Multi-system microvessel co-culture micro-fluidic chip and preparation method thereof

Abstract

The invention provides a multi-system microvessel co-culture micro-fluidic chip and a preparation method thereof, and belongs to the technical field of biomedical engineering and microfluidics. The adipose-derived stem cells are introduced for the first time to participate in the cell culture environment in the chip, the difference between the in-vitro microvascular network micro-fluidic chip and the in-vivo microenvironment of a human body is further reduced, and the in-vivo environment is better simulated in vitro. In addition, different micro-fluidic chip monomers are constructed by using three different representative vascular endothelial cells, namely human brain microvascular endothelial cells (HBMVEC), human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HPMVEC), and the micro-fluidic chip is obtained by connecting all the monomers. The conditions of microvascular endothelium between different systems can be known and compared at the same time.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering and microfluidics, and in particular relates to a multi-system microvascular co-culture microfluidics chip and a preparation method thereof. Background Art

[0002] As people's research on microvessels deepens, the use of animal models for in vivo observation can no longer meet the needs of today's experiments. Therefore, building a microvascular network in vitro that simulates the local environment in vivo has become a hot topic of concern, and has extended to further research on tissue-like and organoid-like. Microfluidic chips are a good platform for in vitro microvascular networks. They can not only provide an environment for microvessels to attach and grow, but also simulate blood perfusion to blood vessels with their fluid control characteristics. Microvascular network microfluidic chips with 3D structures that can mimic blood flow perfusion functions are beneficial to researchers' research on vascular physiology and pathology. If the chip is regarded as an open source system, it can be personalized according to different purposes of use, such as tumor cell metastasis, infection model construction, drug screening, etc. In addition to being used in basic research, using it in clinical practice can achieve individualized, intuitive, and dynamic understanding and detection of changes in the patient's microenvironment, such as diagnosing whether critically ill patients have capillary leakage and evaluating the treatment effect. However, there are still large differences between the construction of in vitro microvascular network microfluidic chips and the composition of the microenvironment in the human body. Moreover, the current vascular-related microfluidic chips cannot realize real-time horizontal comparison of the conditions of multi-system microvascular endothelial cells. How to reduce the difference between the construction of in vitro microvascular network microfluidic chips and the composition of the microenvironment in the human body and realize horizontal comparison of the conditions of multi-system microvascular endothelial cells is an urgent problem to be solved nowadays. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a multi-system microvascular co-culture microfluidic chip and a preparation method thereof. The present invention introduces adipose stem cells for the first time to participate in the formation of the cell culture environment in the chip, which can significantly improve the differences between the construction of in vitro microvascular network microfluidic chips and the composition of the microenvironment in the human body. The present invention constructs different microfluidic chip monomers by using three different representative vascular endothelial cells, namely human brain microvascular endothelial cells (HBMVEC), human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HPMVEC), and connects the monomers to achieve simultaneous lateral comparison of the differences and changes in multi-system microvascular endothelial cells.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a multi-system microvascular co-culture microfluidic chip, wherein the multi-system microvascular co-culture microfluidic chip comprises a plurality of units connected in parallel, and a total sample inlet is respectively connected to an inlet of each unit;

[0006] Each unit includes a microfluidic chip carrier, a biological scaffold disposed on the microfluidic chip carrier, and a sample channel disposed on the biological scaffold;

[0007] The biological scaffold is a tubular structure formed by a fibrin gel mixed solution through a matrix perfusion channel.

[0008] Preferably, the number of the parallel-connected units is 2 to 4.

[0009] Preferably, the matrix perfusion channel comprises an inlet and an outflow channel, and the sample channel comprises an inlet and an outlet.

[0010] Preferably, the fibrin gel mixed solution is formed by mixing fibrinogen with a complete culture medium containing thrombin.

