Vascularized multi-tissue interaction organ chip and construction method and application thereof

Abstract

The invention belongs to the technical field of organ chips and biological manufacturing, and relates to a vascularized multi-tissue interaction organ chip based on a phase change sacrificial structure and a construction method and application thereof. According to the invention, collaborative construction of multiple tissue chambers and a blood vessel-parenchymal tissue biological interface is realized through a continuous phase change process of the sacrificial hydrogel material; according to the organ chip disclosed by the invention, an extra diaphragm or complex processing is not needed, the continuity and the bionic property of a multi-tissue interaction interface are maintained, the simulation of signal exchange and a pathological process among multiple tissues in a body is facilitated, and the organ chip has higher bionic property and applicability in applications such as complex physiological interaction modeling, drug evaluation and disease mechanism research.

Description

Technical Field

[0001] This invention relates to the field of organ-on-a-chip and biomanufacturing technology, and more specifically, to an organ-on-a-chip based on a phase-change sacrificial structure with vascularized multi-tissue interaction, its construction method, and its application. Background Technology

[0002] Developing novel drugs with enhanced safety and efficacy remains a key task for modern medicine and the pharmaceutical industry. However, current drug development often faces challenges such as high costs, long cycles, and high failure rates, necessitating the establishment of more efficient and predictive in vitro evaluation systems to assist in drug screening and mechanism research.

[0003] Animal experiments have long been considered an important validation method for drugs before they enter clinical trials. However, due to species differences, animal models often fail to accurately reflect the true human response to drugs, and also suffer from high experimental costs and significant ethical controversies. In contrast, two-dimensional or three-dimensional cell culture systems offer advantages such as controllable sourcing and ease of mass production, which can improve the human relevance of research results to some extent. However, these models struggle to accurately reproduce the spatial structure of in vivo tissues, the interfaces between multiple tissues, and mechanical stimuli in the microenvironment, thus significantly limiting their accuracy in predicting drug effects.

[0004] In recent years, the rapidly developing organ-on-a-chip technology has provided a new path for constructing in vitro models that are more physiologically relevant to humans. By precisely controlling the spatial arrangement of cells and microenvironmental factors in a microfluidic system, organ-on-a-chip can achieve accurate simulation of tissue structure and physiological function, combining physiological relevance with experimental controllability. It can more accurately predict the efficacy and toxicity of drugs in vivo, thus showing broad application prospects in disease mechanism research, drug screening, and personalized medicine.

[0005] As this field continues to develop, researchers are increasingly recognizing that single-tissue models often fail to fully reproduce the complex physiological responses within the body. Many physiological processes, such as inflammatory responses, cross-barrier transport, tumor metastasis, and metabolic regulation, depend on the exchange of substances and signal interactions between different tissues. Therefore, constructing organ-on-a-chip systems that can simulate multiple tissue interfaces and achieve direct cell-to-cell interactions has become an important direction for development. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an organ-on-a-chip based on a phase change sacrificial structure for vascularized multi-tissue interaction, its construction method and application. By utilizing the solid-liquid reversible phase change behavior of sacrificial hydrogel materials, temporary flow channel occupancy, multi-tissue chambers and vascular-parenchymal biological interfaces are synergistically constructed within the chip, overcoming the problems of insufficient interface continuity, limited tissue configuration and difficulty in establishing a stable perfusion system in existing multi-tissue organ-on-a-chip systems.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a vascularized multi-tissue interaction organ-on-a-chip, comprising a chip substrate, an internal topological structure, and an inlet and outlet connected to the topological structure on the surface of the chip substrate. The topological structure includes a pre-defined flow channel region and a three-dimensional tissue-like chamber region enclosed by the pre-defined flow channel region. The topological structure is used to form temporary flow channel occupants for cells on the pre-defined flow channel region after a sacrificial hydrogel solution carrying vascular-associated cells is introduced into the chip substrate, through a liquid-solid phase transition of the sacrificial hydrogel solution. The structure includes a three-dimensional tissue-like chamber disposed in the region of the three-dimensional tissue-like chamber; the three-dimensional tissue-like chamber is used for perfusing a hydrogel solution carrying parenchymal tissue cells; the temporary flow channel occupancy structure carrying cells is used to allow vascular-associated cells to migrate and attach to the surface of the three-dimensional tissue-like structure through the solid-liquid phase transition of the sacrificial hydrogel solution after the hydrogel solution carrying parenchymal tissue cells has solidified to form a three-dimensional tissue-like structure, thereby forming a vascular-parenchymal tissue biological interface; and after the sacrificial hydrogel solution is removed from the outlet, it is transformed into a perfuse microfluidic channel; the perfuse microfluidic channel is used for delivering nutrients and removing metabolites.

