Perfusion micro-fluidic blood vessel device based on ultrasonic action and preparation method and application of perfusion micro-fluidic blood vessel device

Abstract

The invention belongs to the technical field of micro-fluidic chips, and particularly relates to a perfusion micro-fluidic blood vessel device based on ultrasonic action and a preparation method and application thereof. According to the perfusion microfluidic blood vessel device provided by the invention, the construction of a blood brain barrier model is successfully realized in vitro, meanwhile, a dynamic culture mode is closer to a dynamic environment in a human body, and the perfusion microfluidic blood vessel device realized based on a microfluidic chip technology can be adapted to an ultrasonic transducer; the method is used for screening which pulse parameter is suitable for drug delivery of the blood-brain barrier, so that the technical problem that an in-vitro blood-brain barrier model is difficult to construct by a micro-fluidic chip in the prior art is solved.

Description

Technical Field

[0001] The present application belongs to the technical field of microfluidic chips, and in particular to a perfusable microfluidic vascular device based on ultrasound, and a preparation method and application thereof. Background Art

[0003] Previous animal model studies have shown that the expansion, contraction and collapse of microbubbles under ultrasound induce ultrasonic perforation, opening of cell-cell contacts and endocytosis, which can be used as a variety of vascular drug delivery pathways to help drugs pass through the blood-brain barrier. In animal models, it was found that by shortening the pulse or reducing the pulse repetition frequency, that is, changing different therapeutic ultrasound parameters to reduce the total number of transmitted sound wave cycles, the fast short pulse can help to make the delivered drugs more evenly distributed in space, reduce therapeutic side effects, and cause less tissue damage. This shows that ultrasound is helpful in the clinical development and research of drugs for central nervous system diseases.

[0004] Although animal models can highly simulate the complexity of human physiology and reflect the true state of the microenvironment, it is impossible for animal models to completely reproduce all the characteristics of human central nervous system diseases. Therefore, the development and research results using animal models will encounter obstacles in the process of clinical transformation. In addition, animal models are expensive and difficult to use on a large scale. In addition to animal models, in vitro cell models and computer models are also traditional development and research models for central nervous system disease drugs.

[0005] Ordinary in vitro cell models are difficult to simulate the blood-brain barrier in vitro. In recent years, with the development of microfluidics technology, it has become possible to construct an in vitro blood-brain barrier model on a chip; microfluidics chips, based on semiconductor chip laboratory manufacturing technology, are a scientific technology that constructs micrometer-scale space and manipulates fluids as its main feature. Microfluidics chips can integrate or basically integrate basic operating units such as sample preparation, reaction, separation, detection, cell culture, sorting, and lysis involved in fields such as chemistry and biology onto a chip of a few square centimeters or even smaller, forming a network of microchannels with controllable fluids running through the entire system. Its outstanding advantages are low reagent consumption, high throughput, and the ability to simulate the physical environment of the human body through structural design.

[0006] However, the in vitro cell models currently constructed using microfluidic chips, such as patent applications CN202111546655 and CN20201163361, which use a shaker to control the flow rate of the liquid after inoculating the cultured cells, are difficult to simulate the in vitro blood-brain barrier model. This is because under normal physiological conditions, cells in the human body are subject to the shear force generated by the continuous flow of blood, which makes the cell growth state significantly different from the in vitro cell growth state cultured in CN202111546655 and CN20201163361. The current microfluidic chips are difficult to construct in vitro blood-brain barrier models and are compatible with ultrasound device experiments for in-depth research on preclinical cell experiments. Summary of the invention

[0007] In view of this, the present application provides an ultrasound-based perfusable microfluidic vascular device and a preparation method and application, which are used to solve the technical problem in the prior art that it is difficult to construct an in vitro blood-brain barrier model using a microfluidic chip.

[0008] In a first aspect, the present application provides a perfusable microfluidic blood vessel device, the structure of which includes an input tube, three parallel microchannels of the same size, and an output tube;

[0009] The outlet of the input pipe is communicated with the inlets of the three parallel microchannels of the same size;

[0010] The outlets of the three parallel microchannels of the same size are communicated with the inlet of the output pipe.

[0011] Preferably, the length of a single microchannel in the three parallel microchannels of the same size is 17000 μm, the width is 200 μm, and the height is 100 μm;

[0012] The spacing between adjacent microchannels is 150 μm.

