AN IN VITRO ENDOTHELIAL CELL CULTURE SYSTEM FOR OPTIMIZING PULSATILE WORKING MODES OF THE CONTINUOUS FLOW ARTIFICIAL HEART

Abstract

An in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart belongs to the technical field of artificial organs. The system includes three parts: 1) a cell culture model on a microfluidic chip and an off-chip multielement aortic arch afterload fluid mechanics circulation loop; 2) devices for simulating the power source of a cardiovascular system: a fluid loading device is realized by a pulse blood pump, and an artificial heart device is connected in parallel to both ends of the pulse blood pump; and 3) a peripheral detection and feedback control system, comprising pressure and flow sensors, a fluorescence microscope, a CCD high-speed camera system and a proportional-integral-derivative feedback control system. The system can accurately simulate the real hemodynamics microenvironment of vascular endothelial cells in different parts of the aortic arch.

Claims

1. An in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart, wherein the in vitro endothelial cell culture system comprises three basic units: the first basic unit is a cell culture model on a microfluidic chip and an off-chip multielement aortic arch afterload fluid mechanics circulation loop; wherein the off-chip multielement aortic arch afterload fluid mechanics circulation loop comprises a flow inductance, a resistance valve, a first elastic chamber and a second elastic chamber which are connected in series with the cell culture model,and the first elastic chamber and the second elastic chamber are arranged on both sides of the cell culture model; the second basic unit is a pulse fluid loading device and an artificial heart device for simulating the power source of a cardiovascular system, wherein the fluid loading device is realized by a pulse blood pump,and the artificial heart device is connected in parallel to both ends of the pulse blood pump, and then the pulse fluid loading device and the artificial heart device are connected in series to the off-chip multielement aortic arch afterload fluid mechanics circulation loop; the third basic unit is a peripheral detection and feedback control system, includingan inverted fluorescence microscope, a CCD high-speed camera system, pressure andflow sensors, and a proportional-integral-derivative (PID) feedback control system, wherein the pressure and flow sensors are arranged on both sides of the cell culture model, the fluorescence microscope is located above the cell culture model Rc, the CCD high-speed camera system is connected with the fluorescence microscope, and the CCD high-speed camera system, the pressure and flow sensors are all connected with the PID feedback control system.

2. The in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart according to claim 1, wherein the cell culture model is a cavity with a concave section, an elastic film with the elastic modulus similar to that of an artery is bonded to a cavity, and the cell culture model below the lower surface of the elastic film is full of circulating fluid; air is introduced into cavities on both sides of the upper surface of the elastic film; the middle part of the upper surface of the elastic film is close to the inner surface of the concave part of the cavity in the horizontal direction; and both ends of the middle part of the upper surface of the elastic film are smooth and cambered.

3. The in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart according to claim 1, wherein the in vitro circulatory system is equivalent to a circuit model: the flow resistance of the endothelial cell culture model is equivalent to a resistor, the compliance of the film on the culture model is equivalent to a capacitor, and the compliance, the flow resistance and the flow inductance of an aortic arch downstream vascular bed are equivalent to a capacitor, a resistor and an inductor.

4. The in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart according to claim 1, wherein the off-chip multielement aortic arch afterload fluid mechanics circulation loop shall be designed to keep the pressure, wall shear stress and stretch strain on endothelial cells cultured on the film of the cell culture model consistent with the waveforms of blood pressure, shear stress and stretch strain on endothelial cells of the corresponding part of a heart failure patient implanted with an artificial heart: first, with the waveform of blood pressure p(t), wall shear stress τ.sub.ω (t) and stretch strain ε(t) near local in vivo arterial endothelial cells obtained from the detection and analysis of human or animal experiments as a simulated target, to make the waveforms of blood pressure and shear stress on the endothelial cells cultured on the film of the cell culture model equal to blood pressure and wall shear stress in the in vivo arterial endothelial microenvironment, blood flow q(t) and pressure drop Δp(t) must satisfy: $\begin{array}{cc}q\left(t\right)=\frac{W_c{H}_c^2}{6\eta }{\tau }_{\omega }\left(t\right)& \left(1a\right)\end{array}$ $\begin{array}{cc}\max \left(\frac{\Delta p\left(t\right)}{p\left(t\right)}\right)=\max \left(\frac{2L_c{\tau }_{\omega }\left(t\right)}{H_{c}p\left(t\right)}\right)<<1& \left(1b\right)\end{array}$ wherein η is the viscosity of the cell culture fluid, and Hc, We and Lc are respectively height, width and length of the cell culture model; second, the hemodynamics behavior of the aortic arch afterload is equivalent to a circuit model according to the similarity relationship between the fluid mechanics loop and the circuit, and the circuit model connects the flow inductorL characterizing the hemodynamics characteristics of the aortic arch downstream vascular bed with the flow resistor R in series, with the second elastic chamber C.sub.2 in parallel, then with the flow resistance Rc of the cell culture model in series, and finally with the compliance C.sub.1 of the film on the culture model in parallel, and the parameter values of the above elements in the lumpedparameter circuit model are determined by the system identification method; finally, a multielement in vitro fluid mock circulatory system for simulating the hemodynamics characteristics of the aortic arch afterload is built according to the numerical values of the flow inductanceL, the resistance valve R, the first elastic chamber C.sub.1 and the second elastic chamber C.sub.2.

