CN114107056B - In-vitro blood vessel-like tissue model with fluid environment and application thereof - Google Patents

In-vitro blood vessel-like tissue model with fluid environment and application thereof Download PDF

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CN114107056B
CN114107056B CN202111263803.2A CN202111263803A CN114107056B CN 114107056 B CN114107056 B CN 114107056B CN 202111263803 A CN202111263803 A CN 202111263803A CN 114107056 B CN114107056 B CN 114107056B
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CN114107056A (en
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丁永胜
崔金刚
苏继磊
耿立闯
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University of Chinese Academy of Sciences
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Abstract

The invention discloses an in-vitro blood vessel-like tissue model with a fluid environment and application thereof. The microfluidic chip provided by the invention sequentially comprises an upper deformation constraint layer, a middle PDMS deformation layer and a bottom liquid conveying control layer from top to bottom; at least one micro-channel is arranged on the bottom liquid conveying control layer; a hollowed-out structure with the shape consistent with that of the micro-channel is arranged on the upper deformation constraint layer at the position corresponding to the micro-channel; fluid inlets at two ends of the micro-channel sequentially penetrate through the middle-layer PDMS deformation layer and the upper-layer deformation constraint layer to form a fluid leading-in jack and a fluid leading-out jack. The circulation system of the in-vitro artificial vascular tissue model can effectively control the fluid shearing force, the fluid pressure and the stretching force of the elastic film, namely, a chip with a certain thickness of a middle PDMS deformation layer is arranged, and as the flow rate is increased, the increase of the camber means the increase of the pressure of the fluid to cells; under certain flow conditions, as the thickness of the PDMS in the chip increases, the camber decreases, meaning that the shear force of the fluid on the cells increases.

Description

In-vitro blood vessel-like tissue model with fluid environment and application thereof
Technical Field
The invention relates to an in-vitro blood vessel-like tissue model with a fluid environment and application thereof, belonging to the technical field of biology.
Background
The continuous years of statistical report of world health organization shows that the mortality rate caused by cardiovascular diseases is high in the top of the list worldwide, and the occurrence rate of the diseases is continuously increased and the trend of younger in recent years is presented, so that the health problem of global general concern is caused. Cardiovascular diseases mainly include atherosclerosis, aneurysm, varicose vein, vascular aging, etc. At present, the blood vessel research model mainly adopts various animal models and in-vitro blood vessel models in-vitro fluid environments, and the models all need to sacrifice animal cost, and are long in time consumption, complex in operation and high in cost. Therefore, the development of the in-vitro blood vessel model with the fluid environment has remarkable research and application values.
Disclosure of Invention
The invention aims to provide an in-vitro blood vessel tissue model with a fluid environment, which can provide a fluid environment similar to human blood circulation for co-culture of various cells of an in-vitro blood vessel.
The invention provides a microfluidic chip, which comprises an upper deformation constraint layer, a middle PDMS (polydimethylsiloxane) deformation layer and a bottom liquid conveying control layer from top to bottom in sequence;
at least one micro-channel is arranged on the bottom liquid conveying control layer;
a hollowed-out structure which is consistent with the shape of the micro-channel is arranged on the upper deformation constraint layer and corresponds to the micro-channel in position;
fluid inlets at two ends of the micro-channel sequentially penetrate through the middle-layer PDMS deformation layer and the upper-layer deformation constraint layer to form a fluid leading-in jack and a fluid leading-out jack.
In the microfluidic chip, the thickness of the upper deformation constraint layer is more than 2mm, preferably 2-4 mm;
the upper deformation constraint layer is made of transparent materials;
the transparent material is PDMS or glass.
In the microfluidic chip, the thickness of the middle PDMS deformation layer is 0.05-2 mm, preferably 0.05-1 mm, for cell attachment;
the thickness of the bottom liquid conveying control layer is more than 2mm, preferably 2-4 mm;
the length of the micro-channel is 10-50 mm, the width of the micro-channel is 2-10 mm, and the depth of the micro-channel is 0.1-1 mm;
the bottom liquid conveying control layer is made of PDMS or other transparent organic materials;
the transparent material is PDMS or glass.
