A liver organoid construct and methods of making the same

Abstract

The application discloses a kind of liver organoid constructs and preparation method thereof, belong to liver organoid construction technical field, the construct is multi-layer spatial partition structure, by inside to outside sequentially set inner layer organoid function area, liver sinusoid-Disse gap selective exchange layer, oxygen release-vesselization induction layer and dynamic immune adaptation outer layer, with oxygen and nutrient selective exchange, active oxygen removal, controllable oxygen release, vesselization induction, early immune inflammation regulation function, the application adopts four-layer partition structure, can realize nutrient, oxygen selective exchange, effectively remove active oxygen, protect cell from oxidative damage.It can controllably release oxygen and induce angiogenesis, ensure blood supply after transplantation;Outer layer can adjust immunity, suppress early inflammation, improve biocompatibility and survival rate.The construct forming mode is various, strong adaptability, can be used for liver tissue transplantation, repair, disease treatment and in vitro model construction.

Description

Technical Field

[0001] This invention relates to the field of liver organoid construction technology, and more specifically, to a liver organoid construct and its preparation method. Background Technology

[0002] Liver organoids are miniature tissues formed from stem cells, liver progenitor cells, primary hepatocytes, or various liver-related cells under three-dimensional culture conditions, possessing certain liver tissue structure and liver-specific functions. With the development of stem cell induced differentiation, three-dimensional culture, and tissue engineering materials technologies, liver organoids have significant application prospects in areas such as liver disease model construction, drug screening, hepatotoxicity evaluation, personalized treatment, and liver tissue replacement therapy.

[0003] However, liver organoids still face challenges in long-term survival and functional maintenance during in vivo transplantation. In the early stages of transplantation, the organoids have not yet established an effective connection with the host's vascular system, and oxygen and nutrients mainly rely on diffusion from surrounding tissue fluid, making them prone to internal hypoxia, accumulation of metabolic waste, and decreased cell activity. Hepatocytes are highly sensitive to changes in oxygen supply and the metabolic microenvironment; hypoxia, local pH fluctuations, and stimuli such as inflammatory factors and reactive oxygen species can all affect liver-specific functions such as albumin secretion, urea synthesis, and drug metabolism. Furthermore, if the construct structure is too dense or remains in a closed, embedded state for an extended period, host cells and new blood vessels cannot enter in time, which can also affect vascularization and tissue integration in the later stages of transplantation.

[0004] Therefore, we have made improvements to this by proposing a liver organoid construct and its preparation method. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the present invention aims to provide a liver organoid construct and its preparation method.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows: A method for preparing a liver organoid construct, wherein the construct has a four-layer spatial partition structure, and from the inside out are arranged an inner organoid functional region, a liver sinusoid-Disse gap selective exchange layer, an oxygen-releasing-angiogenic continuation layer, and a dynamic immune adaptation outer layer; the preparation steps are as follows: preparing liver organoids, preparing the inner organoid functional region, constructing each functional layer layer by layer, and cross-linking and curing the whole to obtain the finished construct.

[0007] Preferably, the liver organoids are selected from induced pluripotent stem cell-derived organoids, embryonic stem cell-derived organoids, liver progenitor cell-derived organoids, primary hepatocyte-derived organoids, composite organoids, and bile duct organoids; the composite organoids are prepared by co-culturing liver parenchymal cells with liver sinusoidal endothelial cells, bile duct epithelial cells, stellate cells, and mesenchymal cells, respectively.

[0008] Preferably, the inner organoid functional region is made of a mixture of liver organoids and extracellular matrix material; the extracellular matrix material is selected from gelatin methacrylamide hydrogel, collagen, hyaluronic acid, basement membrane matrix, decellularized liver matrix, fibrin, alginate, and polyethylene glycol hydrogel; when gelatin methacrylamide hydrogel and hyaluronic acid are used in combination, the concentration of gelatin methacrylamide hydrogel is 5% to 15% w / v, and the concentration of hyaluronic acid is 0.1% to 2% w / v.

[0009] Preferably, the matrix of the simulated liver sinusoid-Disse space selective exchange layer is selected from gelatin methacrylamide hydrogel, collagen, hyaluronic acid, alginate, polyethylene glycol hydrogel, liver decellularized matrix, basement membrane matrix, and liver sinusoidal endothelial-associated extracellular matrix components; the thickness of the layer is 10 μm to 5000 μm, and the pore size is 10 nm to 50 μm; reactive oxygen species scavenging / antioxidant components are added to the layer, and the reactive oxygen species scavenging / antioxidant components are selected from catalase, superoxide dismutase, glutathione, ascorbic acid derivatives, cerium dioxide nanoparticles, polydopamine nanoparticles, black phosphorus nanosheets, and manganese dioxide nanoparticles.

[0010] Preferably, catalase and cerium dioxide nanoparticles are simultaneously added to the simulated liver sinusoid-Disse space selective exchange layer; wherein the concentration of catalase is 10 U / mL to 1000 U / mL, and the concentration of cerium dioxide nanoparticles is 0.01 mg / mL to 2 mg / mL.

