WO2021175158A1

WO2021175158A1 - Microfluidic channel, microfluidic chip, and method for preparing vesicles

Info

Publication number: WO2021175158A1
Application number: PCT/CN2021/078091
Authority: WO
Inventors: 杜亚楠; 赵鹏
Original assignee: 清华大学
Priority date: 2020-03-05
Filing date: 2021-02-26
Publication date: 2021-09-10
Also published as: CN111286456A; CN111286456B

Abstract

Provided in the present application is a microfluidic channel. The microfluidic channel comprises an inlet; a crushing area that is connected to the inlet, the crushing area comprising at least one squeezing channel and at least one crushing channel, one squeezing channel being connected to the at least one crushing channel, the squeezing channels being a first predetermined width, the crushing channels being a second predetermined width, and the first predetermined width being greater than the second predetermined width; and an outlet that is connected to the crushing area. Further provided in the present application is a microfluidic chip that comprises the microfluidic channel. The microfluidic channel or the microfluidic chip can be applied to prepare a nano-scale vesicle.

Description

Microfluidic channel, microfluidic chip and method for preparing vesicle

This application requires the priority of a Chinese patent application whose application number is 202010147762.X and the application date is March 05, 2020, and the entire content of this application is quoted in this application.

Technical field

The invention belongs to the field of biomedical engineering, and specifically relates to a microfluidic channel, a microfluidic chip and a method for preparing vesicles.

Background technique

Extracellular vesicles can be divided into 30-150nm exosomes, 200-1000nm microvesicles, and 800-5000nm apoptotic bodies according to their size. According to its source, exosomes are invaginated from the cell membrane, forming endosomes, and then forming multivesicular bodies (MVBs), which are vesicles released outside the cell after fusion with the plasma membrane. Microvesicles are vesicles formed by direct shedding after cell budding and cell membrane fusion. Apoptotic bodies are shrinkage and indentation of the cell membrane, dividing and wrapping the cytoplasm, containing DNA material and organelles, and forming vesicular bodies. Among them, exosomes are an important part of extracellular vesicles. Most cells can secrete exosomes under physiological and pathological conditions. Exosomes have a phospholipid bilayer membrane, which is rich in nucleic acid (microRNA, mRNA, etc.), protein, cholesterol, etc. The surface markers of exosomes mainly include CD63, CD81, CD9, TSG101, HSP27 and so on.

In recent years, exosomal therapy has gradually become an important development direction of modern clinical medicine, and it has become a new generation of nano-drug therapy. Compared with cell therapy, exosomal therapy has the following advantages: 1. It has good biocompatibility and lower immunogenicity. 2. Due to the nano-scale size advantage of exosomes, they have a longer residence time in the blood circulation. When they enter the human pulmonary circulation, they will not block the capillary network of the alveoli and can pass through the dense extracellular matrix, and Cross the thick tissue barrier of the human body, such as the blood-brain barrier. 3. There is no cell activity to avoid the risk of tumor formation. 4. It is more stable than cells and easy to store.

Exosomes can be used as tissue regeneration agents. In April 2018, the U.S. Food and Drug Administration (FDA) approved Aegle's first extracellular vesicles new drug (IND) application for skin injury repair treatment. Exosomes can be used as drug delivery vehicles, Capricor, to obtain exosomes from cardiac spheroid-derived cells (CDC), which have the potential to treat inflammation and fibrosis, and Duchenne muscular dystrophy. Exosomes can be used as tumor diagnostic markers. On January 21, 2016, the world's first cancer diagnostic product based on exosomes was launched in the United States. Exosome Diagnostics launched a cancer liquid biopsy product based on RNA contained in exosomes.

However, the treatment of exosomes also has an important problem. The isolation of exosomes requires a large amount of cell culture supernatant, and the cell source of these cells is often greatly restricted. At present, the extraction of exosomes from small-scale cultured cells is transforming to the extraction of exosomes from large-scale cultured cells. However, its operation is complicated, consumes a lot of manpower and material resources, and costs a lot, which is not conducive to clinical application. In addition, after collecting a large amount of culture supernatant, the exosomes are extracted by centrifugation or density gradient centrifugation and other methods. The total amount of exosomes finally obtained is extremely low, which cannot meet the needs of future clinical applications. Therefore, how to efficiently and controllably prepare nano-scale vesicles similar to exosomes is very important to promote the development of exosomes therapy.

Summary of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the present invention provides a microfluidic channel, a microfluidic chip and a method for preparing vesicles.

