Multiple-organoid co-culture micro-fluidic chip and use method thereof

Abstract

The present invention belongs to the field of organoid chips. Provided is a multiple-organoid co-culture micro-fluidic chip, comprising organoid micro-fluidic chip cells, each organoid micro-fluidic chip cell being divided into an upper layer and a lower layer which are bonded to each other, the upper layer being sequentially provided with an organoid culture medium perfusion channel, a first channel barrier, an organoid culture chamber, a second channel barrier and a vascular endothelial culture channel, and the lower layer being provided with a micro-perfusion network channel. The chip ensures specific nutrition and microenvironment for each organoid and helps to maintain specific phenotypes for same. In addition, the chip can simulate in-vitro blood flowing, so as to perform signal communication and interaction between organoids, thereby fully simulating the in-vivo environment and interaction for multiple-organ diseases.

Description

A multi-organoid co-culture microfluidic chip and its use method Technical Field

[0001] The present invention belongs to the field of organoid chips, and specifically relates to a multi-organoid co-culture microfluidic chip and a method for using the same. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Organoids are microscopic, three-dimensional structures capable of self-assembly formed by pluripotent stem cells (PSCs) and adult stem cells (ASCs) cultured in vitro. Organoids mimic human organs in structure and function, and have great potential for application in regenerative medicine, gene editing, precision medicine, organ development, and disease modeling. They were named "Biotechnology of the Year 2017" by Nature Methods, a subsidiary of Nature magazine. The development of organoid technology was detailed in the New England Journal of Medicine in 2019 and graced the cover of Science magazine.

[0004] Microfluidics, also known as lab-on-chips, is a technology for manipulating fluids at the micrometer scale. In 2017, it was designated a "disruptive technology" by the Ministry of Science and Technology. Organ-on-a-chip technology, which combines microfluidics with cell culture, can simulate the functions of human organ units and was named one of the world's top ten emerging technologies in 2016 by the World Economic Forum.

[0005] The "organ-on-a-chip" technology, which integrates organ-on-a-chip technology with organoids, has unique advantages because it can perform real-time and controllable regulation of signal pathways during organoid development and can fully simulate the in vivo microenvironment and mechanical forces.

[0006] Single organ-on-a-chips offer significant advantages for exploring the function of a single organ. However, single organ-on-a-chip technology cannot simulate the systemic interactions of organs in response to injury, disease, and treatment. This makes it difficult to simulate and study diseases involving multiple organs and vascular flow (such as cancer, metabolism, fibrosis, inflammation, and infection) in in vitro experiments.

[0007] At present, the research on multi-organ chip-related disease models can be roughly divided into three categories. The first category is that in the past, most studies were based on the co-culture of monolayer cell lines from multiple organs. This type of chip can realize the study of the influence and crosstalk between different types of cells, but it cannot give full play to the unique role of organoid technology in organ simulation, and it cannot avoid the mutual mixing of culture media during the co-culture process, which makes it impossible for each cell to maintain its own growth microenvironment and unique phenotype. The second category is represented by the microarray chamber organoid co-culture chip published by Qin Jianhua's team in advanced science in 2022 (IF=17.521). This study used the self-assembly characteristics of organoids to achieve co-culture of organoids in the chip microarray chamber. This type of research has achieved the culture, growth and interaction of organoids in the chip, but it still cannot avoid the mutual mixing of culture media and the unique in vivo microenvironment for organoid growth. The third category is represented by the pluggable multi-organ chip co-culture chip published in Nature Biomedical Engineering in 2022 (IF=25.671) by the Department of Engineering and Medical Center of Columbia University in the United States. For the first time, it simultaneously achieved (1) each organ maintains its own specific microenvironment, (2) interactive communication between different organs through flow in endothelial channels, and (3) separation of vascular channels and organ culture chambers through selective permeability barriers. However, this organ chip is cultured at a large scale and cannot achieve microfluidic liquid perfusion and organoid growth at the micron level.

