Organ chip for measuring contraction force and contraction frequency of myocardial cells as well as preparation method and application of organ chip

Abstract

The invention relates to an organ chip for measuring myocardial cell contraction force and contraction frequency and a preparation method and application thereof.The organ chip comprises a chip cavity body, an elastic thin tube, a middle buffer pool, a rigid microtube, a scale wrapping layer and a supporting table, and the elastic thin tube is arranged in a cavity of the chip body; the elastic slim tube, the middle buffer pool and the rigid microtube are sequentially communicated, and the rigid microtube and the scale wrapping layer are arranged on the supporting table. During measurement, a myocardial cell cluster surrounds the elastic thin tube, cell contraction drives the change of the volume of the elastic thin tube so as to drive the change of the liquid level scale of the measured liquid in the rigid microtube, and the contraction strength and the contraction frequency of myocardial cells are calculated through the variation. According to the measuring method of the chip, scale recording is carried out in a visual inspection and camera shooting mode, the limitation that the contractility of the myocardial cells can be observed only by depending on a microscope is avoided, and the chip is more suitable for the requirement for rapid high-throughput measurement of the contractility and the contractility frequency of the myocardial cells.

Description

Technical Field

[0001] The present invention relates to an organ chip for measuring the contractility and contraction frequency of cardiomyocytes, a preparation method thereof, and an application thereof, belonging to the technical fields of biomedicine and organ chips. Background Art

[0002] When in vitro cultured cardiomyocytes are used for drug screening applications, their contractility and contraction frequency are key important indicators characterizing the activity and function of cardiomyocytes. By measuring the contractility and contraction frequency of cardiomyocytes, the changes in the contraction function of the myocardium in response to drugs can be accurately evaluated.

[0003] Currently, multiple literature reports indicate that models of microfluidic chips and organ chips for detecting the contractility and contraction frequency of cardiomyocytes in vitro have been widely used in pharmacodynamic evaluation and toxicity experiments. Devices for detecting in vitro cardiomyocytes (especially single cardiomyocytes) or heart models are all in the microscale range. Although these devices and detection methods can measure the contractility of microscale cardiomyocytes very precisely, the preparation methods of microdevices are very complex and the process difficulty is relatively high. In addition to complex microdevices, the measurement of contractility also requires the support of mechanical sensors or microscopic imaging systems.

[0004] The existing patent application (CN116256087A) discloses a flexible strain sensor for detecting the contractility of cardiomyocytes. By separating the two friction layers of the flexible strain sensor based on a triboelectric nanogenerator bridge structure with spacers, the weak strain caused by cardiomyocyte contraction can be captured, effectively reducing the collapse caused by gravity and improving the stability and signal-to-noise ratio. Although this device and detection method have good effects in detecting the weak contractility of single cells, they are not suitable for measuring the contractility of clusters of multiple cardiomyocytes and cardiac organoids. The existing patent application (CN118853401A) discloses that two magnetized flexible microbeams distributed perpendicular to each other and corresponding magnetic sensors can detect the contractility of myocardial tissue. However, this device requires a corresponding sensor to detect mechanical changes. However, existing detection devices cannot measure the contractility and contraction frequency of cardiomyocytes simply, quickly, at low cost, and with high throughput in vitro. Summary of the Invention

[0005] (1) Technical Problems to be Solved

[0006] To solve the above problems of the existing technology, the present invention provides an organ chip for measuring the contractility and contraction frequency of cardiomyocytes, a preparation method thereof, and an application thereof.

[0007] (2) Technical Solutions

[0008] To achieve the above object, the main technical solutions adopted by the present invention include:

[0009] An organ-on-a-chip for measuring the contractility and contraction frequency of cardiomyocytes, which comprises a chip body, an elastic capillary tube, an intermediate buffer pool, a rigid microtube, a scale wrapping layer and a support platform. Among them, the chip body, the intermediate buffer pool and the support platform are arranged adjacent to each other in sequence; the center of the chip body is set as a hollow chamber, a sampling port is arranged above the chamber, the elastic capillary tube is arranged in the chamber, and the elastic capillary tube deforms with the contractility of the cardiomyocytes attached to its surface. The elastic capillary tube, the intermediate buffer pool and the rigid microtube are communicated in sequence; the rigid microtube is arranged on the support platform, the scale wrapping layer is arranged on the support platform, the scale wrapping layer wraps the rigid microtube, and a scale ruler for displaying the scale is engraved on the scale wrapping layer for measuring the scale change of the liquid level in the rigid microtube.

[0010] For the organ-on-a-chip as described above, preferably, the structure of the chamber is circular, square or trapezoidal, the volume of its cavity is 200 - 2000 cubic millimeters, and the opening diameter of the sampling port is 0.5 - 5 millimeters.

[0011] For the organ-on-a-chip as described above, preferably, one end of the elastic capillary tube communicates with the intermediate cavity of the intermediate buffer pool, and a plug bolt is arranged at the other end of the elastic capillary tube; the inner diameter of the central cavity of the elastic capillary tube is 1 - 1.5 millimeters, the wall thickness is 0.2 - 1 millimeter, and the length of the elastic capillary tube does not exceed 2 centimeters.

