CN115595268A - Cell co-culture flow cavity device for simulating human body microcirculation vortex in vitro - Google Patents
Cell co-culture flow cavity device for simulating human body microcirculation vortex in vitro Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
Abstract
The invention relates to the field of cell culture, in particular to a cell co-culture flow cavity device for simulating human body microcirculation eddy in vitro, which comprises a first plate, wherein a second plate and a third plate are connected to the first plate, a advection cavity is formed between the second plate and the third plate, a liquid inlet, an inlet flow stabilizing cavity and a liquid outlet are formed in the first plate, a groove is formed in the second plate, and the liquid inlet, the inlet flow stabilizing cavity, the advection cavity and the groove are sequentially communicated with the liquid outlet for flowing of fluid. The cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro can simulate the human body microcirculation vortex environment, provides a culture environment which is closer to a biological entity for cells, solves the problem that the fluid shearing environment of the existing fluid device has larger difference with the biological entity environment, and enables the test result to well reflect the actual situation.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a cell co-culture flow cavity device for simulating human body microcirculation eddy in vitro.
Background
Forces (e.g., shear forces) can regulate cellular functions by affecting the expression of genes and protein synthesis within cells, thereby playing an important role in the physiological and pathological processes of cells.
In the prior art, a fluid device for researching the influence of shearing force stimulation on cells exists, however, the shearing environment and the biological entity environment of the existing device have larger difference, so that the test result cannot well reflect the actual situation.
Disclosure of Invention
The invention provides a cell co-culture flow cavity device for simulating a human body microcirculation vortex in vitro, which can simulate a human body microcirculation vortex environment and provide a culture environment closer to a biological entity for cells, so that the problem that the fluid shearing environment of the conventional fluid device has larger difference with the biological entity environment is solved, and the test result can well reflect the actual situation.
The technical scheme of the invention is that the cell co-culture flow cavity device for simulating the human body microcirculation eddy in vitro comprises a first plate, wherein a second plate and a third plate are connected to the first plate, a horizontal flow cavity is formed between the second plate and the third plate, a liquid inlet, an inlet flow stabilizing cavity and a liquid outlet are formed in the first plate, a groove is formed in the second plate, and the liquid inlet, the inlet flow stabilizing cavity, the horizontal flow cavity and the groove are sequentially communicated with the liquid outlet and are used for flowing fluid;
the cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro, the fluid and the cells meet the following formula:
wherein C is the concentration, D is the effective diffusion coefficient,
is the velocity vector field, P is the pressure, μ is the fluid viscosity,
is the specific gravity of the fluid;
wherein the content of the first and second substances,
is a viscous stress tensor, and T is a transposition tensor;
wherein, OCR represents the oxygen consumption rate,
the maximum oxygen uptake rate is expressed as,
representing the mie constant.
Further, the height of the bottom surface of the inlet flow stabilizing cavity is smaller than that of the bottom surface of the horizontal flow cavity.
Further, an inlet connecting pipe is communicated with the liquid inlet in parallel, and/or an outlet connecting pipe is communicated with the liquid outlet in parallel.
Further, the width depth of the groove is (5) - (1.
Further, the recess is equipped with a plurality ofly along going into the direction of liquid mouth to liquid outlet.
Further, a sealing element is arranged between the first plate and the third plate.
Further, a first transparent area for observing the area between the first plate and the second plate is arranged on the first plate.
Furthermore, a second transparent area for observing the horizontal flow cavity and the groove is arranged on the third plate.
Furthermore, be equipped with on the first plate with second plate matched with boss, the second plate passes through the boss and connects in first plate.
Furthermore, fasteners are arranged on the first plate and the third plate, and the third plate is connected to the first plate through the fasteners.
The invention has the following beneficial effects:
cell culture fluid is let in from the income liquid mouth, then flows through entry steady flow chamber, advection chamber, recess in proper order after flowing through from the liquid outlet outflow. The groove is used for planting cells, the inlet flow stabilizing cavity is used for buffering fluid, the fluid is pressed on the side wall and the bottom surface of the inlet flow stabilizing cavity due to inertia after entering the inlet flow stabilizing cavity, and then flows upwards to the inlet of the flow stabilizing cavity after absorbing most of transverse and vertical speeds, so that a downward flowing part and an upward flowing part are formed in the inlet flow stabilizing cavity due to continuous flow of the fluid, the transverse and vertical speed components of the fluid are further reduced by mutual friction and adhesion between the downward flowing part and the upward flowing part, and the overlarge transverse and vertical speed components influencing the flow stability of the fluid entering the flow stabilizing cavity are avoided.
