Microfluidic wound healing model and implementation method

Abstract

The invention discloses a microfluidic wound healing model. The microfluidic wound healing model comprises a microfluidic chip; the micro-fluidic chip comprises a chip substrate, a partition plate arranged in the middle of the chip substrate, a contrast material area and an experimental material area which are arranged on the chip substrate and located on the two sides of the partition plate, and an upper channel I, an upper channel II, a lower channel II and a lower channel I which are arranged in the chip substrate. According to the method, damage signals released in a damage microenvironment are simulated by using cell debris and matrigel, matrix cells in three-dimensional matrigel and vascular endothelial cells / epithelial cells feel the damage signals and migrate to the damage microenvironment, and the process can simulate interaction of different cells in the real early stage of damage repair; therefore, research on a three-dimensional level has simulation performance, an in-vivo damaged microenvironment can be effectively simulated, and interaction of various cells can be dynamically observed in real time. A control group and an experiment group can be simultaneously arranged on one chip, and the difference and trend of the two groups can be dynamically observed in real time.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic wound healing, and in particular to a microfluidic wound healing model and an implementation method thereof. Background Art

[0002] Existing in vitro wound healing models: 1. Traditional scratch test (two-dimensional) 2. Creating a cell-free area in a microfluidic chip through chemical methods (such as pancreatic enzymes) or physical methods (such as air pumps or mechanical pressure) to simulate the wound healing process (two-dimensional).

[0003] Existing in vitro wound healing models include traditional scratch experiments and the simulation of wound healing process by creating a cell-free area in a microfluidic chip through chemical methods (such as pancreatic enzymes) or physical methods (such as air pumps or mechanical pressure). Both models are only studies at the two-dimensional level and single cell level, while wound healing is a complex dynamic process of multiple cell interactions. Therefore, the present invention designs an in vitro microfluidic wound healing model, which achieves the purpose of being able to dynamically observe cell interactions in the wound healing process in real time by designing a microfluidic chip and using cell fragments and matrix glue to simulate a three-dimensional injury microenvironment. Summary of the invention

[0004] The purpose of the present invention is to provide a microfluidic wound healing model to solve the problem that the prior art cannot simulate the three-dimensional environment in vitro and can dynamically observe and study the cell interactions during the wound healing process in real time.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The present invention provides a microfluidic wound healing model, which includes a microfluidic chip;

[0007] The microfluidic chip comprises a chip substrate, a partition disposed in the middle of the chip substrate, a control material area and an experimental material area disposed on the chip substrate and located on both sides of the partition, and an upper channel I, an upper channel II, a lower channel II, and a lower channel I disposed in the chip substrate;

[0008] A reserved gap I is provided between the microfluidic chambers of the upper channel I and the upper channel II, and a reserved gap II is provided between the upper channel II and the experimental material area and the control material area;

[0009] A reserved gap I is provided between the lower channel II and the lower channel II, and a reserved gap II is provided between the lower channel II and the experimental material area and the control material area.

[0010] Furthermore, the control material area and the experimental material area are respectively injected with required materials through microfluidic tubes located on both sides of the chip substrate;

[0011] The upper channel I, the upper channel II, the lower channel II and the two ends of the lower channel II are respectively provided with a microfluidic injection tube and a microfluidic discharge tube.

[0012] This embodiment provides a method for implementing a microfluidic wound healing model.

[0013] Matrigel and culture medium are injected into the control material area of ​​the chip substrate through an inflow tube;

[0014] Matrigel and cell fragments are injected into the experimental material area of ​​the chip substrate through an inflow tube;

[0015] The upper channel II and the upper channel I are respectively injected with culture medium through microfluidic tubes;

[0016] The lower channel II is injected with matrix cells and endothelial cells through a microfluidic tube;

[0017] The lower channel I is injected with epithelial cells through a microfluidic tube;

[0018] Among them, the real-time dynamic observation of the materials on the chip substrate is carried out based on the microfluidic chip, and the interaction of different types of cells in the wound healing process is observed; cell fragments and matrix glue are used to simulate the damage signals released in the damage microenvironment. The matrix cells and vascular endothelial cells / epithelial cells in the three-dimensional matrix glue sense the damage signals and migrate to the damage microenvironment. This process can simulate the interaction between different cells in the early stage of real damage repair.

