CN118542980A

CN118542980A - Biological printing matrix based on microgel

Info

Publication number: CN118542980A
Application number: CN202310198755.6A
Authority: CN
Inventors: 顾奇; 王新环; 刘鑫
Original assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Current assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2024-08-27

Abstract

The invention relates to the technical field of tissue engineering and biological manufacturing, in particular to a biological printing matrix based on microgel, a preparation method and application thereof. The bioprinting matrices of the present invention comprise a microgel, one or more photocrosslinking molecules, and one or more bioactive substances. The biological printing matrix can be combined with an embedded printing or bio-extrusion printing strategy to regulate and control angiogenesis and angiogenesis behaviors of HUVECs in 2D/3D, so that in-vivo transplantation of multi-scale vascular tissues capable of being directly vascular anastomosed is realized.

Description

Biological printing matrix based on microgel

Technical Field

The invention relates to the technical field of tissue engineering and biological manufacturing, in particular to a biological printing matrix based on microgel, a preparation method and application thereof.

Background

Extrusion bio-3D printing typically mixes hydrogel materials with cells as bio-ink, extrusion printing is performed in air according to a predefined path, and 3D tissues and organs with complex structures are formed by layer-by-layer stacking using multi-material composite printing. For the construction of the vascular system, the sacrificial material is extruded and printed, and the sacrificial material is removed through a solvent core after printing, so that the vascular system is formed. Two printing strategies are necessary for the construction of large-scale tissues and organs (in cm-scale).

Although extrusion type biological 3D printing technology has the potential of manufacturing complex tissue structures, the technology is the mainstream printing technology at present, in the technology, hydrogel materials for wrapping cells are limited by certain viscosity, when the viscosity is low, the cell viability can be ensured, but the printing precision is limited, when the viscosity of the hydrogel is high, the wrapped cells can be subjected to shearing force at a nozzle, and the cell viability is affected. Thus, during extrusion, the magnitude of the shear force is affected by the viscosity and rheological properties of the hydrogel, the greater the viscosity of the hydrogel, the greater the shear force. And the viscosity is too small, which is unfavorable for shape maintenance, extrusion into filaments and reduction of printing resolution. Extrusion bioprinting is greatly limited by the nature and concentration range of hydrogels, and for large volume tissue printing, extrusion printing efficiency affects cell viability at the same time, limiting the application of printing.

On the other hand, the concentration of hydrogel affects the behavior of the encapsulated cells, including 2D attachment, proliferation, cell survival in 3D, proliferation, migration, and self-assembly behavior. For the molecular chains of synthetic and natural polymers, the higher the concentration, the denser the cross-linking between the chains, the smaller the pore structure, and the serious limitation of the extension and migration of cells in the three-dimensional space. In contrast, the lower the concentration, the greater the porosity formed by inter-molecular chain cross-linking, providing room for migration and assembly of cells, but the lower the mechanical properties, the inability to meet the shear force of physiological blood flow, thus limiting the feasibility of in vivo grafting strategies by direct anastomosis with in vivo blood vessels.

Therefore, hydrogel mechanical properties, bioactivity, printing strategies to construct large volumes of vascular tissue, grafting strategies, etc. can limit the transfer, transformation of in vitro constructed tissue. It would be particularly desirable to provide a matrix that is printable, satisfies endothelial cell assembly behavior, and can withstand blood flow shear forces.

Disclosure of Invention

When the implantable vascularized tissue/organ is constructed in vitro in a bionic way, the hydrogel is required to meet the requirements on biological activity and mechanical property, and specifically, the method can ensure angiogenesis and angiogenesis of endothelial cells and can resist shearing force of physiological blood flow on the vessel wall. However, the use of a uniform hydrogel construction strategy cannot meet both of the above requirements.

In view of the above, the present invention provides a composite hydrogel matrix formed of photocrosslinkable microgels with photocrosslinkable molecules and bioactive substances. By utilizing the composite matrix, a biological extrusion printing/embedded printing strategy can be combined, and a core dissolution method is utilized to construct a vascular system, wherein the problem that shearing force affects cell activity when the composite matrix is used as the embedded printing matrix to construct the vascular system does not exist; extrusion printing can utilize a multi-material composite printing strategy, and the composite material can meet the angiogenesis and vasculogenesis of endothelial cells, so that a large-volume vascular tissue with an internal pressure-bearing structure is constructed.

Thus, the present application provides the following applications:

In one aspect, the application provides a bioprinting substrate comprising a microgel, one or more photocrosslinking molecules, and one or more bioactive substances; the microgel is a microgel formed by biocompatible polymers (including but not limited to GelMA, HAMA, chitosan, gelatin, protein or polyethylene glycol microgel); the photo-crosslinking molecules are high polymers containing photo-reactive groups; the bioprinting substrate is capable of forming a hydrogel having a heterogeneous microstructure upon ultraviolet irradiation.

In the present invention, the term "heterogeneous microstructure" means a hydrogel having a differential internal structure, i.e., a differential microenvironment (heterogeneous microenvironment) is formed for a cell. In the hydrogel formed by the bioprinting matrix after illumination, the cross-linked structure inside the particles (microgel particles) is different from the cross-linked structure formed between the particles and the macromolecules (microgel particles and photocrosslinked molecules), so that the differences exist in aspects of pore area, rigidity and the like inside the hydrogel.

In some more specific embodiments, the microgel forms a composite hydrogel with photocrosslinking molecules and bioactive substances through two modes of physical encapsulation and chemical crosslinking, wherein the microgel forms microspheres with compact crosslinking structures inside, and the microgel forms chemical crosslinking with photocrosslinking molecules to form physical encapsulation with bioactive substances. Such a complex forms the heterostructure of the hydrogel.

In some more specific embodiments, the bioprinting matrices of the present invention are photo-crosslinkable composite gel systems based on GelMA microgels. Gelatin (Gelatin) is a product of collagen partial hydrolysis, gelMA (methacryloylated Gelatin) is a double bond modified Gelatin, and can be crosslinked and cured under the action of a photoinitiator through ultraviolet and visible light. GelMA has the characteristics of natural and synthetic biological materials, has a three-dimensional structure suitable for cell growth and differentiation, excellent biocompatibility and cell reaction characteristics, good temperature-sensitive gel characteristics and degradability, and adjustable mechanical properties.

