CN114107056A

CN114107056A - In-vitro vascular tissue-like model with fluid environment and application thereof

Info

Publication number: CN114107056A
Application number: CN202111263803.2A
Authority: CN
Inventors: 丁永胜; 崔金刚; 苏继磊; 耿立闯
Original assignee: University of Chinese Academy of Sciences
Current assignee: University of Chinese Academy of Sciences
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2022-03-01
Anticipated expiration: 2041-10-27
Also published as: CN114107056B

Abstract

The invention discloses an in-vitro vascular tissue-like model with a fluid environment and application thereof. The micro-fluidic chip provided by the invention sequentially comprises an upper deformation restraining layer, a middle PDMS (polydimethylsiloxane) 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 hollow structure with the same shape as the micro-channel is arranged on the upper deformation restraint layer at the position corresponding to the micro-channel; fluid inlets at two ends of the micro-channel sequentially penetrate through the middle PDMS deformation layer and the upper deformation restriction layer to form a fluid inlet jack and a fluid outlet jack. The circulating system of the in-vitro artificial vascular tissue model can effectively control the fluid shear force, the fluid pressure and the tensile force of the elastic film, namely, the arch height of a chip with a certain thickness of the middle-layer PDMS deformation layer is increased along with the increase of the flow, which means the pressure of the fluid on cells is increased; under certain flow conditions, the decrease of arch height with the increase of the thickness of the layer PDMS in the chip means that the shearing force of the fluid on the cells is increased.

Description

In-vitro vascular tissue-like model with fluid environment and application thereof

Technical Field

The invention relates to an in-vitro vascular tissue-like model with a fluid environment and application thereof, belonging to the field of biotechnology.

Background

The continuous years of statistical reports of the world health organization show that the global mortality rate caused by cardiovascular diseases is high at best, and in recent years, the incidence rate of the diseases is continuously increased and the diseases show a youthful trend, so that the diseases become health problems which are generally concerned globally. Cardiovascular diseases mainly include atherosclerosis, aneurysm, varicosity, vascular aging and the like. At present, models for researching blood vessels mainly adopt various animal models and in-vitro blood vessel models in an in-vitro fluid environment, and the models are at the cost of animal sacrifice, long in time consumption, complex in operation and high in cost. Therefore, the development of an in vitro blood vessel model with a fluid environment has significant research and application values.

Disclosure of Invention

The invention aims to provide an in vitro vascular tissue-like model with a fluid environment, which can provide a fluid environment similar to human blood circulation for the co-culture of a plurality of cells of an in vitro blood vessel.

The invention provides a micro-fluidic chip which comprises an upper deformation restraining layer, a middle PDMS (polydimethylsiloxane) deformation layer and a bottom liquid conveying control layer from top to bottom in sequence;

the bottom liquid conveying control layer is provided with at least one microchannel;

a hollow structure with the same shape as the micro-channel is arranged on the upper deformation restraint layer at the position corresponding to the micro-channel;

and fluid inlets at two ends of the microchannel sequentially penetrate through the middle-layer PDMS deformation layer and the upper-layer deformation restriction layer to form a fluid inlet jack and a fluid outlet jack.

In the microfluidic chip, the thickness of the upper deformation restraining layer is greater than 2mm, preferably 2-4 mm;

the upper deformation restricting layer is made of a transparent material;

the transparent material is PDMS or glass.

In the micro-fluidic chip, the thickness of the middle PDMS deformation layer is 0.05-2 mm, preferably 0.05-1 mm, and the PDMS deformation layer is used for cell adhesion;

the thickness of the bottom liquid conveying control layer is larger than 2mm, and 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 microchannel, the middle layer PDMS deformation layer attached with cells is deformed like a pipe wall, and the height of the formed arch deformation is in direct proportion to the flow of the fluid (related to the flow rate of the fluid) and the elastic coefficient of the middle layer PDMS deformation layer (related to the thickness of the middle layer and the proportion of the cross-linking agent).

On the basis of the microfluidic chip, the invention provides a circulating system which has a fluid environment and can bear an in-vitro artificial vascular tissue model, and the circulating system comprises the microfluidic chip, a delivery pump and a liquid storage bottle;

the micro-fluidic chip, the delivery pump and the liquid storage bottle are connected in series to form a closed-loop flow path;

the delivery pump is preferably a peristaltic pump.

The micro-fluidic chip, the delivery pump and the liquid storage bottle are connected through a silicone 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-like model provided by the invention can be used for the research of the vascular disease occurrence mechanism and the evaluation of the drug efficacy.