When fluid that gets into the advection chamber from entry stationary flow chamber passes through the recess, can form the vortex under the fluid flow in the recess, thereby simulation human microcirculation vortex environment, make the cell in the recess can be in the fluid environment who is similar to in the human microcirculation vortex, compare the culture apparatus who more accords with the bioentity in current structure and cultivate the environment, the experiment that the cell receives shear stress stimulation under the realization closely imitates the bioentity environment, thereby there is the problem of great difference in the fluid shear environment of current fluidic device and the bioentity environment, make the experimental result can react actual conditions well.
Drawings
FIG. 1 is a cross-sectional view showing the overall structure of a cell co-culture flow chamber device for simulating the vortex flow of human microcirculation in vitro;
FIG. 2 is a schematic view of the first plate cooperating with the second plate;
FIG. 3 is a schematic view of the first plate member;
FIG. 4 is a schematic view of a second plate member;
FIG. 5 is a schematic structural view of a third plate;
FIG. 6 is a graph of eddy current CFD results for different groove depths;
FIG. 7 is a graph comparing shear forces for different fluid flow rates;
FIG. 8 is a graph of the change in shear protection factor for different groove depths.
Wherein, 1, a first plate; 11. a liquid inlet; 12. an inlet plenum chamber; 13. an outlet flow stabilizing cavity; 14. a liquid outlet; 15. mounting grooves; 16. a boss; 17. a first connection hole; 18. a sealing groove; 19. a first transparent region; 2. a second plate member; 21. a groove; 3. a third plate member; 31. a second connection hole; 32. a second transparent region; 4. a advection chamber; 5. an inlet connection pipe; 6. an outlet connection pipe; 7. a fastener; 8. and a seal.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined or explained in subsequent figures.
In the description of the present invention, it should be noted that, if the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships based on orientations or positional relationships shown in the drawings or orientations or positional relationships that the product of the present invention is usually placed in when used, it is merely for convenience of description and simplification of the description, and it is not intended to indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention. Furthermore, the appearances of the terms "first," "second," and the like in the description of the present invention are only used for distinguishing between the descriptions and are not intended to indicate or imply relative importance.
Furthermore, the terms "horizontal", "vertical" and the like when used in the description of the present invention do not require that the components be absolutely horizontal or overhanging, but may be slightly inclined. For example, "horizontal" merely means that the direction is more horizontal than "vertical" and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.
In the description of the present invention, it should be further noted that unless otherwise explicitly stated or limited, the terms "disposed," "mounted," "connected," and "connected" should be interpreted broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in a specific case to those of ordinary skill in the art.
The embodiment of the application discloses cell coculture flow chamber device of human microcirculation vortex of external simulation, refer to fig. 1, and cell coculture flow chamber device of human microcirculation vortex of external simulation includes first plate 1, second plate 2 and third plate 3, inject into liquid mouth 11, entry steady flow chamber 12, advection chamber 4, recess 21, export steady flow chamber 13 and liquid outlet 14 between first plate 1, second plate 2 and the third plate 3.
Referring to fig. 1 to 5, a rectangular mounting groove 15 is formed in the middle of the upper surface of the first plate 1, the inlet flow stabilizing cavity 12 and the outlet flow stabilizing cavity 13 are both formed in the first plate 1 and are respectively formed at two ends of the mounting groove 15, and the depths of the bottom surfaces of the inlet flow stabilizing cavity 12 and the outlet flow stabilizing cavity 13 are greater than the depths of the bottom surfaces of the mounting groove 15. The liquid inlet 11 and the liquid outlet 14 are respectively arranged at two ends of the first plate 1, the liquid inlet 11 is communicated with the bottom of the inlet flow stabilizing cavity 12, and the liquid outlet 14 is communicated with the bottom of the outlet flow stabilizing cavity 13. The liquid inlet 11 can be communicated with an inlet connecting pipe 5, the liquid outlet 14 can be communicated with an outlet connecting pipe 6, and when the cell culture liquid pipeline is externally connected, the cell culture liquid pipeline can be conveniently connected with a cell co-culture flow cavity device for simulating human body microcirculation eddy currents in vitro.