Microgel refers to gel particles having a crosslinked structure inside, and can also be understood as gel particles comprising colloids consisting of chemically crosslinked three-dimensional polymer networks. The microgel particles may be on the nano-or micro-scale (e.g., 1 to 10nm, 10 to 100nm, 100 to 200nm, 200 to 500nm, 500 to 1000nm, 1 to 10 μm, 10 to 100 μm, 100 to 200 μm, 200 to 500 μm, or 500 to 1000 μm) in size.

In some embodiments, microgels having low yield stress, shear thinning properties may be employed.

The yield stress refers to the yield stress of some non-newtonian fluids, in which the fluid is deformed only when the applied shear stress is small, no flow occurs, and the fluid starts to flow when the shear stress increases to a certain value.

Specifically, after a critical shear stress is applied to the microgel, it begins to flow. This behavior can be analyzed using a rheometer, with the microgel exhibiting a solid when the shear stress is below the yield stress and a fluid when the shear stress is above the yield stress. At this level of applied stress, the microgel undergoes a rapid decrease in viscosity with increasing shear rate, demonstrating the shear thinning properties. While maintaining a shear stress above the critical yield stress, the particles will continue to slide past each other and exhibit fluid-like behavior. When the shear force decreases below the critical yield stress, the microgels reattach to each other by the same force that initially holds them together.

The self-healing property of the printing matrix can be endowed by adopting the material with low yield stress and shear thinning property, so that the requirement of embedded printing is met. In some embodiments, the microgels used in the present invention have shear thinning properties with a yield stress of 10 to 200Pa (e.g., 10 to 20Pa, 20 to 30Pa, 30 to 50Pa, 50 to 100Pa, 100 to 150Pa, or 150 to 200 Pa). The yield stress of the microgel can be measured by using a rheometer, and the measurement method can be a method described in the specification of the rheometer or a method specified by national standard or international standard related to the field.

In some embodiments, the microgel used in the present invention is a GelMA microgel.

The mechanical property of the printing matrix can be regulated and controlled by selecting the modification degree of the GelMA, and the lower the MA content in the GelMA is, the less crosslinking is easy to generate. In some embodiments, the degree of modification of GelMA is 45% -90% (e.g., 45% -50%, 50% -60%, 60% -70%, 70% -75%, 75% -80%, or 80% -90%). The matrix formed by the GelMA microgel with the modification degree within the range has mechanical properties suitable for printing.

Microgels may be prepared by a complex coacervation process comprising: the solubility of the biocompatible polymer in the solvent is reduced by utilizing the complex coacervate, so that the biocompatible polymer is precipitated to form microgel. The material of the complex coacervate may be a polymer that is incompatible with the biocompatible polymer and that can exert an electrostatic effect, for example, gum arabic is incompatible with GelMA, and may be a complex coacervate of GelMA. In some embodiments, gelMA is used to form the microgel, and the solvent used in the complex coacervation process may be a good solvent for GelMA, such as an aqueous ethanol solution.

GelMA microgels may be prepared by a process comprising the steps of:

Step 1: dissolving GelMA with an optional surfactant (e.g., pluronic F-127) in aqueous ethanol to form a GelMA solution; the dissolution may be carried out under heating (e.g. 45-55 ℃) and/or stirring;

step 2: adding complex coacervate (such as acacia gum) into the GelMA solution, dissolving the complex coacervate, regulating the pH value of the solution to 6-7, and stirring the solution to form GelMA microgel;

Step 3: stopping stirring to enable the GelMA microgel to settle; washing and collecting the GelMA microgel. In some embodiments, the process of washing, collecting the GelMA microgel comprises: the GelMA microgel was allowed to settle, the supernatant removed, the GelMA microgel washed with a buffer solution (e.g., PBS) and collected by centrifugation.

After electrostatic interaction is generated on the complex coacervates of GelMA and Arabic gum, the solubility of the GelMA in ethanol aqueous solution is reduced, and microgel is formed and separated out. The higher the modification degree of MA in Gelatin, the higher the solubility of gelMA in ethanol, and the less likely to be precipitated, so that the higher the modification degree of MA, the higher the proportion of ethanol in ethanol aqueous solution is required, the more the microgel can be precipitated, and the larger the scale of the precipitated microgel is.

In some embodiments, in step 1, the GelMA is present in the solution at a mass concentration of 2g/100mL to 5g/100mL (e.g., 2 to 3g/100mL, 3 to 4g/100mL, 4 to 5g/100 mL).

In some embodiments, in step 1, the volume fraction of ethanol in the aqueous ethanol solution is 50% to 80% (e.g., 50% to 60%, 60% to 70%, or 70% to 80%).

In some embodiments, the surfactant is present in the solution at a mass concentration of 0 to 0.5g/100mL (e.g., 0.1 to 0.2g/100mL, 0.2 to 0.25g/100mL, 0.25 to 0.3g/100mL, 0.3 to 0.4g/100mL, or 0.4 to 0.5g/100 mL).

In some embodiments, the complex coacervates are present in the solution at a mass concentration of between 0.02g/100mL and 0.1g/100mL (e.g., between 0.02 and 0.03g/100mL, between 0.03 and 0.05g/100mL, between 0.05 and 0.07g/100mL, or between 0.07 and 0.1g/100 mL).

GelMA is obtained by modifying gelatin with methacrylic anhydride. In some embodiments, gelMA is made by a process comprising the steps of: reacting methacrylic anhydride with gelatin under heating, stirring and in the presence of a solvent. In some embodiments, the humidity of GelMA is 75%.

In order to enhance the mechanical properties of the matrix, the invention uses photocrosslinking molecules to be compounded with microgels to form a gel system. The photocrosslinking molecules used in the invention are polymers containing photoreactive groups, and the microgel can be connected with each other through molecular chains on the photocrosslinking molecules to form a cross-linking system, so that the mechanical strength of the matrix is enhanced.