[0011] Preferably, the oxygen-releasing-angiogenic transition layer is composed of a hydrogel matrix, an oxygen-releasing material, and an angiogenic component; the hydrogel matrix is ​​selected from gelatin methacrylamide hydrogel, alginate, collagen, hyaluronic acid, fibrin, polyethylene glycol, chitosan, and decellularized matrix hydrogel; the oxygen-releasing material is selected from peroxides, oxygen carriers, oxygen-releasing microparticles, and oxygen-releasing nanoparticles, preferably CaO2@PLGA-coated peroxide oxygen-releasing microparticles and CaO2@PCL-coated peroxide oxygen-releasing microparticles; the particle size of the CaO2@PLGA microparticles is 100 nm to 50 μm, preferably 5 μm to 20 μm; the angiogenic component is selected from vascular endothelial growth factor, exosomes, and functional cells; when vascular endothelial growth factor is added, the concentration of vascular endothelial growth factor is 10 ng / mL to 500 ng / mL.

[0012] Preferably, the dynamic immune aptamer outer layer is composed of an immunomodulatory matrix, anti-inflammatory / immunomodulatory factors, antioxidant components, and tissue integration promoting components; the immunomodulatory matrix is ​​selected from gelatin methacrylamide hydrogel, hyaluronic acid, collagen, fibrin, polyethylene glycol, alginate, chitosan, and decellularized matrix hydrogel; the anti-inflammatory and immunomodulatory factors are selected from IL-10, TSG-6, TGF-β, prostaglandin E2, polydopamine nanoparticles, cerium dioxide nanoparticles, antioxidant enzymes, and immunomodulatory cell-derived factors.

[0013] Preferably, the overall morphology of the construct is spherical, near-spherical, sheet-like, columnar, disc-like, blocky, layered, core-shell-like, concentric ring-like, or a customized structure adapted to the transplantation site; when the construct is spherical / near-spherical, the overall diameter is 0.5cm to 8cm; the specific proportions of the dimensions of each structural layer are as follows: the inner organoid functional area accounts for 40% to 70% of the overall diameter, the thickness of the liver sinusoid-Disse gap selective exchange layer accounts for 2% to 15% of the overall diameter, the thickness of the oxygen-releasing-angiogenic continuation layer accounts for 5% to 25% of the overall diameter, and the thickness of the dynamic immune adaptation outer layer accounts for 5% to 25% of the overall diameter; the overall cross-linking and curing method adopts photo-cross-linking, thermosensitive gelation, ionic cross-linking, enzymatic cross-linking, and chemical cross-linking; when photo-cross-linking is used, the cross-linking wavelength is 365nm to 405nm, and the cross-linking time is 10s to 120s.

[0014] Preferably, the molding process of the construct includes mold casting, layer casting, multi-material bioprinting, coaxial printing, microfluidic assembly, sacrificial template, cryogenic casting, support bath printing, and layered photocrosslinking. When using multi-material bioprinting, corresponding printing inks are prepared according to the four-layer structure, and the molding is completed layer by layer from the inside out. The construct prepared by this method can be used to prepare liver organoid transplant materials, liver tissue repair materials, adjuvant treatment materials for liver failure, artificial liver systems, in vitro liver tissue models, and drug screening models.

[0015] Preferably, a liver organoid construct is provided, wherein the construct has a multi-layered spatial partition structure, and from the inside out are arranged an inner organoid functional area, a liver sinusoid-Disse space selective exchange layer, an oxygen release-angiogenic continuation layer, and a dynamic immune adaptation outer layer, which has the functions of selective exchange of oxygen and nutrients, removal of reactive oxygen species, controlled oxygen release, angiogenicity, and regulation of immune inflammation in the early stage of transplantation.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: Employing a four-layer, partitioned composite structure, each functional layer works synergistically. On one hand, it utilizes a selective exchange layer mimicking the sinusoidal-Disse gap to achieve directional exchange of nutrients and oxygen, while effectively scavenging reactive oxygen species with antioxidant components to prevent oxidative damage to cells and maintain the physiological activity of the internal liver organoids in the long term. On the other hand, it provides continuous and controllable oxygen supply through oxygen-releasing materials, combined with angiogenesis-promoting components to induce angiogenesis, solving the problem of insufficient blood supply after transplantation. Simultaneously, the dynamic immune-adaptive outer layer can effectively regulate the early immune response and inhibit inflammation, significantly improving the biocompatibility and in vivo retention of the construct. This construct can be prepared in various forms, adapting to diverse molding processes and different application scenarios. It can not only meet the needs of liver tissue transplantation, damage repair, adjuvant treatment of liver failure, and artificial liver systems, but also construct in vitro liver tissue models and drug screening models. The overall structural design conforms to the natural physiological microenvironment of the liver, and its comprehensive performance is significantly improved compared to traditional liver organoid products, greatly enhancing its application scope and practical value. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of the spherical, space-isolated, oxygen-releasing, and vascularized liver organoid construct with a simulated liver sinusoid-Disse gap interface as described in this invention. The construct comprises, from the inside out, an inner organoid functional region, a simulated liver sinusoid-Disse gap selective exchange layer, an oxygen-releasing and vascularization continuation layer, and a dynamic immune adaptation outer layer. Figure 2 This diagram illustrates the hierarchical structure, function, composition, and interaction of the inner organoid functional region, the liver sinusoid-Disse gap selective exchange layer, the oxygen-releasing-angiogenic continuation layer, and the dynamic immune aptor outer layer in the construct described in this invention. Figure 3 The diagram shows the results of selective exchange, reactive oxygen species buffering, and organoid function maintenance of the selective exchange layer of the liver sinusoid-Disse gap described in this invention; where a is a schematic diagram of selective exchange, b is the result of relative fluorescence intensity of ·OH, c is the result of relative fluorescence intensity of H2O2, d is the result of relative fluorescence intensity of ROS, e is the result of albumin secretion, f is the result of urea production, and g is the result of permeability of substances with different molecular weights. Figure 4 The above figures show the results of in vitro oxygen release and organoid function maintenance of the construct described in this invention; where a is the curve of dissolved oxygen concentration changing over time, b is the result of urea production, c is the result of albumin secretion, d is the result of DCFH-DA relative fluorescence intensity, and e is the result of live cell ratio. Figure 5The images show the vascularization, inflammatory response, and tissue integration results after in vivo transplantation of the construct described in this invention; where a is an in vivo vascularization appearance observation at different time points, b is a CD68 immunofluorescence staining image, c is a CD31 immunofluorescence staining image, d is a result of the number of CD68 positive cells, e is a result of the proportion of CD31 positive area, and f is a result of the collagen volume fraction. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. 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 without creative effort should fall within the scope of protection of the present invention.

