Parallel plate flow cavity system for simulating cross-wall gap flow

Abstract

The invention belongs to the technical field of mechanical biology research, and particularly relates to a parallel plate flow cavity system for simulating cross-wall interstitial flow, the parallel plate flow cavity system comprises a circulation system and a seepage system for simulating cross-wall interstitial flow, the circulation system comprises a flow cavity, a resistance valve, a lower groove, a peristaltic pump, an upper groove and a pulse damper which are connected in sequence and form a loop; the flow cavity comprises a cover plate, a bottom plate and a percolating fluid collecting device which are sequentially connected from top to bottom, and the parallel plate flow cavity capable of simultaneously simulating cross-wall gap flow in a physiological environment and simultaneously loading flow shear force is comprehensively provided by arranging double paths and a height-adjustable liquid storage device; therefore, the stress environment of the vascular cells can be restored more truly, and data visualization processing can be conveniently carried out on shear force distribution in the flowing cavity.

Description

Technical Field

[0001] The invention belongs to the technical field of mechanical biology research, and in particular relates to a parallel plate flow chamber system for simulating cross-wall gap flow. Background Art

[0002] Cell morphology, structure, proliferation, migration, apoptosis and other biological behaviors as well as material metabolism are closely related to the wall shear stress. Flow chamber is one of the main tools for fluid shear stress loading on cells cultured in vitro and is often used in mechanobiology research.

[0003] For example, the Chinese invention patent with application number 2014101175747 discloses a parallel plate flow chamber system for simulating an oscillating blood flow shear stress environment, which includes a parallel plate flow chamber and an oscillating shear stress stimulation generating device. The parallel plate flow chamber has a rectangular cross section, a height of 0.02 to 0.025 cm, a width of 0.002 to 0.0025 cm, and a length of 5 to 10 cm; the oscillating shear stress stimulation generating device includes an upper liquid storage tank, a medical solenoid valve, an elastic chamber, a resistance valve, a lower liquid storage tank, a peristaltic pump, and a silicone tube. By changing the frequency of the medical solenoid valve switch, and the size and ratio of the front and rear flow loads of the parallel plate flow chamber, an oscillating flow environment with different average values, oscillation amplitudes, and oscillation frequencies is achieved inside the parallel plate flow chamber, thereby applying oscillating shear stresses of different characteristics to the vascular endothelial cells cultured at the bottom of the parallel plate flow chamber.

[0004] However, under physiological conditions, there is transmural interstitial flow in blood vessels, which originates from transendothelial flow in blood vessels and is driven by the difference in hydrostatic pressure and osmotic pressure between blood vessels and interstitium. Therefore, vascular cells, especially vascular smooth muscle cells, are also affected by the flow shear force generated by transmural interstitial flow. It is estimated that the average level of shear force exerted by this transmural interstitial flow on smooth muscle cells is 1-3 dyn / cm 2 This level is sufficient to induce biological responses in cells.

[0005] Therefore, with the increasing requirements for experimental accuracy, there is an urgent need for a flow chamber that can simultaneously load transmural interstitial flow under laminar flow, so as to more comprehensively simulate the forces on cells under physiological conditions. Summary of the invention

[0006] The object of the present invention is to provide a parallel plate flow chamber system for simulating transmural interstitial flow and a control method thereof, so as to more comprehensively simulate the stress conditions of vascular cells under physiological environment.

[0007] In order to solve the above-mentioned technical problems, the present invention specifically adopts the following technical solutions: A parallel plate flow chamber system for simulating cross-wall gap flow, comprising a circulation system and a seepage system for simulating cross-wall gap flow, wherein the circulation system comprises a flow chamber, a resistance valve, a lower tank, a peristaltic pump, an upper tank and a pulse damper which are sequentially connected to form a loop; The flow chamber comprises a cover plate, a bottom plate and a seepage liquid collecting device with adjustable height connected in sequence from top to bottom; the cover plate is provided with a water inlet connected to the liquid outlet end of the pulse damper, and a water outlet connected to the liquid inlet end of the resistance valve; The upper surface of the bottom plate is sunken downward to form a groove. When the cover plate is mounted on the bottom plate, the lower surface of the cover plate cooperates with the groove to form a laminar flow area. A through hole penetrating the bottom plate is provided in the groove. The seepage liquid collecting device is provided with a placement tank and a buffer tank which are connected from top to bottom in sequence, the top of the placement tank is provided with an opening corresponding to the through hole, when the cell culture chamber is placed in the placement tank, the porous membrane on the top of the cell culture chamber and the cell layer cultured on the first surface of the porous membrane form a seepage layer located between the through hole and the placement tank; the side wall of the buffer tank is provided with a liquid outlet, and the liquid outlet is connected to a liquid storage device through a seepage pipe; the inner diameter of the liquid storage device is larger than the inner diameter of the seepage pipe, and the liquid level of the upper tank is higher than the liquid level of the liquid storage device, so that the liquid in the upper tank forms a liquid level difference with the liquid in the liquid storage device; When the parallel plate flow chamber system is started, under the action of the resistance valve and the peristaltic pump, part of the liquid flowing through the laminar flow area passes through the water outlet, the resistance valve, the lower tank, the peristaltic pump, the upper tank, the pulse damper and the water inlet in sequence, and returns to the laminar flow area to form a first flow path; at the same time, under the action of the liquid level difference, part of the liquid flowing through the laminar flow area penetrates the seepage layer and then passes through the placement tank, the buffer tank and the seepage pipe in sequence to enter the liquid storage device to form a second flow path.

[0008] As an improvement, the through hole includes a first mounting area and a second mounting area sequentially arranged from top to bottom, the width of the first mounting area is smaller than the width of the second mounting area, at least two fixing holes are arranged at the bottom of the base plate, and at least two mounting holes cooperating with the fixing holes are arranged on the permeate collecting device; When the cell culture chamber is inverted in the placement groove and bolts are passed through the mounting holes and the fixing holes in sequence to fix the permeate collection device to the base plate, the outer wall of the placement groove abuts against the second mounting area, and the outer wall of the cell culture chamber abuts against the first mounting area and the inner wall of the placement groove at the same time, so that the cell culture chamber and the buffer groove are connected to form a flow space.

