preferably 10 μm to 70 μm, preferably 25 μm to 50 μm, for example, a length of 20 μm, 30 μm, 40 μm, 50 μm or 60 μm.

[0015] At least one of the constrictions may have a polygonal cross-section, for example, a rectangular cross-section or a trapezoidal cross-section.

[0016] In one embodiment, at least one of the microchannels includes a plurality of the constrictions, for example, two or more constrictions.

[0017] In one embodiment, the constricted cross-section decreases from one constriction to another constriction in the same microchannel with respect to the fluid flow direction.

[0018] In one embodiment, each of the plurality of microchannels includes one or more of the constrictions.

[0019] In one embodiment, the organelle swelling unit comprises means for bringing cells into contact with a hypotonic aqueous medium.

[0020] In one embodiment, the organelle swelling unit comprises means for bringing cells into contact with a chemical compound, and for example, one or more organelles are vesiculated for a duration in the range of 0.5 hours to 48 hours, preferably 1 hour to 36 hours, more preferably 2 hours to 24 hours.

[0021] In one embodiment, the organelle swelling unit comprises means for bringing cells into contact with bafilomycin-1 at a concentration of 1 nmol / L to 100 μmol / L, preferably 10 nmol / L to 5 μmol / L, more preferably 100 nmol / L to 2 μmol / L for 1 hour to 48 hours, preferably 2 hours to 24 hours, or into contact with methylamine or rapamycin or bafilomycin, and swelling lysosomes and / or endolysosomes and / or endosomes and / or autophagosomes into large vesicles.

[0022] In one embodiment, the organelle swelling unit comprises means for bringing cells into contact with actinomycin D, AZD 5582, AT 101, camptothecin, cisplatin, doxorubicin, etoposide, mitomycin C, MSC 2032964A, nutlin-3, paclitaxel, PRIMA-1MET, staurosporine, vinblastine, and for example inducing cell apoptosis to induce the formation of large vesicles of a plurality of organelles.

[0023] In one embodiment, the organelle swelling unit is formed by a part of the microfluidic chip and / or includes an additional microfluidic chip.

[0024] In one embodiment, the apparatus further comprises a recovery unit configured to recover and / or fractionate the organelle vesicles extracted from cells by the extraction unit.

[0025] In one embodiment, the recovery unit includes a portion of the microfluidic chip and / or additional microfluidic chips.

[0026] In one embodiment, the apparatus further comprises an observation unit configured to observe the organelle vesicles extracted from cells by the extraction unit.

[0027] In one embodiment, the observation unit includes a camera and / or a microscope, such as a confocal microscope.

[0028] Another object of the present invention is a method for recovering organelle vesicles from cells, the method being The steps of swelling the organelle vesicles with the organelle swelling unit of the apparatus defined above; and The procedure includes the step of extracting swollen organelle vesicles from cells using the extraction unit of the apparatus defined above.

[0029] In one embodiment, the method includes, after the extraction step, the step of recovering the organelle vesicles with the recovery unit defined above.

[0030] In one embodiment, the method includes introducing a fluid containing the cells into the microchannel of the microfluidic chip of the extraction unit at a flow rate in the range of 1 μL / min to 500 μL / min, preferably 50 μL / min to 200 μL / min, for example, 75 μL / min, 100 μL / min, 125 μL / min, 150 μL / min, or 175 μL / min.

[0031] In one embodiment, the method further includes the step of observing the organelle vesicles with the observation unit defined above during and / or after the extraction step.

[0032] Another object of the present invention is the extracellular organelle vesicles obtained by the method defined above.

[0033] In one embodiment, the organelle vesicles have a size ranging from 3 μm to 15 μm, for example, from 4 μm to 10 μm.

[0034] Naturally, the above embodiments can be combined with each other.

[0035] The present invention makes it possible to produce large quantities of organelle vesicles for the creation of purified and concentrated samples that can be delivered to the market. Microfluidic extraction according to the present invention is typically ideal for achieving flow rates that continuously extract batches of large organelle vesicles of approximately one million per hour. Furthermore, the high-frequency extraction unit of the present invention can be directly connected to a separator.

[0036] The present invention, and all related aspects, embodiments, and advantages, will become more readily apparent upon consideration of the detailed description of specific embodiments provided below, including the accompanying drawings. [Brief explanation of the drawing]

[0037] Non-limiting embodiments of the present invention will be described below with reference to the following attached schematic diagrams.

