An in-vitro cell culture system and method for studying the regulation of arterial endothelial cell function by external counterpulsation therapy

Abstract

This invention relates to an in vitro cell culture system and method for studying the regulation of arterial endothelial cell function by external counterpulsation therapy, belonging to the technical field of cellular mechanobiology experimental devices. The system includes: 1) an in vitro cell culture microfluidic chip capable of combining pressure, shear stress, and circumferential stress stimulation, and an external afterload hemodynamic multi-element extracorporeal circulation loop, with endothelial cells seeded on an elastic membrane within the cell culture cavity on the chip; 2) a peripheral flow loading device used to simulate the cardiovascular system's power source and the sequential pulsed pressurization effect of external counterpulsation, which can apply hemodynamic signals such as blood pressure and shear stress induced by different counterpulsation modes to the endothelial cells on the chip; and 3) a biochemical signal observation and feedback control system for the peripheral endothelial cells. This system provides an objective, standardized, and quantitative experimental platform for studying the laws governing the regulation of endothelial function by changes in the arterial hemodynamic microenvironment caused by different external counterpulsation modes, as well as the mechanobiological mechanisms involved.

Description

Technical Field

[0001] This invention belongs to the technical field of experimental devices for cell mechanobiology, specifically relating to an in vitro cell culture system and method for studying the regulation of arterial endothelial cell function by in vitro counterpulsation therapy. It is based on hemodynamic principles, machine learning algorithms, microfluidic chips, and intelligent feedback control technology. The design includes an in vitro cell culture microfluidic chip for simultaneously applying pressure, shear force, and circumferential stress; a chip for applying hemodynamic signal stimulation to the arterial endothelial microenvironment under the action of in vitro counterpulsation therapy in different counterpulsation modes; and a multi-element circulation loop and peripheral flow loading system. Background Technology

[0002] Enhanced external counterpulsation (EECP) is the only non-invasive mechanical circulatory support method internationally that has received FDA approval in the United States and is recommended by the American Heart Association / Actress Committee (AHA / ACC), the European Society of Cardiology (ESC), and the Chinese Medical Association clinical guidelines. Due to its significant efficacy, ease of operation, and non-invasive safety, it is widely used in the treatment of ischemic stroke, coronary heart disease, angina pectoris, and other chronic ischemic diseases, and its application is expanding in the prevention and treatment of atherosclerotic cardiovascular diseases and postoperative rehabilitation.

[0003] Over the past two decades, basic and clinical research has suggested that the mechanism of action of EECP (External Electroconvulsive Pulsation) is through vascular mechanobiological effects induced by immediate hemodynamic effects. Specifically, it involves altering the arterial hemodynamic microenvironment caused by different external counterpulsation modes, thereby regulating arterial endothelial cell function to achieve the desired therapeutic and rehabilitative goals. The vascular endothelium, a barrier between blood and the vessel wall, senses changes in hemodynamic signals such as wall shear force, blood pressure, and circumferential strain induced by EECP. These hemodynamic signals are then transmitted to the cellular interior through a series of signaling pathway cascades, causing changes in gene and protein expression. This further induces a series of endothelial functional changes, including the release of the vasodilator nitric oxide (NO) and the vasoconstrictor endothelin-1 (ET-1), oxidative stress, and endothelial growth factor secretion—the endothelial cell mechanobiological effects. Ultimately, this regulates the structural and functional reconstruction of the vascular wall, including vascular smooth muscle. Therefore, EECP's improvement of the local arterial hemodynamic microenvironment and its promotion of the repair of damaged endothelial structure and function has gradually developed into an important new direction for exploring the effective inhibition and delay of the occurrence and progression of cardiovascular and cerebrovascular diseases.

[0004] Animal models and clinical trials are the most direct research methods for exploring the treatment of ischemic cerebrovascular diseases with end-extension pulse embolization (EECP) and its mechanisms of action. In vivo studies on the vascular mechanobiological effects of EECP mainly focus on qualitatively investigating the impact of counterpulsation conditions on endothelial cell function and macroscopic pathological indicators in patients under different physiological and pathological conditions. Due to the complexity of animal and human organisms, the hemodynamic microenvironment of arterial endothelial cells in vivo is very complex and easily interfered with by other factors such as respiration and neural regulation. In addition, the monitoring of in vivo hemodynamic parameters in animal and human clinical trials is limited by the monitoring site and parameters, and there are problems such as long experimental cycles, high risks, high costs, and ethical controversies. Therefore, quantitatively studying the regulation of arterial endothelial function and its cellular and molecular biological mechanisms by hemodynamic signals through in vivo animal and human experiments still faces technical bottlenecks and practical challenges.

