Organ chip and application method thereof

Abstract

The invention discloses an organ chip and an application method thereof, the organ chip comprises a cell culture channel I, a cell culture channel II, a micro-fluidic channel, a cell culture channel III and a cell culture channel IV which are arranged in parallel and in sequence, and round liquid inlets and outlets located at two sides of each channel, and the adjacent cell culture channels are connected by a tiny hollow pipeline. And the micro-fluidic channel and the cell culture channel are separated by a micro-column array. According to the non-contact type design, soluble factors secreted by cells can be transported through a tube cavity, stable cell culture fluid can be applied through the micro-flow pump to simulate a real intravascular microenvironment, proper mechanical stress is provided for angiogenesis, a cell culture channel and a micro-flow control channel are isolated through the micro-column array structure, and the cell culture efficiency is improved. Intelligent control over a fluid path is achieved through surface tension regulation and control, during single-channel perfusion, capillary acting force generated by gaps of the micro-columns can form a stable gas-liquid interface, and fluid channeling is effectively blocked.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic organ chips, and specifically, to an organ chip and an application method thereof. Background Art

[0002] An organ chip is a new biotechnological platform that relies on microfluidic technology to simulate human organs at the micron scale, and can accurately reproduce human physiological and pathological characteristics in aspects such as drug screening and disease research. Compared with traditional animal experiments, the application of organ chips can help save time and costs, and solve the problem that experiments cannot be replicated in humans due to population differences. In recent years, microfluidic technology has been continuously mature, and integrated microfluidic chips covering functions such as micropumps and microvalves have made it possible to precisely control fluids, particles, and cells. This not only expands the application of microfluidic chips in the research field, but also enables microfluidic chips to achieve more complex functions and experimental operations. Microfluidic chips have shown great advantages and potential in aspects such as studying cell-cell interactions, real-time quantitative expression and analysis of cell life information, in vitro tissue reconstruction, and function simulation.

[0003] The combination of 3D cell culture and microfluidic chip technology can significantly improve the physiological relevance of in vitro cell models through precise dynamic regulation of the microenvironment and biomimetic structure design: microfluidic channels can achieve gradient control of nutrients, oxygen, and metabolites in the extracellular matrix, and at the same time apply physiological-level mechanical stimuli such as fluid shear stress and periodic mechanical stress to simulate the in vivo dynamic microenvironment; while the 3D culture structure generated by the gel can reproduce the three-dimensional topology of the organ. The integration of the two not only solves the defects of limited mass exchange and deviation of functional expression in traditional static 3D culture, but also can integrate sensors to real-time monitor parameters such as cell metabolism and barrier integrity, providing a high-fidelity and scalable biomimetic experimental platform for high-throughput drug screening, complex disease modeling, and personalized medicine.

[0004] The current technology has the following technical problems:

[0005] 1) Traditional multi-cell co-culture organ chips mostly adopt a direct contact co-culture mode. Different cell types are prone to spatial competition due to differences in proliferation rates in the shared microenvironment, resulting in a mechanical extrusion effect that changes the morphological structure of adjacent cells.

[0006] 2) In the current field of bone tissue engineering research, there is a lack of a biomimetic microsystem that can dynamically monitor the bone metabolism process. Existing vascularized bone chips mostly adopt a static culture mode and fail to integrate a microfluidic system to simulate capillary-level pulsatile shear stress and physiological flow rate, resulting in a lack of mechanical stress induction in the angiogenesis process and being unable to truly reproduce the angiogenesis process in bone remodeling.

[0007] 3) The traditional co-culture system consumes a large amount of cells and drug reagents, etc. Moreover, the 2D growth mode not only destroys the natural arrangement directionality of cells but also fails to form a real supporting structure, ultimately affecting the fine cooperation mechanism between cells. Summary of the Invention

[0008] The present invention is an invention made in view of the deficiencies of the prior art, and provides an organ chip and a method. The chip has cell culture channels I, II, a microfluidic channel, cell culture channels III, IV arranged in parallel and in sequence, and circular inlet and outlet ports located on both sides of each channel. Adjacent cell culture channels are connected by tiny hollow pipelines, and the microfluidic channel and the cell culture channels are separated by a microcolumn array.

[0009] The specific technical solution of the present invention is as follows:

[0010] The present invention discloses an organ chip, including: cell culture channels I, II, a microfluidic channel, cell culture channels III, IV arranged in parallel and in sequence, and circular inlet and outlet ports located on both sides of each channel. Adjacent cell culture channels are connected by tiny hollow pipelines, and the microfluidic channel and the cell culture channels are separated by a microcolumn array.

