Double-layer hydrogel microsphere encapsulating organoids and preparation method and use thereof

Abstract

The present invention belongs to the technical field of polymer materials, and in particular, relates to a double-layer hydrogel microsphere encapsulating an organoid, and a preparation method and use thereof. The double-layer hydrogel microsphere has an outer shell layer and a core layer, forming a core-shell double-layer structure, and a transplant is encapsulated in the core layer, and the elastic modulus of the outer shell layer and the core layer are different. The double-layer hydrogel microsphere of the present invention can simultaneously meet the requirements of the in vivo transplantation operation for the high elastic modulus of the hydrogel microsphere, and the requirements of the organoid for the low elastic modulus and the biomimetic biochemical microenvironment, thereby improving the function of the organoid.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer materials, and in particular, relates to a double-layer hydrogel microsphere encapsulating an organoid, and a preparation method and application thereof. Background Art

[0002] Hydrogel microspheres based on polymer materials have been widely used in drug delivery. In recent years, hydrogel microspheres composed of polymer materials have immune isolation functions and can be used for the in vivo delivery of organoids or cell therapy drugs, so that the encapsulated allogeneic cell therapy drugs are protected from attacks by the host immune system, thereby achieving long-term therapeutic effects in vivo. In order to ensure the mechanical stability of hydrogel microspheres, the elastic modulus of hydrogel microspheres is much higher than that of pancreatic or liver tissues in the body, resulting in impaired insulin or albumin secretion function, thereby reducing the therapeutic effect of transplanted cell drugs.

[0003] Therefore, an innovative manufacturing scheme for hydrogel microspheres based on polymer materials is urgently needed, so that the hydrogel microspheres have stable mechanical properties and effective immune isolation function, while having adjustable polymer material composition and mechanical properties after cross-linking, so as to obtain bionic extracellular matrix components and elastic modulus matching those of transplanted organoids, so as to enhance the biological activity and therapeutic effect of the transplant. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present invention provides a double-layer hydrogel microsphere encapsulating an organoid constructed based on high-voltage electrostatic non-contact spraying technology, and a preparation method and use thereof.

[0005] According to one aspect of the present invention, a double-layer hydrogel microsphere made of polymer material is provided, wherein the double-layer hydrogel microsphere has an outer shell layer and a core layer to form a core-shell double-layer structure, wherein a transplant is wrapped in the core layer, and the outer shell layer and the core layer have different elastic moduli.

[0006] In the present invention, the elastic modulus of the hydrogel after photocuring or other covalent crosslinking of the core layer material is adjusted by rheometer detection to be equivalent to the elastic modulus of the tissue. The extracellular matrix component of the transplant can be added to the core layer material to improve the biomimetic property of the extracellular matrix of the core layer.

[0007] According to one embodiment of the present invention, the elastic modulus of the core layer is smaller than the elastic modulus of the outer shell layer.

[0008] According to one embodiment of the present invention, the elastic modulus of the core layer is 200 Pa~10 kPa, 150 Pa~3 kPa, 150 Pa~2.5 kPa, 150 Pa~2 kPa, 150 Pa~1.5 kPa, 500 Pa~5 kPa, 800 Pa~3 kPa, 800 Pa~2 kPa, 800 Pa~1500 Pa, 800 Pa~2500 Pa, 1 kPa~3 kPa, 2kPa~3 kPa, 800 Pa~1000 Pa, 800 Pa~1100 Pa, 800 Pa~1200 Pa, 500 Pa~3 kPa, 500 Pa~2 kPa, 500 Pa~1500 Pa, 500 Pa~1300 Pa, 500 Pa~1200 Pa, 500 Pa~1100 Pa, 100 Pa, 150 Pa, 200 Pa, 300 Pa, 400 Pa, 500 Pa, 600 Pa, 700 Pa, 800 Pa, 900 Pa, 1000 Pa, 1100 Pa, 1200 Pa, 1300 Pa, 1400 Pa, 1500 Pa, 2000 Pa, 3000 Pa, 4000 Pa, 5000 Pa, 6000 Pa, 7000 Pa, 8000 Pa, 9000Pa, 10 kPa, etc., as well as any values ​​between them and the ranges formed by these values.

[0009] According to one embodiment of the present invention, the elastic modulus of the outer shell layer is 1 kPa~1 GPa, 1 kPa~100 MPa, 1 kPa~50 MPa, 1 kPa~30 MPa, 1 kPa~10 MPa, 1 kPa~5 MPa, 1 kPa~3MPa, 1 kPa~1 MPa, 1 kPa~800 kPa, 1 kPa~500 kPa, 1 kPa~300 kPa, 1 kPa~200 kPa, 1 kPa~100 kPa, 1 kPa~80 kPa, 1 kPa~50 kPa, 1 kPa~30 kPa, 1 kPa~20 kPa, 1 kPa~10 kPa, 1 kPa~8 kPa, 1 kPa~7 kPa, 1 kPa~6 kPa, 1 kPa~5 kPa, 1 kPa~4 kPa, 1 kPa~3 kPa, 1 kPa~2 kPa, 2 kPa~7 kPa, 2 kPa~5 kPa, 2 kPa~4kPa, 2 kPa~3 kPa, 1 kPa, 2 kPa, 2.5 kPa, 3 kPa, 3.5 kPa, 4 kPa, 4.5 kPa, 5 kPa, 6kPa, 7 kPa, 8 kPa, 9 kPa, 10 kPa, 20 kPa, 50 kPa, 70 kPa, 100 kPa, 300 kPa, 500 kPa, 700kPa, 800 kPa, 1 MPa, etc., and any values ​​between them, as well as the range composed of these values.

[0010] According to one embodiment of the present invention, the elastic modulus of the core layer is comparable to the elastic modulus of the implant in the core layer.

[0011] In the present invention, "equivalent" means that the elastic modulus of the core layer is the same as the elastic modulus of the implant in the core layer, or the difference is very small, for example, a difference of 0.01%-10%, a difference of 0.01%-0.1%, a difference of 0.01%-5%, a difference of 0.1%-3%, a difference of 0.1%-1%, a difference of 0.01%-1%, etc.

[0012] According to one embodiment of the present invention, the double-layer hydrogel microspheres are spherical or approximately spherical in structure as a whole, and the longest axis diameter is 0.2-3 mm.

[0013] According to one embodiment of the present invention, the shape of the core layer is spherical, approximately spherical, triangular pyramidal, T-shaped, spiral, linear or multi-point dispersed.

[0014] According to one embodiment of the present invention, the outer shell material comprises alginate or a derivative thereof.

[0015] According to one embodiment of the present invention, the outer shell layer material comprises alginate or its derivatives and / or other polymer materials.

[0016] According to one embodiment of the present invention, the core layer material is one or more of natural or artificially synthesized proteins, lipids, polysaccharides capable of covalent cross-linking, and polyethers and their derivatives, polyethylene glycol and their derivatives capable of covalent cross-linking.

[0017] According to one embodiment of the present invention, the outer shell layer and the core layer have different compositions of constituent materials.

[0018] According to one embodiment of the present invention, the outer shell layer and the core layer have different concentrations of constituent materials.

[0019] According to one embodiment of the present invention, the shell layer material comprises alginate or its derivatives, including sodium alginate, magnesium alginate, calcium alginate, barium alginate, sodium alginate modified with polycaprolactone, sodium alginate modified with polyethylene glycol, sodium alginate-polyethylene glycol-lipoic acid, sodium alginate-cyclodextrin, sodium alginate-polyethylene glycol-distearoyl phosphatidylethanolamine, sodium alginate-polyethylene glycol-hyaluronic acid, sodium alginate-polyethylene glycol-polyethyleneimine, seaweed One or more of the following: sodium alginate-polyethylene glycol-polylysine, sodium alginate-polyethylene glycol-polyornithine, sodium alginate-polyethylene glycol-polyarginine, dipalmitoylphosphatidylcholine-disulfide bond-sodium alginate, dioleoylphosphatidylcholine-polyethylene glycol-sodium alginate, dioleoylphosphatidylcholine-disulfide bond-sodium alginate, dimyristoylphosphatidylcholine-polyethylene glycol-sodium alginate, amino-polyethylene glycol-sodium alginate, polylactic acid-polyethylene glycol-sodium alginate.

[0020] According to one embodiment of the present invention, the outer shell material comprises natural and / or artificial polymer materials, including but not limited to one or more of polylipoic acid or its derivatives, cyclodextrin or its derivatives, polyethylene glycol or its derivatives, hyaluronic acid or its derivatives, polylysine or its derivatives, polyornithine or its derivatives, polyarginine or its derivatives, polylactic acid or its derivatives, silk fibroin or its derivatives, elastin or its derivatives, hyaluronic acid and its derivatives, heparin or its derivatives, dextran or its derivatives, DNA or its derivatives, chondroitin sulfate or its derivatives, polyethylene oxide-polypropylene oxide or its derivatives, and chitosan or its derivatives.