[0013] Preferably, the input tube has a length of 17000 μm, a width of 400 μm, and a height of 100 μm;

[0014] The output tube has a length of 17000 μm, a width of 400 μm, and a height of 100 μm.

[0015] Preferably, the input tube is used to perfuse the mouse brain microvascular endothelial cell suspension into three parallel microchannels of the same size;

[0016] The three parallel microchannels of the same size are used to attach mouse brain microvascular endothelial cells;

[0017] The output tube is used to output the mouse brain microvascular endothelial cell suspension.

[0018] The second aspect of the present application provides a method for preparing a perfusable microfluidic vascular device, which can be used to prepare the perfusable microfluidic vascular device described in the first aspect. The preparation method comprises the following steps:

[0019] Step S1, using mechanical drawing software to draw a plane figure of the perfusable microfluidic vascular device described in the first aspect;

[0020] Step S2, making the plane pattern of the perfusable microfluidic vascular device into a mask, and obtaining a chip mold corresponding to the perfusable microfluidic vascular device described in the first aspect by photolithography technology;

[0021] Step S3, pouring the polydimethylsiloxane prepolymer mixture onto the chip mold corresponding to the perfusable microfluidic vascular device described in the first aspect;

[0022] Step S4, placing the chip mold cast with the polydimethylsiloxane prepolymer mixture in an oven for curing, peeling, and cleaning in sequence to obtain a perfusable microfluidic vascular device.

[0023] Preferably, in step S4, the curing temperature is 40-60° C. and the curing time is 2-4 hours;

[0024] The cleaning is performed by using a plasma cleaner for 40 to 60 seconds.

[0025] The third aspect of the present application provides a perfusable microfluidic blood vessel device based on ultrasound, the device comprising the perfusable microfluidic blood vessel device described in the first aspect and a ring-shaped ultrasonic transducer;

[0026] The annular ultrasonic transducer is bonded and fixed to the perfusable microfluidic blood vessel device, and the cross section of the annular ultrasonic transducer can completely cover the cross sections of three parallel microchannels of the same size in the perfusable microfluidic blood vessel device.

[0027] Preferably, the medium used by the annular ultrasonic transducer is water.

[0028] The fourth aspect of the present application provides an application of the perfusable microfluidic vascular device described in the first aspect in the field of blood-brain barrier drug delivery.

[0029] The fifth aspect of the present application provides an application of the ultrasound-based perfusable microfluidic vascular device described in the third aspect in the field of blood-brain barrier drug delivery.

[0030] Compared with the prior art, the ultrasound-based perfusable microfluidic vascular device provided by the present application has at least the following beneficial effects.

[0031] 1. This application realizes the construction of an in vitro blood-brain barrier model based on microfluidic chip technology.

[0032] 2. The perfusable microfluidic vascular device implemented in this application based on microfluidic chip technology is a dynamic model. The shear force, physical and chemical gradients, etc. formed by the flow of fluid in the vascular system can be closer to the dynamic environment in the body.

[0033] 3. The perfusable microfluidic vascular device implemented in the present application based on microfluidic chip technology can be adapted to an ultrasonic transducer to screen which pulse parameters are suitable for drug delivery across the blood-brain barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a schematic structural diagram of a perfusable microfluidic vascular device based on ultrasound in Example 1 of the present application;

[0036] Figure 2 It is a cross-sectional schematic diagram of a perfusable microfluidic blood vessel device based on ultrasound, and the reference numerals in the figure include: 1 is an input port, 2 is three parallel microchannels of the same size, 3 is an output port, 4 is an input tube, 5 is an output tube, and 6 is a ring-shaped ultrasonic transducer;

[0037] Figure 3 This is a physical picture of a perfusable microfluidic vascular device based on ultrasound in Example 1 of the present application;

[0038] Figure 4 Schematic diagram of the effect of using a perfusable microfluidic vascular device to culture mouse brain microvascular endothelial cells in Example 2 of the present application, Figure A is a bright field image of static culture, Figure B is a fluorescence image of static culture; Figure C is a bright field image of static culture, and Figure D is a fluorescence image of static culture;