[0011] As a further improvement, the widths of each cell culture channel and the microfluidic channel of the present invention are 500 - 800 µm, and the heights are 80 - 150 µm.

[0012] As a further improvement, between cell culture channel I and cell culture channel II, and between cell culture channel III and cell culture channel IV of the present invention, they are connected by a plurality of cuboid hollow pipelines. The length of the hollow pipelines is 80 - 150 µm, the width is 2 - 10 µm, and the height is 2 - 10 µm.

[0013] As a further improvement, there is a microcolumn array between the cell culture channels and the microfluidic channel of the present invention. The cross-sectional shape of the microcolumns is fan-shaped, trapezoidal, rectangular or regular hexagonal. The spacing between each microcolumn is 50 - 150 µm, and the height is the same as that of the cell culture channels.

[0014] As a further improvement, the sizes of the circular inlet and outlet ports corresponding to both sides of each channel of the present invention are the same.

[0015] As a further improvement, the cells in each cell culture channel of the present invention form three-dimensional cell culture by embedding into a collagen gel scaffold.

[0016] The present invention discloses an application method of an organ chip, including:

[0017] 1) Cultivate mesenchymal stem cells in cell culture channels I and IV to simulate bone tissue;

[0018] 2) Cultivate endothelial cells in cell culture channels II and III to simulate vascular tissue;

[0019] 3) Connect the microfluidic channel with a microfluidic pump device, and apply a uniform and stable cell culture medium to the microfluidic channel through the microfluidic pump device to simulate blood flow, and at the same time apply shear stress to the inner wall of the channel.

[0020] As a further improvement, in step 1) of the present invention, mesenchymal stem cells selected from humans or animals are embedded in a collagen gel, and the gel is introduced into cell culture channels I and IV and left standing for 45 min - 1 h.

[0021] As a further improvement, in step 2) of the present invention, endothelial cells selected from the same species as the mesenchymal stem cells are embedded in a collagen gel, and the gel is introduced into cell culture channels II and III and left standing for 45 min - 1 h.

[0022] As a further improvement, in step 3) of the present invention, the flow rate of the microfluidic pump for simulating blood flow is 5 - 20 µL / h.

[0023] The beneficial effects of the present invention are:

[0024] 1) The different cell channels are connected by a small cuboid hollow pipeline with a length of 80 - 150 µm, a width of 2 - 10 µm, and a height of 2 - 10 µm. This non-contact design not only satisfies the transportation of soluble factors secreted by cells through the lumen, but also avoids the deformation of vascular endothelial cells caused by excessive proliferation of mesenchymal stem cells.

[0025] 2) A stable cell culture medium can be applied through the microfluidic pump to simulate the real microenvironment in blood vessels, and it can provide appropriate mechanical stress for angiogenesis.

[0026] 3) The width of each cell culture channel and the microfluidic channel is 500 - 800 µm, and the height is 80 - 150 µm. This micron-scale precision processed cell culture channel reduces the overall culture unit volume by two orders of magnitude compared with the traditional co-culture system, can reduce the cell usage amount and drug test dose, and can significantly reduce the consumption cost of precious primary cells and expensive drugs.

[0027] 4) A biomimetic tissue microenvironment can be constructed through a three-dimensional collagen gel scaffold, realizing the spatial orderly arrangement and dynamic material exchange of cells, and at the same time supporting the synchronous culture and real-time monitoring of multiple tissue interfaces.

[0028] 5) Isolate the cell culture channel from the microfluidic channel through the microcolumn array structure, and achieve intelligent control of the fluid path through surface tension regulation: during single-channel perfusion, the capillary force generated in the microcolumn gap can form a stable gas-liquid interface, effectively preventing fluid intermixing; when two channels are perfused synchronously, the surface tension balance is broken, and the liquid forms a continuous phase along the microcolumn gap. This structural design that mimics the vascular basement membrane not only maintains the spatial isolation of the independent microenvironment but also realizes cross-channel material exchange activated on demand, providing a new method for constructing a dynamically adjustable inter-organ barrier model. Brief Description of the Drawings

[0029] Figure 1 This is the 2D design drawing of the organ chip provided in the first embodiment of the present invention.

[0030] In the figure: 1 is the inlet of cell culture channel I, 2 is the inlet of cell culture channel II, 3 is the inlet of the microfluidic channel, 4 is the inlet of cell culture channel III, 5 is the inlet of cell culture channel IV, 6 is the outlet of cell culture channel I, 7 is the outlet of cell culture channel II, 8 is the outlet of the microfluidic channel, 9 is the outlet of cell culture channel III, 10 is the outlet of cell culture channel IV, 11 is cell culture channel I, 12 is cell culture channel II, 13 is the microfluidic channel, 14 is cell culture channel III, 15 is cell culture channel IV, 16 is the hollow pipeline, and 17 is the microcolumn.

