Hollow fiber membrane-based vascularized organoid chip and organoid culture method and application

Abstract

The application belongs to the technical field of biomedical engineering, and discloses a vascularized organoid chip based on a hollow fiber membrane, an organoid culture method and application. The vascularized organoid chip comprises a chip shell and a culture cavity arranged in the chip shell, and the culture cavity comprises a hollow fiber membrane, a first flow path, a second flow path, an endothelial cell layer and a basement membrane biomimetic interface layer. The hollow fiber membrane separates the lumen of the hollow fiber membrane and the culture space outside the hollow fiber membrane. The first flow path comprises a lumen-side inlet, the lumen of the hollow fiber membrane and a lumen-side outlet. The second flow path comprises a culture cavity inlet, the culture cavity and a culture cavity outlet. The endothelial cell layer is arranged on the inner wall and / or the outer wall of the hollow fiber membrane. The basement membrane biomimetic interface is arranged between the hollow fiber membrane and the culture cavity. The vascularized organoid chip enables the transmembrane transfer of substances during the organoid culture process, which is very beneficial to the culture of the organoid.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and specifically relates to a vascularized organoid chip based on hollow fiber membrane, as well as organoid culture methods and applications. Background Technology

[0002] Organoids are miniature three-dimensional organs that autonomously differentiate and self-organize from adult stem cells or pluripotent stem cells in an in vitro three-dimensional culture environment. They can reproduce, to a certain extent, the cellular composition, spatial structure, and some physiological functions of human organs. Organoids have been used for developmental research, disease modeling, drug evaluation, and personalized medicine. However, most existing organoids rely on static hydrogels or immersion in culture medium to obtain oxygen and nutrients. As the volume of organoids increases, insufficient supply of oxygen, glucose, and growth factors, as well as the accumulation of metabolic waste, can easily occur inside, leading to problems such as organoid central necrosis, uneven cell differentiation, and functional decline.

[0003] In vivo tissues typically rely on capillary networks to maintain substance exchange. Vascular endothelial cells not only constitute a selective barrier but also develop cell polarity, intercellular connections, and vasoactive phenotypes under the influence of fluid shear forces, and participate in tissue maturation and inflammation regulation through paracrine signals. Traditional organoid culture systems lack an endothelial interface capable of continuous perfusion and withstanding shear forces; simply co-culturing endothelial cells with organoids often results in random, closed, and non-connected vascular-like structures, making it difficult to simulate the real blood-endothelium-basement membrane-tissue mass transfer pathway.

[0004] Current organ-on-a-chip technologies typically utilize planar microchannels or hydrogel microchannels to construct vascular interfaces. Planar microchannels are easy to observe, but their limited surface area makes it difficult to form three-dimensional vascular-like networks within a finite volume. While hydrogel microchannels exhibit good biocompatibility, their long-term mechanical stability, leakage resistance, mass production capabilities, and modular packaging capabilities still require improvement. Existing technologies also include forming three-dimensional interconnected microtube networks using photopolymer materials permeable to liquids and gases, filling the spaces between the microtubes with hydrogel, and culturing cell aggregates. This technology helps to modulate spatial signals within organoids, but it relies on high-resolution photopolymerization manufacturing technology, resulting in high equipment costs and manufacturing complexity.

[0005] In addition, existing hollow fiber membrane cell culture devices mostly use hollow fiber membranes for single nutrient exchange, which cannot ensure that the endothelial cells on the inner or outer wall of the hollow fiber membrane can obtain appropriate shear force, thus failing to effectively form a perfusion vascular network, which is detrimental to organoid culture.

[0006] Therefore, it is necessary to provide an engineerable and scalable vascularized organoid chip that allows endothelial cells located on the inner or outer wall to receive appropriate shear forces, which is helpful for culturing organoids. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a hollow fiber membrane-based vascularized organoid chip, an organoid culture method, and its application. The hollow fiber membrane-based vascularized organoid chip of this invention enables transmembrane transport of oxygen, carbon dioxide, and dissolved nutrients during organoid culture, allowing endothelial cells located on either the inner or outer wall of the hollow fiber membrane to receive appropriate shear forces. Furthermore, the controllable biomimetic interface of the basement membrane and the organoid fixation structure promote endothelial budding and the formation of a perfusionable vascular network, which is highly beneficial for organoid culture.

[0008] Hollow fiber membranes possess high specific surface area, controllable pore structure, excellent mass transfer capacity, and ease of bundling, weaving, and filling, making them suitable as fluid channels, selective mass transfer interfaces, and cell support carriers. However, existing hollow fiber membrane cell culture devices primarily utilize them for single-nutrient exchange, failing to integrate gas transfer, solute transfer, endothelial shear regulation, basement membrane interface construction, and organoid vascularization into a single chip. In particular, when endothelial cells are located on the outer wall of the hollow fiber membrane, flow within the membrane lumen alone cannot provide the outer endothelium with tangential shear force along the cell surface. Furthermore, directly attaching organoids to the endothelial layer can easily lead to local endothelial detachment, decreased mass transfer area, and endothelial polarity disturbances. The vascularized organoid chip described in this invention addresses these technical problems.

