An organoid chip simulating interaction of liver fibrosis microenvironment and application thereof

Abstract

The application discloses a kind of organ chip manufacturing and application of simulating liver fibrosis microenvironment interaction, belong to biomedical engineering technical field.Technical points: the chip includes chip main body, be provided with culture chamber of being divided into upstream chamber and downstream chamber on chip main body, culture chamber upper part is inverted conical column shape, lower part is cylindrical, chip main body is also provided with liquid flow hole, liquid flow channel and check valve, liquid flow channel is connected upstream and downstream chamber, check valve controls liquid flow one-way flow;Chip also includes chip chamber with outer edge and membrane with hole, can place organoid or acellular matrix skeleton.The application places different organoids or acellular matrix skeleton by upstream and downstream chamber, utilizes one-way liquid flow to simulate the action sequence of different components in liver fibrosis process, solves the problem that existing model simulation reality is poor, cannot accurately study cell interaction, long-term culture is difficult, sample separation is difficult, etc., can be used for liver fibrosis mechanism research, drug screening and disease model construction, with simulation real, strong universality, operation is convenient and the like advantages.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to an organoid chip and its applications. Background Technology

[0002] Liver fibrosis is a key intermediate pathological process in the progression of various chronic liver diseases to cirrhosis and liver cancer. Its core mechanism lies in the excessive deposition of extracellular matrix (mainly type I and III collagen) in liver tissue, involving the interaction of various cells such as hepatocytes, hepatic stellate cells, fibroblasts, and macrophages, as well as the dynamic regulation between cells and the microenvironment. Therefore, constructing an in vitro model that can accurately simulate the liver fibrosis microenvironment and cell interaction process is of great significance for the study of the mechanism of liver fibrosis, drug development, and clinical translation.

[0003] Currently, in vitro research on liver fibrosis mainly relies on cell models and animal models. However, both of these models have significant limitations and cannot meet the needs of precise research. Specifically, traditional cell models often employ two-dimensional culture methods using single or mixed cells, which cannot replicate the complex cellular composition and three-dimensional microenvironment structure during liver fibrosis. Furthermore, liver fibrosis is a chronic pathological process, and existing cell models struggle to achieve long-term stable cell culture, failing to simulate the dynamic progression of fibrosis. This results in significant discrepancies between research results and the actual in vivo pathological state, making it difficult to accurately reflect cell-cell interaction mechanisms. While animal models (such as mouse and rat liver fibrosis models) can simulate the pathological process of liver fibrosis to some extent, their in vivo microenvironment is extremely complex, containing various cells, cytokines, metabolites, and tissue structures. This makes it impossible to accurately study single cell types (such as tumor cells, hepatic stellate cells, and fibroblasts) or specific cell-cell interactions. Moreover, animal models suffer from long rearing periods, high costs, and significant species differences, resulting in low clinical translation efficiency of research results.

[0004] Organoids, as a three-dimensional cell culture system, have advantages such as rich cell composition, high structural similarity to in vivo tissues, and the ability to maintain cell characteristics and long-term culture through directed differentiation or the addition of specific factors. They have been increasingly applied to research related to liver fibrosis. However, existing organoid microarrays mostly focus on the simple culture of organoids and are not designed to address the core characteristics of the liver fibrosis microenvironment. Even though some microarrays use Matricene as a support material, they cannot reproduce the real liver fibrosis microenvironment. The main component of Matricene is laminin, while the core components of the extracellular matrix in liver fibrosis are type I and III collagen. The significant differences in their compositions mean that existing organoid microarrays cannot simulate the interaction between cells and specific matrix during fibrosis, nor can they reproduce the sequence in which different cells and factors exert their effects during the fibrosis process, making it difficult to meet the needs of precise research on liver fibrosis.