When the circulatory system of the in-vitro artificial vascular tissue-like model is used, the method can be carried out according to the following steps:

injecting in-vitro cells into the microchannel of the microfluidic chip to enable the in-vitro cells to adhere to and grow on the middle-layer PDMS (polydimethylsiloxane) deformation layer, and starting the delivery pump after fusion is achieved so as to enable fluid to circulate in the closed-loop flow path;

by changing the flow velocity of the fluid, the change of the shearing force, the pressure and the stretching force of the fluid on the inner wall is obtained, and then the influence on the cells is obtained;

changing the shearing force, pressure and stretching force of the fluid on the inner wall by changing the thickness of the middle PDMS deformation layer so as to obtain the influence on the cells;

by changing the composition of the culture medium of the cells, the influence of factors inducing and interfering in the occurrence of the vascular lesion on the cells is obtained.

The circulatory system of the in vitro artificial vascular tissue model can effectively control the fluid shear force, the fluid pressure and the tensile force of the elastic film, in short, the arch height of a chip with a certain thickness of the middle-layer PDMS deformation layer is increased along with the increase of the flow, which means the pressure of the fluid on cells is increased; under certain flow conditions, the decrease of arch height with the increase of the thickness of the layer PDMS in the chip means that the shearing force of the fluid on the cells is increased.

Drawings

FIG. 1 is a schematic structural view (exploded) of a microfluidic chip according to the present invention;

the respective indices in the figure are as follows:

1 upper layer reinforcing and restraining layer, 2 middle layer PDMS deformation layer, 3 bottom liquid conveying control layer, 4 hollow structure, 5 fluid leading-in jack/fluid leading-out jack, 6 ports, 7 micro-channel.

FIG. 2 is a schematic structural diagram of a circulatory system capable of supporting an in vitro artificial vascular tissue model in a fluid environment according to the present invention;

the respective symbols in the figure are as follows: 8 micro-fluidic chip, 9 peristaltic pump, 10 stock solution bottles.

FIG. 3 is a schematic diagram of a cross-section and a longitudinal-section of a static fluid and a cross-section and a longitudinal-section of a dynamic fluid of a microfluidic chip according to the present invention.

Fig. 4 is a schematic diagram of the calculation principle of the fluid shear force.

FIG. 5 is a graph of experimental flow (Q) -camber (h) measurements at a given mid-layer thickness.

FIG. 6 is a graph of the experimental measurements of the mid-layer film thickness (T) versus the crown height (h) at a given flow rate.

FIG. 7 shows the results of laser confocal microscopic identification of bilayer cell structures.

FIG. 8 is a graph showing the degree of intercellular junction and extracellular inflammatory factor levels under relatively high low fluid pressure.

FIG. 9 is a graph showing the degree of foam cell formation under relatively high and low fluid pressures.

Figure 10 is a graph comparing the inhibition of foam cell formation by atorvastatin calcium.

FIG. 11 is a graph showing the degree of foam cell formation under relatively high low fluid shear forces.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up at least 3 independent replicates and the results averaged.

Example 1 microfluidic chip fabrication and circulating System fluid parameter setting

1. Manufacture of microfluidic chip

A. Designing a chip structure and printing a mask.

B. Preparation of silicon wafer template

Soaking a 100mm monocrystalline silicon wafer in Piranha solution (98% concentrated sulfuric acid/30% hydrogen peroxide: 2/1) for 30 min; taking out the silicon wafer, cleaning with a large amount of ultrapure water, and drying at 200 ℃ for 30 min; placing the silicon chip on a spin coater, and coating photoresist SU-82035 to form a film layer with a thickness of about 100 μm; baking the silicon wafer coated with the photoresist at 65 ℃/5min and 90 ℃/30 min; the manufactured mask (the part of the fluid channel is a light-transmitting part) is tightly attached to a silicon wafer coated with photoresist, and is irradiated for 60s by high-strength ultraviolet light; baking the exposed silicon wafer at 65 ℃/5min and 90 ℃/60 min; immersing the silicon wafer after exposure and baking in photoresist developing solution (propyl glycol methyl ether acetate) for treatment for 15 min; and taking out the processed silicon wafer, washing the silicon wafer with isopropanol, baking the silicon wafer at 150 ℃ for 30min, and naturally cooling the silicon wafer to be used as a male die of the microfluidic chip for later use.