[0021] According to one embodiment of the present invention, the core layer material is selected from natural or synthetic polymer materials that can be cross-linked by gelation in a covalent bonding manner, including gelatin or its derivatives, including but not limited to gelatin, thiolated gelatin, and methacrylated gelatin; laminin or its derivatives, including but not limited to laminin, thiolated laminin, and methacrylated laminin; vitronectin or its derivatives, including but not limited to vitronectin, thiolated vitronectin, and methacrylated vitronectin; fibronectin or its derivatives, including but not limited to fibronectin, thiolated fibronectin, and methacrylated fibronectin; fibrin or its derivatives, including but not limited to fibrin, thiolated fibrin, and methacrylated fibrin; type I collagen or its derivatives, including but not limited to type I collagen, thiolated type I collagen, and methacrylated type I collagen; type II collagen or its derivatives, including but not limited to type II collagen, thiolated type II collagen, and methacrylated type II collagen; Type IV collagen or its derivatives, including but not limited to type IV collagen, thiolated type IV collagen, methacryloyl type IV collagen; matrigel; silk protein or its derivatives, including but not limited to silk protein, thiolated silk protein, methacryloyl silk protein; elastin or its derivatives, including but not limited to elastin, thiolated elastin, methacryloyl elastin; hyaluronic acid and its derivatives, including but not limited to hyaluronic acid, thiolated hyaluronic acid, methacryloyl hyaluronic acid; heparin or its derivatives, including but not limited to heparin, thiolated heparin, methacryloyl heparin; dextran or its derivatives, including but not limited to dextran, thiolated dextran, methacryloyl dextran; DNA or its derivatives; lipoic acid or its derivatives; chondroitin sulfate or its derivatives, including but not limited to chondroitin sulfate, thiolated chondroitin sulfate, methacryloyl chondroitin sulfate; polyethylene oxide-polypropylene oxide or its derivatives, including but not limited to Pluronic® F-127, polyether F127 diacrylate; chitosan or its derivatives, including but not limited to one or more of chitosan, thiolated chitosan, and methacryloyl chitosan.

[0022] According to one embodiment of the present invention, the core layer material is selected from natural or synthetic polymer materials that can be gel-crosslinked by covalent bonding, including one or more of methacrylated sodium alginate, thiolated sodium alginate, borneolized sodium alginate, polyether F127 diacrylate, methacrylated hyaluronic acid, methacrylated heparin, gelatin, methacrylated gelatin, laminin, vitronectin, type IV collagen, and type I collagen.

[0023] According to one embodiment of the present invention, the concentration of the constituent material of the outer shell layer is, for example, 0.5%-4% wt / v, 2%-5% wt / v, 2%-4% wt / v, 3%-4% wt / v, 1-3% wt / v, 1-2% wt / v, 1.5%-3%wt / v, 2% wt / v, 2.5% wt / v, 3% wt / v, 3.5% wt / v, 4% wt / v, 4.5% wt / v, 5% wt / v, etc.

[0024] According to one embodiment of the present invention, the concentration of the constituent materials of the core layer is, for example, 0.1%-10% wt / v, 1-10% wt / v, 1-6% wt / v, 1-3% wt / v, 2-5% wt / v, 0.5-8%, 0.5-5%, 0.5-5%, 1% wt / v, 1.5% wt / v, 2% wt / v, 2.5% wt / v, 5% wt / v, etc.

[0025] According to one embodiment of the present invention, the concentration of the constituent material of the core layer is, for example, 0.001-100 μg / mL, 0.01-50 μg / mL, 0.01-20 μg / mL, 0.1-5 μg / mL, 1-10 μg / mL, 1-5 μg / mL, 2-4 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 0.02-8 mg / mL, 1-5 mg / mL, etc.

[0026] According to one embodiment of the present invention, the outer shell layer is formed of a sodium alginate-polyethylene glycol-polyarginine solution, preferably, a sodium alginate-polyethylene glycol-polyarginine solution with a concentration of 0.5-4% wt / v, preferably, a sodium alginate-polyethylene glycol-polyarginine solution with a concentration of 1-3% wt / v.

[0027] According to one embodiment of the present invention, the constituent materials of the core layer are polyether F127 diacrylate, methacrylated heparin, vitronectin and laminin, preferably, the concentration of polyether F127 diacrylate is 1-10% wt / v, the concentration of methacrylated heparin is 0.1-10%, the concentration of vitronectin is 0.001-100 μg / mL and the concentration of laminin is 0.001-100 μg / mL, preferably, 1%-6% wt / v polyether F127 diacrylate, the concentration of methacrylated heparin is 0.5-5%, the concentration of vitronectin is 1-10 μg / mL and the concentration of laminin is 0.1-5 μg / mL.

[0028] According to one embodiment of the present invention, the outer shell layer is formed of a sodium alginate solution, preferably, a sodium alginate solution with a concentration of 0.5%-4% wt / v, preferably, a sodium alginate solution with a concentration of 1%-3% wt / v.

[0029] According to one embodiment of the present invention, the constituent materials of the core layer are methacrylated sodium alginate, type IV collagen and gelatin, preferably, methacrylated sodium alginate with a concentration of 1-3%, type IV collagen with a concentration of 0.02-8 mg / mL and gelatin with a concentration of 1-10% wt / v.

[0030] According to one embodiment of the present invention, the outer shell layer is formed of a material of sodium alginate-polyethylene glycol-lipoic acid, preferably, a sodium alginate-polyethylene glycol-lipoic acid solution with a concentration of 0.5-4% wt / v, preferably, 1-3% wt / v.

[0031] According to one embodiment of the present invention, the constituent material of the core layer is methacrylic anhydride modified gelatin (GelMA), preferably, the concentration of methacrylic anhydride modified gelatin (GelMA) is 1-20% wt / v, preferably, the concentration of methacrylic anhydride modified gelatin (GelMA) is 2-10% wt / v.

[0032] According to one embodiment of the present invention, the transplant is a pancreatic islet organoid, a pancreatic β cell cluster or a pancreatic β cell cluster, a pancreatic α cell cluster or a pancreatic α cell cluster, a liver organoid, a liver parenchymal cell cluster, a kidney organoid, a vascular organoid or a vascular endothelial cell cluster.

[0033] According to one embodiment of the present invention, the transplant is a pancreatic islet organoid or a liver organoid.

[0034] According to one embodiment of the present invention, the transplant is a pancreatic islet organoid.

[0035] According to one embodiment of the present invention, the transplant is a liver organoid.

[0036] According to one embodiment of the present invention, the pancreatic islet organoid has an arbitrary three-dimensional shape, and the diameter of the longest axis in the three-dimensional space is 10-500 μm, preferably 50-250 μm.

[0037] According to one embodiment of the present invention, the pancreatic islet organoid contains one or more cells including pancreatic β cells or pancreatic β cell-like cells having the function of secreting insulin.

[0038] According to one embodiment of the present invention, the pancreatic β cells or pancreatic β cell-like cells are human cells or non-human cells, and the sources of the pancreatic β cells or pancreatic β cell-like cells include one or more of the following:

[0039] i) Derived from the differentiation of induced pluripotent stem cells, embryonic stem cells, endoderm cells, pancreatic endoderm cells, pancreatic precursor cells, pancreatic progenitor cells, mesenchymal stem cells, etc.;

[0040] ii) cells extracted from human or animal pancreatic tissue or embryonic pancreatic tissue;

[0041] iii) Cells obtained through gene editing, virus packaging, establishment or transformation of i) and ii).

[0042] According to one embodiment of the present invention, each of the double-layer hydrogel microspheres encapsulates 1 to 200 pancreatic islet organoids.

[0043] According to one embodiment of the present invention, the liver organoid has an arbitrary three-dimensional shape, and the diameter of the longest axis in the three-dimensional space is 10-500 μm, preferably 40-120 μm.

[0044] According to one embodiment of the present invention, the liver organoid contains one or more cells including hepatocytes or hepatocyte-like cells having metabolic and secretory functions.

[0045] According to one embodiment of the present invention, the hepatocytes or hepatocyte-like cells are human cells or non-human cells, and the sources of the hepatocytes or hepatocyte-like cells include one or more of the following:

[0046] i) Derived from the differentiation of induced pluripotent stem cells, embryonic stem cells, endoderm cells, liver endoderm cells, liver precursor cells, liver progenitor cells, mesenchymal stem cells, etc.;

[0047] ii) cells extracted from human or animal liver tissue or embryonic liver tissue;

[0048] iii) Cells obtained through gene editing, virus packaging, establishment or transformation of i) and ii).

[0049] According to one embodiment of the present invention, each of the double-layer hydrogel microspheres encapsulates 1 to 200 liver organoids.

[0050] According to another aspect of the present invention, there is provided a method for preparing the double-layer hydrogel microspheres as described above, comprising the following steps:

[0051] (1) Preparation of grafts;

[0052] (2) preparing a shell layer polymer hydrogel material mixture and a core layer polymer hydrogel material mixture respectively;

[0053] (3) mixing the graft and the core layer polymer hydrogel material mixture to obtain a mixture A;

[0054] (4) three-dimensionally shaping the mixture obtained in step (2) and step (3) by an engineering method to obtain a double-layer hydrogel microsphere having an outer shell layer and a core layer, and positioning the transplant in the core layer region;

[0055] (5) Cultivating the double-layer hydrogel microspheres obtained in step (4).

[0056] According to one embodiment of the present invention, there is no order for step (1) and step (2). For example, step (1) may be performed first and then step (2), or step (2) may be performed first and then step (1), or step (1) and step (2) may be performed simultaneously.

[0057] According to one embodiment of the present invention, the operation of step (1) is: obtaining pancreatic β cells or cell-like cells thereof or pancreatic islet organoids, or liver cells or cell-like cells thereof or liver organoids, etc. by differentiating stem cells, primary tissue or gene editing. The number of cells constituting the organoids is adjusted by cell digestion and reaggregation so that the number of cells in each cell cluster is 200 to 2000, preferably 400 to 1000 cells per cell cluster. After culturing for 2 days, pancreatic islets or liver organoids with a nearly spherical shape and a longest axis diameter of 10 to 500 μm, preferably 50 to 250 μm, are obtained.

[0058] According to one embodiment of the present invention, the transplant is preferably a pancreatic islet organoid or / and a liver organoid.

[0059] According to one embodiment of the present invention, the operation of step (3) is as follows: in the mixture A, the concentration of the pancreatic islet organoids and / or liver organoids is 1×10 3 / mL~8×10 4 / mL, preferably 1×10 3 / ml~1×10 4 / mL, preferably 1×10 3 / ml~5×10 3 Pieces / mL.

[0060] According to one embodiment of the present invention, the specific operation of step (4) is: using an independently controlled dual-channel injection pump, the shell layer polymer hydrogel material mixture and the core layer polymer hydrogel material mixture mixed with the transplant are loaded into corresponding containers, and by adjusting the injection pump propulsion speed or propulsion pressure and voltage, the high-voltage electrostatic non-contact injection technology is used to position the transplant in the core layer area.