[0008] It is important to understand that the term "solid tissue cells" here includes both parenchymal cells and / or interstitial cells. This is because, considering three-dimensional tissues, i.e., solid tissues, they are actually composed of both parenchymal and interstitial cells. For example, neural tissue can contain parenchymal cells such as neurons and interstitial cells such as glial cells.

[0009] It should also be understood that the directional flow of the sacrificial hydrogel is achieved by the topological constraints and capillary synergy within the chip substrate, allowing the hydrogel solution to flow preferentially along the preset flow channel region.

[0010] Furthermore, the three-dimensional tissue-like chamber region is composed of one or more sub-regions; the three-dimensional tissue-like chamber correspondingly includes one or more sub-chambers for perfusing hydrogel solutions carrying the same or different types of solid tissue cells, which solidify to form one or more three-dimensional tissue-like structures; the perfusing microfluidic channel is also used to enable each of the three-dimensional tissue-like structures to exchange biochemical signals while being physically independent of each other, forming a multi-tissue interaction biological interface.

[0011] It should be noted that even with only one three-dimensional tissue, there is still an interaction interface between the vascular layer and the parenchyma layer. Therefore, it can be considered as an interaction model between vascular tissue and other parenchyma tissues, which meets the definition of multi-tissue interaction organ-on-a-chip in the title of this invention.

[0012] Furthermore, the three-dimensional tissue includes nerve cells, hepatocytes, cardiomyocytes, renal tubular cells, or any combination thereof.

[0013] As can be seen from the above, the three-dimensional tissue-like chamber, when including one or more sub-chambers, can be used to perfuse hydrogel solutions carrying the same or different types of solid tissue cells, and respectively contain one or more three-dimensional tissues formed after solidification, that is, the same or different types of solid tissue cells, including but not limited to nerve cells, hepatocytes, cardiomyocytes, renal tubular cells or any combination thereof, to construct a multi-tissue interaction model.

[0014] Furthermore, the sacrificial hydrogel solution is made from a sacrificial hydrogel material; the sacrificial hydrogel material possesses both liquid-solid phase transition properties and solid-liquid phase transition properties (i.e., the sacrificial hydrogel material has reversible solid-liquid phase transition properties), and is used to transform from a liquid state to a solid state to form a vacancy structure under one triggering condition, and to transform from a solid state back to a liquid state under another triggering condition to achieve sacrificial removal; the sacrificial hydrogel material includes one or more of Pluronic F127, gelatin, sodium alginate, hyaluronic acid, methacrylamide gelatin hydrogel, polyethylene glycol, or a composite thereof.

[0015] Furthermore, the triggering conditions for the sacrificial hydrogel material include temperature changes, ion concentration changes, pH changes, or light changes.

[0016] Furthermore, the vascular-related cells include one or more of the following: endothelial cells (such as human umbilical vein endothelial cells, brain microvascular endothelial cells, lung microvascular endothelial cells, etc.), pericytes, and smooth muscle cells.

[0017] Furthermore, the crosslinking or solidification mechanism of the hydrogel material in the hydrogel solution carrying solid tissue cells is independent of the phase transition or removal mechanism of the sacrificial material; the hydrogel material includes one or more of the following: natural polymer hydrogels (including gelatin, collagen, sodium alginate, hyaluronic acid, fibrin, etc.), synthetic polymer hydrogels (including polyvinyl alcohol, polyethylene glycol and its derivatives, polyacrylamide, etc.) or their composite systems (including methacryloyl gelatin hydrogel (GelMA)-sodium alginate, hyaluronic acid-polyethylene glycol composite hydrogel, collagen-fibrin, etc.).