[0012] For the organ-on-a-chip as described above, preferably, the diameter of the intermediate cavity of the rigid microtube is 10 - 50 micrometers.

[0013] For the organ-on-a-chip as described above, preferably, the volume of the intermediate cavity of the intermediate buffer pool is 100 - 500 microliters.

[0014] For the organ-on-a-chip as described above, preferably, the scale is a length scale, the scale value is 0 - 5 centimeters, and the accuracy is 0.01 centimeter.

[0015] For the organ-on-a-chip as described above, preferably, the horizontal inclination of the upper surface and the lower surface of the support platform does not exceed plus or minus 1°; during measurement, the elastic capillary tube and the intermediate buffer pool are filled with colored liquid.

[0016] For the organ-on-a-chip as described above, preferably, the material of the elastic capillary tube is an elastic silicone rubber material, its elastic modulus is 800 kPa - 1 MPa, and its rebound rate is 75 - 85%; the material of the rigid microtube is a light-transmitting glass or PDMS material, its elastic modulus is 3 - 5 MPa, and its rebound rate < 40%; the material of the intermediate buffer pool is a light-transmitting material, and its light transmittance ≥ 80%; the material of the support platform is PDMS material, metal or hard plastic.

[0017] The preparation method of the organ-on-a-chip as described above includes the following steps:

[0018] S1: Cover the PDMS prepolymer solution on the male mold of the chip body, and place it in an oven for curing to obtain a cured cavity structure made of PDMS material.

[0019] S2: Use a punch to punch holes at the top and side of the PDMS cavity structure; successively clean the punched PDMS cavity structure with absolute ethanol and pure water, and place it in an oven to dry, obtaining a dried chamber structure of the chip body.

[0020] S3: Pour the PDMS prepolymer solution into a rectangular box composed of ultra-clean glass slides, place it in an oven for curing, then remove the rectangular box of ultra-clean glass slides to obtain a rectangular PDMS structure. Then, use a punch to drill a hole in the middle area of the cured rectangular PDMS structure to form an intermediate buffer pool made of PDMS material with a cavity; successively clean the cured intermediate buffer pool with absolute ethanol and pure water, and after cleaning, dry it to obtain a dried intermediate buffer pool with a cavity.

[0021] S4: Clean the thick and thin tubes of two stainless steel hollow tubes with a diameter difference of 100 - 200 microns successively with absolute ethanol and pure water, and dry them after cleaning; evenly spray a release agent inside the thick tube and outside the thin tube and let it dry, then sleeve the thin tube with a smaller diameter in the center of the thick tube with a larger diameter and fix it; pour the mixed prepolymer silicone rubber liquid into the sandwich of the two stainless steel hollow tubes, remove the bubbles by centrifugation and then cure it.

[0022] S5: Pull out the two stainless steel hollow tubes to obtain elastic thin tubes, successively perform ultrasonic cleaning on the elastic thin tubes with absolute ethanol and pure water, and dry them after cleaning to obtain dried elastic thin tubes.

[0023] S6: Fabricate a rigid microtube with glass, successively clean it with absolute ethanol and pure water, and dry it after cleaning to obtain a dried rigid microtube.

[0024] S7: Cover the PDMS prepolymer solution on the graduated female mold, remove the bubbles and then cure it; cut off the cured PDMS male mold layer with graduations, apply color to the graduated area and then dry it to obtain a graduated wrapping layer with length graduations.

[0025] S8: Pour the PDMS prepolymer solution into the model of the support platform, cure it to form a support platform structure, successively clean it with absolute ethanol and pure water, and dry it after cleaning to obtain a dried support platform.

[0026] S9: Place the rigid microtube on the upper surface of the support platform and fix it, then perform plasma treatment on the graduated wrapping layer and the support platform with the rigid microtube made of glass fixed on it, and then wrap the graduated wrapping layer around the periphery of the rigid microtube and implement bonding and encapsulation with the support platform.

[0027] S10: Insert the hollow elastic tube into the openings on both sides of the chamber structure of the chip body and fix it. Use a ultra-clean glass sheet as the base of the chamber, and then irreversibly bond and package the chamber structure of the chip body with the base, so that a chamber for culturing cells is formed in the chip body; Connect one end of the elastic thin tube to the opening on one side of the intermediate buffer pool; Connect the opening on the other side of the intermediate buffer pool to the open end of the rigid microtube;

[0028] S11: Pour the measuring liquid from the side of the chamber connected to the elastic thin tube. Stop pouring when the measuring liquid fills the elastic thin tube and the intermediate buffer pool. Seal the pouring port with a plug bolt, and after sterilization, obtain an organ chip for measuring the contractile force and contraction frequency of cardiomyocytes.

[0029] In a preferred embodiment, the PDMS prepolymer solution is a mixture of PDMS and the curing agent supporting PDMS at a mass ratio of 5:1 to 20:1; In step S4, the diameter difference between the two stainless steel hollow tubes is 200 microns; In step S6, the inner diameter of the rigid microtube is 20 microns.