[0008] There are three main challenges that the above defects fail to solve: First, although the co-culture chips of single-layer cell lines derived from multiple organs and the co-culture chips of microarray chamber organoids are relatively mature, the current solutions have not added endothelial barriers, which cannot completely solve the problem of mixing and interference of culture media during co-culture. Therefore, the unique nutrition and microenvironment of organoids cannot be guaranteed during the culture process. Second, the organoid culture chips represented by the gel drop method have generally encountered the problems of difficult gel filling, high failure rate, and insufficient organoid perfusion, and have failed to become the mainstream solution for organoid chip culture. Third, the current organoid microfluidic chips are difficult to manufacture, and the cost of manufacture and use is high. It is difficult to meet the design requirements of multi-organoid co-culture microfluidic chips at the micron scale.

[0009] Summary of the Invention

[0010] Based on this research background, a multi-organoid co-culture microfluidic chip ensures the specific nutrition and microenvironment of each organ, helping to maintain its specific phenotype. It also simulates blood flow in the body, enabling signal communication and interaction between organoids, fully simulating the in vivo environment and interactions of multi-organ diseases.

[0011] In a first aspect, the present invention provides a multi-organoid co-culture microfluidic chip, comprising an organoid microfluidic chip monomer, wherein the organoid microfluidic chip monomer is divided into an upper and lower layer that are bonded and formed, wherein the upper layer is sequentially provided with an organoid culture medium perfusion channel, a first channel barrier, an organoid culture chamber, a second channel barrier and a vascular endothelial culture channel, and the lower layer is provided with a microperfusion network channel, the organoid culture medium perfusion channel and the microperfusion network channel are connected, the first channel barrier and the second channel barrier are respectively provided with partition holes, and the vascular endothelial culture channels between different organoid co-culture microfluidic chip monomers are connected.

[0012] Furthermore, the organoid culture medium perfusion channel, the first channel barrier, the organoid culture chamber, the second channel barrier and the vascular endothelial culture channel are sequentially arranged on the lower surface of the upper chip and arranged upward, and the microperfusion network channel is arranged downward on the upper surface of the lower chip.

[0013] Furthermore, the organoid culture chamber is perfused with Matrigel containing the organoid suspension.

[0014] Furthermore, the vascular endothelial culture channel is perfused with vascular endothelial culture medium containing vascular endothelial cells.

[0015] Furthermore, the number of monomers in the organoid microfluidic chip is 2, 3 or 4.

[0016] The first channel barrier and the second channel barrier adopt a partition hole design, and the surface tension of the colloid molecules (phase guide) is used to ensure the success rate of glue pouring during glue pouring.

[0017] The endothelial culture channel is designed with a selective permeability barrier formed by endothelial cells, which ensures the independent growth of organoids and signal interaction between organoids.

[0018] The lower layer of the chip is designed with a micro-perfusion network channel to maximize the perfusion efficiency of the organoids, ensuring nutrient supply and the discharge of metabolic waste.

[0019] Furthermore, perfusion holes are provided at the ends of the organoid culture medium perfusion channel, the organoid culture chamber, and the endothelial culture channel. The organoid culture medium perfusion channel and the endothelial culture channel are perfused with organoid culture medium and endothelial culture medium, respectively, while the perfusion holes of the organoid culture chamber are perfused with Matrigel containing the organoid suspension. The design of the perfusion holes facilitates the perfusion of gel or culture medium into the chip and facilitates the connection of external pipelines in the co-culture chip microfluidic system.

[0020] Furthermore, the inner width of the partition hole is less than or equal to the outer width. Furthermore, the inner width of the partition hole is 60 μm, and the outer width of the partition hole is 100 μm.

[0021] After the upper and lower layers of the chip are bonded together, an organoid culture chamber is formed. The organoid culture chamber can be divided into multiple observation spaces, such as four organoid culture observation spaces with a length of 1200μm, a width of 800μm, and a depth of 600μm (300μm each for the upper and lower layers). In the culture chamber, seed cells can grow into organoids through self-assembly in the matrix gel.