The third plate 3 is also detachably connected to the first plate 1, a plurality of first connecting holes 17 are formed in the periphery of the first plate 1, a plurality of second connecting holes 31 are formed in the periphery of the third plate 3, the first plate 1 and the third plate 3 are provided with fasteners 7, the fasteners 7 are screws, the fasteners 7 are in threaded connection with the first connecting holes 17 and the second connecting holes 31, and the fasteners 7 simultaneously penetrate through the first connecting holes 17 and the second connecting holes 31, so that the third plate 3 is installed on the first plate 1.
First plate 1, second plate 2 and third plate 3 are connected from up down in proper order through boss 16 and fastener 7, link together each plate, can conveniently realize the equipment or the dismantlement of each plate, can also avoid the gap weeping between the adjacent plate. In other embodiments, the type of the fastening member 7, the number and the position of the bosses 16 and the fastening members 7 are not limited to these examples, and may be adjusted according to actual conditions, and in order to avoid leakage between the contact surfaces of the panels, the contact surfaces should be close enough to ensure sealing performance after the panels are connected. Of course, the panels may be laminated together in other ways, such as by welding, adhesive bonding, etc.
The groove 21 is opened on the upper surface of the second plate 2 for planting cells. The inner wall of the groove 21 is a cambered surface, and the inner diameter of the cross section of the groove is gradually reduced from top to bottom. The lower surface of the third plate 3 is a plane, after the third plate 3 is connected to the first plate 1, a advection cavity 4 is formed between the second plate 2 and the third plate 3, the advection cavity 4 is positioned above the groove 21, and the advection cavity 4 is communicated with the groove 21 and is communicated with the inlet flow stabilizing cavity 12 and the outlet flow stabilizing cavity 13. The height of the upper surface of the second plate 2 is greater than that of the bottom surface of the inlet flow stabilizing cavity 12, namely the height of the bottom surface of the advection cavity 4 is greater than that of the bottom surface of the inlet flow stabilizing cavity 12.
When the cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro is used, cell culture fluid can be introduced from the fluid inlet 11, then enters the inlet flow stabilizing cavity 12, enters the flow stabilizing cavity 4 in a advective manner after being buffered and stabilized by the inlet flow stabilizing cavity 12, passes through the groove 21, and then flows out from the outlet flow stabilizing cavity 13 and the fluid outlet 14. Fluid can enter the groove 21 when smoothly passing through the advection cavity 4, and fill the groove 21, and apply shearing force to cells planted in the groove 21, thereby realizing loading shearing force stimulation to the cells.
The inlet flow stabilizing cavity 12 is used for buffering fluid, the bottom surface of the inlet flow stabilizing cavity 12 is arranged at a low position, so that a height difference exists between the bottom surface of the inlet flow stabilizing cavity 12 and the advection cavity 4, after the fluid enters the inlet flow stabilizing cavity 12, the side wall and the bottom surface of the inlet flow stabilizing cavity 12 are firstly pressed by inertia, most of the transverse and vertical speeds are absorbed, and then the fluid flows upwards to the inlet of the advection cavity 4, so that a downward flowing part and an upward flowing part are formed in the inlet flow stabilizing cavity 12 due to continuous flow of the fluid, and the transverse and vertical speed components of the fluid are further reduced by mutual friction and adhesion between the downward flowing part and the upward flowing part, so that the excessive transverse and vertical speed components influencing the flow stability of the fluid entering the advection cavity 4 are avoided.
When fluid entering the horizontal flow cavity 4 from the inlet flow stabilizing cavity 12 passes through the groove 21, vortex can be formed in the groove 21 under the fluid flow, so that the human body microcirculation vortex environment is simulated, cells in the groove 21 can be in a fluid environment similar to the human body microcirculation vortex, compared with a culture device with the existing structure, the culture device is more consistent with the culture environment of a biological entity, the experiment that the cells are stimulated by shearing stress under the environment of the biological entity is very closely simulated, the problem of larger difference between the fluid shearing environment of the existing fluid device and the environment of the biological entity is solved, and the actual situation can be well reflected by the test result.