[0039] Figure 5Schematic diagram of the ultrasound microbubble stimulation experiment on the microfluidic vascular channel using the ultrasound-based perfusable microfluidic vascular device in Example 3 of the present application; Figure a is a perfusable microfluidic vascular device based on ultrasound and an enlarged area diagram, and Figure b is two experimental parameters tested, including a long pulse mode (one pulse per second, each pulse consists of a 9.09 millisecond continuous wave, a total of 10 pulses) and a short pulse mode (a total of 60 pulses, one pulse per second, each pulse consists of 10 sub-pulses, the pulse repetition interval is 1 millisecond, and the pulse length is 100 microseconds); the sound pressure parameter is 0.5MPa;

[0040] Figure 6 This is a schematic diagram of the test results of the ultrasound microbubble stimulation experiment on the microfluidic vascular channel using the ultrasound-based perfusable microfluidic vascular device in Example 3 of the present application in the long pulse mode, where green is Calcein-AM indicating cell activity and red is PI indicating cell death;

[0041] Figure 7 This is a schematic diagram of the test results in short pulse mode using an ultrasound-based perfusable microfluidic vascular device to stimulate microfluidic vascular channels using ultrasound in Example 3 of the present application. Green is Calcein-AM, indicating cell activity, and red is PI, indicating cell death. DETAILED DESCRIPTION

[0042] The present application provides an ultrasound-based perfusable microfluidic vascular device and a preparation method and application thereof, which are used to solve the technical problem in the prior art that it is difficult to construct an in vitro blood-brain barrier model using a microfluidic chip.

[0043] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0044] Example 1

[0045] Example 1 of the present application provides a method for preparing a perfusable microfluidic vascular device based on ultrasound, the preparation method comprising the steps of preparing the perfusable microfluidic vascular device and preparing the perfusable microfluidic vascular device based on ultrasound.

[0046] The steps of preparing the perfusable microfluidic vascular device include:

[0047] Step S1, using mechanical drawing software to draw a plane figure of the perfusable microfluidic vascular device described in the first aspect;

[0048] Step S2, using a supplier to make a plane pattern of the perfusable microfluidic vascular device into a mask, and obtaining a chip mold corresponding to the perfusable microfluidic vascular device described in the first aspect through photolithography technology; the photolithography technology can refer to the existing photolithography technology, that is, including pouring a negative photoresist of about the size of a coin on a silicon wafer, and adjusting the rotation speed to the required parameters to obtain the target pattern height; drying at 65°C for 3 minutes according to the required thickness of the poured glue to dry the photoresist; then aligning the mask with the silicon wafer, pressing it with a heavy object, putting it into a photolithography machine, setting the required parameters, and exposing it for 8-10 seconds; drying it at 90°C for 6 minutes after exposure to volatilize organic matter such as residual photoresist; after cooling to room temperature, using a developing agent for development, washing away excess photoresist patterns, leaving only the required patterns; blowing it dry with an air gun, and drying it for use; then placing the developed silicon wafer on a heating plate, heating it at 150 degrees for 30 minutes to complete the chip mold corresponding to the perfusable microfluidic vascular device;

[0049] Step S3, pouring a polydimethylsiloxane prepolymer mixture (PDMS, Sylgard 184 Silicon Elastomer Kit, Dow Corning, 10:1 mixing ratio) onto a chip mold corresponding to the perfusable microfluidic vascular device;

[0050] Step S4, then put it into a vacuum chamber for debubbling treatment, and then put it into an oven for curing at 50° C. for 3 hours; then peel off the cured polydimethylsiloxane, and then use a plasma cleaner for 50 seconds to prepare a perfusable microfluidic vascular device.

[0051] The steps of preparing the perfusable microfluidic vascular device based on ultrasound include: bonding and fixing the prepared perfusable microfluidic vascular device and the purchased annular ultrasonic transducer to the cover glass (25 × 50 mm 2 ), and the cross-section of the annular ultrasonic transducer can completely cover the cross-section of three parallel microchannels of the same size in the perfusable microfluidic vascular device, and water is used as the medium. The schematic diagram of the perfusable microfluidic vascular device based on ultrasound is shown in FIG. Figure 1-2 As shown in the actual picture Figure 3 shown.