[0019] A method for preparing a liver organoid construct, wherein the construct has a four-layer spatial partition structure, and from the inside out are arranged an inner organoid functional region, a liver sinusoid-Disse gap selective exchange layer, an oxygen-releasing-angiogenic continuation layer, and a dynamic immune adaptation outer layer; the preparation steps are as follows: preparing liver organoids, preparing the inner organoid functional region, constructing each functional layer layer by layer, and cross-linking and curing the whole to obtain the finished construct.

[0020] Furthermore, the liver organoids are selected from induced pluripotent stem cell-derived organoids, embryonic stem cell-derived organoids, liver progenitor cell-derived organoids, primary hepatocyte-derived organoids, composite organoids, and bile duct organoids; the composite organoids are prepared by co-culturing liver parenchymal cells with liver sinusoidal endothelial cells, bile duct epithelial cells, stellate cells, and mesenchymal cells, respectively.

[0021] Furthermore, the inner organoid functional area is made of a mixture of liver organoids and extracellular matrix-like materials; the extracellular matrix-like materials are selected from gelatin methacrylamide hydrogel, collagen, hyaluronic acid, basement membrane matrix, decellularized liver matrix, fibrin, alginate, and polyethylene glycol hydrogel; when gelatin methacrylamide hydrogel and hyaluronic acid are used in combination, the concentration of gelatin methacrylamide hydrogel is 5% to 15% w / v, and the concentration of hyaluronic acid is 0.1% to 2% w / v; The inner organoid functional region consists of liver organoids and extracellular matrix-like materials. The liver organoids are formed from one or more of the following: induced pluripotent stem cells, embryonic stem cells, liver progenitor cells, primary hepatocytes, bile duct epithelial cells, hepatic sinusoidal endothelial cells, and mesenchymal cells, through three-dimensional culture, induced differentiation, or co-culture. The extracellular matrix-like materials are one or more combinations of collagen, gelatin methacrylamide hydrogel, hyaluronic acid, basement membrane matrix, decellularized liver matrix, fibrin, and alginate.

[0022] Furthermore, the matrix of the selective exchange layer mimicking the sinusoidal-Disse gap is selected from gelatin methacrylamide hydrogel, collagen, hyaluronic acid, alginate, polyethylene glycol hydrogel, decellularized liver matrix, basement membrane matrix, and sinusoidal endothelial-associated extracellular matrix components; the thickness of this layer is 10 μm to 5000 μm, and the pore size is 10 nm to 50 μm; reactive oxygen species (ROS) scavenging / antioxidant components are added to the layer, and these ROS scavenging / antioxidant components are selected from catalase, superoxide dismutase, glutathione, ascorbic acid derivatives, cerium dioxide nanoparticles, polydopamine nanoparticles, black phosphorus nanosheets, and manganese dioxide nanoparticles; The sinusoidal-Disse gap selective exchange layer is an interface layer with semi-permeability, buffering, and selective exchange capabilities. The material of the sinusoidal-Disse gap selective exchange layer is one or more of the following: gelatin methacrylamide hydrogel, collagen, hyaluronic acid, alginate, polyethylene glycol hydrogel, decellularized liver matrix, basement membrane matrix, and sinusoidal endothelial-associated extracellular matrix components. To enhance the buffering capacity of the liver sinusoidal-Disse gap selective exchange layer for oxygen-releasing byproducts, reactive oxygen species (ROS) scavenging components or antioxidant components may be added to the liver sinusoidal-Disse gap selective exchange layer. These ROS scavenging components or antioxidant components may be one or more of the following: catalase, superoxide dismutase, glutathione, ascorbic acid derivatives, cerium dioxide nanoparticles, polydopamine nanoparticles, and black phosphorus nanosheets. The sinusoidal-Disse space selective exchange layer, mimicking the liver's sinusoidal-Disse space, allows the passage of oxygen, small-molecule nutrients, and metabolic products, while restricting the direct entry of oxygen-releasing particles, some peroxides, reactive oxygen species, or large-molecule inflammatory substances into the inner organoid functional area. This mimicking sinusoidal-Disse space selective exchange layer can be a monolayer, multilayer, microporous, fibrous network, or gradient pore structure.

