Integrated microfluidic chip system and method for mass production and enrichment of engineered extracellular vesicles

Abstract

The present invention belongs to the field of medical micro-nano equipment technology, and specifically relates to an integrated microfluidic chip system and method for mass production and enrichment of engineered extracellular vesicles. The present invention first provides an integrated microfluidic chip system, comprising a microfluidic chip body and pre-placed objects; the microfluidic chip body comprises a cover layer 100, a flow channel layer 200, a liquid reservoir layer 300, and a conductive glass layer 400; the flow channel layer 200, the liquid reservoir layer 300, and the conductive glass layer 400 are integrated together by irreversible bonding, and the cover layer 100 and the flow channel layer 200 are detachably combined; a plurality of reaction zones, micropores, and microchannels are provided on the flow channel layer, and each reaction zone is separately provided and not connected; the pre-placed objects comprise one or more of donor cells, working cells, extracellular vesicle adsorption reagents, or reagents for nano-electroporation, and are provided in the microfluidic chip body.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical and engineering micro-nano equipment, and specifically relates to an integrated microfluidic chip system and method for mass production and enrichment of engineered extracellular vesicles. Background Art

[0002] Cell therapy is increasingly being investigated as a novel treatment approach for disease models and patients. Recent studies have demonstrated that therapeutic benefits for many diseases are driven by extracellular vesicles (EVs) secreted by transplanted cells. EVs have been recognized as important mediators, carrying a rich reservoir of molecular information, including DNA, RNA, and proteins, that serve as vehicles for intercellular communication. Engineered EVs involve the use of engineering techniques to modify EV contents or membrane proteins, imparting specific functions and targeting properties. Engineered EVs offer significant advantages in disease treatment due to their ability to customize their contents to specific disease needs, such as by designing them to encapsulate specific genetic drugs or small molecule compounds. Currently, the most common method for producing engineered EVs is to deliver the desired payload to donor cells, allowing them to secrete EVs containing more of the target payload. This approach can produce EVs with high integrity, but because EV biogenesis and secretion are regulated by cellular mechanisms, existing technologies have limited potential to improve the yield and payload efficiency of EVs secreted by individual cells. In response to this, some studies have proposed that additional stimulation of donor cells producing EVs (such as electrical stimulation, ultrasound, hypoxia and pH change regulation, etc.) can activate the biosynthesis of EVs and accelerate the secretion process to a certain extent. However, there are still many challenges in clinical application: (1) The low secretion amount of EVs from a single cell seriously limits the efficiency of engineering production. Generally, the secretion amount of EVs from a single cell is less than 10 3 particles / cell, and the therapeutic dose of EV required for an adult in clinical practice is generally between 0.5 and 1.4×10 11 Particles / cells, the yield is seriously insufficient. In order to obtain therapeutic doses of EVs, a large number of donor cells need to be cultured. (2) Clinical applications place high demands on the quality of EVs, such as EV integrity, effectiveness of encapsulating target payloads, and batch consistency. (3) The control of the donor cell culture microenvironment during EV production is limited. For example, metabolic waste in the culture environment cannot be removed in real time, resulting in decreased cell activity, EV yield, and changes in EV content. In summary, the current engineered EVs production strategy is still difficult to meet the clinical demand for therapeutic EVs in terms of production efficiency (e.g., EV yield of a single donor cell) and quality (e.g., EV integrity, drug loading capacity, and consistency between different batches). Therefore, advanced systems for efficient and high-quality engineered EVs have always been widely anticipated, but there are still technical difficulties to achieve them.

[0003] The development of microfluidics has provided new insights into the engineering of EVs. For example, patent publication number CN117987467A (20240507), entitled "A method for preparing therapeutic exosomes using nanoelectroporation and other non-endocytic cell transfection methods," discloses a method in which donor cells are placed on the surface of a chip, various plasmids, other transfection vectors, and combinations thereof are added to the chip's buffer, and the cells placed on top of the chip are electroporated, thereby causing the cells to secrete multiple EVs. This patent addresses, to a certain extent, the problem of mass production of EVs from donor cells. Another example is patent publication number CN115228521A (20221025), entitled "Bio-particle separation device and microfluidic chip," which discloses a microfluidic chip device that can be used to separate extracellular membrane vesicles from large samples. This patent combines microfluidics with membrane filtration, addressing, to a certain extent, the problem of improving the separation efficiency and throughput of extracellular membrane vesicles.

[0004] However, existing technologies still find it difficult to achieve high-yield and high-quality EV preparation in the same system. At the same time, existing technologies also have the problem of cumbersome collection steps for engineered EVs. Therefore, it is necessary to propose new methods and strategies. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an integrated microfluidic chip system for mass production and enrichment of engineered extracellular vesicles, as well as a method for producing extracellular vesicles that meet clinical application needs based on this integrated microfluidic chip system, so as to alleviate or partially alleviate the shortcomings of the existing technology. The specific technical solution is as follows.

[0006] An integrated microfluidic chip system for mass production and enrichment of engineered extracellular vesicles, characterized in that the integrated microfluidic chip system comprises a microfluidic chip body and a pre-set; the microfluidic chip body comprises a cover layer 100, a flow channel layer 200, a liquid reservoir layer 300 and a conductive glass layer 400; the flow channel layer 200, the liquid reservoir layer 300 and the conductive glass layer 400 are integrated together by irreversible bonding, and the cover layer 100 and the flow channel layer 200 are detachably combined; the flow channel layer 200 is a liquid reservoir layer 300, and the conductive glass layer 400 is a liquid reservoir layer 400. The channel layer is provided with a plurality of reaction areas, micropores and microchannels, including a production area 201, a capture area 202 and an ammonium ion removal area 203, and each reaction area is separately provided and not connected; the cover layer 100 is provided with a plurality of micropores and microchannels, including an inlet micropore 101 and an outlet micropore 109, which, when combined with the channel layer 200, connect the reaction areas provided on the channel layer 200; the first micropore 102 and the second micropore 103 provided on the cover layer 100 are respectively connected to the production area 201 and the capture area 202 and the ammonium ion removal area 203. The fifth micropore 204 and the sixth micropore 205 provided on the cover layer 1 are connected, the first flow channel 106 provided on the cover layer 100 is connected with the eighth micropore 207 provided on the production area 201 and the fourth flow channel 208 provided on the capture area 202, the second flow channel 107 and the third flow channel 108 provided on the cover layer 100 respectively connect the capture area 202 and the ammonium ion removal area 203; the ammonium ion removal area 203 is configured to include multiple groups of Tesla flow channels, the Tesla flow channels The SiLa flow channel converges at the bottom and sets a ninth micropore 209 to communicate with the outlet micropore 109; the pre-set includes one or more of donor cells, working cells, extracellular vesicle adsorption reagents or reagents for nano-electroporation; the donor cells are arranged in the production area 201; the extracellular vesicle adsorption reagents are arranged in the capture area 202; the working cells are arranged in the ammonium ion removal area 203; the reagents for nano-electroporation are arranged in the liquid storage layer 300.