In some embodiments, the photocrosslinking molecules are biocompatible polymers containing photoreactive groups, including but not limited to: sodium alginate, chitosan, silk fibroin, hyaluronic acid, fibrinogen, chondroitin sulfate, methacryloylated products of albumin (e.g., methacryloylated hyaluronic acid (HAMA), methacryloylated sodium alginate (AlgMA), methacryloylated gelatin (GelMA)), and polyethylene glycol diacrylate (PEGDA), tetra-arm polyethylene glycol acrylate (4-arm-PEG-AC), methacryloylated polyvinyl alcohol (PVAMA)), and the like.

In some embodiments, the biocompatible polymer containing photoreactive groups is selected from the group consisting of: HAMA, PEGDA, gelMA.

In some embodiments, the biocompatible polymer comprising a photoreactive group is selected to be HAMA. In some embodiments, the degree of modification of HAMA is 20% to 25%.

In order to increase the biocompatibility and bioactivity of the matrix, the composite gel system of the invention further comprises bioactive substances. In the present invention, the bioactive substance may be selected from bioactive compounds or combinations thereof, including, but not limited to, polysaccharides, terpenes, sterols, alkaloids, peptides, nucleic acids, proteins, amino acids, glycosides, oils, waxes, resins, plant pigments, mineral elements, enzymes, vitamins, and the like.

In some embodiments, the bioactive substance is a protein (e.g., collagen, fibrinogen, fibronectin) or an extracellular matrix (e.g., matrigel).

In some embodiments, the bioactive substance is a protein at a mass concentration of 0.2g/100mL to 0.5g/100mL (e.g., 0.25g/100 mL).

In some embodiments, the bioactive material is Matrigel, which has a volume to mass ratio of 6-8 ml to 1g of the biocompatible polymer forming the microgel, or which has a volume to microgel ratio of 1:3-5 (e.g., 1:4). In some embodiments, the bioactive agent is a cytokine capable of inducing differentiation of an undifferentiated cell into a smooth muscle cell or endothelial cell, such as TGF- α1, PDGF-BB, VEGF, or b-FGF.

In some embodiments, the bioactive substance is Matrigel, which is a basement membrane matrix, and comprises laminin, type iv collagen, heparan sulfate glycoprotein, entactin, and a plurality of growth factors, matrix metalloproteinases, and the like as a major component. Under the condition of room temperature, matrigel is polymerized to form a three-dimensional matrix with biological activity, and the structure, composition, physical characteristics and functions of in-vivo cell basement membrane are simulated, so that the Matrigel is favorable for in-vitro cell culture and differentiation, and can be used for researching cell morphology, biochemical functions, migration, infection, gene expression and the like. In some embodiments, corning is used for MatrigelThe protein concentration is 8-12mg/mL or 18-22mg/mL.

After the biological printing matrix is irradiated, microgel and photo-crosslinking molecules are initiated to crosslink, so that a stable matrix is formed. In some embodiments, the ultraviolet light irradiation intensity is from 2 to 10mW/cm ² (e.g., from 2 to 4mW/cm ²、4～6mW/cm²、6～8mW/cm² or from 8 to 10mW/cm ²). In some embodiments, ultraviolet light irradiation with wavelengths of 365nm or above 365nm (e.g., 365nm to 405 nm) is used.

The bioprinting matrices of the present invention may also include photoinitiators including, but not limited to: 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (I2959), phenyl (2, 4, 6-trimethylbenzoyl) phosphate lithium salt (LAP), 2-hydroxy-2-methyl-1-phenylpropanone (1173), 1-hydroxycyclohexylphenyl ketone (184), 2-methyl-2- (4-morpholino) -1- [4- (methylthio) phenyl ] -1-propanone (907), 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide (TPO), ethyl 2,4, 6-trimethylbenzoyl phenylphosphonate (TPO-L), 2-dimethylamino-2-benzyl-1- [4- (4-morpholino) phenyl ] -1-butanone (IHT-PI 910), 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (695), methyl Benzoate (MBF), or any combination thereof.

In some embodiments, LAP and I2959 may be selected for use in the cell-containing bioprinting matrix in view of water solubility, photocatalytic efficiency, light source, etc., with LAP being more preferred in view of biocompatibility.

In some embodiments, the concentration of the photoinitiator in the bioprinting matrix is from 0.01g/100mL to 0.1g/100mL (e.g., from 0.01 to 0.03g/100mL, from 0.03 to 0.05g/100mL, from 0.05 to 0.08g/100mL, or from 0.08 to 0.1g/100 mL).

The bioprinting matrices of the present invention may also include one or more cells. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoan cell, a plant cell, an animal cell, an algal cell, a fungal cell, an artificial cell, or any combination thereof; or the cells are selected from stem cells, somatic cells, germ cells, or any combination thereof.

In some embodiments, the stem cells are selected from Embryonic Stem (ES) cells, induced Pluripotent Stem (iPS) cells, mesenchymal stem cells, neural stem cells, muscle stem cells, hematopoietic stem cells, epithelial stem cells, breast stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, or any combination thereof.

In some embodiments, the somatic cell is selected from the group consisting of an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast, an osteoblast, a chondrocyte, a myocyte, a bone cell, a liver cell, a pancreatic cell, an exogenous cell, an endogenous cell, a cardiac myocyte, a skeletal cell, a cardiac myoblast, a skeletal myoblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, or any combination thereof.

In some embodiments, the cells are derived from a tissue selected from the group consisting of: connective tissue (e.g., loose connective tissue, dense connective tissue, elastic tissue, reticulocyte connective tissue, and adipose tissue), muscle tissue (e.g., skeletal muscle, smooth muscle, and cardiac muscle), genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, nerve tissue, and epithelial tissue (e.g., single-layer epithelium and multiple-layer epithelium), endodermal-derived tissue, mesodermal-derived tissue, and ectodermal-derived tissue.

