CN209778896U

CN209778896U - Micro-fluidic chip for evaluating in vitro intravascular stent

Info

Publication number: CN209778896U
Application number: CN201920055693.2U
Authority: CN
Inventors: 徐紫宸; 王贵学
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2019-01-14
Filing date: 2019-01-14
Publication date: 2019-12-13
Anticipated expiration: 2029-01-14

Abstract

The utility model belongs to the technical field of micro-fluidic chip, concretely relates to external vascular support evaluates and uses micro-fluidic chip. The micro-fluidic chip mainly comprises a sample adding valve 1, a circulating pulsation micro-pump 6 and a cell culture pool 4. The micro-fluidic chip can reduce the time cost and the consumption of raw material cost for evaluating the experimental requirements, ensure the identity of the experiment under multiple conditions and ensure the comparability of the experimental results; the adopted ion exchange membrane effectively shields the interference of other ions while ensuring that the concentration of cations in a cell culture area is not changed, and realizes the tightness in the experimental process, so that the experimental reaction and the result detection are integrated, and the method is convenient and fast.

Description

Micro-fluidic chip for evaluating in vitro intravascular stent

Technical Field

The utility model relates to a micro-fluidic chip technical field, concretely relates to external vascular support evaluates and uses micro-fluidic chip.

Background

The prevalence rate of cardiovascular diseases is rising year by year, and according to the aging situation and population growth rate of the population in China, the prevalence rate of cardiovascular diseases in China is predicted to increase to 50% in 2030, and the severity and universality of cardiovascular diseases are not neglected. One of the most important treatment modalities for cardiovascular diseases is intervention, in which the implantation of vascular stents is the most common means in intervention. The research and development of new vascular stents more suitable for the complex internal environment of human bodies have great significance for relieving the incidence of the increasingly severe cardiovascular diseases.

For the relevant study of vascular stents,Evaluating the distance between the stent and the blood flow and the vascular tissue after implantation in a blood vessel The interaction effect is a very critical ring, which is related to the incidence of clinical adverse events after stent implantation and the late heart after surgery The recurrence rate of vascular disease ensures that the stent can perform the therapeutic action as expected and does not produce other fatal later-period side effects The application is as follows.In addition, the research on the force-biology mechanism is also an important thrust for detecting the occurrence and the development of cardiovascular diseases, and the optimal intervention scheme can be constructed by carrying out specific modification and modification on the stent or a stent coating through the related mechanism.

currently, relevant evaluation studies are often limited to animal model testing. The animal experiment has many limiting factors, such as the control of the physiological state and the interventional operation condition of the animal,The cost for raising experimental animals and constructing animal models, etc., Also has the disadvantages of long preparation period, complicated preparation procedure, etc. However, the evaluation test of the stent is indispensable.

Therefore, in order to enable researchers to complete the evaluation experiment of the intravascular stent quickly and conveniently, and to accelerate the research process of the intravascular stent and solve the treatment problem of the closed coronary artery disease, it is necessary to construct a microfluidic circulation cavity device for performing the related evaluation experiment in vitro by using the microfluidic technology.

SUMMERY OF THE UTILITY MODEL

The to-be-solved technical problem of the utility model is to provide an external blood vessel support evaluates and uses micro-fluidic chip to reduce the time cost of experiment demand and the consumption of raw materials cost, ensure the identity of experiment under the multi-condition, ensure the comparability of experimental result.

In order to solve the above problem, the utility model adopts the following scheme:

A micro-fluidic chip for evaluating an in vitro intravascular stent mainly comprises a sample adding valve 1, a circulating pulsation micro pump 6 and a cell culture pool 4.

Further, the chip is circular or rectangular, and 2 cylindrical sample adding valves 1 which are arranged side by side are processed at the center of the chip; each cylindrical sample adding valve 1 is provided with 2 opposite openings on the same section diameter line, and each opening is respectively connected with 1 sample adding pipe 2; each sample inlet pipe 2 is respectively communicated with one cell culture pond 4, and 4 cell culture ponds 4 are formed in total and are communicated with a disc-shaped channel 3 formed at the bottom in each cell culture pond 4.