[0009] As an improvement, an installation slope is provided between the first installation area and the second installation area, and an installation space is formed between the installation slope, the permeate collecting device and the cell culture chamber, and a sealing gasket is provided in the installation space.

[0010] As an improvement, a limiting member is provided at the connection between the placement groove and the buffer groove, and the opening of the cell culture chamber abuts against the limiting member.

[0011] As an improvement, the inner diameter of the buffer groove is larger than the inner diameter of the placement groove.

[0012] As an improvement, the through hole, the opening, the placement groove and the buffer groove are coaxially arranged, and the water inlet and the water outlet are symmetrical about the axis of the through hole.

[0013] As an improvement, the parallel plate flow chamber system further comprises a mounting frame, a lifting mechanism is provided on the mounting frame, and the liquid storage device is connected to the lifting mechanism; When the lifting mechanism is controlled to move up and down, the lifting mechanism drives the liquid storage device to move up and down, so that there are liquid level differences of different sizes between the liquid level in the liquid storage device and the liquid level in the upper tank.

[0014] The present invention also provides a control method for a parallel plate flow chamber system for simulating cross-wall gap flow. Based on the above parallel plate flow chamber system, the control method comprises the steps of: S101, starting the parallel plate flow chamber system and acquiring initialization data, wherein the initialization data at least includes a preset first liquid level difference and a current first liquid level height in the upper tank; S102, controlling the lifting mechanism in the parallel plate flow chamber system to operate according to the first liquid level difference, so that the liquid storage device is adjusted to a first height, so that the liquid level difference between the liquid in the upper tank and the liquid storage device meets the first liquid level difference; S103, after a first preset time period, obtaining a current second liquid level difference between the liquid in the upper tank and the liquid storage device; S104, determining whether a first difference between the second liquid level difference and the first liquid level difference is greater than a first preset threshold value, if so, proceeding to S105, otherwise maintaining the current working state; wherein the working state at least includes the height of the liquid storage device; S105, controlling the lifting mechanism to operate according to the first reduction, adjusting the liquid storage device from the first height to the second height, and executing steps S103 to S105 until a first difference between the second liquid level difference and the first liquid level difference is less than the first preset threshold.

[0015] As an improvement, before executing step S105, the following steps are further included: S41, obtaining the second liquid level in the upper tank; S42, calculating a second difference between the first difference and the first preset threshold, and determining whether the second difference is greater than the second preset threshold, if so, executing S105, otherwise entering S43; wherein the second preset threshold is a third difference between the maximum liquid level in the upper tank and the second liquid level; S43, increase the rotation speed of the peristaltic pump according to the first increment, and adjust the liquid level in the upper tank to a third liquid level, so that the fourth difference between the first liquid level difference and the third liquid level difference is smaller than the first preset difference; wherein the third liquid level difference is the difference between the third liquid level and the current liquid level of the liquid storage device.

[0016] As an improvement, in S104, after determining that the first difference between the second liquid level difference and the first liquid level difference is greater than a first preset threshold, the step of: S106, calculating a liquid level change rate according to the first liquid level difference and the second liquid level difference; S107, determining whether the liquid level change rate meets the preset change rate, if so, maintaining the parallel plate flow chamber system in the current working state, otherwise issuing an early warning.

[0017] The principle and beneficial technical effects of the present invention are: The present invention provides a parallel plate flow chamber that can simultaneously simulate transmural interstitial flow under physiological conditions and simultaneously load flow shear force, so as to more realistically restore the force environment of vascular cells, thereby facilitating data visualization processing of shear force distribution in the flow chamber.

[0018] The fluid in the flow chamber in the prior art is "unidirectional flow", that is, lateral flow. Therefore, whether it is by changing the flow rate to change the magnitude of the oscillating shear stress (for details, please refer to the Chinese invention patent with application number 2014101175747), or by changing the shape of the internal structure of the flow chamber to form vortices, it is impossible to simulate the cross-wall gap flow under the physiological environment. However, the present application sets a "dual path" to enable the fluid in the flow chamber to achieve "bidirectional flow", and the bidirectional flow can be achieved only by the liquid level difference between the liquid storage device and the upper tank, without the need to set up other power devices, and the structure is simple and the cost is low.

[0019] Furthermore, in the present application, the height of the liquid storage device is manually adjusted or automatically adjusted (automatically controlled by the lifting mechanism) so that the liquid level difference between the liquid storage device and the liquid surface in the upper tank is different. On the one hand, it can adapt to permeation layers with different permeabilities (for example, a permeation layer with a single layer of cells, and for example, a permeation layer with a double layer of cells in a combined culture process, or, due to different cell densities, the permeation speed of the permeation layer will also be different). On the other hand, after starting the system for a period of time, when the liquid level difference gradually decreases to below the set threshold, the height of the liquid storage device is lowered so that the liquid level difference meets the requirements, thereby keeping the seepage velocity in the device basically stable, avoiding the seepage velocity gradually slowing down with the decrease of the liquid level difference, which leads to a large difference in flow rate during the culture process.

