Microfluidic 3D bionic heart valve organ-like chip and construction method thereof

Abstract

The invention discloses a microfluidic 3D heart valve organ chip and a construction method thereof, and belongs to the field of microfluidic chips, the key points of the technical scheme are as follows: the organ chip comprises a main body layer and a cover glass layer from top to bottom, and the main body layer comprises a matrigel channel, an endothelial cell channel I and a mesenchymal cell culture medium channel II; the matrigel channel comprises an injection section, a valve culture room section and an outlet section which are arranged in sequence; the endothelial cell channel I comprises an inlet section I, a main body section I and an outlet section I; the mesenchymal cell culture medium channel II comprises an inlet buffer section II, a main body section II and a degassing section II; the main body section I and the main body section II are both communicated with the valve culture room section. The device is mainly used for simulating the microstructures and microenvironments of organ tissues, so that more corresponding micro-physiological functions can be presented; moreover, compared with a 2D cell model and an organ chip in a culture dish, the organ chip is closer to a microstructure and a microenvironment of an organ tissue, so that more corresponding micro-physiological functions can be presented, and compared with a traditional animal model and an organ chip which directly take cells as experimental objects, the cost can be saved, and the organ chip has the advantages of specificity, reliability, rapidness and the like.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chips, and in particular to a microfluidic 3D technology bionic heart valve organ-on-a-chip and a construction method thereof. Background Art

[0002] According to global health statistics, there are more than 100 million people suffering from valvular heart disease (VHD) in the world, and its incidence rate increases with age. Among them, the prevalence of calcific aortic stenosis exceeds about 9.4 million. In addition, studies have reported that the 5-year mortality rates of moderate and severe aortic stenosis heart disease are as high as 56% and 67%, respectively. VHD has become a major and growing threat to human health. At present, the research on the pathological mechanism of heart valve disease is not in-depth enough, especially the problem of heart valve calcification. There is no effective drug treatment. The only way to achieve the purpose of cure is to replace the heart valve, but it will also bring great pain and harm to the patient. Therefore, the development of a microfluidic bionic heart valve organ chip as a research model for heart valve disease has important clinical significance, which is conducive to the study of the mechanism of heart valve calcification and the development of effective therapeutic drugs.

[0003] Microfluidic chip technology uses microchannels, reaction chambers, and other functional components to precisely manipulate fluids, integrating fundamental analytical processes such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analyses. Microfluidic chips offer advantages such as integration, high throughput, low cost, and rapid analysis. Organ-on-a-chip technology, a revolutionary new technology that has emerged in recent years, enables the creation of in vitro microtissue models that more closely resemble the physiological functions of real organs. Organ-on-a-chips do not represent the construction of entire organs, but rather replicate the smallest cell clusters necessary for a specific organ's function. Their advantages include: first, compared to 2D cell models grown in culture dishes, organ-on-chips more closely resemble the microstructure and microenvironment of organ tissues, enabling a more comprehensive representation of corresponding microphysiological functions; second, compared to animal models, organ-on-chips directly utilize cells / tissues as experimental subjects, resulting in cost savings while also offering specificity, reliability, and rapidity. Currently, there are skin organoids, brain organoids, tumor organoids, etc., but the field of microfluidic organoids for studying the mechanism of heart valve calcification under holistic pathological conditions is almost blank. Summary of the Invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide a microfluidic 3D bionic heart valve organoid chip and its construction method. Based on the biological characteristics of heart valve tissue, a bionic heart valve organoid chip is designed, which can simulate the chip of heart valve leaflets and construct heart valve organoids; secondly, the organoid chip can be simulated by infusing growth culture medium and high calcium and phosphate culture medium to explore the expression differences of heart valve-related genes and analyze the possible calcification mechanism; thirdly, different drugs can be added or different levels of shear force can be loaded to detect and analyze the calcification situation to provide experience for the treatment of heart valve calcification; finally, the chip explores the biological response to heart valve calcification when multiple factors coexist through changes in the microenvironment.