[0061] According to one embodiment of the present invention, when a dual-channel syringe pump is used, the propulsion speeds of the outer shell channel syringe pump and the core layer channel syringe pump are 0.06-100 ml / h, the propulsion speed of the outer shell channel syringe pump is preferably 5 ml / h~30 ml / h, and the propulsion speed of the core layer channel syringe pump is preferably 0.1 ml / h~20 ml / h.

[0062] According to one embodiment of the present invention, when printing using a high-voltage electrostatic non-contact printer, the DC high-voltage power supply voltage is preferably 4 kV to 13 kV, and the distance between electrodes is 1 cm to 3 cm.

[0063] According to one embodiment of the present invention, step (4) also includes operations of cross-linking stabilization treatment and sacrificial material removal treatment.

[0064] According to one embodiment of the present invention, the specific operation of step (4) is: using an independently controlled dual-channel injection pump, the shell layer polymer hydrogel material mixture and the core layer polymer hydrogel material mixture mixed with the transplant are loaded into corresponding containers, and by adjusting the injection pump propulsion speed or pushing pressure, voltage, and distance between electrodes, a high-voltage electrostatic non-contact printer is used to print and prepare double-layer hydrogel droplets, and then through stabilization treatment and sacrificial material removal, a double-layer hydrogel microsphere encapsulating the transplant is formed.

[0065] According to one embodiment of the present invention, the stabilization treatment means that the double-layer hydrogel microspheres need to be rapidly cross-linked to fix the shape.

[0066] According to one embodiment of the present invention, the cross-linking method for cross-linking the double-layer hydrogel microspheres can be, for example: (a) using a cross-linking agent containing divalent cations such as calcium ions, barium ions, and iron ions, (b) using a 405 nm light source in combination with a photoinitiator such as phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and / or tris (2,2′-bipyridyl) ruthenium (II) chloride hexahydrate and sodium persulfide for photocross-linking, (c) using one or more of thrombin, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide or their derivatives for cross-linking.

[0067] Preferably, the cross-linking method is:

[0068] (a) using a cross-linking agent containing divalent cations such as calcium ions, barium ions, iron ions, etc., and / or,

[0069] (b) photocrosslinking using a 405 nm light source in combination with lithium phenyl(2,4,6-trimethylbenzoyl)phosphate and / or tri(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfide, and / or,

[0070] (c) Thrombin.

[0071] According to one embodiment of the present invention, the concentration of divalent cations is 1 mM to 500 mM, preferably 10 mM to 200 mM, preferably 10 mM to 100 mM, preferably 10 mM to 50 mM, preferably 10 mM to 30 mM.

[0072] According to one embodiment of the present invention, the divalent cationic cross-linking agent solution comprises the following components: water, and one or more of barium chloride, sodium chloride, and calcium chloride.

[0073] According to one embodiment of the present invention, in the divalent cation crosslinker solution, the barium chloride concentration is 10-20 mM, the calcium chloride concentration is 50-200 mM, the mannitol concentration is 5% wt / v, and the sodium chloride concentration is 0.9 wt / v.

[0074] According to one embodiment of the present invention, the light intensity power of the photocrosslinking is 1-500 mW / cm 2 , preferably 1~200 mW / cm 2 , preferably 10~100 mW / cm 2 , the illumination time is 2 to 600 s, preferably 2 to 500 s, preferably 30 to 120 s, preferably 100 to 300 s.

[0075] According to one embodiment of the present invention, during photocrosslinking, a 405 nm light source and 30-300 mW / cm 2 , 4~300s.

[0076] The photoinitiator concentration is 0.25%~1% wt / v of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and / or tris(2,2′-bipyridyl)ruthenium (II) chloride hexahydrate and sodium persulfide (1~3 mM / 10~30 mM).

[0077] According to one embodiment of the present invention, the sacrificial material removal process refers to removing the structural auxiliary support material in the preparation process.

[0078] The structural auxiliary support material includes a thermosensitive material (such as gelatin, polyoxyethylene polyoxypropylene ether) or a photodegradable material or a material cleaved by oxidative free radicals (such as thioketal modified biomaterials) in a hydrogel microsphere, a photoinitiator (such as phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and / or tris (2,2'-bipyridyl) ruthenium (II) chloride hexahydrate and sodium persulfide), a crosslinking agent (such as thrombin), etc. Preferred sacrificial materials are, for example, gelatin and / or polyoxyethylene polyoxypropylene ether and / or phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and / or thrombin.

[0079] According to one embodiment of the present invention, in the sacrificial material removal process, the solution used may, for example, contain the following components: MCDB131, glucose 0.46 g / L, BSA 21 g / L, GlutaMAX 1% v / v, Heparin 10 mg / L, MEM NEAA 1% v / v.

[0080] According to one embodiment of the present invention, the culturing in step (5) includes static culturing and dynamic culturing.

[0081] According to one embodiment of the present invention, the static culture is carried out in a culture dish, a culture bottle or a multi-well plate;

[0082] The dynamic culture is carried out in an airlift bioreactor, a bubbling bioreactor, a hollow fiber bioreactor, a ceramic rectangular channel honeycomb reactor, a glass bead bed reactor, a fluidized bed reactor, a solidified bed reactor, a pulsating culture device, a microgravity culture device, a stirring culture device, a wave culture device, a chip or a perfusion culture system;

[0083] According to one embodiment of the present invention, the culture in step (5) can be large-scale cell culture, including the use of various forms of large-scale cell expansion platforms and devices such as cell factories.

[0084] According to one embodiment of the present invention, in step (5), the cell culture time is 3 to 60 days.

[0085] According to another aspect of the present invention, there is provided a polymer material composition having a three-dimensional spatial structure, comprising the double-layer hydrogel microspheres as described above.

[0086] According to another aspect of the present invention, there is provided use of the double-layer hydrogel microspheres or the polymer material composition described above in preparing a product for treating pancreatic islet or liver-related diseases.

[0087] According to one embodiment of the present invention, the disease is diabetes, pancreatitis, pancreatic tumor; preferably, the disease is chronic pancreatitis, benign pancreatic tumor, type I diabetes, type II diabetes.

[0088] According to one embodiment of the present invention, the disease is juvenile diabetes, diabetes associated with renal damage, and end-stage diabetes associated with renal failure.

[0089] According to one embodiment of the present invention, the disease is hepatitis, cirrhosis, or liver failure.

[0090] According to one embodiment of the present invention, the disease is alcoholic fatty liver, non-alcoholic fatty liver, alcoholic cirrhosis, non-alcoholic cirrhosis, viral cirrhosis, and drug-induced cirrhosis.

[0091] The double-layer hydrogel microspheres of the present invention have the following beneficial effects:

[0092] (1) The polymer hydrogel material of the microspheres of the present invention is composed of biomaterials with good biocompatibility and has the characteristics of good mechanical properties, low immunogenicity, slow degradation rate and high insulin permeability.

[0093] (2) In the microspheres of the present invention, the biomaterials of the outer shell layer and the core layer have heterogeneous biochemical compositions and physical properties, which can simultaneously meet the requirements of in vivo transplantation operations for high elastic modulus of hydrogel microspheres and the requirements of pancreatic organoids for low elastic modulus and biomimetic biochemical microenvironment, thereby improving the exocrine function of insulin and prolonging the survival rate of organoids.

[0094] (3) In the microspheres of the present invention, the outer shell hydrogel is composed of a polymer hydrogel material with good biostability and biocompatibility, and has a slow degradation rate in the body; it has a high elastic modulus, maintains the mechanical properties of the hydrogel microspheres, and is easy to inject.

[0095] (4) In the microspheres of the present invention, the material composition of the core layer includes extracellular matrix components of pancreatic islet tissue, which provides a bionic microenvironment and reduces cell aging and apoptosis of pancreatic organoids. In addition, the elastic modulus of the core layer hydrogel is equivalent to that of pancreatic islet tissue, which provides a bionic extracellular mechanical microenvironment for pancreatic islet organoids and significantly improves the exocrine capacity of pancreatic islet organoids.

[0096] (5) In the present invention, the pancreatic islet organoids of the double-layer hydrogel microspheres have a highly efficient insulin exocrine function, which meets the requirements of many fields such as precision medicine for pancreatic diseases, pancreatic engineering and regenerative medicine, preclinical drug testing, environmental / toxic substance / pollutant monitoring, pancreatic toxicology testing, development of new drugs related to pancreatic diseases, pancreatic developmental physiology, pancreatic pathology or drug, toxicity and efficacy testing.

[0097] (6) In the microspheres of the present invention, the material of the core layer provides a bionic liver mechanical microenvironment, improves the metabolic function of human primary hepatocytes cultured in vitro, and meets the requirements of many fields such as precision medicine for liver diseases, liver engineering and regenerative medicine, preclinical drug testing, environmental / toxic substance / pollutant monitoring, drug liver injury toxicology testing, liver disease-related new drug development, liver development physiology, liver pathology or drug, toxicity and efficacy testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 The schematic diagram of the double-layer hydrogel microspheres of the present invention encapsulating pancreatic islet organoids, wherein 1 is the outer shell layer, 2 is the core layer, and 3 is the pancreatic islet organoids.

[0099] Figure 2 Immunofluorescence staining analysis of human embryonic stem cells differentiated into pancreatic progenitor cells. Scale bar: 200 μm.

[0100] Figure 3 The shape and characteristics of the hESC-differentiated pancreatic progenitor cell cluster PP2 are shown in Figure 1. Figure 3 A is an optical photograph of the cell cluster formed by pancreatic progenitor cells PP2, scale bar 100 μm; Figure 3 B is Figure 3 Diameter and circularity analysis of cell clusters in A; Figure 3 C is a live-death staining image of the cell cluster formed by pancreatic progenitor cells PP2, Calcein-AM represents live cells, PI represents dead cells, scale bar 100 μm; Figure 3 D is a comparative analysis of the expression of pancreatic progenitor cell marker genes between hESC, hESC-differentiated PP2 monolayer culture cells and PP2 cell clusters. ns in the figure indicates no significance.