[0020] In summary, the present application sets up a "dual path" and a highly adjustable liquid storage device, so that the system can simultaneously provide stable lateral shear force and osmotic shear force to the cells in the cell culture chamber without adding an additional power device. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0022] Figure 1 Schematic diagram of the overall structure of the parallel plate flow chamber system in an embodiment of the present invention; Figure 2 Schematic diagram of the overall structure of the flow chamber in an embodiment of the present invention; Figure 3 It is a schematic diagram of the overall structure of the flow chamber from another angle in an embodiment of the present invention; Figure 4 is a cross-sectional view of a flow chamber in an embodiment of the present invention; Figure 5 is a cross-sectional view of a bottom plate of a flow chamber in an embodiment of the present invention; Figure 6 A top view of the external structure of the flow chamber in an embodiment of the present invention; Figure 7 A top view of the internal structure of the flow chamber in an embodiment of the present invention; Figure 8 Schematic diagram of the distribution of vascular endothelial cells in an inverted cell culture chamber in an embodiment of the present invention; Fig. 9 Schematic diagram of the distribution of vascular endothelial cells and vascular smooth muscle cells in an inverted cell culture chamber in an embodiment of the present invention; Fig.10 is a flow chart of a control method of a parallel plate flow chamber according to an embodiment of the present invention; Fig.11 A schematic diagram of the shear force distribution in a flow chamber of an experiment; Fig.12 This is a schematic diagram of the distribution of RAEC cells on the cell culture chamber in Experiment 2; Fig.13 Schematic diagram of DiI-LDL uptake by RAEC cells at different time periods in Experiment 2.

[0023] Markings in the figure: 1. flow chamber; 2. resistance valve; 3. lower tank; 31. air purifier; 4. peristaltic pump; 5. upper tank; 6. pulse damper; 7. cover plate; 71. water inlet; 72. water outlet; 8. bottom plate; 81. groove; 82. through hole; 821. first installation area; 822. second installation area; 83. fixing hole; 84. sealing gasket; 9. seepage liquid collection device; 91. placement tank; 92. buffer tank; 93. liquid outlet; 10. seepage pipeline; 11. liquid storage device; 12. mounting frame; 13. lifting mechanism; 14. cell culture chamber; 141. porous membrane; 142. vascular epithelial cells; 143. vascular smooth muscle cells; 15. reflux pipeline. Specific implementation method 2 In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] Herein, the suffixes such as "module", "component" or "unit" used to represent elements are used only to facilitate the description of the present invention and have no specific meaning in themselves. Therefore, "module", "component" or "unit" can be used in a mixed manner. Herein, the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", "front", "back", "one end", "the other end", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0026] In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium, or it can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In this document, "plurality" means two or more, that is, it includes two, three, four, five, etc.

[0027] Embodiment 1 This embodiment is basically as shown in the attached Figure 1-Figure 9 As shown: See also Figure 1 The present invention provides a parallel plate flow chamber system for simulating cross-wall gap flow, including a circulation system and a seepage system for simulating cross-wall gap flow, wherein the circulation system includes a flow chamber 1, a resistance valve 2, a lower tank 3, a peristaltic pump 4, an upper tank 5 and a pulse damper 6 which are sequentially connected to form a loop.

[0028] See also Figure 2 and Figure 3 The flow chamber includes a cover plate 7, a bottom plate 8 and a height-adjustable seepage liquid collecting device 9 which are sequentially connected from top to bottom.

[0029] See also Figure 4 The cover plate is provided with a water inlet 71 connected to the liquid outlet end of the pulse damper, and a water outlet 72 connected to the liquid inlet end of the resistance valve; the upper surface of the base plate is recessed downward to form a groove. When the cover plate is installed on the base plate, the lower surface of the cover plate cooperates with the groove to form a laminar flow area, and a through hole penetrating the base plate is provided in the groove.

[0030] The upper surface of the bottom plate refers to the side close to the cover plate, and the lower surface of the cover plate refers to the side close to the bottom plate. In this article, "from top to bottom" means Figure 4 In the direction indicated by the arrow.

[0031] See also Figure 1 and Figure 4, the seepage liquid collection device is provided with a placement tank 91 and a buffer tank 92 which are connected from top to bottom in sequence, the top of the placement tank 91 is provided with an opening corresponding to the through hole, when the cell culture chamber 14 is placed in the placement tank 91, the porous membrane at the top of the cell culture chamber and the cell layer cultured on the first surface of the porous membrane form a seepage layer located between the through hole and the placement tank; the side wall of the buffer tank 92 is provided with a liquid outlet 93, and the liquid outlet 93 is connected to a liquid storage device through a seepage pipe; the inner diameter of the liquid storage device is larger than the inner diameter of the seepage pipe, and the liquid level of the upper tank is higher than the liquid level of the liquid storage device, so that the liquid in the upper tank forms a liquid level difference with the liquid in the liquid storage device. Wherein, the top of the cell culture chamber refers to when the cell culture chamber is inverted, its opening faces downward, and the porous membrane located at the bottom of the cup is located at the "top of the cell culture chamber" at this time.

[0032] When the parallel plate flow chamber system is started, under the action of the resistance valve and the peristaltic pump, part of the liquid flowing through the laminar flow area passes through the water outlet, the resistance valve, the lower tank, the peristaltic pump, the upper tank, the pulse damper and the water inlet in sequence, and returns to the laminar flow area to form a first flow path; at the same time, under the action of the liquid level difference, part of the liquid flowing through the laminar flow area penetrates the seepage layer and then passes through the placement tank, the buffer tank and the seepage pipe in sequence to enter the liquid storage device to form a second flow path.

[0033] Among them, see Figure 8 The cells on the cell culture chamber may be a single layer. For example, when the cells are vascular endothelial cells 142 cultured on the first side (also referred to as the first surface) of the porous membrane, the seepage layer is a seepage layer formed by the vascular endothelial cells 142 and the porous membrane 141 and located between the through hole and the placement groove; see Fig. 9 The cells on the cell culture chamber can also be a double layer. For example, when the cells are vascular endothelial cells 142 cultured on the first side of the porous membrane and vascular smooth muscle cells 143 cultured on the second side of the porous membrane, the seepage layer is a seepage layer formed by the vascular endothelial cells 142, the porous membrane 141 and the vascular smooth muscle cells 143 and located between the through hole and the placement groove.