[0019] Compared with the prior art, the beneficial technical effects of the present invention are:

[0020] In the present invention, cell fragments and matrix gel are used to simulate the damage signals released in the damage microenvironment. The matrix cells and vascular endothelial cells / epithelial cells in the three-dimensional matrix gel sense the damage signals and migrate to the damage microenvironment. This process can simulate the interaction between different cells in the early stage of real damage repair; thus, the research at the three-dimensional level is more realistic, which can effectively simulate the in vivo damage microenvironment and observe the interaction of multiple cells in real time. A chip can set up a control group and an experimental group at the same time, and can observe the difference and trend between the two in real time. It can be used to study the effects of external factors such as drugs and hormones on the wound healing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with the accompanying drawings.

[0022] Figure 1 A schematic diagram of the main body of the microfluidic chip used in the microfluidic wound healing model of the present invention;

[0023] Figure 2This is a schematic diagram of the effect of the damaged microenvironment on the migration ability of five types of cells when the microfluidic wound healing model of the present invention is used;

[0024] Figure 3 Schematic diagram of the effect of fibroblasts on the migration ability of epithelial cells and endothelial cells respectively when used in the microfluidic wound healing model of the present invention;

[0025] Figure 4 This is a schematic diagram of the effect of the damaged microenvironment on the migration ability of fibroblasts treated with Prifenidone when used in the microfluidic wound healing model of the present invention;

[0026] Figure 5 Schematic diagram of the effect of Prifenidone-treated fibroblasts on the migration ability of epithelial cells and endothelial cells respectively when used in the microfluidic wound healing model of the present invention.

[0027] Reference numerals:

[0028] 100. Chip substrate; 101. Upper channel I; 102. Upper channel II; 103. Control material area; 104. Experimental material area; 105. Lower channel II; 106. Lower channel I; 107. Partition; 108. Reserved gap II; 109. Reserved gap I. DETAILED DESCRIPTION

[0029] like Figure 1 As shown, this embodiment discloses a microfluidic wound healing model, which includes a microfluidic chip; wherein the microfluidic chip includes a chip substrate 100, a partition 107 prefabricated in the middle of the chip substrate 100, a control material area 103 and an experimental material area 104 prefabricated on the chip substrate 100 and located on both sides of the partition 107, and an upper channel I 101, an upper channel II 102, a lower channel II 105, and a lower channel I 106 prefabricated in the chip substrate 100; the chip substrate 100 has a microfluidic cavity.

[0030] A reserved gap I109 is designed between the microfluidic chambers of the upper channel I101 and the upper channel II102, and a reserved gap II108 is designed between the upper channel II102 and the experimental material area 104 and the control material area 103; a reserved gap I109 is designed between the lower channel II105 and the lower channel II105, and a reserved gap II108 is designed between the lower channel II105 and the experimental material area 104 and the control material area 103. Specifically, the reserved gap I109 allows a single cell to pass through; the reserved gap II108 allows a group of cells to pass through.

[0031] In this embodiment, the control material area 103 and the experimental material area 104 are respectively injected with required materials through microfluidic tubes located on both sides of the chip substrate 100; the upper channel I 101, the upper channel II 102, the lower channel II 105, and the two ends of the lower channel II 105 are respectively provided with microfluidic injection tubes and microfluidic discharge tubes.

[0032] The present embodiment discloses an implementation method of a microfluidic wound healing model, which applies the above-mentioned microfluidic wound healing model; specifically: matrix glue and culture medium are injected into the control material area 103 of the chip substrate 100 through an inflow tube; matrix glue and cell fragments are injected into the experimental material area 104 of the chip substrate 100 through an inflow tube; the upper channel II 102 and the upper channel I 101 are respectively injected with culture medium through microfluidic tubes; the lower channel II 105 is injected with matrix cells and endothelial cells through a microfluidic tube; the lower channel I 106 is injected with epithelial cells through a microfluidic tube.

[0033] The microfluidic chip is used to dynamically observe the materials on the chip substrate 100 in real time, and the different types of cells interact with each other during the wound healing process; cell fragments and matrix glue are used to simulate the damage signals released in the damage microenvironment, and the matrix cells and vascular endothelial cells / epithelial cells in the three-dimensional matrix glue sense the damage signals and migrate to the damage microenvironment. This process can simulate the interaction between different cells in the early stage of real damage repair. The microfluidic chip is used to dynamically observe the materials on the chip substrate 100 in real time, and the different types of cells interact with each other during the wound healing process.

[0034] The usage process of this embodiment:

[0035] A chip can be used to set up a control group and an experimental group in the middle area at the same time. Cell fragments and matrix glue are used to simulate a three-dimensional injury microenvironment. Culture medium is added to the upper channel to maintain nutrients, and multiple cell types to be studied (stromal cells / epithelial cells / endothelial cells) are injected into the lower channel. The interaction between different types of cells during the wound healing process can be observed in real time and dynamically.