[0011] The present invention also provides a method for preparing a multi-system microvascular co-culture microfluidic chip, comprising the following steps:

[0012] (1) mixing a polydimethylsiloxane prepolymer and a cross-linking agent in a mass ratio of 8 to 12:1, casting the mixture on a surface of a silicon plate with a channel pattern, and curing the mixture to form a chip with a pattern structure;

[0013] (2) removing the chip from the silicon plate surface, and using a punch to make the inlet and outlet channels of the matrix perfusion channel and the inlet and outlet of the sample channel;

[0014] (3) using plasma to bond the chip and glass through chemical bonds to obtain a microfluidic chip carrier;

[0015] (4) placing the microfluidic chip carrier in an oven to restore its hydrophobicity and then sterilizing it with ultraviolet light;

[0016] (5) Using biological 3D printing technology, the printing ink and vascular endothelial cell culture medium are connected to different nozzles of the bioprinter and printed onto the glass winding rod to form a seamless tubular tissue;

[0017] (6) placing the tubular tissue in the microfluidic chip carrier obtained in step (4), injecting the fibrin gel mixed solution into the microfluidic chip carrier from the inflow channel of the matrix perfusion channel, incubating, and obtaining a biological scaffold when a complete tubular structure grows in the matrix perfusion channel;

[0018] (7) pulling out the glass winding rod to form a sample channel, and setting a filter membrane at the inlet of the sample channel to obtain a unit of the microfluidic chip;

[0019] (8) By connecting the units of multiple microfluidic chips, a multi-system microvascular co-culture microfluidic chip can be obtained.

[0020] Preferably, the curing temperature is 60-80° C., and the curing time is 30-60 min.

[0021] Preferably, the method of using plasma to bond the chip and the glass through chemical bonds is: placing the chip and the glass slide in a plasma cleaning machine and evacuating the machine for 1 to 5 minutes.

[0022] Preferably, the vascular endothelial cells are human brain microvascular endothelial cells, human umbilical vein endothelial cells and human lung microvascular endothelial cells.

[0023] Preferably, the fibrin gel mixed solution contains mesenchymal stem cells, adipose stem cells and cytokines, wherein the content of mesenchymal stem cells and adipose stem cells is 1×10 6 ~1×10 8 cells / mL, cytokines include Ang-1, VEGF, and S1P1. The concentration of Ang-1 is 50-100 ng / mL, the concentration of VEGF is 50-200 ng / mL, and the concentration of S1P1 is 100-500 ng / mL.

[0024] Preferably, the pore size of the filter membrane is 0.1-1 μm.

[0025] Compared with the prior art, the present invention has the following beneficial effects: the present invention introduces adipose stem cells for the first time to participate in the cell culture environment in the chip, further reducing the difference between the in vitro microvascular network microfluidic chip and the microenvironment in the human body, and better simulating the in vivo environment in vitro. In addition, the present invention constructs different microfluidic chip monomers by using three different representative vascular endothelial cells, namely human brain microvascular endothelial cells, human umbilical vein endothelial cells and human lung microvascular endothelial cells, and by connecting the monomers, it is possible to simultaneously understand and compare the conditions of the microvascular endothelial cells between different systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flow chart of the multi-system microvascular co-culture microfluidic chip;

[0027] Figure 2 Schematic diagram of the structure of the multi-system microvascular co-culture microfluidic chip. DETAILED DESCRIPTION

[0028] The present invention provides a multi-system microvascular co-culture microfluidic chip, wherein the multi-system microvascular co-culture microfluidic chip comprises a plurality of units connected in parallel, and a total sample inlet is respectively connected to an inlet of each unit;

[0029] Each unit includes a microfluidic chip carrier, a biological scaffold disposed on the microfluidic chip carrier, and a sample channel disposed on the biological scaffold;

[0030] The biological scaffold is a tubular structure formed by a fibrin gel mixed solution through a matrix perfusion channel.

[0031] In the present invention, the number of the units connected in parallel is 2 to 4, preferably 3.

[0032] In the present invention, the matrix perfusion channel includes an inlet and an outflow channel, and the sample channel includes an inlet and an outlet.

[0033] In the present invention, the fibrin gel mixed solution is prepared by diluting 25 mg / mL fibrinogen to a concentration of 10 mg / mL using phosphate buffer and mixing it with DMEM complete culture medium containing 6 U / mL thrombin in a volume ratio of 1:1.