[0018] It should be noted that the crosslinking or curing mechanism of this hydrogel material is independent of the phase change or removal mechanism of the sacrificial hydrogel material, in order to avoid structural instability or dissolution during the removal of the sacrificial hydrogel material. That is, the hydrogel material remains stable after curing and is unaffected by the triggering conditions used to remove the sacrificial material.

[0019] Furthermore, the chip substrate is made of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), photocurable resin, glass, silicon wafer, or any combination thereof.

[0020] This invention also provides a method for constructing the aforementioned vascularized multi-tissue interaction organ-on-a-chip, which utilizes the solid-liquid reversible phase transition properties of sacrificial hydrogel materials to construct temporary flow channel occupant structures, thereby achieving the synergistic construction of multi-tissue chamber structures and blood vessel-parenchymal tissue biological interfaces. The construction method includes: S1. A sacrificial hydrogel solution carrying blood vessel-related cells is introduced into the chip substrate. The solution flows directionally along a preset flow channel region under the topological constraint and capillary synergy of the chip substrate. Its liquid-solid phase transition is triggered by changes in the external environment, thereby forming a temporary flow channel occupancy structure for carrying cells. The occupancy structure and the space around the chip substrate together enclose one or more three-dimensional tissue-like chambers. S2. Introduce a hydrogel solution containing the same or different types of solid tissue cells into one or more three-dimensional tissue-like chambers, and form one or more three-dimensional tissue-like chambers after solidification; S3. The sacrificial hydrogel undergoes a solid-liquid phase transition under external conditions. During the flow sacrifice process, the vascular-associated cells carried in the sacrificial hydrogel migrate and attach to the three-dimensional tissue-like surface, forming a synergistic construction of the vascular-parenchymal tissue biological interface. The cavity formed after removing the sacrificial hydrogel is transformed into a perfusion microfluidic channel for delivering nutrients and removing metabolites. This allows each three-dimensional tissue to exchange biochemical signals while remaining physically independent, thus constructing a multi-tissue interaction biological interface.

[0021] It should be noted that the fluid shear force during the removal of the sacrificial hydrogel needs to be controlled within a range that allows the vascular-associated cells to adhere without being washed away.

[0022] This invention also provides the application of the above-mentioned vascularized multi-tissue interaction organ-on-a-chip in the fields of complex physiological interaction modeling, drug evaluation, and disease mechanism research.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention utilizes the controllable phase transition and flow path design of sacrificial hydrogels to achieve structural separation of multiple tissue chambers without the need for physical diaphragms or rigid interface guiding structures. This separation method maintains the spatial independence of each tissue chamber while enabling the exchange of nutrients, factors, and signaling molecules across chambers through the interconnecting channels formed after the removal of the occupant material. This constructs a biochemical interaction interface that better conforms to the characteristics of in vivo tissue interactions, supporting dynamic bidirectional communication between different types of cells or tissues.

[0024] 2. Achieving in-situ construction of a vascular-plasmic tissue interface based on phase transition processes, improving the physiological biomimicry of the microenvironment. This invention utilizes the solid-liquid reversible phase transition behavior of sacrificial hydrogels to enable vascular-related cells to adhere in situ to a three-dimensional tissue-like surface during the liquefaction and sacrifice of the occupant structure, without physical barriers. This forms a continuous, compact, and physiologically more physiologically relevant vascular-plasmic tissue biological interface, effectively avoiding the interface discontinuities and signal transmission shielding problems caused by physical structural barriers in traditional chip design.

[0025] 3. The pathways formed by the phase-change sacrificial structure significantly improve perfusion efficiency and intra-tissue material exchange capacity: After the sacrificial hydrogel completes its phase transition and is removed, its occupied area transforms into a perfusion microfluidic channel adjacent to the three-dimensional tissue-like structure. The geometric complexity of this channel can be adjusted according to actual needs, allowing the perfusion fluid to form a larger contact area and a more uniform flow field distribution around the solid tissue. This improves the efficiency of nutrient and oxygen transfer to deeper tissues and accelerates the removal of metabolic products, thereby significantly improving the stability and physiological function maintenance of long-term three-dimensional tissue culture.

