CN114260032A - Micro-fluidic chip platform for establishing in-vitro vascular physiology model - Google Patents
Micro-fluidic chip platform for establishing in-vitro vascular physiology model Download PDFInfo
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Abstract
The invention relates to the technical field of microfluidic chips, in particular to a microfluidic chip platform for establishing an in vitro vascular physiology model, which comprises a perfusion liquid tank, a microfluidic peristaltic pump, a substrate and a waste liquid pipe, wherein the perfusion liquid tank, the microfluidic peristaltic pump, the substrate and the waste liquid pipe are sequentially connected with one another through hoses to form a closed pipeline; the substrate includes at least one accommodation space that is used for holding external blood vessel, and sets up on the substrate and all with at least a pair of inlet and liquid outlet that accommodation space is linked together, all be provided with the connector in inlet and the liquid outlet, the one end of connector is used for inserting external blood vessel's tip is in order to prop straight external blood vessel, the other end with the hose links to each other. The microfluidic chip platform provided by the invention can be used for constructing a true blood vessel in-vitro model and can be used as a basic research model of vascular lesions.
Description
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a microfluidic chip platform for establishing an in vitro vascular physiology model.
Background
Microfluidic chips, i.e. microfluidic chip laboratories. The micro-fluidic chip is built with units with proper sizes, and can accommodate molecules, cells, even bionic tissues or organs at the same time. The chip has a special and convenient control system, so that the chip can simultaneously regulate and control physical, chemical and biological parameters and environment of an experiment. Therefore, for twenty-first century, microfluidic chips have become the best platform for biomimetic and precise manipulation of mammalian cells in vitro.
The microfluidic technology has the characteristics of miniaturization, integration and low consumption, and can accurately control a plurality of system parameters, such as chemical concentration gradient, fluid shear force, construction of cell graphical culture, interaction of a tissue-tissue interface and an organ-organ, and the like, so that the complex structure, microenvironment and physiological functions of a human organ are simulated, a plurality of defects of a traditional two-dimensional cell culture mode and an animal experiment are overcome, and the microfluidic technology can become a bionic, efficient and energy-saving physiological research, disease model and drug development tool. The establishment of an in vitro physiological model requires consideration of the authenticity of the external environment parameters. The organ chip technology generated by combining the microfluidic technology with micromachining and cell biology has the capability that other technologies are not comparable in the control of external environment parameters, and cells can respond to physical and chemical stimuli such as fluid shear force, mechanical stress and biochemical concentration gradient to perform self-assembly and show more real physiological functions by generating the physical and chemical stimuli, so that the organ chip technology has special advantages in the establishment of in vitro physiological models.
Vascular disorders, especially diabetic foot, are one of the serious chronic complications leading to disability and death in diabetic patients. The traditional treatment means comprises medicines, vascular bypass, interventional operation and the like, but the clinical effect is poor, and at least 30-40% of diabetic foot patients have to receive amputation operation due to poor microcirculation of lower limbs and complete occlusion of small blood vessels and can not perform surgical revascularization operation. However, most diabetic foot patients are not treated by surgery because of their poor physical fitness, poor cardiopulmonary function and many basic diseases. Therefore, it is a necessary trend to open new therapeutic approaches. The pathological basis of diabetic peripheral arterial lesions is atherosclerosis, severe lesions develop ulcers, bleed and induce thrombosis, resulting in lumen occlusion, which is one of the leading causes of diabetic foot.
However, the existing blood vessel models based on the microfluidic chip platform are simulated blood vessels, and the simulated blood vessels are difficult to be used as physiological models for researching vascular lesions.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: provides a micro-fluidic chip platform capable of constructing a true blood vessel model.