[0034] The present invention also provides a method for preparing a multi-system microvascular co-culture microfluidic chip, comprising the following steps:

[0035] (1) mixing a polydimethylsiloxane prepolymer and a cross-linking agent in a mass ratio of 8 to 12:1, casting the mixture on a surface of a silicon plate with a channel pattern, and curing the mixture to form a chip with a pattern structure;

[0036] (2) removing the chip from the silicon plate surface, and using a punch to make the inlet and outlet channels of the matrix perfusion channel and the inlet and outlet of the sample channel;

[0037] (3) using plasma to bond the chip and glass through chemical bonds to obtain a microfluidic chip carrier;

[0038] (4) placing the microfluidic chip carrier in an oven to restore its hydrophobicity and then sterilizing it with ultraviolet light;

[0039] (5) Using biological 3D printing technology, the printing ink and vascular endothelial cell culture medium are connected to different nozzles of the bioprinter and printed onto the glass winding rod to form a seamless tubular tissue;

[0040] (6) placing the tubular tissue in the microfluidic chip carrier obtained in step (4), injecting the fibrin gel mixed solution into the microfluidic chip carrier from the inflow channel of the matrix perfusion channel, incubating, and obtaining a biological scaffold when a complete tubular structure grows in the matrix perfusion channel;

[0041] (7) pulling out the glass winding rod to form a sample channel, and setting a filter membrane at the inlet of the sample channel to obtain a unit of the microfluidic chip;

[0042] (8) By connecting the units of multiple microfluidic chips, a multi-system microvascular co-culture microfluidic chip can be obtained.

[0043] In the present invention, a polydimethylsiloxane prepolymer and a crosslinking agent in a mass ratio of 8 to 12:1 are mixed, cast on the surface of a silicon plate with a channel pattern, and cured to form a chip with a pattern structure. The mass ratio is preferably 9 to 11:1, and more preferably 10:1; the crosslinking agent is methyl hydrogen siloxane; in the present invention, the crosslinking agent methyl hydrogen siloxane reacts with the vinyl or hydroxyl end groups in the polydimethylsiloxane prepolymer to convert the polydimethylsiloxane prepolymer from a liquid state to a solid with a three-dimensional crosslinked network, which is covered on the surface of the chip; the curing temperature is 60 to 80°C, and the curing time is 30 to 60 minutes.

[0044] In the present invention, the method of using plasma to bond the chip and glass through chemical bonds is as follows: place the bottom surface of the chip with the channel structure and the top surface of the clean glass slide upward in a plasma cleaning machine, evacuate for 1 to 5 minutes, preferably 2 to 4 minutes, and more preferably 3 minutes, adjust the plasma mode to "Mid", treat for 1 to 5 minutes, preferably 2 to 4 minutes, and more preferably 3 minutes, then turn off the air intake knob and the "Mid" mode, slowly release the air and take out the chip and the glass slide, so that the polydimethylsiloxane chip and the glass are irreversibly bonded through chemical bonds to seal the microfluidic chip.

[0045] In the present invention, the vascular endothelial cells are human brain microvascular endothelial cells, human umbilical vein endothelial cells and human lung microvascular endothelial cells.

[0046] In the present invention, the fibrin gel mixed solution contains mesenchymal stem cells, adipose stem cells and cytokines, wherein the content of mesenchymal stem cells and adipose stem cells is 1×10 6 ~1×10 8 cells / mL, cytokines include Ang-1, VEGF, and S1P1. The concentration of Ang-1 is 50-100 ng / mL, the concentration of VEGF is 50-200 ng / mL, and the concentration of S1P1 is 100-500 ng / mL.

[0047] In the present invention, the pore size of the filtration membrane is 0.1-1 μm.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example

[0050] Preparation of a multi-system microvascular co-culture microfluidic chip

[0051] (1) Using soft lithography technology to make a positive mold of a microfluidic chip, including spin coating, pre-baking, exposure, post-baking, development and film solidification;

[0052] (2) Polydimethylsiloxane prepolymer (purchased from Dow Corning (China) Investment Co., Ltd.) and methyl hydrogen siloxane in a mass ratio of 10:1 were mixed and then cast on the surface of the silicon plate with the channel pattern. A dry pump was then used to set the vacuum degree to 1 mTorr for 25±5 min. The bubble removal was observed at the same time. After the bubbles were completely removed, the mixture was cured at 80°C for 60 min.

