Micro-fluidic chip for simulating muscle endothelial connection system
By designing vascular endothelial and smooth muscle cell culture channels with gradually narrowing widths, and combining them with microchannel distribution, the application of microfluidic chips in a simulated myoendothelial connection system was realized. This solves the problem that existing technologies cannot simulate the density differences of vascular tissue and provides an effective in vitro research platform.
Patent Information
- Application Number
- CN202423280895.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-12-26
- Estimated Expiration
- 2034-12-27
AI Technical Summary
Existing microfluidic chips cannot effectively simulate the density differences of myoendothelial connections and vascular networks in different types of vascular tissues, and therefore cannot serve as ideal models for studying myoendothelial connection systems.
A microfluidic chip simulating the endothelial junction system was designed, comprising a cover glass layer and a main body layer. It has vascular endothelial cell culture channels and vascular smooth muscle cell culture channels. The channel width is designed to gradually narrow along the liquid flow direction. The distribution of microchannels conforms to the variation law of vascular lumen and smooth muscle cell layer, realizing the simulation of different types of blood vessels.
It provides a more accurate in vitro model that simulates the in vivo environment, supporting research on the formation mechanism and function of the myoendothelial junction system, and filling a gap in this field.
Smart Images
Figure CN223723144U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model relates to the technical field of muscle endothelial junction research, and particularly relates to a microfluidic chip simulating a muscle endothelial junction system. BACKGROUND
[0002] The muscle endothelial junction in the vascular tissue is a structure formed at the contact site of the vascular endothelial cell or the vascular smooth muscle cell after penetrating the elastic membrane in the blood vessel. The muscle endothelial junction is distributed with various enzymes, receptors, channels and the like, and is an important place for intercellular communication and material exchange. The inventors found in the early stage that the density of the muscle endothelial junction in different types of vascular tissues is different, the lowest in the conductive blood vessel, slightly higher in the resistance blood vessel, and the highest in the volume blood vessel. The muscle endothelial junction in different types of vascular tissues forms a network system and may play an important function. When the muscle endothelial junction system is researched, the gene animal with reduced or increased number of muscle endothelial junctions is the best research model. However, the formation and regulation mechanism of the muscle endothelial junction have not been clarified, and such a model animal cannot be established. In this case, it is an urgent task to establish an in vitro model simulating the muscle endothelial junction system, which is of great significance for the research on the formation mechanism and function of the muscle endothelial junction system.
[0003] At present, the in vitro culture devices capable of forming muscle endothelial junctions include the Transwell cell culture chamber and the microfluidic chip. The Transwell cell culture chamber has a porous membrane, and the vascular endothelial cells and the vascular smooth muscle cells are co-cultured on the upper and lower surfaces of the membrane to form muscle endothelial junctions. However, the Transwell cell culture chamber cannot simulate the blood vessel cavity, and the muscle endothelial junction density of the membrane of different Transwell cell culture chambers and different regions of the same membrane is generally the same, so it cannot be used as a model for researching the muscle endothelial junction system. The microfluidic chip can be freely designed in shape and structure according to the experimental purpose, and the production scale is flexible, which is particularly suitable for frontier research lacking commercial equipment and has become a commonly used in vitro model carrier in life science research. Some microfluidic chips for co-culturing vascular endothelial cells and vascular smooth muscle cells have been developed, and these microfluidic chips can form muscle endothelial junctions, but cannot reflect the density difference of muscle endothelial junctions in different types of blood vessels, can only simulate muscle endothelial junctions and cannot simulate muscle endothelial junction systems, and the simulation of the blood vessel network is also not ideal, which cannot simulate the muscle endothelial junction system. Therefore, it is urgent to develop a microfluidic chip simulating the muscle endothelial junction system to carry out related research in vitro. UTILITY MODEL CONTENTS
[0004] Therefore, it is necessary to provide a microfluidic chip simulating a muscle endothelial junction system in view of the above problems.
[0005] A microfluidic chip simulating an endothelium-smooth muscle cell junction system, comprising a cover glass layer and a main body layer, the cover glass layer and the main body layer being bonded, the main body layer being provided with a blood vessel endothelial cell culture channel and a blood vessel smooth muscle cell culture channel, the blood vessel endothelial cell culture channel and the blood vessel smooth muscle cell culture channel being provided with a liquid inlet and a liquid outlet on the same side, and a plurality of microchannels being connected between the channels,
[0006] The microchannels are distributed in a manner that the interval of the microchannels gradually shortens along the direction from the liquid inlet to the liquid outlet, being 100-500 microns at one end of the liquid inlet and being shortened to 10-100 microns at one end of the liquid outlet.