In order to solve the technical problems, the invention provides a micro-fluidic chip platform for establishing an in vitro vascular physiology model, which comprises a perfusion liquid tank, a micro-fluidic peristaltic pump, a substrate and a waste liquid pipe, wherein the perfusion liquid tank, the micro-fluidic peristaltic pump, the substrate and the waste liquid pipe are sequentially connected with one another through hoses to form a closed pipeline;
the substrate includes at least one accommodation space that is used for holding external blood vessel, and sets up on the substrate and all with at least a pair of inlet and liquid outlet that accommodation space is linked together, all be provided with the connector in inlet and the liquid outlet, the one end of connector is used for inserting external blood vessel's tip is in order to prop straight external blood vessel, the other end with the hose links to each other.
The invention has the beneficial effects that: the microfluidic chip platform for establishing the in vitro vascular physiological model provided by the invention can be used for establishing a true vascular model and simulating arterial blood flow through the microfluidic peristaltic pump, and can be used as a basic research model of vascular lesions. The whole process of occurrence and development of arterial lesions can be dynamically observed by micro-infusing whole blood into the micro-fluidic chip platform, so that a foundation can be provided for discussing a vascular lesion mechanism, and later-stage drug detection and the like.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip platform according to an embodiment of the present invention;
FIG. 2 is a schematic view of a substrate according to an embodiment of the present invention;
FIG. 3 is a partial photograph of a microfluidic chip platform according to an embodiment of the present invention;
FIG. 4 is a photograph showing a 100 magnification of HE staining of blood vessels of a control group according to an embodiment of the present invention;
FIG. 5 is a photograph showing a 400 magnification of HE staining of blood vessels of a control group according to an embodiment of the present invention;
FIG. 6 is a photograph showing the HE staining at 200 times magnification in 96h culture of the blood vessels of the experimental group according to the present invention;
FIG. 7 is a photograph showing the HE staining at a magnification of 800 times in 96h culture of the blood vessels of the experimental group in the embodiment of the present invention;
FIG. 8 is a photograph showing a control group of blood vessels treated by immunohistochemical SP method at a magnification of 200 times in accordance with an embodiment of the present invention;
FIG. 9 is a photograph showing a 400 Xmagnification of blood vessels of a control group treated by immunohistochemical SP method according to an embodiment of the present invention;
FIG. 10 is a photograph showing the results of immunohistochemical SP method treatment on 96h cultured blood vessels in the experimental group according to the present invention at a magnification of 200 times;
FIG. 11 is a photograph showing the magnification of 800 times of the immunohistochemical SP method treatment in the culture of 96h blood vessels in the experimental group according to the present invention;
FIG. 12 is a photograph showing a positive control blood vessel sample treated with TUNEL at 800 times magnification according to an embodiment of the present invention;
FIG. 13 is a photograph showing a control group of blood vessels treated by TUNEL at a magnification of 200 times in accordance with an embodiment of the present invention;
FIG. 14 is a photograph showing the TUNEL method at a magnification of 200 times in 96h of blood vessels in an experimental group according to an embodiment of the present invention;
FIG. 15 is a photograph showing the TUNEL method in 96h culture of the blood vessels of the experimental group according to the present invention at 400 magnification.
Description of reference numerals: 1. a perfusion liquid tank; 2. a microfluidic peristaltic pump; 3. a substrate; 31. a base layer; 32. a cover plate layer; 4. a waste liquid tank; 5. a hose; 6. a connector; 7. an accommodating space; 8. an incubator; 9. an extracorporeal blood vessel; 10. a liquid inlet; 11. and a liquid outlet.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The vascular lesions in the invention are particularly atherosclerosis, hemostasis and thrombosis, inflammatory vascular diseases, tumor metastasis through blood vessels and the like, and are directly related to the shear force generated between the blood vessel wall and the blood flow. Therefore, the in vitro construction of a pathology research model of true blood vessels is of great significance. Compared with the traditional pathology research model with simulated blood vessels, the pathology research model for constructing the true blood vessels has the unique advantage of dynamically observing the whole processes of the occurrence and development of the vascular lesions, and can provide a physiological basis for later-stage medication and medicinal value evaluation.