In some embodiments, the cell is a functional cell, such as a primary stem cell or at least one of a pluripotent stem cell-derived parenchymal cell, a stromal cell, and an endothelial cell.

In some embodiments, the cells are selected from endothelial cells and fibroblasts, such as umbilical cord-derived human vascular endothelial cells.

In some embodiments, the cell is a human mesenchymal stem cell.

In some embodiments, the number of cells contained per volume of bioprinting matrix may be 10 ⁵～10⁷/mL.

The biological printing matrix can be used for embedded printing and also can be used for extrusion printing. When the mass concentration of the microgel is more than or equal to 2.5%, the matrix has higher strength, and can be used as an embedded printing matrix or an extrusion printing matrix; when the mass concentration of the microgel is lower than 2.5%, the strength of the matrix is weaker, and the supporting property cannot meet the requirement of embedded printing, but can be used as an extrusion printing matrix.

In some embodiments, the bioprinting matrix is an embedded printing matrix, the mass concentration of the photocrosslinking molecules is 0.2g/100mL to 0.6g/100mL (e.g., 0.2 to 0.3g/100mL, 0.3 to 0.4g/100mL, 0.4 to 0.5g/100mL, or 0.5 to 0.6g/100 mL), and the mass concentration of the GelMA microgel is 2.5g/100mL to 4g/100mL (e.g., 2.5 to 3g/100mL, 3 to 3.5g/100mL, or 3.5 to 4g/100 mL).

In some embodiments, the bioprinting matrix is an extrusion printing matrix, the mass concentration of the photocrosslinking molecules is 2g/100mL to 4g/100mL (e.g., 2 to 2.5g/100mL, 2.5 to 3g/100mL, 3 to 3.5g/100mL, or 3.5 to 4g/100 mL), and the mass concentration of the GelMA microgel is 0.2g/100mL to 0.6g/100mL (e.g., 0.2 to 0.3g/100mL, 0.3 to 0.4g/100mL, 0.4 to 0.5g/100mL, or 0.5 to 0.6g/100 mL).

According to the composite hydrogel matrix provided by the invention, the addition of photocrosslinking molecules and bioactive substances among microgels provides a heterogeneous microenvironment, so that the self-assembly of endothelial cells wrapped by 3D is promoted to form a capillary network on the basis of ensuring mechanical properties, and a multi-scale complex pipe network can be further constructed. In addition, not only can extrusion printing be used to construct vasculature within the embedded matrix, but extrusion printing can also be used to achieve spatial localization of specific factors or other active molecules.

In one aspect, the application provides a method of preparing a bioprinting substrate of the application, the method comprising: and uniformly mixing all the components contained in the biological printing matrix. In some embodiments, the method comprises: and adding other components contained in the biological printing matrix into the microgel and uniformly mixing.

Optionally, the method further comprises irradiating the bioprinting substrate under ultraviolet light to produce cross-linking. In some embodiments, the ultraviolet light irradiation intensity is from 2 to 10mW/cm ² (e.g., from 2 to 4mW/cm ²、4～6mW/cm²、6～8mW/cm² or from 8 to 10mW/cm ²). In some embodiments, ultraviolet light irradiation with a wavelength of 365nm or above 365nm is used.

In one aspect, the application provides a kit comprising the bioprinting substrate of the application.

In one aspect, the application provides the use of the bioprinting matrices of the application in the preparation of a kit for in vitro 3D bioprinting or in vitro biomimetic structure construction.

In one aspect, the application provides the use of the bioprinting matrices of the application for in vitro 3D bioprinting or in vitro biomimetic structure construction. In some embodiments, the use is for non-diagnostic or therapeutic purposes (e.g., scientific research purposes).

The printing matrix can be used for bioprinting, in some embodiments, cells and a composite matrix containing GelMA microgel are mixed together, the vascular system is constructed by extrusion printing and/or embedded printing, the survival of the cells is not influenced in the printing process, the space heterostructure formed among the microgel matrixes is utilized, the requirements of cell compatibility and mechanical properties are met, and the printing matrix is a novel strategy for constructing large-scale implantable vascular tissues based on the microgel-based bioprinting matrix.

Accordingly, in a further aspect, the present application provides a method of bioprinting comprising the use of a bioprinting substrate or kit of the present application. In some embodiments, the bioprinting method includes extrusion printing and/or embedded printing.

In some embodiments, the method comprises: using the biological print substrate of the present invention as a first printing ink, using a sacrificial ink as a second printing ink, forming a predetermined structure by extrusion printing; irradiating the predetermined structure with ultraviolet light to crosslink the bioprinting substrate; the sacrificial ink is removed to obtain a bioprinting product. In some embodiments, the sacrificial ink is an aqueous gelatin solution (e.g., 10wt% aqueous gelatin solution).

In some embodiments, the method comprises: extruding and printing sacrificial ink into the biological printing substrate of the invention; irradiating the bioprinting substrate with ultraviolet light to produce cross-links; the sacrificial ink is removed to obtain a bioprinting product.

In some embodiments, the bioprinting method is used to prepare blood vessels.

In one aspect, the application provides a product obtained by any one of the above-described methods of bioprinting. In some embodiments, the product is an organoid. In some embodiments, the product is a angioid.

In one aspect, the application provides the use of the bioprinting matrices, kits or products obtained by the bioprinting methods of the application for repair of diseased tissues or organs, drug development and screening, or preparation of pathology research models. In some embodiments, the use is for non-diagnostic or therapeutic purposes (e.g., scientific research purposes). In some embodiments, the tissue or organ is selected from the group consisting of heart, liver, kidney, pancreas, brain, or tissue comprising any of the foregoing organs.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. However, for a better understanding of the present invention, definitions and explanations of related terms in the present specification are provided below. To the extent that the definitions set forth in this specification conflict with the meanings commonly understood by those skilled in the art, the definitions set forth in this specification will control.