[0005] To achieve the above purpose, the technical solutions proposed by the present invention are as follows:

[0006] According to a first aspect of the technical solution of the present invention, a microfluidic 3D bionic heart valve organ chip is provided, wherein the organ chip includes a main layer and a cover glass layer from top to bottom, the main layer includes a matrigel channel, an endothelial cell channel I and a mesenchymal cell culture medium channel II; the endothelial cell channel I and the mesenchymal cell culture medium channel II are distributed on both sides of the matrigel channel as the center, and the matrigel channel, endothelial cell channel I and mesenchymal cell culture medium channel II are all open at the top.

[0007] Furthermore, the matrigel channel includes an injection section, a valve culture chamber section, and an outlet section which are sequentially arranged;

[0008] The end of the injection section is provided with a first channel inlet, and the end of the outlet section is provided with a second channel outlet; both the first channel inlet and the second channel outlet extend to the outside of the top surface of the organ chip;

[0009] The endothelial cell channel I includes an inlet section I, a main section I, and an outlet section I; a channel inlet three is provided at the end of the inlet section I, and a channel outlet four is provided at the end of the outlet section I, and both the channel inlet three and the channel outlet four extend outside the top surface of the organ chip;

[0010] The mesenchymal cell culture medium channel II includes an inlet buffer section II, a main section II and a degassing section II; a channel inlet five is provided at the end of the inlet buffer section II, and a channel outlet six and a channel outlet seven are provided at the end of the degassing section II;

[0011] The main body section I and the main body section II are both connected to the valve culture chamber section.

[0012] Furthermore, the injection section and the outlet section have the same width; the inlet section I and the outlet section I have the same width.

[0013] Furthermore, the width of the injection section / export section is greater than the width of the inlet section I / outlet section I and greater than the width of the degassing section II;

[0014] The widths of the injection section and the outlet section are both 0.8 mm; the widths of the inlet section I and the outlet section I are both 0.5 mm; and the width of the degassing section II is 0.08 mm.

[0015] Here, the injection section I, also known as the outlet section I, is to ensure the smooth infusion of the matrix gel and is relatively large in size; the inlet section I and the outlet section I allow the endothelial cell suspension and its culture medium to circulate smoothly, because it is easier to flow than the colloid and the endothelium only needs to adhere to the gel surface, so the width is slightly smaller; the degassing section removes air when the mesenchymal culture medium is infused to minimize the subsequent circulation of the culture medium, so the width is extremely small.

[0016] Furthermore, the channel inlet 1, channel outlet 2, channel inlet 3, channel outlet 4, channel inlet 5, channel outlet 6 and channel outlet 7 all extend outside the top surface of the organ chip.

[0017] Furthermore, the inlet buffer section II is a flat spiral channel structure, and the total arc length of the spiral area of ​​the flat spiral channel structure is 15 mm.

[0018] Furthermore, a plurality of bluff bodies are provided at intervals on the side of the main body section I and the main body section II close to the valve culture chamber section.

[0019] Furthermore, the bluff body is an elliptical cone-shaped bluff body, the semi-major axis a and semi-minor axis b values ​​of the upper surface ellipse are 0.2mm and 0.12mm respectively, and the semi-major axis a and semi-minor axis b values ​​of the lower surface ellipse are 0.3mm and 0.22mm respectively; the height of the bluff body is 0.25mm.

[0020] Furthermore, a culture chamber is provided in the valve culture chamber section, the height of the culture chamber is 250 μm, and the length of the culture chamber is 3 mm.

[0021] Furthermore, the size of the organ chip is 10 mm×12 mm.

[0022] Furthermore, the main body layer is prepared from polydimethylsiloxane.

[0023] According to a second aspect of the technical solution of the present invention, a method for constructing a microfluidic 3D bionic heart valve organ-on-a-chip according to any of the above aspects is provided, wherein the steps are as follows:

[0024] S1, inject GelMA mixed with target culture cells into the Matrigel channel from the channel inlet 1;

[0025] S2. Inject the endothelial cell suspension and mesenchymal cell culture medium from channel inlet 3 and channel inlet 5, respectively, until the cell suspension and culture medium fill endothelial cell channel I and mesenchymal cell culture medium channel II, respectively, until droplets appear at channel outlets 4, 6, and 7. Place the chip upright in the provided card slot with endothelial cell channel I on the upper side and let it stand for half an hour to allow it to adhere to the adhesive surface and spread and grow.