[0026] 4. Improve the overall stability and application scalability of organ-on-a-chip structures: Due to the high biomimicry and ease of operation of the multi-tissue interaction interface construction method of the present invention, the resulting multi-tissue interaction microsystem can maintain high tissue activity and functional stability during long-term perfusion culture. This allows the present invention to construct in vitro models that are closer to the real physiological environment, providing a more reliable and scalable platform foundation for drug screening, disease mechanism research and precision medicine evaluation. Attached Figure Description

[0027] Figure 1This is a schematic diagram of the structure and cross-section of a vascularized multi-tissue interaction organ-on-a-chip provided by the present invention. Figure 2 A schematic diagram illustrating the method for constructing a vascularized multi-tissue interaction organ-on-a-chip provided by the present invention; Figure 3 The diagram below shows the structure of the vascularized multi-tissue interaction organ-on-a-chip provided in Embodiment 1 of the present invention. It has two semi-cylindrical three-dimensional tissue-like chambers, and the flow channels are constructed in a combination of ring and band shapes. Figure 4 This is a schematic diagram of the structure of the vascularized multi-tissue interaction organ-on-a-chip provided in Embodiment 2 of the present invention. It has an irregularly shaped three-dimensional tissue-like chamber and the flow channel is constructed in the shape of a tai chi symbol. Figure 5 This is a schematic diagram of the structure of the vascularized multi-tissue interaction organ-on-a-chip provided in Embodiment 3 of the present invention. It has four quarter-cylinder-shaped three-dimensional tissue-like chambers, and the flow channels are constructed in a combination of circular and cross-shaped structures.

[0028] In the picture: 1-Chip substrate; 2-Sacrificial hydrogel solution, which forms a temporary flow channel occupancy structure after undergoing a liquid-solid phase transition; 3-Three-dimensional tissue-like chamber; 4-Three-dimensional tissue-like structure; 5-Vascular layer cells. Detailed Implementation

[0029] Currently, common structural designs for achieving multi-tissue interaction within a single chip often have limitations. Researchers have proposed various hydrogel-based multi-tissue interaction organ-on-a-chip structures to replace the traditional method of using elastic porous membranes (such as PDMS membranes) to separate the flow channels, thereby avoiding the membrane blocking intercellular signal transmission.

[0030] Organ-on-a-chip structures based on hydrogels and multi-tissue interactions can be broadly classified into two categories: parallel channel structures and multi-layer stacked structures. The former uses phase-guiding structures or introduces height differences on both sides of the channel to limit the gel flow range, allowing the hydrogel to flow and solidify along the pre-defined channel during perfusion. This creates partitioned spaces capable of accommodating different cell types, enabling direct contact and interaction between vascular and parenchymal cells, forming a biological barrier. While this type of structure expands the direct contact area between cells to some extent and eliminates the discontinuity problem at the cell interaction interface caused by traditional microcolumn flow-limiting methods, its flow-guiding structure relies on a fixed physical structure, which can easily hinder the formation of a continuous biological barrier between the vascular and parenchymal layers. Furthermore, the resulting gel has a uniform geometry, making it difficult to construct complex multi-tissue interaction interfaces. In contrast, the latter involves solidifying cell-loaded hydrogels on a flexible film and seeding cells on its surface to simulate the biological barrier between the vascular and parenchymal layers. While this approach can achieve interlayer cell interaction without introducing phase-guided structures, it fails to provide sufficient perfusion space for the parenchyma, easily leading to necrosis of deep cells due to insufficient material exchange. Furthermore, its geometric configuration is limited, making it difficult to construct complex multi-tissue interaction interfaces. The inventors found that existing hydrogel-based multi-tissue interaction organ-on-a-chip structures still have the following common problems: (1) The phase-guided structure used to define the position or shape of the hydrogel often generates additional physical boundaries, making it difficult to form a continuous contact interface between different tissues, thereby interfering with the microenvironmental integrity required for multi-tissue interaction and weakening the overall biomimetic effect; (2) The internal flow channel geometry of the organ-on-a-chip is simple, making it difficult to construct a multi-tissue interaction interface with spatial hierarchy and tissue heterogeneity; (3) The limited contact area between the constructed tissue and the microchannel makes the exchange of nutrients and metabolites in large-sized or complex-shaped tissue chambers still insufficient, limiting the long-term culture and functional maintenance of tissues.

