Microfluidic device for endothelialization evaluation of tissue engineering valve

Abstract

The invention belongs to the technical field of cell mechanics biology experiment devices for health and rehabilitation engineering, and relates to a microfluidic device for endothelialization evaluation of a tissue engineering valve, which comprises a microfluidic chip, peripheral equipment, a signal acquisition and processing system and a feedback system. The biomechanical principle and the micro-fluidic chip technology are ingeniously combined, the control system, the mechanical loading device and the micro-fluidic chip are connected, a micro-fluidic endothelial cell culture cavity and a peripheral system are constructed, real-time accurate control over aortic valve blood flow is achieved through the PID control system, and the system is suitable for large-scale popularization and application. Shear stress applied to a tissue engineering valve endothelial cell culture area is generated, so that the real stress condition of the tissue engineering valve endothelial cells is simulated, and the method is used for analyzing and researching the influence rule of mechanical loading on the tissue engineering valve endothelial cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell mechanics biology experimental devices for health and rehabilitation engineering, and relates to a microfluidic device for evaluating the endothelialization of tissue engineering valves. Background Art

[0002] Heart valve diseases are common diseases. Generally speaking, these diseases may cause failure of any of the four heart valves, mainly due to stenosis or regurgitation, and can only be treated by surgery or transcatheter intervention. Obviously, any heart valve disease will change its hemodynamic performance, and the purpose of treatment is to restore blood flow to a healthy state. At present, there are two types of surgical artificial valves available: mechanical valves and biological valves. Mechanical valves are very durable and can be used in artificial hearts; however, their main disadvantage is the formation of thrombi due to non-physiological hemodynamics. On the other hand, biological valves mimic the function of natural valves, thus eliminating the need for long-term anticoagulation, but compared with mechanical valves, the problem of their limited durability is currently difficult to overcome.

[0003] Tissue Engineering Heart Valve (TEHV) is a new type of valve in the field of heart valve replacement in recent years and is the future direction of valve development. It uses advanced biotechnology and materials science principles to culture the patient's own cells or tissues on a specific scaffold to form a bioactive valve. The structure of the tissue engineering valve is basically the same as that of the normal heart valve, and it has better hemodynamic characteristics compared with mechanical valves. The tissue engineering valve has biological activity, can grow with the growth and development of the body, is not easy to degenerate and decay, has strong durability, and can effectively avoid secondary surgery. This feature has obvious advantages compared with biological valves (such as porcine aortic valves and bovine pericardial bioprostheses), because the service life of biological valves is relatively short, generally between 10 and 20 years, and secondary surgery may be required due to degenerative changes. In addition, the cell components inside and on the surface of the tissue engineering valve are the same as those of the normal valve and are autologous cells, so there is no immunogenicity, and anticoagulation treatment is not required after implantation, avoiding the bleeding risk and other complications that anticoagulation treatment may bring.

[0004] Endothelialization refers to the process of implanting a seed cell layer equivalent to endothelial cells on the inner surface of the valve scaffold to provide an anticoagulant surface and maintain the normal function of the valve. Endothelial cells have functions such as anti-thrombosis, preventing platelet adhesion, preventing leukocyte adhesion, and inhibiting smooth muscle cell proliferation. The lack of endothelial coverage or the loss of endothelial cell function is the main cause of the aging and calcification of tissue-engineered valves, and it is also the key difficulty in the clinical translation of tissue-engineered valves at present. However, the endothelialization of tissue-engineered valves is a complex process involving multiple factors such as endothelial cell proliferation, blood dynamic flow, regulation of biochemical factor concentration, and the influence of valve material properties. Moreover, there are bottleneck problems such as complex operation, high cost, long cycle, and ethical disputes in clinical and in vivo experiments. Therefore, there is an urgent need to develop an in vitro evaluation device for the endothelialization of tissue-engineered valves with high efficiency and low cost.