2 sample ports 8 are processed in the middle of the chip between 2 side-by-side circular sample adding valves 1, sample channels 9 are processed between the 2 sample ports 8 and are communicated with each other, and stop valves 11 are processed on the communicated sample channels 9; the 2 sample ports 8 are also respectively provided with 2 sample channels 9 which are respectively communicated with the 2 cell culture pools 4.

The sampling tube 2 is respectively provided with 1 one-way circulating tube 10 beside 2 sample adding valves 1 and is communicated with the sampling tubes 2 at two ends of the sample adding valves 1; each one-way circulation pipe 10 is provided with a stop valve 11.

The cylindrical interior of the sample adding valve 1 is a valve body 16, an upper opening is processed in the valve body 16, and an inner valve channel 14 with the size of an end head matched with the sample inlet pipe 2 is formed in the valve body 16; the valve body 16 is provided with a valve knob 15 at the top.

further, the 4 cell culture ponds 4 are uniformly distributed at four corners of the rectangular chip or at four symmetrical edges of the circular chip; 1 circulating pulsation micropump 6 is arranged between a pair of cell culture ponds 4, the circulating pulsation micropump 6 is communicated in series between 2 circulating pipes 5, and the 2 circulating pipes 5 are respectively communicated with the disc-shaped channels 3 in the cell culture ponds 4 at two sides of the circulating pulsation micropump 6.

Further, the circulation tube 5 and the sampling tube 2 are communicated with the disk-shaped channel 3 at the T-shaped port 17 on the side of the cell culture pond 4.

Further, the disc-shaped channel 3 is coiled to the center of the cell culture pool 4 along the bottom surface of the cell culture pool 4 and is communicated with a bracket experiment tube 7 which is processed and fixed at the center; the bracket experiment tube 7 is a hollow tube which is made of elastic materials and is higher than the cell culture pool 4, and a sampling plug 13 is arranged at the opening of the top end of the bracket experiment tube; the sampling plug 13 is internally provided with a pressure sensing probe.

The sampling plug 13 can penetrate the sampling needle for sampling, the diameter of the experimental tube 7 has different specifications, and the vascular stent 12 with different diameters can be attached to the sleeve. The pressure sensing probe in the sampling plug 13 can transmit pressure signals and intervals generated by the circulating pulsation micropump 6 in the bracket experiment tube 7 to a display.

Furthermore, all the sample inlet pipes 2, the disc-shaped channels 3, the circulating pipe 5, the sample channel 9, the one-way circulating pipe 10 and the bracket experiment pipe 7 of the chip are sealed pipelines, so that all the pipelines can be guaranteed to be subjected to pressure generated by the circulating pulsation micropump 6.

The to-be-solved technical problem of the present invention is to provide a disc-shaped channel 3 of the microfluidic chip for in vitro intravascular stent evaluation.

The disk-shaped channel 3 is sealed by an ion exchange membrane, so that the ion solution in the disk-shaped channel 3 and the solution in the cell culture pool 4 can not be mixed; the middle of the disk-shaped channel 3 is provided with a pipeline partition wall 19, the disk-shaped channel 3 is divided into 2 disk-shaped double small pipes 18 from the T-shaped interface 17, and the reagent in the disk-shaped channel 3 can be ensured to circulate under the condition that the strip circulation pipe 5 and the sample inlet pipe 2 are in circulation communication.

The ion exchange membrane is adopted for sealing, so that the ion reagent in the disc-shaped channel 3 can be ensured to permeate into the cell culture pool 4 due to concentration difference, and the ion concentration of the cell culture pool 4 is kept constant. On one hand, the environment state in the chip, especially in the disc-shaped channel, can be stabilized, and the pH value does not fluctuate greatly; on the other hand, in the case of pathological environment simulation, there is also a stabilizing effect, such as simulation of an in vivo state of increased calcium ion level caused by inflammation.

The disk-shaped channel 3 can transmit the pressure generated by the received cyclic pulsation micropump 6 and can add some reagents capable of passing through the ion exchange membrane.

