Double-layer hydrogel microspheres wrapping organoid, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of polymer materials, and particularly relates to double-layer hydrogel microspheres wrapping an organoid, a preparation method therefor, and use thereof. The double-layer hydrogel microspheres have an outer shell layer and a core layer that form a core-shell double-layer structure. A graft is wrapped in the core layer. The outer shell layer and the core layer have different elastic moduli. The double-layer hydrogel microspheres of the present invention can simultaneously meet the requirement of in vivo transplantation operations for a high hydrogel microsphere elastic modulus and the requirements of the organoid for a low elastic modulus and a biomimetic biochemical microenvironment, thereby improving the functions of the organoid.

Description

Double-layer hydrogel microspheres for wrapping organoids and preparation method and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of high polymer materials, and in particular relates to double-layer hydrogel microspheres for wrapping organoids and a preparation method and use thereof. BACKGROUND

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

[0003] Therefore, there is an urgent need for an innovative hydrogel microsphere manufacturing scheme based on high polymer materials, which enables the hydrogel microspheres to have stable mechanical properties and effective immune isolation function, while having adjustable high polymer material composition and mechanical properties after crosslinking, to obtain a biocompatible extracellular matrix composition and elastic modulus matching the transplanted organoids, so as to improve the biological activity and therapeutic effect of the transplants. SUMMARY

[0004] In order to solve the problems of the prior art, the present application provides double-layer hydrogel microspheres for wrapping organoids based on high-voltage electrostatic non-contact jetting technology and a preparation method and use thereof.

[0005] According to an aspect of the present application, there is provided double-layer hydrogel microspheres made of high polymer materials, which have an outer shell layer and a core layer, forming a core-shell double-layer structure, the core layer encapsulating a transplant, and the elastic modulus of the outer shell layer and the core layer being different.

[0006] In the present application, the elastic modulus of the hydrogel after covalent crosslinking initiated by light curing or other methods is detected by a rheometer, so that the elastic modulus is comparable to that of the tissue. The extracellular matrix components of the transplant can be added to the core layer material to improve the extracellular matrix biocompatibility of the core layer.

[0007] According to an embodiment of the present application, the elastic modulus of the core layer is less than that of the outer shell layer.

[0008] According to one embodiment of the present application, the core layer has an elastic modulus of 200 Pa to 10 kPa, 150 Pa to 3 kPa, 150 Pa to 2.5 kPa, 150 Pa to 2 kPa, 150 Pa to 1.5 kPa, 500 Pa to 5 kPa, 800 Pa to 3 kPa, 800 Pa to 2 kPa, 800 Pa to 1500 Pa, 800 Pa to 2500 Pa, 1 kPa to 3 kPa, 2 kPa to 3 kPa, 800 Pa to 1000 Pa, 800 Pa to 1100 Pa, 800 Pa to 1200 Pa, 500 Pa to 3 kPa, 500 Pa to 2 kPa, 500 Pa to 1500 Pa, 500 Pa to 1300 Pa, 500 Pa to 1200 Pa, 500 Pa to 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, 9000 Pa, 10 kPa, or the like, and any value therebetween, and 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 to 1 GPa, 1 kPa to 100 MPa, 1 kPa to 50 MPa, 1 kPa to 30 MPa, 1 kPa to 10 MPa, 1 kPa to 5 MPa, 1 kPa to 3 MPa, 1 kPa to 1 MPa, 1 kPa to 800 kPa, 1 kPa to 500 kPa, 1 kPa to 300 kPa, 1 kPa to 200 kPa, 1 kPa to 100 kPa, 1 kPa to 80 kPa, 1 kPa to 50 kPa, 1 kPa to 30 kPa, 1 kPa to 20 kPa, 1 kPa to 10 kPa, 1 kPa to 8 kPa, 1 kPa to 7 GPa. kPa, ranging from 1 kPa to 6 kPa, from 1 kPa to 5 kPa, from 1 kPa to 4 kPa, from 1 kPa to 3 kPa, from 1 kPa to 2 kPa, from 2 kPa to 7 kPa, from 2 kPa to 5 kPa, from 2 kPa to 4 kPa, from 2 kPa to 3 kPa, from 1 kPa to 2 ...3 kPa, from 1 kPa to 2 kPa, from 2 kPa to 3 kPa, from 3 kPa to 4 kPa, from 4 kPa to 4 kPa, from 5 kPa to 6 kPa, from 2 kPa to 7 kPa, from 2 kPa to 5 kPa, from 2 kPa to 4 kPa, from 2 kPa to 3 kPa, from 1 kPa to 2 kPa, from 2 kPa to 3 kPa, from 3 kPa to 4 kPa, from 4 kPa to 5 kPa, from 3 kPa to 4 kPa, from 4 kPa to 5 kPa, from 3 kPa to 4 kPa, from 4 kPa to 5 kPa, from 1 kPa to 2 kPa, from 4 kPa to 5 kPa, from 1 kPa to 2 kPa, from 2 kPa to 3 kPa, from 1 kPa to 2 kPa, from 2 kPa to 7 kPa, from 2 kPa to 5 kPa, from 2 kPa to 4 kPa, from 2 kPa to 3 kPa, from 1 kPa to 2 kPa, from

[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 within the core layer.

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

[0012] According to one embodiment of the present invention, the bilayer hydrogel microspheres are spherical or nearly spherical in shape, with the longest axis diameter being 0.2 to 3 mm.

[0013] According to one embodiment of the present invention, 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 an embodiment of the present application, the shell layer material comprises alginate or its derivatives and / or other high molecular materials.

[0016] According to an embodiment of the present application, the core layer material comprises one or more than one of natural or artificially synthesized proteins, lipids, polysaccharides, and polyethers and their derivatives, polyethylene glycol and its derivatives, which can be covalently cross-linked.

[0017] According to an embodiment of the present application, the shell layer and the core layer are different in the composition of the materials.

[0018] According to an embodiment of the present application, the shell layer and the core layer are different in the concentration of the materials.

[0019] According to an embodiment of the present application, the shell layer material comprises one or more than one of alginate or its derivatives, including sodium alginate, magnesium alginate, calcium alginate, barium alginate, poly-caprolactone modified sodium alginate, polyethylene glycol modified sodium alginate, sodium alginate-polyethylene glycol-thioctic acid, sodium alginate-cyclodextrin, sodium alginate-polyethylene glycol-distearylphosphatidyl ethanolamine, sodium alginate-polyethylene glycol-hyaluronic acid, sodium alginate-polyethylene glycol-polyethyleneimine, sodium alginate-polyethylene glycol-polylysine, sodium alginate-polyethylene glycol-polyornithine, sodium alginate-polyethylene glycol-polyarginine, dipalmitoylphosphatidylcholine-disulfide-sodium alginate, dioleoylphosphatidylcholine-polyethylene glycol-sodium alginate, dioleoylphosphatidylcholine-disulfide-sodium alginate, dimyristoylphosphatidylcholine-polyethylene glycol-sodium alginate, amino-polyethylene glycol-sodium alginate, polylactic acid-polyethylene glycol-sodium alginate.

[0020] According to an embodiment of the present application, the shell layer material comprises one or more than one of natural and / or artificial high molecular materials, including but not limited to polythioctic 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, chitosan or its derivatives.

[0021] According to one embodiment of the present application, the core layer material is selected from one or more of natural or artificially synthesized high molecular materials that can be gel cross-linked by covalent bonding, including, but not limited to, gelatin or its derivatives, including but not limited to gelatin, thiolated gelatin, methacrylated gelatin; laminin or its derivatives, including but not limited to laminin, thiolated laminin, methacrylated laminin; vitronectin or its derivatives, including but not limited to vitronectin, thiolated vitronectin, methacrylated vitronectin; fibronectin or its derivatives, including but not limited to fibronectin, thiolated fibronectin, methacrylated fibronectin; fibrin or its derivatives, including but not limited to fibrin, thiolated fibrin, methacrylated fibrin; collagen type I or its derivatives, including but not limited to collagen type I, thiolated collagen type I, methacrylated collagen type I; collagen type II or its derivatives, including but not limited to collagen type II, thiolated collagen type II, methacrylated collagen type II; collagen type IV or its derivatives, including but not limited to collagen type IV, thiolated collagen type IV, methacrylated collagen type IV; Matrigel; silk fibroin or its derivatives, including but not limited to silk fibroin, thiolated silk fibroin, methacrylated silk fibroin; elastin or its derivatives, including but not limited to elastin, thiolated elastin, methacrylated elastin; hyaluronic acid and its derivatives, including but not limited to hyaluronic acid, thiolated hyaluronic acid, methacrylated hyaluronic acid; heparin or its derivatives, including but not limited to heparin, thiolated heparin, methacrylated heparin; dextran or its derivatives, including but not limited to dextran, thiolated dextran, methacrylated 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, methacrylated chondroitin sulfate; polyethylene oxide-polypropylene oxide or its derivatives, including but not limited to F-127, polyether F127 diacrylate; chitosan or its derivatives, including but not limited to chitosan, thiolated chitosan, methacrylated chitosan.