At present, the separation, purification and preparation of vesicles faces many problems, which limit the application and related research of vesicles. On the one hand, the yield of natural extracellular vesicles isolated from the supernatant is very small, which greatly affects the function of vesicles; on the other hand, the existing preparation process of in vitro vesicles is very cumbersome. As a result, vesicles can only be prepared on a small scale, which also consumes a lot of manpower and material resources. How to automatically and controllly prepare a large number of vesicles with excellent performance in vitro is the focus of the inventor's research. By designing the microfluidic channel and the microfluidic chip, the inventors of the present invention can crush and break the cells, so that the cell membrane is recombined into nano-scale vesicles. The designed microfluidic channel passes through the crushing zone, through the crushing zone, the cells are squeezed and deformed, and then enters the crushing channel with a narrower space, so that the cells are broken and the cell membrane is recombined into nano-scale vesicles. Through the designed microfluidic channel or microfluidic chip, nano-scale vesicles with uniform size and similar to natural exosomes can be efficiently prepared.

Specifically, the present invention provides the following technical solutions:

In the first aspect of the present invention, the present invention provides a microfluidic channel comprising: an inlet; a crushing zone, the crushing zone is connected to the inlet, and the crushing zone is connected to the inlet through a first connecting zone The crushing zone includes at least one squeezing channel and at least one crushing channel, one of the squeezing channels is respectively connected to at least one of the crushing channels, the squeezing channel is set to a first predetermined width, and the crushing channel It is set to a second predetermined width, the first predetermined width is greater than the second predetermined width; an outlet, the outlet is connected to the crushing zone through a second connecting area. The microfluidic channel provided by the present invention contains an extrusion channel and a crushing channel in the crushing zone. One extrusion channel is connected to at least one crushing channel. The cells pass through the microfluidic channel and are squeezed and deformed in the extrusion channel. Then, after passing through a narrower fragmentation channel, the cells are fragmented and reshaped into nano-scale vesicles. The nano-scale vesicles prepared through the microfluidic channel are similar to natural exosomes. For example, the membrane composition and morphology are similar to those of natural exosomes, and the yield of the prepared nano-scale vesicles is much higher than that of natural exosomes. The production of exosomes. Moreover, the microfluidic channel has a wide range of applications and is suitable for the treatment of various types of cells. For example, in addition to stem cells, other cells such as lymphocytes and tissue cells can all be crushed and broken by using the microfluidic channel to prepare Out of nano-sized vesicles.

According to an embodiment of the present invention, the microfluidic channel provided above may further include the following technical features:

According to an embodiment of the present invention, the first joining area includes at least two inlet flow passages, and the inlet is connected to the crushing zone through the at least two inlet flow passages. The inlet is connected to the crushing zone through at least two inlet flow channels, so that multiple cells can be processed at one time, and the shunting can alleviate the resistance encountered by the cells in the crushing process, so that a large number of nano-scale vesicles can be quickly prepared.

According to an embodiment of the present invention, the inlet is connected to at least two first inlet flow passages, each of the first inlet flow passages is connected to at least two second inlet flow passages, and the second inlet flow passage is connected to the The broken areas are connected. The inlet is connected to at least two first inlet flow channels, and each first inlet flow channel is respectively connected to at least two second inlet flow channels, so that multiple cells can be processed at one time, and the shunting can alleviate cell fragmentation. The resistance encountered in the process can quickly prepare a large number of nano-scale vesicles.

According to an embodiment of the present invention, the second connection area includes at least two outlet flow passages, and the crushing zone is connected to the outlet through the at least two outlet flow passages. As a result, a large number of cells can be processed at the same time.

According to an embodiment of the present invention, the outlet is connected to at least two first outlet flow passages, each of the first outlet flow passages is connected to at least two second outlet flow passages, and the second outlet flow passage is connected to the The broken areas are connected. As a result, a large number of cells can be processed at the same time.

According to an embodiment of the present invention, the first predetermined width is at least twice the second predetermined width, preferably 2-10 times. As a result, the cells can be deformed by squeezing the channel, and then pass through a narrower fragmentation channel, so that the cells are broken and reshaped, and nano-scale vesicles can be obtained.

According to an embodiment of the present invention, the first predetermined width is 5-20 microns, and the second predetermined width is 1-5 microns, preferably 2-5 microns, more preferably 2.5-5 microns, for example 2.5- 4 microns. As a result, the cells can be deformed by squeezing the channel, and then pass through a narrower fragmentation channel, so that the cells are broken and reshaped, and nano-scale vesicles can be obtained.

According to an embodiment of the present invention, a buffer zone is arranged between each of the extrusion channels, and the width of the buffer zone is at least 3 times the first predetermined width. By setting a buffer between the extrusion channels, the flux of cells flowing through the microfluidic channel can be increased, so that more cells can be processed in a short time. According to an embodiment of the present invention, the width of the buffer zone is 30-40 microns. This width is approximately twice the diameter of conventional cells, which enables the cells to converge in the buffer for a short time.