[0034] In some embodiments, the porous membrane of the cell culture chamber is flush with the bottom of the groove; when the cells on the cell culture chamber are a single layer, that is, the vascular endothelial cells located on the first side of the porous membrane, the liquid will generate a lateral shear force on the vascular endothelial cells during the lateral flow process (first flow path), and at the same time, part of the liquid enters the liquid storage device through the seepage layer, and this process will generate a seepage shear force on the vascular endothelial cells. When the cells are a double layer, that is, including the vascular endothelial cells located on the first side of the porous membrane and the vascular smooth muscle cells located on the second side of the porous membrane, the force on the vascular endothelial cells is the same as that in the case of a single layer, and the vascular smooth muscle cells are subjected to the seepage shear force.

[0035] In some embodiments, see Figure 1 The upper tank is provided with a first reflux port, and the lower tank is provided with a second reflux port, and the first reflux port and the second reflux port are connected through a reflux pipe 15; when the liquid in the upper tank reaches the first reflux port, part of the liquid flows back to the lower tank through the reflux pipe 15, so that the liquid level in the upper tank is always flush with the first reflux port, that is, the maximum liquid level is reached.

[0036] In some embodiments, see Figure 1 The lower tank 3 is provided with an air inlet connected to the external environment, and an air purifier 31 is provided at the air inlet.

[0037] In some embodiments, see Figure 5 The through hole includes a first mounting area 821 and a second mounting area 822 arranged in sequence from top to bottom, the width of the first mounting area 821 is smaller than the width of the second mounting area 822, the bottom of the bottom plate is provided with at least two fixing holes 83, and the permeate collecting device is provided with at least two mounting holes matching the fixing holes 83; when the cell culture chamber is inverted in the placement groove, and bolts are sequentially passed through the mounting holes and the fixing holes 83, so that the permeate collecting device is fixed to the bottom plate, see Figure 4 The outer wall of the placement groove abuts against the second installation area, and the outer wall of the cell culture chamber abuts against the first installation area and the inner wall of the placement groove at the same time, so that the cell culture chamber and the buffer groove are connected to form a relatively closed flow space (both sides are closed, and the liquid can only penetrate from the porous membrane of the cell culture chamber and flow out from the liquid outlet located on the side wall of the buffer groove).

[0038] In some embodiments, see Figure 4 and Figure 5An installation slope is arranged between the first installation area 821 and the second installation area 822. The installation slope, the permeate collecting device and the cell culture chamber form an installation space, and a sealing gasket 84 is arranged in the installation space.

[0039] In some embodiments, a limiting member is provided at the connection between the placement tank and the buffer tank, and the opening of the cell culture chamber abuts against the limiting member. Specifically, the limiting member can be a mounting plate provided along the circumference of the inner wall of the cell culture chamber.

[0040] The above structure can ensure the sealing inside the flow chamber, so that after passing through the seepage layer, the liquid can only enter the buffer tank through the internal space of the cell culture chamber and then flow to the seepage pipeline and the liquid storage device.

[0041] In some embodiments, see Figure 4 The inner diameter of the buffer tank 92 is larger than the inner diameter of the placement tank 91. In this way, after the liquid passes through the cell culture chamber and enters the buffer tank, there is enough buffer space, so that the liquid flowing into the permeation pipe maintains a stable flow rate, thereby ensuring the stability of the entire device.

[0042] In some embodiments, the through hole, the opening, the placement groove and the buffer groove are coaxially arranged, and the water inlet and the water outlet are symmetrical about the axis of the through hole.

[0043] In some embodiments, the water inlet and the water outlet are also symmetrically arranged on both sides of the through hole.

[0044] In some embodiments, see Figure 1 The parallel plate flow chamber system also includes a mounting frame 12, on which a lifting mechanism 13 is provided, and the liquid storage device 11 is connected to the lifting mechanism 13; when the lifting mechanism 13 is controlled to rise and fall, the lifting mechanism 13 drives the liquid storage device 11 to rise and fall, so that the liquid in the liquid storage device 11 and the liquid in the upper tank 5 have different liquid level differences.

[0045] In some specific embodiments, the lifting mechanism includes a slide rail arranged along the height direction of the mounting frame, a slider slidably arranged in the slide rail, and a driving motor. The liquid storage device is fixedly connected to the slider. When the driving motor is started to drive the slider to slide up and down in the slide rail, the liquid storage device can move up and down synchronously driven by the slider.

[0046] Of course, in other embodiments, the lifting mechanism may also be a movable plate movably arranged on the mounting frame, and a fixing member fixing the movable plate on the mounting frame, and the liquid storage device is fixedly arranged on the movable plate; when the movable plate is manually moved up and down relative to the mounting frame to a specified position, the two are fixed by the fixing member, so that the liquid storage device on the movable plate can be moved to different heights. In some exemplary embodiments, the mounting frame and the lifting mechanism are iron frames.

[0047] Through the above structure, the present invention provides a parallel plate flow chamber that can simultaneously simulate transmural gap flow under physiological conditions and simultaneously load flow shear force, so as to more realistically restore the force environment of vascular cells, thereby facilitating data visualization processing of shear force distribution in the flow chamber.

[0048] The present application realizes "bidirectional flow" of the fluid in the flow chamber by setting up a "dual path", and the bidirectional flow can be realized only by the liquid level difference between the liquid storage device and the upper tank without the need to set up other power devices. The structure is simple and the cost is low.

[0049] Furthermore, in the present application, the height of the liquid storage device is manually adjusted or automatically adjusted (automatically controlled by the lifting mechanism) so that the liquid level difference between the liquid storage device and the liquid surface in the upper tank is different. On the one hand, it can adapt to permeation layers with different permeabilities (for example, a permeation layer with a single layer of cells, and for example, a permeation layer with a double layer of cells in a co-culture process, or, due to different cell densities, the permeation speed of the permeation layer will also be different). On the other hand, after starting the system for a period of time, when the liquid level difference gradually decreases to below the set threshold, the height of the liquid storage device is lowered so that the liquid level difference meets the requirements, thereby keeping the seepage velocity in the device basically stable, avoiding the situation where the seepage velocity gradually slows down as the liquid level difference decreases.