The beneficial effects of the utility model reside in that:

1) The micro-fluidic chip for evaluating the in vitro intravascular stent can reduce the time cost and the consumption of raw material cost required by evaluation experiments, overcome the feeding and animal model construction costs of experimental animals and the like, and has the defects of long preparation period, complicated preparation procedure and the like;

2) The microfluidic chip for evaluating the in vitro intravascular stent can ensure the identity of experiments under multiple conditions and ensure the comparability of experimental results;

3) The ion exchange membrane adopted by the microfluidic chip for evaluating the in vitro intravascular stent effectively shields the interference of other ions while ensuring the concentration of cations in a cell culture area to be unchanged, realizes the tightness in the experimental process, integrates the experimental reaction and result detection, and is convenient and quick.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the technical solutions in the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

Fig. 1 is a schematic front view of a rectangular chip.

Fig. 2 is a schematic front view of a circular chip.

FIG. 3 is a schematic partial cross-sectional view of a cell culture tank.

fig. 4 is a schematic view of a front view structure of a sample injection valve in an open state.

Fig. 5 is a schematic top view of a sample injection valve in an open state.

Fig. 6 is a schematic view of a front view of a sample adding valve in a closed state.

Fig. 7 is a schematic top view of a sample injection valve in a closed state.

FIG. 8 is a partial schematic view of a T-port of a cell culture well.

Fig. 9 is an SEM scan of the surface of different coatings.

FIG. 10 shows the results of experiments on smooth muscle cell proliferation on the surface of each set of coated stents.

in the figure: 1. a sample adding valve; 2. a sample inlet pipe; 3. a disk-shaped channel; 4. a cell culture pond; 5. a circulation pipe; 6. a circulating pulsation micropump; 7. a bracket test tube; 8. a sample port; 9. a sample channel; 10. a one-way circulation pipe; 11. a stop valve; 12. a vascular stent; 13. a sampling plug; 14. an in-valve passage; 15. a valve knob; 16. a valve body; a T-type interface; 18. a disc-shaped double small tube; 19. pipeline partition wall.

Detailed Description

In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following description is given in conjunction with the embodiments and the accompanying drawings.

The technical solution of the present invention will be described clearly and completely with reference to the accompanying drawings, and obviously, the described embodiments are some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention. In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplification of description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

Example 1A microfluidic chip for in vitro intravascular stent evaluation

A microfluidic rectangular chip for evaluating an in vitro intravascular stent is mainly composed of a sample adding valve 1, a circulating pulsation micropump 6 and a cell culture pool 4, wherein 2 parallel cylindrical sample adding valves 1 are processed at the center positions of the sample adding valves, each cylindrical sample adding valve 1 is processed with 2 opposite openings on the same section diameter line, and each opening is respectively connected with 1 sample adding tube 2; each sample inlet pipe 2 is respectively communicated with one cell culture pond 4, and 4 cell culture ponds 4 are formed in total and are communicated with a disc-shaped channel 3 formed at the bottom in each cell culture pond 4.

The 4 cell culture ponds 4 are uniformly distributed on four symmetrical edges of the circular chip; 1 circulating pulsation micropump 6 is arranged between a pair of cell culture ponds 4, the circulating pulsation micropump 6 is communicated in series between 2 circulating pipes 5, and the 2 circulating pipes 5 are respectively communicated with the disc-shaped channels 3 in the cell culture ponds 4 at two sides of the circulating pulsation micropump 6; the circulating pipe 5 and the sample injection pipe 2 are communicated with the disc-shaped channel 3 at a T-shaped interface 17 at the side of the cell culture pond 4; the middle of the disc-shaped channel 3 is provided with a pipeline partition wall 19, and the disc-shaped channel 3 is divided into 2 disc-shaped double small pipes 18 from the T-shaped interface 17; the disc-shaped channel 3 is coiled to the center of the cell culture pool 4 along the bottom surface of the cell culture pool 4 and is communicated with a bracket experiment tube 7 which is processed and fixed at the center; the bracket experiment tube 7 is a hollow tube which is made of elastic materials and is higher than the cell culture pool 4, and a sampling plug 13 is arranged at the opening of the top end of the bracket experiment tube; the sampling plug 13 is internally provided with a pressure sensing probe.