According to an embodiment of the present invention, two buffer zones are arranged between each extrusion channel, and the length of each extrusion channel formed is 35-200 microns. According to an embodiment of the present invention, the length of each formed extrusion channel may be 40-200 micrometers, for example, it may be 40-150 micrometers. The length of the squeezing channel will affect the elapsed time of the cells in the squeezing channel, which can affect the squeezing effect of the cells. At this length, the effect of the cells through the extrusion treatment can be improved. There are no special requirements for the buffer zone, and it can be set as a circular area or an ellipse. According to an embodiment of the present invention, the buffer zone may be set as a circular area.

According to an embodiment of the present invention, the crushing zone includes 64 to 128 squeezing channels and 64 to 256 crushing channels, and two buffer zones are arranged between each of the squeezing channels. As a result, the shunting of the cells can be effectively realized, and the flux of the cells after the crushing treatment can be improved.

According to an embodiment of the present invention, the length of the first joint area is 1-10 mm, and the width of the first joint area is 0.15-0.5 mm. According to an embodiment of the present invention, the length of the first connecting area is 1.8-8 mm, for example, it may be 1.8-3 mm, preferably 2 mm; the width of the first connecting area is 0.2-0.4 mm, preferably 0.32 mm;

According to an embodiment of the present invention, the length of the second connecting area is 1-10 mm, and the width of the second connecting area is 0.2-0.5 mm. According to an embodiment of the present invention, the length of the second connecting area is 1.2-8 mm, for example, 1.2-2 mm, preferably 1.2 mm; the width of the second connecting area is 0.3-0.4 mm, preferably 0.32 Mm.

According to an embodiment of the present invention, further comprising: a screening area, the screening area is respectively connected with the entrance and the crushing area, the screening area is provided with a barrier, the barrier forming a predetermined in the screening area Clearance channel. The screening zone is set before entering the crushing zone, which can break up or block the larger cell clumps, and the larger-size impurities are blocked, so that the larger-size impurities can be removed.

According to an embodiment of the present invention, according to the liquid flow direction, the screening area is sequentially provided with a first barrier and a second barrier, the first barrier and the second barrier are rhombic columnar structures, and the first barrier The side length of the second barrier is smaller than the side length of the first barrier. By providing the first barrier and the second barrier, it is possible to prevent large cell clusters from blocking the downstream fragmentation zone, and avoid affecting the quality of the prepared nano-scale vesicles.

According to an embodiment of the present invention, the first barrier forms a predetermined gap channel of 70 to 90 microns in the screening area, and the second barrier forms a predetermined gap channel of 30 to 50 microns in the screening area.

In the second aspect of the present invention, the present invention provides a microfluidic chip comprising: a substrate; and a microfluidic channel formed on the substrate, and the microfluidic channel is based The microfluidic channel according to any embodiment of the first aspect of the invention. Through the substrate, the microfluidic channel can be formed on the substrate. The material and height of the substrate are not particularly limited. Some commonly used materials, such as silicon wafers, glass, polycarbonate-polystyrene alloy (PCPS), polymethyl Methyl acrylate (PMMA), etc., can be used as a substrate. The height of the substrate only needs to be suitable for forming the microfluidic channel. The provided microfluidic chip is small in size and easy to carry. The microfluidic chip is used to prepare vesicles with high yield, and vesicles with similar morphology and membrane composition to exosomes can be prepared, and the yield is 8-12 times that of natural exosomes. Moreover, it has a wide range of applications. In addition to stem cells, other cells such as lymphocytes, tissue cells, cancer cells, etc. can be crushed by the microfluidic chip to prepare nano-scale vesicles. According to an embodiment of the present invention, the height of the microfluidic channel is 1-10 microns, for example, 2-10 microns, 3-10 microns, 4-8 microns and so on. Setting a microfluidic channel with a suitable height can allow cells to pass through and process the microfluidic channel to obtain vesicles similar to natural exosomes. The mentioned height of the microfluidic channel is consistent with the height direction of the substrate, which refers to the vertical distance between the top of the microfluidic channel and the bottom in this direction.

In the third aspect of the present invention, the present invention provides a method for preparing vesicles, comprising: using a microfluidic channel or a microfluidic chip to process the cells, wherein the microfluidic channel is the first aspect of the present invention In the microfluidic channel according to any embodiment, the microfluidic chip is the microfluidic chip according to any embodiment of the second aspect of the present invention. According to an embodiment of the present invention, the particle size of the prepared vesicles is 190-250 nanometers, preferably 200-220 nanometers, for example, 200-210 nanometers. According to an embodiment of the present invention, compared with natural exosomes, the yield of the prepared vesicles is at least 5 times, preferably at least 8 times, and more preferably at least 10 times that of natural exosomes. .