[0005] The literature DOI: 10.1016 / j.actbio.2022.11.038 reports a method for preparing acellular matrix scaffolds and organoid implantation to study the interaction between tumor organoids and the extracellular matrix. This technique can well preserve the extracellular matrix characteristics of the original tissue. However, its problem is that the organoid components are directly implanted into the acellular matrix scaffold. After a period of interaction, it is difficult for the two to be completely separated for subsequent identification and detection, which greatly reduces the accuracy and reproducibility of the model. In addition, because all cells and DNA components are removed during the preparation process, it is impossible to restore the pathological characteristics of the high neutrophil extracellular trap network (NETs) in the clinical liver fibrosis microenvironment. NETs are key regulators of liver fibrosis progression and hepatocellular carcinoma invasion and metastasis, resulting in a significant deviation between the model and the real clinical pathological environment. Summary of the Invention

[0006] The technical problem to be solved by this invention is:

[0007] To address the shortcomings of existing technologies, the inventors propose an organoid-on-a-chip fabrication and application that simulates the interactions within the liver fibrosis microenvironment. By designing upstream and downstream dual culture chambers, and placing chambers containing different organoids or (pre-embedded NETs) decellularized matrix skeletons within each chamber, and using one-way valves in the connecting channels to control the flow direction, the sequence in which different cells and matrix components exert their effects during liver fibrosis can be precisely simulated. This solves the problems of existing models being unable to achieve precise interaction studies, difficulties in long-term culture, and difficulties in sample separation, providing an efficient and precise in vitro research tool for studying the mechanisms of liver fibrosis, drug screening, and disease model construction.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention discloses an organoid chip that simulates the interaction of the liver fibrosis microenvironment. Its core structure includes a chip body 1, on which a culture chamber 2 is provided. The culture chamber 2 is divided into two independent but interconnected chambers, an upstream chamber 21 and a downstream chamber 22, for culturing different types of organoids or placing decellularized matrix scaffolds.

[0010] The upper part of the culture chamber 2 is shaped like an inverted frustum, with an upper opening 31 and a lower opening 32. The diameter of the upper opening 31 is slightly larger than the diameter of the lower opening 32. A smooth inclined sidewall 4 is formed between the upper opening 31 and the lower opening 32. The design of the inclined sidewall 4 facilitates the smooth flow of culture medium into the chamber and avoids culture medium residue at the top of the chamber.

[0011] The lower part of the culture chamber 2 is a cylindrical structure. The inner diameter of the cylindrical structure is the same as the diameter of the lower opening 32, forming a stable culture space to accommodate the liquid flow area of ​​the chip chamber 12 and ensure stable liquid flow.

[0012] The chip body 1 is also provided with a liquid flow hole 5, which is divided into an inlet hole 51 and an outlet hole 52. The inlet hole 51 is used to connect fresh culture medium, and the outlet hole 52 is used to discharge culture waste liquid.

[0013] Corresponding to the liquid flow orifice 5, the chip body 1 is also provided with a liquid flow channel 6, which is divided into three parts: an inflow channel 61, an outflow channel 62, and a connecting channel 63. The inflow orifice 51 is connected to the inclined sidewall 4 of the upstream chamber 21 through the inflow channel 61, and the inflow channel 61 has an opening on the inclined sidewall 4 to ensure that fresh culture medium can flow smoothly into the upstream chamber 21 along the inclined sidewall 4. The outflow orifice 52 is connected to the inclined sidewall 4 of the downstream chamber 22 through the outflow channel 62, and the outflow channel 62 has an opening on the inclined sidewall 4 to facilitate the smooth discharge of waste liquid in the downstream chamber 22. The two ends of the connecting channel 63 are respectively opened on the sidewall of the lower part of the upstream chamber 21 and the downstream chamber 22 to connect the lower liquid flow areas of the two chambers and realize the conduction of liquid flow from the upstream chamber to the downstream chamber.

[0014] The connecting channel 63 is equipped with a one-way valve 7. The core function of this one-way valve 7 is to control the direction of fluid flow, ensuring that the fluid can only flow from the upstream chamber 21 to the downstream chamber 22, preventing backflow, thereby accurately simulating the sequence in which different components exert their effects during liver fibrosis and ensuring the accuracy of cell interaction studies. The one-way valve 7 can be any existing design, including passive one-way valves (which do not require external energy and rely on the fluid's own pressure or geometric / material properties to achieve unidirectional flow) and active one-way valves (which require external drive for precise control), etc. This invention is not limited to these types.