[0005] Compared to animal and clinical trials, in vitro endothelial cell culture models (ECCM) can largely eliminate interfering factors, providing a hemodynamic microenvironment similar to that in vivo. This allows for controllable observation of hemodynamic parameters and facilitates the monitoring of endothelial cell biological effects using sensing and imaging technologies. Studying the effects of hemodynamic factors on endothelial function and related mechanobiological mechanisms through ECCM experiments has become a crucial research step before moving to animal models and clinical trials. However, basic research on the application of ECCM in regulating arterial endothelial cell function through the hemodynamic microenvironment induced by EECP has not yet been reported. Therefore, there is an urgent need to design and construct a miniature in vitro circulatory system that can accurately simulate the in vivo arterial endothelial hemodynamic microenvironment. This system should be able to accurately load hemodynamic signals under different in vitro counterpulsation modes and perform online, real-time quantitative monitoring of the mechanobiological effects of arterial endothelial cells within the cell culture chamber of a microfluidic chip. This would facilitate a better analysis of the patterns and mechanobiological mechanisms by which changes in the arterial hemodynamic microenvironment induced by different in vitro counterpulsation modes regulate endothelial function. This would have significant scientific and clinical application value for the scientific and rational selection of counterpulsation conditions and optimization of counterpulsation strategies, thereby promoting the treatment and rehabilitation of cardiovascular and cerebrovascular diseases. Summary of the Invention

[0006] The purpose of this invention is to design and construct an in vitro cell culture microfluidic system and method capable of accurately simulating blood pressure, wall shear stress, and circumferential strain (stress) signals in the arterial endothelial hemodynamic microenvironment induced by different in vitro counterpulsation (IPP) modes, and capable of online and real-time monitoring of the mechanobiological effects on arterial endothelial cells. Combining hemodynamic principles, machine learning algorithms, microfluidic chip technology, and intelligent feedback control technology, an in vitro cell culture microfluidic chip for simultaneously loading pressure, shear force, and circumferential strain is designed. A multi-element lumped parameter model characterizing the hemodynamic properties of the common carotid artery and its afterload is screened using a neural network + PDE-FIND algorithm. Guided by this model, a multi-element extracorporeal circulation loop and peripheral flow loading system are constructed, along with an endothelial cell function peripheral detection and feedback control system. This system simulates the combined effects of pressure, shear stress, and circumferential strain experienced by in vivo arterial endothelial cells under different IPP modes, and is used to study the laws and mechanisms by which differences in hemodynamic signals in the arterial endothelial cell microenvironment induced by different IPP modes regulate arterial endothelial function.

[0007] The technical solution of the present invention is as follows:

[0008] An in vitro cell culture system for studying the regulation of arterial endothelial cell function by external counterpulsation therapy (e.g.) Figure 1 As shown), it includes three basic units: the first basic unit is an in vitro cell culture microfluidic chip and an external afterload hemodynamic multi-element extracorporeal circulation circuit (such as...). Figure 2 (As shown). The in vitro cell culture microfluidic chip contains a cell culture chamber for arterial endothelial cell culture. An external circulation loop connected to the cell culture chamber can apply a combination of pressure, shear stress, and circumferential strain signals to the arterial endothelial cells cultured within the cell culture chamber. The multi-element external circulation loop of the in vitro cell culture microfluidic chip is divided into an upper limb blood circulation loop and a lower limb blood circulation loop. The upper limb blood circulation loop is a fluid circulation loop guided by a multi-element lumped parameter model selected by a neural network + PDE-FIND algorithm, including an elastic cavity C sequentially connected to the in vitro cell culture microfluidic chip. U1 Influenza L U Resistance valve R U1 Elastic cavity C U2 and resistance valve R U2 The lower limb blood circulation circuit includes an elastic cavity C sequentially connected to the microfluidic chip for in vitro cell culture. L1 Influenza L L Resistance valve R L1 Elastic cavity C L2 and resistance valve R L2 .

[0009] The second basic unit is the peripheral flow loading device of the chip (such as...) Figure 2 As shown), specifically including a pulsed fluid loading device simulating the power source of the cardiovascular system and a pulsed pressure loading device simulating external counterpulsation, both of which can employ a programmable pressure controller (PPC). Figure 3 Q(t) and p(t) are realized. A pulsating fluid loading device, serving as a power source for the cardiovascular system, simulates the patient's blood pressure waveform before external counterpulsation therapy. It is connected to the upper and lower limb blood circulation loops via one-way valves. A reservoir collects fluid from both loops to simulate the heart's blood storage function. The pulsating pressure loading device for external counterpulsation is connected to the elastic cavity C of the lower limb blood circulation loop. L2 ( Figure 3 China P e This device is used to simulate the sequential compression effect of counterpulsation airbags on the calves, thighs, and buttocks under different external counterpulsation modes.