[0005] Currently, there are few existing in vitro evaluation devices for tissue-engineered valves, and they are all based on macroscopic in vitro flow culture chambers to fix the valve leaflets in the reaction chamber and ensure that endothelial cells firmly adhere to the surface of the tissue-engineered valve without falling off. For example: a pulsatile flow culture device for cardiac tissue-engineered valves (Patent No.: CN200320129429.8), a flat plate flow reaction chamber for tissue engineering dynamic culture (Patent No.: CN202120468435.4). However, these devices can only achieve simple hemodynamic signals and cannot reproduce complex and real aortic hemodynamic signals, making it difficult to replicate the accurate spatio-temporal distribution of shear stress on the endothelial cells of tissue-engineered valves. The development of microfluidic chip technology provides an effective experimental platform for precisely simulating the extracellular hemodynamic microenvironment and observing and detecting the interaction between the cell microenvironment and cells. At the same time, microfluidic chips have advantages such as a small required sample volume, easy integration, easy optical detection, and good biocompatibility, making them an ideal experimental platform for reproducing the arterial hemodynamic microenvironment in the in vivo blood circulation system. However, an in vitro evaluation device for tissue-engineered valves based on microfluidic chips has not appeared so far. Summary of the Invention

[0006] The design objective of the present invention is to provide a microfluidic chip for the endothelialization of tissue-engineered valves and simulating mechanical loading, which can be used to reproduce the distribution of shear stress on the endothelial cells of the real aortic valve, and based on this, accurately evaluate the endothelialization of tissue-engineered valves. The present invention innovatively introduces microfluidic chip technology, aiming to precisely simulate the biological effects of shear stress on endothelial cells on the human aortic valve, predict the endothelialization performance of tissue-engineered valve endothelial cells on the human aortic valve, and thus provide strong technical support for valve performance evaluation and optimization.

[0007] The present invention ingeniously combines biomechanical principles with microfluidic chip technology, connects a control system, a mechanical loading device, and a microfluidic chip to construct a microfluidic endothelial cell culture chamber and a peripheral system, and realizes real-time and precise control of blood flow in the aortic valve through a PID control system, generating shear stress applied to the endothelial cell culture area of the tissue-engineered valve to simulate the real stress situation of endothelial cells in the tissue-engineered valve, and is used to analyze and study the influence law of mechanical loading on endothelial cells of the tissue-engineered valve.

[0008] The technical solution of the present invention is as follows:

[0009] A microfluidic device for evaluating endothelialization of tissue-engineered valves, comprising a microfluidic chip, peripheral devices, and a signal acquisition and processing system and a feedback system.

[0010] The microfluidic chip includes a microfluidic chip channel, a microfluidic chip inlet, and a microfluidic chip outlet; the microfluidic chip channel is a groove structure etched inside the microfluidic chip and serves as a cell culture chamber. The microfluidic chip inlet and the microfluidic chip outlet are both connected to the cell culture chamber to form a hydrodynamic loop; the tissue-engineered valve is fixed on the upper surface of the cell culture chamber, and endothelial cells are located on the lower surface of the tissue-engineered valve, and the wall shear stress signal in the in-vivo aortic valve endothelial hemodynamic microenvironment is simulated in the cell culture chamber.

[0011] The peripheral devices provide a power flow for the microfluidic chip, including a liquid storage tank, an air pump, and a fluid loading device; the fluid loading device is connected to the liquid storage tank through a pipeline, and the fluid in the fluid loading device is pumped into the liquid storage tank by the air pump; the liquid storage tank is connected to the microfluidic chip inlet and outlet through pipelines to form a fluid circulation loop, and fluid is provided to the cell culture chamber through the microfluidic chip inlet and flows out through the microfluidic chip outlet.

[0012] The signal acquisition and processing system and the feedback system are used for the acquisition, processing, and feedback of hemodynamic signals and intracellular biological and chemical signals on the microfluidic chip, including a flow sensor, a fluorescence microscope, a CCD, and an industrial control computer; the flow sensor, the fluorescence microscope, and the CCD are all connected to the industrial control computer; the flow sensor acquires the flow signals at the front and rear inlets and outlets of the microfluidic chip, and the fluorescence microscope and the CCD camera are combined to detect the dynamic process images of the biological responses of endothelial cells of the tissue-engineered valve; the acquired images are sent to the industrial control computer, and control instructions are formed by the industrial control computer to precisely control the fluid loading device to generate the wall shear stress signal in the real aortic tissue-engineered valve endothelial cell hemodynamic microenvironment in the cell culture chamber of the microfluidic chip.