According to an embodiment of the present invention, the method for preparing a vesicle further includes: preparing a cell suspension from the cells and filling it into a sterile syringe; connecting the sterile syringe with a microfluidic injection pump and the microfluidic syringe respectively The control channel or the microfluidic chip is connected, and the pressure of the microfluidic injection pump is adjusted so that the cell suspension passes through the microfluidic channel or the microfluidic chip. The microfluidic syringe pump can adjust the flow rate of the cell suspension in the microfluidic channel or the microfluidic chip, so that vesicles that are indistinguishable from natural exosomes can be prepared. The provided microfluidic channel or the provided microfluidic chip is highly controllable.

The beneficial effects achieved by the present invention are: the microfluidic flow channel or microfluidic chip provided by the present invention has small size and good portability; strong controllability, and the efficiency of cell disruption can be determined by the concentration of the cell suspension. The size of the squeezing channel and the breaking channel in the zone, the flow rate of the cell suspension in the microfluidic flow channel, etc. are adjusted. Moreover, it has a wide range of applications. In addition to stem cells, other cells such as lymphocytes and tissue cells can be crushed by the microfluidic chip to prepare nanovesicles. Through the provided microfluidic channel or microfluidic chip, nanovesicles with similar morphology and membrane composition to exosomes can be prepared, and the yield is high, which is 10 times that of natural exosomes.

Description of the drawings

Figure 1 is a schematic top view of a microfluidic channel according to an embodiment of the present invention, in which reference number 1 is the entrance, reference number 2 is the first connection area, reference number 3 is the crushing area, reference number 4 is the second connection area, reference number 5 It is the outlet, and the number 301 is the extrusion channel, and the number 302 is the crushing channel.

Fig. 2 is a topographical view of a vesicle provided according to an embodiment of the present invention.

Fig. 3 is a particle size distribution diagram of a vesicle provided according to an embodiment of the present invention.

Fig. 4 is an analysis diagram of membrane components of a vesicle provided according to an embodiment of the present invention.

Fig. 5 is an analysis diagram of the yield of vesicles provided according to an embodiment of the present invention.

Fig. 6 is a diagram showing the results of immunoblotting analysis of vesicles according to an embodiment of the present invention.

Detailed ways

The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals indicate the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention.

In one aspect of the present invention, the present invention provides a microfluidic channel, as shown in FIG. 1. In order to show the structure diagram of the microfluidic channel more clearly, the fragmentation area of the microfluidic channel is enlarged, as shown in Figure 1. Shown in the middle right picture. According to an embodiment of the present invention, the provided microfluidic channel includes: an inlet 1; a crushing zone 3, the crushing zone 3 is connected to the inlet 1, and the crushing zone is connected to the inlet 1 through a first connecting zone 2 The crushing zone 3 includes at least one crushing channel 301 and at least one crushing channel 302. One crushing channel 301 is connected to two crushing channels 302, and the crushing channel 301 is set as the first crushing channel. A predetermined width, the crushing channel is set to a second predetermined width, the first predetermined width is greater than the second predetermined width; an outlet 5, the outlet 5 is connected to the crushing zone 3 through a second connecting area 4.

The first predetermined width mentioned herein refers to setting the squeezing channel to a certain width according to the difference in the diameter of the cells to be processed and the like. The length of the squeezing channel along the direction in which the liquid (for example, cell suspension) flows is taken as the length of the squeezing channel, and the width mentioned here refers to the width of the squeezing channel in the direction perpendicular to the direction of liquid flow. The second predetermined width mentioned herein refers to setting the crushing channel to a certain width according to the difference in the diameter of the cells to be processed and the like. The length of the crushing channel along the direction of the liquid (for example, cell suspension) flow is taken as the length of the crushing channel, and the width mentioned here refers to the width of the crushing channel in the direction perpendicular to the direction of the liquid flow.

According to an embodiment of the present invention, the first predetermined width is at least twice the second predetermined width, preferably 2-10 times. According to an embodiment of the present invention, the first predetermined width is 5 to 20 microns, and the second predetermined width is 1 to 5 microns, preferably 2 to 4 microns. The width of the fragmentation channel in the microfluidic channel will affect the quality and yield of vesicles to a certain extent. When the width of the broken channel is slightly narrower, although the homogeneity of the vesicles will be improved to a certain extent, it will also affect the yield of the vesicles; when the width of the broken channel is slightly wider, the yield of the vesicles will increase, but also Will affect the homogeneity of vesicles. By setting the width of the crushing channel to 1 to 5 microns, high-yield vesicles with uniform particle size can be obtained. At the same time, considering the preparation process of the microfluidic channel, in order to reduce the cost and save the difficulty of preparing the microfluidic channel, the width of the broken channel can be set to 2-5 microns, for example, it can be 2 to 4 microns, or 2.5 ~4 microns or 2.5 to 3 microns.