[0053] (3) peeling off the cured polydimethylsiloxane from the surface of the silicon plate to form a chip with a pattern structure, and making all inlets and outlets using a punch;

[0054] (4) Place the bottom surface of the chip with the channel structure and the top surface of the clean glass slide facing upward in a plasma cleaning machine. After vacuuming for 3 minutes, adjust the plasma mode to "Mid". After processing for 3 minutes, turn off the air intake knob and the "Mid" mode, slowly release the air and take out the chip and glass slide, so that the polydimethylsiloxane chip and the glass are irreversibly bonded through chemical bonds to seal the microfluidic chip, thereby obtaining a microfluidic chip carrier;

[0055] (5) The microfluidic chip carrier is placed in an oven at 80°C for 24 hours to restore its hydrophobicity and then sterilized by ultraviolet irradiation for 30 minutes for later use;

[0056] (6) Using biological 3D printing technology, 500 μL of gelatin solution and 500 μL of sodium alginate solution were kept at 37°C for 25 minutes and mixed as printing ink. The printing inks were connected to different nozzles of the bioprinter with culture fluid of human brain microvascular endothelial cells, human umbilical vein endothelial cells, and human lung microvascular endothelial cells, and printed together on a glass winding rod to form seamless human brain microvascular endothelial cell tubular tissue, human umbilical vein endothelial cell tubular tissue, and human lung microvascular endothelial cell tubular tissue;

[0057] (7) placing the tubular tissue in the microfluidic chip carrier obtained in step (5), injecting the mixed solution of fibrin gel containing mesenchymal stem cells, adipocytes and cytokines from the inflow channel of the matrix perfusion channel into the microfluidic chip carrier using a pipette, incubating in a carbon dioxide incubator at 37° C., 5% CO2 and 95% air mixed gas, and 95% humidity. When the matrix perfusion channel grows into a complete tubular structure, removing the winding rod, and placing the hollow tubular tissue in the chip, thus obtaining a unit of the microfluidic chip;

[0058] (8) Multiple microfluidic chip units are connected through a total sample inlet to obtain a multi-system microvascular co-culture microfluidic chip.

[0059] Chip schematic diagram Figure 2 As shown. If not in use, DMEM complete culture medium must be added from the sample inlet 5 and outlet 6, and fresh culture medium must be replaced every day. When the chip is used, a peristaltic pump is used as the power to establish a circulation perfusion system for the vascular channel, which is convenient for the collection and detection of the perfusion fluid. More than ten components such as pH, nutrients, metabolic waste, compound concentration, signal molecules, and exosomes can be detected and evaluated. Figure 2 The 4 parts are seamless human brain microvascular endothelial cell tubular tissue, human umbilical vein endothelial cell tubular tissue and human lung microvascular endothelial cell tubular tissue, which are equivalent to different blood vessels in the body, and the 3 parts are equivalent to extravascular tissue in the body. Collecting the liquid at 6 and 2 for detection can reflect the permeability of blood vessels and the situation of substances in different blood vessels. This chip can be adapted to the detection methods and real-time dynamic observation of conventional microfluidic chips, and can be integrated with platforms such as live cell imaging technology and fluorescence imaging technology to achieve the effect of 1+1>2.

[0060] The multi-system microvascular co-culture microfluidic chip obtained by the present invention is used for molecular biological detection. Detection method: 5 mL of blood sample to be tested from a sepsis patient is injected into the chip through the sample inlet in the chip to simulate the state of the blood-brain barrier, pulmonary microvessels and other microvessels in the sepsis patient, and the function of the blood-brain barrier, pulmonary microvessels and other microvascular endothelial cells is tested, and the cells are cultured in a cell culture incubator with 5% carbon dioxide, 37°C and 80% humidity for 24 hours, and the liquids at 6 and 2 locations are collected for analysis.