[0010] The third basic unit is the peripheral detection and feedback control system, such as... Figure 1 As shown, it includes an inverted fluorescence microscope, a CCD high-speed imaging system, and a pressure sensor P. i and P o Flow sensor Q, and proportional-integral-derivative feedback control system, pressure sensor P i and P o A flow sensor Q is positioned at the inlet and outlet of the microfluidic chip for in vitro cell culture. It monitors and collects pressure and flow waveforms at the input and output of the cell culture chamber in real time. A fluorescence microscope is located above the microfluidic chip. A high-speed CCD camera system is connected to the fluorescence microscope to collect data on the actual morphology and structure of cells within the cell culture chamber. The CCD camera system, pressure sensor, flow sensor, and programmable pressure controller are all connected to an industrial computer. The industrial computer includes a proportional-integral-derivative (PID) feedback control module. This module controls the acquisition and feedback of pressure and flow waveforms and cell morphology data at both ends of the cell culture chamber by the CCD camera system, pressure sensor, and flow sensor. The PID feedback control module can quantitatively regulate changes in relevant hemodynamic signals. The pressure waveform is set by the industrial computer, and the programmable pressure controller outputs pressure according to the waveform, compressing the air in the reservoir tube and expelling the culture medium.

[0011] The above-mentioned in vitro cell culture microfluidic chips (such as...) Figure 4The chip (shown) employs a three-layer "sandwich" structure. The upper and lower layers are translucent polydimethylsiloxane (PDMS) monolayers containing flat air cavities and cell culture cavities, respectively. These layers are separated by an elastic membrane with the same elastic modulus as the common carotid artery. The upper air cavity is open to the atmosphere, while the lower cell culture cavity connects to the chip's inlet and outlet, forming a fluid circulation pathway. In the experiment, endothelial cells are cultured on the upper surface of the cell culture cavity (corresponding to the lower surface of the elastic membrane). When pulsating flow caused by external counterpulsation is applied to the fluid circulation pathway, the elastic membrane deforms under pressure load, generating circumferential strain. The cultured endothelial cells are simultaneously subjected to a combination of pressure, shear force, and circumferential strain. The selection of the cell culture cavity geometry and the elastic modulus of the elastic membrane must be based on the principles of fluid mechanics and elasticity, determined according to actual conditions to accurately simulate the blood pressure, shear force, and circumferential strain corresponding to different counterpulsation modes at the common carotid artery.

[0012] The in vitro cell culture microfluidic chip afterload hemodynamic multi-element extracorporeal circulation loop is guided by a circuit model determined by a neural network + PDE-FIND algorithm, wherein the flow resistance of the cell culture cavity of arterial endothelial cells is equivalent to a resistance ( Figure 3 Chinese R c The compliance of the elastic cavity connected to the chip outlet is equivalent to a capacitor. Figure 3 C U1 The compliance, flow resistance, and flow rate of the vascular bed in the upper limb circulatory circuit are equivalent to capacitance, resistance, and inductance. Figure 3 C U2 R U1 R U2 and L U In the lower limb blood circulation circuit, the elasticity of the aortic wall is equivalent to capacitance. Figure 3 C L1 The flow resistance of the proximal arteries of the lower extremities is equivalent to inductance and resistance. Figure 3 L L and R L1 The compliance and flow resistance of distal arteries in the lower extremities are equivalent to capacitance and resistance. Figure 3 C L2 and R L2 ).

[0013] Furthermore, the design of the multi-element extracorporeal circulation loop under afterload hemodynamics on the chip needs to ensure that the pressure, wall shear stress, and circumferential strain experienced by the endothelial cells cultured on the cell culture chamber membrane are consistent with the blood pressure, shear stress, and circumferential strain waveforms experienced by the carotid artery endothelial cells during extracorporeal counterpulsation therapy.

[0014] First, the blood pressure p(t) and wall shear stress τ near the local area of ​​arterial endothelial cells in vivo are obtained through human or animal experiments. ω The waveforms of blood flow q(t) and circumferential strain ε(t) are the simulation targets. To ensure that the blood pressure and shear stress waveforms experienced by endothelial cells cultured on the elastic membrane of the cell culture cavity are equal to the blood pressure and wall shear stress in the in vivo arterial endothelial microenvironment, the blood flow q(t) and pressure drop Δp(t) need to satisfy the following:

[0015]

[0016]

[0017] In the formula, η is the viscosity of the cell culture medium, and H c W c and L c These represent the height, width, and length of the cell culture chamber, respectively.

[0018] Secondly, using blood pressure p(t) in the systemic arteries, blood flow Q(t) at the aortic root, and blood flow q(t) in the carotid arteries, the optimal lumped parameter models for the upper and lower limb circulatory systems were selected using a neural network + PDE-FIND algorithm, combined with actual physiological significance. Based on the similarity between fluid dynamics loops and circuits, the equivalent hemodynamic behavior of carotid afterload was then determined. First, the in vivo data was denoised using neural network tools, and a candidate equation database was constructed using automatic differentiation via neural networks. Next, the sparse regression method, i.e., the PDE-FIND algorithm, was used to select the nonlinear terms and derivative terms governing the system operation from the candidate equation database, thereby determining the differential equations that can accurately fit the in vivo data. The physical meaning of the equations was analyzed, and the optimal lumped parameter model (e.g., ...) was constructed. Figure 3 (As shown). Simultaneously, following the equivalence principles of fluid mechanics and electricity, the hemodynamic behavior of the carotid artery afterload is equivalent to a circuit system. The input impedance of the upper limb circulatory system in the fluid mechanics loop is expressed as the frequency domain ratio of the input pressure waveform p(t) and the carotid artery blood flow waveform q(t), expressed as angular frequency ω. n The amplitude and phase characteristics of the corresponding harmonic components of blood pressure and blood flow are as follows:

[0019]

[0020] ∠z(ω n )=∠P(ω n )-∠Q(ω n (2b)

[0021] In the formula, |P(ω n )| and |Q(ω) n The values ​​of blood pressure and blood flow, after Fourier transform, are at an angular frequency ω.n The amplitude at point; ∠P(ω) n ) and ∠Q(ω n () are blood pressure and blood flow, respectively, after Fourier transform, at angular frequency ω n Phase angle at the point; |z(ω) n )| and ∠z(ω n ) are the downstream afterload input impedances of the carotid artery at ω n The amplitude and phase angle at that point. The input impedance of the circuit model of the equivalent upper limb system is expressed as... Figure 3 The complex function formed by the combination of circuit elements in the upper limb blood circulation loop on the left side of the circuit can be used to determine the parameter values ​​of each component in the equivalent lumped parameter circuit model of the upper limb system based on the amplitude-frequency and phase-frequency curves of the input impedance of the upper limb system in the above-mentioned fluid dynamics loop, through the system identification method in Equation 3.

[0022]

[0023] In the formula, and The equivalent input impedance of the upper limb system in the lumped parameter circuit model is ω. n The magnitude and phase angle of the equivalent impedance at that point.

[0024] At the same time, the equivalent lumped parameter circuit model of the lower limb system and the parameter values ​​of each component were determined using the same method.

[0025] Finally, according to influenza (L) U and L L ), resistance valve (R) U1 R U2 R L1 and R L2 ), elastic cavity (C) U1 C U2 C L1 and C L2 Numerical simulation of a multi-element in vitro fluid circulatory system was constructed to simulate the hemodynamic characteristics of carotid artery afterload.

[0026] like Figure 1 and Figure 2 As shown, the circulating fluid in the system is an in vitro vascular endothelial cell culture medium. An elastic cavity simulates arterial compliance (flow capacity), a resistance valve simulates viscous resistance (flow resistance), and an influx element simulates flow inertia. A programmable pressure controller generates the desired blood flow waveform via the lower limb circulatory system and a pulsating blood pump. Once the input blood flow waveform to the cell culture chamber is known, the pressure waveform can be uniquely determined based on the aforementioned equivalent circuit.

[0027] The aforementioned device simulating the external counterpulsation power source is implemented using a pulsed pressure loading device, while the device simulating the cardiovascular system power source is implemented using a pulsed fluid loading device. The pulsed fluid loading device and the pulsed pressure loading device, combined with a PID feedback control device, can be used to simulate blood pressure, wall shear stress, and circumferential strain signals in the in vivo arterial endothelial cell hemodynamic microenvironment before and after external counterpulsation in ischemic stroke patients. The pulsed pressure loading device can be integrated with the lower limb elastic cavity (C... L1 and C L2 ), resistance valve (R) L1 and R L2 ), influenza L L A fluid loading device is connected in series with the upper limb elastic cavity (C). U1 and C U2 ), in vitro cell culture microfluidic chip, resistance valve (R) U1 and R U2 ), influenza L U Connected in parallel to the aforementioned fluid dynamics loop and combined with a PID feedback control device, it can generate hemodynamic signal waveforms of carotid endothelial cells under different external counterpulsation modes. The collected signals are fed back to the PID control device, which can further adjust the fluid loading device and the pressure loading device, thereby quantitatively controlling the changes in the amplitude and frequency of pressure and flow signals acting on the multi-element simulated circulatory system. Ultimately, it generates a combination of blood pressure, shear stress, and circumferential strain based on different external counterpulsation modes in the cell culture chamber of the microfluidic chip.

[0028] A method for studying the regulation of arterial endothelial cell function by external counterpulsation therapy specifically involves using the aforementioned system to study the quantitative relationship between different external counterpulsation modes and hemodynamic signals in the carotid endothelial microenvironment. The steps are as follows:

[0029] Step 1: Passage the primary cultured endothelial cells; passages 2-5 are used for experiments. Adjust the influenza (L) control system in the in vitro cell culture microfluidic system. U and L L ), resistance valve (R) U1 R U2 R L1 and R L2 ), elastic cavity (C) U1 C U2 C L1 and C L2 The magnitude of the value is determined by introducing circulating fluid into the cell culture chamber. By adjusting the programmable pressure controller, various combinations of hemodynamic signals for carotid endothelial cells are applied under different external counterpulsation modes.

[0030] Step 2: Continue to apply the hemodynamic signal stimulation corresponding to the above working mode, and then perform cell activity detection to ensure the effectiveness of the above system.

[0031] Step 3: Collect cell samples from the cell culture chamber of the in vitro cell culture microfluidic chip and detect gene and protein expression levels to analyze the effects of hemodynamic signals such as blood pressure, shear stress, and circumferential strain caused by different in vitro counterpulsation modes on the expression levels of vasoactive substances and pro-inflammatory cytokines genes and proteins.