[0007] The blood vessels are classified according to structure and function, and in the direction of blood flow, they are in turn a conduit vessel, a resistance vessel, a capillary vessel and a capacitance vessel, the lumen of the vessels gradually decreases from the conduit vessel to the capillary vessel, and gradually increases from the capillary vessel to the capacitance vessels. The capillary vessel is composed of endothelial cells, and the other three vessels are composed of endothelial cells, smooth muscle cells and fibroblasts. In the three types of vessel tissues, the endothelial cells are all single-layered, the number of layers of smooth muscle cells gradually decreases from the conduit vessel to the resistance vessel, and gradually increases from the capillary vessel to the capacitance vessels. The number of layers of smooth muscle cells in the same type of vessel tissue is positively correlated with the diameter of the vessel. In addition, the endothelium-smooth muscle cell junction is formed by the contact of endothelial cells and smooth muscle cells, and the density thereof is negatively correlated with the diameter of the vessel. The microfluidic chip simulating the endothelium-smooth muscle cell junction system in the above has a culture channel with a gradually narrowing width, which conforms to the change rule of the lumen of the vessel and the number of layers of smooth muscle cells, so that the chip model can better simulate the environment in which the endothelial cells and the smooth muscle cells are located in the body, and ensure the accuracy of the model simulation.
[0008] In one embodiment, the width of the blood vessel endothelial cell culture channel and the blood vessel smooth muscle cell culture channel gradually narrows along the direction of liquid flow in the channel, being 200-600 microns at one end of the liquid inlet and being narrowed to 30-100 microns at one end of the liquid outlet.
[0009] In one embodiment, the length of the blood vessel endothelial cell culture channel and the blood vessel smooth muscle cell culture channel is 25-60 centimeters, and the distance between the two channels is 5.0-30.0 microns.
[0010] In one embodiment, the interval of the microchannels is 100-300 microns at one end of the liquid inlet of the blood vessel endothelial cell culture channel and is shortened to 20-50 microns at one end of the liquid outlet.
[0011] In one embodiment, the width of the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel is 400-500 microns at the liquid inlet end, and narrows to 40-60 microns at the liquid outlet end.
[0012] In one embodiment, the length of the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel is 45-55 centimeters, and the distance between the two channels is 10.0-20.0 microns.
[0013] In one embodiment, the width of the microchannel is 0.5-5.0 microns, the height is 0.5-5.0 microns, and the length is 5.0-30.0 microns.
[0014] In one embodiment, the width of the microchannel is 2.0 microns, the height is 2.0 microns, and the length is 10.0 microns.
[0015] In one embodiment, two first oxygen electrodes for monitoring the oxygen concentration in the channel are provided in the vascular endothelial cell culture channel, and two second oxygen electrodes for monitoring the oxygen concentration in the channel are provided in the vascular smooth muscle cell culture channel.
[0016] In one embodiment, the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel extend in a serpentine zigzag shape.
[0017] The microfluidic chip simulating the muscle endothelial junction system described above provides an in vitro model for the formation mechanism and function of the muscle endothelial junction system, fills the gap in this field, and enables the research to be carried out at the in vitro level in advance. BRIEF DESCRIPTION OF DRAWINGS
[0018] In order to more clearly illustrate the specific embodiments of the present application, the following will briefly introduce the drawings needed in the specific embodiments. In all the drawings, the elements or parts are not necessarily drawn according to the actual proportions.
[0019] Figure 1 FIG. 1 is a structural schematic diagram of a microfluidic chip simulating an in vivo muscle endothelial junction system according to an embodiment of the present application;
[0020] Figure 2 FIG. 2 is a top view of the microfluidic chip simulating the in vivo muscle endothelial junction system shown in FIG. 1; Figure 1
[0021] Figure 3 FIG. 5 is an enlarged view of the first segment of the serpentine channel to the third segment of the serpentine channel;
[0022] Figure 4 FIG. 6 is an enlarged view of the fourth segment of the serpentine channel to the seventh segment of the serpentine channel;
[0023] Figure 5 For Figure 2 A-A line of the cross-sectional view;
[0024] Figure 6 For the first to seventh segment of the snake-shaped channel cross-sectional view;
[0025] Figure 7 For the first to seventh segment of the snake-shaped channel fluorescence microscope.