[0050] In summary, the present application sets up a "dual path" and a highly adjustable liquid storage device, so that the system can simultaneously provide stable lateral shear force and osmotic shear force to the cells in the cell culture chamber without adding an additional power device.

[0051] Embodiment 2 See also Fig.10 The present invention also provides a control method for a parallel plate flow chamber system for simulating cross-wall gap flow, based on the parallel plate flow chamber system for simulating cross-wall gap flow in the first embodiment, comprising the steps of: S101, starting the parallel plate flow chamber system and acquiring initialization data, wherein the initialization data at least includes a first liquid level difference and a current first liquid level height in the upper tank.

[0052] The first liquid level difference is a liquid level difference selected to match the cells cultured in the cell chamber after a large number of experiments are conducted in advance, according to different types of cells or different conditions of cells (for example, a permeable layer with a single layer of cells, or a permeable layer with a double layer of cells in a co-culture process, or, due to different cell densities, there will be certain differences in the permeation speed of the permeable layer). During the entire experiment, due to the continuous seepage of the liquid, the liquid level in the liquid storage device will slowly increase, and the liquid level in the upper tank will slowly decrease, so that the liquid level difference between the two will gradually decrease. Therefore, the first liquid level difference refers to the initial liquid level difference that can ensure that, under normal circumstances, after the experiment, there is still a certain liquid level difference between the liquid in the liquid storage device and the upper tank.

[0053] S102, controlling the lifting mechanism to operate according to the first liquid level difference, and adjusting the liquid storage device to a first height, so that the liquid level difference between the liquid in the upper tank and the liquid storage device meets the first liquid level difference.

[0054] In some embodiments, the liquid level heights in the liquid storage device and the upper tank can be obtained respectively, and then the liquid level difference can be calculated through the liquid level heights of the two. When obtaining the liquid level height, it can be directly measured by setting sensors in the liquid storage device and the upper tank respectively, or it can be obtained by capturing images and performing image analysis.

[0055] S103, starting the parallel plate flow chamber system, and obtaining a current second liquid level difference between the liquid in the upper tank and the liquid storage device after a first preset time.

[0056] In some embodiments, the first preset time duration may be preset by a user, for example, the current liquid level difference is periodically obtained every half an hour.

[0057] S104, determine whether the first difference between the second liquid level difference and the first liquid level difference is greater than a first preset threshold value (the smaller the first difference is, the closer the liquid level heights are, so no adjustment is required; if the first difference is larger, it means that the liquid level heights are quite different, and the liquid level height in the liquid storage device needs to be adjusted). If so, enter S105, otherwise maintain the current working state; wherein the working state at least includes the height of the liquid storage device.

[0058] Among them, the first preset threshold value can also be set by the user, and can also be directly matched in a pre-constructed database. When the first difference is less than the first preset threshold value, it means that the current liquid level difference is insufficient to meet the needs of subsequent experiments, that is, before the experiment is completed, the liquid level difference between the two may disappear, that is, no seepage will occur in the flow chamber; and when the first difference is greater than the first preset threshold value, it means that the current liquid level difference is sufficient to meet the needs of subsequent experiments, that is, when the experiment is completed, there is still a certain liquid level difference between the two, that is, seepage continues to occur during the entire experiment.

[0059] S105, controlling the lifting mechanism to work according to the first decrement, adjusting the liquid storage device from the first height to the second height, and executing steps S103 to S105 until the first difference between the second liquid level difference and the first liquid level difference is less than the first preset threshold value. The second height is less than the first height.

[0060] Among them, when determining the first reduction, the first difference between the second liquid level difference and the first liquid level difference is based on being able to be less than the first preset threshold. Of course, in the specific control process, due to insufficient adjustment accuracy or other conditions, the second liquid level difference after adjustment may still not meet the requirements, so it may be necessary to adjust multiple times, and finally make the first difference between the second liquid level difference and the first liquid level difference less than the first preset threshold.

[0061] By monitoring the current liquid level difference and judging and adjusting the height of the liquid storage device according to the liquid level difference, the liquid in the upper tank and the liquid storage device can always maintain a certain liquid level difference during the entire experiment, that is, the liquid level in the liquid storage device is always lower than the liquid level in the upper tank, so that the flow chamber always maintains seepage, so as to restore a more realistic cell physiological environment.

[0062] In some embodiments, before executing step S105, the following steps are further included: S41, obtaining the second liquid level in the upper tank; S42, calculate the second difference between the first difference and the first preset threshold, and determine whether the second difference is greater than the second preset threshold. If so, execute S105, otherwise enter S43; wherein the second preset threshold is the third difference between the maximum liquid level height in the upper tank and the second liquid level height.

[0063] When the second difference is greater than the second preset threshold, it means that merely adjusting the liquid level in the upper tank is insufficient to adjust the current liquid level difference to a reasonable range (that is, it cannot be guaranteed that the difference between the first liquid level difference and the adjusted liquid level difference is less than the first difference. At this time, the height of the liquid storage device needs to be adjusted. It should be noted that the second preset threshold is variable, and as the liquid level in the upper tank decreases, the second preset threshold will gradually increase.

[0064] In some embodiments, the maximum liquid level height refers to the liquid level height when the upper tank is full of liquid; in other embodiments, the maximum liquid level height refers to the liquid level height when the liquid level in the upper tank is flush with the first reflux port.

[0065] S43, increase the rotation speed of the peristaltic pump according to the first increment, and adjust the liquid level in the upper tank to a third liquid level, so that the fourth difference between the first liquid level difference and the third liquid level difference is smaller than the first preset difference; wherein the third liquid level difference is the difference between the third liquid level and the current liquid level of the liquid storage device.