When a certain flow of fluid passes through the micro-channel, the middle PDMS deformation layer attached with cells is deformed in a pipe wall shape, and the height of the arched deformation formed by the middle PDMS deformation layer is in direct proportion to the fluid flow (related to the fluid flow rate) and the elastic coefficient of the middle PDMS deformation layer (related to the middle thickness and the cross-linking agent proportion).
On the basis of the microfluidic chip, the invention provides a circulating system capable of bearing an in-vitro artificial vascular tissue model with a fluid environment, which comprises the microfluidic chip, a delivery pump and a liquid storage bottle;
the microfluidic chip, the delivery pump and the liquid storage bottle are connected in series to form a closed loop flow path;
the transfer pump is preferably a peristaltic pump.
The microfluidic chip, the delivery pump and the liquid storage bottle are connected through a silica gel tube.
The fluid leading-in jack and the fluid leading-out jack are respectively connected with the delivery pump and the liquid storage bottle.
The circulatory system of the in-vitro artificial vascular tissue model provided by the invention can be used for researching the mechanism of vascular diseases and evaluating the drug effect.
When the circulatory system of the in-vitro artificial vascular tissue model is used, the circulatory system can be carried out according to the following steps:
injecting in-vitro cells into the micro-channel of the micro-fluidic chip, enabling the in-vitro cells to grow on the middle PDMS deformation layer in an attached mode, starting the conveying pump after fusion is achieved, and enabling fluid to circulate in the closed loop flow path;
obtaining the influence on the cells by changing the flow rate of the fluid to obtain changes in shear force, pressure and stretching force of the fluid on the inner wall;
the thickness of the middle PDMS deformation layer is changed to obtain the change of the shearing force, the pressure and the stretching force of the fluid on the inner wall, so that the influence on the cells is obtained;
the effect of factors that induce and interfere with angiogenesis on the cells is obtained by altering the composition of the culture medium of the cells.
The circulation system of the in-vitro artificial vascular tissue model can effectively control the fluid shearing force, the fluid pressure and the stretching force of the elastic film, in short, the thickness of the middle PDMS deformation layer is a certain chip, and as the flow rate is increased, the increase of the camber means the increase of the pressure of the fluid to cells; under certain flow conditions, as the thickness of the PDMS in the chip increases, the camber decreases, meaning that the shear force of the fluid on the cells increases.
Drawings
Fig. 1 is a schematic structural view (exploded) of a microfluidic chip according to the present invention;
the figures are each marked as follows:
the device comprises a reinforcing constraint layer 1 on the upper layer, a PDMS deformation layer 2 on the lower layer, a liquid conveying control layer 3 on the lower layer, a hollow structure 4, a fluid leading-in jack/fluid leading-out jack 5, a port 6 and a micro-channel 7.
FIG. 2 is a schematic diagram of the circulation system of the present invention with a fluid environment that can carry an in vitro artificial vascular tissue model;
the figures are marked as follows: 8 micro-fluidic chip, 9 peristaltic pump, 10 stock solution bottle.
FIG. 3 is a schematic diagram of the cross-section and the longitudinal section of static fluid and the cross-section and the longitudinal section of dynamic fluid of the microfluidic chip of the present invention.
Fig. 4 is a schematic diagram of the fluid shear force calculation principle.
FIG. 5 is a graph showing the relationship between flow (Q) -camber (h) experimental measurement under a certain thickness of the middle layer.
FIG. 6 is a graph showing experimental measurement of the thickness (T) -camber (h) of the middle layer under a certain flow condition.
FIG. 7 shows the results of confocal laser microscopy of double-layered cell structures.
FIG. 8 is a graph showing the degree of intercellular junctions and extracellular inflammatory factor levels under high and low fluid pressure.
FIG. 9 shows the degree of foam cell formation under relatively high and low fluid pressures.
Fig. 10 is a graph comparing the inhibition of foam cell formation by atorvastatin calcium.