In order to be able to process multiple cells at one time and alleviate the resistance encountered by the cells in the process of crushing, the first connection area and the second connection area can be connected by setting a branch flow channel. It is also possible to set several levels of branch flow channels as required. According to an embodiment of the present invention, the first joining area includes at least two inlet flow passages, and the inlet is connected to the crushing zone through the at least two inlet flow passages. According to an embodiment of the present invention, the inlet is connected to at least two first inlet flow passages, each of the first inlet flow passages is connected to at least two second inlet flow passages, and the second inlet flow passage is connected to the The broken areas are connected. According to an embodiment of the present invention, the second connection area includes at least two outlet flow passages, and the crushing zone is connected to the outlet through the at least two outlet flow passages. For example, the outlet is connected to at least two third outlet flow passages, each of the third outlet flow passages is connected to at least two fourth outlet flow passages, and the fourth outlet flow passage is connected to the crushing zone. According to an embodiment of the present invention, a buffer zone is arranged between each of the extrusion channels, and the width of the buffer zone is at least 3 times the first predetermined width. For example, the width of the buffer zone can be set to 30-40 microns. According to an embodiment of the present invention, two buffer zones are arranged between each extrusion channel, and the length of each extrusion channel formed is 35-200 microns. For example, the crushing zone includes 32 to 128 crushing channels and 64 to 256 crushing channels, and two buffer zones are arranged between each of the crushing channels.

According to an embodiment of the present invention, the provided microfluidic channel may further include a screening area connected to the inlet and the crushing area, respectively, a barrier is arranged in the screening area, and the barrier is The object forms a predetermined gap channel in the screening area. According to an embodiment of the present invention, according to the liquid flow direction, the screening area is sequentially provided with a first barrier and a second barrier, the first barrier and the second barrier are rhombic columnar structures, and the first barrier The side length of the second barrier is smaller than the side length of the first barrier. For example, the first barrier forms a predetermined gap channel of 70 to 90 microns in the screening area, and the second barrier forms a predetermined gap channel of 30 to 50 microns in the screening area.

The present invention also provides a microfluidic chip, the microfluidic chip includes a substrate; and a microfluidic channel, and the microfluidic channel is the microfluidic channel provided above.

The present invention also provides a method for preparing vesicles, which includes: using the microfluidic channel or microfluidic chip provided above to process the cells. The particle size of the vesicles thus prepared is 190-250 nanometers, preferably 200-220 nanometers. Moreover, compared with natural exosomes, the yield of the vesicles is at least 5 times, preferably at least 8 times, and more preferably at least 10 times that of natural exosomes. When preparing the vesicles, the cells can be prepared into a cell suspension, loaded into a sterile syringe, and then the sterile syringe is connected to the microfluidic injection pump and the microfluidic channel or the microfluidic chip respectively, through Adjust the pressure of the microfluidic injection pump so that the cell suspension passes through the microfluidic channel or the microfluidic chip.

The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

Example 1

The design of the microfluidic channel for preparing exosomal nano-scale vesicles of the present invention is shown in FIG. 1. The micro-channel design can be divided into three parts: inlet, crushing zone and outlet. The inlet is directly connected to the crushing zone through a branch flow channel. Specifically, the inlet passes through three first inlet flow channels, and each first inlet flow channel passes through three second inlet flow channels, and each second inlet flow channel respectively. After passing through two third inlet flow passages, each third inlet flow passage is connected to the crushing zone through two fourth inlet flow passages respectively. The length of the first joint area is set to be about 8-9 mm, and the width is set to be about 0.2-0.4 mm. Finally, it branched to form 96 squeezing channels and 192 crushing channels, which merged through the second connecting area and then connected to the exit. Specifically, the outlet passes through three first outlet flow passages, each first outlet flow passage passes through three second outlet flow passages, and each second outlet flow passage passes through two third outlet flow passages. The three outlet flow passages are respectively connected to the crushing zone through two fourth outlet flow passages. The length of the second connecting area is set to be about 8-9 mm, and the width is set to be about 0.3 mm to 0.4 mm. The inlet and outlet are both designed as a circle with a diameter of 0.9mm. The width of the extrusion channel in the crushing zone is designed to be 20 μm, 10 μm, and 5 μm, respectively, and the width of the crushing channel is designed to be 2.5 μm.

The design drawings shown in Figure 1 are handed over to the microfluidic workshop for processing to prepare microfluidic chips containing microfluidic channels. Including the following steps:

1. Submit the design drawings of the present invention to the microfluidic workshop for processing, and mold silicon wafers can be produced. The etching depth of the silicon wafer processing is 10 μm, and the micro flow channel made is 10 μm high.