[0023] Furthermore, catalase and cerium dioxide nanoparticles are simultaneously added to the selective exchange layer of the sinusoidal-Disse space; wherein the concentration of catalase is 10U / mL to 1000U / mL, and the concentration of cerium dioxide nanoparticles is 0.01mg / mL to 2mg / mL.

[0024] Furthermore, the oxygen-releasing-angiogenic transition layer is composed of a hydrogel matrix, oxygen-releasing materials, and angiogenic components. The hydrogel matrix is ​​selected from gelatin methacrylamide hydrogel, alginate, collagen, hyaluronic acid, fibrin, polyethylene glycol, chitosan, and decellularized matrix hydrogel. The oxygen-releasing materials are selected from peroxides, oxygen carriers, oxygen-releasing microparticles, and oxygen-releasing nanoparticles, with CaO2@PLGA-coated peroxide oxygen-releasing microparticles and CaO2@PCL-coated peroxide oxygen-releasing microparticles being preferred. The particle size of CaO2@PLGA microparticles is 100 nm to 50 μm, preferably 5 μm to 20 μm. The angiogenic components are selected from vascular endothelial growth factors, exosomes, and functional cells. When vascular endothelial growth factor is added, the concentration of vascular endothelial growth factor is 10 ng / mL to 500 ng / mL.

[0025] Furthermore, the dynamic immune adaptor outer layer is composed of an immunomodulatory matrix, anti-inflammatory / immunomodulatory factors, antioxidant components, and tissue integration promoting components; the immunomodulatory matrix is ​​selected from gelatin methacrylamide hydrogel, hyaluronic acid, collagen, fibrin, polyethylene glycol, alginate, chitosan, and decellularized matrix hydrogel; the anti-inflammatory and immunomodulatory factors are selected from IL-10, TSG-6, TGF-β, prostaglandin E2, polydopamine nanoparticles, cerium dioxide nanoparticles, antioxidant enzymes, and immunomodulatory cell-derived factors.

[0026] Furthermore, the overall morphology of the construct can be spherical, near-spherical, sheet-like, columnar, disc-like, blocky, layered, core-shell-like, concentric ring-like, or a customized structure adapted to the transplantation site. When the construct is spherical / near-spherical, the overall diameter is 0.5cm to 8cm. The specific proportions of the dimensions of each structural layer are as follows: the inner organoid functional area accounts for 40% to 70% of the overall diameter, the thickness of the liver sinusoid-Disse gap selective exchange layer accounts for 2% to 15% of the overall diameter, the thickness of the oxygen-releasing-angiogenic continuation layer accounts for 5% to 25% of the overall diameter, and the thickness of the dynamic immune adaptation outer layer accounts for 5% to 25% of the overall diameter. The overall cross-linking and curing methods include photocrosslinking, thermosensitive gelation, ionic crosslinking, enzymatic crosslinking, and chemical crosslinking. When photocrosslinking is used, the crosslinking wavelength is 365nm to 405nm, and the crosslinking time is 10s to 120s.

[0027] Furthermore, the molding process of the construct includes mold casting, layer casting, multi-material bioprinting, coaxial printing, microfluidic assembly, sacrificial template, cryogenic casting, support bath printing, and layered photocrosslinking. When using multi-material bioprinting, corresponding printing inks are prepared according to the four-layer structure, and the molding is completed layer by layer from the inside out. The construct prepared by this method can be used to prepare liver organoid transplant materials, liver tissue repair materials, adjuvant treatment materials for liver failure, artificial liver systems, in vitro liver tissue models, and drug screening models.