[0007] Furthermore, the production area 201 is configured to place a nanoporous membrane inside, and the nanoporous membrane is used to plant the donor cells, and the donor cells are cells that produce engineered extracellular vesicles; the production area 201 is also provided with a seventh micropore 206 connected to the inlet micropore 101.

[0008] Furthermore, a liquid storage area 301 is provided inside the liquid storage layer 300 for storing the reagent for nano-electroporation; the liquid storage area 301 is provided below and connected to the production area 201; the 10th micropore 302 and the 11th micropore 303 are provided on the liquid storage area 301 and are connected to the cover layer 100 and the flow channel layer 200 respectively.

[0009] Furthermore, the extracellular vesicle adsorption reagent is a positively charged hydrogel complex; receptor cells or receptor tissues can also be placed in the extracellular vesicle adsorption reagent; the receptor cells or receptor tissues are cells or tissues that receive engineered extracellular vesicles.

[0010] Preferably, the hydrogel complex comprises 1% chitosan solution, 10% GelMA solution and 2.5% LAP solution, with a volume ratio of 10:10:4.

[0011] The hydrogel complex exhibits strong positive charge in the culture solution and can adsorb negatively charged EVs in the liquid flowing out of the P area. EVs are rapidly enriched in this area. At the same time, the hydrogel in this area can also be used as a skeleton for receptor cell culture and tissue culture in vitro. These receptor cells or tissues can directly absorb EVs in this area to conduct corresponding functional studies. In addition, the EV-rich hydrogel complex material has good biosafety and degradability, and can be implanted in the body and used as a carrier for the sustained release of EVs in vivo.

[0012] Furthermore, the working cells are cells capable of adsorbing ammonium ions.

[0013] Furthermore, filter membranes are provided in the microchannels of the cover layer 100 and the channel layer 200 .

[0014] In some embodiments, the working cells are hepatocytes, which have the ability to absorb or adsorb ammonium ions, a metabolic waste product in the culture medium, and convert them into glutamine, which is beneficial to the cells. The ammonium ion removal zone 203 is designed to include multiple Tesla flow channel structures. These structures reduce the flow rate, shear force, and pressure exerted by the fluid on the cells, effectively ensuring ammonium ion removal and maintaining cell integrity.

[0015] On the other hand, the present invention also provides a method for preparing and enriching engineered extracellular vesicles, wherein the preparation method is implemented by the above-mentioned integrated microfluidic chip system, comprising the following steps:

[0016] S01: Inject the donor cell suspension into the production area of ​​the microfluidic chip flow channel layer, inject the working cell suspension into the Tesla flow channel of the flow channel layer, and culture the cells;

[0017] S02: After the donor cells and working cells have adhered to the wall, a nano-electroporation reagent containing a target gene for follicle activation is injected into the liquid storage area of ​​the microfluidic chip reservoir layer. The microfluidic chip is connected to an electroporator, with the negative electrode being a conductive glass layer and the positive electrode being a nano-silver electrode. Electroporation is performed to obtain engineered extracellular vesicles in the production area of ​​the flow channel layer. After the first electroporation operation is completed, the nano-electroporation reagent in the liquid storage area is removed;

[0018] S03: placing the positively charged hydrogel complex into the capture area of ​​the flow channel layer, and injecting receptor cells or receptor tissues therein, then combining and fixing the cover sheet and the flow channel layer, and connecting peristaltic pumps through the inlet and outlet micropores of the cover sheet;

[0019] S04: running the peristaltic pump and setting the power parameter to 5-10 μL / min, so that the engineered cell extracellular membrane vesicles prepared in the production area enter the capture area and are adsorbed by the positively charged hydrogel complex;

[0020] S05: The peristaltic pump continues to operate, allowing the cell culture medium in the production area to circulate throughout the chip. When the cell culture fluid enters the Tesla flow channel and reacts with the working cells to remove ammonium ions, it continues to circulate back to the production area through the peristaltic pump;

[0021] S06: After the first electroporation operation is completed for 8 hours, multiple non-loaded electrical stimulations are continued, wherein the reagent used in the non-loaded electrical stimulation operation is PBS.

[0022] The cell culture fluid purified by the ammonium ion removal zone 203 can flow back to the production zone 201, thereby ensuring a healthy culture environment for the donor cells in the production zone 201, and providing a circulating production system adapted to the yield and quality of EVs.

[0023] Furthermore, the donor cells are ovarian granulosa cells; the working cells are hepatocytes; and the target gene with follicle activation effect is miR-130a.

[0024] In some embodiments, the recipient cell is an ovarian granulosa cell; and the recipient tissue is a mouse ovarian tissue.

[0025] Furthermore, the electroporation operation was performed with a voltage of 30 V, a pulse width of 5 ms, a pulse interval of 0.1 s, and a total of 100 pulse cycles.

[0026] The non-load electrical stimulation operation can further stimulate the secretion of EVs from the donor cells. As a preferred embodiment, the number of non-load electrical stimulations is 1.

[0027] Furthermore, the hydrogel complex includes 1% chitosan solution, 10% GelMA solution and 2.5% LAP solution.

[0028] The hydrogel complex of engineered extracellular vesicles containing miR-130a obtained by the above method.

[0029] Beneficial technical effects:

[0030] The present invention first proposes an integrated microfluidic chip system for mass production and enrichment of engineered extracellular vesicles. Three main functional areas are set in the chip system: production area (P area), capture area (U area) and ammonium ion removal area (RE area). The three functional areas are independently set and do not interfere with each other. They can independently perform specific functions, such as engineered EVs production and preparation, engineered EVs capture, cell culture fluid purification, etc. After the cover of the integrated microfluidic chip is combined, the microporous flow channels and external pumps provided on the cover can connect and connect the functional areas on the microfluidic chip to form a semi-automated circulation system. The semi-automatic means that the system does not need to add additional culture medium and nutrients in the process of producing EVs. It only needs to be electroporated or electrostimulated through an external power supply, and the entire culture system relies on the self-production and purification of cells in the circulation system to perform its functions.