[0066] Wherein, the third liquid level height is less than or equal to the maximum liquid level height, and the third liquid level height is greater than the second liquid level height.

[0067] In some embodiments, step S44 is also included, when the fourth difference between the third liquid level difference between the upper tank and the liquid storage device and the first liquid level difference is less than the first preset difference, the rotation speed of the peristaltic pump is reduced so that the liquid level of the upper tank is maintained at the third liquid level.

[0068] During the operation of the system, with the seepage effect of the flow cavity, part of the liquid will flow from the upper tank into the liquid storage device. Therefore, the liquid level in the upper tank will slowly decrease, and the liquid level in the liquid storage device will slowly increase. In the case of a large change in the liquid level difference, the liquid level difference can be made to meet the conditions again by manually or automatically adjusting the height of the liquid storage device. When the liquid level difference changes slightly, that is, when the liquid level difference needs to be adjusted by a smaller amplitude, the liquid level difference can be "fine-tuned" more accurately by adjusting the speed of the peristaltic pump, thereby avoiding the situation where manual adjustment is difficult to ensure accuracy when the adjustment amplitude is small and repeated adjustments are required.

[0069] In summary, the present application sets up a dual selection control scheme of "coarse adjustment" + "fine adjustment". When the amplitude to be adjusted is small, the "fine adjustment" with higher precision is selected, that is, the liquid level in the upper tank is adjusted by adjusting the speed of the peristaltic pump; when the amplitude to be adjusted is large, a lifting mechanism with lower precision but larger control amplitude is selected for adjustment, which can quickly and accurately adjust the liquid level difference to meet the requirements.

[0070] In some embodiments, in S104, after determining that the first difference between the second liquid level difference and the first liquid level difference is greater than a first preset threshold, the method further includes the following steps: S106, calculating a liquid level change rate based on the first liquid level difference and the second liquid level difference; wherein the liquid level change rate is a first difference between the first liquid level difference and the second liquid level difference divided by a first preset time length.

[0071] S107, determining whether the liquid level change rate meets the preset change rate, if so, maintaining the parallel plate flow chamber system in the current working state, otherwise issuing an early warning.

[0072] The preset change rate refers to the one set in advance after a large number of experiments. When the liquid level change rate does not meet the preset change rate, it means that the experimental requirements cannot be met by adjusting the liquid level height in the upper tank or adjusting the height of the liquid storage device, that is, before the experiment is completed, the liquid level difference between the two may disappear, that is to say, no seepage will occur in the flow chamber.

[0073] When the liquid level difference changes too quickly, there may be a system failure. At this time, simply adjusting the height of the liquid storage device or the liquid level in the upper tank is likely to still not meet the conditions. This solution issues an early warning to prompt the experimenter to repair and thus eliminate the above problem.

[0074] The feasibility of the above scheme is verified through the following experiments: Experiment 1 First, the device in Example 1 is modeled and assembled using 3D modeling software (such as Solidworks 2022). Use Workbench 2021 R2 (ANSYS Inc.) to import the file and perform subsequent processing. The mesh uses the Meshing plug-in that comes with ANSYS, the Target Skewness is set to 0.25, the mesh size is 0.08mm, the inlet and outlet pipes use hexahedral meshes, and the flow chamber uses tetrahedral meshes. When the number of meshes is reduced by 50%, the maximum flow velocity and the maximum wall shear force change by more than 2%; when the number of meshes increases by 50%, the above parameters change by less than 2%. Therefore, the number of mesh units: 33013828, nodes: 7360988 were selected for subsequent experiments.

[0075] Assume that blood is an incompressible Newtonian fluid with no slip on the vascular endothelium. Under these conditions, its flow is governed by the continuity equation and the momentum conservation equation (Navier-Stokes equation): in, , and Represent velocity vector, pressure and time respectively. Represents blood density, which is 1014.5 kg·m -3 ; represents blood viscosity, and its magnitude is 8.62×10 -4 Pa·s. As for the inlet conditions, the flow rate is set to 17.8 ml / min and the flow rate distribution is uniform. The outlet conditions are set to outflow.

[0076] This simulation experiment is based on FLUENT, and uses the "Steady" mode and "Pressure-based" solver to numerically solve the shear force in the perfusion chamber. After the FLUENT solution is completed, the corresponding CFD-Post module in ANSYS is used to post-process and visualize the numerical results.

[0077] Experimental results: like Fig.11 As shown, part A in the figure shows the velocity distribution diagram at the water inlet, the cross section of the laminar flow zone and the water outlet from left to right, and part B shows the wall shear force distribution diagram. It can be seen from the figure that the flow field in the flow cavity in the area where the self-made flow loading system RAEC is located is stable, and the loaded shear force is 0.2±0.005Pa. It can be seen that through this device, a stable flow field can be generated in the flow cavity and a stable shear force can be loaded.

[0078] Experiment 2 This experiment uses the parallel plate flow chamber system for simulating transmural interstitial flow described in Example 1, that is, vascular endothelial cells are exposed to laminar shear force and transmural interstitial flow, simulating pathological shear force levels, and measuring the content of low density lipoprotein (LDL) taken up by cells under this pathological state.

[0079] The reagents used in this experiment are as follows: DMEM culture medium (containing 10% FBS, 1% penicillin-streptomycin mixture), 20 μg / ml fibronectin (Roche), 0.5% glutaraldehyde, paraformaldehyde, 0.9% sodium chloride, 5% glucose aqueous solution, 0.2% silver nitrate, PBS (pH 7.4), developer (KEY-600), 10 mg / ml heparin, 2% bovine serum albumin (BSA), DAPI (Solarbio), and anti-fluorescence quenching sealing solution.

[0080] The instruments and consumables are as follows: T-75 culture flasks, transwell cell culture chambers (referred to as cell culture chambers), scanning electron microscope (JSM-7900F), laser scanning confocal microscope (Dragonfly, Andor), and fluorescence microscope.