[0032] The beneficial effects of this invention are as follows: Based on the above-mentioned in vitro cell culture microfluidic system, this invention can accurately simulate hemodynamic signals such as blood pressure, wall shear stress, and circumferential strain in the arterial endothelial microenvironment caused by different in vitro counterpulsation modes in patients. Furthermore, it uses an in vitro cell culture microfluidic chip with higher integration, fewer consumables, and ease of integration with optical detection and sensing to study the differential effects of combined stimulation of hemodynamic signals under the above modes on arterial endothelial cell function. This provides an efficient and reasonable experimental platform for quantitatively analyzing the influence of hemodynamic signals on arterial endothelial cell function and its molecular biological mechanisms. It also provides a basis for screening hemodynamic conditions that are beneficial to improving or maintaining arterial endothelial function, and for scientifically and rationally selecting counterpulsation conditions and optimizing counterpulsation strategies. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of a microfluidic chip for in vitro endothelial cell culture and its peripheral monitoring system.

[0034] Figure 2 This is a schematic diagram of an in vitro endothelial cell culture microfluidic chip and its peripheral multi-element circulation loop and fluid loading system.

[0035] Figure 3 This is a schematic diagram of the equivalent circuit model of the hemodynamic behavior of carotid afterload.

[0036] Figure 4 This is a schematic diagram of an in vitro cell culture microfluidic chip that simultaneously applies pressure, shear force, and circumferential stress stimulation.

[0037] Figure 5 This is a schematic diagram of the blood pressure, flow rate, and shear stress waveforms experienced by the endothelial cells of the carotid artery before and at a counterpulsation pressure of 0.035 MPa in an in vivo experiment.

[0038] Figure 6The diagram shows the fitting results of the upper and lower limb systems of the above equivalent circuit model to the actual input impedance amplitude and phase angle using Matlab / Simulink; (a): amplitude-frequency curve of input impedance of upper limb system; (b): phase angle-frequency curve of input impedance of upper limb system; (c): amplitude-frequency curve of input impedance of lower limb system; (d): phase angle-frequency curve of input impedance of lower limb system.

[0039] The diagram shows: two programmable pressure controllers (i); a signal acquisition and processing system including an inverted fluorescence microscope, a CCD high-speed imaging system, pressure sensors, and flow sensors (ii); and a proportional-integral-derivative (PID) feedback control system (iii); P i (ii) and P o (ii) is a pressure sensor located at both ends of the cell culture chamber of the microfluidic chip, and Q(ii) is a flow sensor; R c For the flow resistance of the cell culture chamber in a microfluidic chip; R U1 R U2 R L1 and R L2 To connect the pipe flow resistance; C U1 C U2 C L1 and C L2 An elastic air cavity characterizing compliance; L U and L L This refers to the influenza that occurs when pipes are connected during the liquid circulation process. Detailed Implementation

[0040] This paper describes a specific implementation plan for simulating blood pressure in the arterial endothelial hemodynamic microenvironment under external counterpulsation:

[0041] (1) Design the height H of the cell culture chamber of the microfluidic chip c Width W c and length L c The diameters are 0.3 mm, 6 mm, and 15 mm, respectively, and the viscosity η of the cell culture medium is typically 0.001 Pa·s.

[0042] (2)Use Figure 5 Calculate the target input impedance z(ω) of the upper and lower limb systems based on the target carotid artery blood pressure and blood flow, as well as the aortic root blood flow, before external counterpulsation. i Furthermore, machine learning combined with the PDE-FIND algorithm was used to optimize and filter the lumped parameter models of the upper and lower limb systems, ultimately determining that the upper limb system consists of six components and the lower limb system consists of five components, such as... Figure 3 As shown.

[0043] (3) According to relevant circuit theory, the input impedance of the upper limb system of this circuit is known. It can be represented as:

[0044]

[0045] (3) As shown in Formula 4, the equivalent input impedance of the six-element lumped parameter model of the upper limb system is... Combined with the target input impedance z(ω) of the upper limb system i The parameter values ​​of each component can be obtained through system identification methods. Similarly, the parameter values ​​of each component in the lower limb system can be obtained. The parameter values ​​of each component in the fluid dynamics circuit corresponding to this patient are as follows: R c =4.26 mmHg·s / ml, R U1 =2.35 mmHg·s / ml, R U2 =11.51 mmHg·s / ml, R L1 =0.03 mmHg·s / ml, R L2 =1.02 mmHg·s / ml, C U1 =0.0059ml / mmHg, C U2 =0.053ml / mmHg, C L1 =0.59ml / mmHg, C L2 =0.56ml / mmHg, L U =1.48 mmHg·s 2 / ml, L L =0.0015 mmHg·s 2 / ml. For example... Figure 6 As shown, the input impedance curves of the upper and lower limb systems corresponding to the lumped parameter model ( Figure 6 (solid line in the middle) and the target input impedance curve ( Figure 6 The circles in the diagram basically match. Then construct as follows: Figure 2 The fluid dynamic circulation loop of the cell culture chamber based on a microfluidic chip is shown.