[0009] The vascularized organoid chip of this invention is a hollow fiber membrane vascularized organoid chip with gas-liquid dual transmission capability, which solves the problems of limited oxygen and nutrient delivery, lack of physiological fluid shear force in endothelial cells, difficulty in controlling the spatial relationship between the endothelial barrier and organoids, inability to stably connect endothelial vascular-like structures to external perfusion circuits, and difficulty in standardizing and scaling up the device in existing organoid culture systems.

[0010] A first aspect of the present invention provides a vascularized organoid chip based on a hollow fiber membrane.

[0011] Specifically, a vascularized organoid chip based on a hollow fiber membrane includes a chip shell and a culture chamber placed inside the chip shell. The culture chamber includes a hollow fiber membrane, a first flow path, a second flow path, an endothelial cell layer, and a basement membrane biomimetic interface layer. The hollow fiber membrane separates the lumen of the hollow fiber membrane from the culture space outside the hollow fiber membrane; The first flow path includes a lumen-side inlet, a lumen of the hollow fiber membrane, and a lumen-side outlet; The second flow path includes a culture chamber inlet, a culture chamber, and a culture chamber outlet; The endothelial cell layer is disposed on the inner wall and / or outer wall of the hollow fiber membrane; The biomimetic interface of the basement membrane is disposed between the hollow fiber membrane and the culture chamber.

[0012] The first flow path is the cavity formed by the hollow fiber membrane, and the perfusion loop is formed by the cavity inlet and the cavity outlet.

[0013] The first flow path and the second flow path are two different and isolated loops.

[0014] Preferably, the hollow fiber membrane can transfer gases or solutes. During organoid culture, the organoids are located outside the hollow fiber membrane. Oxygen or carbon dioxide can diffuse across the membrane wall through the membrane pores to the outside of the membrane; soluble substances such as glucose, amino acids, vitamins, inorganic salts, and growth factors can be transferred to the outside of the membrane through the membrane pores, providing the necessary nutrients for the growth of the organoids.

[0015] Preferably, the hollow fiber membrane includes any one of a homogeneous porous membrane, an asymmetric porous membrane, or a composite membrane.

[0016] Preferably, when the hollow fiber membrane needs to transfer gas, the material composition of the hollow fiber membrane includes at least one of poly-4-methyl-1-pentene (PMP), polydimethylsiloxane (PDMS), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), or a composite thereof.

[0017] Preferably, when the hollow fiber membrane needs to transfer liquid, the material composition of the hollow fiber membrane includes at least one of polyethersulfone (PES), polyvinylidene ether (PVDF), polysulfone (PSF), polyacrylonitrile (PAN), cellulose acetate (CA), cellulose triacetate (CTA), polyurethane (PU), polyimide (PI), polyetherimide (PEI), or a combination thereof.

[0018] Preferably, the hollow fiber membrane has a wall thickness of 25-400 μm, and more preferably 100-300 μm.

[0019] Preferably, the hollow fiber membrane has an outer diameter of 150-1800 μm, an inner diameter of 50-1000 μm, and a membrane wall thickness of 25-400 μm.

[0020] The first flow path includes a lumen-side inlet, a lumen of the hollow fiber membrane, and a lumen-side outlet; Preferably, both the lumen-side inlet and lumen-side outlet are provided with independent inlet / outlet assemblies between themselves and the lumen of the hollow fiber membrane. A potting seal is provided on the surface of each independent inlet / outlet assembly, and both ends of the hollow fiber membrane are sealed to the chip housing via the potting seal. The potting seal serves to fix and seal, preventing the lumen of the hollow fiber membrane from shifting during organoid culture. It also isolates the first flow path from the second flow path. Liquid can enter the lumen of the hollow fiber membrane through the lumen-side inlet via the independent inlet / outlet assembly in the potting seal, and the liquid can continue to flow through the lumen of the hollow fiber membrane, through the independent inlet / outlet assembly in the potting seal, and out from the lumen-side outlet.

[0021] Preferably, the endothelial cell layer is disposed on the inner or outer wall of the hollow fiber membrane. When the endothelial cells in the endothelial cell layer are located on the inner wall of the hollow fiber membrane, the culture medium on the lumen side (i.e., the lumen of the hollow fiber membrane) flows axially along the lumen formed by the hollow fiber membrane and directly acts on the apical surface of the endothelial cells, causing them to receive tangential shear force. When the endothelial cells in the endothelial cell layer are located on the outer wall of the hollow fiber membrane, the lumen side is used to supply oxygen and nutrients, and the second flow path of the hollow fiber membrane causes the culture medium to flow with low shear along the axial or circumferential direction along the outer wall of the hollow fiber membrane, allowing the endothelial cells to directly receive external shear force.