[0081] Experimental cells: SD rat aortic endothelial cells (hereafter referred to as RAEC).

[0082] Experimental method steps: 1. Cell inoculation and culture: RAEC were cultured in T-75 culture flasks using DMEM medium at 37°C and 5% CO2, with the medium replaced every 2 days. 4 Cells / cm 2 Cells were seeded at a density of 100 μg / ml on the inside or outside of a cell culture chamber pre-coated with 20 μg / ml fibronectin. For cell seeding, the cell culture chamber was inverted with the basal surface facing up and 1 ml of cell suspension was added. After 4 h, the culture dish was returned to its normal orientation and 2 ml of culture medium was added to the inside and outside. Experiments were performed 4 days after seeding, when the cells had reached confluence but had not overgrown.

[0083] 2. Silver staining: On the 4th day, the cells were immersed in fixative (0.5% glutaraldehyde and 1% paraformaldehyde, prepared in PBS) for 5 min, 0.9% NaCl for 2 min, 5% glucose aqueous solution for 10-15 s, 0.2% AgNO3 aqueous solution for 2 min, 5% glucose aqueous solution for 10-15 s, and fixative for 1-2 min, then the cell culture chamber was turned over, the cells were exposed to ultraviolet light for 15 min, and immersed in developer (KEY-600) for 5 s until the membrane darkened. Then, the cell culture chamber was placed in the fixative overnight, and then dehydrated with 50%, 70%, 95%, and 100% ethanol, dehydrating once every 10 min. After drying in the air, the cell-inoculated side was facing up, sprayed with gold, and observed under a scanning electron microscope to determine the cell culture monolayer.

[0084] 3. DiI-LDL incubation: RAEC monolayers were cultured with 5 μg / ml DiI-LDL in DMEM complete medium (without phenol red) for 48 h. After the culture, the monolayers were washed five times with 2% (BSA) in PBS at 4°C to remove unbound LDL, which was defined as time 0.

[0085] 4. Flow shear force loading: The parallel plate flow chamber system in Example 1 was used as a flow shear force loading system to load shear force on the RAEC monolayer.

[0086] The shear force can be estimated by the following formula: ; Where: 𝜏 is the shear force (dyn / cm²), is the viscosity of the medium (8.62×10 -4 Pa·s), Q is the flow rate of the flow chamber (ml / s), H is the channel height (0.04 cm), and W is the channel width (4.8 cm).

[0087] RAEC monolayers were exposed to shear stress of 2 dyn / cm² (pathological shear stress level) for 12 h to determine whether glycocalyx-bound LDL could be confined to this surface layer. The perfusion medium was DMEM without phenol red (containing 5% FBS and 1% penicillin-streptomycin mixture). The entire flow system was placed in a cell culture incubator and cultured at 37°C and 5% CO2.

[0088] 5. Determination of cellular uptake of LDL: RAEC were fixed in 4% paraformaldehyde for 10 min. After rinsing once with PBS, the slides were mounted with anti-fluorescence quenching mounting medium containing DAPI and observed with a 100× oil objective on a confocal laser scanning microscope (Dragonfly). When scanning the confocal imaging system, it should be ensured that all red areas are covered. At least 5 scans were performed for each sample, 1 in the center and 4 in the periphery. In Imaris 9.0.1 software, the "orthogonal" mode was used to obtain the maximum Z-axis projection image, and the confocal image was exported using the software screenshot function. The obtained fluorescence images were analyzed using Image J software, and the projection image with the maximum fluorescence intensity was obtained by Z-axis projection, and the ratio of the total area of ​​DiI-LDL to the number of cells in a given field of view was calculated.

[0089] 6. Statistical analysis: GraphPad Prism 9 software was used for t-test. p < 0.05 was considered statistically significant. The results were expressed as mean ± standard error.

[0090] Experimental results: Scanning electron microscopy results showed that obvious silver lines were generated at the junctions between adjacent RAECs, indicating the formation of a RAEC monolayer (see Fig.12 ).

[0091] like Fig.13 As shown in the figure, the uptake of DiI-LDL by RAEC at 0h and 12h was recorded, and the results were recorded in Table 1. The results showed that the uptake of DiI-LDL by RAEC at 0h (unit: μm 2 / cell) were 180.1±3.444, 79.23±13.45, 113.5±53.15 respectively, and the RAEC intake at 12h were 209.3±24.35, 211.6±21.21, 141.0±13.21 respectively. The intake at 12h was significantly increased compared with that at 0h, and the difference was significant (p<0.05).

[0092] Table 1 (Measurement table of DiI-LDL uptake by RAEC) It can be seen that the laminar shear force (2 dyn / cm 2 ) and transmural interstitial flow (10cm H2O) promoted the uptake of LDL by endothelial cells, which is consistent with the conclusion reported by previous researchers that pathological shear force promotes cholesterol deposition in the vascular endothelium. This further confirms the reliability of this device.