[0030] A method for detecting the contractile force and contraction frequency of cardiomyocytes, characterized in that the organ chip described in any one of claims 1-7 is used for detection; The method includes the following steps:

[0031] 1) Pour a culture medium solution containing collagen with a final concentration of 0.05-0.25 mg / mL into the chamber of the chip body, place it in an incubator at 35-37 °C for incubation, and then remove the culture medium solution to complete the coating in the chamber of the chip body;

[0032] 2) Transplant cardiomyocytes into the chamber of the chip body so that the cardiomyocyte clusters are wrapped around the elastic thin tube;

[0033] 3) Read the change in the liquid level scale of the measuring liquid in the rigid microtube per unit time to obtain the velocity v of the measuring liquid. Record the time period dt within a certain time. The difference in the velocity change of the measuring liquid before and after this time period is dv, and the distance the liquid level moves is L. First, calculate the acceleration a = dv / dt, and then according to W 测 = πr 细 2 ×L×ρ 测 to obtain the mass of the measuring liquid. According to F = W 测 ×a, the contractile force F of the cardiomyocyte clusters can be obtained;

[0034] By counting the number of times T of the change in the liquid level of the measuring liquid within a specific time period t, the contraction frequency per minute H = T / t is obtained;

[0035] wherein, r 细 is the radius of the rigid microtube, ρ 测To measure the density of a liquid.

[0036] (III) Beneficial effects

[0037] The beneficial effects of the present invention are as follows:

[0038] An organ chip for measuring the contractility and contraction frequency of cardiomyocytes provided by the present invention can plant a group of cardiomyocytes or cardiomyocytes mixed in a three-dimensional matrix material in a chamber provided with an elastic capillary tube by designing a chamber with an elastic capillary tube inside, and completely wrap them around the elastic capillary tube in the middle chamber. As the culture time increases, the self-organization behavior of the cell cluster can automatically surround the elastic capillary tube and start spontaneous contraction and relaxation. When contracting, it squeezes the elastic capillary tube, and the measuring liquid in the elastic capillary tube can be pressed out of the capillary tube. This process approximately simulates the process of cardiomyocyte contraction in vivo to pump the blood in the heart out of the heart and into the blood vessels. At the same time, a rigid microtube with a length scale is designed to communicate with the elastic capillary tube through an intermediate buffer pool. The measuring liquid filled in the elastic capillary tube and the intermediate buffer pool constitutes a simple measuring unit that responds to cardiomyocyte contraction. During measurement, the elastic capillary tube and the intermediate buffer pool that are connected in sequence are filled with the measuring liquid, and the cardiomyocyte cluster is surrounded around the elastic capillary tube. The cell contraction drives the change in the volume of the elastic capillary tube, which in turn drives the change in the liquid level scale in the rigid microtube, converting the cardiomyocyte contractility and contraction frequency that cannot be directly measured into the moving distance of the liquid level of the measuring liquid in the hollow rigid microtube. By calculating the moving distance of the liquid level of the measuring liquid, the contractility and contraction frequency of cardiomyocytes are measured. Therefore, the organ chip provided by the present invention does not need to introduce sensors and mechanical force measuring devices anymore, and only needs to calculate the moving distance of the liquid level of the measuring liquid to obtain the contractility and contraction frequency of cardiomyocytes.

[0039] The present invention provides a method for detecting the contractility and contraction frequency of cardiomyocytes. This method is simple to operate, has a low detection cost, and does not require expensive equipment.

[0040] The present invention also provides a preparation method for an organ chip for measuring the contractility and contraction frequency of cardiomyocytes. The raw materials used are easily available. For the prepared organ chip, when in use, the elastic capillary tube that directly responds to cardiomyocyte contraction and the intermediate buffer pool are filled with a colored measuring liquid, and there is a length scale that can be read by the naked eye outside the rigid microtube. When the cardiomyocytes contract and squeeze the elastic capillary tube, the measuring liquid enters the rigid microtube, causing the liquid level to change. Without relying on a microscopic imaging system, the corresponding scale when the measuring liquid level changes can be read by the naked eye or an ordinary photographing device. Moreover, this measuring unit can be prepared into an organ chip with a single measuring unit or an integrated measuring group composed of multiple measuring units according to the use requirements. When prepared into an integrated measuring group, high-throughput measurement of the contractility and contraction frequency of cardiomyocytes can be performed on multiple samples to be measured simultaneously. Brief Description of the Drawings

[0041] Figure 1 FIG. 1 is a schematic structural diagram of an organ chip preferably used for measuring the contractility and contraction frequency of cardiomyocytes according to the present invention;

[0042] Figure 2 FIG. 2 Figure 1 is a schematic structural diagram of the chamber of the chip body and the elastic capillary in the organ chip shown in FIG. 1;

[0043] Figure 3 FIG. 3 Figure 1 is a schematic structural diagram of the intermediate transition pool in the organ chip shown in FIG. 2;

[0044] Figure 4 FIG. 4 Figure 1 is a schematic structural diagram of the support platform and the rigid microtube in the organ chip shown in FIG. 3;

[0045] Figure 5 FIG. 5 Figure 1 is a schematic structural diagram of the graduated wrapping layer in the organ chip shown in FIG. 4;

[0046] Figure 6 FIG. 6 Figure 1 is a schematic diagram of the principle of the method for measuring the contractility and contraction frequency of cardiomyocyte clusters using the organ chip shown in FIG. 5.