[0046] (4) The chip is fabricated using standardized microfabrication methods. An elastic film with a modulus similar to that of an artery is bonded to a cavity made of rigid, transparent PMMA material. The cavity has a concave cross-section. The cell culture cavity below the lower surface of the elastic film is filled with circulating fluid. Air is introduced into the cavities on both sides of the upper surface of the elastic film to provide sufficient space for the film to deform under the pressure of pulsating fluid. The two ends of the cavity in the middle of the upper surface of the elastic film are smooth arcs, and the middle of the upper surface of the film is close to the horizontal inner surface of the concave part of the cavity. This allows the elastic film with attached endothelial cells to undergo only horizontal tensile strain under the traction of both sides. At the same time, the thickness of the concave part of the cavity must be designed to ensure that it can be focused when observing the morphology and structure of endothelial cells under a microscope and will not deform under pulsating pressure. The geometric dimensions of the lower cell culture cavity and the selection of the elastic modulus of the elastic film need to be determined based on the principles of elasticity and by accurately simulating the actual needs of blood pressure, shear stress and circumferential strain waveforms in the endothelial microenvironment of different parts of the carotid artery.

[0047] (5) Establish such Figure 1 The microfluidic chip for in vitro endothelial cell culture and its peripheral monitoring system shown include two programmable pressure controllers (i), a signal acquisition and processing system (ii) composed of various devices, and a proportional-integral-derivative feedback control system (iii). The signal acquisition and processing system (ii) includes an inverted fluorescence microscope, a CCD high-speed imaging system, pressure sensors, and flow sensors, used to monitor and acquire pressure and flow waveforms at the input and output ends of the cell culture chamber in real time, as well as the actual morphological structure of cells within the cell culture chamber of the microfluidic chip. The programmable pressure controller (i) combined with the PID feedback control device (iii) can accurately simulate the patient's resting state conditions. The programmable pressure controller (i) is connected in series in the entire fluid dynamics loop. It can accurately simulate the blood pressure, wall shear stress, and circumferential strain signals borne by the endothelial cells of specific parts of the carotid artery under different external counterpulsation pressures. Finally, it loads a quantitative and controllable pulsatile flow signal onto the multi-element simulated circulatory system. The collected signal is fed back to the PID control device (iii), which can further adjust the programmable pressure controller (i), thereby quantitatively controlling the changes in the amplitude and frequency of the pressure and flow signals acting on the multi-element simulated circulatory system. Finally, it generates the combined effects of blood pressure, shear stress, and circumferential strain under different external counterpulsation modes in the cell culture chamber of the microfluidic chip.

[0048] The pressure within the microfluidic chip can be measured by a pressure sensor; shear stress can be calculated from the flow waveform measured by a flow sensor and the geometry of the cell culture chamber; and the circumferential strain in the horizontal direction of the elastic film within the chip under different pressures can be calibrated on the film using fluorescent microspheres and measured using a fluorescence microscope. By giving different pressures, the corresponding circumferential strain of the elastic film can be obtained, thus establishing a relationship expression between pressure and circumferential strain. Based on this approximate expression, the circumferential strain of the elastic film in actual experiments can be determined when the pressure is known. In addition, the morphology and structure of endothelial cells are detected, recorded, and stored in an industrial control computer using a microscope combined with a CCD high-speed imaging system.

[0049] (6) The specific experimental steps for studying the quantitative relationship between different external counterpulsation modes and hemodynamic signals of the local arterial endothelial microenvironment are as follows:

[0050] Step 1: Primary cultured endothelial cells were passaged in EGM medium, with passages 2-5 used for experiments. During the experiments, endothelial cells were seeded onto the elastic membrane of the microfluidic chip cell culture chamber coated with Fibronection, ensuring cell adhesion and confluence of over 90%.

[0051] Step two involves applying a combination of hemodynamic signals corresponding to different in vitro counterpulsation modes to the arterial endothelial cells; cell activity is then assessed using the NucView™-488 cell activity assay reagent to ensure the effectiveness of the in vitro simulated circulatory system.

[0052] Step 3: Collect cell samples from the cell culture chamber of the in vitro cell culture microfluidic chip to detect gene and protein expression levels, thereby determining the effects of hemodynamic signals such as blood pressure, shear stress, and circumferential strain on the gene and protein expression levels of vasoactive substances and pro-inflammatory cytokines under different in vitro counterpulsation modes.

[0053] This invention can successfully reproduce the blood pressure, wall shear stress, and circumferential strain signals experienced by in vivo arterial endothelial cells under different modes of external counterpulsation, and can monitor the differential effects on the function of cultured arterial endothelial cells under the stimulation of the above-mentioned hemodynamic signal combinations in real time.