[0022] Preferably, the first flow path and the second flow path are independent of each other.

[0023] Preferably, the first flow path further includes a first liquid reservoir, a first drive pump, a first pipeline, a first sampling port, and a first flow regulating device.

[0024] Preferably, the second flow path further includes a second liquid reservoir, a second drive pump, a second pipeline, a second sampling port, and a second flow regulating device.

[0025] Preferably, the first drive pump and the second drive pump are each independently selected from peristaltic pumps, syringe pumps, pressure-driven pumps, diaphragm pumps, or other microfluidic drive devices capable of providing stable microflow rates.

[0026] Preferably, the first drive pump and the second drive pump are each used independently in conjunction with the microfluidic chip, the fluid controller, and the automated perfusion device.

[0027] Preferably, by independently adjusting the flow rate and pressure of the first flow path and the second flow path, the shear force on the inner wall endothelial cells or the outer wall endothelial cells of the hollow fiber membrane can be controlled while maintaining a low transmembrane pressure difference. Continuous perfusion, intermittent perfusion, pulsed perfusion, or flow gradient regulation can be achieved according to culture requirements, thereby reducing cell shedding or matrix loss caused by excessive transmembrane convection.

[0028] Preferably, the endothelial cells in the endothelial cell layer are selected from human umbilical vein endothelial cells, microvascular endothelial cells, organ-specific endothelial cells, endothelial progenitor cells, induced pluripotent stem cell-derived endothelial cells, and combinations thereof.

[0029] More preferably, pericytes, vascular smooth muscle cells, mesenchymal matrix cells, fibroblasts, or immune cells are added to the endothelial cell layer to stabilize the endothelial network or construct a tissue-specific microenvironment.

[0030] Preferably, when the vascularized organoid chip is used to culture organoids, the organoids are located outside the hollow fiber membrane and in the culture chamber, and the biomimetic interface of the basement membrane is disposed between the endothelial cell layer and the organoids.

[0031] Preferably, the biomimetic interface of the basement membrane comprises collagen, laminin, fibronectin, gelatin, hyaluronic acid, Matrigel, GelMA, decellularized extracellular matrix, and combinations thereof.

[0032] Preferably, the biomimetic interface of the basement membrane is a membrane surface coating layer, a thin layer of hydrogel, or an organoid fixation scaffold with micropores.

[0033] Preferably, the biomimetic interface layer of the basement membrane has a structure that allows endothelial cell migration and matrix remodeling. By adjusting collagen concentration, gel crosslinking density, matrix degradability, and the spatial distribution of VEGF, FGF, Ang-1, S1P, or organ-specific angiogenesis factors, endothelial cells on the outer wall of the hollow fiber membrane are induced to bud towards the organoid, and the budding structure enters the organoid interior. The budding structure can connect with endogenous endothelial cells or microvascular-like structures of the organoid.

[0034] Preferably, the hollow fiber membrane is a single fiber, arranged in a parallel array, woven, or cross-arranged. Different hollow fibers or membrane filament groups can be connected to independent lumen-side inlets and lumen-side outlets for delivering basal culture medium, oxygenated culture medium, gas, mixed gas, drugs, or cytokines, respectively.

[0035] The vascularized organoid chip can be customized to meet the culture needs of different cells, tissues, and organoids by adjusting the hollow fiber membrane's material, pore size, wall thickness, gas and liquid permeability, perfusion parameters, and the composition of the biomimetic interface layer of the basement membrane. It is suitable for the culture and vascularization of various tissues or organoids, including liver, kidney, lung, heart, intestine, brain, pancreas, and tumors. Its applications can be expanded to include organ-on-a-chip construction, disease model establishment, drug screening and toxicity evaluation, research on tissue development and angiogenesis mechanisms, cell therapy product culture, regenerative medicine, and personalized precision medicine. The chip can be used as an independent culture unit or combined with other tissue culture modules, sensing and monitoring modules, and culture medium processing modules to form a single-organ or multi-organ in vitro biomimetic culture and evaluation platform.

[0036] Preferably, when culturing organoids using the vascularized organoid microarray, the organoids are cultured outside a hollow fiber membrane, and during the culture process, the culture chamber also includes an organoid fixation structure. The organoids are fixed near the endothelial cell layer by means of hydrogel embedding, micropore trapping, or other methods.

[0037] Preferably, the distance from the surface of the organoid to the outer wall of the hollow fiber membrane is 20-500 μm, more preferably 50-100 μm. This allows some endothelial cell surfaces to remain in direct contact with the fluid outside the hollow fiber membrane.

[0038] A second aspect of the present invention provides a method for culturing organoids.

[0039] Specifically, an organoid culture method includes the following steps: The organoid is placed in the culture chamber of the vascularized organoid chip, and the organoid is outside the hollow fiber membrane.