[0022] Furthermore, a recess is provided on the lower layer of the chip at the position of the observation space.

[0023] In another aspect, the present invention provides a method for using a multi-organoid co-culture microfluidic chip, comprising the following steps:

[0024] 1) Turn the chip upside down and perfuse Matrigel mixed with organoid suspension from bottom to top through the perfusion hole of the organoid culture chamber below;

[0025] 2) After the Matrigel solidifies, place the chip upright to allow the Matrigel to solidify further;

[0026] 3) The organoid culture medium perfusion channel is perfused with organoid culture medium, and the endothelial culture channel is perfused with endothelial culture medium containing endothelial cells, and the perfusion is allowed to stand to allow the endothelial cells to adhere to the surface of the endothelial culture channel;

[0027] 4) The organoid culture medium perfusion channel and the vascular endothelial culture channel are respectively connected to external peristaltic pumps to circulate the culture medium. The organoids self-assemble in the organoid culture chamber and gradually grow into mature organoids.

[0028] Another aspect of the present invention provides a circulating perfusion system for a multi-organoid co-culture microfluidic chip. The perfusion holes at both ends of the organoid culture medium perfusion channel and the vascular endothelial culture channel are connected to a peristaltic pump and culture medium via pipes. The peristaltic pump and culture medium are also connected via pipes. Powered by the peristaltic pump, a circulating perfusion system for the organoid nutrient channel and vascular channel is established, facilitating the collection and detection of perfusate.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This multi-organoid co-culture microfluidic chip can simultaneously meet the co-culture and free combination of 2-4 different organoids at the micron scale. It can be widely used to simulate multi-organ systemic diseases involving two or more organoids (such as cancer, metabolism, inflammatory infection, etc.) and multi-organ drug effects and toxicity assessments, which are difficult to simulate in vitro experiments. It fills a gap in the industry and has broad application value.

[0031] The addition of microperfusion channels greatly increases the efficiency of organoid perfusion, improves the exchange efficiency of nutrients and metabolites, and can meet the needs of long-term organoid culture.

[0032] The addition of channel barriers ensures the specific nutrition and microenvironment of each organ, helping to maintain its specific phenotype. At the same time, each organ can use circulatory channels through the selective permeability barrier to simulate blood flow in the body, allowing signal communication and interaction between organoids, fully simulating the in vivo environment and interactions of multi-organ diseases.

[0033] This chip is adaptable to conventional microfluidic chip detection methods and real-time dynamic observation. The presence of a liquid storage tube and the circulation of culture medium facilitates the replacement of culture medium at any time and can detect and evaluate more than ten components of the circulating culture medium, including pH, nutrients, metabolic waste, compound concentrations, signaling molecules, and exosomes, greatly enriching detection methods.

[0034] This multi-organoid microfluidic organoid co-culture chip maximizes the advantages of soft lithography technology, uses high-precision silicon wafers as lithography molds, and uses PDMS as a matrix for processing. It can be mass-produced, and the single-chip production cost is low and the yield rate is high, which has great commercial potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1: Schematic diagram of the structure of a multi-organoid co-culture microfluidic chip;

[0037] Figure 2: Schematic diagram of the upper structure;

[0038] Figure 3: Schematic diagram of the lower microperfusion network channel;

[0039] Figure 4: Schematic diagram of the partition hole structure;

[0040] Figure 5: Schematic diagram of vascular endothelial cell adhesion;

[0041] Figure 6: Schematic diagram of a dual-organoid microfluidic co-culture chip;

[0042] Figure 7: Actual image of the dual-organoid microfluidic co-culture chip;

[0043] Figure 8: Schematic diagram of chip usage;

[0044] Figure 9: Schematic diagram of the chip's circulating perfusion system;

[0045] Among them: 1 is the organoid culture medium perfusion channel, 2 is the first channel barrier, 3 is the organoid culture chamber, 4 is the second channel barrier, 5 is the vascular endothelial culture channel, 6 is the microperfusion network channel, 7 is the partition hole, 8 is the observation space, and 9 is the perfusion hole. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0049] Example:

[0050] As shown in Figures 1-7, a multi-organoid co-culture microfluidic chip includes an organoid microfluidic chip monomer, which is divided into two bonded layers, the upper layer of which is sequentially provided with an organoid culture medium perfusion channel 1, a first channel barrier 2, an organoid culture chamber 3, a second channel barrier 4 and a vascular endothelial culture channel 5, and the lower layer is provided with a microperfusion network channel 6. The organoid culture medium perfusion channel 1 and the perfusion network channel 6 are connected, the first channel barrier 2 and the second channel barrier 4 are respectively provided with partition holes 7, and the vascular endothelial culture channels 5 between different organoid microfluidic chip monomers are connected.

[0051] Organoid culture chamber 3 is perfused with Matrigel containing organoid suspension.

[0052] Endothelial culture channel 5 is perfused with endothelial culture medium containing endothelial cells.

[0053] Furthermore, the number of monomers in the organoid microfluidic chip is 2, 3 or 4.

[0054] The first channel barrier 2 and the second channel barrier 4 are designed with a partition hole 7, which uses the surface tension of the colloidal molecules (phase guide) to ensure the success rate of glue injection. The width of the partition hole 7 is smaller than or equal to the width of the outer side. More specifically, the inner width of the partition hole 7 is 60μm and the outer width of the partition hole 7 is 100μm.

[0055] Endothelial culture channel 5 is designed with a selective permeability barrier formed by endothelial cells, which ensures the independent growth of organoids and the signal interaction between organoids.

[0056] The lower layer of the chip is designed with a micro-perfusion network channel 6, which maximizes the perfusion efficiency of the organoids and ensures the supply of nutrients and the discharge of metabolic waste.

[0057] Furthermore, perfusion holes 9 are provided at the ends of the organoid culture medium perfusion channel 1, the organoid culture chamber 3, and the endothelial culture channel 5. Organoid culture medium and endothelial culture medium are perfused into the organoid culture medium perfusion channel 1 and the endothelial culture channel 5, respectively, while the perfusion holes of the organoid culture chamber 3 are perfused with Matrigel containing the organoid suspension. The design of the perfusion holes 9 facilitates the perfusion of gel or culture medium into the chip and facilitates the connection of external pipelines in the co-culture chip microfluidic system.

[0058] After the upper and lower layers of the chip are bonded together, an organoid culture chamber 3 is formed. The organoid culture chamber 3 can be divided into multiple observation spaces 8, for example, four organoid culture observation spaces 8 with a length of 1200 μm, a width of 800 μm, and a depth of 600 μm (300 μm each for the upper and lower layers). In the culture chamber, seed cells can grow into organoids through self-assembly in the matrix gel.

[0059] The lower layer of the chip is provided with a recess at the position of the observation space 8 .

[0060] As shown in FIG8 , a method for using a multi-organoid co-culture microfluidic chip includes the following steps:

[0061] 1) Invert the chip and perfuse Matrigel mixed with the organoid suspension from the bottom up through the perfusion holes in the lower organoid culture chamber 3. Figure 8A shows a schematic diagram of the upright chip, where the numbers indicate specific locations and dimensions in μm. Figure 8B shows a schematic diagram of Matrigel perfusion in the inverted chip, with Matrigel being perfused into the organoid culture chamber 3.

[0062] 2) After the Matrigel solidifies, place the chip upright to allow the Matrigel to solidify further;

[0063] 3) Organoid culture medium is perfused into organoid culture channel 1, and endothelial culture medium containing endothelial cells is perfused into endothelial culture channel 5. The cells are allowed to stand for a while, allowing the endothelial cells to adhere to the surface of the endothelial culture channel. Figure 8C is a schematic diagram of the chip perfused with organoid culture medium (red shading) and endothelial culture medium (blue shading), and Figure 8D is a schematic diagram of endothelial cell adhesion to the chip (blue lines are shown outside the blue shading area). Endothelial cells adhere to the inner wall of endothelial culture channel 5 and septum 7, as shown in Figure 5, forming a selectively permeable barrier.