2. Using Dow corning SYLGARD ^TM 184 pouring sealant (ie polydimethylsiloxane, hereinafter referred to as PDMS) as the raw material, the crosslinking agent is mixed at a ratio of 10:1 and then fully stirred, and placed in a vacuum tank for processing Vacuum until there are no visible bubbles;

3. Drop a circle of trimethylchlorosilane around the silicon wafer, and use trimethylchlorosilane to volatilize the model silicon wafer for 10 minutes;

4. Pour the PDMS onto the surface of the model silicon wafer for inverting the mold, and vacuumize it in a vacuum tank for 1 hour;

5. Transfer the model silicon wafer and PDMS to an oven at 65°C overnight for cross-linking;

6. Remove the PDMS after the down-mold, cut according to the pattern arrangement, and punch holes at the entrance and exit with a No. 18 hole punch (inner diameter 0.9mm, outer diameter 1.3mm);

7. Prepare 75% (v/v) alcohol with absolute ethanol and deionized water, surpass the slide (76mm length, 25mm width) and PDMS after punching, ultrasonically clean for 10 minutes;

8. Completely air-dry the slides and PDMS in the ultra-clean table, transfer them to the plasma cleaner for surface treatment, immediately bond the PDMS and the slides to make a microfluidic chip after taking it out, and put it in a 65°C oven overnight to promote Interface bonding.

9. Take out the bonded microfluidic chip and store it at room temperature.

Example 2

Example 2 provides a method for preparing nano-scale vesicles, and the specific operation steps include:

1. Connect the syringe with the microfluidic chip prepared in Example 1 with a polytetrafluoroethylene (PTFE) catheter with an outer diameter of 0.9mm and an inner diameter of 0.5mm, first rinse the microfluidic channel with 75% alcohol, and then rinse with PBS Microfluidic channel, and finally exhaust the microfluidic channel.

2. After digestion and centrifugation of mesenchymal stem cells (MSC), resuspend them in sterile phosphate buffered saline (PBS) to ^{a cell suspension with a density of 1x10 6} mL ^-1 and transfer to a 1 mL sterile syringe;

3. Load the syringe on the microsyringe pump, set the size of the 1mL syringe (inner diameter 4.69mm), and then perform a bolus injection at a linear speed of 50μL/min.

4. Collect the crushed vesicles from the outlet.

5. Collect the vesicle suspension and perform differential centrifugation for purification and separation. First, centrifuge at 300g for 10 minutes to remove the cell pellet. Then centrifuge at 2000g for 10 minutes to remove the dead cell pellet. After centrifugation at 10,000 g for 30 min, the dead cell pellet was removed.

6. Collect the supernatant after differential centrifugation, and perform ultra-high-speed centrifugation to purify and separate nano-sized vesicles. First, centrifuge at 100000g for 2h to collect the precipitate. Then the vesicles were resuspended in 1ml sterile phosphate buffered saline (PBS) and passed through a 0.45μm filter membrane. Centrifuge again at 100000g for 1h, at which time the precipitate is pure nano-sized vesicles.

The prepared nano-scale vesicles were characterized as follows:

(1) Use transmission electron microscopy to characterize the morphology of the prepared nano-sized vesicles, as shown in Figure 2, the scale is 100nm, and the arrow on the left in Figure 2 refers to the nano-sized vesicles obtained from mesenchymal stem cells. The arrow on the right picture indicates natural exosomes derived from mesenchymal stem cells.

(2) Detect the particle size distribution of nano-sized vesicles by dynamic light scattering (DLS), as shown in Figure 3 (the whitish part surrounded by the circle in the upper figure in Figure 3 is the vesicle). The average particle size of the obtained nano-sized vesicles was 201 nm.

(3) The cell membrane of nano-scale vesicles was characterized by Nikon super-resolution laser scanning confocal microscope. As shown in Fig. 4, the scale is 1 μm. The result shows that the cell membrane of the prepared nano-scale vesicles is a phospholipid bilayer.

(5) using the BCA kit nanoscale vesicles total protein detection, cells were counted before extrusion crushing, the final ¹⁰⁷ cells in terms of the total amount of protein fragmentation extruded nanoscale vesicles were compared. In Figure 5, 1, 2, and 3 are three repeated experiments. The results showed that the yield of nano-sized vesicles was 20-40 μg vesicles ^{per 1×10 7 cells.} The yield is 10 times that of natural exosomes (refer to ACS Nano, 2014, 8(1): 7698-7710. Sci Rep, 2018, 8(1): 2471.).

At the same time, the hepatic sinusoidal endothelial cells (LSEC) were replaced with the above-mentioned mesenchymal stem cells, and the microfluidic chip provided in Example 1 was used for the same treatment, and it was verified that nano-scale vesicles can be prepared. The yield of vesicles is 9 times that of natural exosomes. Moreover, the performance of the prepared nano-scale vesicles is not significantly different from that of natural exosomes.