[0026] Reference signs:
[0027] 10-cover glass layer, 20-body layer, 21-vascular endothelial cell culture channel, 22-vascular smooth muscle cell culture channel, 23-microchannel, 24-liquid inlet, 25-liquid outlet, 26-first oxygen electrode, 27-second oxygen electrode, 31-first segment of the snake-shaped channel, 32-second segment of the snake-shaped channel, 33-third segment of the snake-shaped channel, 34-fourth segment of the snake-shaped channel, 35-fifth segment of the snake-shaped channel, 36-sixth segment of the snake-shaped channel, 37-seventh segment of the snake-shaped channel. DETAILED DESCRIPTION
[0028] In order to make the above-mentioned purposes, features and advantages of the utility model more obvious and easy to understand, the specific embodiments of the utility model are described in detail below. In the following description, a lot of specific details are set forth in order to fully understand the utility model. However, the utility model can be implemented in many other ways different from the description herein, and those skilled in the art can make similar improvements without departing from the connotation of the utility model, so the utility model is not limited by the following disclosed specific implementation.
[0029] It should be noted that when an element is referred to as being "fixed" to another element, it can be directly on the other element or there can be intervening elements. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements can be present. The terms "vertical", "horizontal", "left", "right", and the like as used herein are for purposes of illustration and description only and are not intended to be limiting.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the utility model belongs. The terminology used in the description of the utility model herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the utility model.
[0031] Please refer to Figures 1 to 3 The microfluidic chip simulating the myoendothelial junction system in an embodiment comprises a cover glass layer 10 and a body layer 20, and the body layer 20 is arranged on the cover glass layer 10.
[0032] The main body layer 20 is provided with a vascular endothelial cell culture channel 21 and a vascular smooth muscle cell culture channel 22, which are designed side by side and spaced apart from each other. A plurality of microchannels 23 are connected between the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22.
[0033] In this embodiment, the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 are designed as a pair of channels. It can be understood that in other embodiments, a plurality of pairs of vascular endothelial cell culture channels 21 and vascular smooth muscle cell culture channels 22 can be designed on the main body layer 20 for control experiments and the like.
[0034] The vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 are provided with a liquid inlet 24 and a liquid outlet 25, and the liquid inlet 24 and the liquid outlet 25 of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 are designed on the same side, that is, the liquid inlet 24 of the vascular endothelial cell culture channel 21 and the liquid inlet 24 of the vascular smooth muscle cell culture channel 22 are located on one side of the main body layer 20, and the liquid outlet 25 of the vascular endothelial cell culture channel 21 and the liquid outlet 25 of the vascular smooth muscle cell culture channel 22 are located on the other side of the main body layer 20.
[0035] The vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 gradually narrow in width along the direction from the liquid inlet 24 to the liquid outlet 25, and are widest at the liquid inlet 24 end of the two channels and narrowest at the liquid outlet 25 end of the two channels.
[0036] According to the structure and function classification, the blood vessels are in turn conduit vessels, resistance vessels, capillary vessels and capacitance vessels in the direction of blood flow, and the lumen gradually decreases from the conduit vessels to the capillary vessels and gradually increases from the capillary vessels to the various levels of capacitance vessels. The capillary vessels are composed of vascular endothelial cells, and the other three types of vessels are composed of vascular endothelial cells, vascular smooth muscle cells and vascular fibroblasts. In these three types of vessel tissues, the vascular endothelial cells are single-layered, the number of layers of the vascular smooth muscle cells gradually decreases from the conduit vessels to the resistance vessels and gradually increases from the capillary vessels to the heart capacitance vessels, and the number of layers of the vascular smooth muscle cells in the same type of vessel tissue is positively correlated with the diameter of the vessel. In addition, the myoendothelial junction is formed by the contact between the vascular endothelial cells and the vascular smooth muscle cells, and the density thereof is negatively correlated with the diameter of the vessel. Therefore, the culture channel with gradually narrowing width conforms to the change rule of the vessel lumen and the number of layers of the vascular smooth muscle, so that the chip model can better simulate the environment in which the vascular endothelial cells and the vascular smooth muscle cells are located in vivo.
[0037] In an embodiment, the length of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 is 25-60 cm, and the distance between the two channels is 5-30 microns. Among them, the longer cell culture channel can better simulate the structural characteristics of the vascular network and replicate the gradually decreasing oxygen partial pressure trend in the vascular network; the appropriate width of the cell culture channel can ensure the smoothness of the channel while making the number of cultured vascular smooth muscle cells closer to the in vivo vascular tissue. The length of the myoendothelial junction formed by the in vivo vascular endothelial-smooth muscle cells is about 0.5-10 microns. Considering the processing difficulty of the microfluidic chip and the strength of the chip, the distance between the two channels is between 5-30 microns. Preferably, the length of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 is 45-55 cm, and the distance between the two channels is 10.0-20.0 microns.