[0026] S3. Connect the injection pump to channel inlet three and channel inlet five to infuse the culture medium. By changing the culture medium composition and the flow rate of the injection pump, the effects of different drugs and shear forces on the calcification of the heart valve can be evaluated.

[0027] Furthermore, in S2, the concentration of the endothelial cell suspension is 1×10 7 / ml.

[0028] Beneficial effects of the above scheme:

[0029] 1. The present invention constructs an organ chip that can simulate heart valves and establishes heart valve organoids with multiple adjustable microenvironmental parameters. 5% GelMA material is used as the culture matrix to perform 3D co-culture of endothelial cells and mesenchymal cells.

[0030] 2. Due to the parallel culture chamber structure, this Organ-Chip can simulate the effects of various chemical factors used in clinical practice and heart valve calcification research by adding drugs or cytokines to the endothelial culture medium. Because the endothelium is directly attached to the Matrigel interface, simulating a biological barrier, it can also be subjected to fluid shear forces under physiological and pathological conditions, simulating the shear force experienced by heart valve leaflets in clinical practice, and exploring the possible mechanisms of pathological changes such as calcified stenosis or incomplete opening and closing of heart valve leaflets.

[0031] 3. In this organ chip, due to the bionic three-channel structure, the fibrous layer in the heart valve leaflet structure in vivo can be simulated by growing endothelial cells on the endothelial cell channel I and interacting with mesenchymal cells in the matrix gel channel, so a microfluidic bionic heart valve organ chip can be constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1A schematic structural diagram of a bionic heart valve organ-on-a-chip provided by an exemplary embodiment of the present invention;

[0034] Figure 2 for Figure 1 A top view of the chip structure shown;

[0035] Figure 3 A physical picture of a bionic heart valve organ-on-a-chip provided by an exemplary embodiment of the present invention; DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0037] The technical solution of the present invention generally provides a microfluidic 3D biomimetic heart valve organ-on-a-chip and its construction method. The chip consists of three parts: an endothelial cell culture unit on one side, a mesenchymal cell 3D culture unit in the middle area, and a channel for infusing mesenchymal cell culture medium on the other side. By attaching and culturing endothelial cells on the surface of the middle mesenchymal cell 3D culture unit and contacting them with mesenchymal cells to form a co-culture system, the fibrous layer structure of the human heart valve leaflet is simulated, fully restoring the signal transmission and contact between cells in the valve leaflet, providing a highly controllable technical platform for studying heart valve calcification.

[0038] Specifically, the technical solution of the present invention first provides a microfluidic 3D bionic heart valve organ chip, wherein the organ chip includes a main layer and a slide layer from top to bottom, and the main layer includes a matrix gel channel, an endothelial cell channel I, a mesenchymal cell culture medium channel II and a mesenchymal cell culture medium degassing channel; the endothelial cell channel I and the mesenchymal cell culture medium channel II are distributed on both sides of the matrix gel channel as the center; and the matrix gel channel, endothelial cell channel I, mesenchymal cell culture medium channel II and the mesenchymal cell culture medium degassing channel are all open at the top.

[0039] In a preferred embodiment, the matrix gel channel includes an injection section, a valve culture chamber section, and an outlet section arranged in sequence; the end of the injection section is provided with a channel inlet 1, and the end of the outlet section is provided with a channel outlet 2; the channel inlet 1 and the channel outlet 2 both extend to the outside of the top surface of the organ chip.

[0040] In a preferred embodiment, the endothelial cell channel I includes an inlet section I, a main section I and an outlet section I; a channel inlet three is provided at the end of the inlet section I, and a channel outlet four is provided at the end of the outlet section I, and both the channel inlet three and the channel outlet four extend to the outside of the top surface of the organ chip.