[0013] The beneficial effects of the present invention:

[0014] The present invention can conveniently carry out experiments on the interaction between hemodynamic signals and tissue-engineered valve endothelial cells under real physiological conditions outside the cell, accurately and completely replicating the hemodynamic microenvironment at the endothelial cells of the real aortic tissue-engineered valve. It has a higher integration level, less consumables, and can observe and collect the cell mechanobiological response in real time, providing an efficient and powerful experimental platform for studying the quantitative relationship between hemodynamic signals and the mechanobiological mechanism of tissue-engineered valve endothelial cells.

[0015] The device of the present invention integrates advanced computational fluid dynamics (CFD) simulation technology, microfluidic chip and biomedical engineering technology, and can realize in vitro simulation, accurate evaluation and in-depth analysis of the mechanobiology research of tissue-engineered valve endothelial cells, providing a scientific basis and technical support for the treatment of valve diseases. Brief Description of the Drawings

[0016] Figure 1 It is a schematic diagram of the microfluidic chip.

[0017] Figure 2 It is a schematic cross-section of the cell culture chamber.

[0018] Figure 3 It is a structural diagram of the in vitro simulation circulation system at the microfluidic chip level.

[0019] In the figure: 1-1 inlet hole of the microfluidic chip; 1-2 outlet hole of the microfluidic chip; 1-3 upper layer of PDMS; 1-4 lower layer of PDMS; 1-5 tissue-engineered valve; 1-6 endothelial cells; 1-7 microfluidic chip channel; 2-1 air pump; 2-2 fluid loading device; 2-3 liquid storage tank; 2-4 microfluidic chip; 2-5 inlet flow sensor of the microfluidic chip; 2-6 outlet flow sensor of the microfluidic chip; 2-7 fluorescence microscope; 2-8 CCD camera; 2-9 industrial control computer. Detailed Embodiment

[0020] The technical solution of the present invention will be further described below in combination with specific embodiments and drawings.

[0021] A microfluidic device and method for evaluating the endothelialization of tissue-engineered valves. The system consists of three basic units: The first basic unit is a microfluidic chip that simulates the wall shear stress signal in the in-vivo aortic valve endothelial hemodynamic microenvironment in the cell culture chamber, including the cell culture chamber and the tissue-engineered valve, forming a multi-component hydrodynamic circuit. The second basic unit is a peripheral device that provides the power flow for the microfluidic chip, including a liquid storage tank, an air pump, and a fluid loading device. Among them, the liquid storage tank is connected to the inlet and outlet of the microfluidic chip to form a fluid circulation circuit, and the air pump is connected to the liquid storage tank through the fluid loading device. The third basic unit is a hemodynamic signal, intracellular biological and chemical signal acquisition, processing system, and feedback system on the microfluidic chip, including sensors, a fluorescence microscope, a CCD, and an industrial control computer. The signal enters the industrial control computer through the sensor, fluorescence microscope, or CCD, and after being processed by the industrial control computer, control instructions are formed to precisely control the fluid loading device to generate the wall shear stress signal in the target aortic tissue-engineered valve endothelial hemodynamic microenvironment in the cell culture chamber on the microfluidic chip.

[0022] The cell culture chamber on the described microfluidic chip is as Figure 2 shown. The upper and lower layers of PDMS with flat cubic channels are separated by the tissue-engineered valve to form the cell culture chamber, and the lower cell culture chamber is connected to the circulation path. The length L c 、width W c and height H c of the culture chamber satisfy: H c << W c and H c << L c . When the fluid is driven by pressure, the tissue-engineered valve is stretched, and the endothelial cells cultured on the top of the tissue-engineered valve in the lower culture chamber are subjected to the action of the wall shear stress τ w (t). Since the height of the cell culture chamber is much smaller than its width and length, the flow Reynolds number Re and Womersley number α in the cell culture chamber satisfy Re << 1 and α << 1, meeting the quasi-steady assumption. Further assuming that the tissue-engineered valve undergoes small deformation after being stretched, the wall shear stress τ w (t) borne by the endothelial cells should satisfy:

[0023]

[0024] In the formula, η is the viscosity of the cell culture medium, and q(t) is the volume flow rate through the cell culture chamber. Therefore, the magnitude τ of the shear stress applied to the endothelial cells of the tissue-engineered valve is proportional to the fluid flow rate q(t) through the channel, and the magnitude of the shear stress can be quantitatively regulated by controlling the flow rate q(t).