[0093] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0094] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A parallel plate flow chamber system for simulating cross-wall gap flow, characterized in that: It includes a circulation system and a seepage system for simulating cross-wall gap flow, wherein the circulation system includes a flow chamber, a resistance valve, a lower tank, a peristaltic pump, an upper tank and a pulse damper which are sequentially connected to form a loop; The flow chamber comprises a cover plate, a bottom plate and a seepage liquid collecting device with adjustable height connected in sequence from top to bottom; the cover plate is provided with a water inlet connected to the liquid outlet end of the pulse damper, and a water outlet connected to the liquid inlet end of the resistance valve; The upper surface of the bottom plate is sunken downward to form a groove. When the cover plate is mounted on the bottom plate, the lower surface of the cover plate cooperates with the groove to form a laminar flow area. A through hole penetrating the bottom plate is provided in the groove. The seepage liquid collecting device is provided with a placement tank and a buffer tank which are connected from top to bottom in sequence, the top of the placement tank is provided with an opening corresponding to the through hole, when the cell culture chamber is placed in the placement tank, the porous membrane on the top of the cell culture chamber and the cell layer cultured on the first surface of the porous membrane form a seepage layer located between the through hole and the placement tank; the side wall of the buffer tank is provided with a liquid outlet, and the liquid outlet is connected to a liquid storage device through a seepage pipe; the inner diameter of the liquid storage device is larger than the inner diameter of the seepage pipe, and the liquid level of the upper tank is higher than the liquid level of the liquid storage device, so that the liquid in the upper tank forms a liquid level difference with the liquid in the liquid storage device; When the parallel plate flow chamber system is started, under the action of the resistance valve and the peristaltic pump, part of the liquid flowing through the laminar flow area passes through the water outlet, the resistance valve, the lower tank, the peristaltic pump, the upper tank, the pulse damper and the water inlet in sequence, and returns to the laminar flow area to form a first flow path; at the same time, under the action of the liquid level difference, part of the liquid flowing through the laminar flow area penetrates the seepage layer and then passes through the placement tank, the buffer tank and the seepage pipe in sequence to enter the liquid storage device to form a second flow path.

2. A parallel plate flow chamber system for simulating cross-wall gap flow according to claim 1, characterized in that: The through hole includes a first mounting area and a second mounting area sequentially arranged from top to bottom, the width of the first mounting area is smaller than the width of the second mounting area, at least two fixing holes are arranged at the bottom of the bottom plate, and at least two mounting holes matching with the fixing holes are arranged on the permeate collecting device; When the cell culture chamber is inverted in the placement groove and bolts are passed through the mounting holes and the fixing holes in sequence to fix the permeate collection device to the base plate, the outer wall of the placement groove abuts against the second mounting area, and the outer wall of the cell culture chamber abuts against the first mounting area and the inner wall of the placement groove at the same time, so that the cell culture chamber and the buffer groove are connected to form a flow space.

3. A parallel plate flow chamber system for simulating cross-wall gap flow according to claim 2, characterized in that: An installation slope is arranged between the first installation area and the second installation area. The installation slope, the permeate collecting device and the cell culture chamber enclose an installation space, and a sealing gasket is arranged in the installation space.

4. A parallel plate flow chamber system for simulating cross-wall gap flow according to claim 1, characterized in that: A limiting piece is arranged at the connection between the placement groove and the buffer groove, and the opening of the cell culture chamber abuts against the limiting piece.

5. A parallel plate flow chamber system for simulating cross-wall gap flow according to claim 1, characterized in that: The inner diameter of the buffer groove is larger than the inner diameter of the placement groove.

6. A parallel plate flow chamber system for simulating cross-wall gap flow according to claim 1, characterized in that: The through hole, the opening, the placement groove and the buffer groove are coaxially arranged, and the water inlet and the water outlet are symmetrical about the axis of the through hole.

7. A parallel plate flow chamber system for simulating cross-wall gap flow according to claim 1, characterized in that: The parallel plate flow chamber system further comprises a mounting frame, a lifting mechanism is arranged on the mounting frame, and the liquid storage device is connected to the lifting mechanism; When the lifting mechanism is controlled to move up and down, the lifting mechanism drives the liquid storage device to move up and down, so that there are liquid level differences of different sizes between the liquid level in the liquid storage device and the liquid level in the upper tank.

8. A control method for a parallel plate flow chamber system for simulating cross-wall gap flow, wherein the parallel plate flow chamber system is the parallel plate flow chamber system as claimed in any one of claims 1 to 7, characterized in that: The control method comprises the steps of: S101, starting the parallel plate flow chamber system and acquiring initialization data, wherein the initialization data at least includes a preset first liquid level difference and a current first liquid level height in the upper tank; S102, controlling the lifting mechanism in the parallel plate flow chamber system to operate according to the first liquid level difference, so that the liquid storage device is adjusted to a first height, so that the liquid level difference between the liquid in the upper tank and the liquid storage device meets the first liquid level difference; S103, after a first preset time period, obtaining a current second liquid level difference between the liquid in the upper tank and the liquid storage device; S104, determining whether a first difference between the second liquid level difference and the first liquid level difference is greater than a first preset threshold value, if so, proceeding to S105, otherwise maintaining the current working state; wherein the working state at least includes the height of the liquid storage device; S105, controlling the lifting mechanism to operate according to the first reduction, adjusting the liquid storage device from the first height to the second height, and executing steps S103 to S105 until a first difference between the second liquid level difference and the first liquid level difference is less than the first preset threshold.

9. The control method of a parallel plate flow chamber system for simulating cross-wall gap flow according to claim 8, characterized in that: Before executing step S105, the method further includes the following steps: S41, obtaining the second liquid level in the upper tank; S42, calculating a second difference between the first difference and the first preset threshold, and determining whether the second difference is greater than the second preset threshold, if so, executing S105, otherwise entering S43; wherein the second preset threshold is a third difference between the maximum liquid level in the upper tank and the second liquid level; S43, increase the rotation speed of the peristaltic pump according to the first increment, and adjust the liquid level in the upper tank to a third liquid level, so that the fourth difference between the first liquid level difference and the third liquid level difference is smaller than the first preset difference; wherein the third liquid level difference is the difference between the third liquid level and the current liquid level of the liquid storage device.

10. The control method of a parallel plate flow chamber system for simulating cross-wall gap flow according to claim 8, characterized in that: In S104, after determining that the first difference between the second liquid level difference and the first liquid level difference is greater than a first preset threshold, the method further includes the following steps: S106, calculating a liquid level change rate according to the first liquid level difference and the second liquid level difference; S107, determining whether the liquid level change rate meets the preset change rate, if so, maintaining the parallel plate flow chamber system in the current working state, otherwise issuing an early warning.

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