[0061] By detecting biomarkers related to endothelial cell damage (such as DNA, RNA, protein, etc.), the pathophysiological changes of the blood-brain barrier, pulmonary microvessels and other microvessels can be understood. The multi-system microvascular co-culture microfluidic chip obtained by the present invention can detect target molecules as low as picomolar (pM) level, and the cross-reaction rate is low (<1%). It can be seen that the detection sensitivity of the present invention is high and the specificity is strong. By detecting cell activity, proliferation and apoptosis, in vitro cell analysis can be performed in real time. And the cell capture efficiency of the multi-system microvascular co-culture microfluidic chip obtained by the present invention can reach more than 90%, and real-time counting of cells in each microfluidic channel can be achieved. When performing chemical analysis to detect the concentration of chemical substances (such as ions, molecules) in the solution, the concentration detection limit (<1nM) of nanomolar (nM) or even lower can be reached, and the detection can be repeated many times, and the relative standard deviation (RSD) of the experiment is less than 5%.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A multi-system microvascular co-culture microfluidic chip, characterized in that: The multi-system microvascular co-culture microfluidic chip includes a plurality of units connected in parallel, and the total sample inlet is connected to the inlet of each unit respectively; Each unit includes a microfluidic chip carrier, a biological scaffold disposed on the microfluidic chip carrier, and a sample channel disposed on the biological scaffold; The biological scaffold is a tubular structure formed by a fibrin gel mixed solution through a matrix perfusion channel.

2. The multi-system microvascular co-culture microfluidic chip according to claim 1, characterized in that: The number of the parallel-connected units is 2 to 4.

3. The multi-system microvascular co-culture microfluidic chip according to claim 1, characterized in that: The matrix perfusion channel includes an inlet and an outlet, and the sample channel includes an inlet and an outlet.

4. The multi-system microvascular co-culture microfluidic chip according to claim 1, characterized in that: The fibrin gel mixed solution is formed by mixing fibrinogen with a complete culture solution containing thrombin.

5. A method for preparing a multi-system microvascular co-culture microfluidic chip, characterized in that: The following steps are involved: (1) mixing a polydimethylsiloxane prepolymer and a cross-linking agent in a mass ratio of 8 to 12:1, casting the mixture on a surface of a silicon plate with a channel pattern, and curing the mixture to form a chip with a pattern structure; (2) removing the chip from the silicon plate surface, and using a punch to make the inlet and outlet channels of the matrix perfusion channel and the inlet and outlet of the sample channel; (3) using plasma to bond the chip and glass through chemical bonds to obtain a microfluidic chip carrier; (4) placing the microfluidic chip carrier in an oven to restore its hydrophobicity and then sterilizing it with ultraviolet light; (5) Using biological 3D printing technology, the printing ink and vascular endothelial cell culture medium are connected to different nozzles of the bioprinter and printed onto the glass winding rod to form a seamless tubular tissue; (6) placing the tubular tissue in the microfluidic chip carrier obtained in step (4), injecting the fibrin gel mixed solution into the microfluidic chip carrier from the inflow channel of the matrix perfusion channel, incubating, and obtaining a biological scaffold when a complete tubular structure grows in the matrix perfusion channel; (7) pulling out the glass winding rod to form a sample channel, and setting a filter membrane at the inlet of the sample channel to obtain a unit of the microfluidic chip; (8) By connecting the units of multiple microfluidic chips, a multi-system microvascular co-culture microfluidic chip can be obtained.

6. The preparation method according to claim 5, characterized in that: The curing temperature is 60-80° C., and the curing time is 30-60 minutes.

7. The preparation method according to claim 5, characterized in that: The method of using plasma to bond the chip and the glass through chemical bonds is as follows: placing the chip and the glass slide in a plasma cleaning machine and evacuating the machine for 1 to 5 minutes.

8. The preparation method according to claim 5, characterized in that: The vascular endothelial cells are human brain microvascular endothelial cells, human umbilical vein endothelial cells and human lung microvascular endothelial cells.

9. The preparation method according to claim 5, characterized in that: The fibrin gel mixed solution contains mesenchymal stem cells, adipose-derived stem cells, and cytokines, with the content of mesenchymal stem cells and adipose-derived stem cells both at 1×10 6 ~1×10 8 cells / mL, cytokines include Ang-1, VEGF, and S1P1. The concentration of Ang-1 is 50-100 ng / mL, the concentration of VEGF is 50-200 ng / mL, and the concentration of S1P1 is 100-500 ng / mL.

10. The preparation method according to claim 5, characterized in that: The pore size of the filter membrane is 0.1-1 μm.

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