As used herein, the term "bioprinting" refers to: printing with biological materials (including, but not limited to, biomolecules such as proteins, lipids, nucleic acids and metabolites, cells such as cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates and multicellular bodies, subcellular structures such as organelles and cell membranes, molecules associated with biomolecules such as synthetic biomolecules or analogs of biomolecules). As used herein, the term "printing" refers to a process of depositing material in a predetermined pattern. In the present invention, the bio-printing is preferably achieved by a method that matches an automated or semi-automated, computer-aided three-dimensional prototype device (e.g., a bio-printer). However, in the present invention, "printing" (e.g., bioprinting) may be performed by a variety of methods, including, but not limited to, printing using a printer (e.g., a 3D printer or a bioprinter); printing using automated or non-automated mechanical processes (rather than printers); printing is performed by manual placement or manual deposition (e.g., using a pipette).

As used herein, the term "bioprinting substrate" refers to a material that can be used for bioprinting to produce a particular planar and/or layered geometry; and preferably the resulting planar and/or layered geometries can be further stacked to form a three-dimensional construct having a particular shape and structure. In addition, cells in the bioprinting matrix are capable of performing various desired vital activities before, during, and/or after bioprinting. In some cases, the bioprinting substrate is an ink (bio-ink) used for bioprinting.

As used herein, the term "biocompatible material" refers to a material that is non-toxic to cells (and degradation products thereof) and that is compatible with a host after implantation into the host (e.g., human body) without causing significant or serious side effects, e.g., without causing toxic effects to the host (e.g., human tissue), without causing immune rejection, allergic or inflammatory reactions, and the like in the host.

As used herein, the term "polymer" refers to compounds having a relative molecular mass of from a few thousand to millions, including natural polymers and synthetic polymers.

As used herein, the term "photoinitiator" is also known as a photosensitizer (photosensitizer) or photo-curing agent (photocuring agent) and is a class of compounds that absorb energy of a wavelength in the ultraviolet or visible region to generate free radicals, cations, etc., to initiate polymerization and/or crosslinking of monomers for curing.

As used herein, the term "organoid" refers to a three-dimensional (3D) cell culture that contains some of the key characteristics that it represents an organ, belonging to an in vitro culture system. Organoids comprise a self-renewing stem cell population that can differentiate into a plurality of organ-specific cell types that possess similar spatial organization to the corresponding organ and are capable of reproducing a portion of the function of the corresponding organ, thereby providing a highly physiologically relevant system.

Advantageous effects

The invention provides a composite matrix formed by compositing gel microspheres, photo-crosslinking molecules and bioactive substances, which can simultaneously meet the mechanical property and bioactivity required by biological printing and can be used as embedded printing ink or extrusion printing ink according to the concentration of the microgel. Compared with the existing biological ink, the ink has the following advantages and outstanding effects, and is shown in the following:

(1) The gel microsphere-based composite matrix has the characteristics of shear thinning, low yield stress and self-healing, can be directly used as an embedded printing matrix under the condition of not adding a rheology modifier, realizes the characteristic that conventional gel materials such as collagen, matrigel, active molecules and the like are difficult to print at an ultralow concentration, and endows the difficult-to-print hydrogel material with excellent printability, so that a biological ink reservoir is greatly expanded; the cells can be directly mixed with the embedded matrix, so that the tissue is constructed in a bionic way, the damage of shearing force to the cells is avoided, and the self-assembly or the realization of tissue functions is facilitated; meanwhile, bioactive factors such as VEGF and the like can be specifically distributed in any space of the embedded matrix by extrusion printing, and specifically induce migration and assembly of cells.

(2) The gel microsphere is compounded with the photo-crosslinking molecules to form a heterogeneous microstructure at 37 ℃, wherein the high-modulus sphere formed by the compact crosslinking of the microgel is favorable for cell adhesion at a 3D interface, the macroporous structure formed by the weak crosslinking between the microgel is favorable for cell migration and assembly, and the heterogeneous structure can simultaneously show biological performance and mechanical property in a matrix and lay a foundation for the transplantation of tissues/organs constructed in vitro and the perfusion of a constructed vascular system.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 is an inverted microscopic field plot of the GelMA microgel of example 1.

FIG. 2 is a graph showing the rheological properties of GelMA microgels of example 1.

Fig. 3 shows the printing of the helical piping network of example 1.

FIG. 4 is a graph of heterogeneous microscopic characterization of the crosslinked microgel of example 2, including cryo-scanning electron microscopy (left) and microscopic heterogeneous stiffness (right).

FIG. 5 is a fluorescent characterization of HUVEC endothelialization, 3D HUVEC self-assembled capillary networks in example 4.

Figure 6 shows the vascular tissue model printed in example 4 as matched to blood vessels in rats and blood perfusion.

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents used in the examples of the present invention are all commercially available unless otherwise specified.

Example 1

The embodiment provides a preparation method of GelMA microgel, which comprises the following steps:

Preparation of GelMA with 75% ma modification: 10g of gelatin was dissolved in PBS to prepare a 10g/100mL solution, which was stirred at a constant temperature of 50 ℃. After the gelatin was completely dissolved, 5mL of methacrylic anhydride was dropwise added to the solution. After the completion of the dropwise addition, stirring at constant temperature for 3 hours. And (3) dialyzing, freeze-drying to obtain methacrylic anhydride modified gelatin (GelMA). The degree of modification of 75% was determined using nuclear magnetic resonance identification.

2G of the 75% MA modified GelMA polymer and 0.25g of F127 were added to 100mL of 70% aqueous ethanol at final concentrations of 2g/100mL and 0.25g/100mL, respectively, heated in a 50℃water bath with a magnetic stirring speed of 250rpm; after complete dissolution, 0.5g of acacia gum is added, and stirring is continued until dissolution; stopping heating after dissolution, adjusting the pH of the solution to 6.5, and stirring overnight at room temperature; stopping stirring, settling GelMA microgel, removing supernatant, dispersing with PBS, centrifuging for 10min under 1000rpf centrifugal force, collecting GelMA microgel, washing with PBS, centrifuging for 3 times, and collecting GelMA microgel aggregate. The morphology of the microgel was observed in the open field using an inverted microscope, and the results are shown in FIG. 1.

The GelMA microgel obtained in this example had an average gel size of 143.5. Mu.m.