[0041] In a preferred embodiment, the mesenchymal cell culture medium channel II includes an inlet buffer section II, a main section II and a degassing section II, wherein the inlet buffer section II can allow more mesenchymal cell culture medium to enter the matrix gel channel, and the degassing section II can remove the residual gas originally in the cavity and minimize the loss of culture medium; the end of the inlet buffer section II is provided with a channel inlet five, and the end of the degassing section II is provided with a channel outlet six and a channel outlet seven; the channel inlet five, channel outlet six and channel outlet seven all extend to the outside of the top surface of the organ chip.

[0042] In a preferred embodiment, the main body segment I and the main body segment II are both connected to the valve culture chamber segment.

[0043] In a preferred embodiment, the injection section and the outlet section have the same width; the inlet section I and the outlet section I have the same width.

[0044] In a preferred embodiment, the width of the injection section / export section is greater than the width of the inlet section I / outlet section I and greater than the width of the degassing section II;

[0045] The widths of the injection section and the outlet section are both 0.8 mm; the widths of the inlet section I and the outlet section I are both 0.5 mm; and the width of the degassing section II 18 is 0.08 mm.

[0046] In a preferred embodiment, the channel inlet one, channel outlet two, channel inlet three, channel outlet four, channel inlet five, channel outlet six and channel outlet seven all extend outside the top surface of the organ chip.

[0047] In a preferred embodiment, the inlet buffer section II is a flat spiral channel structure, and the total arc length of the spiral region of the flat spiral channel structure is 15 mm.

[0048] In a preferred embodiment, a plurality of elliptical cone-shaped bluff bodies are provided at intervals on one side of the main body segment I and the main body segment II close to the valve culture chamber segment, and the semi-major axis a and semi-minor axis b values ​​of the upper surface ellipse are 0.2 mm and 0.12 mm respectively, and the semi-major axis a and semi-minor axis b values ​​of the lower surface ellipse are 0.3 mm and 0.22 mm respectively, and the height of the bluff body is 0.25 mm.

[0049] In a preferred embodiment, a culture chamber is provided in the valve culture chamber section, the height of the culture chamber is 0.25 mm, and the length of the culture chamber is 3 mm.

[0050] In a preferred embodiment, the organ chip has a size of 10 mm × 12 mm, the width of the matrix gel channel injection section and outlet section is 0.8 mm, the width of the inlet section I and outlet section I of the endothelial cell channel I is 0.5 mm, the arc length of the inlet buffer section II of the mesenchymal cell culture medium channel II is 12 mm, and the width of the degassing section II is 0.08 mm.

[0051] In a preferred embodiment, the main layer is made of polydimethylsiloxane.

[0052] Based on this microfluidic chip design, the technical solution of the present invention also provides a method for constructing a microfluidic bionic heart valve organ-on-a-chip, the specific steps of which are as follows:

[0053] 1) Gel construction: First, 5% GelMA mixed with mesenchymal cells was injected into the Matrigel channel from the channel inlet. After the solution filled the slow channel, it was cured using a 405 nm wavelength UV lamp for 30 seconds;

[0054] 2) Static infusion of mesenchymal cell culture medium: inject the mesenchymal cell culture medium from channel inlet 5 to fill the mesenchymal cell culture medium channel II until droplets appear at channel outlet 6 and channel outlet 7;

[0055] 3) Endothelial cell attachment: 1% fibrin solution was injected from channel inlet 3 to coat the adhesive surface, filling the endothelial cell channel I until droplets appeared at channel outlet 4. After half an hour, phosphate buffered saline (PBS) was passed through to wash away the residue, and 1×10 7 An endothelial cell suspension of 100 cells / ml was injected from channel inlet 3 until the cell suspension filled endothelial cell channel I until a droplet appeared at channel outlet 4. The chip was then placed upright in a predetermined slot with endothelial cell channel I positioned on the upper side to ensure that the endothelial cells adhered to the surface of the hydrogel constructed in step 1). The cell was then incubated for half an hour to allow for full adhesion to the surface.