[0025] Further, the industrial control computer uses the PID algorithm to achieve precise real-time control of mechanical loading. The formula used when adjusting the relationship between PID parameters and output is as follows:

[0026]

[0027] In the formula, e(t) is the deviation, that is, the difference between the set value and the actual output value. u(t) is the output voltage, dt is the time change, and K P is the proportionality coefficient, K i is the integral coefficient, K d is the differential coefficient. Adjusting these three parameters makes the PID control more precise and rapid.

[0028] The fluid circuit on the microfluidic chip discharges gas into the liquid storage tank through a fluid loading device. Due to the pressure change, an equal amount of liquid is discharged. The liquid enters the chip through the microfluidic chip inlet, leaves the chip through the microfluidic chip outlet, and returns to the liquid storage tank for recycling. The design of the hydrodynamic circuit on the microfluidic chip needs to make the wall shear stress borne by the endothelial cells cultured on the tissue engineering valve at the top of the cell culture chamber consistent with the wall shear stress τ w (t) waveform of the aortic valve endothelial microenvironment.

[0029] The fluid loading device uses a programmable air pump manufactured by Elveflow. Relevant programs are written based on the Labview platform to generate the target arterial endothelial microenvironment flow rate waveform q(t) in the cell culture chamber of the microfluidic chip. Thus, according to Equation (1), it can be known that the endothelial cells on the tissue engineering valve at the top of the cell culture chamber are subjected to the action of the wall shear stress τ w (t).

[0030] The fluid flow rate waveform and shear stress waveform of the target arterial endothelial microenvironment can be obtained through computational fluid dynamics (CFD) simulation technology.

[0031] The signal acquisition and processing system and the feedback control system are as Figure 3 shown. The signal acquisition and processing system includes an inverted fluorescence microscope, a CCD industrial camera, and a flow sensor, which are used to monitor and collect the flow waveforms q(t) at the input and output ends of the cell culture chamber in real time, and the actual state of the cell mechanobiological response in the chip cell culture chamber. The collected signals are fed back to the industrial control computer to further adjust the fluid loading device, quantitatively control the changes in the wall shear stress and flow signals acting on the circulation simulation system, and finally generate the action of the human real wall shear stress τ w (t) in the cell culture chamber of the microfluidic chip. In addition, the dynamic process of the mechanobiological response of aortic valve endothelial cells is detected, recorded, and saved to the industrial control computer by a microscope combined with a CCD camera.

[0032] AsFigure 1 and Figure 2 As shown in Figure 2 , a microfluidic device for precisely simulating the endothelialization of tissue-engineered valves according to the present invention includes an upper layer of PDMS 1-3 and a lower layer of PDMS 1-4, and the upper layer of PDMS 1-3 is bonded to the lower layer of PDMS 1-4. PDMS is prepared from a prepolymer A glue and a cross-linking agent B glue. Before fabricating the chip, a preliminary experiment is first conducted on the ratio of the prepolymer A glue to the cross-linking agent B glue, aiming to select the most suitable ratio for mechanical loading, so as to ensure appropriate elastic deformation under mechanical loading. Vertically arranged inlet holes 1-1 and outlet holes 1-2 are etched inside the upper layer of PDMS 1-3. The cell culture chamber on the microfluidic chip is as Figure 2 shown. The tissue-engineered valve 1-5 separates the upper and lower layers of PDMS to form a cell culture chamber 1-7, and endothelial cells 1-6 are cultured on the tissue-engineered valve.

[0033] As Figure 3 shown, it is a system diagram of a microfluidic chip for precisely simulating the endothelialization of tissue-engineered valves according to the present invention. The microfluidic chip system includes a microfluidic chip, a peripheral system (an air pump 2-1, a fluid loading device 2-2, and a liquid storage tank 2-3), a signal acquisition and processing system, and a feedback system (an inlet flow sensor 2-5, an outlet flow sensor 2-6, a fluorescence microscope 2-7, a CCD camera 2-8, and an industrial control computer 2-9). The peripheral system is used to generate shear stress applied to the cell culture area of the aortic tissue-engineered valve and observe its effect on endothelial cells.