The rheological property test is carried out on the GelMA microgel prepared by the embodiment, and the result is shown in figure 2, which shows that the GelMA microgel has low yield stress and self-healing property of shear thinning and can be used as an embedded printing matrix.

Further, the GelMA microgel aggregate prepared in this example was centrifuged at 1000rpf to collect the GelMA microgel. The storage modulus of the GelMA microgel at 4 ℃ is tested to be 100Pa.

GelMA microgel containing 0.1g/100mL LAP was used as an embedded print substrate, 10% Gelatin was added as a print substrate (sacrificial ink) to a cartridge connected to a 250 μm needle, the position of the print needle in the substrate was calibrated by loading the print path of the spiral tube structure on a printer (Cellink), and a spiral tube network was printed in the GelMA microgel at a speed of 3mm/s under an extrusion pressure of 320kPa (FIG. 3). After printing, turning on an ultraviolet lamp of a surface light source, setting the wavelength to 365nm and the power to 10mW/cm ², placing the printed substrate at the center of the ultraviolet lamp, and irradiating for 2min to carry out photo-crosslinking. The storage modulus of the matrix after crosslinking was 200Pa and was able to exist stably at 37 ℃. Adding the crosslinked matrix into PBS, placing in a water bath at 37 ℃, dissolving and removing the Gelatin printed in the matrix to form a hollow pipeline structure, and obtaining the spiral vascular structure.

Example 2

This example prepares a mechanical property-enhanced methacrylic anhydride modified hyaluronic acid (HAMA)/Matrigel/GelMA microgel with high bioactivity.

Preparation of HAMA: 2.5g of sodium hyaluronate (MW=90 kDa) was dissolved in 125ml of deionized water. The pH was adjusted to 8.5 using 1.0M NaOH while stirring in an ice bath environment. Then 7mL of MA was added dropwise, the pH was adjusted repeatedly to 8.5 during the addition, and the reaction was carried out under stirring in an ice bath environment for 4 hours. Next, the mixture was vigorously stirred at room temperature overnight. After dialysis and freeze drying, HAMA is obtained, and the modification degree of MA is about 20% -25%.

The GelMA microgel prepared in example 1 was freeze-dried to determine that the mass fraction concentration of the GelMA microgel was 4g/100mL, and HAMA, LAP and Matrigel stock solution (Corning, HC-Standard, 354248) were added to the GelMA microgel, the volume of the GelMA microgel being 4 times the volume of the Matrigel stock solution. HAMA mass fraction concentration is 0.4g/100mL, matrigel volume concentration is 20% of Matrigel stock solution, photosensitive molecule LAP final concentration is 0.1g/100mL, gelMA microgel final concentration is 3g/100mL, ultraviolet light is irradiated for 2min at 10mW/cm ², after crosslinking, the mixture is placed at 37 ℃ for incubation for 10min, PBS is added to soak hydrogel, and then microstructure is observed by utilizing a low-temperature cryoelectron microscope, as shown in the left side of fig. 4, heterogeneous space structure is characterized by SEM, and although photosensitive molecule active molecules cannot be distinguished in the heterogeneous space, microsphere formed by compact crosslinking inside the microsphere and microsphere-microsphere crosslinking degree difference can be clearly seen.

The effective Young's modulus of the hydrogels was tested using a nanoindenter (Piuma, optics 11). The hydrogel was fixed on a glass slide, and the whole was immersed in PBS to perform a subsurface indentation test. Firstly, the indentation probe gradually descends to the surface of a sample, a multipoint indentation test (1 mu m) is carried out at a random position to obtain a curve of force and indentation depth, and the effective Young modulus of the hydrogel is obtained through Hertz model fitting. Control group with 3% GelMA was used as the same concentration. Preparation of 3% gelma: 0.5g of GelMA powder with the modification degree of 75% MA in example 1 is weighed and dissolved in 10mL of PBS to prepare 5g/100mL of stock solution, HAMA, matrigel stock solution (Corning, HC-Standard, 354248) and LAP (total mass fraction concentration of HAMA) are added into the GelMA, the mass fraction concentration of HAMA is 0.4g/100mL, the volume concentration of Matrigel is 20% of Matrigel stock solution, the final concentration of light-sensitive molecule LAP is 0.1g/100mL, the final concentration of GelMA is 3g/100mL, the solution is irradiated for 2min under ultraviolet light of 10mW/cm ², and after crosslinking, the solution is incubated for 10min at 37 ℃, PBS is added to soak the hydrogel.

Statistical analysis (right in fig. 4) demonstrates that the young's modulus of the matrix after cross-linking of the 3% gelma microgel is not only significantly improved, but also has significant modulus heterogeneity. Thus, the rigidity difference of the microstructure can be determined, and the microgel of the heterogeneous microenvironment can be obtained.

The same microgel formulation as described above was used to test the biocompatibility of the microgel with green protein-labeled human mesenchymal stem cells GFP-hMSC (from national stem cell resource pool). Firstly, adding cell suspension into prepolymer of the same microgel formula, then placing the prepolymer in an ultraviolet light source, irradiating the prepolymer for 2min at ultraviolet light of 10mW/cm ², after crosslinking, incubating the prepolymer at 37 ℃ for 10min, adding a culture medium of MSCs (DMEM basic culture medium, adding 15% FBS (v/v) +1% MEM NEAA (v/v) +1% gluta MAX (v/v) +1% penicillin/streptomycin, mixing the mixture to form a culture medium), and culturing the mixture in an incubator. The 3D-packed GFP-hMSC is 1X 10 ⁶/mL hydrogel, the culture is carried out for 48 hours, the cell morphology is observed by using a fluorescence microscope, the biocompatibility of the material is determined, and the result shows that the cells obviously proliferate and spread in the hydrogel, the fluorescence intensity is increased along with the increase of the culture time, and the cells are connected and spread with each other.

Example 3

This example prepared a methacrylic anhydride modified gelatin/Fibrin/GelMA microgel with enhanced mechanical properties with high bioactivity.