[0031] Figure 2 This is the 3D design drawing of the organ chip provided in the first embodiment of the present invention.

[0032] Figure 3 This is the effect diagram after inoculating bone marrow mesenchymal stem cells wrapped in collagen gel in the cell culture channel in the second embodiment of the present invention.

[0033] Figure 4 This is the effect diagram after inoculating human umbilical vein endothelial cells wrapped in collagen gel in the cell culture channel in the second embodiment of the present invention.

[0034] Figure 5 This is the fluorescence image of CD31 expression of human umbilical vein endothelial cells in the bone microvascular organ chip in the second embodiment of the present invention.

[0035] Figure 6 This is the fluorescence image of VEGFA expression of human umbilical vein endothelial cells in the microvascular organ chip in the second embodiment of the present invention.

[0036] Figure 7 This is the fluorescence image of RUNX2 expression of human bone marrow mesenchymal stem cells in the microvascular organ chip in the second embodiment of the present invention. Detailed Embodiments

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0039] The present invention is an organ chip, and its structure includes cell culture channels I11, cell culture channels II12, microfluidic channels 13, cell culture channels III14, cell culture channels IV15 arranged in parallel and in sequence, and circular inlet and outlet ports located on both sides of each channel; adjacent cell culture channels are connected by tiny hollow pipelines 16, and the microfluidic channel 13 and the cell culture channels are blocked by a microcolumn 17 array.

[0040] Embodiment 1: Structure of the organ chip

[0041] As Figure 1 shown, a 2D design diagram of an organ chip structure provided by an embodiment of the present invention. The organ chip includes five channels arranged in parallel. There are rectangular hollow pipelines 16 between adjacent cell culture channels, a microcolumn 17 array between the cell culture channels and the microfluidic channel 13, and ten inlet and outlet ports, including the inlet port 1 of cell culture channel I, the inlet port 2 of cell culture channel II, the inlet port 3 of the microfluidic channel, the inlet port 4 of cell culture channel III, the inlet port 5 of cell culture channel IV, the outlet port 6 of cell culture channel I, the outlet port 7 of cell culture channel II, the outlet port 8 of the microfluidic channel, the outlet port 9 of cell culture channel III, and the outlet port 10 of cell culture channel IV.

[0042] In this embodiment, considering the comprehensive manufacturing limitations and ease of use of the cell culture channels and the microfluidic channel 13, the width is preferably set to 800 μm and the height is set to 100 μm.

[0043] In this embodiment, in order to isolate the direct contact of cells between adjacent cell channels, as Figure 2 shown, rectangular hollow pipelines 16 are provided for connection, with a length of 100 µm, a width of 4 µm, and a height of 4 µm.

[0044] In this embodiment, the microcolumn 17 array between the cell culture channel and the microfluidic channel 13 adopts a fan-shaped structure with a spacing of 100 µm. The purpose of this design is that when there is liquid in a single channel, a stable gas-liquid interface can be formed through capillary action in the gaps between the microcolumns 17, effectively preventing fluid intermixing; when there is liquid in two adjacent channels simultaneously, the surface tension balance is broken, and the liquid can interact through the gaps between the microcolumns 17.

[0045] In this embodiment, the sizes of the liquid inlets and outlets of the cell culture channel I11, the cell culture channel II12, the microfluidic channel 13, the cell culture channel III14, and the cell culture channel IV15 are the same.

[0046] Example 2: Construction of a bone microvascular organ chip

[0047] In this embodiment, the organ chip model in Example 1 is adopted. The cell culture channel I11 and the cell culture channel IV15 are designed as bone cell culture units, and bone tissue is constructed by inoculating human bone marrow mesenchymal stem cells. Since human bone marrow mesenchymal stem cells have multi-directional differentiation potential and play a core role in bone regeneration, implanting human bone marrow mesenchymal stem cell collagen gel in the cell culture channel can better simulate the processes of bone formation and bone repair, and can be used for subsequent research on bone-related diseases. As Figure 3 shown, the human bone marrow mesenchymal stem cells embedded in the collagen gel have a rhomboid or irregular triangular growth shape and relatively long cell tails after growing in the injection channel for 24 hours, indicating good cell growth status.