C. Making the top and bottom PDMS films

Upper and lower layer PDMS films: the PDMS monomers and curing agent were mixed as follows 10: 1, degassing, pouring the mixture on two templates respectively containing upper and lower channels, and heating and curing at 65 ℃ for 60 min. And after cooling, stripping the lower PDMS film containing the micro-fluidic channel print from the template for later use. And cutting the stripped upper PDMS film according to the strip-shaped print to form a strip-shaped hollow part with the width of 6mm and the length of 30mm for later use.

D. Middle and upper layer PDMS film production

Taking a certain amount of PDMS monomer and curing agent according to the weight ratio of 10: 1, degassing, uniformly coating the mixture on a clean silicon wafer by using a spin coater to form a film with the thickness of about 100mm, and heating the film in a 65 ℃ oven for 10 min. Spreading the prepared upper PDMS film on the middle film which is solidified for 10min, and placing the film into a 65 ℃ oven to be continuously baked for 60min to form a middle and upper PDMS film combination body.

E. The upper and middle layers are combined with the lower PDMS film

And placing the middle upper layer PDMS film and the lower layer PDMS film together in a plasma cleaner for processing for 30s, quickly taking out the middle upper layer PDMS film and the lower layer PDMS film, and aligning the hollow parts of the upper layers of PDMS to the micro-fluidic channel print of the lower layer to extrude together, thereby forming the three-layer sealed integrated PDMS micro-fluidic chip.

The schematic structural 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 transport control layer 3 which are sequentially stacked together. 4 microchannels 7 are arranged on the bottom layer liquid conveying control layer 3, a hollow structure 4 which is consistent with the position and the shape of the microchannels 7 is arranged on the upper layer deformation restraining layer 1, and two ports 6 at two ends of the microchannels 7 sequentially penetrate through the upper layer deformation restraining layer 1 and the middle layer 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 upper deformation confined layer 1 is 2 ~ 4mm, and the thickness of middle level PDMS deformation layer 2 is 0.05 ~ 1mm, and the thickness of bottom liquid transport control layer 3 is 2 ~ 4mm, and the length of microchannel 7 is 30mm, and the width is 6mm, and the degree of 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/outlet 5 is connected to the peristaltic pump 9 and the liquid storage bottle 10 through a silicone tube, as shown in fig. 2.

When fluid flows in the microchannel 7, the middle PDMS deformation layer 2 undergoes an arch deformation, as shown in fig. 3, which is a schematic cross-sectional and longitudinal-sectional view of the microfluidic chip when the fluid is not connected to the circulation system and when the fluid is connected to the circulation system and flows, as shown in fig. 3.

And calculating the fluid shear force according to the arch height and the flow rate. The mathematical derivation process in conjunction with the fluid shear force calculation method of fig. 4 is as follows:

the height (h) of the arch-shaped deformation generated by the PDMS layer with a certain elastic coefficient (E) is in direct proportion to the flow (Q) of the passing liquid,

h is EQ formula (1)

When h is less than r, the relation between h and the radius (r) of the circle where the arch is located and the width (w) of the strip-shaped cavity meets the straking theorem,

(r-h)²+(w/2)²＝r²formula (2)

Knowing w and h, the arc length sandwich angle theta and the arched transverse cutting area A can be obtained,

the rate of change of the flow velocity (u) in the vertical plane of the flow direction by the distance (y) of descent, as defined by the shear force (τ) of the fluid,

and the fluid shear force of the tubular structure,

under the condition of same flow rate, according to the principle of physical approximate equivalence, the area of fluid passing through is replaced by an arched cross-sectional area (A) instead of a circular cross-sectional area (pi r)²) Replacing the circular radius (r) with half (h/2) of the arch height of the flow velocity reduction distance to obtain an approximate formula of the fluid shearing force in the arch chamber,

in a specific experiment, the flow rate (Q) was obtained by actually measuring the volume of the liquid flowing out per unit time, the height (h) of the dome shape was obtained by observing the distance of the focal plane moving up and down by a microscope, the dome cross-sectional area (A) was calculated in combination with the width (w) of the known strip-shaped chamber, and the viscosity (μ) of the cell culture medium (DMEM + 10% FBS) at 37 ℃ was 9.3X 10^-3Dyne seconds/square centimeter. And measuring the arch height (h) of the middle layer PDMS bulge with the thickness of 0.1mm according to different flow rates, and substituting the arch height (h) into the formula (6) to obtain the corresponding fluid shear force. FIG. 5 shows the camber height of the middle elastic film, corresponding to the fluid shear force and the pressure of the liquid on the inner wall, with the change of the fluid flow under the condition of the middle film with a certain thickness. FIG. 6 shows the height of the middle elastic film arch, the corresponding fluid shear force and the pressure of the liquid on the inner wall, which are changed along with the change of the thickness of the middle elastic film under a certain fluid flow rate condition.