[0015] The chip body 1 is also provided with a chamber sealing connection part 8, which surrounds the upper opening 31 and protrudes upward from the surface of the chip body 1. A detachable sealing cap assembly 9 is detachably connected to the chamber sealing connection part 8. The detachable connection method includes threaded connection, snap-fit ​​connection, screw-on connection, flange connection, etc., and is not limited to any particular method. After the detachable sealing cap assembly 9 is closed, the culture chamber 2 can be sealed, preventing external contamination during the culture process and maintaining a stable culture environment within the chamber.

[0016] The organoid chip also includes a matching chip chamber 12. The chip chamber 12 has an outer edge 121 at the top and a perforated membrane 122 at the bottom. When the chip chamber 12 is placed inside the culture chamber 2, the outer edge 121 can rest stably on the edge of the lower opening 32, so that the bottom of the chip chamber 12 does not contact the bottom of the sub-chamber. The perforated membrane 122 divides the sub-chamber into the chip chamber cavity 14 and the working fluid buffer area 13 of the simulated liver fibrosis microenvironment located in the lower part of the culture chamber 2. This can ensure the exchange of substances between the organoids or decellularized matrix skeleton in the chip chamber and the lower fluid flow area, and can also prevent the organoids or scaffolds from falling into the lower part of the chamber, thus ensuring the stability of the culture process. The inner cavity of the chip chamber 12 is used to carry the target culture, which can be flexibly selected according to research needs, including but not limited to liver organoids, normal liver decellularized scaffolds, and fibrotic liver decellularized scaffolds; wherein, the fibrotic liver decellularized scaffold is preferably a fibrotic liver decellularized scaffold with pre-embedded NETs, ​​which can accurately reproduce the fibrotic pathological microenvironment with high NETs in clinical liver diseases and improve the in vivo similarity of the model.

[0017] The present invention has the following beneficial technical effects:

[0018] This invention provides an organ-on-a-chip that simulates the interactions of the liver fibrosis microenvironment. Its structure is scientifically designed and easy to use. Compared to existing technologies, it offers the following significant advantages and effectively addresses the technical shortcomings of existing models:

[0019] 1. Easier and more accurate sample acquisition: This invention, through the design of detachable culture chambers and one-way valves, effectively separates the upstream culture, upstream culture medium, downstream culture, and downstream culture medium. When interaction studies are needed, the fluid flow can connect the upstream and downstream cultures in series; when sample testing is required, the fluid flow can be stopped, and the upstream culture, upstream culture medium, downstream culture, and downstream culture medium can all be individually removed for subsequent testing, including microscopic observation, fluorescence staining, ELISA, and sequencing. This results in more accurate results and significantly improves model reproducibility and reliability.

[0020] 2. Precise simulation of interactions between different cell populations during liver fibrosis: When it is necessary to study the interactions between different cell populations in the liver during fibrosis, chip chambers 12 containing organoids of different cell populations can be placed in the upstream chamber 21 and the downstream chamber 22, respectively. Fresh culture medium flows from the upstream chamber to the downstream chamber through a one-way liquid path (controlled by a one-way valve 7). Researchers can accurately determine the crosstalk effect of upstream organoids on downstream organoids by detecting changes in the composition of cytokines, metabolites, etc. in the incoming fresh culture medium, the liquid flow in the lower part of the upstream chamber, the liquid flow in the lower part of the downstream chamber, and the outflow waste liquid, combined with the detection results of organoid morphology, activity, gene expression, etc., thus restoring the dynamic interaction mechanism between cells during liver fibrosis and solving the problem that existing models cannot accurately study cell interactions.