[0028] The structure employs a four-layer gradient spatial partitioning structure, consisting of an inner organoid functional area, a liver sinusoid-Disse gap selective exchange layer, an oxygen-releasing and angiogenesis-promoting continuation layer, and a dynamic immune adaptation outer layer, from the inside out. The entire structure is prepared through a process of preparing liver organoids, constructing each functional layer, and cross-linking and solidifying the whole structure. Each layer relies on a dedicated matrix, functional components, and structural parameters to work together to simulate the natural liver microenvironment, comprehensively ensuring organoid activity, material exchange, in vivo compatibility, and application effects. The inner organoid functional region is the core functional component of the construct. It uses induced pluripotent stem cells, embryonic stem cells, liver progenitor cells, primary hepatocytes, composite organoids, and bile duct organoids as functional cell sources. The liver organoids are mixed with various extracellular matrix materials such as gelatin methacrylamide hydrogel, collagen, and hyaluronic acid to form the structure. The gelatin methacrylamide hydrogel and hyaluronic acid compound system has a defined concentration range. This region mainly undertakes the inherent physiological functions of the liver, while relying on the extracellular matrix to simulate the in vivo cell growth microenvironment to maintain the normal survival and physiological activity of hepatocytes and various composite cells. The selective exchange layer mimicking the sinusoidal-Disse space of the liver, located adjacent to the core area, uses various hydrogels and liver-derived matrices as substrates. The layer thickness is strictly limited to 10μm~5000μm and the pore size to 10nm~50μm, simulating the physiological structure of the human liver sinusoidal and Disse space to achieve selective permeable exchange of oxygen, nutrients, and metabolic products. This layer is compounded with catalase, cerium dioxide nanoparticles, and other reactive oxygen species scavengers and antioxidants, with limited concentrations of two core antioxidant components. This allows for the timely removal of reactive oxygen species generated within the system, preventing oxidative stress damage to internal liver organoids and ensuring stable cell survival in the core functional area. The oxygen-releasing and angiogenesis-promoting continuation layer uses various hydrogels as a matrix, combined with peroxides, oxygen carriers, and micro / nano oxygen-releasing particles as oxygen-releasing materials. It preferentially uses CaO2@PLGA and CaO2@PCL coated microparticles with specific particle size ranges to achieve long-term and controllable oxygen release, continuously supplying oxygen to the internal cells. At the same time, it adds angiogenesis-promoting components such as vascular endothelial growth factors, exosomes, and functional cells. Under the premise of limiting the concentration of vascular endothelial growth factors, it induces angiogenesis and helps the construct establish vascular communication with the surrounding tissue, solving the problem of oxygen supply and nutrient delivery after transplantation. The outermost dynamic immune adaptor layer, relying on the composite hydrogel matrix, is compounded with anti-inflammatory and immunomodulatory factors such as IL-10, TGF-β, nano-antioxidant materials, and antioxidant enzymes. On the one hand, it exerts an antioxidant effect, and on the other hand, it precisely regulates the local immune response, inhibits the inflammatory rejection response in the early stage of transplantation. At the same time, it promotes the fusion of the construct with the host tissue with the help of tissue integration components, which greatly improves the biocompatibility and survival rate after transplantation. This construct can be processed into various shapes, including spheres, sheets, and columns. The spherical / near-spherical products clearly define the size proportions of the four-layer structure. The entire structure can be cured through various methods such as photocrosslinking and ionic crosslinking, with photocrosslinking having a specific wavelength and duration. During the production stage, various mature processes such as mold casting, multi-material bioprinting, and microfluidic assembly can be used to adapt to different preparation scenarios. The resulting liver organoid construct integrates multiple functions, including substance exchange, anti-oxidation, oxygen supply and angiogenesis promotion, and immune regulation. It can be widely used in liver organoid transplantation, liver tissue repair, adjuvant therapy for liver failure, artificial liver system construction, and the construction of in vitro liver tissue models and drug screening models.

[0029] Furthermore, a liver organoid construct is provided, which has a multi-layered spatial partition structure. From the inside out, it is arranged as follows: an inner organoid functional area, a selective exchange layer mimicking the liver sinusoid-Disse gap, an oxygen release-angiogenic continuation layer, and a dynamic immune adaptation outer layer. It has the functions of selective exchange of oxygen and nutrients, removal of reactive oxygen species, controlled oxygen release, angiogenicity, and regulation of immune inflammation in the early stage of transplantation.

[0030] The construct employs a four-layer spatial partitioning structure arranged from the inside out, with each layer performing its specific function and working synergistically to fully simulate the liver's physiological microenvironment and achieve multiple core functions: the inner organoid functional zone serves as the functional core, fulfilling the inherent functions of liver cells; the middle sinusoid-Disse space selective exchange layer replicates the physiological space structure of the liver, enabling selective permeable exchange of oxygen, nutrients, and metabolites, while simultaneously scavenging reactive oxygen species and reducing oxidative damage; the oxygen-releasing and angiogenesis-promoting continuation layer continuously and controllably releases oxygen, ensuring oxygen supply to internal cells and inducing angiogenesis, establishing a material transport pathway between the construct and body tissues; the outermost dynamic immune adaptation layer regulates the immune response and inflammatory state in the early stages of transplantation, reducing the risk of rejection and inflammation. These four layers work together, integrating material exchange, antioxidation, oxygen supply, angiogenesis, and immune regulation, comprehensively ensuring the construct's cell activity, in vivo survival capacity, and biological functional stability. Among them, a four-layer spatially partitioned liver organoid transplantation construct was prepared using 3D printing. The spatial partitioning of the inner organoid functional region, the sinusoidal-Disse space selective exchange layer, the oxygen-releasing and angiogenesis-promoting continuation layer, and the dynamic immune adaptation outer layer was achieved through multi-material 3D printing. S1. Preparation of printing ink: The first printing ink was prepared as the printing ink for the functional areas of the inner organoids. The first printing ink consisted of 8% w / v GelMA, 0.5% w / v hyaluronic acid, 1%–5% v / v extracellular matrix components, 0.05% w / v LAP photoinitiator, and 100 liver organoids / mL.

[0031] A second printing ink was formulated as a selective exchange layer printing ink mimicking the sinusoidal-Disse space of the liver. The second printing ink comprises 5% w / v PEG-based hydrogel, 0.5%–1.0% w / v polysaccharide component, 100 U / mL–200 U / mL Catalase, 20 μg / mL–50 μg / mL LCeO2 nanoparticles, and 0.05% w / v LAP photoinitiator.

[0032] A third printing ink was formulated as the printing ink for the oxygen-releasing and angiogenesis-promoting continuation layer. The third printing ink consisted of 8% w / v GelMA, 0.5% w / v hyaluronic acid, 0.3 mg / mL to 0.5 mg / mL CaO2@PLGA microparticles, 50 ng / mL VEGF, 30 ng / mL to 50 ng / mL LFGF, and 0.05% w / v LAP photoinitiator.