Meanwhile, the cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro, the fluid and the cells meet the following formula:
wherein C is the concentration, D is the effective diffusion coefficient,
is the velocity vector field, P is the pressure, μ is the fluid viscosity,
is the specific gravity of the fluid;
wherein the content of the first and second substances,
is the viscous stress tensor, T is the transposition tensor;
wherein, the OCR tableThe oxygen consumption rate is shown,
the maximum oxygen uptake rate is expressed as,
representing the mie constant.
In a preferred embodiment, the inlet 11 is in parallel communication with the inlet connection 5 and the outlet 14 is in parallel communication with the outlet connection 6. The liquid inlet 11 and the inlet connecting pipe 5 are arranged in parallel, so that the flow velocity of the transversely inflowing fluid flows through the advection cavity 4 in a horizontal flow direction, and the flow of the fluid flowing through the advection cavity 4 is ensured to meet the requirement. The liquid outlet 14 and the outlet connecting pipe 6 are arranged in parallel, so that fluid can be rapidly discharged out of the cell co-culture flow cavity device simulating the human body microcirculation vortex, and in other embodiments, the liquid outlet 14 does not need to be communicated with the outlet connecting pipe 6 in parallel.
The cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro is characterized by low Reynolds number and almost always laminar flow of flow. At reynolds number 0, the nonlinear inertial term disappears, simplifying the fluid mechanics equation, and for steady state flow, an unmixed streamline is obtained. Referring to fig. 6, the flow lines for different depth recesses 21 in the perfusion chamber are shown, H denoting the height of the advection chamber (4); d denotes the depth of the groove 21; l represents the length of the groove 21. At low reynolds numbers, these streamlines are independent of flow rate. As shown in fig. 6, for shallow troughs the flow may partly follow the cross-sectional change and laminar flow is carried through from the main channel to the trough bottom (< 10 μm), whereas at the corners moffatt vortices are observed. As the depth of the groove 21 increases (> 10 μm and <50 μm), the extraction flow of the main flow separates from the bottom of the groove 21, and the vortices in the corners expand towards the center of the groove 21. For deeper grooves 21 (> 70 μm and <140 μm), corner vortex cells and counter-rotating main vortices are formed below the extracted main flow. The size of the main vortex increases with the depth of the groove 21. As the groove 21 deepens (> 140 μm), two counter-rotating vortices are formed.
Through CFD simulation analysis, when the width-depth ratio of the groove 21 is less than 10:3, eddy currents occur, which increase with increasing depth. When the width-depth ratio of the groove 21 is less than 5:7, two vortexes rotating in opposite directions can be formed, and the mechanical characteristics of the human microcirculation vortexes are not met. The depth of the grooves 21 is too shallow to facilitate the formation of a vortex, and too deep the fluid pattern is too complex. Preferably, the optimum range of the width-depth ratio of the groove 21 is 5:3 to 1:1, a main vortex of reverse rotation appears at the moment, and the characteristics of the human body microcirculation vortex are met. The depth-width ratio of the groove 21 in this embodiment is 1:1, the depth to width ratio may also be either 5:4 or 10:9, etc. Wherein, the width of the groove 21 depends on the width of the culture slide, and the depth of the groove 21 is optimized after CFD simulation.
Specifically, a plurality of recesses 21 on the second plate 2 have been seted up, and a plurality of recesses 21 are along the even distribution in length direction interval of second plate 2 on second plate 2, and the length direction of recess 21 is perpendicular with the length direction of second plate 2, and the both ends of recess 21 extend to the both sides of second plate 2. A plurality of recesses 21 are arranged along the direction of going into liquid mouth 11 to liquid outlet 14, and a plurality of recesses 21 set gradually along the flow direction of fluid in advection chamber 4 promptly, and the metabolite of the cell in the both sides that different recesses 21 correspond can exchange under the fluid flow drives like this to form the coculture environment that accords with biological entity environment more, recess 21 homoenergetic is covered by fluid moreover, make full use of space, raise the efficiency.