[0021] 3. Enables precise research on the direct impact of specific cell populations on liver fibrosis: When it is necessary to study the direct impact of specific cell populations (such as tumor cells and macrophages) on liver fibrosis, the organoids in the chip chamber 12 of the downstream chamber 22 can be replaced with normal liver decellularized matrix scaffold. At the same time, hepatic stellate cells, primary fibroblasts, macrophages, and other cell populations involved in fibrosis regulation can be seeded on this decellularized matrix scaffold. Through the translocation of metabolites from the organoids in the upstream chamber 21, the activation, proliferation, and matrix secretion of cells on the downstream decellularized matrix scaffold can be observed, clarifying the regulatory role of specific cell populations on liver fibrosis and providing precise experimental evidence for the study of fibrosis mechanisms.

[0022] 4. Enables research on the direct impact of the fibrotic microenvironment on specific cell populations: When it is necessary to study the direct impact of the liver fibrotic microenvironment on specific cell populations (such as tumor cells and hepatocytes), the organoids in the upstream chamber 21 can be replaced with the decellularized matrix scaffold of the fibrotic liver. At the same time, microenvironmental factors commonly found in the fibrosis process, such as extracellular traps for neutrophils, can be added to the decellularized matrix scaffold. The components related to the fibrotic microenvironment can be transported to the organoids of specific cell populations in the downstream chamber 22 through fluid flow. The morphological, activity, and functional changes of the organoids can be observed to clarify the regulatory mechanism of the fibrotic microenvironment on cell fate.

[0023] 5. Support for long-term stable culture of organoids and related cells: The sealed design of the chip (the detachable sealing cap assembly 9 docks with the chamber sealing connection part 8) can maintain the stability of the culture environment. At the same time, organoids can maintain their cell characteristics by relying on the directed differentiation culture system in the chip chamber 12 or by adding specific factors. Combined with a stable unidirectional fluid supply, long-term stable culture of organoids and cells involved in fibrosis regulation can be achieved, perfectly matching the chronic pathological process of liver fibrosis and solving the defect that existing cell models cannot be cultured for a long time.

[0024] 6. Precisely recreates the real clinical pathological microenvironment: This invention can be used with a pre-embedded NETs-rich decellularized liver matrix scaffold, which can not only recreate the type I and III collagen matrix environment of the core of liver fibrosis, but also simulate the pathological characteristics of high NETs in clinical liver diseases. It fully recreates the in vivo processes in which NETs participate in the progression of liver fibrosis, regulate cell interactions, and affect tumor cell behavior. Compared with existing technologies, the realism and accuracy of the microenvironment simulation are greatly improved, and the clinical translational value of the research results is significantly enhanced. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of an organoid-on-a-chip that simulates the interaction of the liver fibrosis microenvironment.

[0026] Figure 1 In the middle: 1 chip body, 12 chip chamber, 2 culture chamber, 31 upper layer port, 32 lower layer port, 4 sloping sidewall, 51 inlet hole, 52 outlet hole, 6 liquid flow channel, 7 one-way valve;

[0027] Figure 2 A schematic diagram of the key internal structure of an organoid-on-a-chip that simulates the interaction of the liver fibrosis microenvironment;

[0028] Figure 2 In the middle: 12 chip chambers, 121 outer edge, 122 perforated membrane, 21 upstream chamber, 22 downstream chamber, 31 upper layer port, 32 lower layer port, 4 oblique sidewalls, 51 inlet port, 52 outlet port, 61 inlet channel, 62 outlet channel, 63 connecting channel, 7 one-way valve;

[0029] Figure 3 This is a schematic diagram of the fluid flow path and chamber placement of an organoid on-chip that simulates the interaction of the liver fibrosis microenvironment.

[0030] Figure 3 In the middle: 121 outer edge, 122 perforated membrane, 13 working fluid buffer area, 14 chip chamber, 8 sealing connection, 9 sealing cap assembly. Detailed Implementation

[0031] The following combination Figure 1-3 Specific embodiments are provided to illustrate the manufacturing and application of the organoid chip of the present invention in detail. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. It should be noted that cell culture conditions, reagent concentrations, etc., not explicitly stated in the following embodiments are conventional experimental conditions and can be adjusted according to actual needs; specific parameters not explicitly stated (such as cell seeding density) can be determined by those skilled in the art using conventional technical means.