Example 2 in vitro vascular-like tissue model construction in circulating fluid Environment

Injecting cells into a micro-channel 7 of the micro-fluidic chip to ensure that the cells attach and grow on the middle PDMS deformation layer 2, and connecting the cells into a fluid circulation system after fusion. The flow rate or thickness of the middle PDMS deformation layer 2 is varied to obtain the corresponding fluid shear, fluid pressure and tensile forces acting on the cells. Factors inducing and interfering in the development of vascular lesions are introduced by altering the composition of the culture medium.

The experimental procedure was as follows:

cell lines: the vascular smooth muscle cell is T/G HA-VSMC; endothelial cells were ea.h926; the monocyte is THP-1;

cell culture medium composition: DMEM basal medium, 10% fetal bovine serum, penicillin (100IU/mL), streptomycin (100. mu.g/mL), insulin (40IU/mL), heparin (40IU/mL) and 1% of the non-essential amino acid NEAA;

cell culture conditions: constant temperature incubator 5% CO₂，37℃。

The chip pretreatment method comprises the following steps: ultrasonically cleaning the microfluidic chip, the silica gel pipeline and the liquid storage bottle with absolute ethyl alcohol and ultrapure water, placing the chip in an oven, drying the chip at 65 ℃, packaging the chip with new tin foil paper, placing the chip in an autoclave, sterilizing the chip at 121 ℃ for 30min, then placing the chip in the oven, drying the chip at 65 ℃, and transferring the chip to a clean bench for later use.

Fixing and coating the microfluidic chip: the microfluidic chip was mounted on a sterile 100mm cell culture dish, and the microfluidic channel was filled with 20. mu.g/ml fibronectin solution and placed in an incubator overnight.

Cell inoculation: vascular smooth muscle cells (2.0X 10) in good culture condition were cultured⁵Pieces/ml) is injected into the coated microfluidic channel, and is statically cultured for 24 hours by using an inoculation culture medium (DMEM + 10% FBS +50 mu g/ml ascorbic acid) to form a layer of tightly connected vascular smooth muscle cells; endothelial cells (3.0X 10) in good culture were cultured⁵One/ml) is injected into the microfluidic channel forming the smooth muscle cell layer, and static culture is continued for 24 hours by using an inoculation culture medium to form a 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 membranous films formed by co-culture of vascular smooth muscle cells and endothelial cells are shown in fig. 7 (red fluorescence is an endothelial marker, VE-cadherin, green fluorescence is a smooth muscle cell marker, SM-alpha-actin, blue fluorescence, DAPI, is a nuclear marker, and the upper part and the right side of the figure are respectively a confocal microscope z-axis scanning horizontal orthogonal segmentation figure). The SM-alpha-actin antibody and the VE-Cadherin antibody are adopted to mark smooth muscle cells and endothelial cells respectively, and the observation under a laser confocal microscope shows that clear double-layer cell structural characteristics are formed on the middle-layer PDMS deformation layer 2.

2. Higher low fluid pressure endothelial cell connectivity and inflammatory factor expression

After high and low fluid pressure (cell culture medium without ascorbic acid) is obtained to continuously act on cells for 24 hours under the condition of setting different flow rates, the contents of IL-6 and MCP-1 inflammatory factors in the culture medium are measured by ELISA; immunofluorescence was used to characterize the expression of the cellular pro-inflammatory molecules VE-cadherin and CD 31. The results are shown in FIG. 8(A) shows immunofluorescence detection of endothelial cell-to-cell junction molecules VE-cadherin (green fluorescence), CD31 (red fluorescence), histogram shows fluorescence intensity statistics, and FIG. 8(B) shows ELISA detection of extracellular inflammatory factors MCP-1 and IL-6), and it can be seen that the expression of the cellular inflammatory factors at low fluid pressure is higher than that at high fluid pressure.