FIG. 11 shows the degree of foam cell formation under relatively high and low fluid shear forces.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative tests in the following examples were each set up for at least 3 independent replicates and the results averaged.
Example 1 microfluidic chip fabrication and circulation System fluid parameter set-up
1. Fabrication of microfluidic chips
A. Chip structure and print mask are designed.
B. Preparation of a silicon wafer template
Soaking a monocrystalline silicon wafer of 100mm in Piranha solution (98% concentrated sulfuric acid/30% hydrogen peroxide=2/1) for 30min; taking out the silicon wafer, cleaning the silicon wafer with a large amount of ultrapure water, and drying the silicon wafer at 200 ℃ for 30min; placing a silicon wafer on a spin coater to coat photoresist SU-82035 to form a film layer with the thickness of about 100 mu m; placing the silicon wafer coated with the photoresist in a baking process at 65 ℃/5min and 90 ℃/30 min; the mask (the fluid channel part is a light-transmitting part) is tightly attached to a silicon wafer coated with photoresist, and is irradiated by high-intensity ultraviolet light for 60 seconds; the silicon wafer after exposure treatment is placed in a baking treatment at 65 ℃/5min and 90 ℃/60 min; immersing the exposed and baked silicon wafer into photoresist shadow washing liquid (propylene glycol methyl ether acetate) for 15min; taking out the treated silicon chip, washing with isopropanol, baking at 150deg.C for 30min, and naturally cooling to obtain the final die of microfluidic chip.
C. Manufacturing upper and bottom PDMS film
Upper and lower PDMS films: PDMS monomer and curing agent were mixed at 10:1, uniformly mixing, degassing, pouring the mixture on two templates respectively containing an upper layer channel and a lower layer channel, and heating and curing the mixture at 65 ℃ for 60 minutes. And (3) stripping the lower PDMS film containing the microfluidic channel print from the template after cooling for later use. Cutting the stripped upper PDMS film according to a strip print to form a strip-shaped hollow with the width of 6mm and the length of 30mm for later use.
D. Production of middle and upper PDMS film
Taking a certain amount of PDMS monomer and curing agent according to the weight ratio of 10:1, uniformly mixing, degassing, uniformly coating on a clean silicon wafer by using a spin coater to form a film with the thickness of about 100mm, and heating in an oven at 65 ℃ for 10min. Spreading the prepared upper PDMS film on the middle layer film solidified for 10min, and placing into a 65 ℃ oven for continuously baking for 60min to form the middle and upper PDMS film combination.
E. The upper middle layer is combined with the lower layer PDMS film
And (3) placing the middle-upper PDMS film and the lower PDMS film together in a plasma cleaner for 30s, rapidly taking out and extruding more than two PDMS hollow parts aiming at the bottom microfluidic channel print together to form the three-layer sealed integrated PDMS microfluidic chip.
The structural schematic diagram of the microfluidic chip manufactured by the invention is shown in fig. 1, and the microfluidic chip comprises an upper deformation constraint layer 1, a middle PDMS deformation layer 2 and a bottom liquid conveying control layer 3 which are sequentially overlapped. The bottom liquid conveying control layer 3 is provided with 4 micro-channels 7, the upper deformation constraint layer 1 is provided with a hollow structure 4 which is consistent with the micro-channels 7 in position and shape, and two ports 6 at two ends of the micro-channels 7 sequentially penetrate through the upper deformation constraint layer 1 and the middle PDMS deformation layer 2 through metal pipe joints to form a fluid leading-in jack/a fluid leading-out jack 5.
The micro-fluidic chip manufactured by the invention has the following dimensions: the thickness of the upper deformation constraint layer 1 is 2-4 mm, the thickness of the middle PDMS deformation layer 2 is 0.05-1 mm, the thickness of the bottom liquid conveying control layer 3 is 2-4 mm, the length of the micro channel 7 is 30mm, the width is 6mm, and the depth is 0.05-0.1 mm.