All the sample inlet pipes 2, the disc-shaped channels 3, the circulating pipe 5, the sample channels 9, the one-way circulating pipe 10 and the bracket experiment pipe 7 of the chip are sealed pipelines; the disk-shaped channel 3 is closed by an ion exchange membrane; the cylindrical interior of the sample adding valve 1 is a valve body 16, an upper opening is processed in the valve body 16, and an inner valve channel 14 with the size of an end head matched with the sample inlet pipe 2 is formed in the valve body 16; the valve body 16 is provided with a valve knob 15 at the top.

the sampling plug 13 can penetrate into a sampling needle to sample; the sampling plug 13 is internally provided with a pressure sensing probe which can output the liquid pressure in the bracket experiment tube 7 to a display; the diameter size of the bracket experiment tube 7 has different specifications, and the blood vessel brackets 12 with different pipe diameters can be attached to the sleeve.

Embodiment 2 a microfluidic chip for in vitro intravascular stent evaluation

A micro-fluidic circular chip for evaluating an in vitro vascular stent is mainly composed of a sample adding valve 1, a circulating pulsation micro pump 6 and a cell culture pool 4, wherein 2 parallel cylindrical sample adding valves 1 are processed at the center of the chip, each cylindrical sample adding valve 1 is processed with 2 opposite openings on the same section diameter line, and each opening is respectively connected with 1 sample adding tube 2; each sample inlet pipe 2 is respectively communicated with one cell culture pond 4, and 4 cell culture ponds 4 are formed in total and are communicated with a disc-shaped channel 3 formed at the bottom in each cell culture pond 4.

The 4 cell culture ponds 4 are uniformly distributed at four corners of the rectangular chip, 1 circulating pulsation micropump 6 is processed and installed between a pair of cell culture ponds 4, the circulating pulsation micropump 6 is communicated in series between 2 circulating pipes 5, and the 2 circulating pipes 5 are respectively communicated with the disc-shaped channels 3 in the cell culture ponds 4 at two sides of the circulating pulsation micropump 6; the circulating pipe 5 and the sample injection pipe 2 are communicated with the disc-shaped channel 3 at a T-shaped interface 17 at the side of the cell culture pond 4; the middle of the disc-shaped channel 3 is provided with a pipeline partition wall 19, and the disc-shaped channel 3 is divided into 2 disc-shaped double small pipes 18 from the T-shaped interface 17; the disc-shaped channel 3 is coiled to the center of the cell culture pool 4 along the bottom surface of the cell culture pool 4 and is communicated with a bracket experiment tube 7 which is processed and fixed at the center; the bracket experiment tube 7 is a hollow tube which is made of elastic materials and is higher than the cell culture pool 4, and a sampling plug 13 is arranged at the opening of the top end of the bracket experiment tube; the sampling plug 13 is internally provided with a pressure sensing probe.

Example 3 platelet adhesion experiment

By using the microfluidic chip for the rectangular intravascular stent experiment described in example 1, the intravascular stent 12 with different coating components (coating components can be dopamine, PEI and drugs) processed on the surface is placed on the matched stent experiment tube 7. Adding the same culture medium, and adding platelet-rich plasma with the same concentration into the device to enable the plasma to flow through the surface of the stent at a constant speed. The experiment temperature is 37 ℃, the experiment time is 1h, the same cells are inoculated, and then the related experiment is carried out. Evaluation of the results of the subsequent experiments was performed using Scanning Electron Microscopy (SEM). Six fields of magnification 3000 were randomly selected for each concentration sample in this assay, as shown in FIG. 9, where A is a 2mg/ml dopamine +5mg/ml PEI group; b is a group of 2mg/ml dopamine +10mg/ml PEI; c is a group of 2mg/ml dopamine +15mg/ml PEI; d is a blank control group, namely a 316L stainless steel bracket group without a coating, 1 represents an unloaded medicine group, and 2 is a loaded medicine group.

As is clear from the analysis of the platelet morphology and the number of platelets in the visual field frame, in the method for producing the hybrid membrane, the number of platelets in the visual field frame was smaller in the drug-loaded composition than in the drug-unloaded composition, and more importantly, the morphology of the adhered platelets was not changed much and no false feet were protruding. Among the three drug-carrying groups, the B-2 group, namely the drug-carrying combination of 2mg/ml dopamine and 10mg/ml PEI, has the least adhesion quantity, most adhered platelets are in a round normal state, the activation degree is low, and the effect in a platelet adhesion test is relatively good.