[0022] According to one embodiment of the present application, the core layer material is selected from one or more of natural or artificially synthesized high molecular materials that can be gel cross-linked by covalent bonding, including methacrylated sodium alginate, thiolated sodium alginate, ice alkenyl sodium alginate, polyether F127 diacrylate, methacrylated hyaluronic acid, methacrylated heparin, gelatin, methacrylated gelatin, laminin, vitronectin, collagen type IV, collagen type I.

[0023] According to an embodiment of the present application, the concentration of the material constituting the 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 an embodiment of the present application, the concentration of the material constituting 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 an embodiment of the present application, the concentration of the material constituting 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 an embodiment of the present application, the material constituting the shell layer is 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 with a concentration of 1-3% wt / v.

[0027] According to an embodiment of the present application, the material constituting the core layer is polyether F127 diacrylate, methacrylated heparin, vitronectin and laminin, preferably, polyether F127 diacrylate with a concentration of 1-10% wt / v, methacrylated heparin with a concentration of 0.1-10%, vitronectin with a concentration of 0.001-100 μg / mL and laminin with a concentration of 0.001-100 μg / mL, preferably, polyether F127 diacrylate with a concentration of 1-6% wt / v, methacrylated 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.

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

[0029] According to an embodiment of the present application, the material of the core layer is methyl methacrylate anhydride modified sodium alginate, type IV collagen and gelatin, preferably, methyl methacrylate anhydride modified 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 an embodiment of the present application, the material of the shell layer is sodium alginate-polyethylene glycol-thioctic acid, preferably, sodium alginate-polyethylene glycol-thioctic acid with a concentration of 0.5-4% wt / v, preferably, sodium alginate-polyethylene glycol-thioctic acid solution with a concentration of 1-3% wt / v.

[0031] According to an embodiment of the present application, the material of the core layer is methyl methacrylate anhydride modified gelatin (GelMA), preferably, methyl methacrylate anhydride modified gelatin (GelMA) with a concentration of 1-20% wt / v, preferably, methyl methacrylate anhydride modified gelatin (GelMA) with a concentration of 2-10% wt / v.

[0032] According to an embodiment of the present application, the transplant is pancreatic islet organoid, pancreatic islet beta cell cluster or pancreatic islet beta-like cell cluster, pancreatic islet alpha cell cluster or pancreatic islet alpha-like cell cluster, liver organoid, liver parenchymal cell cluster, kidney organoid, blood vessel organoid or blood vessel endothelial cell cluster.

[0033] According to an embodiment of the present application, the transplant is pancreatic islet organoid or liver organoid.

[0034] According to an embodiment of the present application, the transplant is pancreatic islet organoid.

[0035] According to an embodiment of the present application, the transplant is liver organoid.

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

[0037] According to an embodiment of the present application, the pancreatic islet organoid contains one or more cells, including pancreatic islet beta cells or pancreatic islet beta-like cells with insulin secretion function.

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

[0039] i) cells differentiated from 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 tissues or embryonic pancreatic tissues;

[0041] iii) cells obtained by genetic editing, virus packaging, cell line construction or modification of the cells of i) and ii).

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

[0043] According to an embodiment of the present application, the liver organoids have a three-dimensional arbitrary shape, and the longest axis diameter of the three-dimensional space is 10-500 μm, preferably 40-120 μm.

[0044] According to an embodiment of the present application, the liver organoids contain one or more cells including hepatocytes or hepatocyte-like cells having metabolic and secretory functions.

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

[0046] i) cells differentiated from 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 tissues or embryonic liver tissues;

[0048] iii) cells obtained by genetic editing, virus packaging, cell line construction or modification of the cells of i) and ii).

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

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

[0051] (1) preparing a graft;

[0052] (2) respectively prepare a mixture of high-molecular hydrogel material of the shell layer and a mixture of high-molecular hydrogel material of the core layer;

[0053] (3) mix the graft and the mixture of high-molecular hydrogel material of the core layer to obtain mixture A;

[0054] (4) three-dimensionally shape the mixture obtained in step (2) and step (3) by an engineering method to obtain a double-layer hydrogel microsphere with a shell layer and a core layer, and position the graft in the core layer region;

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

[0056] According to an embodiment of the present application, step (1) and step (2) have no sequence, for example, step (1) can be performed first and then step (2) is performed, or step (2) can be performed first and then step (1) is performed, or step (1) and step (2) are performed simultaneously.

[0057] According to an embodiment of the present application, the operation of step (1) is to obtain pancreatic beta cells or its cell-like cells or islet organoids, or liver cells or its cell-like cells or liver organoids by differentiating stem cells, primary tissues or gene editing. The number of cells in the organoid is adjusted by cell digestion and re-aggregation, so that the number of cells in each cell cluster is 200-2000, preferably 400-1000 cells. After 2 days of culture, islets or liver organoids with approximate spherical shape and longest axis diameter of 10-500 μm are obtained, preferably 50-250 μm.

[0058] According to an embodiment of the present application, the graft is preferably islet organoids or / and liver organoids.

[0059] According to an embodiment of the present application, the operation of step (3) is that the concentration of the islet organoids or / and liver organoids in the mixture A is 1×10 3 6 / mL-8×10 4 6 / mL, preferably 1×10 3 6 / ml-1×10 4 6 / mL, preferably 1×10 3 6 / ml-5×10 3 6 / mL.

[0060] According to an embodiment of the present application, the specific operation of step (4) is: using independently controlled double-channel injection pumps, the shell layer high polymer hydrogel material mixture and the core layer high polymer hydrogel material mixture mixed with the implant are loaded into corresponding containers, and the implant is positioned in the core layer area by adjusting the injection pump pushing speed or pushing pressure, voltage, and using high-voltage electrostatic non-contact spraying technology.

[0061] According to an embodiment of the present application, when using double-channel injection pumps, the pushing speed of the shell layer channel injection pump and the core layer channel injection pump is 0.06-100 ml / h, and the pushing speed of the shell layer channel injection pump is preferably 5 ml / h-30 ml / h, and the pushing speed of the core layer channel injection pump is preferably 0.1 ml / h-20 ml / h.

[0062] According to an embodiment of the present application, when printing using a high-voltage electrostatic non-contact printer, the direct-current high-voltage power supply voltage is preferably 4 kV-13 kV, and the electrode distance is 1 cm-3 cm.

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

[0064] According to an embodiment of the present application, the specific operation of step (4) is: using independently controlled double-channel injection pumps, the shell layer high polymer hydrogel material mixture and the core layer high polymer hydrogel material mixture mixed with the implant are loaded into corresponding containers, and the implant is positioned in the core layer area by adjusting the injection pump pushing speed or pushing pressure, voltage, electrode distance, and using a high-voltage electrostatic non-contact printer to print to prepare a double-layer hydrogel droplet, and then through stabilization treatment and sacrificial material removal, a double-layer hydrogel microsphere wrapping the implant is formed.

[0065] According to an embodiment of the present application, the stabilization treatment refers to that the double-layer hydrogel microsphere needs to be quickly cross-linked to be fixed and shaped.

[0066] According to an embodiment of the present application, the cross-linking method when the double-layer hydrogel microsphere is cross-linked and shaped may 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 and a photo initiator such as lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and / or tris (2,2'-bipyridine) ruthenium (II) chloride hexahydrate and sodium persulfate for photo cross-linking, and (c) using one or more of thrombin, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide or its 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, and the like, and / or,

[0069] (b) using a 405 nm light source in combination with phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and / or tris(2,2'-bipyridyl) ruthenium (II) chloride hexahydrate and sodium persulfate for photo-crosslinking, and / or,

[0070] (c) thrombin.