The GelMA microgel prepared in example 1 was subjected to freeze drying to determine that the mass fraction concentration of the GelMA microgel was 4%, fibrinogen, gelMA microgel and LAP were added to methacrylic anhydride modified gelatin, the mass fraction concentration of methacrylic anhydride modified gelatin was 3%, the mass fraction concentration of fibrinogen was 0.25%, the final concentration of light-sensitive molecule LAP was 0.1% (w/v), the concentration of GelMA microgel was 0.4% (w/v), 50U/mL thrombin was added after mixing, 2min was irradiated with ultraviolet light 10mW/cm ², and after crosslinking, the mixture was incubated at 37℃for 10min, and the hydrogel was soaked in PBS, and then the microstructure was observed by a low-temperature cryoelectron microscope. The same microgel formulation as described above was tested for biocompatibility of the microgel using green protein-labeled human mesenchymal stem cells GFP-hMSC. Firstly, adding cell suspension into prepolymer with the same formula, mixing, adding 50U/mL thrombin, then placing in an ultraviolet light source, irradiating for 2min at ultraviolet light of 10mW/cm ², cross-linking, incubating for 10min at 37 ℃, adding a culture medium of MSCs (adding 15%FBS+1%MEM NEAA+1% Gluta MAX +1% penicillin/streptomycin into a DMEM basal medium, mixing to form a culture medium), and culturing in an incubator. Wherein the concentration of the 3D-wrapped RFP-hMSC is 1X 10 ⁶/mL hydrogel, after culturing for 48 hours, the cell morphology is observed by using a fluorescence microscope, and the result shows that obvious proliferation and extension of cells occur in the hydrogel.

Example 4: the hydrogel composite system prepared in example 2 was subjected to 2D (endothelialization) and 3D culture (vascularization) of endothelial cells HUVEC, and the vascular anastomosis transplantation and application prospects of vascular tissues were examined.

The surface of the hydrogel prepared in example 2 was dropped with a red protein-labeled umbilical cord-derived human vascular endothelial cell RFP-HUVEC (from the excellent center of innovation of molecular cell science, china academy of sciences) suspension having a suspension density of 4X10 ⁴/50. Mu.L of a culture medium (EGM-2 culture medium), after overnight surface attachment, the culture medium (EGM-2 culture medium) was added, and the mixture was cultured in an incubator at 37℃with 5% carbon dioxide, and the attachment of the cells to the surface of the hydrogel was observed by a confocal fluorescence microscope. The results showed that the cells were completely attached and spread on the surface of the hydrogel, and the cells were in contact with each other.

Under the same conditions, using a die, placing the inner wall of the formed pipeline in the hydrogel by using an 800 mu m needle, adding cell suspension, attaching the surface of the pipeline, and observing the attachment and angiogenesis condition of cells in the pipeline after the attachment. Cells are attached to the inner wall of the whole pipeline, cells are connected with each other, and migrate, assemble and bud from the surface of the pipeline to the inside of the hydrogel.

The ability of cells to self-assemble into capillary-like vessels within hydrogels (3D) was examined using cell aggregates (HUVEC: 4X 10 ⁵/cell aggregates, HFF5.times.10 ⁴/cell aggregates) formed by the aggregation of endothelial cells and fibroblasts (HFF, from national stem cell resource pool) in low-attachment well plates. As shown in fig. 5, the cells formed a dense capillary-like network within the hydrogel.

HFF cell culture medium: 10% FBS, 1% penicillin/erythromycin was added to DMEM basal medium.

HUVEC-HFF cell aggregates: 8X 10 ⁴ RFP-HUVECs and 2X 10 ⁴ HFFs were separately collected and resuspended in 1.5mL EGM-2 medium and gently mixed by pipetting. The cell suspension was uniformly dropped into one well of a low-adhesion 24-well plate, and cultured in a 5% CO ₂ cell incubator at 37 ℃. After 18h 400 HUVEC-HFF aggregates/well were harvested.

1000 Cell aggregates are added into each 50uL hydrogel, EGM-2 culture medium containing growth factors VEGF (40 ng/mL) and bFGF (40 ng/mL) is added for culture, and the situation that endothelial cells self-assemble into capillary vessels in the gel is examined.

The method comprises the steps of printing a large-volume tissue of a blood vessel by one-in-two with a 10% gelatin aqueous solution (w/v) as printing ink and a composite matrix before photo-crosslinking as an embedded printing matrix, and dissolving a core after printing to form a hollow vascular system. The microgel composite prepolymer of example 2 was used as an embedded print substrate, 10% gelatin was added as a print substrate (sacrificial ink) to a cartridge connected to a 250 μm needle, the print path was loaded in one-half-four through a printer (Cellink), the position of the print needle in the substrate was calibrated, and one-half-four tubing was printed in GelMA microgel at an extrusion pressure of 320kPa and a speed of 3 mm/s. After printing, turning on an ultraviolet lamp of a surface light source, setting the wavelength to 365nm and the power to 10mW/cm ², placing the printed substrate at the center of the ultraviolet lamp, and irradiating for 2min to carry out photo-crosslinking. Incubate at 37℃for 10min. Adding the crosslinked matrix into PBS, placing in a water bath at 37 ℃, dissolving and removing the Gelatin printed in the matrix to form a hollow pipeline structure, and obtaining the hydrogel with a one-to-two-to-four vascular structure.

The vascular structure hydrogel tissue obtained above was encapsulated with PDMS shell, PU tube connection (Alzet, 0007770) and photosensitive PDMS, and the printed vascular tissue was connected to the rat body in a vascular anastomosis manner by means of PU tube connection to the rat carotid artery and jugular vein, for direct blood perfusion (fig. 6).