[0032] Example 1: Study on the Interrelationships Between Organoids

[0033] This embodiment is used to study the crosstalk between two different cell populations of organoids during liver fibrosis. The specific application method is as follows:

[0034] 1. Chip preparation: According to the chip structure described above, an organoid chip simulating the interaction of the liver fibrosis microenvironment is prepared. The integrity of the chip body 1, culture chamber 2, fluid channel 6 and one-way valve 7 is checked to ensure that the fluid channel is unobstructed and the one-way valve 7 can work normally (only allowing fluid to flow from the upstream chamber 21 to the downstream chamber 22). The perforated membrane 122 of the chip chamber 12 is undamaged and has a uniform pore size (preferably 0.4-1.0 μm, which allows nutrients and metabolites to pass freely and avoids organoid cell shedding).

[0035] 2. Organoid preparation: Culture two organoids related to liver fibrosis (organoid A and organoid B, for example, organoid A is a hepatocyte organoid and organoid B is a hepatic stellate cell organoid) separately, and culture them to the logarithmic growth phase to ensure that the organoids are morphologically intact and have good activity (viability ≥90%).

[0036] 3. Chip Implantation: Place the chip chamber 12 containing organoid A stably in the upstream chamber 21, ensuring that the outer edge 121 of the chip chamber 12 is tightly against the lower opening 32, and that the perforated membrane 122 is well separated from the lower space of the upstream chamber 21. Place the chip chamber 12 containing organoid B stably in the downstream chamber 22, in the same manner as the upstream chamber. After implantation, align the removable sealing cap assembly 9 with the chamber sealing connection part 8 to seal the culture chamber 2.

[0037] 4. Fluid flow control: Connect the micro-liquid pump to the inlet 51 using a sterile thin tubing, and connect the outlet 52 to the waste collection bottle using another sterile thin tubing; adjust the flow rate of the micro-liquid pump to control the flow rate range of 5-20 μL / min (this speed range ensures that the fresh culture medium can fully nourish the organoids, while avoiding mechanical damage to the organoids caused by excessively fast flow rate, and accumulation of metabolites caused by excessively slow flow rate), and supply fresh culture medium by continuous pumping.

[0038] 5. Cultivation and Detection: Fresh culture medium flows through inlet 51, then through inlet channel 61, and then through the opening on the inclined sidewall 4 into the upper part of upstream chamber 21. It then flows smoothly into the chip chamber 12 containing organoid A, where nutrients are absorbed and utilized by organoid A. At the same time, the metabolites produced by organoid A pass through the perforated membrane 122 with the liquid flow and enter the lower part of upstream chamber 21. Under the control of one-way valve 7, the liquid carrying organoid A metabolites flows through connector 63 into the lower part of downstream chamber 22, and then through the perforated membrane 122 into chip chamber 12 containing organoid B. Nutrients and organoid A metabolites are absorbed and utilized by organoid B, while the metabolites produced by organoid B enter the upper part of downstream chamber 22. Finally, the waste liquid carrying organoid B metabolites flows through the opening on the inclined sidewall 4 through outlet channel 62, and then through outlet 52 into the waste liquid collection bottle via a flexible tube.

[0039] 6. Sample Collection and Analysis: Fresh culture medium flowing in, fluid flow from the lower part of the upstream chamber 21, fluid flow from the lower part of the downstream chamber 22, and waste fluid flowing out were collected periodically to detect changes in the levels of cytokines related to liver fibrosis (such as TGF-β1, COL1A1, α-SMA, etc.). Simultaneously, the chip chamber 12 was periodically removed to observe the morphological changes of organoid A and organoid B. The expression levels of fibrosis-related genes in the organoids were detected by immunofluorescence, real-time quantitative PCR, and other methods to analyze the crosstalk between organoid A and organoid B and clarify the mutual influence mechanism between the two cell populations in the process of liver fibrosis.