[0047] Description of the Reference Numerals in the Drawings

[0048] 1: Chip body;

[0049] 2: Elastic capillary;

[0050] 3: Intermediate buffer pool;

[0051] 4: Rigid microtube;

[0052] 5: Graduated wrapping layer;

[0053] 6: Support platform;

[0054] 7: Sampling port;

[0055] 8: Sealing bolt;

[0056] 9: Hollow pool;

[0057] 10: Opening;

[0058] 11 Chamber;

[0059] 12: Cardiomyocyte cluster;

[0060] 13: Deformation;

[0061] 14: Liquid level. Detailed Embodiments

[0062] An organ chip designed and prepared according to the present invention for measuring the contractility and contraction frequency of cardiomyocytes is composed of a chip body, an elastic capillary tube, an intermediate buffer pool, a rigid microtube, a scale wrapping layer and a support platform. The elastic capillary tube is arranged in the cavity of the chip body. The elastic capillary tube, the intermediate buffer pool and the rigid microtube are connected in sequence. The measurement liquid is filled in the elastic capillary tube and the intermediate buffer pool. The scale wrapping layer wraps the rigid microtube and is arranged on the support platform to form a measurement unit. During measurement, a cluster of cardiomyocytes is planted around the elastic capillary tube. The spontaneous contraction of the cell cluster drives the change of the hollow volume of the elastic capillary tube, squeezing out the measurement liquid therein and entering the intermediate buffer pool. Then, the measurement liquid in the intermediate buffer pool is squeezed into the rigid microtube, causing the scale of the liquid level in the rigid microtube to change. The contractility and contraction frequency of the cardiomyocytes can be calculated through the amount of scale change. The measurement method of this chip records the scale by visual inspection or photography, avoiding the limitation of relying on a microscope to observe the contractility of cardiomyocytes, and is more suitable for the requirements of rapid high-throughput measurement of the contractility and contraction frequency of cardiomyocytes.

[0063] In order to better explain the present invention for easy understanding, the present invention will be described in detail below with reference to the accompanying drawings through specific embodiments.

[0064] Example 1

[0065] An organ-on-a-chip for measuring the contractility and contraction frequency of cardiomyocytes, the organ-on-a-chip comprising a chip body 1, an elastic capillary 2, an intermediate buffer pool 3, a rigid microtube 4, a scale wrapping layer 5 and a support 6, and the chip body 1, the intermediate buffer pool 3 and the support 6 are arranged adjacent to each other in sequence. Among them, the outer shape of the chip body 1 can be set as a cube, the center of the chip body 1 is provided with a hollow chamber, a sampling port 7 is opened at the top of the hollow chamber of the chip body 1 for adding cardiomyocytes and their solutions into the chamber, and an elastic capillary 2 is arranged in the chamber. The elastic capillary 2 can deform with the contractility of the cardiomyocytes attached to its surface. Both the elastic capillary 2 and the rigid microtube 4 are hollow in the middle. One end of the middle cavity of the elastic capillary 2 communicates with one end of the middle cavity of the intermediate buffer pool 3, and the other end of the middle cavity of the intermediate buffer pool 3 communicates with the central cavity of the rigid microtube 4. The rigid microtube 4 is arranged on the support 6, and the scale wrapping layer 5 is arranged on the support 6. The scale wrapping layer 5 wraps the rigid microtube 4, and the scale wrapping layer 5 is used to display the scale change of the liquid level in the rigid microtube 4. On the scale wrapping layer 5, the scale mark close to the intermediate buffer pool 3 is marked as 0, and the scale of the rigid microtube away from the intermediate buffer pool 3 is marked as 5 cm. The elastic capillary 2 and the hollow rigid microtube 4 are communicated through the intermediate buffer pool 3. When performing the detection work, the elastic capillary 2 and the intermediate buffer pool 3 are both filled with the measuring liquid; the measuring liquid is a colored solution, for example, it can be pure water or mercury with red color. The cardiomyocytes to be measured are planted around the elastic capillary 2 in the chip body 1 of the organ-on-a-chip. The contractility of the cardiomyocytes compresses the elastic capillary 2, causing the measuring liquid in the elastic capillary 2 to flow into the intermediate buffer pool 3, and squeezing the measuring liquid in the intermediate buffer pool 3 into the connected rigid microtube 4, causing the liquid level of the rigid microtube 4 to change. By reading the liquid level change displayed on the scale of the scale wrapping layer 5, the contractility and contraction frequency of the cardiomyocytes can be calculated.