[0056] 4) Dynamic Infusion of Endothelial Cell Culture Medium and Mesenchymal Cell Culture Medium: Syringe pumps were connected to channel inlet 3 and channel inlet 5 to infuse the culture medium. By varying the medium composition and the flow rate of the syringe pump, the effects of different drugs and shear stress on heart valve calcification were evaluated. The Matrigel mixed with the target cultured cells in this step was a mixture of the target cultured cell type and the 3D culture scaffold matrix in a specific ratio.

[0057] Using the above technical solution, an organ chip was fabricated using polydimethylsiloxane (PDMS). The chip size was 10 mm × 12 mm, and the culture chamber height was 250 μm and the length was 3 mm. 5% GelMA mixed with mesenchymal cells was injected from channel inlet one to channel outlet two. Under the action of the bluff body, the GelMA was well confined within the valve culture chamber. Cell culture medium was injected from channel inlets three and five, respectively, and the cell culture medium filled endothelial cell channel I and mesenchymal cell culture channel II until droplets appeared at channel outlets four, six, and seven, to ensure that no bubbles remained in endothelial cell channel I and mesenchymal cell culture channel II.

[0058] Example

[0059] A microfluidic 3D heart valve organ-on-a-chip, such as Figure 1 As shown, the organ chip includes a main body layer 1 and a cover glass layer 2 from top to bottom. Figures 1 to 3 As shown, the main body layer 1 includes a matrix glue channel 3 ( Figure 1 The dotted line shows the part), endothelial cell channel I4 ( Figure 1 The dotted line shows the part) and the mesenchymal cell culture medium passage II 5 ( Figure 1 The dotted line shows the part), where Figure 1 and Figure 2 It shows the effect of bulkhead from blank to filler. Figure 3This is the overall effect achieved by the chip. Endothelial cell channel I4 and mesenchymal cell culture channel II5 are arranged on either side of Matrigel channel 3. All three channels are open at the top. The organ chip measures 10 mm x 12 mm. The main body layer 1 is made of polydimethylsiloxane. Matrigel channel 3 includes an injection section 6, a valve culture chamber section 7, and an outlet section 8, arranged in that order. The end of injection section 6 is provided with channel inlet 1 9, and the end of outlet section 8 is provided with channel outlet 2 10. Both sections are 0.8 mm wide. Both inlet 1 9 and outlet 2 10 extend beyond the top surface of the organ chip. The endothelial cell channel I4 includes an inlet section I11, a main section I12, and an outlet section I13. The end of inlet section I11 is provided with a channel inlet 3 14, and the end of outlet section I13 is provided with a channel outlet 4 15. Both inlet section I11 and outlet section I13 are 0.5 mm wide. Both channel inlet 3 14 and channel outlet 4 15 extend beyond the top surface of the organ chip. The mesenchymal cell culture medium channel II5 includes an inlet buffer section II16, a main section II17, and a degassing section II18. The end of inlet buffer section II16 is provided with a channel inlet 5 19, which extends beyond the top surface of the organ chip. The end of degassing section II18 is provided with a channel outlet 6 20 and a channel outlet 7 21, which both extend beyond the top surface of the organ chip. The degassing section II18 is 0.08 mm wide. Both main sections I12 and II17 are connected to the valve culture chamber section 7. The outer walls of the main body section I 12 and the main body section II 17 near the valve culture chamber section 7 are both spaced apart with a plurality of elliptical cone-shaped bluff bodies 22, the upper surface ellipse a and b values ​​of which are 0.2mm and 0.12mm respectively, the lower surface ellipse a and b values ​​of which are 0.3mm and 0.22mm respectively, and the bluff body height is 0.25mm; the valve culture chamber section 7 is provided with a culture chamber 23 ( Figure 1 The height of the culture chamber 23 is 0.25 mm, and the length of the culture chamber 23 is 3 mm.

[0060] In summary, the present invention discloses a microfluidic heart valve organ-on-chip and a construction method thereof, which are mainly used to simulate the microstructure and microenvironment of organ tissues, so as to present more corresponding microphysiological functions; and compared with the 2D cell model in the culture dish, the organ chip is closer to the microstructure and microenvironment of organ tissues, so it can present more corresponding microphysiological functions. Compared with the traditional animal model, the organ chip directly uses cells as experimental objects, which can save costs and has the advantages of specificity, reliability and rapidity.