[0038] [Figure 1] Figure 1 is a schematic representation of the apparatus according to the present invention, which comprises a preparation unit, an extraction unit, a recovery unit, and an observation unit. [Figure 2] Figure 2 shows a schematic representation of the microfluidic chip according to the present invention. [Figure 3] Figure 3 shows a schematic representation of the microchannel of the present invention in the first cross-sectional plane. [Figure 4] Figure 4 is a schematic representation of the microchannel in Figure 3 using a second cross-sectional plane perpendicular to the first cross-sectional plane. [Figure 5] Figure 5 shows a schematic representation of a microchannel according to one embodiment of the present invention. [Figure 6] Figure 6 shows a schematic representation of a microchannel according to another embodiment of the present invention. [Figure 7]Figure 7 shows a schematic representation of a microchannel according to a further embodiment of the present invention. [Modes for carrying out the invention]

[0039] Figure 1 schematically shows apparatus 1 according to a non-limiting embodiment of the present invention. Apparatus 1 is typically intended for recovering organelle vesicles from cells.

[0040] Generally, cells may be mammalian, plant, and / or bacterial cells, such as COS-7 cells, HeLa cells, HEK cells, CHO cells, fibroblasts, erythrocytes, T cells, neuroblastoma cells, stem cells, and iPSC (induced pluripotent stem cell) derived cell types, such as iPSC-derived neural stem cells, iPSC-derived mesenchymal stem cells, iPSC-derived monocyte stem cells, iPSC-derived cardiomyocyte stem cells, iPSC-derived microglia stem cells, and iPSC-derived myotubular stem cells. These cells can be obtained from the market, laboratories, and / or hospitals, and / or recovered from organoids, biopsies, tissues, organs, and / or living organisms derived from healthy humans or patients with diseases.

[0041] In this specification, organelles are defined as differentiated biological materials such as lysosomes, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, chloroplasts, autophagosomes, autolysosomes, endosomes, endolysosomes, peroxisomes, and polyvesicles.

[0042] The apparatus 1 in Figure 1 comprises a preparation unit 2 configured to prepare cells and intracellular organelle vesicles, an extraction unit 3 configured to extract organelle vesicles from cells, a recovery unit 4 configured to collect the extracted organelle vesicles, and an observation unit 5 configured to observe the extracted organelle vesicles.

[0043] <Cell preparation> In this embodiment, the preparation unit 2 includes means (not shown) for bringing cells into contact with a hypotonic aqueous medium to produce swollen and compartmentalized intracellular organelles (these are called organelle vesicles).

[0044] Therefore, preparation unit 2 may include an organelle swelling unit, also called a "swelling unit," and is configured to produce such intracellular organelle vesicles. These organelle vesicles may be, in particular, intracellular bilayer-bound organelles, lipid droplets, ribosomes, or cytoskeleton. Preferably, the organelle vesicles are not cell nuclei.

[0045] In this embodiment, the cells are brought into contact with the hypotonic aqueous medium, which may be any aqueous solution having an osmotic pressure in the range of 0.1 mOsm / L to 200 mOsm / L, preferably 0.1 mOsm / L to 100 mOsm / L, preferably 1 mOsm / L to 50 mOsm / L, more preferably 5 mOsm / L to 50 mOsm / L, and most preferably 10 mOsm / L to 40 mOsm / L, for 0.5 to 30 minutes, for example, 0.5 minutes, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes, or 20 to 30 minutes.

[0046] For example, aqueous solutions can be derived from buffer solutions, such as diluted Dulbecco's phosphate-buffered saline (DPBS), diluted cell culture media, such as Dulbecco's modified Eagle medium (DMEM), ionic solutions, and salt solutions, such as CaCl2 or KCl solutions. These types of solutions can be used to obtain stable and functional organelle vesicles.

[0047] In the example protocol, Cos7, HeLa, HEK, CHO, and fibroblasts are maintained in DMEM supplemented with 10% thermoinactivated fetal bovine serum and 1% penicillin-streptomycin. Prior to the swelling step, cells are cultured in DMEM medium at 5% CO2 and 37°C for 48 hours. Cells are transfected for 24 hours with plasmids indicated for examination of various organelles before extraction and harvesting, and are cultured attached to Matek dishes with or without pretreatment with adhesive.