Example 4 vascular smooth muscle cell proliferation assay

1. 4 groups of brackets (316LSS groups, 5mg/ml PEI + DA + GO + DTX coating groups, 10mg/ml PEI + DA + GO + DTX coating groups and 15mg/ml PEI + DA + GO + DTX coating groups) of different experimental types are sterilized by ultraviolet front and back irradiation in a clean bench for 12 hours;

2. Placing the sterilized stent in a pore plate, and adding a smooth muscle cell suspension to allow cells to adhere to the surface of the stent;

3. Sterilizing the chip, placing the bracket in the chip, adding a cell culture medium, and culturing in a 5% CO2 incubator at 37 ℃;

4. Taking out the chip at the specified time (1d, 3d and 5d), taking out the bracket, putting the bracket into a new pore plate, adding 500 mu L of culture medium and 50 mu L of LMTS detection solution into each pore, and putting the pore plate back to the cell culture box at 37 ℃ for wet incubation for 2 h;

5. The well plate is taken out, the reaction solution is shaken up and 100 microlitres of the reaction solution is absorbed and added into a 96 well plate, the well plate is placed at the 490nm wavelength of an enzyme labeling instrument, and the OD value of absorbance is measured.

As shown in fig. 10, there was a significant difference (p < 0.05) between the OD values of the three drug-loaded scaffolds and the 316LSS bare chip scaffold at all 1, 3, 5d, with a very significant difference (p < 0.01) between the three drug-loaded scaffolds and the 316LSS bare chip scaffold at 5 d. The OD value of the drug-loaded stent is far lower than that of a non-drug-loaded stent and a bare-die stent, which shows that DTX (drug) carrying of the group of coatings is successful and the coating has a remarkable inhibiting effect on proliferation of smooth muscle cells.

the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the scope of the embodiments of the present invention, and are intended to be covered by the claims and the specification.

Claims

1. A micro-fluidic chip for evaluating an in vitro intravascular stent is characterized by mainly comprising a sample adding valve (1), a circulating pulsation micro pump (6) and a cell culture pool (4); 2 sample adding valves (1) which are arranged side by side are processed at the center of the chip; each sample adding valve (1) is provided with 2 opposite openings on the same section diameter line, and each opening is respectively connected with 1 sample adding pipe (2); each sample inlet pipe (2) is respectively communicated with a respective cell culture pond (4), 4 cell culture ponds (4) are formed in total, and the sample inlet pipes are communicated with a disc-shaped channel (3) processed at the bottom in each cell culture pond (4); 1 circulating pulsation micropump (6) is arranged between the pair of cell culture ponds (4), the circulating pulsation micropump (6) is communicated in series between 2 circulating pipes (5), and the 2 circulating pipes (5) are respectively communicated with the disc-shaped channel (3).

2. The microfluidic chip according to claim 1, wherein the circulation tube (5) and the sample inlet tube (2) are commonly connected to the disc-shaped channel (3) at a T-shaped interface (17) at the side of the cell culture chamber (4).

3. The microfluidic chip according to claim 2, wherein the disc-shaped channel (3) spirals along the bottom surface of the cell culture tank (4) to the center of the cell culture tank (4) and is communicated with a bracket test tube (7), the bracket test tube (7) is a hollow tube which is higher than the cell culture tank (4), a sampling plug (13) is installed at an opening at the top end of the bracket test tube, and a pressure sensing probe is installed inside the sampling plug (13).

4. The microfluidic chip according to claim 1, wherein the sample inlet tube (2), the disc-shaped channel (3), the circulating tube (5), the sample channel (9), the one-way circulating tube (10) and the rack experiment tube (7) are sealed tubes.

5. microfluidic chip according to claim 1, characterized in that the disk-shaped channels (3) are closed with ion exchange membranes.

6. The microfluidic chip according to claim 5, wherein the disc-shaped channel (3) has a channel partition (19) in the middle, and the disc-shaped channel (3) is divided into 2 disc-shaped double small tubes (18) from the T-shaped interface (17).