Example 3

In Example 3, Western blot was used to characterize the CD63, CD105, and Actin of the cells before treatment and the nano-sized vesicles prepared in Example 2. CD63 is highly expressed in vesicles, but low or not expressed in MSC and LESC. Therefore, CD63, as a universal marker of vesicles, can be used to indicate vesicles; CD105 is not expressed or low expressed in vesicles. It is highly expressed in MSC and LESC, so CD105, as a marker of MSC and LSEC, can be used to indicate MSC and LSEC. The source of the vesicles can be further verified by analyzing the markers on the vesicles from different sources and the corresponding source cells.

The experimental results are shown in Figure 6. For MSC, Actin showed high expression in MSC cells and MSC vesicles (referring to vesicles obtained by MSC treatment); CD63 showed high expression in MSC vesicles, and the expression level in MSC cells was low; CD105 High expression in MSC cells, low expression in MSC vesicles. It shows that MSC vesicles are derived from MSC.

For LSEC, Actin is highly expressed in LSEC cells and low in LSEC vesicles (referring to vesicles obtained by LSEC treatment); CD63 is highly expressed in LSEC vesicles, and the expression level is low in LSEC cells; CD105 is expressed in LSEC cells Medium and high expression, low expression in LSEC vesicles. It shows that LSEC vesicles are derived from LSEC.

For the first time, the present invention designs microfluidic channels and microfluidic chips to squeeze and break cells by using narrow space constraints to recombine them into nano-scale vesicles. Using the provided microfluidic channel or microfluidic chip to prepare nano-scale vesicles is a new method for preparing exosomal nano-scale vesicles, which avoids the difficulty of large-scale production of natural exosomes and can replace natural exosomes. Exosomes for downstream applications.

Vesicles have strong solubilization ability, and their double-layer membranes have good firmness and stability. The prepared vesicles can be used as carriers of drug delivery systems for targeted therapy.

In addition, vesicles prepared by using microfluidic channels can also promote the regeneration of liver and other tissues; can be used for communication between cells; can be used to regulate intracellular molecular levels and improve cell activity; and can be used to treat cardiovascular disease , Tumors and other diseases. Therefore, the technology of preparing vesicles using microfluidic channels or microfluidic chips is more suitable for industrial production and application, and can be applied to various fields such as clinical and big data research. Moreover, it provides a maneuverable means for the mass generation and preparation of vesicles, and the prepared vesicles exhibit uniform characteristics and have excellent performance.

In the description of the present invention, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", " "Back", "Left", "Right", "Vertical", "Horizontal", "Top", "Bottom", "Inner", "Outer", "Axial", "Radial", "Circumferential", etc. The indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the pointed device or element must have a specific orientation or a specific orientation. The structure and operation cannot therefore be understood as a limitation of the present invention.

In addition, the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. . Thus, the features defined with "first", "second", "third", and "fourth" may explicitly or implicitly include at least one of the features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

In the present invention, unless otherwise clearly specified and limited, the terms "connected", "connected", "fixed" and other terms should be understood in a broad sense. For example, they may be fixedly connected, detachably connected, or integrated. ; It can be mechanically connected, or electrically connected or communicable with each other; it can be directly connected, or indirectly connected through an intermediate medium, it can be the internal communication of two components or the interaction relationship between two components, unless otherwise specified The limit. For those of ordinary skill in the art, the specific meanings of the above-mentioned terms in the present invention can be understood according to specific situations.

In the present invention, unless expressly stipulated and defined otherwise, the “on” or “under” of the first feature on the second feature may be in direct contact with the first and second features, or the first and second features may be indirectly through an intermediary. touch. Moreover, the "above", "above" and "above" of the first feature on the second feature may mean that the first feature is directly above or diagonally above the second feature, or it simply means that the level of the first feature is higher than that of the second feature. The “below”, “below” and “below” of the second feature of the first feature may mean that the first feature is directly below or obliquely below the second feature, or it simply means that the level of the first feature is smaller than the second feature.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structures, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above-mentioned terms are not necessarily directed to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims

A microfluidic channel is characterized in that it comprises:

Entrance;

A crushing zone, the crushing zone is connected to the inlet, the crushing zone is connected to the inlet through a first connecting area, the crushing zone includes at least one squeezing channel and at least one crushing channel, one of the squeezing channels Respectively connected to at least one of the crushing channels, the squeezing channel is set to a first predetermined width, the crushing channel is set to a second predetermined width, the first predetermined width is greater than the second predetermined width;