[0031] Furthermore, the present invention also provides a method for preparing and enriching engineered extracellular vesicles. This method relies on the collaborative cooperation of the three functional areas of the chip system to increase the EV secretion of single cells (12 times higher than that of normal cells) and the content of target nucleic acids in EVs by approximately 30 times. Without introducing any chemicals potentially toxic to cells throughout the process, the controllability of the cell culture microenvironment is guaranteed, ensuring the quality of the engineered EVs. In addition, the engineered EVs obtained by the method of the present invention do not need to be enriched through steps such as centrifugation and purification. The engineered EVs obtained by adsorption enrichment in the hydrogel complex through charge interaction can be directly used for application. That is, the final product of the present invention does not deviate from the system used to produce the product, making it more convenient to operate.

[0032] In particular, the present invention proposes a method of combining loaded electroporation with unloaded electrical stimulation, which not only enables customized preparation of EV contents but also significantly increases EV yield. Furthermore, the method ultimately produces a hydrogel composite containing engineered EVs, which can be implanted in vivo, exhibits high safety, and exhibits sustained EV release, suggesting promising medical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0034] Summary of reference numerals:

[0035] Cover layer 100, inlet micropore 101, first micropore 102, second micropore 103, third micropore 104, fourth micropore 105, first flow channel 106, second flow channel 107, third flow channel 108, outlet micropore 109; flow channel layer 200, production area 201, capture area 202, ammonium ion removal area 203, fifth micropore 204, sixth micropore 205, seventh micropore 206, eighth micropore 207, fourth flow channel 208, ninth micropore 209, twelfth micropore 210, thirteenth micropore 211; liquid storage layer 300, liquid storage area 301, tenth micropore 302, eleventh micropore 303; conductive glass layer 400.

[0036] Figure 1 An exploded view of an integrated microfluidic chip body in one embodiment of the present invention;

[0037] Figure 2 A schematic diagram of an integrated microfluidic chip cover layer, a flow channel layer, and a liquid reservoir layer in one embodiment of the present invention;

[0038] Figure 3 A schematic diagram of the preparation of an integrated microfluidic chip in one embodiment of the present invention;

[0039] Figure 4 A schematic diagram of the design principle and working process of an integrated microfluidic chip in one embodiment of the present invention;

[0040] Figure 5 This is the numerical simulation result of the mechanical stress in the ammonium ion removal area of ​​different flow channel layers in one embodiment of the present invention;

[0041] Figure 6 This is the result verification of producing engineered EVs by electro-stimulation in the flow channel layer production area in one embodiment of the present invention;

[0042] Figure 7 The effect of the hydrogel complex in the capture zone of the flow channel layer on the uptake of engineered EVs by recipient cells in one embodiment of the present invention;

[0043] Figure 8 This is an in vitro experiment on promoting granulosa cell proliferation using the PURE chip system in one embodiment of the present invention;

[0044] Figure 9 This is an experiment on activating primordial follicles in ovarian tissue in vitro using a PURE chip in one embodiment of the present invention;

[0045] Figure 10 This is a process of in situ implantation of a hydrogel complex containing engineered EVs in a mouse in one embodiment of the present invention;

[0046] Figure 11This is a verification of the effect of improving ovarian function after in situ implantation of a hydrogel complex containing engineered EVs in mice in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0047] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0049] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0050] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0051] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0053] Detailed description of some drawings

[0054] Figure 4 :AC are schematic diagrams of the key structures of the EV production area (A), capture area (B) and ammonium ion removal (RE) area (C); D is an overview schematic diagram of the PURE platform; E is the nanopore electroporation of the target nucleic acid drug (miR-130a) into the donor cells in the production area, promoting the release of engineered EVs encapsulated with high-abundance therapeutic miR-130a; F is the porous hydrogel sponge in the capture area that collects engineered EVs based on electrostatic attraction; G is the hepatocytes located in the Tesla flow channel in the ammonium ion removal area that removes ammonium ions from the extracellular environment.

[0055] Figure 5 : A and B are finite element analyses of the shear stress distribution of the fluid in the PURE chip with Tesla flow channels or S-shaped channels, respectively; C is a finite element analysis of the compressive stress distribution in the PURE chip with Tesla flow channels or S-shaped channels; D is that the Tesla flow channel has a stronger effect on reducing ammonium ions in the system than the S-shaped flow channel.

[0056] Figure 6 : A is the efficiency of electro-delivery of miR-130a in the P region of the PURE chip system; B is the comparison of the number of EVs secreted by single donor cells in the PURE, Lipo transfection and negative control groups; C is the exploration of the conditions for stimulating EV secretion by empty-load electroporation: the horizontal axis is the number of empty-load electroporations; D is the comparison of the miR-130 content in the engineered EVs produced by the optimal electric field combination: 1 loaded electro-delivery + 1 unloaded electro-stimulation and the Lipo transfection scheme.

[0057] Figure 7 : A is the laser scanning confocal microscopy (LSCM) fluorescence and bright field images showing the engineered EVs captured in the GelMA-chitosan hydrogel in the uptake area, with the red arrow indicating the engineered EVs; B is the transmission electron microscopy (TEM) image of the engineered EVs (marked by red arrows) immobilized by the GelMA-chitosan hydrogel (gray background); C is the time-dependent efficiency of EV uptake by the GelMA-chitosan hydrogel; D is the fluorescence images and quantitative analysis of the fluorescence intensity of the recipient cells (GCs) after PKH67-EV uptake in the U zone with or without the hydrogel at different time schedules.

[0058] Figure 8A shows the expression of mTOR signaling pathway-related proteins in recipient and donor cells after transfection with miR-130a or NC. B and C show the proliferation capacity assessment of recipient cells cultured on the PURE chip. After 48 hours of culture on the corresponding PURE platform, recipient cells were removed for proliferation capacity testing, including Edu staining to count the proportion of Edu-positive cells (B) and MTT staining at different time points using a microplate reader (C). D shows the comparison of apoptosis rates in recipient cells after treatment with engineered EVs (miR-130a) or control EVs (NC). E shows the comparison of the proportion of recipient cells and cell cycle stages after treatment with engineered EVs (miR-130a) or control EVs (NC).

[0059] Figure 9 A shows an in vitro activation model for primordial follicles: neonatal mouse ovarian tissue was encapsulated in a hydrogel in the capture region (U region) and cultured for 24 hours before HE staining. P region (-): donor cells were transfected with a negative control oligonucleotide; P region (+): donor cells were transfected with miR-130a; RE region (-): hepatocytes were absent in the depleted region; RE region (+): hepatocytes were present in the depleted region. Yellow arrows indicate activated follicles, green arrows indicate primordial follicles, and white arrows indicate apoptotic follicles. B and C show the percentages of activated follicles (B) and apoptotic follicles (C) in primordial follicles of mouse ovaries at the end of 24 hours in the capture region of the tissue chip.