[0040] Example 2: Study on the impact of tumor organoids on the fibrotic microenvironment

[0041] This embodiment is used to study the direct impact of liver tumor organoids on the liver fibrotic microenvironment and to clarify the regulatory role of tumor cells in the process of liver fibrosis. The specific application method is as follows:

[0042] 1. Chip preparation: The chip preparation method is consistent with that in the first embodiment, ensuring that all chip components are intact, well sealed, and have smooth fluid flow, and that the perforated membrane of the chip chamber 12 is undamaged.

[0043] 2. Sample preparation: (1) Culture liver tumor organoids (such as hepatocellular carcinoma organoids) to the logarithmic growth phase to ensure that the organoids are morphologically intact and have good activity (viability ≥90%); (2) Prepare normal liver decellularized matrix scaffold (the preparation method is based on the literature DOI: 10.1016 / j.actbio.2022.11.038, rich in type I and III collagen, adapted to the simulation of liver fibrosis microenvironment); (3) Seed hepatic stellate cells and fibroblasts (both types of cells are core cells involved in the regulation of liver fibrosis) on normal liver decellularized matrix scaffold, with a seeding density of 1×10^5-5×10^5 cells / mL, and culture for 24h to allow the cells to adhere to the scaffold surface.

[0044] 3. Chip implantation: The chip chamber 12 containing liver tumor organoids is placed in the upstream chamber 21 in the same manner as in the first embodiment; the normal liver decellularized matrix skeleton seeded with hepatic stellate cells and fibroblasts is placed in the chip chamber 12, and then the chip chamber 12 is placed in the downstream chamber 22 to ensure that the chip chamber is firmly fixed and well sealed.

[0045] 4. Fluid flow control: Consistent with the first embodiment, a micro-liquid pump, inlet 51 and outlet 52, and waste liquid collection bottle are connected with a sterile thin tubing. The fluid flow rate is adjusted to 5-20 μL / min to continuously pump in fresh culture medium, ensuring that the fluid flows unidirectionally from the upstream chamber to the downstream chamber.

[0046] 5. Culture and detection: Fresh culture medium enters the upstream chamber 21 through inlet channel 61 to nourish liver tumor organoids. Metabolic products and secreted cytokines produced by the tumor organoids pass through the perforated membrane into the lower part of the upstream chamber, and then flow into the lower part of the downstream chamber through connector channel 63 (controlled by a one-way valve), and then penetrate into the normal liver decellularized matrix skeleton, acting on the hepatic stellate cells and fibroblasts on the scaffold.

[0047] 6. Sample Collection and Analysis: Fluid samples were collected periodically at each stage to detect changes in the levels of fibrosis-related cytokines (TGF-β1, α-SMA, COL3A1, etc.). The decellularized matrix scaffold of the downstream chamber was periodically removed, and the activation level of hepatic stellate cells, the proliferation capacity of fibroblasts, and the amount of collagen secretion on the scaffold were detected by immunohistochemistry and Western blot. The morphological and activity changes of upstream tumor organoids were observed, and the regulatory effects of factors secreted by tumor organoids on hepatic stellate cells, fibroblasts, and the fibrotic microenvironment were analyzed to clarify the mechanism of action of tumor organoids in the process of liver fibrosis.

[0048] Example 3: Study on the impact of fibrotic microenvironment on tumor organoids

[0049] This embodiment is used to study the direct impact of the liver fibrosis microenvironment on the morphology, activity, and invasiveness of liver tumor organoids, providing experimental evidence for the synergistic study of liver cancer and liver fibrosis. The specific application method is as follows:

[0050] 1. Chip preparation: Consistent with the first and second embodiments, ensure that all components of the chip are normal and well-sealed, the fluid flow channels are unobstructed, and the one-way valve is working properly.

[0051] 2. Sample preparation: (1) Prepare a decellularized scaffold for fibrotic liver, preferably a decellularized scaffold for fibrotic liver with pre-embedded extracellular neutrophil traps (NETs) (the preparation method is described in the applicant's prior invention patent application (application number: 2026102610813, application date: March 5, 2026), which will not be described again in this application). The scaffold is pre-embedded with extracellular neutrophil traps at a concentration of 10-50 μg / mL in the collagen matrix, which can accurately simulate the liver fibrosis microenvironment with high NETs in vivo; (2) Culture liver tumor organoids to the logarithmic growth phase to ensure that the organoids are morphologically intact and have good activity (viability ≥90%).