[0033] A fourth printing ink was formulated as the outer printing ink for dynamic immunoadaptor printing. The fourth printing ink consisted of 8% w / v GelMA, 0.5% w / v hyaluronic acid, 50 ng / mL IL-10, 100 ng / mL–200 ng / mL TSG-6, and 0.05% w / v LAP photoinitiator.

[0034] Once all printing inks are prepared, store them at 4°C away from light and use them within 2 hours. S2. Printing Model Design: A spherical four-layer construct model was established using 3D modeling software. The overall diameter D of the construct is 13 mm. The model parameters are as follows: the inner organoid functional region has a diameter of 7 mm; the selective exchange layer mimicking the hepatic sinusoid-Disse gap has a thickness of 0.6 mm; the oxygen-releasing and angiogenesis-promoting continuation layer has a thickness of 1.2 mm; and the dynamic immune adaptation outer layer has a thickness of 1.2 mm. S3, Multi-material Printing: To improve the molding stability of centimeter-scale spherical hydrogel constructs, the printing process can be carried out in a sterile support bath, with removable sacrificial support material, or in a hemispherical auxiliary mold. The support bath can be a gelatin microgel support bath, a Pluronic F127 support system, an alginate microgel support system, or other biocompatible removable support systems. After printing and cross-linking are complete, the support material can be removed by temperature changes, buffer replacement, or mechanical assistance to obtain a spherical or near-spherical four-layer spatially partitioned construct.

[0035] The first, second, third, and fourth printing inks were each loaded into an independent sterile printing syringe. The printing temperature was controlled at 18℃–25℃, the printing platform temperature at 10℃–20℃, the nozzle diameter at 200μm–500μm, the printing pressure at 20kPa–100kPa, the printing speed at 2mm / s–10mm / s, and the layer height at 150μm–400μm. First, the first printing ink was used to form an inner organoid functional region with a diameter of 7mm. After printing, cross-linking was performed using 365nm–405nm light for 10s–30s to maintain structural stability. Then, the second printing ink was used to print a 0.6mm thick selective exchange layer mimicking the liver sinusoid-Disse gap outside the inner organoid functional region. After printing, photocross-linking was performed for 10s–30s. Finally, the third printing ink was used to print a 1.2mm thick oxygen-releasing and angiogenesis-promoting continuation layer outside the selective exchange layer mimicking the liver sinusoid-Disse gap. After printing, photocrosslinking is performed for 20-40 seconds. Finally, the process switches to the fourth printing ink to print a 1.2 mm thick dynamic immune adaptor outer layer outside the oxygen-releasing, angiogenesis-promoting continuation layer. After printing, overall photocrosslinking is performed for 30-60 seconds to allow the construct to solidify. After crosslinking, the construct is gently washed 1-2 times with sterile PBS to remove any uncrosslinked residue. S4. In vitro culture of the printed construct: The printed four-layer spatial partitioned construct was placed in a sterile culture container, and sufficient liver organoid culture medium was added. It was then cultured at 37℃ and 5% CO2. The culture medium was changed every 24-48 hours during the culture period. The morphology of the construct was observed after 1-3 days of pre-culture. A qualified construct should maintain a spherical or near-spherical structure, with complete four-layer partitions, uniform internal organoid distribution, and no obvious cracking, collapse, or interlayer separation.

[0036] Therefore, the effect verification examples are as follows: Example 1: Buffering and Functional Maintenance of the Selective Exchange Layer in the Hepatic Sinusoidal-Disse Space To verify the selective exchange capacity, reactive oxygen species (ROS) buffering capacity, and maintenance effect of the liver sinusoid-Disse gap selective exchange layer on liver organoid function, four comparison groups were set up: Control group, CaO2 group, DLI group, and DLI(-ROS) group. The Control group served as the control group for conventional organoid culture; the CaO2 group was an oxygen-releasing group with direct addition of CaO2 particles; the DLI group consisted of a construct with a liver sinusoid-Disse gap selective exchange layer containing ROS scavenging components; and the DLI(-ROS) group consisted of a liver sinusoid-Disse gap selective exchange layer construct without ROS scavenging components.

[0037] Experimental results showed that the CaO2 group had higher levels of ·OH, H2O2, and total ROS, indicating that oxidative stimulation may accompany the oxygen release process of CaO2. The DLI group had the lowest levels of these reactive oxygen species-related signals, indicating that the selective exchange layer mimicking the liver sinusoid-Disse gap can weaken the oxidative stimulation of oxygen release byproducts on organoid functional areas. The ROS-related signal in the DLI(-ROS) group was higher than that in the DLI group, indicating that reactive oxygen species scavenging components play an important role in reducing oxidative stress. (See attached figures). Figure 3 b~ Figure 3 d. Further examination of liver organoid function revealed that the DLI group showed superior albumin secretion and urea production compared to the CaO2 and DLI(-ROS) groups. This indicates that the construct of this invention, through the synergistic effect of a semi-permeable buffer interface and reactive oxygen species scavenging components, can better maintain the protein synthesis and nitrogen metabolism functions of liver organoids under oxygen-releasing conditions. The results are shown in [see attached table]. Figure 3 e and Figure 3 f. Simultaneously, permeability experiments using molecules of different molecular weights revealed that, compared to ordinary hydrogels, the DLI interface exhibits differentiated permeability characteristics to substances of different molecular weights, indicating that it is not merely a physical encapsulation structure but possesses selective exchange capabilities. The results are shown in [see figure]. Figure 3 g.