[0048] According to this example protocol, cells are transfected with various plasmids fused with fluorescent protein constructs, such as red fluorescent protein (RFP) or blue fluorescent protein (BFP), 24 hours prior to organelle vesicle extraction. These plasmids help express proteins that report on various organelles. The Kdel-RFP and Mito-BFP plasmids can be used to identify the endoplasmic reticulum and mitochondria, respectively.

[0049] The cell culture medium can be diluted with H2O at pH 7.4 at 37°C and 5% CO2 before the cells reach confluence. Naturally, the osmotic gradient of the swollen solution can be controlled and adjusted according to the cell type.

[0050] As an indicator, in a specific embodiment where the cells are COS-7 cells, the cytoplasmic osmotic pressure is approximately 300 mOsm / L. Therefore, in such embodiments, any aqueous solution having an osmotic pressure of less than approximately 100 mOsm / L and greater than 0.1 mOsm / L can be used to produce organelle vesicles.

[0051] The above-described type of hypotonic aqueous medium allows for the immediate application of a sufficiently non-destructive, rapid, and effective osmotic shock to cells and their intracellular organelles, enabling the formation of spherically swollen cells and enlarged organelle vesicles.

[0052] Several molecules can be added to promote the breakdown of the cytoskeleton and reduce the lysis tension of the cell. For example, cytoskeletal disruptors such as nocodazole, navelbine, latranculine A, latranculine B, and cytochalasin, and / or kinesin, myosin, and / or dynein motor inhibitors such as brevistatin, benzyltoluenesulfonamide, butanedione, and monoxime.

[0053] The hypotonic aqueous medium may also contain one or more molecules, such as protease inhibitors, molecular motor inhibitors, organelle-cytoskeleton contact inhibitors, cytoskeleton disruptors, and surfactants, that regulate the distribution of the surface area-to-volume ratio of extracellular organelle vesicles, while preventing their degradation and lowering the surface tension value to dissolve the plasma membrane.

[0054] To accelerate cell swelling, several molecules can also be added. These include ion channel modulators / blockers such as thapsigargin, caffeine, and benzothiazepines; extracellular matrix disruptors such as trypsin; protein transport inhibitors; protein signaling inhibitors such as xerospondin; and chemical surfactants such as Triton-X-100, octyl glucoside, DDM, and / or carboxylic acids.

[0055] The hypotonic aqueous medium may contain one or more molecules selected from the group including nocodazole, latranculines, trypsin, misaquinolides, micalorides, aprilonides, vinblastine, rotenone, swinholides, jaspraquinolides, vincristine, demecolsin, cytochalasins, colchicine, vinca alkaloids, dihydropyridine, phenylalkylamines, benzothiazepines, gabapentinoids, brevistatin, benzyltoluenesulfonamide, butanedione monoxime, thapsigargin, xerospondin, Triton X-100, Tween, SDS, Brij, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, and magnesium.

[0056] Thus, the preparation unit 2 can be configured to produce the intracellular organelle vesicles having a reduced surface-to-volume ratio (surface divided by the volume of the geometric shape, most often spherical) compared to the original form. For example, 0.15 μm -1 to 3 μm -1 Preferably, 0.15 μm -1 to 2 μm -1 More preferably, 0.15 μm -1 to 1.5 μm -1 Most preferably, 0.15 μm -1 to 1.2 μm -1 having a surface-to-volume ratio in the range, and / or having a size greater than 4 μm intracellularly. Thus, such organelle vesicles can be referred to as "giant" organelle vesicles.

[0057] In one embodiment, the preparation unit 2 comprises additional means (not shown) for treating the cells before contacting them with the hypotonic aqueous medium, in particular so that the extracellular organelle vesicles can have specific biochemical properties. For example, this treatment can be carried out in order to prevent proteolysis and / or to regulate biochemical reactions on the organelles. In other words, the metabolic conditions and structure of the cells and their organelles can be modified by chemicals and / or by modifying the expression levels of proteins that affect the structure and / or metabolic conditions and / or surface-to-volume ratio and / or relative position and / or arrangement of the organelles relative to each other of the cells and / or their organelles.

[0058] For example, before the osmotic swelling step described above, the surface-to-volume ratio of the extracellular organelle vesicles to be produced in the future can be adjusted by the following means. Changing the expression levels of proteins that affect the shape of the organelles and the contact sites with other organelles, the plasma membrane and the cytoskeleton; and / or Treating cells with molecules that alter both the activity of molecular transport proteins and signaling proteins localized to the plasma membrane and organelles, including the cytoskeleton and molecular motors, organelle contact sites (contact sites with other organelles, the plasma membrane, and the cytoskeleton); and / or Altering cellular metabolic pathways that affect the number and / or shape of organelles; and / or Any treatment that mediates a change in the surface-to-volume ratio of organelles and / or inter-organelle contact.