[0048] In this embodiment, the organ chip model in Example 1 is adopted. The cell culture channel II12 and the cell culture channel III14 are designed as bone microvascular endothelial cell culture units, and a vascular microenvironment is constructed by inoculating human umbilical vein endothelial cells. The three-dimensional dynamic simulation of the vascular network is realized by using a microfluidic pump and the method of culturing cells embedded in collagen gel, and angiogenesis and vascular lesions can be observed in real time, which can be used for subsequent research on vascular-related diseases. As Figure 4 shown, the human umbilical vein endothelial cells embedded in the collagen gel have an oval or spindle shape after growing in the injection channel for 24 hours, and microfilaments are formed between multiple cells and cross-linked with each other to form a clearly visible vascular cavity structure.

[0049] In this embodiment, the flow rate of the microfluidic pump is set to 10 µL / h. Under this condition, blood flow can be better simulated, and shear stress can be provided for angiogenesis.

[0050] In this embodiment, functional protein immunofluorescence characterization is performed on the human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells in the cell culture channel, as Figure 5 and Figure 6As shown, both CD31 and VEGFA of human umbilical vein endothelial cells in the bone microvascular organ chip are highly expressed, indicating that the vascular endothelial cells in the chip are in the stage of continuous development and angiogenesis, and the cell - cell connections are tight. As Figure 7 , RUNX2 is highly expressed in bone marrow mesenchymal stem cells, indicating that bone marrow mesenchymal stem cells in the bone microvascular organ chip can undergo osteogenic differentiation.

[0051] Example 3: Structure of the organ chip

[0052] In the implementation of the present invention, the organ chip includes five parallel - arranged channels, a rectangular hollow pipeline 16 between adjacent cell - culture channels, an array of micro - pillars 17 between the cell - culture channels and the microfluidic channel 13, and ten liquid inlets and outlets.

[0053] In this embodiment, considering the manufacturing limitations and ease of use, the cell - culture channels and the microfluidic channel 13 are preferably set with a width of 500 μm and a height of 150 μm.

[0054] In this embodiment, in order to isolate the direct contact of cells between adjacent cell channels, a rectangular hollow pipeline 16 is set for connection, with a length of 80 µm, a width of 8 µm, and a height of 8 µm.

[0055] In this embodiment, the array of micro - pillars 17 between the cell - culture channels and the microfluidic channel 13 adopts a regular - hexagon structure with a spacing of 150 µm. The purpose of this design is that when there is liquid in a single channel, a stable gas - liquid interface can be formed through capillary action in the gaps between the micro - pillars 17, effectively blocking the inter - flow of fluids; when there is liquid in two adjacent channels at the same time, the surface - tension balance is broken, and the liquid can interact through the gaps between the micro - pillars 17.

[0056] In this embodiment, the sizes of the liquid inlets and outlets of cell - culture channel I11, cell - culture channel II12, microfluidic channel 13, cell - culture channel III14, and cell - culture channel IV15 are the same.

[0057] Example 4: Structure of the organ chip

[0058] In the implementation of the present invention, the organ chip includes five parallel - arranged channels, a rectangular hollow pipeline 16 between adjacent cell - culture channels, an array of micro - pillars 17 between the cell - culture channels and the microfluidic channel 13, and ten liquid inlets and outlets.

[0059] In this embodiment, considering the manufacturing limitations and ease of use, the cell - culture channels and the microfluidic channel 13 are preferably set with a width of 650 μm and a height of 80 μm.

[0060] In this embodiment, in order to isolate the direct contact of cells between adjacent cell channels, a rectangular hollow pipeline 16 is provided for connection, with a length of 150 µm, a width of 5 µm, and a height of 5 µm.

[0061] In this embodiment, the micro-column 17 array between the cell culture channel and the microfluidic channel 13 adopts a square structure with a spacing of 80 µm. The purpose of this design is that when there is liquid in a single channel, a stable gas-liquid interface can be formed through capillary action in the gaps between the micro-columns 17, effectively blocking the inter-channel fluid flow; when there is liquid in two adjacent channels simultaneously, the surface tension balance is broken, and the liquid can interact through the gaps between the micro-columns 17.

[0062] In this embodiment, the sizes of the liquid inlets and outlets of the cell culture channel I11, the cell culture channel II12, the microfluidic channel 13, the cell culture channel III14, and the cell culture channel IV15 are the same.

[0063] Comparative Example 1: Organ-on-a-chip structure

[0064] In this comparative example, the rectangular hollow pipeline 16 is not provided, and each cell channel is directly connected. The rest of the structure is the same as that of Example 1. In this design, different cells can make direct contact, resulting in serious deformation of the cells. Through comprehensive analysis, it is more appropriate to choose the structure of the cuboid hollow pipeline 16 to connect adjacent cell channels.