3. The effect of a relatively high low fluid pressure on foam cell formation

The medium containing the suspension of monocyte THP-1 was used as a circulating fluid to compare the foam cell formation after 24 hours at different flow rates. And (3) identifying foam cells by fixing paraformaldehyde for 15min, dyeing with oil red for 1 hour, fixing with 60% isopropanol for 3min, dyeing with hematoxylin for 3min, adding water to turn blue, buckling, sealing, and observing the number of red lipid drops under a microscope. Oil red staining causes lipid droplets to appear red, cells with lipid droplets aggregated are identified as foam cells, and foam cell formation is judged by observing the amount of oil red. The results are shown in fig. 9, where it can be seen that the number of foam cells at low fluid pressure is significantly greater than at high fluid pressure.

4. Effect of atorvastatin on foam cell formation under Low fluid pressure

The statin drugs not only have the effect of reducing blood fat, but also have the functions of improving endothelial cells, resisting inflammation, inhibiting smooth muscle cell proliferation, resisting thrombosis, resisting platelet and the like. The in vitro efficacy of atorvastatin calcium was investigated using the in vitro artificial blood vessel tissue model of the invention. The experiment used low fluid pressure conditions comparing 20ng/ml atorvastatin calcium medium as a circulating fluid cultured cells for 24 hours. The results are shown in fig. 10, and it can be seen that atorvastatin calcium significantly inhibits foam cell formation.

5. The effect of higher low fluid shear on foam cell formation

The culture medium containing the suspended monocyte THP-1 is used as circulating fluid, and under the condition of fixed flow rate, the formation of foam cells after 24 hours of high and low shearing force action is formed by the medium-layer membrane chips with different thicknesses is compared. The foam cell identification adopts paraformaldehyde for fixation for 15min, oil red for 1 hour, 60% isopropanol for fixation for 3min, hematoxylin for 3min, water for returning blue, buckle, seal, and observe the quantity of red lipid droplets under a microscope. Oil red staining causes lipid droplets to appear red, cells with lipid droplets aggregated are identified as foam cells, and foam cell formation is judged by observing the amount of oil red. The results are shown in FIG. 11, where it can be seen that the number of foam cells under low fluid shear conditions is significantly greater than the high fluid pressure.

The experimental results show that the micro-fluidic chip provided by the invention is used for constructing an in vitro vascular tissue model, can develop various blood circulation system influence factors such as fluid shearing force, fluid pressure, tube wall elasticity, stretching force, fluid composition and the like, can simulate various physiological or pathological environments of blood vessels, and provides a reliable in vitro model and research means for the research of cardiovascular diseases.

Claims

1. A micro-fluidic chip comprises an upper deformation restraining layer, a middle PDMS deformation layer and a bottom liquid conveying control layer from top to bottom in sequence;

and fluid inlets at two ends of the micro-channel sequentially penetrate through the middle PDMS deformation layer and the upper deformation restriction layer to form a fluid inlet jack and a fluid outlet jack.

2. The microfluidic chip of claim 1, wherein: the thickness of the upper deformation restraining layer is more than 2 mm;

the upper deformation restricting layer is made of a transparent material;

the transparent material is PDMS or glass.

3. The microfluidic chip according to claim 1 or 2, wherein: the thickness of the middle PDMS deformation layer is 0.05-2 mm;

the thickness of the bottom liquid conveying control layer is more than 2 mm;

the bottom liquid transport control layer is made of a transparent material;

the transparent material is PDMS or glass.

4. A circulatory system provided with a fluid environment and capable of bearing an in-vitro artificial vascular tissue model, comprising the microfluidic chip, a delivery pump and a liquid storage bottle of any one of claims 1 to 3;

the micro-fluidic chip, the delivery pump and the liquid storage bottle are connected in series to form a closed-loop flow path.

5. The circulatory system of the in vitro artificial vascular-like tissue model according to claim 4, wherein: the micro-fluidic chip, the delivery pump and the liquid storage bottle are connected through a silicone tube.

6. Use of the microfluidic chip of any one of claims 1 to 3, or the circulatory system of the in vitro artificial vascular tissue-like model of claim 4 or 5 in the research of the mechanism of vascular disease development and the evaluation of drug efficacy.

7. The method for using the circulatory system of the in vitro artificial vascular tissue-like model according to claim 4 or 5, comprising the steps of:

changing the flow velocity of the fluid to obtain the change of the shearing force, the pressure and the stretching force of the fluid on the inner wall, thereby obtaining the influence on the cells;

by changing the composition of the culture medium of the cells, the influence of factors inducing and interfering in the occurrence of the vasculopathy on the cells is obtained.

8. Use according to claim 7, characterized in that: the ex vivo cells include smooth muscle cells and endothelial cells.