2. Circulation system establishment and fluid parameter setting
The microfluidic chip 8 manufactured in this embodiment is connected in series with the peristaltic pump 9 and the liquid storage bottle 10 to form a closed loop flow path, wherein the fluid inlet jack/fluid outlet jack 5 is connected with the peristaltic pump 9 and the liquid storage bottle 10 through a silicone tube, as shown in fig. 2.
When fluid circulates in the micro-channel 7, the middle PDMS deformation layer 2 will be arched deformed, as shown in FIG. 3, and in FIG. 3, the schematic diagrams of the transverse and longitudinal sections of the micro-fluidic chip when the fluid of the non-connected circulation system is stationary and the transverse and longitudinal sections when the fluid of the connected circulation system flows are shown.
Fluid shear force is calculated based on arch height and flow. The mathematical derivation of the fluid shear force calculation method in connection with fig. 4 is as follows:
the height (h) of the arch-shaped deformation of the PDMS layer with a certain elastic coefficient (E) is proportional to the flow rate (Q) of the passing liquid,
h=eq formula (1)
When h is less than r, the relation between h and the radius (r) of the circle where the arch is positioned and the width (w) of the strip-shaped cavity meets Pythagorean theorem,
(r-h) 2 +(w/2) 2 ＝r 2 formula (2)
Knowing w and h, the arc length sandwich angle θ and arch cross-sectional area a can be obtained,
the rate of change of the flow velocity (u) in the vertical plane of the fluid direction by the distance (y) is defined by the fluid shear force (tau),
the shear force of the fluid in the tubular structure,
under the same flow rate, the area of fluid passing through is replaced by the arched cross-sectional area (A) instead of the circular cross-sectional area (pi r) according to the principle of physical approximation 2 ) The flow velocity falling distance is replaced by half (h/2) of the arch height to obtain an archThe fluid shear force within the chamber approximates a formula,
in a specific experiment, the flow rate (Q) was obtained by actually measuring the volume of the effluent liquid per unit time, the height (h) of the dome was obtained by observing the distance of the focal plane moving up and down by a microscope, and the dome-shaped cross-sectional area (A) was calculated by combining the width (w) of a known bar-shaped chamber, and the viscosity (μ) of the cell culture medium (DMEM+10% FBS) at 37℃was 9.3X10 -3 Dyne seconds per square centimeter. And (3) according to different flow measurement thicknesses, the arch height (h) of the PDMS bulge in the middle layer is 0.1mm, and the corresponding fluid shear force is obtained by substituting the formula (6). FIG. 5 shows the height of the camber of the middle elastic membrane with the change of fluid flow under the condition of a middle membrane with a certain thickness, corresponding to the change of fluid shearing force and pressure of liquid on the inner wall. FIG. 6 shows the height of the camber of the middle elastic membrane with the thickness of the middle elastic membrane under a certain fluid flow condition, corresponding to the fluid shear force and the pressure change of the liquid on the inner wall.
Example 2 in vitro vascular-like tissue model construction in circulating fluid Environment
And injecting cells into the micro-channel 7 of the micro-fluidic chip, enabling the cells to be attached and grown on the middle PDMS deformation layer 2, and after the cells are fused, connecting the cells into a fluid circulation system. The thickness of the middle PDMS deformation layer 2 was varied by varying the flow rate to obtain the corresponding fluid shear force, fluid pressure and stretching force acting on the cells. Factors that induce and interfere with vasculopathy occurrence are introduced by changing the composition of the medium.
The experimental procedure was as follows:
cell lines: vascular smooth muscle cells are T/G HA-VSMC; endothelial cells were ea.h926; the mononuclear cells are THP-1;
cell culture medium composition: DMEM basal medium, 10% fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), insulin (40 IU/mL), heparin (40 IU/mL) and 1% nonessential amino acid NEAA;
cell culture conditions:constant temperature incubator 5% CO 2 ，37℃。
The chip pretreatment method comprises the following steps: and ultrasonically cleaning the microfluidic chip, the silica gel pipeline and the liquid storage bottle by absolute ethyl alcohol and ultrapure water, placing the materials in an oven, drying at 65 ℃, packaging the chips by using new tinfoil paper, placing the chips in an autoclave, sterilizing at 121 ℃ for 30min, then placing the chips in the oven again, drying at 65 ℃, and transferring the chips into an ultra-clean workbench for standby.