[0060] Figure 10 : A shows the surgical process of implanting the EV-rich hydrogel complex produced by the PURE platform into mice; B shows the degradation process of the EV-rich hydrogel complex in vivo.

[0061] Figure 11 A: HE-stained sections of ovaries treated with hydrogels containing EVs produced under the indicated conditions; P region (-): donor cells transfected with a negative control oligonucleotide; P region (+): donor cells transfected with miR-130a; RE region (-): absence of hepatocytes in the depleted region; RE region (+): presence of hepatocytes in the depleted region; B: percentage of healthy and atretic follicles in the ovaries of each treatment group. Healthy follicles include primary follicles, secondary follicles, and cystic follicles; C: anti-Müller hormone (AMH) levels in the ovaries of each treatment group. D: in vitro fertilized oocytes obtained from ovaries of each treatment group and their in vitro development into blastocysts; E: statistical statistics of the probability of in vitro blastocyst formation in oocytes obtained by superovulation from ovaries of different treatment groups.

[0062] Glossary

[0063] The "engineered extracellular vesicles" described in the present invention refer to engineered extracellular vesicles containing target genes obtained by modifying the contents of extracellular vesicles using technical means, and have specific functions.

[0064] Example 1

[0065] This embodiment provides an example of the structure of an integrated microfluidic chip for mass production and enrichment of engineered extracellular vesicles.

[0066] The integrated microfluidic chip system includes a microfluidic chip body and pre-sets; the microfluidic chip body includes a cover layer 100, a flow channel layer 200, a liquid reservoir layer 300 and a conductive glass layer 400; the pre-sets include one or more of donor cells, working cells, extracellular vesicle adsorption reagents or reagents for nano-electroporation.

[0067] The flow channel layer 200, the liquid storage layer 300 and the conductive glass layer 400 are integrated together by irreversible bonding; a plurality of reaction zones, micropores and microchannels are provided on the flow channel layer, including a production zone 201, a capture zone 202 and an ammonium ion removal zone 203, and each reaction zone is separately provided and not connected; a plurality of micropores and microchannels are provided on the cover layer 100, including an inlet micropore 101 and an outlet micropore 109, which, when reversibly bonded to the flow channel layer 200, connect and penetrate each reaction zone provided on the flow channel layer 200, including the first micropore 102 and the second micropore 103 provided on the cover layer 100. The ammonium ion removal zone 203 is connected to the 5th micropore 204 and the 6th micropore 205 provided on the production zone 201 respectively, the 1st flow channel 106 provided on the cover layer 100 is connected to the 8th micropore 207 provided on the production zone 201 and the 4th flow channel 208 provided on the capture zone 202, the 2nd flow channel 107 and the 3rd flow channel 108 provided on the cover layer 100 respectively connect the capture zone 202 and the ammonium ion removal zone 203; the ammonium ion removal zone 203 is configured to include a plurality of Tesla flow channels, which converge at the bottom and set the 9th micropore 209 to communicate with the outlet micropore 109.

[0068] The donor cells are arranged in the production area 201 ; the extracellular vesicle adsorption reagent is arranged in the capture area 202 ; the working cells are arranged in the ammonium ion removal area 203 ; and the reagent for nano-electroporation is arranged in the liquid reservoir 300 .

[0069] In some embodiments, the plurality of micropores on the cover layer 100 are configured to be approximately 0.8 mm in diameter, wherein the inlet micropore 101 and the outlet micropore 109 are used to connect to the steel needle on the peristaltic pump connecting tube.

[0070] In some embodiments, the production area 201 is configured as a square chamber with a size of 33*33 mm, with a nanoporous membrane placed inside. The production area 201 is also provided with a seventh micropore 206 communicating with the inlet micropore 101 .

[0071] In some embodiments, the capture chamber 202 is configured as two square chambers with a size of 12.8*12.8, and the cells and the hydrogel complex are placed inside.

[0072] In some embodiments, the ammonium ion removal zone 203 is configured as four groups of Tesla flow channels with a width of 0.43 mm, and the four groups of Tesla flow channels are connected and connected at the bottom.

[0073] In some embodiments, the liquid reservoir layer 300 is provided with a liquid storage area 301, which is located below and connected to the production area 201 and is used to store the reagents used for nano-electroporation. The liquid reservoir 301 is configured as a square chamber with dimensions of 33*33 mm. In addition, the 10th micropore 302 and the 11th micropore 303 provided in the liquid reservoir 301 are connected to the 3rd micropore 104 and the 4th micropore 105 provided in the cover layer 100, respectively.

[0074] In some embodiments, filter membranes are provided in the microchannels of the cover layer 100 and the flow channel layer 200 to filter impurities and cell debris.

[0075] In some embodiments, the conductive glass layer 400 is ITO conductive glass.

[0076] In some embodiments, the flow channel layer 200 may further be provided with a 12th micropore 210 and a 13th micropore 211 , which are connected to the 3rd micropore 104 and the 4th micropore 105 upwards and to the 10th micropore 302 and the 11th micropore 303 downwards.

[0077] Example 2

[0078] This embodiment provides an example of a method for preparing the integrated microfluidic chip of embodiment 1.

[0079] The PURE chip's various regions and flow channels were designed using AutoCAD software. A polyester photofilm mask was used for photolithography, and the mold was fabricated in a micro-nano cleanroom. The main process steps are as follows.

[0080] 1. Cleaning the silicon wafer

[0081] Select a 4-inch single-polished silicon wafer as the substrate and visually inspect it for impurities. If impurities are present, wipe the wafer with a dust-free cloth soaked in acetone to remove any surface impurities before proceeding to the next step. If no impurities are present, proceed directly to the next step. Place the wafer in a glass dish filled with acetone (the wafer should be submerged in the acetone solution) and use maximum-power ultrasonic cleaning to remove any residual organic matter on the surface. Then, ultrasonically clean the wafer using anhydrous ethanol and then ultrapure water. After cleaning, blow dry the wafer with a nitrogen gun until no visible water marks remain on the surface.

[0082] 2. Baking the silicon wafer

[0083] Bake at 150°C for 45 minutes.