[0101] Figure 4 This is the elastic modulus diagram of the cross-linked shell layer and core layer polymer hydrogel material for rheological analysis, where **** p <0.0001.

[0102] Figure 5 The shape and characteristics of the double-layer hydrogel microspheres encapsulating hESC differentiated pancreatic islet organoids. Figure 5 A is the image of double-layer hydrogel microspheres; Figure 5 B is a micrograph of hESC-differentiated pancreatic islet organoids located in the core area of ​​the double-layer hydrogel microsphere; Figure 5 C is a statistical diagram of the diameter size distribution of double-layer hydrogel microspheres; Figure 5 D is a fluorescent staining live-death image of hESC islet organoids encapsulated in double-layer hydrogel; scale bar 200 μm.

[0103] Figure 6The effect of different elastic moduli of the core layer on the maturation of stem cell differentiated pancreatic islet organoids. Figure 6 A is the relative expression of INS marker genes under different elastic moduli; Figure 6 B is an immunofluorescence staining analysis of the expression of marker proteins in the double-layer hydrogel-encapsulated hESC differentiated pancreatic islet organoids. Among them, NKX6.1 and INS are marker proteins for differentiated mature pancreatic β cells, Hochest33342: cell nucleus; scale 50 μm; * p <0.05, **** p <0.0001, ns, not significant.

[0104] Figure 7 In vivo transplantation safety evaluation of double-layer hydrogel microspheres in C57BL / 6J mice. Figure 7 A Bilayer hydrogel microspheres 14 days after in vivo transplantation; Figure 7 B Double-layer hydrogel microspheres removed from the mesenteric transplantation site; Figure 7 C Masson staining of the mesentery embedded with double-layer hydrogel microspheres; Figure 7 D Routine blood test of mice after transplantation of double-layer hydrogel microspheres.

[0105] Figure 8 This is the ELISA detection image of insulin exocrine secretion of pancreatic islet organoids encapsulated in double-layer hydrogel microspheres.

[0106] Fig. 9 is the elastic modulus diagram of the outer shell and core layer polymer hydrogel materials based on single-layer hydrogel microspheres and double-layer hydrogel microspheres, where **** p <0.0001.

[0107] Fig.10 This is a biological comparison analysis of double-layer hydrogel microspheres and single-layer hydrogel microspheres encapsulating rat primary pancreatic islet cells. Fig.10 A: On the left is the optical microscope image of β-galactosidase chemical staining of rat primary pancreatic islets encapsulated by double-layer hydrogel microspheres and single-layer hydrogel microspheres, and on the right is a bar graph based on image statistical analysis, with a scale of 200 μm; Fig.10 B is a flow cytometric analysis of apoptosis and death of rat primary pancreatic islet cells; Fig.10 C is an ELISA analysis of insulin exocytosis of rat primary islet cells encapsulated by double-layer hydrogel microspheres and single-layer hydrogel microspheres; Fig.10 D is the insulin exocrine detection diagram under different elastic moduli of the core layer. * p <0.05,** p <0.01.

[0108] Fig.11This is a graph showing the results of a study on lowering blood sugar by transplanting rat primary pancreatic islet cells encapsulated in double-layer hydrogel microspheres in vivo. Fig.11 A: Hydrogel microspheres were transplanted into the mesentery of hyperglycemic mice. Fig.11 B. Blood sugar lowering experiment in which double-layer hydrogel microspheres encapsulating rat primary pancreatic islet cells were transplanted into the peritoneal cavity of hyperglycemic mice. The X-axis represents the number of days of in vivo transplantation. Fig.11 C Comparison of the blood glucose lowering efficiency of double-layer and single-layer hydrogel microspheres encapsulating rat primary pancreatic islet cells.

[0109] Fig.12 is the elastic modulus diagram of the shell layer and core layer polymer hydrogel material, where **** p <0.0001.

[0110] Fig.13 The expression diagram of liver organoid markers encapsulated by double-layer hydrogel microspheres. Fig.13 A is the transcriptional expression level of CYP1A1 gene expressed in hepatic parenchymal cells, Fig.13 B is the expression level of ALB protein and CYP1A1 protein, ** p <0.01,*** p <0.001, ns means no significant difference.

[0111] Fig.14 This is a graph showing the bile salt transport capacity of hepatic parenchymal cell clusters wrapped in double-layer hydrogel microspheres. Fig.14 A is after CLF incubation, Fig.14 B. Fig.14 C is after CLF is released.

[0112] Fig.15 This is a graph showing the fat accumulation ability of liver parenchymal cell clusters wrapped in double-layer hydrogel microspheres. Fig.15 A is a double-layer hydrogel microsphere encapsulating a liver parenchymal cell cluster, and Fig.15 B is the in situ staining of 3D suspension cultured cell pellets with Nile red, and DAPI represents the cell nucleus. Fig.15 C is the analysis result of Nile red fluorescence area ratio to cell nuclear area. ns means no significant difference. DETAILED DESCRIPTION

[0113] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0114] Example 1

[0115] This embodiment provides a method for preparing a double-layer hydrogel microsphere made of a polymer material based on a high-voltage electrostatic non-contact electrospraying technique, and encapsulating a pancreatic islet organoid (such as a pancreatic islet organoid) differentiated from stem cells. Figure 1 As shown, 1 is the outer shell layer, 2 is the core layer, and 3 is the pancreatic islet organoid). The outer shell layer of the hydrogel microspheres of this embodiment provides structural stability and immune isolation for the in vivo transplantation of pancreatic islet organoids differentiated from stem cells, and the core layer provides bionic extracellular matrix components for pancreatic islet organoids differentiated from stem cells, and can also provide a bionic mechanical microenvironment by controlling the degree of covalent cross-linking, thereby promoting the functional maturation of pancreatic islet organoids derived from stem cells. The details are as follows:

[0116] 1. Preparation of grafts

[0117] 1) Directed differentiation of human embryonic stem cells (ESCs) into pancreatic progenitor cells.

[0118] (a) hESCs were seeded into cell culture vessels and cultured to 100% confluence. They were first cultured for 3 to 5 days in induction medium 1, which included RPMI-1640 (Thermo Fisher, 11875093), 1×B27 (Gibco, insulin-free version, A1895601), 100 ng / mL Activin A (R&D, 338-AC) and 20 ng / mL WNT3A (R&D, 5036-WN). Restricted endoderm cells can be obtained at this stage.

[0119] (b) The cells were then cultured in induction medium 2 for 2 to 3 days. The ingredients included RPMI-1640, 1×B27, 50 ng / mL KGF (R&D, 5028-KG), and 10 ng / mL FGF10 (R&D, 345-FG).

[0120] (c) The cells were then cultured in induction medium 3 for 2-3 days, which included DMEM-F12 (Thermo Fisher, 11320033), 1×ITS (Gibco, 41400045), 50 ng / ml, 0.25 μM Sant1 (Selleck, S7092), 2 μM RA (Selleck, s1653), 200 nM LDN193189 (Selleck, s2618), and 500 nM PdBU (Yeasen, 53352ES03). At this stage, pancreatic progenitor cells PP1 were obtained.

[0121] (d) The cells were then cultured for 2 days in induction medium 4, which included DMEM-F12, 1×ITS, 50 ng / ml KGF, 0.25 μM Sant1, 100 nM RA, and 5 ng / mL Activin A. At this stage, pancreatic progenitor cells PP2 were obtained.

[0122] (e) Cell morphology observation and biological detection

[0123] Immunofluorescence staining was used to identify the expression of marker proteins in cells at different differentiation stages, and laser scanning confocal microscopy was used to observe and record the results. Figure 2 Among them, SOX17 and FOXA2 are restricted endoderm marker proteins; PDX1 is a pancreatic progenitor cell PP1 marker protein; PDX1 and NKX6.1 are pancreatic progenitor cell PP2 marker proteins.

[0124] During the differentiation of hESC into pancreatic progenitor cells, the restricted endoderm marker proteins SOX17 and FOXA2 were highly expressed, the pancreatic progenitor cell PP1 marker protein PDX1 was highly expressed, and the differentiation efficiency was as high as over 90%, and the pancreatic progenitor cell PP2 marker protein PDX1 and NKX6.1 double-positive cells were as high as over 60%, indicating that the present invention obtained PDX1 and NKX6.1-positive pancreatic progenitor cells.

[0125] 2) Preparation of pancreatic progenitor cell PP2 clusters

[0126] (a) Pancreatic progenitor cells PP2 were digested into single cells using Accutase and cultured at a rate of 3 × 10 6 The cells were inoculated into 6-well Aggrewell400 plates (Stemcell, 34421) per well and cultured statically for 2 days, and then cultured using induction medium 4 for 2 days.

[0127] (b) Next, the cells were replaced with induction medium 5, which contained MCDB 131 (Thermo Fisher, 10372019), 0.36 g / mL glucose (Selleck, S3131), 20 g / L FAF-BSA (Capricorn-Scientific, BSA-FAF-1U), 1% v / v GlutaMAX (Thermo Fisher, 35050061), 10 mg / L Heparin (MedChemExpress, HY-17567), 1% MEM NEAA (Thermo Fisher, 10370088), 168 μg / L ZnSO4 (Sigma, 221376), 0.25 μM Sant1 (Selleck, S7092), 100 nM RA (Selleck, s1653), 1 μM XXI (Sigma, 565790), and 10 μM Alk5i II (Selleck, S7223), 1 μM T3 (Selleck, S5726) and 20 ng / ml Betacellulin (Gibco, 100-50). At this stage, pancreatic progenitor cell pellets were obtained.