[0040] Preferably, the organoid culture method includes the following steps: (1) Prepare the first flow path and sterilize it; (2) Modify the lumen formed by the hollow fiber membrane using dopamine hydrochloride solution and protein, then inoculate endothelial cells and culture them to form the endothelial cell layer; (3) A basement membrane biomimetic interface is provided outside the hollow fiber membrane, and organoids are fixed thereon; (4) Set up the second flow path, then introduce culture medium into the first flow path and introduce organoid culture medium into the second flow path to culture organoids.

[0041] A third aspect of the present invention provides the application of the above-described hollow fiber membrane-based vascularized organoid chip.

[0042] The above-mentioned hollow fiber membrane-based vascularized organoid microarrays have applications in tissue development, disease modeling, drug screening and toxicity evaluation, angiogenesis mechanism research, or cell product culture.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention utilizes hollow fiber membranes to form vascularized organoid chips, which can realize the transfer of oxygen, carbon dioxide, water and dissolved nutrients, shorten the distance between the cells inside the organoid and the mass transfer interface, and help maintain oxygen and nutrient homeostasis during long-term culture.

[0044] (2) Endothelial cells can be selectively placed on the inner or outer wall of the hollow fiber membrane according to the needs of organoids; the inner wall endothelium receives shear force on the lumen side, and the outer wall endothelium receives independent extra-membrane low-shear perfusion, thereby overcoming the problem that the outer wall cells cannot obtain tangential shear force when there is only lumen flow.

[0045] (3) The biomimetic interface layer of the basement membrane forms a controllable spatial coupling between the hollow fiber membrane, the endothelial cell layer and the organoid, avoiding direct compression of the endothelial cell layer by the organoid, and providing a structural channel for endothelial cell migration, budding and vascular network invasion.

[0046] (4) Hollow fiber membranes have high mechanical strength and can be used to construct replaceable culture cartridges using mature membrane fiber bundling, weaving and filling processes, making it easy to achieve parallel connection of multiple membrane fibers, scale-up and batch standardization.

[0047] (5) Independent perfusion circuits are used on the lumen side and the membrane side, which can control shear force, pressure, gas composition and irritant concentration respectively, and are suitable for studies on organ development, tumor microenvironment, drug transendothelial transport and toxicity evaluation.

[0048] (6) By connecting the endothelial cell layer on the outer wall with the vascular-like structure inside the organoid, the perfusion channel, endothelial barrier and organoid tissue can form a continuous functional unit, providing an engineering basis for constructing perfusion-capacitated vascularized organoids. Attached Figure Description

[0049] Figure 1 A schematic diagram of the structure of a vascularized organoid chip; Figure 2 Schematic diagrams showing two scenarios where the endothelial cell layer is located on the inner and outer walls of a hollow fiber membrane; Figure 3 A schematic diagram of the biomimetic interface of the basement membrane and the budding of the endothelial cell layer on the outer wall of the hollow fiber membrane into organoids; Figure 4 Schematic diagram of different arrangements of hollow fiber membranes and independent group perfusion; Figure 5 This is a schematic diagram of the overall structure of a vascularized organoid on-chip. Figure 6 This is a schematic diagram of vascularized organoid microarray culture of liver organoids in Example 1; Figure 7 This is a schematic diagram of vascularized organoid microarray culture of lung organoids in Example 2; Figure 8 This is a diagram showing the staining results of the cytoskeleton and nuclei of the outer wall of the hollow fiber membrane in Example 2. Detailed Implementation

[0050] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0051] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0052] The component names corresponding to all the reference numerals used in the figures of this invention are uniformly shown in Table 1 below.

[0053] Table 1

[0054] Figure 1 A schematic diagram of the structure of a vascularized organoid chip; Figure 2 The diagram illustrates two scenarios: the endothelial cell layer is located on the inner wall and the outer wall of the hollow fiber membrane. Diagram A shows the endothelialization pattern on the inner wall, and diagram B shows the endothelialization pattern on the outer wall. Figure 3 This is a schematic diagram of the biomimetic interface of the basement membrane and the budding of the endothelial cell layer on the outer wall of the hollow fiber membrane into organoids.

[0055] A culture chamber 2 is formed inside the chip housing 1. Multiple hollow fiber membranes 3 form an array. Both ends of each hollow fiber membrane 3 are fixed and sealed by a potting seal 25. The potting seal 25 separates the lumen 4 of the hollow fiber membrane 3 from the culture chamber 2, so that the lumen-side inlet 5, each lumen 4 and the lumen-side outlet 6 form a continuous first flow path, and the membrane-side inlet 8, the culture chamber 2 and the membrane-side outlet 7 form a continuous second flow path.

[0056] When the first flow path is working, the pre-oxygenated culture medium enters from the lumen-side inlet 5, and after being distributed in the filling area where the filling seal is located, it enters the lumen 4 of each hollow fiber membrane 3 and flows axially along the lumen 4, passes through the filling area where the filling seal is located at the other end, and finally collects at the lumen-side outlet 6 for discharge.