[0031] In view of this, the present invention provides a vascularized multi-tissue interactive organ-on-a-chip, its construction method and application.

[0032] like Figure 1 As shown, the vascularized multi-tissue interaction organ-on-a-chip includes a chip substrate 1, the interior of which is provided with a topological structure, and the surface of the chip substrate 1 is also provided with an inlet and an outlet connected to the topological structure. The topology includes a pre-defined flow channel region and a three-dimensional tissue-like chamber region enclosed by the pre-defined flow channel region; The topological structure is used to form a temporary flow channel occupancy structure for carrying blood vessel-related cells on a predetermined flow channel region and a three-dimensional tissue-like chamber 3 on a three-dimensional tissue-like chamber region after the sacrificial hydrogel solution 2 carrying blood vessel-related cells is introduced into the chip substrate 1 through the liquid-solid phase transition of the sacrificial hydrogel solution 2. That is, after the sacrificial hydrogel solution 2 carrying blood vessel-related cells is introduced into the chip substrate 1 from the inlet, the sacrificial hydrogel solution 2 flows directionally along the predetermined flow channel region under the topological constraint and capillary synergy. The liquid-solid phase transition is triggered by changes in the external environment, thereby forming the temporary flow channel occupancy structure for carrying cells and the three-dimensional tissue-like chamber 3. Three-dimensional tissue-like chamber 3 is used for perfusion of hydrogel solutions carrying parenchymal tissue cells; The temporary flow channel occupancy structure carrying cells is used to allow vascular-related cells to migrate and attach to the surface of the three-dimensional tissue-like tissue 4 after the hydrogel solution carrying parenchymal tissue cells is solidified to form a three-dimensional tissue-like tissue 4 through the solid-liquid phase transition of the sacrificial hydrogel solution. This is the morphology of vascular layer cells 5 attaching to the three-dimensional tissue-like tissue 4, forming a vascular-parenchymal tissue biological interface; and after the sacrificial hydrogel solution 2 is removed from the outlet, it is transformed into a perfusion microfluidic channel. Perfusion-compatible microfluidic channels are used to deliver nutrients and remove metabolites.

[0033] like Figure 2 As shown, the present invention provides a method for constructing a vascularized multi-tissue interaction organ-on-a-chip, the method comprising: S1. A sacrificial hydrogel solution carrying blood vessel-related cells is introduced into the chip substrate. Through the combined effects of topological constraints and capillary action, the solution flows directionally along a predetermined path in a liquid state. A liquid-solid phase transition is triggered by changes in the external environment, forming a stable temporary occupant channel structure for carrying cells. This solidified occupant structure, together with the chip substrate, encloses one or more three-dimensional tissue-like chambers for accommodating solid tissue cells.

[0034] S2. Introduce and solidify a hydrogel carrying solid tissue cells into the three-dimensional tissue-like chamber to construct a three-dimensional tissue-like structure with a specific cellular composition. Since the cross-linking or solidification mechanism of the hydrogel carrying solid tissue cells is independent of the phase transition behavior of the sacrificial hydrogel, the structure remains stable and undisturbed during the subsequent removal of the sacrificial material.

[0035] S3. By regulating external conditions, the sacrificial hydrogel is induced to transform from a solid state back into a liquid state and then removed. During this process, vascular-associated cells in the temporary occupant structure migrate and adhere to the three-dimensional tissue-like surface under controllable fluid shear forces, thereby forming a continuous and tightly adhered vascular-plasmic tissue biological interface without physical septa. The cavity left after the removal of the sacrificial material is transformed into a perfusion microfluidic channel, which can be used for continuous nutrient delivery and metabolite removal, while allowing direct exchange of biochemical signals while maintaining physical spatial independence for each three-dimensional tissue-like structure, thus constructing an organ-on-a-chip that can simulate the multi-tissue interaction behavior in vivo.