[0052] Example 2

[0053] In Example 2 of the present application, a mouse brain microvascular endothelial cell culture experiment was performed on the perfusable microfluidic vascular device provided in Example 1. The culture experiment was divided into a static culture period and a dynamic culture period.

[0054] The steps of the static culture period include:

[0055] bEnd.3 cells (mouse brain microvascular endothelial cells.3) were cultured in DMEM medium (Gibco, C11995500BT) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2 under conventional culture conditions for 5 to 12 generations; bEnd.3 cells (mouse brain microvascular endothelial cells.3) were then trypsinized and resuspended in pre-warmed (37°C) culture medium to obtain a cell density of 1x10 7 / mL of mouse brain microvascular endothelial cell suspension;

[0056] The microchannels of the perfusable microfluidic vascular device were pre-wetted with 1XPBS for 5 minutes, then coated with 50 µg / mL fibronectin (Roche, 10838039001) at 37°C for 15 minutes, and washed with 1x DPBS (Gibco, Ca2+) before cell seeding. 2+ ) for washing; then, the mouse brain microvascular endothelial cell suspension was introduced into three parallel microchannels of the same size through the input tube using a 1 ml sterile syringe; when the injected cells reached the required density (60-70%) in the microchannel, the input and output tubes of the microchannel were clamped, and the device was placed in an incubator for 0.5 hours to allow the cells to attach under static conditions.

[0057] The steps of the dynamic culture period included: forming a cell monolayer in three parallel microchannels of the same size using a syringe pump (KDS, R462) at a perfusion flow rate of 2 µL / min with DMEM growth medium, and culturing the cells for about 6 hours.

[0058] In this example, in order to better observe the cell growth status, fluorescent labeling was performed. The cells in the microchannel were perfused with DMEM growth medium containing 5μg / ml Hoechst 33342 (biosharp, BL803A) and 1μM calcein-AM (Invitrogen, 2525771), which were used to mark the cell nucleus and indicate cell viability, respectively; after incubation at 37°C in the dark for 15 minutes, the unloaded dye was washed with 1X DPBS at a speed of 2μL / min for 1 minute; then the microfluidic chip containing cells was mounted on a microscope, and 1X DPBS (containing Ca 2+) containing microbubbles (1:20 v / v dilution) and propidium iodide (PI, Thermo Fisher Scientific, P21493) was perfused at a flow rate of 75μL / min using a syringe pump. 2+ ), with a target concentration of 100 µg / mL.

[0059] The steps of static culture and the effects of dynamic culture are as follows Figure 4 As shown, by comparing Figure A and Figure B of static culture and Figure C and Figure D of dynamic culture, it can be clearly seen that under the flow shear force conditions of dynamic culture, mouse brain microvascular endothelial cells underwent obvious longitudinal growth, which makes the dynamic culture cell growth closer to the physiological state of the blood-brain barrier and can better simulate the blood-brain barrier in vitro. Therefore, the blood-brain barrier model constructed using a perfusable microfluidic vascular device in this application is helpful for the study of blood-brain barrier drug delivery.

[0060] Example 3

[0061] Example 3 of the present application conducts an ultrasound-based perfusion microfluidic vascular device provided in Example 1 to perform an ultrasound microbubble stimulation experiment on a microfluidic vascular channel; the experiment includes a long pulse parameter test and a short pulse parameter test. The results of the long pulse parameter test are shown in FIG. Figure 6 The results of the short pulse parameter test are shown in Figure 7 shown.

[0062] The process of long pulse parameter test or short pulse parameter test is as follows Figure 5 As shown, the system includes: the syringe pump is used to input the flow rate of 75 µL / min into three parallel microchannels of the same size through the input tube, and the bright field and fluorescence signals of the cells are captured through a 5x microscope objective (N-Achroplan 5x / 0.15); the exposure output signal of the PCO camera is used to trigger the digital delay generator, which issues a command to trigger the ultrasound system after 20 seconds of fluorescence baseline recording; the fluorescence image (total recording time is 380 seconds, and the exposure time of PI and calcein channels is 100 milliseconds) is recorded through Manager 2.0 version, which can communicate and coordinate the operation sequence between the Cool LED fluorescence excitation device, the microscope filter turret and the PCO camera to achieve simultaneous imaging of calcein and PI.