[0057] When the second flow path is in operation, the organoid culture medium enters the culture chamber 2 through the outer inlet 8 of the membrane, flows along the outer surface of the hollow fiber membrane 3, and then exits from the outer outlet 7 of the membrane. Figure 1The inlet 8 is located at the bottom and the outlet 7 is located at the top. This is for illustration only. In actual encapsulation, it can be set at the diagonal position of the culture chamber 2 to reduce short-circuit flow and dead zone. However, the flow direction should always be clearly defined as 8 entering and 7 exiting.

[0058] Organoid 19 is fixed near the hollow fiber membrane 3 by organoid fixation structure 20 and maintains a predetermined distance from the outer wall of the hollow fiber membrane 3. The oxygen and permeable solute contained in the culture medium in the lumen 4 pass sequentially through the lumen-side fluid, the membrane wall of the hollow fiber membrane 3, the endothelial cell layer, and the biomimetic interface layer of the basement membrane along the transmembrane transport path 23. Figure 1 The endothelial cell layer and the biomimetic interface layer of the basement membrane are not shown in the drawing. See [link / reference]. Figure 3 The diagram shows an endothelial cell layer 17, a basement membrane biomimetic interface layer 18, and an organoid fixation structure 20, ultimately leading to an organoid 19. Carbon dioxide and permeable metabolites produced by the organoid 19 can either enter the lumen 4 in reverse or be discharged from the outer membrane outlet 7 along with the extracellular culture medium.

[0059] In the endothelialization pattern of the inner wall (e.g.) Figure 2 (Figure A in the diagram), endothelial cell layer 17 is located on the inner wall of hollow fiber membrane 3 and directly receives the shear force generated by the flow on the luminal side; in the external wall endothelialization mode (e.g. Figure 2 (Figure B in the diagram) The endothelial cell layer 17 is located on the outer wall of the hollow fiber membrane 3 and directly receives the low shear force generated by the flow outside the membrane. The biomimetic interface layer 18 of the basement membrane is disposed between the endothelial cell layer 17 and the organoid 19 (e.g., Figure 3 (As shown), and retains part of the endothelial cell layer surface in direct contact with the external culture medium of the hollow fiber membrane, from Figure 3 It can also be seen that the endothelial budding structure 21 extends from the endothelial cell layer toward the organoid 19, which is conducive to promoting the formation of endogenous microvascular-like structures 22 inside the organoid 19.

[0060] Figure 4 The diagram shows different arrangements of hollow fiber membranes and independent group perfusion. Figure A shows a single membrane filament, Figure B shows a parallel array, Figure C shows a cross array (Figures B and C are both hollow fiber membrane arrays), and Figure D shows an independent membrane filament group and a replaceable culture cartridge, including a filling and sealing component 25, a replaceable culture cartridge 26, a first hollow fiber membrane group 28, a second hollow fiber membrane group 29, and an independent inlet and outlet assembly 30.

[0061] Figure 5 This is a schematic diagram of the overall structure of a vascularized organoid on-chip.

[0062] The chip housing 1 contains a culture chamber 2, and multiple hollow fiber membranes 3 are disposed in the culture chamber 2. The two ends of the hollow fiber membranes 3 are respectively sealed to the chip housing 1 through potting seals 25, so that the tube and the culture chamber 2 are isolated from each other. The tube side inlet 5 and the tube side outlet 6 are connected to the tube 4; the membrane side inlet 8 and the membrane side outlet 7 are connected to the culture chamber 2, thereby forming a first flow path and a second flow path that do not cross-contaminate.

[0063] During lumen-side perfusion, pre-oxygenated endothelial cell culture medium, endothelial-organoid compatible culture medium, or culture medium containing a predetermined stimulant is output from the lumen-side reservoir 9 and enters the lumen-side perfusion line 13 under the action of the lumen-side drive pump 10. It then passes sequentially through the first sampling port 15 and the first flow regulating device 16 located on the lumen-side perfusion line 13, and finally enters the lumen 4 of each hollow fiber membrane 3 through the lumen-side inlet 5. The culture medium flows axially along the hollow fiber membrane 3 from the lumen-side inlet 5 to the lumen-side outlet 6, and during the flow, it flows towards the endothelial cell layer located on the inner wall of the lumen 4 (…). Figure 5 Endothelial cell layer 17 is not shown; see [link to relevant documentation]. Figure 2 The endothelial cell layer 17 in the culture medium is subjected to tangential shear force; dissolved oxygen, carbon dioxide, glucose, amino acids, inorganic salts, and permeable protein factors in the culture medium can pass through the membrane wall of the hollow fiber membrane 3 along the transmembrane transport pathway and enter the basement membrane biomimetic interface layer, organoid fixation structure, and organoid ( Figure 5 The biomimetic interface layer 18 of the basement membrane, the organoid fixation structure 20, and the organoid 19 are not shown; please refer to [link to relevant documentation]. Figure 3 The culture medium is located in the region containing the biomimetic interface layer 18 of the basement membrane, the organoid fixation structure 20, and the organoid 19. The culture medium then flows out from the lumen-side outlet 6, returns to the lumen-side reservoir 9 via the lumen-side return perfusion line 13, forming a closed-loop circulation on the lumen side. The first sampling port 15 is used for aseptic collection of the culture medium, the first flow regulating device 16 is used to regulate or limit the flow rate, and the sensing and monitoring module 27 is used to monitor the pressure, flow rate, temperature, pH, and dissolved oxygen within the first flow path or the vascularized organoid chip.