[0128] (c) Cell morphology observation and biological detection

[0129] (c1) Morphological observation and cell survival rate detection: In order to detect the size of pancreatic progenitor cell clusters, light microscopy and image measurement were used to detect the longest axis of the cell clusters. Figure 3 As shown, pancreatic progenitor PP2 cells form nearly spherical and compact cell clusters with the longest axis of about 150 μm ( Figure 3 A, 3B). 2 μM Calcein-AM and 4.5 μM PI staining solution were used for incubation for 15 minutes, and then the viability of cells in the cell clusters was detected using a laser scanning confocal microscope (LSCM, Nikon, Z2). Calcein AM is metabolized by living cells and produces fluorescent signals in cells, while PI can only produce fluorescent signals in dead cells. The results are shown in Figure 3 As shown, the cell survival rate in the cell clusters was good ( Figure 3 C).

[0130] (c2) Detection of pancreatic progenitor cell marker genes: qPCR technology was used to detect the transcription levels of pancreatic progenitor cell PP2 marker genes (such as PDX1 and NKX6.1) in the cell clusters. Figure 3As shown in D, compared with the monolayer culture cells before the pancreatic progenitor cell PP2 clusters formed, the PDX1 and NKX6.1 gene expression levels of the pancreatic progenitor cell PP2 clusters co-cultured under 3D conditions did not change significantly, indicating that the pancreatic progenitor cell PP2 clusters maintained the stem cell properties of pancreatic progenitor cells.

[0131] 2. Prepare the shell layer polymer hydrogel material mixture and the core layer polymer hydrogel material mixture respectively

[0132] 1) Preparation of the outer shell hydrogel mixture

[0133] Sodium alginate-polyethylene glycol-polyarginine was prepared into a solution with a concentration of 3% wt / v using 0.9% sodium chloride solution.

[0134] 2) Preparation of covalently cross-linkable core layer hydrogel mixture

[0135] Pluronic F127 diacrylate at a concentration of 5% wt / v, methacrylated heparin at a concentration of 1% wt / v, vitronectin (Sigma, 5051) at a concentration of 2 μg / mL, laminin (Sigma, L2020) at a concentration of 2 μg / mL, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) at a concentration of 0.25% wt / v.

[0136] 3) Material rheology testing

[0137] The results are as follows Figure 4 As shown, based on the rheological mechanical testing of the shell layer and the core layer mixture after cross-linking, the elastic modulus of the shell layer after ionic cross-linking is 3.155±0.210 kPa, and by optimizing the exposure time of the 405 nm light source, the elastic modulus of the core layer is 0.964±0.056 kPa. The elastic modulus of the shell layer hydrogel mixture is significantly greater than that of the core layer.

[0138] 3. Mix the graft and the core layer polymer hydrogel material mixture to obtain a mixture A:

[0139] Pancreatic progenitor cell PP2 cell clusters are added to the core layer hydrogel mixture in an amount of 50-100000 cells / mL, preferably 1000-5000 cells / mL.

[0140] 4. Preparation of double-layer hydrogel microspheres

[0141] 1) Use a dual-channel independently controlled microinjection pump and an 18G / 21G (outer layer / inner layer) coaxial stainless steel needle. The outer shell layer hydrogel mixture was loaded into the outer shell layer barrel, connected to the outer layer of the coaxial needle; the core layer material was loaded into the core layer barrel, connected to the inner layer of the coaxial needle, and then high-voltage electrostatic non-contact electrospray was used to prepare double-layer hydrogel microspheres. The DC high-voltage power supply voltage was set to 12 kV, the outer shell channel injection pump propulsion speed was 15 ml / h, the core channel injection pump propulsion speed was 5 ml / h, and the distance between electrodes was 2.5 cm. The hydrogel microsphere cross-linking solution was added to the plastic receiving dish, and the ingredients included water, barium chloride, sodium chloride, and mannitol, wherein the concentration of barium chloride (Sigma, 342920) was 20 mM, the concentration of mannitol (Sigma, BP1007) was 5%wt / v, the concentration of sodium chloride (Sigma, S7653) was 0.9 wt / v, and LAP was 0.25% wt / v. The hydrogel microspheres enter the receiving dish and solidify instantly through ionic coordination bonds.

[0142] Next, the hydrogel microspheres in the receiving dish were collected into a transparent 15 mL centrifuge tube and illuminated using a 405 nm light source with a power of 50 mw / cm 2 , irradiated for 60 seconds. This step completes the covalent cross-linking of the core layer of the hydrogel microspheres.

[0143] Next, the microspheres were washed three times with a buffer solution to remove sacrificial materials such as LAP and barium chloride. The buffer solution contained: MCDB131, glucose 0.46 g / L, BSA 21 g / L, GlutaMAX 1% v / v, heparin 10 mg / L, MEMNEAA 1% v / v. This step resulted in a double-layer hydrogel microsphere, with the pancreatic progenitor cell PP2 cell cluster located in the core layer.

[0144] 2) Size morphology of double-layer hydrogel microspheres: The collected double-layer hydrogel microspheres were observed and photographed using an inverted phase contrast microscope, and the diameter of the double-layer microspheres was analyzed by image analysis. Figure 5 As shown in AC, the double-layer hydrogel microspheres after printing and curing are approximately spherical, with an approximate diameter of about 1.5 mm.

[0145] 3) Cell live / dead staining detection: Use a mixed solution of 2 μM Calcein-AM (Sigma, 17783) and 4.5 μM PI (Sigma, GF16233836) to stain live / dead cells respectively. The staining is performed in the dark for 15 minutes. Use a laser scanning confocal microscope (LSCM, Nikon, Z2) to observe and record. The live / dead staining photos are statistically analyzed, such as Figure 5 As shown in D, the pancreatic progenitor cell PP2 cell clusters in the core layer of the double-layer hydrogel microspheres after printing have a high cell survival rate and almost no cell death.

[0146] 5. Cultivation of double-layer hydrogel microspheres

[0147] The double-layer hydrogel microspheres obtained in step 4 were added to a dynamic cell culture tank (Corning, 430825) for culture at a speed of 30 rpm / min, a culture environment of 37°C, a humidified environment, and 5% CO2. The culture medium contained MCDB131, glucose 0.46 g / L, BSA 21 g / L, GlutaMAX 1% v / v, Heparin 10 mg / L, MEM NEAA 1% v / v, Alk5i II (Selleck, S7223) 10 μM, and T3 1 μM. The medium was changed every other day, and the culture time was 7-20 days. In this step, pancreatic islet organoids encapsulated by double-layer hydrogel microspheres with insulin secretion function were obtained.

[0148] 6. Biological evaluation of pancreatic islet organoids encapsulated by double-layer hydrogel microspheres

[0149] 1) Double positive detection of NXK6.1 and INS.

[0150] By adjusting the illumination time and light intensity of the 405 nm light source and the content of the photoinitiator, the core layer with different elastic moduli can be obtained through rheological testing. Based on the above illumination conditions and initiators, double-layer hydrogel microspheres wrapped with the core layer of pancreatic islet organoids were made. After 7-20 days of culture, the cells were collected for qRT-PCR detection. The expression level of INS in pancreatic progenitor cells PP1 cells was used as the standard calculation to analyze the changes in the expression level of INS gene under different elastic moduli. The results are shown in Figure 6 As shown in A, when the elastic modulus is in the range of 150 Pa-3000 Pa, the expression level of INS gene increases significantly. At the same time, the expression level of INS shows a downward trend with the increase of elastic modulus. When the elastic modulus of the core layer is higher than 4000 Pa, the gene expression level of INS is not significantly increased compared with the pancreatic progenitor cell PP1, indicating that high elastic modulus hinders the development and maturation of pancreatic islet cells, and appropriate elastic modulus plays an important regulatory role in the development and maturation of pancreatic islet cells.

[0151] The pancreatic islet organoids encapsulated in double-layer hydrogel microspheres with a core layer elastic modulus of 850 Pa were stained in situ using cell immunofluorescence staining and NKX6.1 / INS antibody mixture. Hoechst 33342 was used to stain the cell nucleus. Laser confocal microscopy and 3D image analysis software were used to analyze the expression of NKX6.1 (Abcam, ab268088) / INS (suncell, SNL-332) proteins and the proportion of cells. The immunofluorescence staining method was as follows: the hydrogel microspheres were collected on the 15th day of culture, fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed three times with Hanks buffer, each for 5 minutes; blocked with a mixture containing 0.3% Triton-X (Sigma, X100) and 5% bovine serum albumin (Bovine Serum Albumin, BSA) (Solerbo, A8020) for 1 hour; the blocking buffer was aspirated, and the diluted primary antibody (containing 0.1% Triton-X and 1% BSA) was added, and incubated overnight at 4℃. Use Hanks buffer 3 times, 5 minutes each time; add the corresponding secondary antibodies (Abcam, ab205718) and (Abcam, ab205719), incubate at room temperature in the dark for 2 hours, wash with Hanks buffer 3 times, 5 minutes each time; then add Hoechst33342 to stain the cell nucleus, incubate at room temperature in the dark for 5 minutes. Observe and record with a laser confocal microscope (LSCM, Nikon, Z2).

[0152] The results are as follows Figure 6 As shown in B, the islet organoids encapsulated in the double-layer hydrogel microspheres highly expressed the islet tissue development and maturation markers NKX6.1 and INS proteins, indicating that after culture, the pancreatic progenitor cell PP2 cell clusters differentiated from pluripotent stem cells can develop and mature into islet organoids in the double-layer hydrogel microspheres and highly express islet maturation markers. This shows that the core layer of the double-layer hydrogel microspheres provides a suitable microenvironment for the maturation of the PP2 cell clusters into islet organoids.

[0153] 2) Safety testing of double-layer hydrogel microspheres for in vivo transplantation.