Claims

1. An in vitro cell culture system for studying the regulation of arterial endothelial cell function by in vitro counterpulsation therapy, characterized in that, The in vitro cell culture microfluidic system comprises three basic units: The first basic unit is an in vitro cell culture microfluidic chip and an external afterload hemodynamic multi-element extracorporeal circulation circuit. The in vitro cell culture microfluidic chip contains a cell culture chamber for arterial endothelial cell culture. The external circulation circuit, connected to the cell culture chamber, can apply a combination of pressure, shear stress, and circumferential strain signals to the arterial endothelial cells cultured within the cell culture chamber. The external multi-element extracorporeal circulation circuit is divided into an upper limb blood circulation circuit and a lower limb blood circulation circuit. The upper limb blood circulation circuit includes an elastic cavity C sequentially connected to the in vitro cell culture microfluidic chip. U1 Influenza L U Resistance valve R U1 Elastic cavity C U2 and resistance valve R U2 The lower limb blood circulation circuit includes an elastic cavity C sequentially connected to the microfluidic chip for in vitro cell culture. L1 Influenza L L Resistance valve R L1 Elastic cavity C L2 and resistance valve R L2 ; The second basic unit is the peripheral fluid loading device for the chip, specifically including a pulsed fluid loading device simulating the power source of the cardiovascular system and a pulsed pressure loading device simulating external counterpulsation, both of which can be implemented using a programmable pressure controller. The pulsed fluid loading device, as the power source of the cardiovascular system, is used to simulate the patient's blood pressure waveform before external counterpulsation therapy, and is connected to the upper limb and lower limb blood circulation loops respectively through one-way valves. A reservoir collects fluid from the upper and lower limb blood circulation loops to simulate the heart's blood storage function. The pulsed pressure loading device for external counterpulsation is connected to the elastic cavity C of the lower limb blood circulation loop. L2 It is used to simulate the sequential compression effect of counterpulsation airbags on the calves, thighs and buttocks under different external counterpulsation modes; The third basic unit is the peripheral detection and feedback control system, which includes an inverted fluorescence microscope, a CCD high-speed imaging system, and a pressure sensor P. i and P o Flow sensor Q, and proportional-integral-derivative feedback control system, pressure sensor P i and P o The inlet and outlet of the microfluidic chip for in vitro cell culture are respectively located at the inlet and outlet. A flow sensor Q is located at the outlet of the microfluidic chip for in vitro cell culture, used to monitor and acquire pressure and flow waveforms at the input and output ends of the cell culture chamber in real time. A fluorescence microscope is located above the microfluidic chip for in vitro cell culture. A CCD high-speed camera system is connected to the fluorescence microscope to acquire the actual morphological structure of cells within the cell culture chamber of the microfluidic chip. The CCD high-speed camera system, pressure sensor, flow sensor, and programmable pressure controller are all connected to an industrial control computer. The industrial control computer includes a proportional-integral-derivative (PID) feedback control module, which controls the acquisition and feedback of pressure and flow waveforms and cell morphological structure data at both ends of the cell culture chamber by the CCD high-speed camera system, pressure sensor, and flow sensor. The PID feedback control module can quantitatively regulate changes in relevant hemodynamic signals. The pressure waveform is set by the industrial control computer, and the programmable pressure controller outputs pressure according to the pressure waveform. Force is applied to compress the air in the reservoir tube of the storage tank, squeezing out the culture medium. The in vitro cell culture microfluidic chip adopts a three-layer "sandwich" structure. The upper and lower layers of the chip are transparent polydimethylsiloxane monolayers containing flat air cavities and cell culture cavities, respectively. The upper and lower layers are separated by an elastic film with the same elastic modulus as the common carotid artery. The upper air cavity is connected to the atmosphere, and the lower cell culture cavity is connected to the inlet and outlet of the chip to form a fluid circulation pathway. In the experiment, endothelial cells are cultured on the upper surface of the cell culture cavity, which corresponds to the lower surface of the elastic film. When pulsating flow caused by in vitro counterpulsation is applied in the fluid circulation pathway, the elastic film deforms under pressure load and generates circumferential strain. The cultured endothelial cells are simultaneously subjected to the combined effects of pressure, shear force, and circumferential strain. The selection of the geometric dimensions of the cell culture cavity and the elastic modulus of the elastic film should be based on the principles of fluid mechanics and elasticity, and determined according to actual conditions, using the blood pressure, shear force, and circumferential strain corresponding to different counterpulsation modes at the common carotid artery as the standard. The in vitro cell culture microfluidic chip-based afterload hemodynamic multi-element extracorporeal circulation loop is guided by a circuit model determined by a neural network + PDE-FIND algorithm, wherein the flow resistance of the cell culture cavity for arterial endothelial cells is equivalent to a resistance R. c The compliance of the elastic cavity connected to the chip outlet is equivalent to the capacitance C. U1 The compliance, flow resistance, and influenza of the vascular bed in the upper limb circulatory circuit are equivalent to capacitance C. U2 Resistance R U1 Resistance R U2 and inductor L U The elasticity of the aortic wall in the lower limb blood circulation loop is equivalent to the capacitance C. L1 The flow resistance and inductance of the proximal arteries of the lower extremities are equivalent to the inductance L. L and resistance R L1 The compliance and flow resistance of the distal arteries of the lower extremities are equivalent to the capacitance C. L2 and resistance R L2 .