[0034] The specific implementation method of the microfluidic device for precisely simulating the endothelialization of tissue-engineered valves according to the present invention is as follows:

[0035] Step 1: Conduct a preliminary experiment on the PDMS material ratio. Both layers of PDMS need to have a suitable elastic modulus. Verify the following ratios: the volume ratios of the prepolymer A glue to the cross-linking agent B glue of PDMS are 10:1, 8:1, and 6:1 respectively. The smaller the ratio of the prepolymer A glue to the cross-linking agent B glue, the greater the elasticity of PDMS and the easier it is to deform. Select the most suitable ratio for mechanical loading through the preliminary experiment to ensure that the chip can have appropriate elastic deformation under mechanical loading.

[0036] Step 2: Design a "sandwich" microfluidic chip. The inlet hole 1-1 and the outlet hole 1-2 of the microfluidic chip are both round holes with a diameter of 2 mm, and the holes are through holes in the upper layer of PDMS; the height H c 、width W c and length L cThey are 0.5 mm, 10 mm, and 15 mm respectively. Usually, the viscosity η of the cell culture medium is 0.001 Pa·s. Each channel and chamber structure of the microfluidic chip is fabricated using standardized microfabrication methods. The chip material uses PDMS material and is bonded and sealed with a clean glass slide to form a transparent glass-PDMS chip with good biocompatibility. The etching height of all microfluidic channels is 150 μm.

[0037] Step 3: Fabricate a microfluidic chip based on the design in Step 1. The microfluidic chip can be processed using standard soft lithography technology and laser engraving technology. In the device structure made of polydimethylsiloxane (PDMS) material, a 150-μm-thick polyimide film is obtained by cutting with a laser engraving machine and pasted on the silicon wafer. Place the silicon wafer in a glass dish, pour in the prepared PDMS solution, then let it stand in a vacuum for 20 min to remove air bubbles, put it in an oven, bake at 80 °C for 90 min to cure the PDMS. After that, take out the glass dish. After its temperature drops, separate the PDMS from the mold to obtain PDMS with a microfluidic channel structure. Cut off the PDMS with the used microfluidic channels, use a punch to punch through the channel entrances and exits. After cleaning, sandwich the tissue engineering valve between the PDMS and the clean lower PDMS and bond them together. Heat at 120 °C for 15 - 20 min to make the bond more stable. Then insert the catheter into the holes punched on the chip and evenly apply glue at the junction of the catheter and the hole to avoid liquid leakage. After applying the glue, place the chip in an oven and bake at 80 °C overnight. Finally, connect the catheter and the fluid loading device. Thus, the preparation of the device is completed.

[0038] Step 4: Put the fabricated microfluidic chip into water to evacuate air bubbles, and then place it in a high-temperature and high-pressure sterilizer for sterilization after the air bubbles in the channels are exhausted. The sterilized microfluidic chip is placed in a laminar flow hood for standby.

[0039] Step 5: Passage culture the primary cultured aortic valve endothelial cells using the endothelial cell-specific EGM medium (Lonza Benelux). The 2nd - 5th passages are used for experiments. During the experiment, seed the endothelial cells on the lower surface of the tissue engineering valve located in the cell culture chamber of the chip coated with Fibronection, so that the cells adhere to the wall and the confluence reaches more than 90% as Figure 1 shown.

[0040] Step 6: Collect the shear force waveform received by the aortic valve during normal human conditions as the shear force waveform received by the aortic tissue engineering valve through fluid dynamics (CFD) simulation technology. Calculate the target flow waveform and input the proportional coefficient K in the three-dimensional mechanical loading PID control into the industrial control computer p1 and the integral coefficient K i1, Differential coefficient K d1 The initial value is output to the high-precision fluid loading device 2-2, and the fluid loading device 2-2 precisely controls the air pump 2-1 to achieve the compression force, tensile force, and shear force on the microfluidic chip. The microfluidic chip feeds back the inlet and outlet flow rates to the inlet flow sensor 2-5 and the outlet flow sensor 2-6. The flow sensors can accurately detect the magnitude of the flow rate on the microfluidic chip and transmit the flow rate signal back to the industrial control computer in real time. The industrial control computer calculates the initial value and the error signal, and recalculates the input quantity through the PID calculation formula to achieve closed-loop real-time control. If the returned flow rate signal is inappropriate, the proportionality coefficient K can be adjusted manually in a timely manner p1 , Integral coefficient K i1 , Differential coefficient K d1 to reproduce the target mechanical signal quickly, precisely, and with high signal-to-noise ratio