[0084] 3. Make the base layer

[0085] Place the baked silicon wafer in an oxygen plasma cleaner and clean it with oxygen plasma for 3 minutes. Immediately after completion, place it in the center of a spin coater and activate the vacuum pump to secure the wafer. Then, pipette 2 mL of SU8-2010, which has been returned to room temperature, onto the center of the wafer and begin spin coating, ultimately achieving a uniform 10 μm thick SU8-2010 base layer. Bake the wafer containing the SU8-2010 base layer on a heat plate at 95°C for 3 minutes, then place it in a photolithography machine for exposure and curing. After exposure, place the wafer back on a heat plate and bake it at 95°C for 3 minutes to complete the base layer.

[0086] 4. Make the structural layer

[0087] The silicon wafer with the finished base layer was placed in a spin coater for secondary coating, using spin coating to obtain a uniform thickness of 200 μm SU8-2075.

[0088] After coating, let the wafer rest for 5 minutes to reduce any residual prestress in the photoresist. Then, begin the pre-bake process. The pre-bake parameters are: 65°C for 6 minutes, followed by 95°C for 40 minutes. The wafer is then placed in the photolithography machine for exposure and curing.

[0089] Set the exposure conditions and perform the exposure. Immediately after the exposure, place the silicon wafer on a hot plate for intermediate baking. The specific parameters are: first bake at 65°C for 5 minutes, then bake at 95°C for 14 minutes.

[0090] After post-baking, remove the silicon wafer, cool it to room temperature, and then develop it. Place the silicon wafer in a completely clean glass dish and pour PM-THINNER negative photoresist developer into it until it covers the wafer by approximately 2mm. Shake continuously until the structural layer is fully developed. Remove the wafer with wafer tweezers and rinse it repeatedly with isopropyl alcohol, then rinse it with ultrapure water, and finally blow dry with nitrogen. Then, harden the film. Use wafer tweezers to transfer the wafer to a hotplate and bake it at 150°C for 10 minutes.

[0091] 5. Surface modification

[0092] The processed silicon wafer can be used as a master mold for subsequent soft lithography chip production. The silicon wafer mold and trichlorosilane are placed in a vacuum desiccator at a pressure of 100 mbar overnight for silanization. After silanization, the surface-modified silicon wafer mold is obtained.

[0093] PURE chip preparation:

[0094] The PURE chip is a multi-layer PDMS device. Each layer is molded using photolithography, then replicated in batches using soft lithography. Finally, individual components are obtained through cutting and punching. The main steps of soft lithography are as follows.

[0095] 1. Glue preparation

[0096] Prepare the PDMS with a base component to curing agent mass ratio of 10:1. The flexibility of the molded PDMS can be adjusted by varying the base component to curing agent mass ratio according to experimental requirements. Generally, a larger ratio results in a softer finished adhesive.

[0097] 2. Glue spreading

[0098] Use a glass rod to stir the two-component mixture in a clockwise direction until the mixture produces uniform and dense small bubbles. Then place the plastic cup in a vacuum pump to evacuate the air until all bubbles are removed. Store in a refrigerator at 4°C before use to avoid immediate cross-linking.

[0099] 3. Casting

[0100] Pour the prepared PDMS two-component mixture into the mold, then place the mold in a vacuum pump to evacuate. After the vacuum is completed, visually inspect for bubbles. If there are no bubbles, place the mold directly in an oven and bake at 70°C for 120 minutes to ensure that the PDMS is fully cross-linked.

[0101] 4. Draft cutting

[0102] After the PDMS in the mold is completely cross-linked and cooled to room temperature, it is peeled off from the mold and then cut along the pre-marked lines on the PDMS structure.

[0103] 5. Punch

[0104] Use a needle puncher and a customized probe reservoir puncher to punch holes: Use a needle puncher to punch out the sample inlet and outlet ports on each layer. For the sample inlet and outlet ports connected to the chip, the size of the hole should be smaller than the size of the steel needle for the sample inlet and outlet connection, so that the steel needle can completely fill the sample inlet and outlet ports to avoid leakage; secondly, use a needle puncher to reserve a position for inserting the nanosilver wire top electrode in the cover layer in advance to facilitate subsequent electroporation; finally, use a customized plasmid reservoir puncher to punch out the storage area in the PDMS reservoir layer.

[0105] 6. Final integration of the chip

[0106] Device integration is achieved through oxygen plasma activated bonding.

[0107] (1) Bonding of the liquid storage layer and the conductive glass layer (ITO glass substrate)

[0108] (2) Bonding of the flow channel layer and the PC nanoporous membrane

[0109] First, the PDMS was immersed in a 1% volume ratio AEAPS aqueous solution for surface chemical modification for 15 minutes, and then the nanoporous membrane was treated with oxygen plasma (0.75mbr, 18W, 50s). Then, the PDMS chip was taken out, and the surface to be bonded was aligned with the PC nanomembrane. After a light test of bonding, it was placed in an oven (110℃, 60min) to complete the bonding process.

[0110] (3) Bonding of flow channel layer and liquid reservoir layer

[0111] The flow channel layer bonded with the nanoporous membrane is bonded to the liquid storage layer bonded with the ITO glass substrate by oxygen plasma activation, and the specific steps are the same as above.

[0112] (4) The cover layer is fixedly assembled with the integrated chip body

[0113] Align the filter membrane with the inlet / outlet of the U zone and RE zone. The filter membrane is used to filter cell debris and large vesicles and can be replaced at any time. Cover the cover layer on the flow channel layer and align it with each outlet / inlet. In order to build a closed system, the multilayer structure is finally clamped together with uniform stress by a customized polymethyl methacrylate (PMMA) clamp, connected to a sterilized soft catheter and steel needle, and other accessories are assembled at the same time. Finally, the chip assembly is completed to obtain an integrated PURE chip system. See the schematic diagram Figure 3 .

[0114] Hydrogel preparation process

[0115] 1. Prepare 1% chitosan solution:

[0116] Add 1g of chitosan powder (viscosity: approximately 100-200mPa·s) to 100mL of ultrapure water, followed by 15mL of 1% HCl solution. Dissolve the chitosan using a high-speed magnetic stirrer. After dissolution, adjust the pH of the chitosan solution to approximately 6 using Na2HPO4 (pH=9). Sterilize the prepared chitosan solution by placing it under strong UV radiation overnight.

[0117] 2. Prepare 10% GelMA solution:

[0118] Dissolve 0.5 g of GelMA solid in 5 mL of ultrapure water and heat the solution in a 60°C water bath for 30 min to ensure complete dissolution of the solid.