[0038] On the basis of the above-mentioned embodiments, further, the width of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 is 200-600 microns at one end of the liquid inlet 24, then gradually narrows, and becomes 30-100 microns at one end of the liquid outlet 25. Preferably, the width of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 is 400-500 microns at one end of the liquid inlet 24, then gradually narrows, and becomes 40-60 microns at one end of the liquid outlet 25.
[0039] In an embodiment, the distance between the microchannels 23 gradually shortens along the direction of liquid flow in the vascular endothelial cell culture channel 21, being the largest at one end of the liquid inlet 24 and the smallest at one end of the liquid outlet 25. After the vascular endothelial cells and the vascular smooth muscle cells are co-cultured in the cell culture channel, they will contact and form myoendothelial junctions through the microchannels 23, and the appropriate channel distance is an important feature of the myoendothelial junction system. The distance of the myoendothelial junctions will gradually shorten from one end of the liquid inlet 24 of the vascular endothelial cell culture channel 21 to the smallest at one end of the liquid outlet 25, which can simulate the distribution characteristics of the myoendothelial junction system in vivo, i.e., the density gradually increases from the aorta to the arteriole, the microartery, and the microvein.
[0040] In the present embodiment, the distance between the two adjacent microchannels 23 is 100-500 microns at one end of the liquid inlet 24 of the vascular endothelial cell culture channel 21 and 10-100 microns at one end of the liquid outlet 25. Preferably, the distance between the microchannels 23 is 100-300 microns at one end of the liquid inlet 24 of the vascular endothelial cell culture channel 21 and shortens to 20-50 microns at one end of the liquid outlet 25. The width of the microchannels 23 is 0.5-5.0 microns, the height is 0.5-5.0 microns, and the length is 5.0-30.0 microns.
[0041] Specifically, the width of the microchannel 23 is 2.0 microns, the height is 2.0 microns, and the length is 10.0 microns. The parameters of the microchannel 23 comprehensively consider the size of the myoendothelial junction and the machining precision of the microfluidic chip, and can simulate the in vivo myoendothelial junction system and have operability.
[0042] In an embodiment, two first oxygen electrodes 26 are arranged in the vascular endothelial cell culture channel 21, one near the liquid inlet 24 and one near the liquid outlet 25, for monitoring the oxygen concentration in the channel. Two second oxygen electrodes 27 are arranged in the vascular smooth muscle cell culture channel 22, one near the liquid inlet 24 and one near the liquid outlet 25, for monitoring the oxygen concentration in the channel. The oxygen electrodes arranged near the liquid inlets 24 and liquid outlets 25 of the two culture channels can accurately measure the oxygen partial pressure data in the two culture channels in real time, improving the accuracy of the data and the reliability of the conclusions.
[0043] In an embodiment, the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 extend in a serpentine shape. The vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 are in a serpentine shape, which can meet the length of the channel in a limited area, reduce the size of the microfluidic chip, and save costs.
[0044] Please refer to Figures 5 to 7 In a specific embodiment, the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 are serpentine channels with a length of 55 centimeters and a width gradually decreasing from 500 microns at the liquid inlet 24 end to 50 microns at the liquid outlet 25 end. The width of the first serpentine channel 31 is 500 microns, the width of the second serpentine channel 32 is 500 microns, the width of the third serpentine channel 32 is 400 microns, the width of the fourth serpentine channel 34 is 300 microns, the width of the fifth serpentine channel 35 is 200 microns, the width of the sixth serpentine channel 36 is 100 microns, and the width of the seventh serpentine channel 37 is 50 microns.
[0045] In another embodiment, the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 are serpentine channels with a length of 42 centimeters and a width gradually decreasing from 102 microns at the liquid inlet 24 end to 98 microns at the liquid outlet 25 end. The width of the first serpentine channel 31 is 102 microns, the width of the second serpentine channel 32 is 102 microns, the width of the third serpentine channel 33 is 101 microns, the width of the fourth serpentine channel 34 is 101 microns, the width of the fifth serpentine channel 35 is 100 microns, the width of the sixth serpentine channel 36 is 99 microns, and the width of the seventh serpentine channel 37 is 98 microns.