[0063] The test results of long pulse parameter test are as follows: Figure 6 As shown, from Figure 6 It can be seen that the time series of long pulse sound parameters affect cell damage. Red is PI, indicating whether the cell is perforated, and green is Calcein-AM, indicating cell activity. It can be seen from the figure that during ultrasonic stimulation (0-20s), cell activity decreases rapidly, green fluorescence decreases, and red fluorescence increases rapidly. At 170 and 192s, cell detachment and displacement can be seen due to serious damage to the cells caused by cavitation.

[0064] The test results of short pulse parameter test are as follows: Figure 7 As shown, from Figure 7It can be seen that the time series of short pulse sound parameters affect cell damage. Red is PI, indicating whether the cell is perforated, and green is Calcein-AM, indicating cell activity. It can be seen from the figure that during ultrasonic stimulation (0-20s), cell activity decreases rapidly, green fluorescence decreases, and red fluorescence increases rapidly, but no cell detachment or displacement is observed.

[0065] Comparing the test results of the long pulse parameter test and the short pulse parameter test, it can be seen that the ultrasound perforation occurring under the long pulse parameter / short pulse parameter can be used to screen the drug delivery pathway under ultrasound to determine which pulse parameter is suitable for drug delivery across the blood-brain barrier.

[0066] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A perfusable microfluidic vascular device, characterized in that: It includes an input pipe, three parallel microchannels of the same size, and an output pipe; The outlet of the input pipe is communicated with the inlets of the three parallel microchannels of the same size; The outlets of the three parallel microchannels of the same size are communicated with the inlet of the output pipe.

2. A perfusable microfluidic vascular device according to claim 1, characterized in that: The length of a single microchannel in the three parallel microchannels of the same size is 17000 μm, the width is 200 μm, and the height is 100 μm; The spacing between adjacent microchannels is 150 μm.

3. The perfusable microfluidic vascular device according to claim 1, characterized in that: The input tube has a length of 17000 μm, a width of 400 μm, and a height of 100 μm; The output tube has a length of 17000 μm, a width of 400 μm, and a height of 100 μm.

4. The perfusable microfluidic vascular device according to claim 1, characterized in that: The input tube is used to perfuse the mouse brain microvascular endothelial cell suspension into three parallel microchannels of the same size; The three parallel microchannels of the same size are used to attach mouse brain microvascular endothelial cells; The output tube is used to output the mouse brain microvascular endothelial cell suspension.

5. A method for preparing a perfusable microfluidic vascular device according to any one of claims 1 to 4, characterized in that: Includes steps: Step S1, using mechanical drawing software to draw a plane figure of the perfusable microfluidic vascular device according to any one of claims 1 to 4; Step S2, making the plane pattern of the perfusable microfluidic vascular device into a mask, and obtaining a chip mold corresponding to the perfusable microfluidic vascular device according to any one of claims 1 to 4 by photolithography technology; Step S3, pouring the polydimethylsiloxane prepolymer mixture onto a chip mold corresponding to the perfusable microfluidic vascular device according to any one of claims 1 to 4; Step S4, placing the chip mold cast with the polydimethylsiloxane prepolymer mixture in an oven for curing, peeling, and cleaning in sequence to obtain a perfusable microfluidic vascular device.

6. The method for preparing a perfusable microfluidic vascular device according to claim 5, characterized in that: In step S4, the curing temperature is 40-60°C and the curing time is 2-4 hours; The cleaning is performed by using a plasma cleaner for 40 to 60 seconds.

7. A perfusable microfluidic vascular device based on ultrasound, characterized in that: A perfusable microfluidic vascular device comprising any one of claims 1 to 4 and a ring-shaped ultrasonic transducer; The annular ultrasonic transducer is bonded and fixed to the perfusable microfluidic blood vessel device, and the cross section of the annular ultrasonic transducer can completely cover the cross sections of three parallel microchannels of the same size in the perfusable microfluidic blood vessel device.

8. The ultrasound-based perfusable microfluidic vascular device according to claim 7, characterized in that: The medium used by the annular ultrasonic transducer is water.

9. Use of the perfusable microfluidic vascular device according to any one of claims 1 to 4 in the field of blood-brain barrier drug delivery.

10. Use of the ultrasound-based perfusable microfluidic vascular device according to any one of claims 7 to 8 in the field of blood-brain barrier drug delivery.

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