[0119] 3. Preparation of hydrogel complex (GelMA-Chitosan-Hydrogel):

[0120] Mix 2 mL of 1% chitosan solution, 2 mL of 10% GelMA solution, and 400 μL of 2.5% LAP solution in a light-protected environment. Stir the mixture in a 60°C water bath for 10 minutes to ensure thorough mixing. Pour the GelMA-Chitosan-Hydrogel liquid mixture into the designated mold and expose it to ultraviolet (UV) light for 30 seconds. After the solution solidifies, freeze it at -20°C overnight and then transfer it to a freeze dryer to dry for 2 days. After drying, remove the GelMA-Chitosan-Hydrogel from the mold.

[0121] Example 3

[0122] This example provides an example of a method for producing, preparing, and enriching engineered EVs based on the integrated microfluidic chip system of Example 1.

[0123] S01: Inject the donor cell suspension (ovarian granulosa cells, including cell culture medium) into the production area of ​​the microfluidic chip flow channel layer, inject the liver cell suspension into the Tesla flow channel of the flow channel layer, and incubate the cells in a cell culture incubator overnight to allow them to adhere;

[0124] S02: After the donor cells and working cells are attached to the wall, a nano-electroporation reagent containing miR-130a is injected into the liquid storage area of ​​the liquid storage layer of the microfluidic chip. The fluidic chip body is connected to the electroporator, with the negative electrode being the conductive glass layer and the positive electrode being the nano-silver electrode. The electroporation operation is performed with a voltage of 30V, a pulse width of 5ms, and a pulse interval of 0.1s, for a total of 100 pulse cycles. Engineered extracellular vesicles are obtained in the production area of ​​the flow channel layer. After the electroporation operation is completed, the nano-electroporation reagent containing miR-130a is removed.

[0125] S03: Place the positively charged hydrogel complex into the capture area of ​​the flow channel layer and inject receptor cells or receptor tissues into it. Then, combine the cover sheet and the flow channel layer and fix them using a customized fixture. The inlet and outlet micropores of the cover sheet are respectively connected to the peristaltic pump needles.

[0126] S04: running the peristaltic pump and setting the power parameter to 10 μL / min, so that the engineered cell extracellular membrane vesicles prepared in the production area enter the capture area and are adsorbed by the positively charged hydrogel complex;

[0127] S05: The peristaltic pump continues to operate, and the cell culture fluid in the production area enters the Tesla flow channel and reacts with the liver cells (ammonium ions, a product of cell metabolism, pass through the liver cells in the flow layer of the Tesla flow channel, decelerate in this area, fully contact with the liver cells, and can be absorbed and converted into glutamine that can be used by the cells to provide nutrients beneficial to cell growth). The fluid then continues to circulate back to the production area through the peristaltic pump (the purified culture medium and the converted glutamine flow out through the micropores and are transported to the P area through the peristaltic pump pipeline for recycling. The cell culture medium then flows from the P area to the U area and then to the RE area).

[0128] S06: After 8 hours of the first electroporation operation, a non-loaded electrical stimulation is continued. The reagent used for the non-loaded electrical stimulation is PBS.

[0129] The working flow diagram of this embodiment is shown in Figure 4 .

[0130] Example 4

[0131] This example provides performance verification of the method of Example 3.

[0132] The sterilized soft catheter (inner diameter 0.5 mm) and steel needle (outer diameter 0.9 mm) were connected to the inlet micropores of the integrated microfluidic chip (hereinafter referred to as the PURE platform) for cell inoculation. 5 The suspension of ovarian granulosa cells was injected into the nanoporous membrane of the square pool through the corresponding micropores in the P area; the prepared liver cell suspension was injected into the Tesla flow channel through the corresponding micropores in the RE area, and the cells were incubated in a cell culture incubator overnight to allow them to adhere to the wall.

[0133] 200nM miRNA-130a mimics or corresponding negative control oligonucleotides were suspended in Opti-MEM and injected into the reservoir below the P area. The positive electrode of the electroporator was clamped on the top silver wire (0.2mm), and the negative electrode was clamped on a custom ITO-coated glass slide. A voltage of 30V (99 pulses, each pulse lasting 5ms, with an interval of 0.1s) was applied between the positive and negative electrodes for delivery. After delivery, the miRNA solution was removed from the storage area and the storage reservoir was cleaned with 5mg / mL RNase A for 10min to completely remove residual miRNA.

[0134] Effect verification:

[0135] (1) PURE chip removes NH4 + Performance evaluation.

[0136] The ammonium ion removal area 203 provided in the flow channel layer 200 in Example 1 was set as an S-shaped flow channel as a control group. The width of the S-shaped flow channel was 0.43 mm, and the rest of the structure remained the same.

[0137] The results showed that the shear force and pressure of the fluid in the Tesla flow channel were less than those in the S-type flow channel, indicating less interference with the cells. In addition, the liquid flow rate in the Tesla flow channel was significantly reduced, so the output flow rate on the same area was 70% lower than that of the S-type channel. Under the action of the Tesla flow channel structure, the liver cells in the RE area of ​​the PURE platform were able to effectively convert NH4 + The concentration is reduced from 10mM to 1mM. Therefore, the design of the present invention can achieve the reduction of ammonium ions, reduce its impact on the cell viability of donor cells, and ensure that the cell viability of donor cells is at a high level during the whole culture process. Figure 5 .

[0138] (2) Verification of the PURE chip system’s ability to mass-produce engineered EVs.

[0139] See Figure 6 .like Figure 6 As shown in A, the PURE chip system achieved a transfection efficiency of approximately 90% for miR-130a-mimics labeled with a FAM fluorescent tag in donor GC cells. Compared with natural secretion and liposome transfection (as a benchmark method), Figure 6 As shown in B, the PURE chip system increased the production of EVs by approximately 4.8 times and approximately 3.8 times, respectively. The cells were grouped according to the number of non-load electrical stimulation treatments, with the loaded electroporation as the control group 0. The EVs produced under different non-load electrical stimulations were obtained for nanoparticle size analysis, as shown in Figure 6 As shown in C, one additional non-load electrical stimulation using the PURE chip system resulted in the highest EV yield (up to 1.5 × 10 per cell).5 EVs), which is 10 times higher than the natural secretion level of the cell line. As the number of non-load electrical stimulation treatments increases, the EV production shows a trend of first increasing and then gradually decreasing. Therefore, the mode with the highest production is one load electroporation followed by one non-load electrical stimulation. Finally, according to this optimal mode, Figure 6 The qPCR results in D showed that the level of the therapeutic component (miR-130a) in the engineered EVs prepared by the PURE chip system was increased by approximately 150-fold and approximately 30-fold compared with the natural secretion and liposome transfection methods, respectively.