[0061] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A microfluidic 3D bionic heart valve organ-on-a-chip, characterized in that: The organ chip includes a main layer and a cover glass layer from top to bottom, and the main layer includes a matrix gel channel, an endothelial cell channel I and a mesenchymal cell culture medium channel II; the endothelial cell channel I and the mesenchymal cell culture medium channel II are distributed on both sides of the matrix gel channel as the center, and the matrix gel channel, endothelial cell channel I and mesenchymal cell culture medium channel II are all open at the top.

2. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 1, characterized in that: The matrix gel channel comprises an injection section, a valve culture chamber section and an outlet section which are arranged in sequence; The end of the injection section is provided with a first channel inlet, and the end of the outlet section is provided with a second channel outlet; both the first channel inlet and the second channel outlet extend to the outside of the top surface of the organ chip; The endothelial cell channel I includes an inlet section I, a main section I, and an outlet section I; a channel inlet three is provided at the end of the inlet section I, and a channel outlet four is provided at the end of the outlet section I, and both the channel inlet three and the channel outlet four extend outside the top surface of the organ chip; The mesenchymal cell culture medium channel II includes an inlet buffer section II, a main section II and a degassing section II; a channel inlet five is provided at the end of the inlet buffer section II, and a channel outlet six and a channel outlet seven are provided at the end of the degassing section II; The main body section I and the main body section II are both connected to the valve culture chamber section.

3. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 2, characterized in that: The injection section and the outlet section have the same width, and the inlet section I and the outlet section I have the same width; The width of the injection section / export section is greater than the width of the inlet section I / outlet section I and greater than the width of the degassing section II. The widths of the injection section and the outlet section are both 0.8 mm; the widths of the inlet section I and the outlet section I are both 0.5 mm; and the width of the degassing section II is 0.08 mm.

4. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 2, characterized in that: The channel inlet 1, channel outlet 2, channel inlet 3, channel outlet 4, channel inlet 5, channel outlet 6 and channel outlet 7 all extend outside the top surface of the organ chip.

5. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 2, characterized in that: The inlet buffer section II is a flat spiral channel structure.

6. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 2, characterized in that: A plurality of bluff bodies are provided at intervals on one side of the main body section I and the main body section II close to the valve culture chamber section.

7. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 6, characterized in that: The bluff body is an elliptical cone-shaped bluff body, and the elliptical area of ​​the upper surface is smaller than the elliptical area of ​​the lower surface; The semi-major axis a and semi-minor axis b of the upper surface ellipse are 0.2 mm and 0.12 mm respectively, and the semi-major axis a and semi-minor axis b of the lower surface ellipse are 0.3 mm and 0.22 mm respectively; the height of the bluff body is 0.25 mm.

8. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 2, characterized in that: A culture chamber is provided in the valve culture chamber section; Wherein, the height of the culture chamber is 250 μm, and the length of the culture chamber is 3 mm.

9. The microfluidic 3D bionic heart valve organ-on-a-chip according to claim 2, characterized in that: The main body layer is prepared from polydimethylsiloxane; The size of the organ chip is 10 mm × 12 mm.

10. A method for constructing a microfluidic 3D bionic heart valve organ-on-a-chip according to any one of claims 1 to 9, characterized in that: The following steps are involved: S1, inject GelMA mixed with target culture cells into the Matrigel channel from the channel inlet 1; S2. Inject the endothelial cell suspension and mesenchymal cell culture medium from channel inlet 3 and channel inlet 5, respectively, until the cell suspension and culture medium fill endothelial cell channel I and mesenchymal cell culture medium channel II, respectively, until droplets appear at channel outlets 4, 6, and 7. Place the chip upright in the provided card slot with endothelial cell channel I on the upper side and let it stand for half an hour to allow it to adhere to the adhesive surface and spread and grow. S3. Connect the injection pump to channel inlet three and channel inlet five to infuse the culture medium. By changing the culture medium composition and the flow rate of the injection pump, the effects of different drugs and shear forces on the calcification of the heart valve can be evaluated.

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