Therefore, referring to fig. 1 to 3, the present invention provides a microfluidic chip platform for establishing a physiological model of an extracorporeal blood vessel 9, comprising a perfusion liquid tank 1, a microfluidic peristaltic pump 2, a substrate 3 and a waste liquid pipe, which are connected to each other sequentially through a hose 5 to form a closed pipeline; substrate 3 includes at least one accommodation space 7 that is used for holding external blood vessel 9, and sets up substrate 3 go up and all with at least a pair of inlet 10 and liquid outlet 11 that accommodation space 7 is linked together, all be provided with connector 6 in inlet 10 and the liquid outlet 11, the one end of connector 6 is used for inserting the tip of external blood vessel 9 is in order to prop straight external blood vessel 9, the other end with hose 5 links to each other.
The micro-fluidic peristaltic pump 2 is used as a basis for simulating vascular hydrodynamics, and the flexible pipe 5 is extruded through the pulley of the micro-fluidic peristaltic pump to simulate the flow of blood in a blood vessel and reproduce arterial blood flow. Meanwhile, the pulley rotating speed of the microfluidic peristaltic pump 2 can be adjusted to change the perfusion volume, perfusion time and perfusion pressure in real time, adjust the relevant vascular lesion influence factors such as vascular shearing force and the like, and allow the research on the formation and development of various vascular lesions. The microfluidic chip platform of the in vitro blood vessel 9 physiological model is formed by combining the microfluidic peristaltic pump 2 and the substrate 3, and fluid (perfusate) is only contacted with the inner hose 5 and the outer hose 5 of the microfluidic peristaltic pump 2 and is not contacted with other structures of the microfluidic peristaltic pump 2, so that fluid pollution can be effectively avoided, and the microfluidic chip platform has the advantages of high repeatability precision and stability precision, good self-priming capability, capability of idling or avoiding fluid backflow and the like.
The substrate 3 has at least one receiving space 7 for receiving an extracorporeal blood vessel 9. The number of the accommodating spaces 7 may be one or more, and when the technical scheme of the plurality of accommodating spaces 7 is adopted, the arrangement mode of the plurality of accommodating spaces 7 may be a single-row mode or an array mode. The receiving space 7 may be formed entirely inside the substrate 3 or formed on the surface of the substrate 3.
Each receiving space 7 should have at least one pair of inlet 10 and outlet 11 ports in it to allow perfusion fluid to be pumped from the perfusion fluid tank 1 through the microfluidic peristaltic pump 2 into the blood vessel located in the receiving space 7 and through the outlet 11 into the waste fluid tank 4. Meanwhile, each accommodating space 7 also allows a plurality of pairs of liquid inlets 10 and liquid outlets 11, and different distances exist between the plurality of pairs of liquid inlets 10 and liquid outlets 11, so as to allow the blood vessels to be connected with the different liquid inlets 10 and liquid outlets 11 through the connectors 6, thereby realizing the application of the blood vessels with different lengths.
In order to maintain the sealing between the end of the external blood vessel 9 and the connector 6 in the actual operation process, a sterile line can be used for ligation, or the connector 6 with a spherical end can be used for sealing through the expansion action of the external blood vessel 9 and the spherical end.
It should be noted that, in order to avoid the toxic effect on the external blood vessel 9 during the experiment, the components should be made of medical grade materials or sterile non-toxic materials, such as medical grade peristaltic pump silicone tubes and standard connectors 6 which are sterilized and disinfected under strict high pressure. The medical peristaltic pump silicone tube should have high transparency so as to observe the flow of the perfusion fluid in the hose 5.
The perfusion fluid tank 1 is used for storing perfusion fluid, which may be whole blood, or a blood product subjected to specific processing, such as a blood product added with a specific drug or specific components (lipid, sugar, etc.), or reduced by certain specific components (lipid, sugar, etc.), or a specific cell culture medium, such as a DEME medium, so as to simulate lesion formation and development of blood vessels under specific blood conditions.