[0064] 4) The organoid culture medium perfusion channel 1 and the vascular endothelial culture channel 5 are respectively connected to external peristaltic pumps to circulate the culture medium. The organoids self-assemble in the organoid culture chamber 3 and gradually grow into mature organoids.

[0065] Figure 9 shows a circulating perfusion system for a multi-organoid co-culture microfluidic chip. The perfusion holes 9 at both ends of the organoid culture medium perfusion channel 3 and the vascular endothelial culture channel 5 are connected to a peristaltic pump and culture medium via tubing. The peristaltic pump and culture medium are also connected via tubing. Powered by the peristaltic pump, a circulating perfusion system for the organoid nutrient channel and vascular channel is established, facilitating the collection and detection of perfusate.

[0066] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A multi-organ co-culture microfluidic chip, characterized in that: The invention comprises an organoid microfluidic chip monomer, wherein the organoid microfluidic chip monomer is divided into an upper and a lower layer which are bonded and formed, wherein the upper layer is provided with an organoid culture medium perfusion channel, a first channel barrier, an organoid culture chamber, a second channel barrier and a vascular endothelial culture channel in sequence, and the lower layer is provided with a microperfusion network channel, the organoid culture medium perfusion channel and the microperfusion network channel are connected, the first channel barrier and the second channel barrier are respectively provided with partition holes, and the vascular endothelial culture channels between different organoid co-culture microfluidic chip monomers are connected.

2. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: The organoid culture medium perfusion channel, the first channel barrier, the organoid culture chamber, the second channel barrier and the vascular endothelial culture channel are sequentially arranged on the lower surface of the upper chip and arranged upward, and the microperfusion network channel is arranged downward on the upper surface of the lower chip.

3. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: The organoid culture chamber is perfused with matrix gel containing organoid suspension; the endothelial culture channel is perfused with endothelial culture medium containing endothelial cells.

4. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: The number of monomers in the organoid microfluidic chip is 2, 3 or 4.

5. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: The vascular endothelial culture channel is designed with a selective permeability barrier formed by vascular endothelial cells.

6. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: The inner width of the partition hole is less than or equal to the outer width.

7. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: The inner width of the partition hole is 60 μm, and the outer width of the partition hole is 100 μm.

8. The multi-organoid co-culture microfluidic chip according to claim 1, characterized in that: After the upper and lower layers of the chip are bonded together, an organoid culture chamber is formed, which can be divided into multiple observation spaces. A method for using a multi-organoid co-culture microfluidic chip, Its characteristic is that the steps are: 1) Turn the chip upside down and perfuse the matrix gel mixed with the organoid suspension from bottom to top through the perfusion hole of the organoid culture chamber below; 2) After the matrix gel solidifies, the chip is placed upright to allow the matrix gel to solidify further; 3) The organoid culture medium perfusion channel is perfused with the organoid culture medium, and the endothelial culture channel is perfused with the endothelial culture medium containing endothelial cells, and the channels are allowed to stand to allow the endothelial cells to adhere to the surface of the endothelial culture channel; 4) The organoid culture medium perfusion channel and the vascular endothelial culture channel are respectively connected to external peristaltic pumps to circulate the culture medium. The organoids self-assemble in the organoid culture chamber and gradually grow into mature organoids.

9. A circulating perfusion system for a multi-organ co-culture microfluidic chip, characterized in that: The perfusion holes at both ends of the organoid culture medium perfusion channel and the vascular endothelial culture channel are respectively connected to a peristaltic pump and the culture medium through pipelines, and the peristaltic pump and the culture medium are connected through pipelines.

10. Using a peristaltic pump as the power source, establish a circulating perfusion system for the organoid nutrition channel and vascular channel to facilitate the collection and detection of the perfusion fluid.

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