In order to improve the sealing performance between the plates, a rectangular sealing groove 18 is formed in the upper surface of the first plate 1, and the sealing groove 18 surrounds the inlet flow stabilizing cavity 12, the advection cavity 4, the groove 21 and the outlet flow stabilizing cavity 13. An elastic sealing element 8 is arranged in the sealing groove 18, the sealing element 8 is a sealing gasket, and the outer diameter of the sealing element 8 under the unstressed condition is larger than the diameter of the sealing groove 18, so that the elastic sealing element 8 and the sealing groove 18 form interference fit. The seal 8 can be in contact with the lower surface of the third plate 3 when the third plate 3 is mounted on the first plate 1. The sealing element 8 is arranged to ensure the sealing performance of the contact surface between the first plate 1 and the third plate 3 and avoid the leakage of the outer surface of the cell co-culture flow cavity device which simulates the human body microcirculation vortex in vitro. Alternatively, the sealing member 8 may be a silicone gasket or a gasket of another suitable material.
Furthermore, a first transparent area 19 is arranged in the middle of the first plate 1 corresponding to the position of the advection cavity 4, and the first transparent area 19 is made of quartz glass and is of a transparent structure so as to serve as an observation window. The degree of closeness of cooperation of first plate 1 and second plate 2 can be observed from first plate 1 bottom through first transparent area 19, observes whether there is the clearance between first plate 1 and the second plate 2, prevents that the clearance from influencing fluid flow rate etc. to influence the cultivation condition of cell, and all-round three-dimensional observation experimentation among the experimentation. The area of the projection of the first transparent area 19 on the first plate 1 in this embodiment is equal to the area of the projection of the advection cavity 4 on the first plate 1, and in other embodiments, the area of the projection of the first transparent area 19 on the first plate 1 may be larger than the area of the projection of the advection cavity 4 on the first plate 1, as long as the advection cavity 4 can be covered in principle.
Furthermore, a second transparent area 32 is arranged in the middle of the third plate 3 corresponding to the position of the advection cavity 4, and the second transparent area 32 is also made of quartz glass and is of a transparent structure to serve as an observation window. The advection cavity 4 and the groove 21 can be observed from the top of the third plate 3 through the second transparent area 32, so that the cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro can better observe the test process in real time. Similarly, the area of the second transparent area 32 is only required to cover the advection cavity 4.
Because mammalian cells under the micro-circulation vortex environment have high requirements on culture conditions (shearing force, oxygen level, pH value, nutrient substances and the like), the constant-flow system of the planar micro-channel biological culture device is difficult to culture the mammalian cells under the vortex environment. The device with the grooves 21 can utilize the micro-topographic characteristics to weaken the adverse effect of the fluid shear stress, protect sensitive cells and simulate the special mechanical environment (such as a vortex environment) of a human body to culture. Theoretical analysis is carried out on operating conditions and design parameters through a numerical model, the complex flow state of the grooves 21 with different depths is compared with the plane micro-channel, and the shearing protection coefficient and the vortex simulation degree of the groove are evaluated. The material transmission problem is coupled with the fluid mechanics problem, and under the condition of assuming a certain shear stress limit, different tank types are simulated to evaluate the perfusion condition of oxygen in different tank types. Besides theoretical model results, experimental studies on Human Foreskin Fibroblasts (HFFs) cultured in grooves 21 with different depths under different perfusion flow rates are also carried out, and the experimental studies are compared with the cell culture in a conventional flat-plate microchannel. The results show that the grooves 21 improve the maximum perfusion rate by an order of magnitude while simulating a vortex environment compared to a planar microchannel. In conclusion, the research shows that the device can well realize cell culture in the human body microcirculation vortex environment.
(1) Cell survival culture in microdevices is regulated by shear stress and nutrient transport. Therefore, shear stress and mass transport within the microchannels should be adequately simulated. The device simulates the fluid dynamics and solute transmission in the biomechanical environment of human microcirculation vortex by using the cell culture flow cavity with the groove 21 or the hole substrate. The formula is obtained through research:
c is the concentration, D is the effective diffusion coefficient,
is the velocity vector field, P is the pressure, μ is the fluid viscosity,
is the specific gravity of the fluid.