In one embodiment, the substrate 3 is further provided with a culture window, which is in communication with the receiving space 7. The culture window is arranged on the substrate 3 and communicated with the accommodating space 7 to form a groove for accommodating an extracorporeal blood vessel 9 on the surface of the substrate 3. The culture window is convenient for the experimenter to observe the external blood vessel 9, and is also convenient for the experimenter to embed the external blood vessel 9 into the substrate 3 and to insert the end part of the external blood vessel 9 into one end of the connector 6 and ligate.
The substrate 3 has at least one gas outlet hole communicating with the receiving space 7. The air outlet hole is not only used to balance the air pressure outside the accommodating space 7 and the substrate 3, but also to promote the circulation of air inside the accommodating space 7.
Preferably, the material of the substrate 3 is polydimethylsiloxane.
It should be noted that the substrate 3 may be injection molded by using hydrogel as well as polydimethylsiloxane.
In one embodiment, referring to fig. 2, the substrate 3 includes a substrate layer 31 and a cover plate layer 32, which are stacked, the substrate layer 31 and the cover plate layer 32 are stacked, the viewing window is disposed on the surface of the cover plate layer 32, and the substrate layer 31 and the cover plate layer 32 are connected by a plasma oxidation method. Specifically, the substrate 3 is formed by photoetching and casting a substrate layer 31 and a cover plate layer 32, then the substrate layer 31 and the cover plate layer 32 are respectively cut by a hollow tube cutting method and a pressing puncher so as to form at least one pair of a liquid inlet 10 and a liquid outlet 11 and at least one air outlet, and then the substrate layer 31 and the cover plate layer 32 are compounded, irradiated by plasma for several seconds and rapidly pressed and thermally set so as to form the substrate 3. The substrate layer 31 and the cover plate layer 32 are connected by a plasma oxidation method, so that the connectivity between the substrate layer and the cover plate layer can be effectively improved, and the overall mechanical strength of the substrate 3 is improved. Before use, the substrate 3 should be strictly sterilized at high pressure and high temperature, specifically, the accommodating space 7, the liquid inlet 10, the liquid outlet 11 and the air outlet in the substrate 3 are cleaned by isopropanol, and then the substrate is cleaned by ultrasonic waves and then is thoroughly sterilized by using an autoclave.
Further, the microfluidic chip platform further comprises an incubator 8, and the substrate 3 is positioned in the incubator 8 so as to maintain the substrate 3 in a working environment with a temperature of 37 ℃ and a carbon dioxide concentration of 5%. The culture condition of the in vitro blood vessel 9 is strictly controlled by the incubator 8, so that the reduction of the experiment precision of the microfluidic chip platform caused by the change of the culture environment condition is avoided.
Example 1
Referring to fig. 1 to 3, the microfluidic chip platform for establishing a physiological model of an extracorporeal blood vessel 9 comprises a perfusion liquid tank 1, a microfluidic peristaltic pump 2, a substrate 3 and a waste liquid pipe, which are sequentially connected with each other through a hose 5 to form a closed pipeline;
the substrate 3 comprises at least one accommodating space 7 for accommodating an external blood vessel 9, and a pair of a liquid inlet 10 and a liquid outlet 11 which are arranged on the substrate 3 and communicated with the accommodating space 7, wherein connectors 6 are arranged in the liquid inlet 10 and the liquid outlet 11, one end of each connector 6 is used for being inserted into the end part of the external blood vessel 9 to straighten the external blood vessel 9, and the other end of each connector is connected with the hose 5;
the substrate 3 is also provided with a culture window which is communicated with the accommodating space 7;
the substrate 3 has an air outlet hole communicating with the accommodating space 7;
the substrate 3 is made of polydimethylsiloxane;
the substrate 3 comprises a base layer 31 and a cover plate layer 32 which are arranged in a stacked mode, wherein the base layer 31 is connected with the cover plate layer 32 through a plasma oxidation method;
the microfluidic chip platform further comprises an incubator 8, and the substrate 3 is positioned in the incubator 8 so as to keep the substrate 3 at 37 ℃ in a working environment with 5% carbon dioxide concentration.