[0071] According to an embodiment of the present application, the concentration of the 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, and preferably 10 mM to 30 mM.

[0072] According to an embodiment of the present application, the divalent cation cross-linking agent solution contains water and one or more of barium chloride, sodium chloride, and calcium chloride.

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

[0074] According to an embodiment of the present application, the light intensity for photo-crosslinking is 1 to 500 mW / cm 2 , preferably 1 to 200 mW / cm 2 , and preferably 10 to 100 mW / cm 2 , and the irradiation time is 2 to 600 s, preferably 2 to 500 s, preferably 30 to 120 s, and preferably 100 to 300 s.

[0075] According to an embodiment of the present application, the light source for photo-crosslinking is preferably a 405 nm light source, 30 to 300 mW / cm 2 , and 4 to 300 s.

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

[0077] According to an embodiment of the present application, the sacrificial material removal process refers to the removal of a structural auxiliary support material in the preparation process.

[0078] The structural auxiliary support material comprises a temperature-sensitive material (such as gelatin, polyoxyethylene polyoxypropylene ether) or a light-degradable material or a material cut by oxidative free radicals (such as ketone thiol-modified biomaterials), a photoinitiator (such as lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and / or tris (2,2'-bipyridyl) ruthenium (II) chloride hexahydrate and sodium persulfate), a crosslinking agent (such as thrombin), and the like. Preferred sacrificial materials are, for example, gelatin and / or polyoxyethylene polyoxypropylene ether and / or lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and / or thrombin.

[0079] According to an embodiment of the present application, the solution used in the sacrificial material removal process may, for example, comprise 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 an embodiment of the present application, the culture in step (5) comprises static culture and dynamic culture.

[0081] According to an embodiment of the present application, 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 a lift-type bioreactor, a bubble-type bioreactor, a hollow fiber bioreactor, a ceramic rectangular channel honeycomb reactor, a glass bead bed reactor, a fluidized bed reactor, a solid bed reactor, a pulsatile culture device, a microgravity culture device, a stirring culture device, a wave culture device, a chip or a perfusion culture system.

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

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

[0085] According to another aspect of the present application, a high molecular material composition with a three-dimensional spatial structure is provided, comprising the double-layer hydrogel microspheres as described above.

[0086] According to another aspect of the present application, the use of the double-layer hydrogel microspheres as described above or the high molecular material composition as described above in the preparation of a product for treating pancreatic islet or liver-related diseases is provided.

[0087] According to an embodiment of the present application, 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 an embodiment of the present application, the disease is juvenile diabetes, diabetes with kidney damage, end-stage diabetes with kidney failure.

[0089] According to an embodiment of the present application, the disease is hepatitis, cirrhosis, liver failure.

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

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

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

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

[0094] (3) In the microspheres of the present application, the outer shell hydrogel is composed of high-molecular-weight hydrogel materials with good biostability and good biocompatibility, and has a slow degradation rate in vivo; 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 application, the material composition of the core layer contains extracellular matrix components of islet tissue, providing a biomimetic microenvironment to alleviate the cell aging and apoptosis of islet organoids; in addition, the elastic modulus of the core layer hydrogel is comparable to that of islet tissue, providing a biomimetic extracellular mechanical microenvironment for islet organoids, significantly improving the exocrine capacity of islet organoids.

[0096] (5) In the present application, the islet organoids of the double-layer hydrogel microspheres have high-efficiency insulin excretion function, meeting the requirements of precision medicine, pancreatic engineering and regenerative medicine, drug preclinical testing, environmental / toxicity / pollutant monitoring, pancreatic toxicology testing, pancreatic disease-related new drug research and development, pancreatic development physiology, pancreatic pathology, or drug, toxicity and efficacy testing in many fields.

[0097] (6) The material of the core layer in the microsphere of the present application provides a mechanical microenvironment of a biomimetic liver, improves the metabolic function of in vitro cultured human primary liver parenchymal cells, and meets the requirements of many fields such as precision medicine for liver diseases, liver engineering and regenerative medicine, preclinical detection of drugs, environmental / toxic substances / pollutant monitoring, drug liver injury toxicology detection, research and development of new drugs related to liver diseases, liver development physiology, liver pathology, or drug, toxicity and efficacy tests. BRIEF DESCRIPTION OF DRAWINGS

[0098] Figure 1 is a schematic diagram of the double-layer hydrogel microspheres of the present application encapsulating islet organoids. Wherein 1 is the shell layer, 2 is the core layer, and 3 is the islet organoid.

[0099] Figure 2 is an immunofluorescence staining analysis diagram of human embryonic stem cells being directionally differentiated into pancreatic progenitor cells. The scale is 200 μm.

[0100] Figure 3 is a shape and feature related characterization diagram of hESC differentiated pancreatic progenitor cell clusters PP2. Among them, Figure 3A is an optical photograph of the cell cluster formed by the pancreatic progenitor cells PP2, the scale is 100 μm; Figure 3B is a diameter and roundness analysis diagram of the cell cluster in Figure 3A; Figure 3C is a cell live and dead staining diagram of the cell cluster formed by the pancreatic progenitor cells PP2, Calcein-AM represents live cells, PI represents dead cells, the scale is 100 μm; Figure 3D is a comparative analysis diagram of the expression of pancreatic progenitor cell marker genes between hESC, hESC differentiated PP2 monolayer cultured cells and PP2 cell clusters, ns in the diagram represents no significant.

[0101] Figure 4 is a diagram of the elastic modulus of the cross-linked shell layer and core layer high molecular hydrogel material in rheological analysis, wherein ****p<0.0001.

[0102] Figure 5 is a shape and feature related characterization diagram of the double-layer hydrogel microspheres encapsulating hESC differentiated islet organoids. Among them, Figure 5A is a double-layer hydrogel microsphere image; Figure 5B is a microscopic image of the hESC differentiated islet organoids located in the core area of the double-layer hydrogel microspheres; Figure 5C is a statistical diagram of the diameter size distribution of the double-layer hydrogel microspheres; Figure 5D is a fluorescence staining live and dead diagram of the double-layer hydrogel encapsulating hESC islet organoids; the scale is 200 μm.

[0103] Figure 6 is the influence of different elastic modulus of the core layer on the maturation of stem cell differentiated islet organoids. Figure 6A is the relative expression of INS marker genes under different elastic modulus; Figure 6B is an immunofluorescence staining analysis diagram of the expression of marker proteins of the double-layer hydrogel encapsulating hESC differentiated islet organoids. Among them, NKX6.1 and INS are marker proteins of differentiated mature islet beta cells, Hochest33342: nucleus; the scale is 50 μm; *p<0.05, ****p<0.0001, ns no significant.

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

[0105] Figure 8 is the insulin excretion ELISA detection chart of double-layer hydrogel microspheres wrapped islet organoids.

[0106] Figure 9 is the elastic modulus chart of the outer shell and core layer of the high molecular hydrogel material of the single-layer hydrogel microspheres and the double-layer hydrogel microspheres, wherein ****p<0.0001.

[0107] Figure 10 is a biological comparison analysis chart of double-layer hydrogel microspheres and single-layer hydrogel microspheres wrapped rat primary islet cells.

[0108] Among them, Figure 10A is the β-galactosidase chemical staining optical microscope chart of double-layer hydrogel microspheres and single-layer hydrogel microspheres wrapped rat primary islet cells on the left, and the column chart based on image statistical analysis on the right, the scale is 200 μm; Figure 10B is a flow cytometry analysis chart of rat primary islet cell apoptosis and death; Figure 10C is an ELISA analysis chart of insulin excretion of double-layer hydrogel microspheres and single-layer hydrogel microspheres wrapped rat primary islet cells; Figure 10D is an insulin excretion detection chart under different elastic modulus of the core layer. *p<0.05, **p<0.01.

[0109] Figure 11 is a graph of the results of in vivo transplantation of double-layer hydrogel microspheres wrapped rat primary islet cells for hypoglycemic research. Figure 11A is the transplantation of hydrogel microspheres to the mesentery of hyperglycemic mice. Figure 11B is the hypoglycemic experiment of double-layer hydrogel microspheres wrapped rat primary islet cells transplanted into the abdominal cavity of hyperglycemic mice, and the X-axis is the number of days of in vivo transplantation. Figure 11C is a comparison of the hypoglycemic efficiency of double-layer and single-layer hydrogel microspheres wrapped rat primary islet cells.