[0036] It should be noted that by regulating external conditions to induce the sacrificial hydrogel to change from solid to liquid again and achieve sacrificial removal, the sacrificial removal specifically refers to the process of the sacrificial hydrogel being guided out of the core after liquefaction. To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and thoroughly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art should fall within the protection scope of the present invention.

[0037] Example 1 like Figure 3 As shown, this embodiment provides a vascularized multi-tissue interaction organ-on-a-chip based on a phase change sacrificial structure. The chip substrate is made of PDMS material.

[0038] S1. At room temperature, a 2% (w / v) gelatin solution containing brain microvascular endothelial cells and pericytes is infused into the chip substrate. Under the combined action of the substrate topology and capillary forces, the gelatin solution undergoes self-guided flow along a predetermined path, preferentially filling the target flow channel region. After it completely occupies the predetermined space, the chip is placed in a 4°C refrigerator for about 10 minutes to allow the gelatin to undergo a liquid-solid phase transition. The cured gelatin forms a temporary flow channel occupancy structure with a combination of annular and strip-shaped structures, which, together with the chip substrate, enclose two semi-circular three-dimensional tissue-like chambers.

[0039] S2. A cell suspension carrying neurons and astrocytes was mixed with a collagen solution and injected into one of the chambers to construct a brain parenchyma-like tissue. A collagen hydrogel carrying microglia was injected into the other chamber to form an immune cell zone. The chip was placed in a 37°C incubator for about 1 hour to allow the collagen solution to gel and form a stable three-dimensional tissue-like structure.

[0040] S3. Simultaneously, cerebral microvascular endothelial cells and pericytes located in the temporary flow channel occupant material migrate to the tissue-like surface and assemble to form a vascular layer; as the temperature rises, the gelatin undergoes a solid-liquid phase transition. Subsequently, the gelatin solution is slowly removed (after liquefaction, it is guided out from the core matrix), leaving a cavity that serves as a channel structure for continuous perfusion. The two sides of the three-dimensional tissue-like material can exchange biochemical signals through this channel, thereby forming a stable neuro-immune cell interaction interface.

[0041] During use, culture medium containing pro-inflammatory factors can be introduced into the perfusion channel. Real-time imaging or immunolabeling analysis can be used to observe behaviors such as microglia migration to the interaction interface, activation, release of inflammatory factors, and cross-boundary penetration. This organ-on-a-chip can be used to construct neuroinflammation models, study the bidirectional regulatory mechanisms between neural and immune cells, evaluate the regulatory effects of candidate drugs on inflammatory responses, and explore the role of the multicellular microenvironment in the early stages of neurodegenerative diseases.

[0042] Example 2 like Figure 4 As shown, this embodiment provides a vascularized multi-tissue interaction organ-on-a-chip based on a phase change sacrificial structure. The chip substrate is made of PDMS material.

[0043] S1. At room temperature, a 20% (w / v) F127 solution containing brain microvascular endothelial cells and pericytes is infused into the chip substrate. Under the combined action of the substrate topology and capillary forces, the gelatin solution undergoes self-guided flow along a predetermined path, preferentially filling the target flow channel region. After it completely occupies the predetermined space, the chip is placed in a 37°C incubator for about 10 minutes to allow the gelatin to undergo a liquid-solid phase transition. The cured gelatin forms a temporary flow channel occupancy structure with a combination of circular and S-shaped (Tai Chi) shapes, which, together with the chip substrate, enclose two irregularly shaped three-dimensional tissue-like chambers.

[0044] S2. A cell suspension containing neurons and astrocytes was mixed with a 5% GalMA solution and injected into one of the chambers to construct brain parenchyma tissue; the other chamber was injected with a 5% GalMA solution containing oligodendrocytes or fibroblasts to simulate different types of brain tissue or scarred tissue areas. Photocuring caused the GalMA solution to cross-link and form a stable three-dimensional tissue-like structure.

[0045] S3. Simultaneously, cerebral microvascular endothelial cells and pericytes located in the temporary flow channel spacer material migrate to the tissue-like surface and assemble to form a vascular layer. The chip is then transferred to a 4°C freezer, removed after 10 minutes, and the F127 solution is slowly removed, leaving a cavity that serves as a channel structure for continuous perfusion. The two sides of the three-dimensional tissue-like structure can exchange biochemical signals through this channel.