[0140] (3) Verification of the ability of the PURE chip to capture EVs in situ.

[0141] After running the PURE chip system for 48 hours, the captured EVs were qualitatively and quantitatively analyzed. Figure 7 As shown in A and B, fluorescence confocal microscopy and transmission electron microscopy (TEM) can both observe the presence of a large number of intact EVs trapped in the hydrogel complex. Further quantitative experiments have shown that Figure 7 As shown in C, at approximately 24 h, the U region captured approximately 90% of the total EVs generated from the P region.

[0142] To further explore the efficiency of the collected engineered EVs (miR-130a-EVs) taken up by recipient cells, e.g. Figure 7 As shown in D. The donor cells in the P region were stained with PKH67, so the membrane skeleton of the EVs obtained after nano-electroporation was also labeled with green fluorescence. Ovarian granulosa cells (GC) were used as recipient cells and seeded on the hydrogel scaffold in the U region for in situ culture on the chip. Figure 7 The confocal fluorescence image in Figure C shows that when the hydrogel complex is placed in the U region, the recipient cells take up EVs faster and in higher amounts, indicating that the hydrogel complex placed in the U region not only enriches EVs around the recipient cells but also promotes their uptake.

[0143] Example 5

[0144] (1) The PURE chip system promotes granulosa cell proliferation in vitro:

[0145] Ovarian follicles are composed of eggs and granulosa cells. The first step in primordial follicle activation is granulosa cell proliferation. Therefore, promoting granulosa cell proliferation is the first step in improving and activating primordial follicles. Activation of the mTOR signaling pathway is a core step in granulosa cell proliferation. Currently, small molecule compounds based on mTOR activation are the mainstream in vitro primordial follicle activation strategies. However, their limited efficiency in cellular entry and safety concerns limit their clinical application.

[0146] Based on this, the PURE chip system provided by the present invention team can realize the production of extracellular vesicles with specific contents. Specifically, miR-130a, which has a follicle-stimulating and activating effect, is delivered into extracellular vesicles through electroporation. The extracellular vesicles can further efficiently enter the recipient cells and significantly activate the mTOR signaling pathway in the recipient granulosa cells. The self-produced biological components with low immunogenicity and high safety have great application potential in the process of primordial follicle activation.

[0147] This example analyzes the effect of miR-130a-EV on the target protein TSC1 and its downstream key effectors in the mTOR signaling pathway in recipient cells. Figure 8 As shown in A, Western blot results showed a significant downregulation of TSC1, and upregulation of phosphorylation of mTOR, p70S6K, and 4E-BP1, indicating an increased activation level of the mTOR signaling pathway.

[0148] Subsequently, the recipient cells were subjected to cell proliferation assay using the classic Edu assay ( Figure 8 B) and MTT assay ( Figure 8 C) As shown in the figure, the proliferation rate of recipient GC cells that took up miR-130a-EV produced by PURE was significantly increased.

[0149] The apoptosis level of the recipient cells was detected, such as Figure 8 As shown in D, the flow cytometry results showed that the early and late apoptosis levels of recipient GCs treated with miR-130a-EVs were significantly downregulated compared with the control group. The cell cycle of recipient cells was detected. Figure 8 As shown in Figure E, flow cytometry results showed that miR-130a-EVs caused 65.5% of recipient cells to enter the S and G2 / M phases, the proliferation phases of the cell cycle, an increase of 17.5% compared to the control group, suggesting that it promoted the cell cycle. Overall, the in situ cell experiments confirmed that the engineered EVs mass-produced by the PURE chip can efficiently deliver target nucleic acid drugs into target cells. At the same time, this miR-130a-EV can significantly promote the activation of the mTOR signaling pathway in granulosa cells, thereby promoting the proliferation of granulosa cells.

[0150] (2) PURE chip activates primordial follicles in ovarian tissue in vitro:

[0151] After verifying the proliferation-promoting effect on recipient cells, the activation of miR-130a-EVs on ovarian tissue was verified at the tissue level. Newborn mouse ovaries were placed in the U region of the PURE chip for three-dimensional culture to examine the effect of miR-130a-EVs on the three-dimensional follicles.

[0152] The results showed that after the activation and proliferation of granulosa cells in the primordial follicles, the enlargement of the oocytes surrounded by granulosa cells indicated the development of primordial follicles. The results of HE staining of ovarian tissue sections showed that Figure 9 As shown, compared with the control group, the PURE chip-produced engineered EVs significantly increased the activation rate of primordial follicles by approximately 30%. At the same time, EVs significantly reduced the apoptosis rate of primordial follicles from 42% (control group) to <5%. Overall, the PURE chip system provides a safe and efficient alternative method for the in vitro activation of primordial follicles.

[0153] (3) In situ implantation of hydrogel complexes containing engineered EVs in vivo:

[0154] After capturing mass-produced engineered EVs (miR-130a-EVs), the hydrogel in the U region was implanted in situ into the ovaries of 8-month-old aged mice (implantation process as shown in Figure 10 A).

[0155] The results of surgical implantation of the hydrogel first confirmed the strong bond between the hydrogel and the ovary. After 21 days of observation, the hydrogel completely degraded and continuously released EVs during the degradation process. Figure 10 B).

[0156] (4) Verification of the effect after in situ implantation of hydrogel complexes containing engineered EVs in vivo:

[0157] Three weeks after implantation, ovarian function was systematically measured and evaluated to determine the recovery of ovarian function, including the ratio of healthy and atrophic follicles, anti-Müllerian hormone (AMH) levels, and oocyte developmental potential. Figure 11 As shown in AB, the ovaries of the aging mice in the negative control group (P(-)RE(-) group) showed a decrease in healthy follicles at all stages (primordial follicles, primary follicles, secondary follicles and mature follicles), accompanied by signs of aging ovaries such as oocyte degeneration, peripheral granulosa cell apoptosis and follicle atrophy. However, the PURE chip mass-produced engineered EVs (P(+)RE(+) group) significantly improved the quality of follicles in aging ovaries, such as an increase in the proportion of healthy oocytes (an increase of 45.8% compared to the P(-)RE(-) group) and a decrease in the proportion of atrophic follicles (a decrease of 68.4% compared to the P(-)RE(-) group). The anti-Müllerian hormone (AMH) level in ovarian tissue was tested, and the results showed that the AMH level in the P(+)RE(+) group was 3.4 times higher than that in the P(-)RE(-) group, further confirming that the ovarian function of aging mice was restored after miR-130a-EV treatment (as shown in Figure 2). Figure 11 C).