[0110] Figure 12 is a chart of the elastic modulus of the outer shell and core layer of the high molecular hydrogel material, wherein ****p<0.0001.

[0111] Figure 13 is a chart of the expression of liver organoid markers wrapped by double-layer hydrogel microspheres. Figure 13A is the transcriptional expression level of CYP1A1 gene expressed by liver parenchymal cells, Figure 13B is the ALB protein expression level and CYP1A1 protein expression level, **p<0.01, ***p<0.001, ns represents no significant difference.

[0112] Figure 14 is a graph showing the detection of bile salt transport capacity of double-layer hydrogel microspheres encapsulated hepatocytes. Figure 14A is after CLF incubation, Figure 14B and Figure 14C are after CLF release.

[0113] Figure 15 is a graph showing the detection of lipid accumulation capacity of double-layer hydrogel microspheres encapsulated hepatocytes. Figure 15A is double-layer hydrogel microspheres encapsulated hepatocytes, and Figure 15B is Nile red in situ staining of 3D suspension culture cell clusters, DAPI represents cell nuclei. Figure 15C is the analysis result of Nile red fluorescence area ratio to cell nuclei area. ns represents no significant difference. DETAILED DESCRIPTION

[0114] The following examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Unless otherwise indicated, the techniques employed in the examples are standard techniques commonly used in the art. The materials used in the examples are commercially available.

[0115] Example 1

[0116] This example provides a process for preparing double-layer hydrogel microspheres based on high-voltage electrostatic non-contact electrospraying technology, and encapsulating stem cell differentiated pancreatic islet organoids (as shown in Figure 1, where 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 example provides structural stability and immune isolation for the in vivo transplantation of stem cell differentiated pancreatic islet organoids, and the core layer provides a biomimetic extracellular matrix component for stem cell differentiated pancreatic islet organoids, while also providing a biomimetic mechanical microenvironment by controlling the degree of covalent cross-linking, thereby promoting the functional maturation of stem cell derived pancreatic islet organoids. The details are as follows:

[0117] 1. Preparation of the transplant

[0118] 1) Direct differentiation of human embryonic stem cells (hESC) into pancreatic progenitor cells.

[0119] (a) Seed hESC into cell culture vessels and culture to 100% confluency. First use induction medium 1 for 3-5 days, which contains RPMI-1640 (Thermo Fisher, 11875093), 1x B27 (Gibco, insulin-free version, A1895601), 100 ng / mL Activin A (R&D, 338-AC), and 20 ng / mL WNT3A (R&D, 5036-WN). This stage can obtain restricted endoderm cells.

[0120] (b) Next, culture with induction medium 2 for 2-3 days, the components include RPMI-1640, 1x B27, 50 ng / mL KGF (R&D, 5028-KG), 10 ng / mL FGF10 (R&D, 345-FG).

[0121] (c) Next, culture with induction medium 3 for 2-3 days, the components include DMEM-F12 (Thermo Fisher, 11320033), 1x ITS (Gibco, 41400045), 50 ng / ml, 0.25 µM Sant1 (Selleck, S7092), 2 µM RA (Selleck, s1653), 200 nM LDN193189 (Selleck, s2618), 500 nM PdBU (Yeasen, 53352ES03). Pancreatic progenitor cells PP1 are obtained in this stage.

[0122] (d) Next, culture with induction medium 4 for 2 days, the components include DMEM-F12, 1x ITS, 50 ng / ml KGF, 0.25 µM Sant1, 100 nM RA, 5 ng / mL Activin A. Pancreatic progenitor cells PP2 are obtained in this stage.

[0123] (e) Cell morphology observation and biological detection

[0124] The expression of marker proteins of cells at different differentiation stages is identified by using cell immunofluorescence staining, and the observation record is obtained by using a laser scanning confocal microscope, and the results are shown in FIG. 2. Among them, SOX17 and FOXA2 are restrictive endoderm marker proteins; PDX1 is a pancreatic progenitor cell PP1 marker protein; PDX1 and NKX6.1 are pancreatic progenitor cell PP2 marker proteins.

[0125] During the differentiation of hESC into pancreatic progenitor cells, the restrictive endoderm marker proteins SOX17 and FOXA2 are highly expressed, the pancreatic progenitor cell PP1 marker protein PDX1 is highly expressed, and the differentiation efficiency is as high as more than 90%, and the pancreatic progenitor cell PP2 marker proteins PDX1 and NKX6.1 double-positive cells are as high as more than 60%, indicating that the pancreatic progenitor cells positive for PDX1 and NKX6.1 are obtained.

[0126] 2) Preparation of pancreatic progenitor cell PP2 cell clusters

[0127] (a) The pancreatic progenitor cell PP2 is digested into single cells by using Accutase, and is inoculated into an Aggrewell400 6-well plate (Stemcell, 34421) at 3x10 6 / well, and is incubated for 2 days, and is cultured for 2 days by using induction medium 4.

[0128] (b) Next, change to induction medium 5, the components include, 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 pg / L ZnS04(Sigma, 221376), 0.25 pM Santl (Selleck, S7092), 100 nM RA (Selleck, s1653), 1 pM XXI (Sigma, 565790), 10 pM Alk5i II (Selleck, S7223), 1 pM T3 (Selleck, S5726) and 20 ng / ml Betacellulin (Gibco, 100-50). The pancreatic progenitor cell clusters are obtained in this stage.

[0129] (c) Cell morphology observation and biological detection

[0130] (c1) Morphological observation and cell survival rate detection: In order to detect the size of the pancreatic progenitor cell clusters, the longest axis of the cell cluster was detected by light microscopy and image measurement. The results are shown in Figure 3, the pancreatic progenitor cell PP2 cells form a nearly spherical tight cell cluster, the longest axis is about 150 pm (Figure 3A, 3B). Using 2 pM Calcein-AM and 4.5 pM PI staining solution, incubate for 15 minutes, then use laser scanning confocal microscope (LSCM, Nikon, Z2) to detect the survival rate of cells in the cell cluster. Calcein AM is metabolized by living cells to produce a fluorescent signal in the cells, while PI can only produce a fluorescent signal in dead cells. The results are shown in Figure 3, the cell survival rate in the cell cluster is good (Figure 3C).

[0131] (c2) Detection of pancreatic progenitor cell marker genes: qPCR technology was used to detect the transcription level of pancreatic progenitor cell PP2 marker genes (such as PDX1 and NKX6.1) in the cell cluster. The results are shown in Figure 3D, compared with the single-layer culture cells before the pancreatic progenitor cell PP2 formed a cluster, the PDX1 and NKX6.1 gene expression levels of the pancreatic progenitor cell PP2 cell cluster co-cultured in 3D conditions did not change significantly, indicating that the pancreatic progenitor cell PP2 cell cluster maintained the stem cell properties of pancreatic progenitor cells.

[0132] 2) Preparation of the core layer hydrogel mixture

[0133] 1) Preparation of the core layer hydrogel mixture

[0134] Sodium alginate-polyethylene glycol-polyarginine was prepared in a 0.9% sodium chloride solution at a concentration of 3% wt / v.

[0135] 2) Preparation of the core layer hydrogel mixture

[0136] Polyether F127 diacrylate at a concentration of 5% wt / v, 1% wt / v methacrylated heparin, vitronectin (Sigma, 5051) at a concentration of 2 μg / mL, laminin (Sigma, L2020) at a concentration of 2 μg / mL, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) at a concentration of 0.25% wt / v.

[0137] 3) Measurement of the rheological mechanics of the material

[0138] The results are shown in Figure 4, based on the rheological mechanics of the shell and core layer mixture after cross-linking, the elastic modulus of the shell after ionic cross-linking was 3.155 ± 0.210 kPa, and by optimizing the exposure time of the 405 nm light source, the elastic modulus of the core layer was 0.964 ± 0.056 kPa, and the elastic modulus of the shell hydrogel mixture was significantly greater than that of the core layer.

[0139] 3) Mixing of the graft and the core layer hydrogel mixture to obtain mixture A:

[0140] The core layer hydrogel mixture was added to pancreatic progenitor PP2 cell clusters in an amount of 50-100,000 cells / mL, preferably 1,000-5,000 cells / mL.