[0154] 10-50 hydrogel microspheres were transplanted into the mesentery of C57BL / 6J male mice. In order to evaluate the in vivo transplantation safety of the double-layer hydrogel microspheres, 14 days after in vivo transplantation ( Figure 7 A), Blood was collected from mice by orbital bleeding to detect the inflammatory response of mice, and the hydrogel microspheres in the peritoneal cavity of mice were removed for optical microscopy and histological examination.

[0155] The results are as follows Figure 7 As shown in the figure, the hydrogel microspheres removed from the mesentery were intact and undamaged, and there was no obvious deposition of mouse immune cells and fibroblasts on the surface of the hydrogel microspheres ( Figure 7 B) Histological section staining results showed that there was no obvious collagen deposition around the hydrogel microspheres ( Figure 7 C), these results show that the double-layer hydrogel microspheres constructed in this example did not cause obvious foreign substance immune response in mice. Blood test results showed that the number and percentage of immune cells such as white blood cells, lymphocytes, and monocytes in mice were within the normal range ( Figure 7 D). The above results indicate that the double-layer hydrogel microspheres constructed in this example have good in vivo transplantation safety.

[0156] 3) Insulin exocrine detection.

[0157] High glucose was used to stimulate the pancreatic islet organoids encapsulated in double-layer hydrogel microspheres, and the insulin exocrine detection kit (Alpco_80_INSHUU_E01.1) was used to detect the insulin exocrine function of the organoids in response to high glucose stimulation.

[0158] The method for detecting insulin exocrine secretion is as follows:

[0159] (a) Sample preparation: Centrifuge at 80 g to collect 1×10 6 The original culture medium was removed from the single-layer or double-layer hydrogel microspheres printed with cells, washed 3 times with Krebs buffer, cultured for 2 hours with Krebs buffer containing low glucose (2 mM / L), washed 3 times with Krebs buffer, and then cultured for 0.5 hours with Krebs buffer containing low glucose (2 mM / L), and the supernatant of the culture medium was collected and stored in a 4°C refrigerator for detection. Then the hydrogel microspheres were collected, washed once with Krebs buffer, cultured for 0.5 hours with Krebs buffer containing high glucose (20 mM / L), and the supernatant was collected and stored in a 4°C refrigerator for detection.

[0160] (b) Preparation of standards: After the kit is taken out of the refrigerator, it should be equilibrated at room temperature (25~28℃) for 20 minutes. Prepare an appropriate amount of washing solution: dilute the washing solution (20X) to 1X with double distilled water or deionized water. Add the standard diluent to 1 bottle of standard according to the volume marked on the standard label and incubate at room temperature for 15 minutes. Then gently mix and pipette several times to completely dissolve the standard. Usually, 2 wells are required for each concentration of standard, and the amount of standard in each well is 100 μl, resulting in a total of seven standard concentrations of 200, 100, 50, 25, 12.5, 6.25, and 0 μL U / mL. Finally, the diluted standards are added to the pre-coated plate wells in turn, for a total of seven standard concentrations.

[0161] (c) Detection: Add samples or standards of different concentrations to the corresponding wells at 25 μl / well. Then immediately add horseradish peroxidase-labeled human insulin antibody at 100 μl / well. Seal the reaction wells with a sealing film (transparent) and incubate at room temperature for 120 minutes. Then wash the plate 5 times, and pat dry on thick absorbent paper for the last time. Finally, add the color developer TMB solution at 100 μl / well, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 10 minutes. When the room temperature is low, the incubation time needs to be appropriately extended. At this time, the incubation can be continued until the standard and sample show a very significant color change. Add the stop solution at 50 μl / well, mix well and measure the A450 value immediately.

[0162] (d) Result analysis: The absorbance value of the background calibration well should be subtracted from the absorbance value of each standard or sample (if no calibration well is made, no subtraction is required). Draw a standard curve. Use the standard concentration as the horizontal axis and the A450 value as the vertical axis, and connect the coordinate points of each standard with a smooth line. Calculate the corresponding concentration of the sample through the absorbance value of the sample and the standard curve.

[0163] The results are as follows Figure 8 As shown, pancreatic islet organoids have a response effect to changes in glucose concentration, and the insulin secretion under high glucose concentration stimulation is greater than that of the low glucose concentration stimulation group. In addition, the insulin secretion of pancreatic islet organoids wrapped with 100 microspheres under high glucose stimulation is as high as 60 μL U / mL, which is much higher than the 20 μL U / mL of the positive control of the insulin test in the test kit. This result shows that the core layer of the double-layer hydrogel microspheres promotes the maturation of the exocrine insulin function of pancreatic islet organoids differentiated from stem cells.

[0164] Example 2

[0165] Hydrogel microspheres provide a physical barrier for the graft, reducing the immune rejection of allogeneic pancreatic islet tissue transplantation. Since the extracellular matrix and cell-cell interaction are destroyed during the islet tissue extraction process, the survival rate and exocrine function of pancreatic islet tissue cells in vivo transplantation are impaired.

[0166] This embodiment provides a preparation process for preparing double-layer hydrogel microspheres based on high-voltage electrostatic non-contact electrospray and polymer materials to encapsulate primary pancreatic islet tissue in rats; the extracellular matrix components of pancreatic islet tissue are added to the core layer material, and the mechanical properties of the matrix are adjusted, thereby reconstructing the interaction between pancreatic islet tissue and extracellular matrix, and improving the survival rate and insulin exocrine function of pancreatic islet tissue transplantation in vivo. This embodiment provides comparative data on the insulin exocrine function of traditional single-layer hydrogel microspheres and double-layer hydrogel microspheres, as well as the therapeutic effect on hyperglycemic mice, to illustrate that compared with the prior art, double-layer hydrogel microspheres have significant improvements.

[0167] The details are as follows:

[0168] 1. Preparation of rat primary pancreatic islet cell clusters.

[0169] Rat primary pancreatic islet cells were cultured at 3 × 10 6 The cells were inoculated into 6-well Aggrewell400 plates (Stemcell, 34421) per well and cultured for 2 days. The culture medium contained RPMI1640, 10% v / v fetal bovine serum, and 50 μM mercaptoethanol.

[0170] 2. Preparation of monolayer hydrogel microspheres encapsulating primary rat pancreatic islet cells

[0171] 1) Preparation of hydrogel mixture

[0172] Sodium alginate solution, type IV collagen solution, gelatin and rat primary pancreatic islet cells were mixed. Sodium alginate used SLG-20 (Sigma, 4202001) with a concentration of 2.5% wt / v, type IV collagen solution with a concentration of 2 mg / mL (Sigma), gelatin with a concentration of 2%, and cell microspheres with a number of 3000 / mL.

[0173] 2) Preparation of single-layer hydrogel microspheres

[0174] Use a microinjection pump and an 18G stainless steel needle to connect. Load the hydrogel mixture into the barrel of the syringe pump, and then use a high-voltage electrostatic non-contact printer to prepare a single-layer hydrogel microsphere. The DC high-voltage power supply voltage is set to 12 kV, the syringe pump advances at a speed of 12 ml / h, and the distance between electrodes is 2.5 cm. Add a hydrogel microsphere cross-linking solution to the plastic receiving dish, the ingredients include water, barium chloride, sodium chloride, and mannitol, where the concentration of barium chloride (Sigma, 342920) is 20 mM, the concentration of mannitol (Sigma, BP1007) is 5% wt / v, and the concentration of sodium chloride (Sigma, S7653) is 0.9 wt / v. The hydrogel microspheres enter the receiving dish and the ion coordination bonds are instantly solidified, completing the stabilization of the hydrogel microspheres. Next, the microspheres are washed three times with a buffer solution to remove sacrificial materials such as barium chloride.

[0175] 3) Cultivation of hydrogel microspheres

[0176] The buffer solution contains: MCDB131, glucose 0.46 g / L, BSA 21g / L, GlutaMAX 1% v / v, Heparin 10 mg / L, MEM NEAA 1% v / v. Then the cells were placed in a culture dish for static culture for 10 days. The culture medium contains RPMI1640, fetal bovine serum 10% v / v, and mercaptoethanol 50 μM.

[0177] 3. Preparation of double-layer hydrogel microspheres encapsulating primary rat pancreatic islet cells

[0178] 1) Preparation of shell layer hydrogel mixture: 2.5% wt / v sodium alginate SLG 20 solution was used for the shell layer.

[0179] 2) Preparation of core layer hydrogel mixture: The core layer is composed of 1% methacrylic anhydride sodium alginate, 2 mg / mL type IV collagen, 5% gelatin, tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfide (2 mM / 20 mM). Then, rat primary pancreatic islet cell clusters are added in an amount of 1000 / mL to 5000 / mL.

[0180] 3) Rheological testing of materials after cross-linking

[0181] After the single-layer polymer hydrogel material is cross-linked and the shell layer and core layer mixture of the double-layer hydrogel microsphere are cross-linked, the elastic modulus is measured by rheometer. The results are as follows Fig. 9 As shown, the elastic modulus of the single-layer hydrogel microsphere material after cross-linking is 2.654±0.016 kPa, the elastic modulus of the outer shell layer material of the double-layer hydrogel microsphere is 2.631±0.214 kPa, and the elastic modulus of the core layer material is 0.845±0.027 kPa.

[0182] 4) Preparation process: Use a dual-channel independently controlled microinjection pump and an 18G / 21G (outer layer / inner layer) coaxial stainless steel needle. Load the outer shell layer hydrogel mixture into the outer shell layer barrel, connecting the outer layer of the coaxial needle; load the core layer material into the core layer barrel, connecting the inner layer of the coaxial needle, and then use a high-voltage electrostatic non-contact printer to prepare double-layer hydrogel microspheres. The DC high-voltage power supply voltage is set to 12 kV, the outer shell channel injection pump propulsion speed is 15 ml / h, the core channel injection pump propulsion speed is 10 ml / h, and the distance between electrodes is 2.5 cm. A hydrogel microsphere crosslinking solution was added to a plastic receiving dish, the ingredients of which included water, barium chloride, sodium chloride, and mannitol, wherein the concentration of barium chloride (Sigma, 342920) was 20 mM, the concentration of mannitol (Sigma, BP1007) was 5% wt / v, the concentration of sodium chloride (Sigma, S7653) was 0.9 wt / v, tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfide (2 mM / 20 mM). The hydrogel microspheres entered the receiving dish and instantly solidified by ionic coordination bonds. The hydrogel microspheres were then collected and irradiated with a 405 nm light source for 4 minutes (50 mW / cm 2 ), thus completing the stabilization process. Next, the microspheres were washed three times with a buffer solution to remove sacrificial materials such as barium chloride.