[0066] Among them, the calculation formula of the contractility is as follows:

[0067] L = L 2 - L 1 (1), where: L 1 refers to the scale value close to 0 on the read scale, indicating the state when the myocardium is not contracted, and the scale value of the measuring liquid does not change at this time. L 2 refers to the scale value far from 0 on the read scale, indicating the distance that the measuring liquid in the elastic capillary is squeezed by the contraction of the myocardium and is pressed from the intermediate buffer pool into the scaled rigid microtube 4. This formula obtains the absolute value L of the liquid level change of the measuring liquid compressed by the myocardial pulsation in the rigid microtube. For different pulsation forces, this absolute value is different.

[0068]

[0069] Among them, W 测is the mass of the liquid level change in the graduated rigid microtube 4, r 细 is the radius of the rigid microtube 4, ρ 测 is the density of the measured liquid. This formula obtains the mass of the measured liquid squeezed into the rigid microtube 4 by the myocardial pulsation. According to Newton's second law F = Ma, the contraction force F of the myocardial cell can be derived 测 = W 测 *a, where a is the acceleration. The acceleration a can be obtained by the calculation formula (3) in this measurement scheme:

[0070]

[0071] where: v is the velocity of the liquid level movement of the measured liquid in the graduated hollow rigid microtube 4, and t is the movement time. Usually, since the pulsation frequency of the myocardium is below 100 times per minute, the value of v can be obtained by taking an ordinary video of the liquid level distance change and extracting the time of each frame of the video. The distance that the liquid level moves within one second can obtain the velocity v. This method does not require a high-speed imaging device and does not need to be taken under a microscope

[0072] The calculation method of the contraction frequency is to count the number of times T of the liquid level change of the measured liquid within a specific time period t (t is in minutes), and the contraction frequency H per minute can be obtained as H = T / t (times / min)

[0073] The structure of the chamber inside the chip body 1 can be set as a cavity with an indefinite shape, which can be circular, square, trapezoidal, etc. The volume of the chamber can be set within the range of 200 - 2000 cubic millimeters according to needs. Specifically, as Figure 2 shown, there is one or more sample addition ports 7 for adding cells on the top surface of the chamber; the opening diameter of the sample addition port 7 is 0.5 - 5 millimeters. There is a measurement liquid perfusion port on the side wall of the chamber, and the perfusion port has a matching sealing bolt 8; there is an opening for installing the elastic capillary 2 on the other side wall of the chamber. The outer wall thickness of the chamber is set above 0.5 millimeters. The elastic capillary 2 is a circular hollow tube with an elastic tube wall, which is prepared from a highly elastic silicone rubber material. The inner diameter of the tube is 1 - 1.5 millimeters, the wall thickness of the tube is 0.2 - 1 millimeter, and the length of the elastic capillary 2 does not exceed 2 centimeters; one end of the elastic capillary 2 is encapsulated in the measurement liquid perfusion port on the side wall of the chamber and sealed with a plugging bolt 8, and the other end is connected to the intermediate buffer pool

[0074] As Figure 3 shown, the intermediate buffer pool 3 is a connecting component between the elastic capillary and the rigid microtube. The middle of the intermediate buffer pool 3 is a hollow pool 9, and the volume of the hollow pool 9 can be set within the range of 100 - 500 microliters according to needs. There is an opening 10 penetrating through the middle cavity region on each of the two side walls of the hollow pool; the intermediate buffer pool can be prepared from a highly transparent material

[0075] like Figure 4 As shown, the rigid microtube 4 is a hollow microtube whose lumen structure does not produce deformation. The inner diameter of the microtube is smaller than the inner diameter of the elastic capillary 2, and the inner diameter of the rigid microtube 4 is 10 to 50 microns; one end of the rigid microtube 4 is an open port, which is connected to the intermediate buffer tank, and the other end is a sealed port; the rigid microtube 4 is made of high-transmittance glass or PDMS material. Generally, the transmittance is measured according to the national standard GB / T 2410─2008.3.2. In the present invention, high transmittance is defined as a transmittance of not less than 80%.

[0076] The rigid microtube 4 is arranged above the support 6, which is a base made of a material selected from PDMS, metal, and hard plastic. In order to ensure the accuracy of the measurement results, the horizontal inclination of its upper and lower surfaces does not exceed plus or minus 1°, and is used to place the rigid microtube 4 and the scale wrapping layer.

[0077] like Figure 5 As shown, the scale wrapping layer 5 is a highly light-transmitting material, wrapped around the rigid microtube 4, and a length scale is provided on the scale wrapping layer 5; the rigid microtube 4 is placed in the center of the scale wrapping layer 5, and scales are marked on both sides of the scale wrapping layer, with a scale value of 0 to 5 cm, 0 cm near the central buffer pool, and a normal scale far from the central buffer pool, with a total length of 5 cm and an accuracy of 0.01 cm. An amplifying film coating may also be provided above the scale value of the scale wrapping layer 5.