[0059] For example, overexpression of CLIMP-63 before osmotic swelling can result in larger extracellular organelle vesicles emerging from the endoplasmic reticulum with a size greater than 30 μm. Similarly, overexpression of Mfn2 before osmotic swelling can result in 0.75 μm -1 Larger extracellular organelle vesicles can be generated from mitochondria with a smaller surface-to-volume ratio. Pre-treating cells with nocodazole or latranculin A for 1 to 90 minutes before swelling allows for the formation of larger organelles from the endoplasmic reticulum. Adding rapamycin 12 to 24 hours before swelling allows for the formation of larger extracellular organelle vesicles from endosomes, lysosomes, autolysosomes, and polyvesicles. Adding bafilomycin before swelling can also yield more extracellular organelle vesicles derived from autophagosomes.

[0060] In a further embodiment, the preparation unit 2 comprises additional means (not shown) for processing cells after osmotic swelling by moving the cells through a hypotonic aqueous medium at a speed ranging from about 0.01 m / s to 10 m / s for, for example, from about 0.01 seconds to 10 minutes, thereby disrupting both the cytoskeleton and extracellular matrix of the cells. This optional treatment can reduce the mechanical restraint required to be applied to the cells by the extraction unit 3 (see below) to lyse them. Indeed, the cytoskeleton gives the cell's plasma membrane further resistance. Therefore, by exposing the cells to such movement without lysing them, the lysis tension can be further reduced without damaging the intracellular organelle vesicles still inside.

[0061] <Extraction of organelle vesicles from cells> According to the present invention, the extraction unit 3 comprises a microfluidic chip 11, as shown in the non-limiting example of Figure 2.

[0062] As is known in itself, the microfluidic chip 11 comprises a body 12 made of a material such as polydimethylsiloxane (PDMS), and an array 13 of microchannels 14 formed by the body 12 (13 microchannels 14 in this example), which is fluidically connected to at least one inlet 15 and at least one outlet 16.

[0063] Examples of manufacturing the chip 11 are described further below. Naturally, the main body 12 and the chip 11 can be manufactured using any technology and / or materials.

[0064] Figures 3 and 4 schematically show the longitudinal portion of the microchannel 14 according to the present invention and provide a reference frame defining the longitudinal direction D1 and the transverse directions D2 and D3, where the D1, D2 and D3 directions are orthogonal to each other. Figure 3 is a diagram of the portion of the microchannel 14 in a cross-sectional plane from D1-D2. Figure 4 is a diagram of the same portion of the microchannel 14 in a cross-sectional plane from D1-D3.

[0065] In this specification, the terms “upstream” and “downstream” refer to the flow direction F1 along D1 of the fluid flowing through the microchannel 14 during the extraction step.

[0066] The vertical portion of the microchannel 14 shown in Figures 3 and 4 typically includes an upstream section 21, a downstream section 22, and an intermediate section 23 between the upstream section 21 and the downstream section 22.

[0067] The intermediate portion 23 of the microchannel 14 itself comprises various parts along the direction D1, namely an upstream portion 25, a downstream portion 26, and an intermediate portion 27 between the upstream portion 25 and the downstream portion 26.

[0068] In this embodiment, the microchannel 14 in Figures 3 and 4 is defined by wall members 31-46.

[0069] Referring to Figure 3, wall members 31, 33, 35, 37, and 39 define the first lateral surface of the microchannel 14, and wall members 32, 34, 36, 38, and 40 define the second lateral surface of the microchannel 14. The first and second lateral surfaces are spaced apart from each other in direction D2.

[0070] Referring to Figure 4, wall member 41 defines the third lateral surface of the microchannel 14, and wall members 42, 43, 44, 45, and 46 define the fourth lateral surface of the microchannel 14. The third and fourth lateral surfaces are spaced apart from each other in direction D3.

[0071] In this embodiment, each of the wall members 31-40 extends parallel to direction D2, and each of the wall members 41-46 extends parallel to direction D2.

[0072] Therefore, the portion of the microchannel 14 shown in Figures 3 and 4 has, in this case, a rectangular cross-section or a square cross-section, also known as a cross-section, at any coordinate along direction D1.