[0183] 5) Microsphere collection and culture: The solution contains: MCDB131, glucose 0.46 g / L, BSA 21 g / L, GlutaMAX 1% v / v, Heparin 10 mg / L, MEM NEAA 1% v / v. Then put it in a culture dish for static culture for 10 days. The culture medium contains RPMI1640, fetal bovine serum 10% v / v, and mercaptoethanol 50 μM.

[0184] 4. Comparative analysis of the biological functions of two hydrogel microspheres encapsulating primary rat pancreatic islet cells

[0185] 1) Cell senescence detection. After 10 days of culture of the two microspheres, the β-galactosidase in situ staining kit (Biyuntian, RG0039) was used to detect the senescence of the cells encapsulated by the two microspheres. The detection method is as follows: collect the hydrogel microspheres on the 10th day of culture, fix them with the kit fixative at room temperature for 15 minutes, and wash them with Hanks buffer 3 times, 5 minutes each time. Stain with the staining working solution and incubate at 37°C for 20 minutes to 2 hours, or longer. Observe under an ordinary optical microscope.

[0186] The results are as follows Fig.10 As shown in A, the coloring of the cell clusters of rat primary pancreatic islet cells encapsulated by double-layer hydrogel microspheres is lighter than that of the cell clusters encapsulated by single-layer hydrogel microspheres, indicating that the aging effect of double-layer hydrogel microspheres on cells is lighter than that of single-layer hydrogel microspheres.

[0187] 2) Apoptosis detection. The detection method is as follows: collect hydrogel microspheres on the 10th day of culture, hydrate the microspheres with citric acid-EDTA solution, collect rat primary pancreatic islet cell clusters, and use a mixture of trypsin-EDTA and collagenase to digest the cell clusters into single cells. Then, flow cytometry and Annexin V-FITC apoptosis detection kit (Biyuntian, C1062S) were used to quantitatively detect the apoptosis of the cell clusters. Gently resuspend the single cells into a suspension with staining buffer, add 5 μL Annexin V-FITC, and 10 μl propidium iodide (PI) staining solution, and gently mix. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, and then analyze by flow cytometry.

[0188] The results are as follows Fig.10 As shown in B, the non-apoptotic and necrotic cells of rat primary pancreatic islet cells encapsulated by double-layer hydrogel microspheres accounted for 93.2%, apoptotic cells accounted for 3.8% (2.96% + 0.84%), and dead cells accounted for 3%. The non-apoptotic and necrotic cells encapsulated by single-layer hydrogel microspheres accounted for 82.04%, apoptotic cells accounted for 9.83% (7.69% + 2.14%), and dead cells accounted for 8.13%. The results show that the cell apoptosis and mortality of rat primary pancreatic islet cells encapsulated by double-layer hydrogel microspheres are lower than those of single-layer hydrogel microspheres.

[0189] 3) Insulin exocrine detection.

[0190] (a) Sample preparation: Preparation of double-layer hydrogel and single-layer hydrogel microspheres to encapsulate rat primary pancreatic islet cells. Based on the rheological test results, the hydrogel microspheres were irradiated with a 405 nm light source for 4 minutes and 10 minutes to obtain double-layer hydrogel microspheres with core layer elastic moduli of 1120 Pa and 4150 Pa, respectively. The hydrogel microspheres containing 1×10 6 The original culture medium was removed from the single-layer or double-layer hydrogel microspheres printed with cells, washed 3 times with Krebs buffer, cultured for 2 hours with Krebs buffer containing low glucose (2 mM / L), washed 3 times with Krebs buffer, and then cultured for 0.5 hours with Krebs buffer containing low glucose (2 mM / L), and the supernatant of the culture medium was collected and stored in a 4°C refrigerator for detection. Then the hydrogel microspheres were collected, washed once with Krebs buffer, cultured for 0.5 hours with Krebs buffer containing high glucose (20 mM / L), and the supernatant was collected and stored in a 4°C refrigerator for detection.

[0191] (b) Preparation of standards: After the kit is taken out of the refrigerator, it should be equilibrated at room temperature (25~28℃) for 20 minutes. Prepare an appropriate amount of washing solution: dilute the washing solution (20X) to 1X with double distilled water or deionized water. Add the standard diluent to 1 bottle of standard according to the volume marked on the standard label and incubate at room temperature for 15 minutes. Then gently mix and pipette several times to completely dissolve the standard. Usually, 2 wells are required for each concentration of standard, and the amount of standard in each well is 100 μL, resulting in a total of seven standard concentrations of 200, 100, 50, 25, 12.5, 6.25, and 0 μL U / mL. Finally, the diluted standards are added to the pre-coated plate wells in turn, for a total of seven standard concentrations.

[0192] (c) Detection: Add samples or standards of different concentrations to the corresponding wells at 25 μL / well. Then immediately add horseradish peroxidase-labeled human insulin antibody at 100 μL / well. Seal the reaction wells with a plate sealer (transparent) and incubate at room temperature for 120 minutes. Then wash the plate 5 times, and pat dry on thick absorbent paper for the last time. Finally, add the color developer TMB solution at 100 μL / well, seal the reaction wells with a plate sealer (white), and incubate at room temperature in the dark for 10 minutes. When the room temperature is low, the incubation time needs to be appropriately extended. At this time, the incubation can be continued until the standard and sample show a very significant color change. Add the stop solution at 50 μL / well, mix well, and measure the A450 value immediately.

[0193] (d) Result analysis: The absorbance value of the background calibration well should be subtracted from the absorbance value of each standard or sample (if no calibration well is made, no subtraction is required). Draw a standard curve. Use the standard concentration as the horizontal axis and the A450 value as the vertical axis, and connect the coordinate points of each standard with a smooth line. Calculate the corresponding concentration of the sample through the absorbance value of the sample and the standard curve.

[0194] The results are as follows Fig.10 As shown in C, under the induction of high concentration of glucose, the insulin secretion of the double-layer hydrogel microspheres was about 59.1 μL U / mL, which was much higher than the insulin secretion of the single-layer hydrogel microspheres, which was 39.5 μL U / mL. p <0.01, with significant difference. The results show that the biochemical and physical properties of the hydrogel in the core area of ​​the double-layer hydrogel microspheres provide a suitable microenvironment for insulin exocytosis of rat primary pancreatic islet cells. Fig.10 As shown in Figure D, the islet secretion of the double-layer microspheres with a low core layer modulus (1120 Pa) is higher than that of the double-layer microspheres with a high core layer modulus (4150 Pa), which indicates that the appropriate elastic modulus can promote the insulin exocytosis of pancreatic islet cells.

[0195] 5. Evaluation of the hypoglycemic ability of two hydrogel microspheres encapsulating primary rat pancreatic islet cells

[0196] Package 1×10 6 / mL rat primary pancreatic islet cells monolayer hydrogel microspheres or double-layer hydrogel microspheres were transplanted into the peritoneal mesentery of hyperglycemic mice ( Fig.11 A). Before transplantation, the blood glucose level of mice was higher than 11 mM / L, which was a typical feature of hyperglycemia. The day of transplantation was recorded as day 0. On the second day of transplantation, the blood glucose level of mice dropped below 11 mM / L, and the blood glucose level continued to drop for 14 days ( Fig.11 B). Moreover, the double-layer hydrogel microsphere group had a higher blood sugar lowering efficiency, with blood sugar reduced from 22.9 mM / L to 7.1 mM / L, but the single-layer hydrogel microsphere group had a blood sugar lowering efficiency of only 56%, from 14.4 mM / L to 8.1 mM / L ( Fig.11 C). These results indicate that the double-layer hydrogel has a better hypoglycemic effect.

[0197] Example 3

[0198] 1. Preparation of hepatocyte pellet.

[0199] Human primary hepatocytes cultured on a flat surface were digested into single cells using trypsin and plated at a rate of 3 × 10 6The cells were inoculated into 6-well plates of Aggrewell400 (Stemcell, 34421) per well and cultured statically for 2 days. After the cell clusters were collected, they were resuspended in complete culture medium for human hepatocytes (Pricella, CM-H043). Then, the cells were cultured in a dynamic culture shaker at 80 rpm at 37°C and 5% carbon dioxide in a humidified cell culture incubator for 3 to 10 days.

[0200] 2. Preparation of double-layer hydrogel microspheres encapsulating hepatocyte clusters

[0201] 1) Preparation of the outer shell layer hydrogel mixture: The outer shell layer uses an aqueous solution of sodium alginate-polyethylene glycol-lipoic acid with a concentration of 2.5% wt / v.

[0202] 2) Preparation of photo-crosslinked responsive core layer hydrogel mixture: 5% wt / v methacrylic anhydride-modified gelatin (GelMA), photoinitiator tri(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfide were added at a concentration of 0.5 mM / 5 mM. Then, hepatocytes were added at a number of 3000 / mL.

[0203] 3) Material rheology test: After the shell layer and core layer mixture of the double-layer hydrogel microspheres were cross-linked, the elastic modulus was tested using a rheometer. The results are as follows: Fig.12 As shown, the elastic modulus of the shell layer material of the double-layer hydrogel microspheres after cross-linking is 2.754±0.017 kPa, and the elastic modulus of the core layer material after photo-cross-linking is 0.964±0.016 kPa. Therefore, the elastic modulus of the shell layer hydrogel is significantly greater than that of the core layer hydrogel.