Fixing and coating a microfluidic chip: the microfluidic chip was fixed in a sterile 100mm cell culture dish, and 20. Mu.g/ml fibronectin solution was filled in the microfluidic channel and placed in an incubator overnight.
Cell inoculation: vascular smooth muscle cells (2.0X10) in good culture were isolated 5 Injecting the mixture into the coated microfluidic channel, and statically culturing for 24 hours with an inoculation culture medium (DMEM+10% FBS+50 mug/ml ascorbic acid) to form a layer of closely connected vascular smooth muscle cells; endothelial cells (3.0X10) were cultured well 5 And/ml) was injected into the microfluidic channel forming the smooth muscle cell layer, and static culture with the seeding medium was continued for 24 hours, to form vascular smooth muscle and endothelial cell co-culture tissue.
The experimental results are as follows:
1. double-layer film structure formed by vascular smooth muscle cells and endothelial cells
The results of the double-layer membrane formed by co-culturing vascular smooth muscle cells and endothelial cells are shown in FIG. 7 (red fluorescence is endothelial marker VE-cadherin, green fluorescence is smooth muscle cell marker SM-alpha-actin, blue fluorescence DAPI is cell nucleus marker, and the upper and right sides of the figure are respectively confocal microscope z-axis scanning horizontal orthogonal partition diagrams). Smooth muscle cells and endothelial cells are respectively marked by adopting SM-alpha-actin antibody and VE-Cadherin antibody, and the results are observed under a laser confocal microscope, so that clear double-layer cell structural characteristics are formed on the middle PDMS deformation layer 2.
2. Comparing the effect of the degree of endothelial cell attachment and inflammatory factor expression at high and low fluid pressures
Under the condition of different flow rates, the continuous action of high and low fluid pressure (cell culture medium without ascorbic acid) on cells is obtained for 24 hours, and then the content of IL-6 and MCP-1 inflammatory factors in the culture medium is measured by ELISA; immunofluorescence was used to characterize the expression of the cellular pro-inflammatory molecules VE-cadherein and CD 31. The results are shown in FIG. 8 (A) shows the immunofluorescence detection of the endothelial cell-to-endothelial cell connecting molecule VE-cadherein, CD31 (green fluorescence), the statistical histogram shows the fluorescence intensity statistics, and FIG. 8 (B) shows the ELISA detection of extracellular inflammatory factors MCP-1 and IL-6), and it can be seen that the expression of low fluid pressure cytokines is higher than high fluid pressure.
3. Comparing the effect of higher and lower fluid pressure on foam cell formation
The formation of foam cells after 24 hours at different flow rates was compared using medium containing suspended monocytes THP-1 as circulating fluid. Foam cell identification adopts paraformaldehyde for fixation for 15min, oil red for 1 h, 60% isopropanol for fixation for 3min, hematoxylin for 3min, water for blue returning, buckling, sealing and observing the number of red lipid drops under a microscope. The oil red staining causes lipid droplets to appear red, and cells with aggregated lipid droplets are identified as foam cells, and foam cell formation is judged by observing how much oil red is. As a result, as shown in fig. 9, it can be seen that the number of foam cells at low fluid pressure is significantly greater than at high fluid pressure.
4. Influence of atorvastatin on foam cell formation under low fluid pressure
The statin has the functions of reducing blood lipid, improving endothelial cells, resisting inflammation, inhibiting smooth muscle cell proliferation, resisting thrombosis, resisting blood platelet, etc. The in vitro artificial vascular tissue model of the invention is utilized to examine the in vitro drug effect of atorvastatin calcium. The experiment used low fluid pressure conditions and compared 20ng/ml atorvastatin calcium medium as circulating fluid for cell culture for 24 hours. The results are shown in fig. 10, and it can be seen that atorvastatin calcium significantly inhibited foam cell formation.