[0073] As shown in Figures 3 and 4, the cross-section of the intermediate portion 27 is smaller than the cross-sections of the upstream portion 21 and the downstream portion 22, taking into account the respective orientations of the wall members.

[0074] In this embodiment, wall members 31, 32, 35, 36, 39, 40, 41, 42, 44, and 46 are parallel to direction D1, while wall members 33, 34, 37, 38, 43, and 45 are oblique to direction D1.

[0075] More specifically, referring to Figure 3, the wall member 33 is perpendicular to direction D1 and defines an imaginary cross-sectional plane P1 and angle A1 that separates the upstream portion 21 of the microchannel 14 from the intermediate portion 23. The wall member 34 also defines an angle A2 with the cross-sectional plane P1.

[0076] Similarly, the wall member 37 is perpendicular to direction D1 and defines an angle A3 with an imaginary cross-sectional plane P2 that separates the downstream portion 22 of the microchannel 14 from the intermediate portion 23. The wall member 38 also defines an angle A4 with the cross-sectional plane P2.

[0077] Referring to Figure 4, wall member 43 defines an angle A5 with the transverse plane P1, and wall member 45 defines an angle A6 with the transverse plane P2.

[0078] In this embodiment, the wall members 33, 34, 41, and 43 are configured such that the cross-section of the upstream portion 25 decreases monotonically from the upstream portion 21 of the microchannel 14 to the intermediate portion 27 of the intermediate portion 23 of the microchannel 14. Similarly, the wall members 37, 38, 41, and 45 are configured such that the cross-section of the downstream portion 26 increases monotonically from the intermediate portion 27 of the intermediate portion 23 of the microchannel 14 to the downstream portion 22 of the microchannel 14.

[0079] The term "monotonically" should be understood in a mathematical sense, and a monotonic function is defined as a function whose first derivative does not change in sign.

[0080] In this particular embodiment, the upstream portion 25 and the downstream portion 26 are symmetric with respect to a hypothetical cross-sectional plane (not shown) that extends equidistant from planes P1 and P2.

[0081] In light of the above, and considering that the cross-section of the intermediate portion 27 is smaller than the cross-sections of the upstream portion 21 and the downstream portion 22, the intermediate portion 27 forms a portion of the microchannel 14 called the "contracted portion".

[0082] Referring to Figures 3 and 4, the upstream portion 21, the contraction portion 27, and the downstream portion 22 of the microchannel 14 have a first size in the direction D2 indicated by references X1, X2, and X3, and a second size in the direction D3 indicated by references X4, X5, and X6, respectively. The first and second sizes are also called width and height, respectively.

[0083] Furthermore, the upstream portion 25, the contraction portion 27, and the downstream portion 26 of the microchannel 14 have a size in direction D1, also called length, as indicated by references X7, X8, and X9, respectively.

[0084] The above non-restrictive geometric parameters can have the following values: X1: in the range of 30 μm to 200 μm, preferably in the range of 50 μm to 150 μm, for example, 100 μm. X2: in the range of 4 μm to 15 μm, preferably in the range of 7 μm to 13 μm, for example, 8 μm, 9 μm, 10 μm, 11 μm, or 12 μm. X3: in the range of 30 μm to 200 μm, preferably in the range of 50 μm to 150 μm, for example, 100 μm. X4: in the range of 10 μm to 100 μm, preferably in the range of 20 μm to 50 μm, for example, 30 μm. X5: in the range of 5 μm to 100 μm, preferably in the range of 10 μm to 30 μm, for example 25 μm, 30 μm, 35 μm, or 40 μm. X6: in the range of 10 μm to 100 μm, preferably in the range of 20 μm to 50 μm, for example, 30 μm. X7: in the range of 5 μm to 500 μm, preferably in the range of 50 μm to 300 μm, for example, 200 μm. X8: in the range of 1 μm to 200 μm, preferably in the range of 10 μm to 70 μm, preferably in the range of 25 μm to 50 μm, for example 20 μm, 30 μm, or 40 μm. X9: Range from 1 μm to 500 μm, preferably in the range of 5 μm to 200 μm, for example 10 μm, 30 μm, 50 μm, 70 μm, 90 μm, 110 μm, 130 μm, 150 μm, 170 μm, or 190 μm. A1: A range from 91° to 179°, preferably from 99° to 170°, for example, 105° or 130°. A2: A range from 91° to 179°, preferably from 99° to 170°, for example, 105° or 130°. A3: A range from 91° to 180°, preferably a range from 120° to 179°, for example, 150° or 170°. A4: A range from 91° to 180°, preferably a range from 120° to 179°, for example, 150° or 170°. A5: A range of 90° to 170°, preferably a range of 90° to 160°, for example, 95° or 120°. A6: A range of 90° to 170°, preferably a range of 90° to 160°, for example, 95° or 120°.