5. The in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart according to claim 1, wherein the cell culture model provides circulating fluid for cells in the cell culture model through matching of the resistance valve and the reservoir.

6. The in vitro endothelial cell culture system for optimizing the pulsatile working mode of a continuous flow artificial heart according to claim 2, wherein the pulse fluid loading device can be used in combination with the PID feedback control device to simulate signals of blood pressure, wall shear stress and stretch strain in the hemodynamics microenvironment of in vivo arterial endothelial cells under normal and heart failure physiological conditions, the artificial heart device and the fluid loading device are connected in parallel and then connected in series to the above fluid mechanics circulation loop, and can produce hemodynamics signal waveforms of different parts of the aortic arch under different pulsatile working modes of the artificial heart pump speed in combination with the PID feedback control device; and the acquired signals are fed back to the PID control device to further regulate the fluid loading device and the artificial heart, so as to quantitatively regulate changes in the amplitude and frequency of pressure and flowsignals on the multielement mock circulatory system, and finally produce the combined effect of blood pressure, shear stress and stretch strain under different pulsatile working modes of the artificial heart pump speed in the cell culture model on a microfluidic chip.

Description

DESCRIPTION OF DRAWINGS

[0028] FIG. 1 is a structural schematic diagram of an in vitro endothelial cell culture model and a periphery monitoring system.

[0029] FIG. 2 is a schematic diagram of a fluid mechanics circulation loop of an in vitro endothelial cell culture model.

[0030] FIG. 3 is a schematic diagram of an equivalent circuit model of hemodynamics behaviors of aortic arch afterload.

[0031] FIG. 4 is a schematic diagram of a cell culture model on a microfluidic stretch chip.

[0032] FIG. 5 is a schematic diagram showing the waveforms of blood pressure and shear stress on vascular endothelial cells of the aortic arch obtained through in vivo experiments and the waveform of blood flow in the culture model obtained by inverse solution according to the waveform of shear stress and the size of the cell culture model in normal, heart failure and asynchronous modulation modes.

[0033] FIG. 6 is a schematic diagram showing the results of fitting the amplitude and the phase angle of the actual input impedance by Matlab/Simulink in normal, heart failure and asynchronous modulation modes of the above equivalent circuit model and the results of comparing the output voltage of the model as the simulation target with the blood pressure in FIG. 5 with the blood flow information as current excitation; (a-1): an amplitude-frequency curve of the input impedance under normal physiological conditions; (a-2): a phase angle-frequency curve of the input impedance under normal physiological conditions; (a-3): a comparison diagram of the output voltage of the model and the blood pressure under normal physiological conditions; (b-1): an amplitude-frequency curve of the input impedance under heart failure conditions; (b-2): a phase angle-frequency curve of the input impedance under heart failure conditions; (b-3): a comparison diagram of the output voltage of the model and the blood pressure under heart failure conditions; (c-1): an amplitude-frequency curve of the input impedance under the asynchronous pulsatile working mode of the artificial heart pump speed; (c-2): a phase angle-frequency curve of the input impedance under the asynchronous pulsatile working mode of the artificial heart pump speed; and (c-3): a comparison diagram of the output voltage of the model and the blood pressure under the asynchronous pulsatile working mode of the artificial heart pump speed.