[0064] During extracellular perfusion, the organoid culture medium is output from the extracellular reservoir 11 and enters the extracellular perfusion line 14 under the action of the extracellular drive pump 12. It then enters the culture chamber 2 through the extracellular inlet 8. Within the culture chamber 2, the culture medium flows uniformly axially and circumferentially along the outer surface of the hollow fiber membrane 3, applying low shear force to the endothelial cell layer on the outer wall of the hollow fiber membrane 3, while simultaneously supplying nutrients to the organoids and removing metabolic waste. The extracellular culture medium finally flows out through the extracellular outlet 7 and returns to the extracellular reservoir 11 via the extracellular perfusion line 14, forming a closed loop. 31 is the second sampling port, and 32 is the second flow rate regulating device. The culture medium in the second flow path can be sampled and observed as needed, and the flow rate can be adjusted by regulating the second flow rate regulating device. The sensing and monitoring module 27 can also monitor the parameters of the second flow path or the culture chamber 2 online.

[0065] During operation, the difference between the average pressure on the lumen side and the average pressure on the outside of the membrane is maintained within a set range by adjusting the lumen-side drive pump 10, the membrane-side drive pump 12, and the flow regulating device 16, thereby reducing excessive transmembrane convection. The liquids in the two flow paths do not mix directly; gases and permeable solutes are transferred only through the membrane wall of the hollow fiber membrane 3. Metabolic products can enter the first flow path in the opposite direction to nutrients, or they can be discharged from the membrane-side outlet 7 along with the external culture medium.

[0066] When using the direct gas supply mode, the lumen-side reservoir 9 can be replaced with a sterile gas supply and humidity control unit. Under the action of the lumen-side drive pump 10 or gas pressure source, the gas enters the lumen 4 through the lumen-side perfusion line 13, flow regulating device 16, and lumen-side inlet 5. After the gas is transferred to the culture chamber 2 through the membrane wall, the gas that does not permeate enters the gas recovery end through the lumen-side outlet 6 or is discharged through the sterile filter. Example 1 Figure 6 This is a schematic diagram of culturing liver organoids on an vascularized organoid microarray, as shown in Example 1. Figure 6 Figure C in the diagram is a schematic diagram of the overall structure, Figure A is a magnified view of a part, and Figure B is a further magnified view of a part.

[0067] A method for culturing liver organoids in the endothelial model of vascularized organoid microarrays includes the following steps: (1) Chip preparation and sterilization: 20 PES hollow fiber membranes are arranged in parallel inside the chip housing 1. The two ends of the hollow fiber membrane lumen are filled with medical grade polyurethane to form a filling seal 25. The end sealing layer of the hollow fiber membrane is removed so that the lumen 4 is connected to the lumen-side inlet 5 and the lumen-side outlet 6 respectively. After assembly, the chip is sterilized with ethylene oxide and fully analyzed, and then rinsed with sterile PBS. (2) Modification of the inner surface of the lumen: 2 mg / mL dopamine hydrochloride solution (10 mmol / L Tris-HCl, pH 8.5) was injected into lumen 4 and reacted at room temperature for 2 h. The lumen was then rinsed with PBS. A coating solution containing 50 μg / mL fibronectin, 50 μg / mL type IV collagen and 20 μg / mL laminin was injected and incubated at 4℃ for 12 h. The lumen was then rinsed with sterile PBS. This step was used to improve the uniformity of endothelial cell adhesion in the lumen. (3) Endothelial cell seeding: Human microvascular endothelial cells were used, and the concentration was prepared at 3×10⁻⁶. 6 A cell suspension with a cell count / mL was slowly injected in 50 μL through inlet 5 on the luminal side, resulting in approximately 1.5 × 10⁻⁶ cells entering the vascularized organoid microarray. 5 After sealing the inlet and outlet, the vascularized organoid chip is rotated 90° along the axis every 15 minutes, for a total of 4 rotations, and then left to stand for 4 hours to adhere. (4) Pre-culture of luminal endothelium: After inoculation, keep the cell line static for 0-24 h, continuously perfuse at 1.0 μL / min for 24-36 h, increase to 3.0 μL / min for 36-48 h, increase to 7.5 μL / min after 48 h and maintain, and after 72 h of culture, use CD31, VE-cadherin and ZO-1 staining to confirm the continuous endothelial cell layer; (5) Basement membrane biomimetic interface and liver organoid fixation: A local basement membrane biomimetic interface layer 18 with a thickness of 70 μm is formed on the outside of the hollow fiber membrane 3. The layer consists of type I collagen 1.0 mg / mL, type IV collagen 50 μg / mL, laminin 20 μg / mL, fibrinogen 5 mg / mL and thrombin 2 U / mL. Ten liver organoids with a diameter of 250±50 μm are embedded in 5% (w / v) GelMA hydrogel (GelMA hydrogel serves as organoid fixation structure 20), so that the distance from the surface of the organoid to the outer wall of the hollow fiber membrane is 75±25 μm, and at least 50% of the outer surface of the hollow fiber membrane is kept in direct contact with the extracellular culture medium. (6) Dual-flow culture: 5 mL of pre-oxygenated endothelial-liver organoid compatible culture medium was added to the lumen-side reservoir 9 and continuously circulated at 7.5 μL / min; 3 mL of liver organoid culture medium was added to the membrane-side reservoir 11 and circulated at 20 μL / min for the first 12 h after inoculation, followed by continuous circulation at 50 μL / min; before starting perfusion, the actual liquid levels of the lumen-side reservoir 9 and the membrane-side reservoir 11 were adjusted to be basically the same height so that the average pressure on both sides was basically the same, in order to reduce excessive transmembrane convection. 50% of the culture medium in the two reservoirs was replaced every 48 h, and samples were taken through sampling port 15 on days 0, 3, 7, and 14. (7) Status evaluation: On days 7 and 14, the morphology, diameter, and retention rate of liver organoids were measured. Calcein-AM / PI and HIF-1α were used to evaluate survival, hypoxia, and necrosis. Albumin, urea, and CYP3A4 activity were measured to evaluate liver function. CD31, VE-cadherin, ZO-1, and vWF were measured to evaluate the integrity of the luminal endothelium. When the organoid survival rate was ≥85%, the hypoxic or necrotic area was ≤15%, and the liver function remained stable or increased, the organoid was considered to be in good growth status.