(2) The object is subjected to fluid shear stress due to the fluid viscosity and tangential stress exerted by the flow on the surface of the object. When the shear force exceeds certain criteria (depending on the cell type, substrate interaction and duration), the cells will detach from the substrate. Therefore, when culturing cells in a cell culture flow chamber, the shear stress acting on the cells must be kept below the cell separation threshold. Deriving a viscous stress tensor (T) expression:
(3) Oxygen is the most important nutrient and also an important regulator of cell function. The transportation of oxygen in the cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro is important for cell culture, and is the key point of focusing on optimization during model design and optimization. In the device, the oxygen intake of the cells is researched to obtain an expression:
the OCR means the rate of oxygen consumption,
the maximum oxygen uptake rate is expressed as,
representing the mie constant.
Experiment: the oxygen transport and shear stress acting on the cells was studied using a finite element multiphysics hydrodynamic and mass transport model. A model containing microchannel and microgroove geometries was assembled. The wall surface adopts the condition of no-slip boundary, the inlet axial velocity is uniform, and the medium is assumed to be Newtonian fluid. Except at the bottom of the cell, all surfaces imposed a zero mass flux boundary condition (without disturbing the fluid form and intensity) where oxygen uptake was consistent with Michaelis-Menten kinetics, assuming constant oxygen concentration at the inlet.
Shearing protection: referring to FIG. 7, HFF culture of human foreskin fibroblasts was compared to cell culture in conventional flat microchannels. HFFs were subjected to shear stress for long periods of time in simple flat microchannels, and it was found that these cells could withstand shear stress up to 10MPa. In the pre-experimental microchannel configuration, this shear stress threshold corresponds to a flow rate of 0.1 mL/h. At a flow rate of 1mL/h, the cells were washed off the surface of the main channel and proliferated only at the bottom of the groove 21, where they were shear-protected. (a: 0.1mL/h, b:1 mL/h)
The cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro has the characteristics of low Reynolds number and almost always laminar flow of flow. At reynolds number 0, the nonlinear inertial term disappears and for steady state flow, a non-mixed streamline is obtained. Referring to fig. 6, streamlines for different depth recesses 21 in the perfusion chamber are shown. At low reynolds numbers, these streamlines are independent of flow velocity. As shown in fig. 6, for shallow troughs the flow may partly follow the cross-sectional change and laminar flow is carried through from the main channel to the trough bottom (< 10 μm), whereas at the corners moffatt vortices are observed. As the depth of the groove 21 increases (> 10 μm and <50 μm), the extraction flow of the main flow separates from the bottom of the groove 21, and the vortex in the corner expands toward the center of the groove 21. For deeper grooves 21 (> 70 μm and <140 μm), corner vortex cells and counter-rotating main vortices are formed below the extracted main flow. The size of the main vortex increases with the depth of the groove 21. As the groove 21 deepens (> 140 μm), two counter-rotating vortices are formed.
Shear protection coefficient:
we define the shear protection factor as the ratio of the maximum shear stress at the bottom of the groove 21 to the shear stress at the bottom of the flat microchannel. In the cell co-culture flow chamber device for simulating the human body microcirculation eddy in vitro, because Re <1, the complete Navier-Stokes equation is simplified into a linear differential equation for incompressible Newtonian fluid, and therefore the ratio is not influenced by the flow velocity. FIG. 8 shows the shear protection factor as a function of microgroove depth: as the depth of the groove 21 increases, the whole tends to rise.