2. The in vitro cell culture system for studying the regulation of arterial endothelial cell function by in vitro counterpulsation therapy according to claim 1, characterized in that, The design of the multi-element extracorporeal circulation loop for microfluidic chip-based afterload hemodynamics in in vitro cell culture must ensure that the pressure, wall shear stress, and circumferential strain experienced by the endothelial cells cultured on the cell culture membrane are consistent with the blood pressure, shear stress, and circumferential strain waveforms experienced by the carotid artery endothelial cells during extracorporeal counterpulsation therapy. First, the blood pressure p(t) and wall shear stress near the local area of ​​arterial endothelial cells in vivo are obtained through human or animal experiments. and circumferential strain The waveform is the simulation target. To ensure that the blood pressure and shear stress waveforms experienced by endothelial cells cultured on the elastic membrane of the cell culture cavity are equal to the blood pressure and wall shear stress in the in vivo arterial endothelial microenvironment, the blood flow q(t) and pressure drop Δp(t) need to satisfy the following: (1a) ； (1b) ； In the formula, η is the viscosity of the cell culture medium, and H c W c and L c These are the height, width, and length of the cell culture chamber, respectively. Secondly, using blood pressure p(t) in the systemic arteries, blood flow Q(t) at the aortic root, and blood flow q(t) in the carotid arteries, the optimal lumped parameter models for the upper and lower limb circulatory systems were selected based on the actual physiological significance using a neural network + PDE-FIND algorithm. Furthermore, based on the similarity between fluid dynamics loops and circuits, the equivalent hemodynamic behavior of the carotid artery afterload was determined. Firstly, neural network tools were used to denoise the in vivo data, and a database of candidate equations was constructed using automatic differentiation via neural networks. Next, the sparse regression method, specifically the PDE-FIND algorithm, is used to select the nonlinear and derivative terms governing the system operation from the candidate equation database. This determines the differential equations that can accurately fit the in vivo data, analyzes the physical meaning of the equations, and then constructs the optimal lumped parameter model. Simultaneously, following the equivalence principle between fluid mechanics and electricity, the carotid artery afterload hemodynamic behavior is equivalent to a circuit system. The input impedance of the upper limb circulatory system in the fluid mechanics loop is expressed as the frequency domain ratio of the input pressure waveform p(t) and the carotid artery blood flow waveform q(t), expressed using the angular frequency ω. n The amplitude and phase characteristics of the corresponding harmonic components of blood pressure and blood flow are as follows: (2a) ； (2b) ； In the formula, | | and | | These are blood pressure and blood flow, respectively, after Fourier transform, at angular frequency ω. n The amplitude at point; ∠P(ω) n ) and ∠Q(ω n () are blood pressure and blood flow, respectively, after Fourier transform, at angular frequency ω n Phase angle at the point; |z(ω) n )| and ∠z(ω n ) are the downstream afterload input impedances of the carotid artery at ω n The amplitude and phase angle at the point; the input impedance of the equivalent upper limb system circuit model is expressed as a complex function of each circuit element in the upper limb blood circulation loop. Based on the amplitude-frequency and phase-frequency curves of the upper limb system input impedance in the above-mentioned fluid dynamics loop, the parameter values ​​of each component in the equivalent lumped parameter circuit model of the upper limb system can be determined by the system identification method in Equation 3. (3) ； In the formula, and The equivalent input impedance of the upper limb system in the lumped parameter circuit model is ω. n The magnitude and phase angle of the equivalent impedance at that point; At the same time, the equivalent lumped parameter circuit model of the lower limb system and the parameter values ​​of each component were determined using the same method; Finally, according to influenza L U and L L Resistance valve R U1 R U2 R L1 and R L2 Elastic cavity C U1 C U2 C L1 and C L2 A multi-element in vitro fluid simulation circulatory system was numerically constructed to simulate the hemodynamic characteristics of carotid artery afterload.

3. A method for studying the regulation of arterial endothelial cell function by in vitro counterpulsation therapy using the in vitro cell culture microfluidic system according to any one of claims 1-2, characterized in that, The steps are as follows: Step 1: Passage the primary cultured endothelial cells; passages 2-5 are used for experiments; adjust the influenza L in the in vitro cell culture microfluidic system. U and L L Resistance valve R U1 R U2 R L1 and R L2 Elastic cavity C U1 C U2 C L1 and C L2 The magnitude of the value is determined by introducing circulating fluid into the cell culture chamber and adjusting the programmable pressure controller to load a combination of various hemodynamic signals for carotid endothelial cells under different external counterpulsation modes. Step 2: Continue to apply the hemodynamic signal stimulation corresponding to the working mode of Step 1, and then perform cell activity detection to ensure the effectiveness of the above system; Step 3: Collect cell samples from the cell culture chamber of the in vitro cell culture microfluidic chip and detect gene and protein expression levels to analyze the effects of hemodynamic signals such as blood pressure, shear stress, and circumferential strain caused by different in vitro counterpulsation modes on the gene and protein expression levels of vasoactive substances and pro-inflammatory cytokines.

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