5. Comparing the effect of high and low fluid shear on foam cell formation
The culture medium containing suspended mononuclear cells THP-1 is used as circulating fluid, and the formation of foam cells after the high-low shearing force action is performed for 24 hours by comparing the film chips with different thicknesses under the condition of fixed flow. Foam cell identification adopts paraformaldehyde for fixation for 15min, oil red for 1 h, 60% isopropanol for fixation for 3min, hematoxylin for 3min, water for blue returning, buckling, sealing and observing the number of red lipid drops under a microscope. The oil red staining causes lipid droplets to appear red, and cells with aggregated lipid droplets are identified as foam cells, and foam cell formation is judged by observing how much oil red is. As a result, as shown in fig. 11, it can be seen that the number of foam cells under low fluid shear conditions is significantly greater than that of high fluid pressure.
The experimental result shows that the micro-fluidic chip provided by the invention is utilized to construct an in-vitro blood vessel tissue model, so that various blood circulation system influencing factors such as fluid shear force, fluid pressure, tube wall elasticity, stretching force, fluid composition and the like can be developed, various physiological or pathological environments of blood vessels can be simulated, and a reliable in-vitro model and research means can be provided for the research of cardiovascular diseases.

Claims (5)

1. Application of circulatory system of in vitro artificial vascular tissue model in research of vascular disease generation mechanism and evaluation of drug effect;
the circulation system of the in-vitro artificial blood vessel tissue model comprises a microfluidic chip, a delivery pump and a liquid storage bottle;
the microfluidic chip, the delivery pump and the liquid storage bottle are connected in series to form a closed loop flow path;
the microfluidic chip is sequentially provided with an upper deformation constraint layer, a middle PDMS deformation layer and a bottom liquid conveying control layer from top to bottom;
at least one micro-channel is arranged on the bottom liquid conveying control layer;
a hollowed-out structure which is consistent with the shape of the micro-channel is arranged on the upper deformation constraint layer and corresponds to the micro-channel in position;
fluid inlets at two ends of the micro-channel sequentially penetrate through the middle-layer PDMS deformation layer and the upper-layer deformation constraint layer to form a fluid leading-in jack and a fluid leading-out jack;
the thickness of the middle PDMS deformation layer is 0.05-2 mm;
the length of the micro-channel is 10-50 mm, the width of the micro-channel is 2-10 mm, and the depth of the micro-channel is 0.1-1 mm;
the application method of the circulatory system of the in-vitro artificial blood vessel tissue model comprises the following steps:
injecting in-vitro cells into the micro-channel of the micro-fluidic chip, enabling the in-vitro cells to grow on the middle PDMS deformation layer in an attached mode, starting the conveying pump after fusion is achieved, and enabling fluid to circulate in the closed loop flow path;
obtaining the influence on the cells by changing the flow rate of the fluid to obtain changes in shear force, pressure and stretching force of the fluid on the inner wall;
the thickness of the middle PDMS deformation layer is changed to obtain the change of the shearing force, the pressure and the stretching force of the fluid on the inner wall, so that the influence on the cells is obtained;
the effect of factors that induce and interfere with angiogenesis on the cells is obtained by altering the composition of the culture medium of the cells.
2. The use according to claim 1, characterized in that: the thickness of the upper deformation constraint layer is more than 2mm;
the upper deformation constraint layer is made of transparent materials;
the transparent material is PDMS or glass.
3. Use according to claim 1 or 2, characterized in that: the thickness of the bottom liquid conveying control layer is more than 2mm;
the bottom liquid delivery control layer is made of a transparent material;
the transparent material is PDMS or glass.
4. The use according to claim 1, characterized in that: the microfluidic chip, the delivery pump and the liquid storage bottle are connected through a silica gel tube;
the fluid leading-in jack and the fluid leading-out jack are respectively connected with the delivery pump and the liquid storage bottle.
5. The use according to claim 1, characterized in that: the ex vivo cells include smooth muscle cells and endothelial cells.
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