[0036] The real-time dynamic observation in this embodiment is to observe the migration status of cells by taking pictures every 24 hours under an inverted fluorescence microscope for one week and counting the migration distance of cells, and then making a cell migration trajectory map and a cell migration quantitative analysis map after the statistical data are collected.

[0037] The working principle of this embodiment:

[0038] Cell fragments and matrix gel are used to simulate the damage signals released in the damage microenvironment. The matrix cells and vascular endothelial cells / epithelial cells in the three-dimensional matrix gel sense the damage signals and migrate to the damage microenvironment. This process can simulate the interaction between different cells in the early stage of real damage repair.

[0039] In this embodiment, the tissues at the skin and lung injury sites are in a three-dimensional horizontal state in vivo, and the various cells distributed therein are in different planes. Traditional studies (such as scratch experiments) are conducted based on the morphology of cell adhesion, which cannot effectively simulate the real cell existence state in vivo. However, our model uses matrigel to enable the simulated injury microenvironment and cells to be in a three-dimensional plane.

[0040] Research at the three-dimensional level is more simulated. Its working principle is as follows: the left side of the middle area of ​​the chip is composed of a mixture of matrix gel (matrigel) and culture medium (1:1), the simulated damage microenvironment on the right side is composed of a mixture of matrix gel and culture medium containing cell fragments (1:1), and the cell channel under the chip is also added with a mixture of matrix gel and cell fluid (1:1), which can make the cells in a three-dimensional environment (rather than the attached cell state) to more realistically simulate the three-dimensional state in vivo.

[0041] In this embodiment, Figure 1 As shown in FIG. 1 , it is a schematic diagram of a microfluidic model: Matrigel and culture medium (mixed solution in a ratio of 1:1) are added to the control material area 103. Matrigel and a solution containing 4.4% cell debris (cell density of cell debris: 1*10 4 / μL) in a 1:1 mixture. The upper channel I 101 and the upper channel II 102 are added with culture medium to maintain nutrition. The lower channel II 105 and the lower channel I 106 can be added with a single cell or multiple cells to be studied.

[0042] like Figure 2 As shown, using the model of the present invention, we first tested the response of human dermal epithelial cells (HaCaT), human lung epithelial cells (BEAS-2B) and human vascular endothelial cells (HUVEC), as well as human skin fibroblasts (HDF) and human lung fibroblasts (HFL) to the wound microenvironment in the model. After these five types of cells were injected into the lower channel II105, they were photographed and observed every 24 hours and the cell migration distance was counted. It can be found that compared with the control group on the left, after adding cell fragments on the right, compared with HaCaT (Figure A), BEAS-2B (Figure B) and HUVEC cells (Figure C), HDF (Figure D) and HFL (Figure E) migrated faster to the injury microenvironment, indicating that fibroblasts are more sensitive to the injury stimulus simulated by cell fragments.

[0043] Figure A: The left side is the migration status diagram of HaCaT cells at 120h, and the right side is the quantitative diagram of migration distance of HaCaT cells from 0h to 120h.

[0044] Figure B: The left side is the migration status diagram of BEAS-2B cells at 120h, and the right side is the quantitative diagram of migration distance of BEAS-2B cells from 0h to 120h.

[0045] Figure C: The left side is the migration status diagram of HUVEC cells at 120h, and the right side is the quantitative diagram of HUVEC cell migration distance from 0h to 120h.

[0046] Figure D: The left side is the migration status diagram of 120h HDF cells, and the right side is the quantitative diagram of migration distance of 0h-120h HDF cells.

[0047] Figure E: The left side is the migration status diagram of HFL cells at 120h, and the right side is the quantitative diagram of migration distance of HFL cells from 0h to 120h.

[0048] like Figure 3 As shown, HFL and HUVEC, HDF and HUVEC, and HFL and BEAS-2B were mixed and injected into the chip, and the cell migration distance was counted and quantitatively analyzed every 24 hours. It can be found that compared with the control group, the fibroblasts in the cell fragment group can promote the migration of epithelial cells and endothelial cells to the damaged microenvironment.

[0049] Figure A: HFL was mixed with HUVEC at a ratio of 1:1, and then mixed with matrix gel at a ratio of cells: matrix gel (1:1) and injected into the lower channel II 105. The left side is a graph of HUVEC cell migration trajectory from 0 to 120 hours, and the right side is a quantitative graph of HUVEC cell migration distance from 0 to 120 hours.

[0050] Figure B: HDF and HUVEC were mixed at a ratio of 1:1, and then mixed with matrix gel at a ratio of cells: matrix gel (1:1) and injected into the lower channel II 105. The left side is a 0-120h HUVEC cell migration trajectory map, and the right side is a 0-120h HUVEC cell migration distance quantitative map.