[0046] Based on this geometric and organizational layout, further research on the interactions between brain tissues exhibiting left-right functional heterogeneity can be achieved. For example, it can be used to simulate the regulation of neural circuits between different brain regions, analyze the spatial dependence mechanism of oligodendroglial cells during myelination, study the boundary effects between scarred and healthy tissues after brain injury, and explore the interaction patterns of cell migration, inflammation regulation, and signal transduction pathways during the repair process. This organ-on-a-chip can also be used to evaluate the effectiveness and cellular response characteristics of damage repair strategies, myelin regeneration drugs, or anti-scarring therapies.

[0047] Example 3 like Figure 5 As shown, this embodiment provides a vascularized multi-tissue interaction organ-on-a-chip based on a phase change sacrificial structure. The chip substrate is made of PDMS material.

[0048] S1. At room temperature, a 1.5% (w / v) sodium alginate solution containing endothelial cells is infused into the chip substrate. Under the synergistic effect of the substrate topology and capillary forces, the sodium alginate solution undergoes self-guided flow along a predetermined path, preferentially filling the target flow channel region. Once it has completely occupied the predetermined space, a Ca-containing solution is introduced to the outside of the chip. 2+ The cross-linking buffer solution allows sodium alginate to rapidly form an ionic cross-linking gel. The solidified sodium alginate forms a temporary flow channel occupancy structure composed of a combination of circular and cross-shaped structures, which, together with the chip substrate, enclose four quarter-cylindrical three-dimensional tissue-like chambers.

[0049] S2. Different types of parenchymal tissue cells were mixed with collagen solution and injected into four chambers to construct a multi-organ interaction model. A three-dimensional liver-like tissue containing hepatocytes was constructed in the first chamber; a three-dimensional kidney-like tissue containing renal tubular epithelial cells was constructed in the second chamber; a three-dimensional intestinal-like tissue containing intestinal epithelial cells was reconstructed in the third chamber; and an immune regulatory zone carrying immune cells was formed in the fourth chamber. The chip was placed in a 37°C incubator for approximately one hour to allow the collagen solution to gel and form a stable three-dimensional tissue-like structure.

[0050] S3. Simultaneously, endothelial cells located in the sodium alginate temporary flow channel occupant migrate and assemble on the three-dimensional tissue-like surfaces to form a vascular-encapsulating layer. Subsequently, by injecting a sodium-containing chelation buffer, the sodium alginate is decrosslinked and liquefied, allowing for the slow removal of sacrificial material and the formation of a continuous perfusion channel structure spanning all four zones. This channel can mediate the exchange of biochemical signals and metabolites between the liver, kidneys, intestines, and immune modules, thus enabling the study of multi-tissue responses to drug metabolites, differential drug penetration behavior across different tissues, and the mechanisms of transregional diffusion and multidirectional signal regulation of inflammatory factors.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An organ-on-a-chip with vascularized multi-tissue interaction, characterized in that, The vascularized multi-tissue interaction organ-on-a-chip includes a chip substrate, the interior of which is provided with a topological structure, and the surface of the chip substrate is also provided with an inlet and an outlet connected to the topological structure. The topology includes a preset flow channel region and a three-dimensional tissue-like chamber region enclosed by the preset flow channel region; The topology is used to form a temporary flow channel occupancy structure for carrying cells on the preset flow channel region and a three-dimensional tissue-like cavity on the three-dimensional tissue-like cavity region by liquid-solid phase change of the sacrificial hydrogel solution after introducing a sacrificial hydrogel solution carrying blood vessel-related cells into the chip substrate. The three-dimensional tissue-like chamber is used for perfusing a hydrogel solution carrying parenchymal tissue cells; The temporary flow channel occupancy structure carrying cells is used to allow vascular-associated cells to migrate and attach to the surface of the three-dimensional tissue-like structure through the solid-liquid phase transition of the sacrificial hydrogel solution after the hydrogel solution carrying parenchymal tissue cells has been solidified to form a three-dimensional tissue-like structure, thereby forming a vascular-parenchymal tissue biological interface; and to transform into a perfusionable microfluidic channel after the sacrificial hydrogel solution is removed from the outlet. The perfusion-capable microfluidic channel is used to deliver nutrients and remove metabolites.