[0078] Methods for detecting the contractility and contraction frequency of cardiomyocytes using the chip, such as Figure 6 As shown, before measurement, the elastic capillary 2 and the middle buffer tank are filled with colored liquid as the measuring liquid. The principle of the detection method is to transplant the cardiomyocytes into the chamber 11 of the chip body, so that the cardiomyocyte cluster 12 is wrapped around the elastic capillary 2, and the cardiomyocytes contract and compress the wall of the elastic capillary 2, so that the elastic capillary 2 is deformed 13, and the measuring liquid therein is squeezed into the middle buffer tank 3, and then the measuring liquid in the middle buffer tank 3 enters the rigid microtube 4, and the scale of the scale wrapping layer 5 before and after the contraction of the cardiomyocytes is obtained by reading the liquid level 14 of the measuring liquid, and the scale difference is calculated to obtain the strength and contraction frequency of the contraction force of the cardiomyocytes.

[0079] Specifically, a method for detecting the contractility and contraction frequency of myocardial cells is specifically performed as follows:

[0080] 1) A culture medium solution with a final concentration of 0.1 mg / mL type I rat tail collagen was perfused into the chip body cavity, and the chip was placed in a 37°C incubator for incubation for 2 hours, and then the culture medium solution was removed to complete the coating of the chip body cavity;

[0081] 2) Transplant cardiomyocytes into the chamber of the chip body, and through cultivation, make the cardiomyocyte clusters wrap around the elastic capillary;

[0082] 3) Read the change in the liquid level scale of the measuring liquid in the rigid microtube per unit time (s) to obtain the velocity v of the measuring liquid, record it within a certain time dt, and the difference in the velocity change of the measuring liquid before and after this period of time is dv = v 后 -v 前 , and the distance the liquid level moves is L;

[0083] First calculate the acceleration a = dv / dt, and then according to W 测 = πr 细 2 × L × ρ 测 to obtain the mass of the measuring liquid, and according to F = W 测 × a to obtain the contractile force F of the cardiomyocyte clusters;

[0084] By counting the number of times T of the change in the measuring liquid level within a specific time period t, the contraction frequency per minute H = T / t is obtained;

[0085] Among them, r 细 is the radius of the rigid microtube, and ρ 测 is the density of the measuring liquid.

[0086] Example 2

[0087] This example provides a preparation method of an organ chip for detecting the contractile force and contraction frequency of cardiomyocytes. The structure of the prepared organ chip is as shown in Example 1 Figure 1 and includes the following steps: S1: Cover the PDMS (polydimethylsiloxane) prepolymer solution (a commercially available PDMS component and a PDMS fixing solution are completely mixed at a mass ratio of 10:1) on the male mold of the chip body. After removing the bubbles in the PDMS prepolymer solution in a vacuum drying oven, put it into an oven at 60 °C for curing for 4 hours to obtain a cured PDMS material cavity structure. Among them, the male mold of the chip body can be prepared by hot pressing of polytetrafluoroethylene, 3D printing, or other injection molding and laser engraving methods to obtain a male mold of the chip body with a convex structure made of polytetrafluoroethylene, PLC, or metal material.

[0088] S2: Peel the cured PDMS cavity structure from the male mold, and punch holes at the top and side of the PDMS cavity structure with a punch; Wash the punched PDMS cavity structure with absolute ethanol and pure water in turn, and the washing time is not less than 5 minutes. After washing, put it into an oven at 60 °C for drying to obtain a dried chip body with a PDMS chamber structure.

[0089] S3: Pour the PDMS prepolymer solution (prepared by completely mixing commercially available PDMS components and PDMS fixing solution at a mass ratio of 5:1) into a rectangular box composed of ultra-clean glass slides. After removing the bubbles in the PDMS prepolymer solution in a vacuum drying oven, place it in an oven at 60 °C for curing for 4 hours. Remove the rectangular box composed of ultra-clean glass slides to obtain a rectangular PDMS structure. Then, use a punch to drill holes in the middle area of the cured rectangular PDMS structure to form an intermediate buffer pool made of PDMS with a cavity. Wash the cured PDMS successively with absolute ethanol and pure water for no less than 5 minutes. After washing, place it in an oven at 60 °C to dry, and obtain a dry intermediate buffer pool.

[0090] S4: Wash two stainless steel hollow tubes with a diameter difference of 200 microns, namely the thick tube and the thin tube, successively with absolute ethanol and pure water for no less than 5 minutes. After washing, dry them. Uniformly spray a release agent inside and outside the tubes and then let it dry. Sleeve the stainless steel hollow tube with a smaller diameter (the thin tube) in the center of the stainless steel hollow tube with a larger diameter (the thick tube) and fix it. Pour the mixed prepolymer silicone rubber liquid (PDMS is Dow Corning 184 type, and the mixing ratio of the rubber and the curing agent is 15:1 by mass) into the sandwich of the two stainless steel hollow tubes, remove the bubbles by centrifugation and then cure it.

[0091] S5: Pull out the two stainless steel hollow tubes to obtain an elastic thin tube with a wall thickness of 200 microns. Ultrasonically wash the elastic hollow thin tube successively with absolute ethanol and pure water for no less than 1 minute. After washing, dry it to obtain a dry elastic thin tube.

[0092] S6: Wash the rigid microtubes with a square cross-section of 50-micron width made by photolithography successively with absolute ethanol and pure water for no less than 5 minutes. After washing, dry them to obtain dry rigid microtubes.