[0051] Figure C: HFL and matrix gel were mixed at a ratio of 1:1 and injected into the lower channel II 105, and BEAS-2B cell solution was injected into the lower channel I 106. The left side is the migration trajectory of BEAS-2B cells from 0 to 96 hours, and the right side is the quantitative migration distance of BEAS-2B cells from 0 to 96 hours.

[0052] like Figure 4As shown in the figure, after HFL cells were treated with an inhibitor (Prifenidone) for 48 hours, they were mixed with matrix gel at a ratio of 1:1 and injected into channel II105 under the chip. The cell migration distance was observed and photographed every 24 hours. We can find that the migration ability of HFL cells in the cell debris group did not change compared with the control group after the addition of inhibitors, and the damaged microenvironment can still stimulate the migration of HFL cells. The left side shows the migration status of HFL cells at 120 hours, and the right side shows the quantitative graph of the migration distance of HFL cells from 0 to 120 hours.

[0053] like Figure 5 As shown, Prifenidone-treated HFL was mixed with HUVEC and BEAS-2B and injected into the chip, and continuous photography was taken to observe and count the cell migration distance. It can be found that Prifenidone-treated HFL has no effect on the migration speed of HUVEC and BEAS-2B, which further verifies that in the wound healing model, fibroblasts can promote the migration of epithelial cells and endothelial cells to the injured microenvironment.

[0054] Figure A: Prifenidone-treated HFL was mixed with HUVEC at a ratio of 1:1, and then mixed with matrix gel at a ratio of cells: matrix gel (1:1) and injected into the lower channel II 105. The left side is the migration trajectory of HUVEC cells from 0 to 96 hours, and the right side is the quantitative migration distance of HUVEC cells from 0 to 96 hours.

[0055] Figure B: Prifenidone-treated HFL was mixed with Matrigel at a ratio of 1:1 and injected into lower channel II 105, and BEAS-2B cell solution was injected into lower channel I 106. The left side is a graph of BEAS-2B cell migration trajectory from 0 to 96 hours, and the right side is a quantitative graph of BEAS-2B cell migration distance from 0 to 96 hours.

[0056] The above embodiments are only descriptions of the preferred methods of the invention, and are not intended to limit the scope of the invention. Without departing from the design spirit of the invention, various modifications and improvements made to the technical solutions of the invention by ordinary technicians in this field should fall within the protection scope of the claims of the invention.

Claims

1. A microfluidic wound healing model, characterized in that: It includes a microfluidic chip; The microfluidic chip comprises a chip substrate (100), a partition (107) arranged in the middle of the chip substrate (100), a control material area (103) and an experimental material area (104) arranged on the chip substrate (100) and located on both sides of the partition (107), and an upper channel I (101), an upper channel II (102), a lower channel II (105), and a lower channel I (106) arranged in the chip substrate (100); A reserved gap I (109) is provided between the microfluidic cavity of the upper channel I (101) and the upper channel II (102), and a reserved gap II (108) is provided between the upper channel II (102) and the experimental material area (104) and the control material area (103); A reserved gap I (109) is provided between the lower channel II (105) and the lower channel II (105), and a reserved gap II (108) is provided between the lower channel II (105) and the experimental material area (104) and the control material area (103).

2. The microfluidic wound healing model according to claim 1, characterized in that: The control material area (103) and the experimental material area (104) are respectively injected with required materials through microfluidic tubes located on both sides of the chip substrate (100); The upper channel I (101), the upper channel II (102), the lower channel II (105), and the two ends of the lower channel II (105) are respectively provided with a microfluidic injection tube and a microfluidic discharge tube.

3. A method for implementing a microfluidic wound healing model, using the microfluidic wound healing model described in any one of claims 1 or 2; characterized in that: The control material area (103) of the chip substrate (100) is injected with matrix gel and culture medium through an inflow tube; Injecting matrix glue and cell fragments into the experimental material area (104) of the chip substrate (100) through an inflow tube; The upper channel II (102) and the upper channel I (101) are respectively injected with culture medium through microfluidic tubes; The lower channel II (105) is injected with matrix cells and endothelial cells through a microfluidic tube; The lower channel I (106) is injected with epithelial cells through a microfluidic tube; Among them, the materials on the chip substrate are observed dynamically in real time based on the microfluidic chip, and different types of cells interact with each other during the wound healing process. Cell fragments and matrix gel are used to simulate the damage signals released in the damage microenvironment. The matrix cells and vascular endothelial cells / epithelial cells in the three-dimensional matrix gel sense the damage signals and migrate to the damage microenvironment. This process simulates the interaction between different cells in the early stages of real damage repair.

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