2. The vascularized multi-tissue interaction organ-on-a-chip according to claim 1, characterized in that, The three-dimensional tissue-like cavity region is composed of one or more sub-regions; The three-dimensional tissue-like chamber includes one or more sub-chambers for infusing hydrogel solutions containing the same or different types of solid tissue cells, which solidify to form one or more three-dimensional tissue-like structures. The perfusion-enabled microfluidic channel is also used to enable the three-dimensional tissues to exchange biochemical signals while remaining physically independent, forming a multi-tissue interaction biological interface.

3. The vascularized multi-tissue interaction organ-on-a-chip according to claim 2, characterized in that, The three-dimensional tissues include nerve cells, hepatocytes, cardiomyocytes, renal tubular cells, or any combination thereof.

4. The vascularized multi-tissue interaction organ-on-a-chip according to claim 1, characterized in that, The sacrificial hydrogel solution is made of a sacrificial hydrogel material; the sacrificial hydrogel material has both liquid-solid phase change properties and solid-liquid phase change properties, and is used to change from liquid to solid under one triggering condition to form a vacancy structure, and change from solid to liquid again under another triggering condition to achieve sacrificial removal. The sacrificial hydrogel material includes one or more of Pluronic F127, gelatin, sodium alginate, hyaluronic acid, methacrylamide gelatin hydrogel, polyethylene glycol, or a combination thereof.

5. The vascularized multi-tissue interaction organ-on-a-chip according to claim 4, characterized in that, The triggering conditions for the sacrificial hydrogel material include temperature changes, ion concentration changes, pH changes, or light changes.

6. The vascularized multi-tissue interaction organ-on-a-chip according to claim 1, characterized in that, The vascular-associated cells include one or more of endothelial cells, pericytes, and smooth muscle cells.

7. The vascularized multi-tissue interaction organ-on-a-chip according to claim 1, characterized in that, The cross-linking or solidification mechanism of the hydrogel material in the hydrogel solution carrying solid tissue cells is independent of the phase change or removal mechanism of the sacrificial material. The hydrogel material includes natural polymer hydrogels, synthetic polymer hydrogels, or composite systems thereof.

8. The vascularized multi-tissue interaction organ-on-a-chip according to claim 1, characterized in that, The chip substrate is made of polydimethylsiloxane, polymethyl methacrylate, cyclic olefin copolymer, photocurable resin, glass, silicon wafer, or any combination thereof.

9. A method for constructing an organ-on-a-chip with vascularized multi-tissue interaction according to any one of claims 1 to 8, characterized in that, The construction method includes: S1. A sacrificial hydrogel solution carrying blood vessel-related cells is introduced into the chip substrate. The solution flows directionally along a preset flow channel region under the topological constraint and capillary synergy of the chip substrate. Its liquid-solid phase transition is triggered by changes in the external environment, thereby forming a temporary flow channel occupancy structure for carrying cells. The occupancy structure and the space around the chip substrate together enclose one or more three-dimensional tissue-like chambers. S2. Introduce a hydrogel solution containing the same or different types of solid tissue cells into one or more three-dimensional tissue-like chambers, and form one or more three-dimensional tissue-like chambers after solidification; S3. The sacrificial hydrogel undergoes a solid-liquid phase transition under external conditions. During the flow sacrifice process, the vascular-associated cells carried in the sacrificial hydrogel migrate and attach to the three-dimensional tissue-like surface, forming a synergistic construction of the vascular-parenchymal tissue biological interface. The cavity formed after removing the sacrificial hydrogel is transformed into a perfusion microfluidic channel for delivering nutrients and removing metabolites. This allows each three-dimensional tissue to exchange biochemical signals while remaining physically independent, thus constructing a multi-tissue interaction biological interface.

10. The application of an organ-on-a-chip with vascularized multi-tissue interaction according to any one of claims 1 to 8 in the fields of complex physiological interaction modeling, drug evaluation, and disease mechanism research.

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