Detection example 1
The method comprises the steps of taking a white New Zealand rabbit and a Thailand piglet as samples to obtain arteries, taking blood vessels with the caliber of about 1mm at the lower limb arteries and carotid arteries of animals, trimming two ends of the blood vessels, removing necrotic tissues and clotted blood, immediately implanting the blood vessels into a low-temperature whole-cell nutrient solution containing antibiotics, and flushing for 3 times to remove necrotic parts. The sample blood vessels are divided into 1-1.5 cm long sections, and are implanted into an incubator at 37 ℃ and 5% carbon dioxide concentration for later use as experimental blood vessels (experimental group) and a control group (no treatment).
Referring to fig. 3, the blood vessel for experiment is placed in the microfluidic chip, the end of the blood vessel is sleeved on the connector by using the autoclaved forceps and tweezers, and the two ends of the blood vessel are ligated by using a sterile line to ensure that the perfusate flows smoothly and does not leak.
In the experiment, the perfusate of the experimental group adopts DMEM culture medium containing 10% of fetal bovine serum, 1% of penicillin and streptomycin and 5% of serum of the corresponding group, and is filtered by a 0.22 mu m filter head before use.
Observing and cleaning the microfluidic chip every 12h, respectively taking 24h perfusate, 48h perfusate, 72h perfusate and 96h perfusate for freezing and storing at minus 80 ℃, changing the perfusate every 48h, taking blood vessels every 96h, fixing in formaldehyde fixing solution, slicing, and respectively carrying out HE dyeing, immunohistochemical experiment by using SP method and TUNEL detection on the blood vessels, wherein experimental pictures are respectively shown in figures 4 to 15.
Fig. 4 shows a 100-fold magnification photograph of control group vessels under HE staining. In the figure, the blood vessel is composed of 3 layers of structures, the inner membrane, the middle layer (muscular layer) and the outer membrane (trophoblast) are bounded by the inner elastic plate and the outer elastic plate, and the tissue structure is clear and complete. Figure 5 shows a 400-fold magnification photograph of control vessels under HE staining. The inner vessel endothelium is clearly visible and intact, the inner elastic plate is in a wave-fold shape, the middle layer of multiple layers of smooth muscle cells annularly surround the lumen, and a few elastic fibers are visible in the middle layer of the inner elastic plate.
Fig. 6 shows a 200-fold magnification photograph of HE staining of experimental group vessels under microfluidic chip platform culture for 96 h. The three layers of tissue structures in the figure are complete and clear, the cells in each layer are well arranged, and the cell structures are not obviously abnormal. Fig. 7 shows an 800-fold magnification photograph of HE staining of experimental group vessels under microfluidic chip platform culture for 96 h. The vascular endothelial cells are clearly visible in the figure, the inner elastic plate is wavy, and the structure of the medial smooth muscle cells is normal.
FIG. 8 shows a photograph at 200 times magnification of blood vessels of a control group treated by the immunohistochemical SP method. In the figure, the staining of the antibody to vascular smooth muscle alpha-actin shows that the medial smooth muscle cells in the blood vessels have positive reaction. FIG. 9 shows a photograph at 400 times magnification of blood vessels of a control group treated by the immunohistochemical SP method. In the figure, when the antibody of the vascular smooth muscle alpha-actin is stained, the medial smooth muscle cells of the blood vessels show positive reaction, and the vascular endothelium and the adventitial stroma show negative reaction.
Fig. 10 shows a 200-fold photograph of experimental group blood vessels treated by immunohistochemical SP method under microfluidic chip platform culture for 96 h. In the figure, the antibody staining to the antibody alpha-actin of the medial smooth muscle of the blood vessel shows that the medial smooth muscle has positive reaction. FIG. 11 shows 800-fold photographs of experimental group blood vessels treated by immunohistochemical SP method under microfluidic chip platform culture for 96 h. In the figure, when the antibody of alpha-actin is stained on the medial smooth muscle, it can be seen that the medial smooth muscle is positively reacted, and the vascular endothelial cell is clearly seen and negatively reacted.