Material transportation: the flow pattern shown in fig. 6 may affect the transport of material at the bottom of the groove 21. For shallow troughs, the flow lines can emanate from the main channel to the trough bottom (< 20 μm) for mass transport by convection. For deeper grooves 21 (> 40 μm), the presence of eddy currents limits the mass transport. We analyzed the spatial distribution of oxygen in the cells at different depths in the device at different flow rates. The results show that for grooves 21 having a depth greater than 50 μm, the isoconcentration contours in the grooves 21 are flat and unaffected by the flow, i.e. the predominant transport form of the species is diffusion, consistent with the species exchange characteristics in a turbulent environment. In conventional flow chamber cell culture devices, there is an oxygen gradient in the direction of flow as oxygen is consumed by the cells. In certain applications, it is important that the cell population throughout the device maintain a stable oxygen concentration. Therefore, minimization of the oxygen gradient is critical. The research result of the model shows that the device can effectively reduce the oxygen gradient and is beneficial to cell culture. In contrast, cells of different oxygen consumption types are cultured, and it is found that according to the characteristics of the oxygen consumption rate, the device can realize shearing force protection and long-term better cell culture in a vortex environment by adjusting the depth of the groove 21. For example, for osteoblasts with moderate oxygen consumption, the use of 30 μm grooves 21 can increase the number of cells by a factor of 30 compared to planar channels. Hepatocytes with high oxygen consumption could not be cultured in planar channels under the above conditions (channel 100 mm, shear stress less than 10 MPa), however, they could be cultured using grooves 21 and the maximum cell culture area could be obtained in 40 μm grooves 21.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A cell co-culture flow chamber device for simulating human microcirculation vortex in vitro is characterized in that: the plate comprises a first plate (1), wherein a second plate (2) and a third plate (3) are connected to the first plate (1), a advection cavity (4) is formed between the second plate (2) and the third plate (3), a liquid inlet (11), an inlet flow stabilizing cavity (12) and a liquid outlet (14) are formed in the first plate (1), a groove (21) is formed in the second plate (2), and the liquid inlet (11), the inlet flow stabilizing cavity (12), the advection cavity (4), the groove (21) and the liquid outlet (14) are sequentially communicated for a fluid to flow through;
the cell co-culture flow cavity device for simulating the human body microcirculation vortex in vitro, the fluid and the cells meet the following formula:
wherein C is the concentration, D is the effective diffusion coefficient,
is the velocity vector field, P is the pressure, μ is the fluid viscosity,
is the specific gravity of the fluid;
wherein, the first and the second end of the pipe are connected with each other,
is the viscous stress tensor, T is the transposition tensor;
wherein, OCR represents the oxygen consumption rate,
the maximum oxygen uptake rate is expressed as,
representing the mie constant.
2. The in vitro cell co-culture flow chamber device for simulating the human body microcirculation vortex as claimed in claim 1, wherein: the height of the bottom surface of the inlet flow stabilizing cavity (12) is smaller than that of the bottom surface of the horizontal flow cavity (4).
3. The in vitro cell co-culture flow chamber device for simulating the human body microcirculation vortex as claimed in claim 1 or 2, wherein: an inlet connecting pipe (5) is communicated with the liquid inlet (11) in parallel, and/or an outlet connecting pipe (6) is communicated with the liquid outlet (14) in parallel.
4. The cell co-culture flow chamber device for simulating the human body microcirculation vortex in vitro as claimed in claim 1, wherein: the width-depth ratio of the groove (21) is (5.
5. The in vitro cell co-culture flow chamber device for simulating the human body microcirculation vortex as claimed in claim 1 or 4, wherein: a plurality of grooves (21) are formed in the direction from the liquid inlet (11) to the liquid outlet (14).
6. The in vitro cell co-culture flow chamber device for simulating the human body microcirculation vortex as claimed in claim 1, wherein: a sealing element (8) is arranged between the first plate (1) and the third plate (3).
7. The in vitro cell co-culture flow chamber device for simulating the human body microcirculation vortex as claimed in claim 1, wherein: the first plate (1) is provided with a first transparent area (19) for observing the area between the first plate (1) and the second plate (2).
8. The device of claim 7, wherein the device comprises: and a second transparent area (32) for observing the horizontal flow cavity (4) and the groove (21) is arranged on the third plate (3).
9. The cell co-culture flow chamber device for simulating the human body microcirculation vortex in vitro as claimed in claim 1, wherein: be equipped with on first plate (1) with second plate (2) matched with boss (16), second plate (2) are connected in first plate (1) through boss (16).
10. The in vitro cell co-culture flow chamber device for simulating the human body microcirculation vortex as claimed in claim 1, wherein: the first plate (1) and the third plate (3) are provided with fasteners (7), and the third plate (3) is connected to the first plate (1) through the fasteners (7).
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