[0041] Step 7: After adjusting and stabilizing the mechanical environment of the microfluidic chip according to Step 6, observe the flow of nanoparticles under transient conditions through an inverted fluorescence microscope 2-7. The CCD camera collects video images at a shooting rate of n frames per second, and the images can be observed and collected on the screen of the industrial control computer 2-9

Claims

1. A microfluidic device for evaluating endothelialization of tissue-engineered valves, characterized in that: The microfluidic device for evaluating endothelialization of tissue-engineered valves comprises a microfluidic chip, peripheral equipment, and a signal acquisition and processing system and a feedback system; The microfluidic chip comprises a microfluidic chip channel, a microfluidic chip inlet and a microfluidic chip outlet; the microfluidic chip channel is a groove structure etched inside the microfluidic chip, which serves as a cell culture chamber, and the microfluidic chip inlet and the microfluidic chip outlet are both connected to the cell culture chamber to form a fluid mechanics circuit; the tissue engineering valve is fixed on the upper surface of the cell culture chamber, and the endothelial cells are located on the lower surface of the tissue engineering valve, and the wall shear stress signal in the in vivo aortic valve endothelial hemodynamic microenvironment is simulated in the cell culture chamber; The peripheral device provides power flow for the microfluidic chip, including a liquid storage tank, an air pump and a fluid loading device; the fluid loading device is connected to the liquid storage tank through a pipeline, and the fluid in the fluid loading device is pumped into the liquid storage tank through the air pump; the liquid storage tank is connected to the inlet and outlet of the microfluidic chip through a pipeline to form a fluid circulation loop, and the fluid is provided to the cell culture chamber through the inlet of the microfluidic chip, and flows out through the outlet of the microfluidic chip; The signal acquisition and processing system and feedback system are used for the acquisition, processing and feedback of hemodynamic signals and intracellular biological and chemical signals on the microfluidic chip, including a flow sensor, a fluorescence microscope, a CCD and an industrial computer; the flow sensor, the fluorescence microscope and the CCD are all connected to the industrial computer; the flow sensor acquires flow signals at the front and rear inlets and outlets of the microfluidic chip, and the fluorescence microscope and the CCD camera are combined to detect the dynamic process images of the biological response of the tissue engineering valve endothelial cells; the acquired images are sent to the industrial computer, and the industrial computer controls the fluid loading device to generate the wall shear stress signal in the real aortic tissue engineering valve endothelial cell hemodynamic microenvironment in the cell culture chamber on the microfluidic chip.

2. A microfluidic device for evaluating endothelialization of tissue-engineered valves according to claim 1, characterized in that: Cell culture chamber length L c , Width W c and height H c Satisfaction: H c < <W c And H c < <L c ; Wall shear stress τ on endothelial cells w (t) shall meet the following requirements: Where η is the viscosity of the cell culture fluid, q(t) is the volume flow rate through the cell culture chamber; the shear stress τ loaded on the tissue engineered valve endothelial cells is proportional to the fluid flow q(t) flowing through the channel, and the shear stress can be quantitatively controlled by controlling the flow q(t).

3. A microfluidic device for evaluating endothelialization of tissue-engineered valves according to claim 1, characterized in that: The industrial computer uses the PID algorithm to achieve accurate real-time control of mechanical loading. The formula used to adjust the PID parameters and output relationship is: Where e(t) is the deviation, i.e. the difference between the set value and the actual output value, u(t) is the output voltage, dt is the time change, K P is the proportionality coefficient, K i is the integration coefficient, K d It is the differential coefficient. Adjusting these three parameters makes PID control more accurate and rapid.

4. A microfluidic device for evaluating endothelialization of tissue-engineered valves according to claim 1, characterized in that: The fluid circuit on the microfluidic chip discharges gas into the liquid storage tank through the fluid loading device, and discharges an equal amount of liquid due to the pressure change. The liquid enters the chip through the microfluidic chip inlet, leaves the chip through the microfluidic chip outlet and returns to the liquid storage tank for recycling.

5. A microfluidic device for evaluating endothelialization of tissue-engineered valves according to claim 1, characterized in that: The fluid loading device adopts a programmable air pump.

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