Figure 12 shows a 800-fold magnification photograph of a positive control blood vessel sample treated by the TUNEL method. When the positive control blood vessel sample was treated with DNase1, it was found that the medial smooth muscle cells were mostly positive by TUNEL staining. FIG. 13 shows a photograph of a control group of blood vessels treated by TUNEL at 200 times magnification. In the figure, it can be seen that the vascular endothelial cells and the medial smooth muscle cells are mostly negative when stained by TUNEL method.
FIG. 14 shows a 200-fold photograph of experimental group vessels treated by TUNEL method under microfluidic chip platform culture for 96 h. It can be seen that vascular endothelial cells, medial smooth muscle cells and adventitial interstitium were mostly negative when stained by TUNEL method. FIG. 15 shows a 400-fold photograph of experimental group vessels treated by TUNEL method under microfluidic chip platform culture for 96 h. In the figure, the TUNEL method staining of vascular endothelial cells and medial smooth muscle cells is mostly negative.
Therefore, the activity (pulsation), tissue structure and cell morphology of the blood vessels of the experimental group cultured by the microfluidic chip platform at different time have no obvious difference compared with the blood vessels of the control group; immunohistochemical staining shows that the blood vessels of the experimental group at different periods show positive reaction of the alpha-actin of the smooth muscle cells; the medial smooth muscle cells of each artery have a certain proliferation capacity (a small part of cells proliferate to have positive nuclear antigen staining); the vascular wall cells showed no apparent apoptotic features (TUNEL staining negative).
Based on the above experiments, it is shown that the blood vessels of the experimental group are not obviously different from the blood vessels of the control group in morphology and function, that is, the micro-fluidic chip platform for establishing an in vitro blood vessel physiological model provided by the invention can be effectively used as a physiological model of vasculopathy, and has important significance for researching the vasculopathy.
In conclusion, the microfluidic chip platform for establishing the in vitro vascular physiological model provided by the invention can be used for establishing a true vascular model and simulating arterial blood flow through the microfluidic peristaltic pump, and can be used as a basic research model of vascular lesions. The whole process of occurrence and development of arterial lesions can be dynamically observed by micro-infusing whole blood into the micro-fluidic chip platform, so that a foundation can be provided for discussing a vascular lesion mechanism, and later-stage drug detection and the like.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (6)
1. The microfluidic chip platform is used for establishing an in vitro vascular physiology model and is characterized by comprising a perfusion liquid tank, a microfluidic peristaltic pump, a substrate and a waste liquid pipe which are sequentially connected with one another through a hose to form a closed pipeline;
the substrate includes at least one accommodation space that is used for holding external blood vessel, and sets up on the substrate and all with at least a pair of inlet and liquid outlet that accommodation space is linked together, all be provided with the connector in inlet and the liquid outlet, the one end of connector is used for inserting external blood vessel's tip is in order to prop straight external blood vessel, the other end with the hose links to each other.
2. The microfluidic chip platform for establishing a model of in vitro vascular physiology according to claim 1, wherein the substrate is further provided with a culture window, the culture window being in communication with the receiving space.
3. The microfluidic chip platform for modeling in vitro vascular physiology of claim 1, wherein the substrate has at least one gas outlet hole in communication with the receiving space.
4. The microfluidic chip platform according to claim 1, wherein the substrate is polydimethylsiloxane.
5. The microfluidic chip platform for creating in vitro vascular physiology models according to claim 1, wherein the substrate comprises a substrate layer and a cover plate layer arranged in a stack, and the substrate layer and the cover plate layer are connected by plasma oxidation.
6. The microfluidic chip platform for creating a model of in vitro vascular physiology according to claim 1, further comprising an incubator in which the substrate is positioned to maintain the substrate in a working environment of 37 ℃ and 5% carbon dioxide concentration.
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