[0052] 3. Chip implantation: The chip chamber 12 containing the decellularized matrix framework of fibrotic liver with added extracellular trapping net of neutrophils is placed in the upstream chamber 21, and the placement method is the same as in the previous embodiment; the chip chamber 12 containing liver tumor organoids is placed in the downstream chamber 22, ensuring that the chip chamber is firmly fixed and well sealed.

[0053] 4. Fluid flow control: As in the previous embodiment, connect a micro-liquid pump, inlet port, outlet port and waste liquid collection bottle, adjust the fluid flow rate to 5-20 μL / min, continuously pump in fresh culture medium, maintain unidirectional fluid flow, and ensure that the components related to the fibrotic microenvironment can be translocated to downstream tumor organoids.

[0054] 5. Culture and detection: Fresh culture medium enters the upstream chamber 21 through the inflow channel 61, permeates into the decellularized matrix skeleton of the fibrotic liver, carries the collagen components, extracellular traps of neutrophils and fibrosis-related factors secreted by the scaffold itself, passes through the perforated membrane into the lower part of the upstream chamber, and then flows into the lower part of the downstream chamber through the connecting channel 63 (controlled by a one-way valve), and then permeates into the chip chamber 12 containing tumor organoids, acting on the tumor organoids.

[0055] 6. Sample Collection and Analysis: Fluid samples were collected periodically at each stage to detect changes in the levels of fibrosis-related factors and tumor-related factors (such as VEGF, MMP-9, AFP, etc.). Tumor organoids from downstream chambers were periodically removed to observe changes in their morphology and size. The activity and proliferation capacity of the organoids were detected using the CCK-8 assay, and the invasive capacity of the tumor organoids was detected using the Transwell assay (adapted chip chamber). The expression levels of invasion and metastasis-related genes in tumor organoids were detected using real-time quantitative PCR, Western blot, and other methods to analyze the effects of the fibrotic microenvironment (collagen, NETs, ​​etc.) on the growth, invasion, and metastasis of tumor organoids, and to clarify the synergistic relationship between liver fibrosis and liver cancer progression.

[0056] The inventors could not exhaustively describe all possible implementation methods and embodiments. Although the present invention has been disclosed above, the scope of protection disclosed herein is not limited thereto. Various changes to the chip structure, modifications and alterations to the process parameter ranges during application, made by those skilled in the art without departing from the technical concept disclosed herein, will all fall within the scope of protection of this invention.

Claims

1. An organ-on-a-chip that simulates the interactions of the liver fibrosis microenvironment, characterized in that, The main structure includes a chip body (1), on which a culture chamber (2) is provided. The culture chamber (2) includes multiple sub-chambers arranged upstream and downstream. The chip body (1) also includes a liquid flow channel (6), an inlet hole (51), and an outlet hole (52). The liquid flow channel (6) includes an inlet channel (61), an outlet channel (62), and a connecting channel (63). The inlet hole (51) is connected to the upstream sub-chamber through the inlet channel (61), and the outlet hole (52) is connected to the downstream sub-chamber through the outlet channel (62). The connecting channel (63) is arranged in series between the multiple sub-chambers upstream and downstream. The organoid chip also includes multiple chip chambers (12) for holding different cell groups of liver, organoids, and / or extracellular matrix skeletons. Each sub-chamber has a corresponding chip chamber (12).

2. The organ-on-a-chip according to claim 1, characterized in that, Each chip chamber (12) has an outer edge (121) at the top and a perforated membrane (122) at the bottom. The perforated membrane (122) divides the corresponding sub-chamber into a chip chamber cavity (14) and a working fluid buffer zone (13) of a simulated liver fibrosis microenvironment located at the bottom of the culture chamber (2). The pore size of the perforated membrane (122) is 0.4-1.0 μm.