[0141] 4) Preparation of the double-layer hydrogel microspheres

[0142] 1) A dual-channel independently controlled microinjection pump and an 18G / 21G (outer / inner) coaxial stainless steel needle were used. The outer shell hydrogel mixture was loaded into the outer shell tube, connecting it to the outer layer of the coaxial needle; the core layer material was loaded into the core layer tube, connecting it to the inner layer of the coaxial needle. Bilayer hydrogel microspheres were then prepared using high-voltage electrostatic non-contact electro-spraying. The DC high-voltage power supply was set to 12kV, the outer shell channel injection pump advance rate was 15ml / h, the core layer channel injection pump advance rate was 5ml / h, and the electrode distance was 2.5cm. A hydrogel microsphere crosslinking solution was added to a plastic receiving dish, consisting of water, barium chloride, sodium chloride, and mannitol, with barium chloride (Sigma, 342920) concentration of 20mM, mannitol (Sigma, BP1007) concentration of 5% wt / v, sodium chloride (Sigma, S7653) concentration of 0.9 wt / v, and LAP concentration of 0.25% wt / v. The hydrogel microspheres solidify ionic coordination bonds instantaneously upon entering the receiving dish.

[0143] Next, the hydrogel microspheres in the receiving dish were collected into a transparent 15 mL centrifuge tube and centrifuged using a 405 nm light source with a power of 50 mW / cm². 2 Irradiate for 60 seconds. This step completes the covalent cross-linking of the core layer of the hydrogel microspheres.

[0144] 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, and MEM NEAA 1% v / v. This step yielded bilayer hydrogel microspheres with pancreatic progenitor cell (PP2) clusters located in the core layer.

[0145] 2) Size and morphology of bilayer hydrogel microspheres: The collected bilayer hydrogel microspheres were observed and photographed using an inverted phase-contrast microscope, and the diameter of the microspheres was analyzed through image analysis. As shown in Figures 5A-C, the printed and cured bilayer hydrogel microspheres are approximately spherical with an approximate diameter of about 1.5 mm.

[0146] 3) Cell viability / deadness detection: Live and dead cells were stained separately using a mixed solution of 2 μM Calcein-AM (Sigma, 17783) and 4.5 μM PI (Sigma, GF16233836) in the dark for 15 minutes. Observation and recording were performed using a laser scanning confocal microscope (LSCM, Nikon, Z2). Data analysis of the stained images was performed, as shown in Figure 5D. The pancreatic progenitor cell (PP2) cell clusters in the core layer of the printed bilayer hydrogel microspheres showed high cell viability with almost no cell death.

[0147] 5. Cultivation of bilayer hydrogel microspheres

[0148] The double-layer hydrogel microspheres obtained in step 4 were added to a dynamic cell culture tank (Corning, 430825) for culture, with a culture rotation speed of 30 rpm / min, a culture environment of 37°C, a humid environment, and 5% CO2. The culture solution 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. This step obtained double-layer hydrogel microspheres-encapsulated pancreatic islet organoids with insulin secretion function.

[0149] 6. Biological evaluation of double-layer hydrogel microspheres-encapsulated pancreatic islet organoids

[0150] 1) NXK6.1 and INS double-positive detection.

[0151] By adjusting the light exposure time and light intensity of the 405 nm light source and the content of the photoinitiator, different elastic moduli of the core layer can be obtained through rheological detection. Based on the above light exposure conditions and initiators, double-layer hydrogel microspheres encapsulating pancreatic islet organoids were prepared, and after 7-20 days of culture, the cells were collected for qRT-PCR detection. The expression level of INS in pancreatic progenitor PP1 cells was used as a standard to calculate the expression level of INS under different elastic moduli. The results are shown in FIG. 6A. When the elastic modulus is in the range of 150 Pa-3000 Pa, the expression level of INS gene is significantly increased, and the expression level of INS decreases with the increase of elastic modulus. When the elastic modulus of the core layer is higher than 4000 Pa, the expression level of INS gene is not significantly increased compared with that of pancreatic progenitor PP1, indicating that high elastic modulus hinders the development and maturation of islet cells, and appropriate elastic modulus plays an important regulatory role in the development and maturation of islet cells.

[0152] The pancreatic islet organoids encapsulated by the double-layer hydrogel microspheres with the core layer elastic modulus of 850 Pa were stained in situ by cell immunofluorescence staining and NKX6.1 / INS antibody mixture, the cell nucleus was stained by Hoechst 33342, and the expression status of NKX6.1 (Abeam, ab268088) / INS (sunncell, SNL-332) protein and the proportion of cells were analyzed by laser confocal microscope and 3D image analysis software. The immunofluorescence staining method is as follows: collect the hydrogel microspheres cultured for 15 days, fix with 4% polyformaldehyde at room temperature for 15 minutes, and wash with Hanks buffer for 3 times, 5 minutes each time; block for 1 hour by using a mixture containing 0.3% Triton-X (Sigma, X100) and 5% bovine serum albumin (BSA) (Solebao, A8020); aspirate the blocking buffer, add the diluted primary antibody (containing 0.1% Triton-X and 1% BSA), and incubate overnight at 4°C. Wash with Hanks buffer for 3 times, 5 minutes each time; add the corresponding secondary antibody (Abeam, ab205718) and (Abeam, ab205719), incubate at room temperature for 2 hours in the dark, and then wash with Hanks buffer for 3 times, 5 minutes each time; then add Hoechst 33342 to stain the cell nucleus, and incubate at room temperature for 5 minutes in the dark. Observe and record by laser confocal microscope (LSCM, Nikon, Z2).

[0153] The results are shown in FIG. 6B. The pancreatic islet organoids encapsulated by the double-layer hydrogel microspheres highly express the pancreatic islet tissue development and maturation markers NKX6.1 and INS protein, which indicates that after culture, the pancreatic progenitor cell PP2 cell clusters obtained by differentiation of pluripotent stem cells can develop and mature into pancreatic islet organoids in the double-layer hydrogel microspheres, and highly express the pancreatic islet maturation markers, which indicates that the core layer of the double-layer hydrogel microspheres provides a suitable microenvironment for the maturation of PP2 cell clusters into pancreatic islet organoids.

[0154] 2) Safety detection of double-layer hydrogel microspheres in vivo transplantation.

[0155] 10-50 hydrogel microspheres were transplanted into the mesentery of C57BL / 6J male mice. In order to evaluate the safety of the double-layer hydrogel microspheres in vivo transplantation, the blood of the mice was collected by orbital blood collection 14 days after in vivo transplantation (FIG. 7A), the inflammatory response of the mice was detected, and the hydrogel microspheres in the abdominal cavity of the mice were taken out for optical microscope detection and histological detection.

[0156] The results are shown in Figure 7. The hydrogel microspheres taken from the mesentery were intact without breakage, and there was no obvious deposition of mouse immune cells and fibroblasts on the surface of the hydrogel microspheres (Figure 7B). The results of histological section staining showed that there was no obvious collagen deposition around the hydrogel microspheres (Figure 7C), indicating that the double-layer hydrogel microspheres constructed in this example did not cause obvious immune response to foreign substances in mice. The results of blood tests 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 7D). The above results indicate that the double-layer hydrogel microspheres constructed in this example have good in vivo transplantation safety.

[0157] 3) Insulin excretion detection.

[0158] The double-layer hydrogel microspheres encapsulating the pancreatic islet organoids were stimulated using high glucose, and an insulin excretion detection kit (Alpco_80_INSHUU_E01.1) was used to detect the insulin excretion function of the organoids under high glucose stimulation.

[0159] The insulin excretion detection method is as follows:

[0160] (a) Sample preparation: 1 x 10 6 cells were collected by centrifugation at 80g, the original culture medium was removed, and the hydrogel microspheres were washed 3 times with Krebs buffer, cultured in Krebs buffer containing low glucose (2mM / L) for 2 hours, washed 3 times with Krebs buffer, and then cultured in Krebs buffer containing low glucose (2mM / L) for 0.5 hours. The supernatant was collected and stored in a 4°C refrigerator for detection. Then the hydrogel microspheres were collected, washed 1 time with Krebs buffer, cultured in Krebs buffer containing high glucose (20mM / L) for 0.5 hours, and the supernatant was collected and stored in a 4°C refrigerator for detection.