[0085] Clearly, the upstream section 21, the downstream section 22 and the intermediate section 23, as well as the upstream section 25, the downstream section 26 and the contraction section 27, are fluidically connected to one another, and so the fluid flowing into the upstream section 21 can flow through these various sections and regions of the microchannel 14 in the flow direction F1.

[0086] In this embodiment, the wall members 31-46 are integrally formed from the same material and form the body of a microfluidic chip, such as the chip 11 in Figure 2.

[0087] Referring again to Figure 2, in one embodiment, each of the microchannels 14 of the chip 11 comprises one microchannel portion similar to those described above with reference to Figures 3 and 4. In another embodiment, each of the microchannels 14 of the chip 11 comprises multiple microchannel portions similar to those described above with reference to Figures 3 and 4, thus forming a series of shrinkage portions 27.

[0088] Naturally, the chip 11 in Figure 2, or any other type of microfluidic chip, may comprise one or more microchannels having one or more parts similar to those described above with reference to Figures 3 and 4, and / or one or more microchannels having other geometric features. In particular, the size and / or shape of any other part or region of the contraction portion 27 and / or microchannel 14 may differ from those described above. Specifically, the above geometric parameters of the microchannel 14 can be combined with various values.

[0089] Furthermore, any other portion or region of the shrinkage section 27 and / or the microchannel 14 may have a cross-section that is not rectangular or square, such as a trapezoidal cross-section or other polygonal cross-section.

[0090] In some embodiments, as shown in Figures 5-7, the upstream portion 25 is longer than the downstream portion 26. In a non-limiting representation, the ratio of the upstream portion 25 to the downstream portion 26 is approximately 2.4 in the embodiment of Figure 6 and higher than 50 in the embodiments of Figures 5 and 7.

[0091] The above description applies by analogy to the embodiments shown in Figures 5-7.

[0092] In the embodiment shown in Figure 5, the microchannel 14 has the following geometric parameters: X1=X3=100μm; X2=13μm; X4=X5=X6=25μm; X8=38μm; A1=A2=110°; A3=A4=1°; A5=A6=90°.

[0093] In the embodiment shown in Figure 6, the microchannel 14 has the following geometric parameters: X1=X3=100μm;X2=7μm;X4=X5=X6=25μm;X8=9μm;A1=A2=111°;A3=A4=64°;A5=A6=90°.

[0094] In the embodiment shown in Figure 7, the microchannel 14 has the following geometric parameters: X1=X3=100μm; X2=11μm; X4=X5=X6=25μm; X8=6μm; A1=A2=100°; A3=A4=1°; A5=A6=90°.

[0095] Referring to the embodiment in Figure 1, the microfluidic chip 11 of the extraction unit 3 is connected to the preparation unit 2 in this example and receives a fluid stream containing the cells and intracellular organelle vesicles within the microchannel 14.

[0096] The fluid has a flow rate of, for example, about 100 μL / min and 10 per mL. 5 From the cell 10 7 It can be introduced into the microchannel 14 at concentrations between cells.

[0097] The contractile portion 27 of the microchannel 14 mechanically restrains the cell as it passes through it. This, in this example, dissolves and removes the cell plasma membrane, allowing the release of stable and functional extracellular organelle vesicles into a hypotonic medium without lysis.

[0098] Therefore, the apparatus 1 and, in particular, the extraction microfluidic unit 3, enable the production of extracellular organelle vesicles typically having an average size ranging from approximately 3 μm to 15 μm.

[0099] <Collection of extracted organelle vesicles> In this example, the recovery unit 4 of the apparatus 1 is configured to separate organelle vesicles according to their type and to individually extract membrane fragments coming out of the extraction unit 3.

[0100] In one embodiment, the recovery unit 4 includes an additional microfluidic chip (not shown).