3. The organ-like chip according to claim 2, characterized in that, The upper part of the culture chamber (2) is an inverted frustum shape, with an upper opening (31) and a lower opening (32). The diameter of the upper opening (31) is larger than the diameter of the lower opening (32). A smooth inclined sidewall (4) is formed between the upper opening (31) and the lower opening (32). When the chip chamber (12) is placed inside the culture chamber (2), the outer edge (121) can rest smoothly on the edge of the lower opening (32), so that the bottom of the chip chamber (12) does not contact the bottom of the sub-chamber.

4. The organ-like chip according to claim 3, characterized in that, The inflow channel (61) and outflow channel (62) open into the inclined sidewall (4); the connecting channel (63) opens into the sidewall of the lower part of the culture chamber (2).

5. The organ-on-a-chip according to any one of claims 1 to 4, characterized in that, The multiple sub-chambers arranged along the upstream and downstream sides consist of two chambers.

6. The organ-on-a-chip according to any one of claims 1 to 4, characterized in that, A one-way valve (7) is provided inside the connecting channel (63).

7. The organ-on-a-chip according to any one of claims 1 to 4, characterized in that, The chip body (1) is provided with a chamber sealing connection part (8) protruding from the upper edge of the top opening of each sub-chamber, and a sealing cover assembly (9) is detachably installed at the chamber sealing connection part (8).

8. The organ-on-a-chip according to any one of claims 1 to 4, characterized in that, The inner diameter of the liquid flow channel (6) is 0.1-0.5 mm to ensure stable liquid flow and prevent eddy currents.

9. The organ-on-a-chip according to any one of claims 1 to 4, characterized in that, The target culture contained in the chip chamber (12) is any one or more combinations of different liver cell populations, organoids, and extracellular matrix skeleton. The extracellular matrix framework is a liver extracellular matrix framework with pre-embedded NETs.

10. An application of an organoid microarray as described in any one of claims 1 to 8 in studying the interaction relationships between different cell populations of organoids during liver fibrosis, characterized in that, Includes the following steps: Chip chambers (12) containing different organoids were placed in the upstream chamber (21) and downstream chamber (22), respectively. Fresh culture medium was continuously pumped in through the inlet hole (51) by a micro-liquid pump. The liquid flowed into the sub-chamber of the upstream chamber (21) through the inlet channel (61), and then flowed naturally into the chip chamber cavity (14). It flowed into the working fluid buffer zone (13) through the perforated membrane (122), and then into the working fluid buffer zone (13) of the downstream chamber (22) through the connecting channel (63). It flowed into the downstream chip chamber cavity (14) through the perforated membrane (122), and then flowed out through the outlet hole (52) through the outlet channel (62). The one-way valve (7) controlled the liquid flow to flow only from the upstream chamber (21) to the downstream chamber (22). The liquid composition and the morphology, activity and gene expression changes of the organoids at each stage were detected, and the crosstalk between organoids was analyzed.

11. An application of an organoid microarray as described in any one of claims 1 to 8 in studying the effects of tumor organoids on the liver fibrotic microenvironment, characterized in that, Includes the following steps: The chip chamber (12) containing tumor organoids was placed in the upstream chamber (21), and the normal liver decellularized matrix skeleton with hepatic stellate cells and fibroblasts was placed in the chip chamber (12) and placed in the downstream chamber (22). The metabolites of tumor organoids were transferred through unidirectional fluid flow, and the changes in fibrosis-related factors and cells on the scaffold were detected to analyze the regulatory effect of tumor organoids on the fibrotic microenvironment.

12. An application of an organoid microarray as described in any one of claims 1 to 8 in studying the effects of the liver fibrosis microenvironment on tumor organoids, characterized in that, Includes the following steps: A chip chamber (12) containing a decellularized matrix skeleton of fibrotic liver with added NETs was placed in the upstream chamber (21), and a chip chamber (12) containing tumor organoids was placed in the downstream chamber (22). The fibrotic microenvironment-related components were transferred by unidirectional fluid flow, and the morphology, activity, invasiveness and related gene expression changes of tumor organoids were detected. The regulatory effect of the fibrotic microenvironment on tumor organoids was analyzed.

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