[0161] (b) Standard preparation: After taking the kit out of the refrigerator, it should be placed at room temperature (25-28°C) for 20 minutes. Prepare the appropriate amount of washing solution: dilute the washing solution (20X) with double distilled water or deionized water to 1X. 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 mix gently and blow with a pipette several times to completely dissolve the standard. Usually 2 wells are needed for each concentration of standard, and the amount of standard used for each well is 100μl, resulting in 200, 100, 50, 25, 12.5, 6.25, 0μLU / mL, a total of seven standard concentrations. Finally, the diluted standard is added to the pre-coated plate wells in turn, a total of seven standard concentrations.

[0162] (c) Detection: Add 25 μΐ / well of sample or different concentrations of standard into the corresponding wells. Then add 100 μΐ / well of horseradish peroxidase labeled human insulin antibody immediately. Seal the reaction wells with sealing film (transparent) and incubate at room temperature for 120 minutes. Then wash the plate for 5 times and finally pat dry on thick absorbent paper. Finally, add 100 μΐ / well of color developing TMB solution, seal the reaction wells with sealing film (white) and incubate at room temperature for 10 minutes in the dark. If the room temperature is low, the incubation time should be appropriately extended. When the standard and sample show very significant color change, measure the A450 value immediately after adding 50 μΐ / well of stop solution.

[0163] (d) Result analysis: The absorbance value of each standard or sample should be subtracted by the absorbance value of the background correction well (if no correction well is made, no subtraction is needed). Draw the standard curve. Connect the coordinate points of each standard with a smooth line with the standard concentration as the horizontal coordinate and the A450 value as the vertical coordinate. Calculate the corresponding concentration of the sample through the absorbance value of the sample and the standard curve.

[0164] The results are shown in Figure 8. The islet organoids have a responsive effect to the change of glucose concentration. The insulin secretion amount under high glucose concentration stimulation is greater than that under low glucose concentration stimulation. The insulin secretion amount of the islet organoids encapsulated by 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 test kit. This result shows that the core layer of the double-layer hydrogel microspheres promotes the maturation of the exocrine insulin function of the stem cell differentiated islet organoids.

[0165] Example 2

[0166] The hydrogel microspheres provide a physical barrier for the graft, reducing the immune rejection of the allogeneic islet tissue transplantation. Because the extracellular matrix and the interaction between cells are destroyed during the extraction process of the islet tissue, the survival rate and exocrine function of the islet tissue cells in the in vivo transplantation are impaired.

[0167] This example provides a preparation process for preparing double-layer hydrogel microspheres based on high-voltage electrostatic non-contact electrospinning and high molecular material manufacturing, and encapsulating rat primary islet tissue. The extracellular matrix component of the islet tissue is added to the core layer material, and the mechanical properties of the matrix are adjusted, so as to reconstruct the interaction between the islet tissue and the extracellular matrix, and improve the survival rate and insulin exocrine function of the in vivo transplantation of the islet tissue. This example provides comparative data of the insulin exocrine function of the traditional single-layer hydrogel microspheres and the double-layer hydrogel microspheres and the treatment effect on hyperglycemic mice, to show that the double-layer hydrogel microspheres have significant progress compared with the prior art.

[0168] The specific process is as follows:

[0169] 1. Preparation of rat primary islet cell clusters.

[0170] Rat primary islet cells were seeded at 3 x 10 6 cells / well in 6-well plates of Aggrewell 400 (Stemcell, 34421) and incubated for 2 days, the medium composition included RPMI1640, fetal bovine serum 10% v / v, mercaptoethanol 50 mM.

[0171] 2. Preparation of single-layered alginate microspheres encapsulating rat primary islet cells

[0172] 1) Preparation of hydrogel mixture

[0173] Sodium alginate solution, type IV collagen solution, gelatin and rat primary islet cells were mixed. Among them, sodium alginate was SLG-20 (Sigma, 4202001), the concentration was 2.5% wt / v, the concentration of type IV collagen solution was 2 mg / mL (Sigma), the concentration of gelatin was 2%, and the number of cell microspheres was 3000 / mL.

[0174] 2) Preparation of single-layered alginate microspheres

[0175] A microsyringe pump and an 18G stainless steel needle were connected. The hydrogel mixture was loaded into the syringe of the microsyringe pump, and then single-layered alginate microspheres were prepared using a high-voltage electrostatic non-contact printer. The voltage of the direct current high-voltage power supply was set to 12 kV, the advancing speed of the syringe pump was 12 ml / h, and the distance between the electrodes was 2.5 cm. A plastic receiving dish was added with a hydrogel microsphere cross-linking solution, which included water, barium chloride, sodium chloride, and mannitol, among which the concentration of barium chloride (Sigma, 342920) was 20 mM, the concentration of mannitol (Sigma, BP1007) was 5% wt / v, and the concentration of sodium chloride (Sigma, S7653) was 0.9 wt / v. The hydrogel microspheres were instantly solidified by ionic coordination bonds when they entered the receiving dish, and the stabilization treatment of the hydrogel microspheres was completed. Next, the microspheres were washed three times with a buffer solution to remove the sacrificial materials such as barium chloride.

[0176] 3) Culture of alginate microspheres

[0177] The buffer solution composition included: MCDB131, glucose 0.46 g / L, BSA 21 g / L, GlutaMAX 1% v / v, Heparin 10 mg / L, MEM NEAA 1% v / v. Then it was placed in a culture dish for static culture for 10 days, and the medium composition included RPMI1640, fetal bovine serum 10% v / v, mercaptoethanol 50 mM.

[0178] 3. Preparation of double-layered alginate microspheres encapsulating rat primary islet cells

[0179] 1) Preparation of the shell layer hydrogel mixture: The shell layer was prepared using a 2.5% wt / v sodium alginate SLG 20 solution.

[0180] 2) Preparation of the core layer hydrogel mixture: The core layer was prepared using the following materials: 1% methacrylated sodium alginate, 2 mg / mL type IV collagen, 5% gelatin, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate (2 mM / 20 mM). Rat primary islet cell clusters were then added to the mixture at a concentration of 1000-5000 cells / mL.

[0181] 3) Rheological testing of the crosslinked material

[0182] After the single layer of hydrogel material and the shell and core layer mixture of the double layer hydrogel microspheres were crosslinked, the elastic modulus of the material was tested using a rheometer. The results are shown in Figure 9. The elastic modulus of the crosslinked single layer of hydrogel microspheres was 2.654 ± 0.016 kPa, the elastic modulus of the shell layer material of the double layer hydrogel microspheres was 2.631 ± 0.214 kPa, and the elastic modulus of the core layer material was 0.845 ± 0.027 kPa.

[0183] 4) Preparation process: A double-channel independent control microsyringe pump and an 18G / 21G (outer / inner) coaxial stainless steel needle were used. The shell layer hydrogel mixture was loaded into the shell layer syringe, which was connected to the outer layer of the coaxial needle; the core layer material was loaded into the core layer syringe, which was connected to the inner layer of the coaxial needle, and then a high-voltage electrostatic non-contact printer was used to prepare the double layer hydrogel microspheres. The voltage of the direct current high-voltage power supply was set to 12 kV, the shell layer channel syringe pump was set to 15 ml / h, the core layer channel syringe pump was set to 10 ml / h, and the distance between the electrodes was 2.5 cm. A hydrogel microsphere crosslinking solution was added to a plastic receiving dish, and the composition included water, barium chloride, sodium chloride, mannitol, barium chloride (Sigma, 342920) at a concentration of 20 mM, mannitol (Sigma, BP1007) at a concentration of 5% wt / v, sodium chloride (Sigma, S7653) at a concentration of 0.9 wt / v, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate (2 mM / 20 mM). The hydrogel microspheres were instantly ionically coordinated and cured in the receiving dish, and then the hydrogel microspheres were collected and irradiated with a 405 nm light source for 4 minutes (50 mW / cm 2 ), thereby completing the stabilization treatment. Next, the microspheres were washed three times with a buffer solution to remove the sacrificial materials such as barium chloride.

[0184] 5) Microspheres collection and culture: Solution components include: 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 into culture dish for static culture for 10 days, culture medium components include, RPMI1640, fetal bovine serum 10% v / v, mercaptoethanol 50 μΜ.

[0185] 4. Comparative analysis of biological functions of rat primary pancreatic islet cells encapsulated by two kinds of hydrogel microspheres

[0186] 1) Cell aging detection. After two kinds of microspheres were cultured for 10 days, the aging of cells encapsulated by two kinds of microspheres was detected by β-galactosidase in situ staining kit (Bi Yun Tian, RG0039). The detection method is as follows: collect the hydrogel microspheres cultured for 10 days, fix the microspheres with the fixing solution of the kit at room temperature for 15 minutes, and then wash with Hanks buffer for 3 times, 5 minutes each time. Use the staining working solution to stain, incubate at 37°C for 20 minutes to 2 hours, or longer. Observe under an ordinary optical microscope.