[0065] Comparative Example 2: Organ-on-a-chip structure

[0066] In this embodiment, the micro-column 17 array between the cell culture channel and the microfluidic channel 13 adopts a fan-shaped structure, with different lengths of intervals set. The rest of the structure is the same as that of Example 1.

[0067] The test results are shown in Table 1:

[0068] Microcolumn 17 Spacing Distance / µm Flow Condition Assessment 25 Abnormal Flow 50 Normal Flow 75 Normal Flow 100 Normal Flow 125 Normal Flow 150 Normal Flow 175 Abnormal Flow

[0069] As can be seen from Table 1, this interval design fails to form a stable gas-liquid interface when it is less than 50 µm or greater than 150 µm, and cannot block the inter-channel fluid flow. Through comprehensive analysis, it is more appropriate to choose the spacing of the micro-columns 17 to be 50 - 150 µm.

[0070] Comparative Example 3: Construction of a bone microvascular organ-on-a-chip

[0071] In this comparative example, different flow rates are set for the microfluidic pump, and the rest of the design is the same as that of Example 2.

[0072] The test results are shown in Table 2:

[0073] Microfluidic Pump Flow Rate / µL / h Angiogenesis Condition Assessment 0 No Angiogenesis 5 Angiogenesis 10 Angiogenesis 15 Angiogenesis 20 Angiogenesis 25 No Angiogenesis

[0074] From Table 2, under this flow rate condition, the cells in the endothelial cell culture channel were relatively loose and failed to form a stable vascular network. Comprehensive analysis showed that the microfluidic pump flow rate of 5-20μL / h was more appropriate.

[0075] Finally, it should be noted that what is listed above is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiment, and there may be many variations. All variations that can be directly derived or associated with the contents disclosed by ordinary technicians in this field should be considered to be within the scope of protection of the present invention.

Claims

1. An organ chip, characterized in that: include: The cell culture channel I, cell culture channel II, microfluidic channel, cell culture channel III, and cell culture channel IV are arranged in parallel and in sequence, and circular inlets and outlets are located on both sides of each channel. Adjacent cell culture channels are connected by tiny hollow pipes, and the microfluidic channel and the cell culture channel are separated by a microcolumn array.

2. The organ chip according to claim 1, characterized in that: The cell culture channels and microfluidic channels have a width of 500-800 µm and a height of 80-150 µm.

3. The organ chip according to claim 2, characterized in that: The cell culture channel I and the cell culture channel II, and the cell culture channel III and the cell culture channel IV are connected by a plurality of rectangular hollow pipes, and the hollow pipes are 80-150µm in length, 2-10µm in width, and 2-10µm in height.

4. The organ chip according to claim 3, characterized in that: There is a microcolumn array between the cell culture channel and the microfluidic channel. The cross-sectional shape of the microcolumns is fan-shaped, trapezoidal, rectangular or regular hexagonal. The spacing between each microcolumn is 50-150µm, and the height is consistent with the cell culture channel.

5. The organ chip according to claim 1, 2, 3 or 4, characterized in that: The corresponding circular inlet and outlet ports on both sides of each channel have the same size.

6. The organ chip according to claim 5, characterized in that: The cells in each cell culture channel are embedded in a collagen gel scaffold to form a three-dimensional cell culture.

7. An application method of the organ chip according to claim 1 or 2 or 3 or 4 or 6, characterized in that: include: 1) Cultivating mesenchymal stem cells in the cell culture channel I and the cell culture channel IV to simulate bone tissue; 2) Cultivating endothelial cells in the cell culture channel II and the cell culture channel III to simulate vascular tissue; 3) By connecting the microfluidic channel with the microfluidic pump device, the microfluidic pump device applies a uniform and stable cell culture fluid to the microfluidic channel to simulate blood flow, and at the same time applies shear stress to the inner wall of the channel.

8. The application method of the organ chip according to claim 7, characterized in that: In the step 1), mesenchymal stem cells selected from humans or animals are embedded in collagen gel, and the gel is passed into cell culture channel I and cell culture channel IV and allowed to stand for 45 minutes to 1 hour.

9. The application method of the organ chip according to claim 8, characterized in that: In the step 2), endothelial cells of the same species as the mesenchymal stem cells are embedded in the collagen gel, and the gel is passed into the cell culture channel II and the cell culture channel III and allowed to stand for 45 minutes to 1 hour.

10. The application method of the organ chip according to claim 8 or 9, characterized in that: In the step 3), the flow rate of the microfluidic pump simulating blood flow is 5-20 µL / h.

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