[0046] Please refer to Figure 3 andFigure 4 In one embodiment, the first section of the serpentine channel 31 at the liquid inlet 24 end of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 is not provided with the microchannels 23, the second section of the serpentine channel 32 is provided with the microchannels 23 at a spacing of 200 microns, the third section of the serpentine channel 33 is provided with the microchannels 23 at a spacing of 150 microns, the fourth section of the serpentine channel 34 is provided with the microchannels 23 at a spacing of 150 microns, the fifth section of the serpentine channel 35 is provided with the microchannels 23 at a spacing of 100 microns, the sixth section of the serpentine channel 36 is provided with the microchannels 23 at a spacing of 50 microns, and the seventh section of the serpentine channel 37 is provided with the microchannels 23 at a spacing of 50 microns.
[0047] In another embodiment, the first section of the serpentine channel 31 at the liquid inlet 24 end of the vascular endothelial cell culture channel 21 and the vascular smooth muscle cell culture channel 22 is not provided with the microchannels 23, the second section of the serpentine channel 32 is provided with the microchannels 23 at a spacing of 100 microns, the third section of the serpentine channel 33 is provided with the microchannels 23 at a spacing of 100 microns, the fourth section of the serpentine channel 34 is provided with the microchannels 23 at a spacing of 50 microns, the fifth section of the serpentine channel 35 is provided with the microchannels 23 at a spacing of 50 microns, the sixth section of the serpentine channel 36 is provided with the microchannels 23 at a spacing of 20 microns, and the seventh section of the serpentine channel 37 is provided with the microchannels 23 at a spacing of 20 microns.
[0048] The microfluidic chip simulating the myoendothelial junction system provides an in vitro model for the formation mechanism and function of the myoendothelial junction system, fills the gap in the field, and enables the research to be carried out in advance at the in vitro level.
[0049] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.
Claims
1. A microfluidic chip simulating an endothelial-myocyte junction system, characterized by: The cover glass layer and the main body layer are bonded, the main body layer is provided with a vascular endothelial cell culture channel and a vascular smooth muscle cell culture channel, the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel are provided with a same side liquid inlet and a liquid outlet, and a plurality of microchannels are connected between the channels, The distribution mode of the microchannels is that the interval of the microchannels gradually shortens along the direction from the liquid inlet to the liquid outlet, is 100-500 microns at one end of the liquid inlet, and is shortened to 10-100 microns at one end of the liquid outlet.
2. The microfluidic chip simulating an endothelial cell junction system according to claim 1, wherein, The width of the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel gradually narrows along the direction of liquid flow in the channel, is 200-600 microns at one end of the liquid inlet, and is narrowed to 30-100 microns at one end of the liquid outlet.
3. The microfluidic chip simulating an endothelial cell-connection system according to claim 1, wherein, The length of the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel is 25-60 centimeters, and the distance between the two channels is 5.0-30.0 microns.
4. The microfluidic chip simulating an endothelial cell-connection system according to claim 1, wherein, The interval of the microchannels is 100-300 microns at one end of the liquid inlet of the vascular endothelial cell culture channel, and is shortened to 20-50 microns at one end of the liquid outlet.
5. The microfluidic chip simulating an endothelial cell-connection system according to claim 2, wherein, The width of the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel is 400-500 microns at one end of the liquid inlet, and is narrowed to 40-60 microns at one end of the liquid outlet.
6. The microfluidic chip simulating an endothelial cell-connection system according to claim 3, wherein, The length of the vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel is 45-55 centimeters, and the distance between the two channels is 10.0-20.0 microns.
7. The microfluidic chip simulating an endothelial cell-connection system according to claim 1, wherein, The width of the microchannels is 0.5-5.0 microns, the height is 0.5-5.0 microns, and the length is 5.0-30.0 microns.
8. The microfluidic chip simulating an endothelial cell- muscle cell junction system according to claim 7, wherein, The width of the microchannels is 2.0 microns, the height is 2.0 microns, and the length is 10.0 microns.
9. The microfluidic chip simulating an endothelial cell-connection system according to claim 1, wherein, The vascular endothelial cell culture channel is provided with two first oxygen electrodes for monitoring the oxygen concentration in the channel, and the vascular smooth muscle cell culture channel is provided with two second oxygen electrodes for monitoring the oxygen concentration in the channel.
10. The microfluidic chip simulating an endothelial cell-connection system according to claim 1, wherein, The vascular endothelial cell culture channel and the vascular smooth muscle cell culture channel extend in a serpentine zigzag shape.