[0093] S7: Prepare a negative template with length scales using the SU-8 process. Cover the negative template with the PDMS prepolymer solution, remove the bubbles and then cure it. Cut off the cured PDMS positive mold layer, apply color to the scale area and then dry it to obtain a wrapping layer with length scales.

[0094] S8: Pour a rectangular structure model with the PDMS prepolymer solution (prepared by completely mixing commercially available PDMS components and PDMS fixing solution at a mass ratio of 5:1). After curing, form a platform structure. Wash it successively with absolute ethanol and pure water for no less than 5 minutes. After washing, dry it to obtain a dry platform.

[0095] S9: Place the rigid microtube on the upper surface of the carrier and fix it. Then, perform plasma treatment on the scale wrapping layer and the carrier with the rigid microtube made of glass fixed thereon to modify its surface. After that, wrap the scale wrapping layer around the periphery of the rigid microtube and perform irreversible bonding encapsulation with the carrier.

[0096] S10: Insert the hollow elastic tube into the openings on both sides of the chamber of the chip body and fix it. Then, perform irreversible bonding encapsulation on the chamber base of the chip body to form a chamber for culturing cells in the chip body; Connect one end of the elastic thin tube to the opening on one side of the middle buffer pool; Connect the opening on the other side of the middle buffer pool to the open end of the rigid hollow microtube. Among them, the chamber base can adopt the structure of a super-clean glass slide or a cured PDMS sheet.

[0097] S11: Pour the pure water with red color, i.e., the measurement liquid, from the side of the chamber of the chip body cavity connected to the elastic thin tube. Stop pouring when the measurement liquid fills the elastic thin tube and the middle buffer pool. Seal the pouring port with a plug bolt to obtain the overall structure of the organ chip for measuring the contractile force and contraction frequency of cardiomyocytes. It should be noted that the role of the buffer pool is to control the measurement liquid to only fill the buffer pool when pouring the measurement liquid. As can be observed through a stereomicroscope, the excess liquid can be drawn out from the pouring port. There is a small section without scale in the part where the rigid microtube is connected to the middle buffer pool. Even if some measurement liquid enters the rigid microtube as long as it does not exceed the scale area, it will not affect the subsequent measurement.

[0098] S12: To ensure a sterile environment, soak the overall structure of the organ chip in absolute ethanol for 30 minutes and then soak it in sterile pure water for 1 hour in sequence; Take out the overall structure of the organ chip and dry it at a low temperature (20 - 40 degrees Celsius) in a sterile environment, and then irradiate it under ultraviolet light for 30 minutes to obtain the sterilized organ chip for measuring the contractile force and contraction frequency of cardiomyocytes.

[0099] The organ chip for measuring the contractile force and contraction frequency of cardiomyocytes provided by the present invention can be prepared into a measuring device integrated with a single measuring unit and multiple measuring units. The chip preparation cost is low, the detection experiment operation is simple, and the measuring device integrated with multiple measuring units can also achieve high-throughput detection. It can achieve rapid detection by observing the change of the scale with the naked eye within one minute, that is, calculate the contractile force and contraction frequency of cardiomyocytes through the moving distance of the liquid surface. The whole detection process does not need to rely on a microscopic imaging system to obtain the liquid surface change amount.

[0100] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art can use the disclosed technical content above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An organ chip for measuring the contractility and contraction frequency of cardiomyocytes, characterized in that: The invention comprises a chip body, an elastic capillary, an intermediate buffer tank, a rigid microtube, a scale wrapping layer and a support platform, wherein the chip body, the intermediate buffer tank and the support platform are arranged adjacent to each other in sequence, the center of the chip body is a hollow chamber, a sample addition port is arranged above the chamber, and the elastic capillary is arranged in the chamber; the elastic capillary is deformed by the contraction force of the myocardial cells attached to its surface, and the elastic capillary, the intermediate buffer tank and the rigid microtube are connected in sequence; the rigid microtube is arranged on the support platform, the scale wrapping layer is arranged on the support platform, the scale wrapping layer wraps the rigid microtube, and a ruler showing the scale is engraved on the scale wrapping layer, which is used to measure the scale change of the liquid level in the rigid microtube.

2. The organ chip according to claim 1, characterized in that: The structure of the chamber is circular, square or trapezoidal, the volume of the chamber is 200 to 2000 cubic millimeters, and the opening diameter of the sample adding port is 0.5 to 5 millimeters.

3. The organ chip according to claim 1, characterized in that: One end of the elastic capillary is connected to the middle cavity of the middle buffer tank, and the other end of the elastic capillary is provided with a plugging bolt; the inner diameter of the central cavity of the elastic capillary is 1 to 1.5 mm, the tube wall thickness is 0.2 to 1 mm, and the length of the elastic capillary does not exceed 2 cm.

4. The organ chip according to claim 1, characterized in that: The diameter of the middle cavity of the rigid microtube is 10 to 50 micrometers; the cavity volume of the middle buffer pool is 100 to 500 microliters.

5. The organ chip according to claim 1, characterized in that: The scale is a length scale with a scale value of 0 to 5 cm and an accuracy of 0.01 cm.