[0038] Example 2: Effects of a spatially isolated oxygen release system on the maintenance of organoid function To verify the effect of a spatially isolated oxygen release system on the long-term functional maintenance of organoids, four comparison groups were set up: DirectCaO2, CaO2@PLGA, Directmixing, and DLIsystem. The DirectCaO2 group was a direct CaO2 oxygen release group; the CaO2@PLGA group was a PLGA-coated oxygen-releasing microparticle group; the Directmixing group was a group where oxygen-releasing materials were directly mixed with organoids; and the DLIsystem group was the complete spatially partitioned construct containing a selective exchange layer mimicking the liver sinusoid-Disse gap, as described in this invention. Dissolved oxygen detection results showed that the DirectCaO2 group exhibited a rapid increase in oxygen concentration in the early stages, followed by a relatively rapid decrease; the CaO2@PLGA group showed a more stable oxygen release, indicating that PLGA coating can reduce the burst release effect of CaO2 and prolong the oxygen release time. (See attached figures). Figure 4 a. Further examination of organoid function revealed that, compared with the Directmixing group, the DLIsystem group maintained higher levels of urea production and albumin secretion during culture. This indicates that spatially isolating the oxygen-releasing material from the organoid functional region is beneficial for maintaining the metabolic and protein synthesis functions of the liver organoids. (See results below.) Figure 4 b and Figure 4 c.

[0039] Furthermore, the relative fluorescence intensity of DCFH-DA in the DLIsystem group was lower than that in the Directmixing group, while the proportion of viable cells was higher. This indicates that the construct of this invention can reduce intracellular oxidative stress caused by direct contact of oxygen-releasing materials with organoids and improve organoid cell survival. The results are shown in [Figure number missing]. Figure 4 d and Figure 4 e.

[0040] Example 3: Evaluation of vascularization, inflammatory response, and tissue integration after in vivo transplantation. To verify the inflammatory response, vascularization and tissue integration of the construct after in vivo transplantation, the construct prepared in Example 1 or Example 2, or a scaled-down construct with the same hierarchical structure, material composition and similar layer thickness ratio, was transplanted into animals, and the tissue reaction around and inside the construct was observed on days 3, 7, 14 and 28 after transplantation.

[0041] In vivo and external observations showed that with prolonged transplantation time, the number of vascular-like structures around the construct gradually increased and gradually approached or grew into the construct region. This indicates that the oxygen-releasing-angiogenic continuation layer and dynamic immune adaptor outer layer described in this invention can provide a favorable microenvironment for host vascularization continuation. (See results below.) Figure 5 a. Further immunofluorescence staining of the transplanted tissue was performed. CD68 staining results showed that there were certain inflammatory cell responses around the construct at different time points after transplantation, but no persistent excessive inflammatory accumulation was observed, indicating that the dynamic immune adaptor outer layer helps buffer the early host inflammatory response after transplantation. (See attached results). Figure 5 b and Figure 5 d. CD31 staining results showed that with prolonged transplantation time, the number of CD31-positive vascular structures in the transplantation area gradually increased, and the proportion of CD31-positive area improved. This indicates that the construct of this invention can support the ingrowth of host blood vessels into the construct region and promote the transition from early material oxygen supply to later host blood vessel oxygen supply. (See attached figures.) Figure 5 c and Figure 5 e. Collagen volume fraction analysis showed that collagen deposition around the construct was within an observable range at different time points after transplantation, indicating that the construct could gradually integrate with the host tissue. (See attached image.) Figure 5 f.

[0042] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0043] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A method of preparing a liver organoid construct, the method comprising, The construct is a four-layer spatial partition structure, consisting of an inner organoid functional region, a liver sinusoid-Disse gap selective exchange layer, an oxygen-releasing and angiogenesis-promoting continuation layer, and a dynamic immune adaptation outer layer, arranged sequentially from the inside out. The preparation steps are as follows: preparing liver organoids, preparing the inner organoid functional region, constructing each functional layer layer by layer, and cross-linking and curing the whole structure to obtain the finished construct.

2. The method for preparing a liver organoid construct according to claim 1, characterized in that, The liver organoids are selected from induced pluripotent stem cell-derived organoids, embryonic stem cell-derived organoids, liver progenitor cell-derived organoids, primary hepatocyte-derived organoids, composite organoids, and bile duct organoids; the composite organoids are prepared by co-culturing liver parenchymal cells with liver sinusoidal endothelial cells, bile duct epithelial cells, stellate cells, and mesenchymal cells, respectively.

3. The method for preparing a liver organoid construct according to claim 1, characterized in that, The inner organoid functional region is made of a mixture of liver organoids and extracellular matrix materials; the extracellular matrix materials are selected from gelatin methacrylamide hydrogel, collagen, hyaluronic acid, basement membrane matrix, decellularized liver matrix, fibrin, alginate, and polyethylene glycol hydrogel; when gelatin methacrylamide hydrogel and hyaluronic acid are used in combination, the concentration of gelatin methacrylamide hydrogel is 5% to 15% w / v, and the concentration of hyaluronic acid is 0.1% to 2% w / v.