[0187] The results are shown in Figure 10A. The color of the cell cluster of rat primary pancreatic islet cells encapsulated by double-layer hydrogel microspheres is lighter than that of the cell cluster encapsulated by single-layer hydrogel microspheres, indicating that the double-layer hydrogel microspheres have less impact on cell aging than the single-layer hydrogel microspheres.

[0188] 2) Apoptosis detection. The detection method is as follows: collect the hydrogel microspheres cultured for 10 days, hydrate the microspheres with citric acid-EDTA solution, collect the rat primary pancreatic islet cell clusters, and digest the cell clusters into single cells using a mixture of trypsin-EDTA and collagenase. Then use flow cytometry and Annexin V-FITC apoptosis detection kit (Bi Yun Tian, C1062S) to quantitatively detect the apoptosis of the cell clusters. Resuspend the single cells into a suspension with staining buffer, add 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) staining solution, and mix gently. Incubate at room temperature (20-25°C) for 10-20 minutes in the dark, and then detect and analyze using flow cytometry.

[0189] The results are shown in Figure 10B. The non-apoptotic and necrotic cells of rat primary pancreatic islet cells encapsulated by double-layer hydrogel microspheres account for 93.2%, apoptotic cells account for 3.8% (2.96% + 0.84%), and dead cells account for 3%. The non-apoptotic and necrotic cells of rat primary pancreatic islet cells encapsulated by single-layer hydrogel microspheres account for 82.04%, apoptotic cells account for 9.83% (7.69% + 2.14%), and dead cells account for 8.13%. The results show that the apoptosis and death rate of rat primary pancreatic islet cells encapsulated by double-layer hydrogel microspheres is lower than that of single-layer hydrogel microspheres.

[0190] 3) Insulin exocytosis detection.

[0191] (a) Sample preparation: preparation of double-layer hydrogel and single-layer hydrogel microspheres encapsulating rat primary pancreatic islet cells. Based on the results of rheological detection, the hydrogel microspheres were irradiated with a 405 nm light source for 4 minutes and 10 minutes, respectively, to obtain double-layer hydrogel microspheres with core layer elastic modulus of 1120 Pa and 4150 Pa, respectively. The single-layer or double-layer hydrogel microspheres containing 1 x 10 6 cells were collected by centrifugation at 80 g, the original culture medium was removed, washed with Krebs buffer for 3 times, cultured with Krebs buffer containing low glucose (2 mM / L) for 2 hours, washed with Krebs buffer for 3 times, then cultured with Krebs buffer containing low glucose (2 mM / L) for 0.5 hours, the supernatant was collected and stored in a 4°C refrigerator for detection. Then the hydrogel microspheres were collected, washed with Krebs buffer for 1 time, cultured with Krebs buffer containing high glucose (20 mM / L) for 0.5 hours, and the supernatant was collected and stored in a 4°C refrigerator for detection.

[0192] (b) Standard preparation: the kit should be taken out of the refrigerator and placed at room temperature (25-28°C) for 20 minutes. Prepare the appropriate amount of washing solution: dilute the washing solution (20X) with double distilled water or deionized water to 1X. Add the standard diluent to the standard bottle according to the volume marked on the standard label, and incubate at room temperature for 15 minutes. Then mix gently and blow with a pipette several times to completely dissolve the standard. Usually 2 wells are needed for each concentration of standard, and the amount of standard used in each well is 100 μL, resulting in 200, 100, 50, 25, 12.5, 6.25, 0 μLU / mL, a total of seven standard concentrations. Finally, the diluted standard is added to the pre-coated plate wells in turn, a total of seven standard concentrations.

[0193] (c) Detection: add the sample or different concentrations of standard to the corresponding wells according to 25 μL / well. Then immediately add the horseradish peroxidase-labeled human insulin antibody according to 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 the last time is placed on thick absorbent paper to dry. Finally, add the color developing agent TMB solution according to 100 μL / well, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 10 minutes. If the room temperature is low, the incubation time needs to be appropriately extended, at which time the standard and sample can be incubated until a very significant color change occurs. Add the stop solution according to 50 μL / well, mix well, and immediately measure the A450 value.

[0194] (d) Result analysis: The absorbance value of each standard or sample should be subtracted by the absorbance value of the background correction well (if no correction well is made, no subtraction is needed). Draw the standard curve. Plot the standard concentration as the horizontal coordinate and the A450 value as the vertical coordinate, 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.

[0195] The results are shown in Figure 10C. Under high glucose induction, the insulin secretion amount of the double-layer hydrogel microspheres was about 59.1 μLU / mL, which was much higher than the insulin secretion amount of the single-layer hydrogel microspheres, 39.5 μLU / mL, p<0.01, with a significant difference. The results show that the hydrogel biochemical and physical properties of the core region of the double-layer hydrogel microspheres provide a suitable microenvironment for the insulin excretion of rat primary islet cells. As shown in Figure 10D, the insulin secretion amount of the double-layer microspheres with a low core layer modulus (1120 Pa) was higher than that of the double-layer microspheres with a high core layer modulus (4150 Pa), which shows that a suitable elastic modulus can promote the insulin excretion of islet cells.

[0196] 5. Evaluation of the hypoglycemic ability of rat primary islet cells encapsulated by two kinds of hydrogel microspheres

[0197] 1 x 10 6 The single-layer hydrogel microspheres or double-layer hydrogel microspheres encapsulating 1 x 10 6 The single-layer hydrogel microspheres or double-layer hydrogel microspheres encapsulating 1 x 10

[0198] Example 3

[0199] 1. Preparation of liver parenchymal cell clusters.

[0200] The planar cultured human primary liver parenchymal cells were digested into single cells using trypsin, and 3 x 10 6 / Holes inoculated in Aggrewell 400 6-well plates (Stemcell, 34421), static culture for 2 days, after collecting the cell mass, the cell mass was resuspended using human liver parenchymal cell complete culture medium (Pricella, CM-H043), and then cultured in a cell culture incubator at 37°C, 5% carbon dioxide and humidified conditions, using a dynamic culture shaker at a speed of 80 rpm, and cultured for 3-10 days.

[0201] 2. Preparation of double-layer hydrogel microspheres wrapped with liver parenchymal cell mass

[0202] 1) Preparation of shell layer hydrogel mixture: the shell layer is selected from a 2.5% wt / v aqueous solution of sodium alginate-polyethylene glycol-thioctic acid.

[0203] 2) Preparation of core layer hydrogel mixture with light cross-linking response: 5% wt / v methacrylic anhydride modified gelatin (GelMA) is added with a light initiator tris(2,2'-bipyridyl) ruthenium (II) chloride hexahydrate and sodium persulfate at a concentration of 0.5 mM / 5 mM. Then add the liver parenchymal cell mass, the number is 3000 / mL.

[0204] 3) Rheological detection of materials: after the shell layer and core layer mixture of the double-layer hydrogel microspheres are cross-linked, the elastic modulus is detected by a rheometer, and the results are shown in Figure 12. 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 light 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.

[0205] 4) Preparation process: Dual-channel independent control microsyringe pump and 18G / 23G (outer / inner) coaxial stainless steel needle were used. The outer shell layer hydrogel mixture was loaded into the outer shell layer syringe, connecting the outer layer of the coaxial needle; the core layer material was loaded into the core layer syringe, connecting the inner layer of the coaxial needle, and then the dual-layer hydrogel microspheres were prepared using a high-voltage electrostatic non-contact printer. The voltage of the direct current high-voltage power supply was set to 12 kV, the outer shell layer channel syringe pump pushing speed was 20 ml / h, the core layer channel syringe pump pushing speed was 10 ml / h, and the electrode distance was 2.5 cm. The hydrogel microspheres were added to the crosslinking solution in the plastic receiving dish, which included water, barium chloride, sodium chloride, and mannitol, with a barium chloride (Sigma, 342920) concentration of 20 mM, a mannitol (Sigma, BP1007) concentration of 5% wt / v, and a sodium chloride (Sigma, S7653) concentration of 0.9 wt / v. The hydrogel microspheres were instantaneously ionically coordinated to solidify in the receiving dish, and then a 405 nM light source was used for irradiation for 5 minutes, thereby completing the stabilization treatment. Next, the microspheres were washed three times with a buffer solution to remove barium chloride and sacrificial materials such as the photo-initiator tris(2,2'-bipyridyl)ruthenium(II) hexahydrate and sodium persulfate.