[0204] 4) Preparation process: Use a dual-channel independently controlled microinjection pump and an 18G / 23G (outer layer / inner layer) coaxial stainless steel needle. Load the outer shell layer hydrogel mixture into the outer shell layer barrel, connecting the outer layer of the coaxial needle; load the core layer material into the core layer barrel, connecting the inner layer of the coaxial needle, and then use a high-voltage electrostatic non-contact printer to prepare double-layer hydrogel microspheres. The DC high-voltage power supply voltage is set to 12 kV, the outer shell channel injection pump propulsion speed is 20 ml / h, the core channel injection pump propulsion speed is 10 ml / h, and the distance between electrodes is 2.5 cm. Add hydrogel microsphere cross-linking solution to the plastic receiving dish, the ingredients include water, barium chloride, sodium chloride, and mannitol, where the concentration of barium chloride (Sigma, 342920) is 20 mM, the concentration of mannitol (Sigma, BP1007) is 5% wt / v, and the concentration of sodium chloride (Sigma, S7653) is 0.9 wt / v. The hydrogel microspheres enter the receiving dish and solidify by ionic coordination bonds instantly, and then are irradiated with a 405 nM light source for 5 minutes to complete the stabilization process. Next, the microspheres are washed three times with a buffer solution to remove sacrificial materials such as barium chloride and photoinitiator tri(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfide.

[0205] 5) Microsphere collection and culture: The solution contains complete culture medium for human hepatocytes (Pricella, CM-H043). Then, the microspheres are cultured in a cell culture incubator at 37°C, 5% carbon dioxide and humidified conditions using a dynamic culture shaker at 80 rpm for 3 to 10 days.

[0206] 3. Biological function detection of hepatocytes encapsulated by double-layer hydrogel microspheres

[0207] 1) Expression of liver organoid markers encapsulated by double-layer hydrogel microspheres.

[0208] After culturing the double-layer hydrogel microspheres encapsulating the hepatic parenchymal cell clusters for 7 days, the cell clusters in the hydrogel microspheres were collected for hepatic parenchymal cell clusters, and the gene and protein expression levels of ALB and CYP1A1, markers related to hepatic parenchymal cell function, were detected by qRT-PCR and cell immunofluorescence staining.

[0209] like Fig.13 As shown, Fig.13 A indicates that the gene expression level of CYP1A1, a marker of liver detoxification function, in the hepatocytes encapsulated by the double-layer hydrogel microspheres was significantly higher than that in the human primary hepatocytes and single-layer hydrogel microspheres cultured in conventional plane (2D). The results of cell immunofluorescence staining further confirmed that ( Fig.13B) The ALB and CYP1A1 proteins of the hepatocytes encapsulated by the double-layer hydrogel microspheres were highly expressed. These results indicate that the double-layer hydrogel microspheres can better maintain the functions and cell characteristics of human primary hepatocytes than the single-layer hydrogel microspheres and 2D culture.

[0210] 2) Detection of bile salt transport capacity of hepatocytes encapsulated by double-layer hydrogel microspheres

[0211] After culturing the double-layer hydrogel microspheres encapsulating the hepatic parenchymal cell clusters for 7 days, cholecystokinin (CLF) was added to the culture medium for co-incubation, and then the culture medium was replaced with fresh culture medium without CLF to release CLF. The uptake and transport capacity of the hepatic parenchymal cell clusters for CLF was determined by detecting the fluorescence signal of CLF in the cell clusters.

[0212] like Fig.14 As shown, the hepatocytes encapsulated by the double-layer hydrogel microspheres efficiently took up CLF ( Fig.14 A arrow), after immersion in the culture medium for 4 hours, the CLF signal decreased, indicating that the hepatic parenchymal cell clusters encapsulated by the double-layer hydrogel microspheres have the function of absorbing and releasing CLF ( Fig.14 B and Fig.14 C).

[0213] 3) Detection of fat accumulation ability of hepatic parenchymal cell clusters wrapped in double-layer hydrogel microspheres

[0214] After 7 days of culture of the double-layer hydrogel microspheres encapsulating the hepatocyte clusters, the hydrogel microspheres were fixed in situ and stained in situ with Nile red and nucleus DAPI staining solution and image quantitative analysis. The control group was a 3D primary hepatocyte cluster cultured in a conventional Aggrewell microplate. The effects of the double-layer hydrogel microsphere material system and engineering manufacturing process on the fat storage function of human primary hepatocyte clusters were analyzed by image quantitative comparison.

[0215] like Fig.15 As shown, Nile red fat staining signals can be seen in the hepatic parenchymal cell clusters encapsulated by the double-layer hydrogel microspheres ( Fig.15 A), and there was no significant difference in the fat staining signal between the primary human hepatocytes and the primary human hepatocytes ( Fig.15 B and Fig.15 C). This indicates that the material system and engineering manufacturing process of the double-layer hydrogel microspheres have no significant damage to the function of human liver cells.

[0216] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A double-layer hydrogel microsphere, characterized in that: The double-layer hydrogel microspheres have an outer shell layer and a core layer to form a core-shell double-layer structure, wherein a graft is wrapped in the core layer, and the outer shell layer and the core layer have different elastic moduli; the elastic modulus of the core layer is 500 Pa-1100 Pa, and the elastic modulus of the outer shell layer is 2 kPa-4 kPa; the outer shell layer is made of: sodium alginate-polyethylene glycol-polyarginine with a concentration of 1-3% wt / v, a sodium alginate solution with a concentration of 1%-3% wt / v, or a sodium alginate-polyethylene glycol-lipoic acid solution with a concentration of 1-3% wt / v; the core layer is made of: polyether F127 diacrylate with a concentration of 1%-6% wt / v, methacrylic anhydride heparin with a concentration of 0.5-5%, vitronectin with a concentration of 1-10 μg / mL, and laminin with a concentration of 0.1-5 μg / mL, methacrylic anhydride sodium alginate with a concentration of 1-3%, and 0.02-8 mg / mL of type IV collagen and 1-10% wt / v gelatin, or 2-10% wt / v methacrylic anhydride-modified gelatin; The preparation method of the double-layer hydrogel microspheres comprises the following steps: (1) Preparation of grafts; (2) preparing a shell layer polymer hydrogel material mixture and a core layer polymer hydrogel material mixture respectively; (3) mixing the graft and the core layer polymer hydrogel material mixture to obtain a mixture A; (4) three-dimensionally shaping the mixture obtained in step (2) and step (3) by a high-voltage electrostatic non-contact spraying method to obtain a double-layer hydrogel microsphere having an outer shell layer and a core layer, and positioning the transplant in the core layer region; (5) culturing the double-layer hydrogel microspheres obtained in step (4); The specific operation of step (4) is as follows: using an independently controlled dual-channel syringe pump, the shell layer polymer hydrogel material mixture and the core layer polymer hydrogel material mixture mixed with the transplant are loaded into corresponding containers, and by adjusting the syringe pump pushing speed or pushing pressure, voltage, and distance between electrodes, a high-voltage electrostatic non-contact printer is used to print and prepare double-layer hydrogel droplets, and then a stabilization treatment and sacrificial material removal are performed to form double-layer hydrogel microspheres encapsulating the transplant; The stabilization treatment means that the double-layer hydrogel microspheres need to be rapidly cross-linked to fix the shape; The cross-linking method for the cross-linking of double-layer hydrogel microspheres is: (a) using a cross-linking agent containing divalent cations such as calcium ions, barium ions, and iron ions, and (b) using a 405 nm light source in combination with a photoinitiator such as phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and / or tri(2,2′-bipyridyl) ruthenium (II) chloride hexahydrate and sodium persulfide for photocross-linking.

2. The double-layer hydrogel microsphere according to claim 1, characterized in that: The elastic modulus of the core layer is comparable to the elastic modulus of the implant.

3. The double-layer hydrogel microsphere according to claim 1 or 2, characterized in that: The double-layer hydrogel microsphere is spherical or approximately spherical in structure as a whole, and the longest axis diameter is 0.2-3 mm; the shape of the core layer is spherical, approximately spherical, triangular pyramidal, "T"-shaped, spiral, linear or multi-point dispersed.

4. The double-layer hydrogel microsphere according to claim 1 or 2, characterized in that: The transplant is a pancreatic islet organoid, a pancreatic β cell cluster or a pancreatic β cell cluster, a liver organoid, or a liver parenchymal cell cluster.

5. The double-layer hydrogel microsphere according to claim 4, characterized in that: The pancreatic islet organoid has an arbitrary three-dimensional shape, and the longest axis diameter of the three-dimensional space is 10-500 μm; the pancreatic islet organoid contains one or more cells including pancreatic β cells or pancreatic β-like cells with insulin secretion function.

6. The double-layer hydrogel microsphere according to claim 4, characterized in that: The pancreatic β cells or pancreatic β-like cells are human or non-human cells, and the sources of the pancreatic β cells or pancreatic β-like cells include one or more of the following: i) Induced pluripotent stem cells, embryonic stem cells, endoderm cells, pancreatic endoderm cells, pancreatic precursor cells, and pancreatic progenitor cells; ii) cells extracted from human or animal pancreatic tissue or embryonic pancreatic tissue; iii) Cells obtained through gene editing, virus packaging, establishment or transformation of i) and ii).

7. The double-layer hydrogel microsphere according to claim 1, characterized in that: The transplant is an artificial pancreatic islet organoid.

8. The double-layer hydrogel microsphere according to claim 7, characterized in that: In mixture A, the concentration of the artificial pancreatic islet organoids is 1×10 3 / mL~8×10 4 Pieces / mL.

9. A polymer material composition having a three-dimensional spatial structure, comprising the double-layer hydrogel microspheres according to any one of claims 1 to 8.

10. Use of the double-layer hydrogel microspheres according to any one of claims 1 to 8 or the polymer material composition according to claim 9 in preparing products for treating pancreatic islet or liver-related diseases.

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