[0034] FIG. 1 includes a fluid loading device—a pulse blood pump (i) and an artificial heart (i); a signal acquisition and processing system (ii) comprising an inverted fluorescence microscope, a CCD high-speed camera system, pressure and flow sensors, and a proportional-integral-derivative (PID) feedback control system (iii); A(ii) and B(ii) are pressure and flow sensors located on both ends of the cell culture model on a microfluidic chip; Rc is the flow resistance of a cell culture model on a microfluidic chip; C.sub.1 is the compliance of a film; R is the flow resistance of the connecting tube; C.sub.2 is an elastic air chamber characterizing compliance; and L is the flow inductance of the connecting tube in the fluid circulation process.

DETAILED DESCRIPTION

[0035] The specific implementation solution of simulating blood pressure in the arterial endothelial hemodynamics microenvironment under different pulsatile working modes of the artificial heart pump speed is described as follows:

[0036] (1) The height Hc, the width We and the length Lc of the cell culture model on a microfluidic chip are respectively designed to be 0.3 mm, 6 mm and 15 mm, and the viscosity η of the cell culture fluid is usually 0.001 Pa.Math.s; and three target input impedances z(ω.sub.i) are respectively calculated according to the target blood pressure and blood flow in three physiological conditionsin FIG. 5;

[0037] (2) The hemodynamics characteristics of the in vitro mock circulatory system can be characterized by the five-element equivalent circuit model shown in FIG. 3, and it can be known from the related circuit theory that the input impedance {circumflex over (z)}(ω.sub.i) of the circuit can be expressed as follows:

[00004]$\begin{array}{cc}\hat{z}\left({\omega }_i\right)=\frac{R_{c}L{C_2\left(j{\omega }_i\right)}^2+\left(R_{c}R{C}_2+L\right)\left(j{\omega }_i\right)+R_1+R_2}{\begin{array}{c}{R}_c{C}_1{C}_2{L\left(j{\omega }_i\right)}^3+\left(R_{c}R{C}_1{C}_2+C_{1}L+C_{2}L\right){\left(j{\omega }_i\right)}^2+\\ \left(R_c{C}_1+R{C}_1+R{C}_2\right)\left(j{\omega }_i\right)+1\end{array}}& \left(4\right)\end{array}$

[0038] (3) Formula 4 shows the equivalent input impedance {circumflex over (z)}(ω.sub.i) of the five-element lumpedparameter model, and the parameter value of each element can be obtained through the system identification method in combination with thetarget input impedance z(ω.sub.i), wherein the parameter value of each element in the corresponding fluid mechanics loop in the normal physiological status is respectively Rc=8.6 kPa.Math.s/ml, R=113.06 kPa.Math.s/ml, C.sub.1=0.0053 ml/kPa, C.sub.2=0.0097ml/kPa and L=19.2972 kPa.Math.s.sup.2/ml,and the parameter value of each element in the corresponding fluid mechanics loop in the heart failure status and under the asynchronous pulsatile working mode of the pump speed after implantation of an artificial heart is respectively Rc=13 kPa.Math.s/ml, R=109 kPa.Math.s/ml, C.sub.1=0.005 ml/kPa, C.sub.2=0.009 ml/kPa and L=1 kPa.Math.s.sup.2/ml. As shown in FIG. 6, the input impedance curve (solid line in FIG. 6) corresponding to the five-element lumpedparameter model and the target input impedance curve (circle in FIG. 6) basically coincide. Based on the parameter values of the elements in the above three different physiological conditions, after the corresponding input blood flow waveform is given, the blood pressure waveform obtained by Matlab/simulink simulation is basically consistent with the corresponding blood pressure waveform in FIG. 5, as shown in FIG. 6, and the root mean square errors are 0.237, 0.401 and 0.625 respectively; and then the fluid mechanics circulation loop based on the cell culture model on a microfluidic chip as shown in FIG. 2 is constructed;