[0158] To further evaluate the quality and developmental potential of oocytes, oocytes from aged mice obtained by superovulation were subjected to in vitro fertilization. The developmental potential of oocytes in the P(-)RE(-) group after fertilization was very low, with only 10% of zygotes able to develop into blastocysts. However, the blastocyst formation rate in the P(+)RE(+) group was increased by more than 4 times, indicating that the treatment of PURE chip-produced engineered EVs significantly improved the quality of oocytes (e.g., Figure 11 In addition, the blastocyst formation rate of the P(+)RE(+) group was significantly higher than that of the P(+)RE(-) and P(-)RE(+) groups, further verifying the importance of the two functional regions in the efficient production of engineered EVs.

[0159] In summary, the PURE chip system can produce hydrogel complexes containing engineered EVs with promising in vivo applications. These hydrogel complexes can be implanted in the body and slowly release EVs, showing promising medical application prospects.

[0160] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. An integrated microfluidic chip system for mass production and enrichment of engineered extracellular vesicles, characterized in that: The integrated microfluidic chip system comprises a microfluidic chip body and a prefabricated object; the microfluidic chip body comprises a cover layer (100), a flow channel layer (200), a liquid storage layer (300) and a conductive glass layer (400); the flow channel layer (200), the liquid storage layer (300) and the conductive glass layer (400) are integrated together by irreversible bonding, and the cover layer (100) and the flow channel layer (200) are detachably combined; a plurality of reaction areas, micropores and microchannels are arranged on the flow channel layer, including a production area (201) , capture zone (202) and ammonium ion removal zone (203), and each reaction zone is separately arranged and not connected; a plurality of micropores and microchannels are arranged on the cover layer (100), including an inlet micropore (101) and an outlet micropore (109), which, when combined with the flow channel layer (200), enable each reaction zone arranged on the flow channel layer (200) to be connected; the first micropore (102) and the second micropore (103) arranged on the cover layer (100) are respectively connected to the fifth micropore (204) and the second micropore (105) arranged on the production zone (201). The 6 micropores (205) are connected, the 1st flow channel (106) provided on the cover layer (100) is connected with the 8th micropore (207) provided on the production area (201) and the 4th flow channel (208) provided on the capture area (202), the 2nd flow channel (107) and the 3rd flow channel (108) provided on the cover layer (100) respectively connect the capture area (202) and the ammonium ion removal area (203); the ammonium ion removal area (203) is configured to include a plurality of Tesla flow channels, the Tesla flow channels The SiLa flow channel converges at the bottom and sets a ninth micropore (209) to communicate with the outlet micropore (109); the preset includes one or more of donor cells, working cells, extracellular vesicle adsorption reagents or reagents for nano-electroporation; the donor cells are arranged in the production area (201); the extracellular vesicle adsorption reagents are arranged in the capture area (202); the working cells are arranged in the ammonium ion removal area (203); and the reagents for nano-electroporation are arranged in the liquid storage layer (300).

2. The integrated microfluidic chip system according to claim 1, characterized in that: The production area (201) is configured to have a nanoporous membrane placed therein, and the nanoporous membrane is used to plant the donor cells, which are cells that produce engineered extracellular vesicles; the production area (201) is also provided with a seventh micropore (206) connected to the inlet micropore (101).

3. The integrated microfluidic chip system according to claim 1, characterized in that: The liquid storage layer (300) is provided with a liquid storage area (301) for placing the reagent for nano-electroporation; the liquid storage area (301) is provided below and connected to the production area (201); the liquid storage area (301) is provided with a tenth micropore (302) and an eleventh micropore (303) which are respectively connected to the cover layer (100) and the flow channel layer (200).

4. The integrated microfluidic chip system according to claim 1, wherein: The extracellular vesicle adsorption reagent is a positively charged hydrogel complex; receptor cells or receptor tissues are placed in the extracellular vesicle adsorption reagent; the receptor cells or receptor tissues are cells or tissues that receive engineered extracellular vesicles.

5. The integrated microfluidic chip system according to claim 1, wherein: The working cells are cells capable of adsorbing ammonium ions.

6. A method for preparing and enriching engineered extracellular vesicles, characterized in that: The method is implemented based on the integrated microfluidic chip system according to any one of claims 1 to 5, comprising the following steps: S01: Inject the donor cell suspension into the production area of ​​the microfluidic chip flow channel layer, inject the working cell suspension into the Tesla flow channel of the flow channel layer, and culture the cells; S02: After the donor cells and working cells have adhered to the wall, a nano-electroporation reagent containing a target gene for follicle activation is injected into the liquid reservoir layer of the microfluidic chip. The liquid reservoir layer is provided with a liquid storage area for storing the nano-electroporation reagent; the microfluidic chip is connected to an electroporator, with the negative electrode being a conductive glass layer and the positive electrode being a nanosilver electrode, and an electroporation operation is performed to obtain engineered extracellular vesicles in the production area of ​​the flow channel layer. After the first electroporation operation is completed, the nano-electroporation reagent in the liquid reservoir is removed; S03: placing an extracellular vesicle adsorption reagent, which is a positively charged hydrogel complex, into the capture area of ​​the flow channel layer, and injecting receptor cells or receptor tissues therein, then combining and fixing the cover sheet and the flow channel layer, and connecting peristaltic pumps through the inlet and outlet micropores of the cover sheet layer; S04: running the peristaltic pump and setting the power parameter to 5-10 μL / min, so that the engineered cell extracellular membrane vesicles prepared in the production area enter the capture area and are adsorbed by the positively charged hydrogel complex; S05: The peristaltic pump continues to operate, allowing the cell culture fluid in the production area to circulate throughout the chip. When the cell culture fluid enters the Tesla flow channel and reacts with the working cells, it removes ammonium ions and then continues to circulate back to the production area through the peristaltic pump. S06: After 8 hours of the first electroporation operation, multiple non-loaded electrical stimulations are continued, wherein the reagent used for the non-loaded electrical stimulation is PBS.

7. The method according to claim 6, wherein The donor cells are ovarian granulosa cells; the working cells are liver cells; and the target gene with follicle activation effect is miR-130a.

8. The method according to claim 7, wherein The electroporation operation was performed with a voltage of 30 V, a pulse width of 5 ms, a pulse interval of 0.1 s, and a total of 100 pulse cycles.

9. The method according to claim 7, wherein The hydrogel composite includes 1% chitosan solution, 10% GelMA solution and 2.5% LAP solution.

10. A hydrogel complex of engineered extracellular vesicles containing miR-130a obtained by the method according to any one of claims 7 to 9.

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