[0101] In another embodiment, a single identical microfluidic chip (not shown) forms both a microchannel for extracting organelle vesicles from cells and a further microchannel for separating and recovering the extracted organelle vesicles. In other words, a single identical microfluidic chip can form both the extraction unit 3 and the recovery unit 4, or a portion of these units.

[0102] In embodiments in which the recovery unit 4 is equipped with microchannels, these microchannels may be spiral microchannels known in the art.

[0103] In another embodiment, the recovery unit 4 is configured to purify organelle vesicles using fluorescence-activated cell sorting (FACS) techniques also known in the art.

[0104] Various types of sorting and / or recovery technologies (including, but not limited to, microfluidic technologies and FACS technologies) can be combined.

[0105] <Observation of organelle vesicles> The observation unit 5 of the apparatus 1 is configured to allow observation of organelle vesicles extracted using any appropriate technique.

[0106] In one embodiment, the observation unit 5 includes an ultra-high-speed camera configured to achieve, for example, up to 10,000 frames per second, which typically enables live visualization of organelle vesicle extraction. For example, recording of the extraction can be done at a resolution of 2,000 to 5,000 frames per second to produce video with a slow-motion factor of 80 to 210 times.

[0107] In one embodiment, the observation unit 5 is equipped with a confocal microscope, for example, a microscope known as "LSM800 Zeiss," which typically enables visualization of extracellular organelle vesicles emerging from the extraction unit 3.

[0108] Observation unit 5 can be equipped with various combinations of control devices (including, but not limited to, a high-speed camera and / or a confocal microscope).

[0109] <Manufacturing of microfluidic chips> The microfluidic chip according to the present invention can be manufactured as follows.

[0110] In the first step, a photomask is created using SU-8 2025 or SU-8 2050 negative resin to manufacture a chip wafer using photolithography technology. Thus, patterns with thicknesses of 25 μm or 50 μm can be obtained, respectively. The table below provides protocols for 25 μm and 50 μm thick wafers. [Table 1]

[0111] The wafers thus obtained are subjected to gas-phase methyltrichlorosilane treatment to cure the resin adhering to the wafer and facilitate the removal of polymerized PDMS from the wafer. Specifically, 15 μL of methyltrichlorosilane is dropped onto the wafer and a Petri dish. The Petri dish is tightly sealed with Parafilm before the reagent evaporates excessively. The treatment is carried out for approximately 15 minutes to allow for the precipitation of hydrophobic residues.

[0112] In this example, the PDMS chips are manufactured using elastomer silicon marketed as "Sylgard 184" and a 184 polymerization initiator with an elastomer-to-crosslinking agent ratio of 10:1. The mixture is vigorously homogenized, degassed in a vacuum chamber, and carefully deposited onto a wafer surrounded by an aluminum mold. After pouring the PDMS to a predetermined height, the entire assembly is placed in an incubator at 70°C for 2 hours. Once the PDMS has hardened, the aluminum mold is detached and the PDMS is removed from the wafer. The PDMS is then cut to the desired chip size and perforated at levels to provide an inlet and outlet. After cutting, perforating, and cleaning the PDMS components, they are exposed to a glass slide and subjected to 1 minute of exposure to air plasma (plasma chamber air pressure between 0.6 mbar and 0.8 mbar) to allow for irreversible adhesion between the PDMS and the glass slide.

[0113] Before being used for organelle vesicle extraction, the tips are preferably subjected to air plasma exposure to enhance their hydrophobicity.

Claims

1. A device (1) for recovering organelle vesicles from cells, Organelle swelling unit (2) configured to produce swollen intracellular organelle vesicles; and, An extraction unit (3) configured to extract swollen organelle vesicles from cells. Equipped with, The apparatus (1) comprises an extraction unit (3) having a microfluidic chip (11) having one or more microchannels (14) configured to receive a fluid flow containing the cells, the microchannels (14) having one or more contraction sections (27) configured to mechanically restrain the cells as they pass through it.

2. At least one of the shrinking portions (27) is connected to the upstream portion (25) and the downstream portion (27) of the same microchannel (14), The upstream portion (25) has a cross-section that preferably decreases monotonically from the upstream portion (21) of the microchannel (14) toward the contraction portion (27), The apparatus (1) according to claim 1, wherein the downstream portion (26) has a cross-section that preferably increases monotonically from the contraction portion (27) toward the downstream portion (22) of the microchannel (14).