[0039] (4) The chip is made through a standardized micromachining method, an elastic film with the elastic modulus similar to that of an artery is bonded to a cavity which is made of hard and transparent PMMA and has a concave section, and the cell culture model below the lower surface of the elastic film is full of circulating fluid; air is introduced into cavities on both sides of the upper surface of the elastic film to provide enough space for the deformation of the film at the cavities on both sides under the action of pulsating fluid pressure; both ends of a cavity in the middle of the upper surface of the elastic film are smooth and cambered, the middle part of the upper surface of the film is close to the inner surface of the concave part of the cavity in the horizontal direction, which enables the lower elastic film adhering to endothelial cells to produce horizontal stretch strain under the action of stretch of both sides, and the concave thickness of the cavity shall be designed to ensure that the microscope can focus when being used for observing the morphological structure of endothelial cells and the cavity will not deform under the action of pulsating pressure; and the geometric size of the lower cell culture model and the elastic modulus of the elastic film shall be selected according to the principle of elastic mechanics, and determined by accurately simulating the actual need of the waveform of blood pressure, shear stress and stretch strain in the endothelial microenvironment of different parts of aorta;

[0040] (5) An in vitro endothelial cell culture model and a periphery monitoring system shown in FIG. 1 is established, includinga pulse blood pump (i), an artificial heart (i), a signal acquisition and processing system (ii) composed of a plurality of components, and a PID feedback control system (iii). The signal acquisition and processing system (ii) comprises an inverted fluorescence microscope, a CCD high-speed camera system, pressure and flow sensors, and is used for real-time monitoring and acquisition of pressure and flow waveforms of the input end A and the output end B of the cell culture model, and the actual morphological structure of cells in the cell culture model on a microfluidic chip. The pulse blood pump (i) can accurately simulate normal and heart failure physiological conditions in combination with the PID feedback control device (iii), and the artificial heart device (i) is connected in parallel to both ends of the pulse blood pump, and can accurately simulate signals of blood pressure, wall shear stress and stretch strain on vascular endothelial cells of specific parts of the aortic arch under different pulsatile working modes of the artificial heart pump speed in combination with the PID feedback control device (iii), to finally load quantitative and controllable pulsating flow signals into the multielement mock circulatory system; The acquired signals are fed back to the PID control device (iii) to further regulate the pulse blood pump (i) and the artificial heart (i), so as to quantitatively regulate changes in the amplitude and frequency of pressure and flow signals on the multielement mock circulatory system, and finally produce the combined effect of blood pressure, shear stress and stretch strain under different pulsatile working modes of the artificial heart pump speed in the cell culture model on a microfluidic chip.

[0041] The pressure in the microfluidic chip can be measured by the pressure sensor; the shear stress can be calculated according to the flow measured by the flow sensor and the geometric size of the cell culture model; and the horizontal stretch strain of the elastic film in the chip under different pressures can be calibrated on the film by using fluorescent microspheres and measured by the fluorescence microscope. The strain corresponding to the elastic film is obtained by giving different pressures, so as to establish a relational expression between the pressures and the stretch strain. According to the approximate expression, the stretch strain of the elastic film in the actual experiment is determined under the condition that the pressure is known. In addition, the morphological structure of endothelial cells is detected and recorded by the microscope in combination with the CCD high-speed camera system and saved to the computer.

[0042] (6) The specific experimental steps for studying the quantitative relationship between different pulsatile working modes of the pump speed of the continuous flow artificial heart and hemodynamics signals of a local arterial endothelial microenvironment are as follows: [0043] Step 1: carrying out subculturing on primarily cultured endothelial cells by an EGM culture medium, wherein the 2.sup.nd to 5.sup.th generations are used for experiments. During experiments, endothelial cells are planted on an elastic film of a cell culture model on a microfluidic chip coated with Fibronection so that the cells adhere to the wall and the degree of fusion is more than 90%. [0044] Step 2: loading combined stimulation of hemodynamics signals corresponding to different pulsatile working modes of the artificial heart pump speed on arterial endothelial cells; and using NucView™-488 cell activity detection reagent for cell activity detection to ensure the effectiveness of the in vitro mock circulatory system. [0045] Step 3: collecting cell samples from the cell culture model on a microfluidic chip to determine gene and protein expression levels, so as to obtain the influence of hemodynamics signals such as blood pressure, shear stress and stretch strain corresponding to different pulsatile working modes of the artificial heart pump speed on the gene and protein expression levels of vasoactive substances and proinflammatory cytokines.

[0046] The present invention can successfully reproduce signals of blood pressure, wall shear stress and stretch strain on in vivo arterial endothelial cells in different pulsatile working modes of the artificial heart pump speed, and monitor the differentiated influence of the functions of arterial endothelial cells cultured under the combined stimulation of the above hemodynamics signals in real time.