[0068] Example 2 Figure 7 This is a schematic diagram of culturing lung organoids on a vascularized organoid microarray, as shown in Example 2. Figure 7 Figure C in the diagram is a schematic diagram of the overall structure, Figure A is a magnified view of a part, and Figure B is a further magnified view of a part.

[0069] A method for culturing lung organoids using the endothelial model of the outer wall of a vascularized organoid microarray includes the following steps: (1) Using the same chip shell, hollow fiber membrane array and potting method as in Example 1, the outer surface of the hollow fiber membrane 3 was treated with 30 W oxygen plasma for 60 s, then wetted with sterile PBS, and incubated at 37 °C for 2 h with a coating solution containing 50 μg / mL fibronectin, 50 μg / mL type IV collagen and 20 μg / mL laminin; (2) Prepare human pulmonary microvascular endothelial cells to a concentration of 1.5 × 10⁻⁶. 6 160 μL of cell suspension was injected into culture chamber 2 at a cell count / mL. The vascularized organoid chip was rotated 90° every 30 min for a total of 4 times to allow the cells to adhere evenly to the outer surface of the hollow fiber membrane in 3 weeks. Then, the cells were incubated statically for 12 h. (3) Run at 50 μL / min for 12-24 h after inoculation, increase to 150 μL / min for 24-36 h, and increase to 300 μL / min after 36 h and maintain it; (4) A locally remodelable basement membrane biomimetic interface layer 18 with a thickness of 75±25 μm is formed on the side of the outer wall endothelial cell layer 17 near the lung organoid 19. The basement membrane biomimetic interface layer 18 is composed of fibrinogen 5 mg / mL, thrombin 2 U / mL, type IV collagen 50 μg / mL, laminin 20 μg / mL and 3% (w / v) GelMA hydrogel containing matrix metalloproteinase sensitive peptides. Ten lung organoids with a diameter of 200±50 μm are fixed at the organoid fixation structure 20 at a distance of 75±25 μm from the outer wall of the hollow fiber, and at least 50% of the area of ​​the outer wall endothelial cell layer is exposed to the extracellular fluid. (5) Add VEGF-A 50 ng / mL, FGF2 20 ng / mL, Ang-1 100 ng / mL and S1P 1 μmol / L to the organoid fixation structure 20. Add 5 mL of pre-oxygenation compatible culture medium to the lumen-side reservoir 9 and circulate continuously at 10 μL / min. Add 3 mL of lung organoid culture medium to the membrane-side reservoir 11 and circulate at 300 μL / min. Before starting perfusion, adjust the actual liquid levels of the lumen-side reservoir 9 and the membrane-side reservoir 11 to be basically the same height so that the average pressure on both sides is basically the same, so as to reduce excessive transmembrane convection. Replace 50% of the culture medium every 48 h. (6) Status evaluation and judgment: Live cell imaging and confocal reconstruction were performed on days 3, 7, 14 and 21 to evaluate endothelial cell budding, branching, coverage area, connection with organoid endogenous microvascular-like structures 22 and fluorescent dextran perfusion connectivity; organoid survival, hypoxia / necrosis and expression of EPCAM, SFTPC, AQP5 and PDPN were detected at the same time. When the organoid retention rate was ≥80%, the live cell count was ≥85%, the hypoxia / necrosis was ≤15%, the markers showed no significant decrease and perfusion connectivity was formed on day 21, the growth status was judged to be good.