[0206] 5) Microsphere collection and culture: The solution composition included human liver parenchymal cell complete culture medium (Pricella, CM-H043). Then, under the conditions of 37°C, 5% carbon dioxide, and humidity in the cell culture box, the culture was carried out using a dynamic culture shaker at a speed of 80 rpm for 3-10 days.

[0207] 3, Biological function detection of liver parenchymal cell clusters wrapped by double-layer hydrogel microspheres

[0208] 1) Marker expression of liver organoids wrapped by double-layer hydrogel microspheres

[0209] After culturing the double-layer hydrogel microspheres wrapped liver parenchymal cell clusters for 7 days, the cell clusters in the hydrogel microspheres were collected to detect the gene and protein expression levels of the liver parenchymal cell function-related markers ALB and CYP1A1 by qRT-PCR and cell immunofluorescence staining methods.

[0210] As shown in FIG. 13, FIG. 13A shows that the gene expression level of the liver detoxification function marker CYP1A1 of the double-layer hydrogel microsphere-wrapped liver parenchymal cell cluster was significantly higher than that of the conventional planar culture (2D) human primary liver parenchymal cells and single-layer hydrogel microspheres. The cell immunofluorescence staining results further confirmed (FIG. 13B) that the double-layer hydrogel microsphere-wrapped liver parenchymal cell cluster had high expression of ALB and CYP1A1 proteins. These results indicate that the double-layer hydrogel microspheres better maintain the function and cell characteristics of human primary liver parenchymal cells than the single-layer hydrogel microspheres and 2D culture.

[0211] 2) Bile salt transport ability detection of double-layer hydrogel microsphere-encapsulated hepatocyte spheroids

[0212] After culturing the double-layer hydrogel microsphere-encapsulated hepatocyte spheroids for 7 days, cholate-lysylfluorescein (CLF) was added into the culture medium for co-incubation, and then the culture medium was replaced with fresh culture medium without CLF for CLF release. The uptake and transport ability of hepatocyte spheroids to CLF was determined by detecting the fluorescence signal of CLF in the spheroids.

[0213] As shown in Figure 14, the double-layer hydrogel microsphere-encapsulated hepatocyte spheroids efficiently uptook CLF (Figure 14A arrow), and the signal of CLF decreased after 4 hours of soaking in the culture medium, indicating that the double-layer hydrogel microsphere-encapsulated hepatocyte spheroids had the function of uptaking and releasing CLF (Figure 14B and Figure 14C).

[0214] 3) Fatty accumulation ability detection of double-layer hydrogel microsphere-encapsulated hepatocyte spheroids

[0215] After culturing the double-layer hydrogel microsphere-encapsulated hepatocyte spheroids for 7 days, the hydrogel microspheres were fixed in situ, and Nile red and cell nucleus DAPI staining solution were used for in situ staining and image quantitative analysis. The control group was the 3D primary hepatocyte spheroids cultured in the conventional Aggrewell microplate. The influence of the double-layer hydrogel microsphere material system and the engineering manufacturing process on the fatty storage function of human primary hepatocyte spheroids was analyzed by image quantitative comparison.

[0216] As shown in Figure 15, the fatty staining signal of Nile red was visible in the double-layer hydrogel microsphere-encapsulated hepatocyte spheroids (Figure 15A), and there was no significant difference with the fatty staining signal of human primary hepatocyte spheroids (Figure 15B and Figure 15C). This indicates that the material system and the engineering manufacturing process of the double-layer hydrogel microspheres have no obvious damage to the function of human-derived hepatocytes.

[0217] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. These modifications or improvements made on the basis of the spirit of the present application are within the scope of the present application.

Claims

1. Double-layered hydrogel microspheres, characterized in that, The double-layer hydrogel microspheres have an outer shell layer and a core layer, forming a core-shell double-layer structure, the core layer encapsulating a graft, and the outer shell layer and the core layer having 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 material constituting the outer shell layer is: 1-3 % wt / v sodium alginate-polyethylene glycol-polyarginine, 1 %-3 % wt / v sodium alginate solution or 1-3 % wt / v sodium alginate-polyethylene glycol-α-lipoic acid solution; the material constituting the core layer is: 1 %-6 % wt / v polyether F127 diacrylate, 0.5-5 % methacrylated heparin, 1-10 μg / mL vitronectin and 0.1-5 μg / mL laminin, 1-3 % methacrylated sodium alginate, 0.02-8 mg / mL type IV collagen and 1-10 % wt / v gelatin, or 2-10 % wt / v methacrylated gelatin; The preparation method of the double-layer hydrogel microspheres comprises the following steps: (1) preparing a graft; (2) respectively preparing a mixture of outer shell layer polymer hydrogel material and a mixture of core layer polymer hydrogel material; (3) mixing the graft and the mixture of core layer polymer hydrogel material to obtain a mixture A; (4) three-dimensionally forming the mixture obtained in steps (2) and (3) by a high-voltage electrostatic non-contact spraying method to obtain double-layer hydrogel microspheres having an outer shell layer and a core layer, and positioning the graft in the core layer region; (5) culturing the double-layer hydrogel microspheres obtained in step (4); The specific operation of step (4) is: using independently controlled double-channel injection pumps, the mixture of outer shell layer polymer hydrogel material and the mixture of core layer polymer hydrogel material mixed with the graft are loaded into corresponding containers, the injection pump advancing speed or pushing pressure, voltage, electrode distance are adjusted, a high-voltage electrostatic non-contact printer is used to print to prepare double-layer hydrogel droplets, and then a stabilization treatment and sacrificial material removal are performed to form double-layer hydrogel microspheres encapsulating the graft; The stabilization treatment refers to that the double-layer hydrogel microspheres need to be rapidly crosslinked to be fixed and formed; The crosslinking method when the double-layer hydrogel microspheres are crosslinked and formed is: (a) using a crosslinking agent containing divalent cations such as calcium ions, barium ions and iron ions, (b) using a 405 nm light source and a photo initiator such as lithium phenyl (2,4,6-trimethylbenzoyl) phosphate and / or chlorotris (2,2'-bipyridine) ruthenium (II) hexahydrate and sodium persulfate for photo crosslinking.

2. The double-layered hydrogel microspheres according to claim 1, wherein, The elastic modulus of the core layer is comparable to the elastic modulus of the graft.

3. The double-layer hydrogel microspheres according to claim 1 or 2, characterized in that, The double-layer hydrogel microspheres are in a spherical or approximately spherical structure, 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-layered hydrogel microspheres according to claim 1 or 2, characterized in that, The transplant is an islet organoid, an islet beta cell cluster or an islet beta-like cell cluster, a liver organoid, a liver parenchymal cell cluster.

5. The double-layered hydrogel microspheres according to claim 4, wherein, The islet organoid has a three-dimensional arbitrary shape, and a longest axis diameter of a three-dimensional space is 10-500 μm; the islet organoid contains one or more cells including islet beta cells or islet beta-like cells having an insulin secretion function.

6. The double-layered hydrogel microspheres according to claim 4, wherein, The islet beta cells or islet beta-like cells are human or non-human cells, and the islet beta cells or islet beta-like cells are derived from one or more of the following: i) induced pluripotent stem cells, embryonic stem cells, endoderm cells, pancreatic endoderm cells, pancreatic precursor cells, pancreatic progenitor cells differentiated from; ii) cells extracted from human or animal pancreatic tissues or embryonic pancreatic tissues; iii) cells obtained by gene editing, virus packaging, line construction or modification of i) and ii).

7. The double-layered hydrogel microspheres according to claim 1, wherein, The transplant is an artificial islet organoid.

8. The double-layered hydrogel microspheres according to claim 7, wherein, In the mixture A, the concentration of the artificial pancreatic islet organ is 1 x 10 3 to 8 x 10 4 to 8 x 10 9. A polymer material composition having a three-dimensional space structure, comprising the double-layer hydrogel microspheres of any one of claims 1-8.

10. Use of the double-layer hydrogel microspheres of any one of claims 1-8 or the polymer material composition of claim 9 in the preparation of a product for treating an islet or liver related disease.

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