3. The aforementioned upstream portion (25) At least one first wall member (33, 34) defining an angle (A1, A2) in the range of 91° to 179°, preferably 99° to 170°, for example, 105° or 130°, with respect to a transverse plane (P1); and / or It is formed by at least one second wall member (43) that defines an angle (A5) between the transverse plane (P1) and the transverse plane (P1) in the range of 90° to 170°, preferably 90° to 160°, for example, an angle of 95° or 120°, and / or The aforementioned downstream portion (26) At least one first wall member (37, 38) defining an angle (A3, A4) in the range of 91° to 180°, preferably 120° to 179°, for example, 150° or 170°, with respect to a transverse plane (P2); and / or The transverse plane (P2) is formed by at least one second wall member (45) that defines an angle (A6) in the range of 90° to 170°, preferably 90° to 160°, for example, 95° or 120°. The apparatus (1) according to claim 2.

4. The apparatus (1) according to claim 2 or 3, wherein the upstream portion (25) is longer than the downstream portion (26) with respect to the direction (F1) of the fluid flow.

5. The shrinking portion (27) 100 μm ２ From 2000 μm ２ Preferably 170 μm ２ From 650 μm ２ Cross-section within the range of 225 μm ２ Alternatively, 330 μm ２ The cross-section, and / or Width (X2) in the range of 0.5 μm to 15 μm, preferably 2 μm to 15 μm, preferably 4 μm to 15 μm, preferably 7 μm to 13 μm, for example, width (X2) of 8 μm, 9 μm, 10 μm, 11 μm or 12 μm, and / or Heights (X5) in the range of 0.5 μm to 15 μm, preferably 2 μm to 100 μm, preferably 5 μm to 100 μm, preferably 10 μm to 30 μm, for example, heights (X5) of 25 μm, 30 μm, 35 μm or 40 μm, and / or Length (X8) in the range of 1 μm to 200 μm, preferably 10 μm to 70 μm, preferably 25 μm to 50 μm, for example, having a length (X8) of 20 μm, 30 μm, 40 μm, 50 μm or 60 μm. Apparatus (1) according to any one of claims 1 to 4.

6. The apparatus (1) according to any one of claims 1 to 5, wherein at least one of the contraction portions (27) has a polygonal cross-section, for example, a rectangular cross-section or a trapezoidal cross-section.

7. The apparatus (1) according to any one of claims 1 to 6, wherein at least one of the microchannels (14) comprises a plurality of shrinkage portions (27).

8. The apparatus (1) according to any one of claims 1 to 7, wherein each of the multiple microchannels (14) comprises one or more shrinkage portions (27).

9. The apparatus (1) according to any one of claims 1 to 8, wherein the organelle swelling unit (2) comprises means for bringing the cells into contact with a hypotonic aqueous medium.

10. Furthermore, the system includes a recovery unit (4) configured to recover and / or separate the organelle vesicles extracted from the cells by the extraction unit (3), The apparatus (1) according to any one of claims 1 to 9, wherein the recovery unit (4) may be formed from a portion of the microfluidic chip (11) and / or may include additional microfluidic chips.

11. Furthermore, the system includes an observation unit (5) configured to observe the organelle vesicles extracted from cells by the extraction unit (3), The apparatus (1) according to any one of claims 1 to 10, wherein the observation unit (5) may include a camera and / or a microscope.

12. A method for recovering organelle vesicles from cells, The step of swelling the organelle vesicles with the organelle swelling unit (2) of the apparatus (1) according to any one of claims 1 to 11; and Step of extracting swollen organelle vesicles from cells using the extraction unit (3) of the apparatus (1) according to any one of claims 1 to 11. Includes, The method preferably includes, after the extraction step, the step of recovering the organelle vesicles with a recovery unit of the apparatus (1) according to claim 10.

13. The method according to claim 12, comprising introducing a fluid containing the cells into the microchannel (14) of the microfluidic chip of the extraction unit (3) at a flow rate in the range of 1 μL / min to 500 μL / min, preferably 50 μL / min to 200 μL / min, for example, 75 μL / min, 100 μL / min, 125 μL / min, 150 μL / min, or 175 μL / min.

14. The method according to claim 12 or 13, further comprising the step of observing the organelle vesicles with the observation unit (5) of the apparatus (1) according to claim 11 during and / or after the extraction step.

15. Extracellular organelle vesicles obtained by the method according to any one of claims 12 to 14, wherein the organelle vesicles have a size in the range of 3 μm to 15 μm, for example, 4 μm to 10 μm.

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