An outlet, which is connected to the crushing zone through a second connecting area.
The microfluidic channel according to claim 1, wherein the first connecting area comprises at least two inlet flow channels, and the inlet is connected to the crushing zone through the at least two inlet flow channels.
The microfluidic channel according to claim 2, wherein the inlet is connected to at least two first inlet flow channels, and each of the first inlet flow channels is connected to at least two second inlet flow channels, The second inlet flow channel is connected to the crushing zone.
The microfluidic channel according to claim 1, wherein the second connection area comprises at least two outlet flow channels, and the crushing zone is connected to the outlet through the at least two outlet flow channels.
The microfluidic channel of claim 4, wherein the outlet is connected to at least two first outlet flow channels, and each of the first outlet flow channels is connected to at least two second outlet flow channels, The second outlet flow channel is connected to the crushing zone.
The microfluidic channel according to claim 1, wherein the first predetermined width is at least twice the second predetermined width.
The microfluidic channel according to claim 1, wherein the first predetermined width is 2-10 times the second predetermined width.
The microfluidic channel according to claim 1, wherein the first predetermined width is 5-20 microns, and the second predetermined width is 1-5 microns.
The microfluidic channel according to claim 1, wherein the first predetermined width is 5-20 microns, and the second predetermined width is 2-5 microns.
The microfluidic channel according to claim 1, wherein the first predetermined width is 5-20 mm, and the second predetermined width is 2.5-5 microns.
The microfluidic channel according to claim 1, wherein a buffer zone is arranged between each of the extrusion channels, and the width of the buffer zone is at least 3 times the first predetermined width.
The microfluidic channel according to claim 11, wherein the width of the buffer zone is 30-40 microns.
The microfluidic channel according to claim 1, wherein two buffers are arranged between each of the extrusion channels, and the length of each formed extrusion channel is 35-200 microns.
The microfluidic channel according to claim 1, wherein the crushing zone includes 32 to 128 squeezing channels and 64 to 256 crushing channels, and two squeezing channels are arranged between each Buffers.
The microfluidic channel according to claim 1, wherein the length of the first connecting area is 1-10 mm; the width of the first connecting area is 0.15-0.5 mm.
The microfluidic channel according to claim 1, wherein the length of the first joint area is 1.8-8 mm; the width of the first joint area is 0.2-0.4 mm.
The microfluidic channel according to claim 1, wherein the length of the second connecting area is 1-10 mm; the width of the second connecting area is 0.2-0.5 mm.
The microfluidic channel according to claim 1, wherein the length of the second connecting area is 1.2-10 mm; the width of the second connecting area is 0.3-0.4 mm.
The microfluidic channel of claim 1, further comprising:

A screening area, the screening area is respectively connected with the entrance and the crushing area, a barrier is arranged in the screening area, and the barrier forms a predetermined gap channel in the screening area.
The microfluidic channel according to claim 19, characterized in that, according to the direction of liquid flow, the screening area is sequentially provided with a first barrier and a second barrier, the first barrier and the second barrier The object has a rhombic columnar structure, and the side length of the second barrier is smaller than the side length of the first barrier.
22. The microfluidic channel according to claim 21, wherein the first barrier forms a predetermined gap channel of 70-90 microns in the screening area, and the second barrier forms 30 to 30 microns in the screening area. ~50 micron predetermined gap channel.
A microfluidic chip, characterized in that it comprises:

Substrate; and

A microfluidic channel, the microfluidic channel is formed on the substrate, and the microfluidic channel is the microfluidic channel according to any one of claims 1-21.
The microfluidic chip of claim 22, wherein the height of the microfluidic channel is 1-10 microns.
The microfluidic chip according to claim 22, wherein the substrate comprises at least one selected from the group consisting of silicon wafer, glass, polycarbonate-polystyrene alloy, and polymethyl methacrylate.
A method for preparing vesicles, which is characterized in that it comprises:

The cells are processed using a microfluidic channel or a microfluidic chip, wherein the microfluidic channel is the microfluidic channel of any one of claims 1-21, and the microfluidic chip is claim 22 The microfluidic chip of any one of ~24.
The method according to claim 25, wherein the particle size of the vesicle is 190-250 nanometers.
The method according to claim 25, wherein the particle size of the vesicle is 200-220 nanometers.
The method according to claim 25, characterized in that, compared with the natural exosomes, the yield of the vesicles is at least 5 times that of the natural exosomes.
The method according to claim 25, wherein the vesicle has a yield of at least 8 times that of the natural exosomes compared with the natural exosomes.
The method according to claim 25, wherein the vesicle has a yield of at least 10 times that of the natural exosomes compared with the natural exosomes.
The method according to claim 25, further comprising:

Preparing a cell suspension from the cells and filling them into a sterile syringe;

The sterile syringe is respectively connected with a microfluidic injection pump and the microfluidic channel or the microfluidic chip, and the pressure of the microfluidic injection pump is adjusted to make the cell suspension pass through the microfluidic channel Or the microfluidic chip.