6. The organ chip according to claim 1, characterized in that: The horizontal inclination of the upper surface and the lower surface of the support platform does not exceed plus or minus 1°; during measurement, the elastic capillary and the middle buffer tank are filled with colored liquid.

7. The organ chip according to claim 1, characterized in that: The material of the elastic capillary is elastic silicone rubber material, whose elastic modulus is 800kPa~1MPa and whose rebound rate is 75~85%; the material of the rigid microtube is light-transmitting glass or low-elastic PDMS material, whose elastic modulus is 3~5MPa and whose rebound rate is less than 40%; the material of the intermediate buffer tank is light-transmitting material, whose transmittance is ≥80%; the material of the support is PDMS material, metal or hard plastic.

8. The method for preparing an organ chip according to any one of claims 1 to 7, characterized in that: It includes the following steps: S1: Covering the PDMS prepolymer solution on the positive mold of the chip body, putting it into an oven for curing, and obtaining a cavity structure of a cured PDMS material; S2: using a puncher to punch holes on the top and side of the PDMS cavity structure; cleaning the punched PDMS cavity structure with anhydrous ethanol and pure water, and drying it in an oven to obtain a dry cavity structure of the chip body; S3: preparing a rectangular PDMS structure, drilling a hole in the middle region thereof with a puncher to form an intermediate buffer pool of PDMS material with a cavity; washing it with anhydrous ethanol and pure water, and drying it after washing to obtain a dry intermediate buffer pool; S4: Spray the release agent evenly on the inside and outside of the thick tube and thin tube of two clean stainless steel hollow tubes with a diameter difference of 100 to 200 microns, and then dry them. Put the thin tube with a smaller diameter into the center of the thick tube with a larger diameter and fix it; pour the mixed pre-polymerized silicone rubber liquid into the sandwich of the two stainless steel hollow tubes, centrifuge to remove bubbles, and then cure; S5: extracting the two stainless steel hollow tubes to obtain elastic capillaries; ultrasonic cleaning and drying to obtain dry elastic capillaries; S6: making a rigid microtube from glass, washing it with anhydrous ethanol and pure water in turn, and drying it after washing to obtain a dry rigid microtube; S7: Cover the PDMS prepolymer solution on the graduated negative template and solidify it; The solidified PDMS positive mold layer with scale is cut off, and the scale area is painted with color and then dried to obtain a scale wrapping layer with length scale; S8: casting a model of a support platform with a PDMS prepolymer solution, forming a support platform structure after solidification, and then washing and drying to obtain a dry support platform; S9: placing the rigid microtube on the upper surface of the support and fixing it, then subjecting the scale wrapping layer and the support fixed with the rigid microtube made of glass to plasma treatment, and then wrapping the scale wrapping layer around the rigid microtube and bonding it to the support for packaging; S10: inserting an elastic capillary into the openings on both sides of the cavity of the chip body and fixing them, using an ultra-clean glass sheet as the base of the cavity, and then irreversibly bonding and encapsulating the cavity structure of the chip body and the base, so that the chip body forms a cavity for culturing cells; connecting one end of the elastic capillary to an opening on one side of the middle buffer tank; connecting the other opening of the middle buffer tank to the open end of the rigid microtube; S11: The measuring liquid is poured into the chamber from one side of the elastic capillary. When the measuring liquid fills the elastic capillary and the intermediate buffer tank, the perfusion is stopped. The perfusion port is sealed with a plugging bolt. After sterilization, an organ chip for measuring the contractile force and contraction frequency of cardiomyocytes is obtained.

9. The preparation method according to claim 8, characterized in that: The PDMS prepolymer solution is a mixture of PDMS and a curing agent matching PDMS at a mass ratio of 5:1 to 20:1; in step S4, the diameter difference between the two stainless steel hollow tubes is 200 microns; in step S6, the inner diameter of the rigid microtube is 20 microns.

10. A method for detecting the contractility and contraction frequency of myocardial cells, characterized in that: The method of using the organ chip according to any one of claims 1 to 7 for detection comprises the following steps: 1) A culture medium solution containing a final concentration of 0.05 to 0.25 mg / mL collagen is perfused into the chip body cavity, and the chip is placed in a 35 to 37° C. incubator for incubation, and then the culture medium solution is removed to complete the coating of the chip body cavity; 2) Transplanting cardiomyocytes into the chamber of the chip body so that the cardiomyocyte clusters are wrapped around the elastic capillaries; 3) Read the change of the liquid level scale per unit time in the rigid microtube to obtain the velocity v of the measured liquid, record it in a certain time dt, the difference of the velocity change of the measured liquid before and after this period of time is dv, and the distance moved by the liquid surface is L, first calculate the acceleration a=dv / dt, and then according to W 测 =πr 细 2 ×L×ρ 测 Obtain the mass of the measured liquid according to F = W 测 ×a is to obtain the contractile force F of the myocardial cell cluster; By counting the number of times T that the liquid level changes within a specific time period t, the contraction frequency per minute H = T / t is obtained; Among them, r 细 is the radius of the rigid microtubule, ρ 测 To measure the density of liquids.

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