4. The method for preparing a liver organoid construct according to claim 1, characterized in that, The matrix of the simulated liver sinusoid-Disse space selective exchange layer is selected from gelatin methacrylamide hydrogel, collagen, hyaluronic acid, alginate, polyethylene glycol hydrogel, liver decellularized matrix, basement membrane matrix, and liver sinusoidal endothelial-associated extracellular matrix components; the thickness of this layer is 10 μm to 5000 μm, and the pore size is 10 nm to 50 μm; reactive oxygen species scavenging / antioxidant components are added to the layer, and the reactive oxygen species scavenging / antioxidant components are selected from catalase, superoxide dismutase, glutathione, ascorbic acid derivatives, cerium dioxide nanoparticles, polydopamine nanoparticles, black phosphorus nanosheets, and manganese dioxide nanoparticles.

5. The method for preparing a liver organoid construct according to claim 1, characterized in that, The selective exchange layer of the simulated liver sinusoid-Disse gap is simultaneously enriched with catalase and cerium dioxide nanoparticles; wherein the concentration of catalase is 10U / mL to 1000U / mL, and the concentration of cerium dioxide nanoparticles is 0.01mg / mL to 2mg / mL.

6. The method for preparing a liver organoid construct according to claim 1, characterized in that, The oxygen-releasing-angiogenic transition layer is composed of a hydrogel matrix, an oxygen-releasing material, and an angiogenic component. The hydrogel matrix is ​​selected from gelatin methacrylamide hydrogel, alginate, collagen, hyaluronic acid, fibrin, polyethylene glycol, chitosan, and decellularized matrix hydrogel. The oxygen-releasing material is selected from peroxides, oxygen carriers, oxygen-releasing microparticles, and oxygen-releasing nanoparticles, preferably CaO2@PLGA-coated peroxide oxygen-releasing microparticles and CaO2@PCL-coated peroxide oxygen-releasing microparticles. The particle size of the CaO2@PLGA microparticles is 100 nm to 50 μm, preferably 5 μm to 20 μm. The angiogenic component is selected from vascular endothelial growth factor, exosomes, and functional cells. When vascular endothelial growth factor is added, the concentration of vascular endothelial growth factor is 10 ng / mL to 500 ng / mL.

7. The method for preparing a liver organoid construct according to claim 1, characterized in that, The dynamic immune aptamer outer layer is composed of an immunomodulatory matrix, anti-inflammatory / immunomodulatory factors, antioxidant components, and tissue integration promoting components; the immunomodulatory matrix is ​​selected from gelatin methacrylamide hydrogel, hyaluronic acid, collagen, fibrin, polyethylene glycol, alginate, chitosan, and decellularized matrix hydrogel; the anti-inflammatory and immunomodulatory factors are selected from IL-10, TSG-6, TGF-β, prostaglandin E2, polydopamine nanoparticles, cerium dioxide nanoparticles, antioxidant enzymes, and immunomodulatory cell-derived factors.

8. The method for preparing a liver organoid construct according to claim 1, characterized in that, The overall morphology of the construct can be spherical, near-spherical, sheet-like, columnar, disc-like, blocky, layered, core-shell-like, concentric ring-like, or a customized structure adapted to the transplantation site. When the construct is spherical / near-spherical, the overall diameter is 0.5cm to 8cm. The specific proportions of the dimensions of each structural layer are as follows: the inner organoid functional area accounts for 40% to 70% of the overall diameter, the thickness of the liver sinusoid-Disse gap selective exchange layer accounts for 2% to 15% of the overall diameter, the thickness of the oxygen-releasing-angiogenic continuation layer accounts for 5% to 25% of the overall diameter, and the thickness of the dynamic immune adaptation outer layer accounts for 5% to 25% of the overall diameter. The overall cross-linking and curing methods include photocrosslinking, thermosensitive gelation, ionic crosslinking, enzymatic crosslinking, and chemical crosslinking. When photocrosslinking is used, the crosslinking wavelength is 365nm to 405nm, and the crosslinking time is 10s to 120s.

9. The method for preparing a liver organoid construct according to claim 1, characterized in that, The molding process of the construct includes mold casting, layer casting, multi-material bioprinting, coaxial printing, microfluidic assembly, sacrificial template, cry casting, support bath printing, and layered photocrosslinking. When using multi-material bioprinting technology, the corresponding printing inks are prepared according to the four-layer structure, and the molding is completed by printing layer by layer from the inside out. The constructs prepared using this method can be used to prepare liver organoid transplant materials, liver tissue repair materials, adjuvant treatment materials for liver failure, artificial liver systems, in vitro liver tissue models, and drug screening models.

10. A liver organoid construct, characterized in that, The construct is prepared by the preparation method according to any one of claims 1 to 9. The construct is a multi-layered spatial partition structure, which is arranged from the inside out as follows: an inner organoid functional area, a liver sinusoid-Disse gap selective exchange layer, an oxygen release-angiogenic continuation layer, and a dynamic immune adaptation outer layer. It has the functions of selective exchange of oxygen and nutrients, removal of reactive oxygen species, controlled oxygen release, angiogenicity, and regulation of early transplantation immune inflammation.

Images