[0070] Comparative Example 1 Compared with Example 2, the only difference in Comparative Example 1 is that the basement membrane biomimetic interface layer 18 is not provided in Comparative Example 1, and all other contents are the same as in Example 2.

[0071] The comparison of endothelial cell budding, branching, budding coverage area, organoid endogenous microvascular-like structure connection ratio, perfusion connectivity, organoid survival rate, hypoxia or necrosis area, and lung epithelial marker expression between Example 2 and Comparative Example 1 was used to evaluate the effect of the basement membrane biomimetic interface layer on endothelial cell budding, vascular connectivity, and lung organoid growth status.

[0072] Figure 8 This image shows the staining results of the cytoskeleton and nuclei on the outer wall of the hollow fiber membrane in Example 2, which serves as a verification result of the endothelialization of the outer wall of the hollow fiber membrane in Example 2. After treating and coating the outer surface of the hollow fiber membrane 3 according to the method described in Example 2, endothelial cells were seeded onto its outer surface. After culture, the cytoskeleton and nuclei were fluorescently stained, and the results were observed using a fluorescence microscope. Figure 8 .Depend on Figure 8 It is evident that endothelial cells can stably adhere to and fully spread on the outer surface of the hollow fiber membrane, exhibiting a good growth state. This indicates that it is feasible to inoculate and culture endothelial cells on the outer surface of the hollow fiber membrane, thus providing experimental evidence for the external wall endothelialization culture mode described in Example 2.

Claims

1. A vascularized organoid on-chip, characterized in that, It includes a chip housing and a culture chamber placed inside the chip housing, wherein the culture chamber includes a hollow fiber membrane, a first flow path, a second flow path, an endothelial cell layer, and a basement membrane biomimetic interface layer; The hollow fiber membrane separates the lumen of the hollow fiber membrane from the culture space outside the hollow fiber membrane; The first flow path includes a lumen-side inlet, a lumen of the hollow fiber membrane, and a lumen-side outlet; The second flow path includes a culture chamber inlet, a culture chamber, and a culture chamber outlet; The endothelial cell layer is disposed on the inner wall and / or outer wall of the hollow fiber membrane; The biomimetic interface of the basement membrane is disposed between the hollow fiber membrane and the culture chamber.

2. The vascularized organoid chip according to claim 1, characterized in that, The hollow fiber membrane includes any one of a homogeneous porous membrane, an asymmetric porous membrane, or a composite membrane.

3. The vascularized organoid chip according to claim 1, characterized in that, When the hollow fiber membrane needs to transfer gas, the material composition of the hollow fiber membrane includes at least one of poly-4-methyl-1-pentene, polydimethylsiloxane, polypropylene, polyethylene, polytetrafluoroethylene, or a composite thereof.

4. The vascularized organoid chip according to claim 1, characterized in that, When the hollow fiber membrane needs to transfer liquid, the material composition of the hollow fiber membrane includes at least one of polyethersulfone, polyvinylidene ethylene, polysulfone, polyacrylonitrile, cellulose acetate, cellulose triacetate, polyurethane, polyimide, polyetherimide, or a composite of these materials.

5. The vascularized organoid chip according to claim 1, characterized in that, The hollow fiber membrane has an outer diameter of 150-1800 μm and an inner diameter of 50-1000 μm.

6. The vascularized organoid chip according to claim 1, characterized in that, The lumen-side inlet and lumen-side outlet are each provided with independent inlet / outlet components between themselves and the lumen of the hollow fiber membrane. A potting seal is provided on the surface of the independent inlet / outlet components, and both ends of the hollow fiber membrane are respectively sealed to the chip housing through the potting seal.

7. The vascularized organoid chip according to claim 1, characterized in that, The first flow path further includes at least one of a first reservoir, a first drive pump, a first pipeline, a first sampling port, and a first flow regulating device; and / or, the second flow path further includes at least one of a second reservoir, a second drive pump, a second pipeline, a second sampling port, and a second flow regulating device.

8. The vascularized organoid chip according to claim 1, characterized in that, When the vascularized organoid chip is used to culture organoids, the organoids are located outside the hollow fiber membrane and in the culture chamber, and the biomimetic interface of the basement membrane is disposed between the endothelial cell layer and the organoids.

9. A method for culturing organoids, characterized in that, Includes the following steps: The organoid is placed in the culture chamber of the vascularized organoid chip according to any one of claims 1-8, and the organoid is outside the hollow fiber membrane.

10. The application of the vascularized organoid microarray according to any one of claims 1-8 in the fields of tissue development, disease modeling, drug screening and toxicity evaluation, angiogenesis mechanism research or cell product culture.

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