PDMS shell: PDMS is mixed, placed on a mixing and defoaming stirrer, subjected to mixing and defoaming treatment for 30s at a speed of 2,000g, and repeated for 3 times; drawing and generating a model in an STL format by using Solidworks, and introducing Ultimaker S a printing die to manufacture a reverse die of the shell; sticking the printed counter mould at the center of a 10cm glass dish, pouring PDMS prepared in advance, and measuring the PDMS to be 1.2mm higher than the top end of the mould by using a graduated scale; vacuum-pumping in a vacuum drying oven for 30min to remove bubbles; oven curing at 50 ℃ for 12 hours; taking down the solidified PDMS by using blunt forceps, and cutting the PDMS to a proper size according to a preset edge line; punching at the preset hole line by using a 2mm puncher to obtain the transplantable PDMS shell, and cleaning, drying and sterilizing the transplantable PDMS shell for later use.

Preparation of photosensitive PDMS encapsulation Material dimethyldiethoxysilane (80.55 g,0.67 mol) and mercaptopropyl methyl dimethoxysilane (12.08 g,0.067 mol) were mixed with H ₂ O (26.53 g,1.47 mol) and 1.0wt% HCl in a round bottom flask and placed on a heatable magnetic stirrer. After the mixture of the previous step was kept at 70℃for 5 hours, PDMS-SH was isolated and washed with 75wt% ethanol. PDMS-SH is dried in vacuum at 130 ℃ to obtain transparent viscous liquid.

Tissue assembly: placing vascular tissues into a PDMS shell, and respectively inserting PU connecting pipes into holes at two ends of the PDMS shell to keep the PU in a hydrogel tissue pipeline; pouring the packaging material, so that the packaging material is enabled to permeate through the functional vascular tissue and fill the inside of the PDMS shell, and curing and forming the packaging material under the catalysis of an ultraviolet light source; after the encapsulation is completed, the PU connection pipes are respectively connected with the carotid artery and the jugular vein at the carotid artery of the rat.

Example 5: the hydrogel composite system prepared in example 3 was subjected to 2D (endothelialization) and 3D culture (vascularization) of endothelial cells HUVEC, and the application prospect of establishing vascular tissues was examined.

The surface of the hydrogel prepared in example 3 was dropped with red protein-labeled umbilical cord-derived human vascular endothelial cell RFP-HUVEC suspension (national stem cell resource library) having a suspension density of 4X 10 ⁴/50. Mu.L, and after overnight surface attachment, the culture medium (EGM-2 medium) was added, and the mixture was cultured in a 5% carbon dioxide incubator at 37℃to observe the attachment of the cells to the surface of the hydrogel by confocal fluorescence microscopy. Cells attach and extend on the surface of the hydrogel, and connection between cells occurs.

Under the same conditions, using a die, adding cell suspension into the inner wall of a pipeline formed by casting the 800 mu m needle occupying place, attaching the surface of the pipeline, and observing the attachment and angiogenesis condition of cells in the pipeline after the attachment. Cells attach along the inner wall of the pipeline and completely spread on the inner surface of the pipeline, and cell-cell connection occurs.

Cell aggregates formed by the aggregation of endothelial cells and fibroblasts (HFF) in low-attachment well plates (HUVEC: 4X 10 ⁵/cell aggregates, HFF 5X 10 ⁴/cell aggregates) were used to examine the ability of cells to self-assemble into capillary-like vessels within hydrogels (3D). 1000 cell aggregates are added into each 50uL hydrogel, a culture medium containing a growth factor VEGF (40 ng/mL) and bFGF (40 ng/mL) is added for culture, and the situation that endothelial cells self-assemble into capillary vessels in the gel is examined. Endothelial cells form a dense capillary-like network within the hydrogel.

The hydrogel formulation prepolymer of example 3 was a printing ink, 10% gelatin was a printing ink, and the printing was performed using SIA pro printer developed by shenyang automation in the department of chinese sciences, using 10% gelatin as a core-dissolving ink, and the composite matrix before photocrosslinking as an extrusion matrix, using multi-material composite extrusion printing. G-codes are generated in a printer, printing ink is respectively placed in two printing cartridges, and the blood vessel large-volume tissue printing with one-to-two combination is carried out according to the G-codes. Adding 50U/mL thrombin into the printed matrix to catalyze fibrinogen to crosslink to form fibrin, irradiating for 2min at ultraviolet light of 10mW/cm ², incubating for 10min at 37 ℃ after crosslinking, adding PBS to soak hydrogel for dissolving core, and forming hollow vascular system hydrogel.

The PDMS shell, the PU tube and the photosensitive PDMS are used for packaging, the PDMS shell is connected with the pipelines at the two ends of the hydrogel containing the vessel, the printed vascular tissue is connected into a rat body in a vascular anastomosis mode by means of the connection of the PU tube with the carotid artery and the jugular vein, direct blood perfusion is carried out, and the crosslinked matrix meets physiological blood flow shearing force and can meet the direct blood flow perfusion.

Preparation of photosensitive PDMS encapsulation Material dimethyldiethoxysilane (80.55 g,0.67 mol) and mercaptopropyl methyl dimethoxysilane (12.08 g,0.067 mol) were mixed with H ₂ O (26.53 g,1.47 mol) and 1.0wt% HCl in a round bottom flask and placed on a heatable magnetic stirrer. After the step 1 mixture was kept at 70℃for 5 hours, PDMS-SH was isolated and washed with 75wt% ethanol. PDMS-SH was dried in vacuo at 130℃to give a clear viscous liquid.

From the combination of fig. 1 and 2, it can be seen that the spherical structure, self-healing properties of GelMA microgels, as an embedded print matrix, print the vascular-like tubing capability. And combining the addition of a macromolecular chain HAMA and a biocompatible matrix Matrigel to construct a microenvironment with rigidity heterogeneity caused by structural microscopic heterogeneity. FIGS. 4 and 5 show the possibility of gelMA microgel-based hydrogels as a matrix for implantable blood vessels, satisfying both vascular endothelialization, angiogenesis and vasculogenesis of endothelial cells, as well as direct in vivo transplantation with vascular anastomosis.

In conclusion, the composite hydrogel constructed by combining the photosensitive polymer and the bioactive molecules based on the GelMA microgel can meet the application prospect of constructing vascularized parenchymal tissues by printing.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A bioprinting matrix comprising a microgel, one or more photocrosslinking molecules, and one or more bioactive substances; the microgel is a microgel formed by biocompatible polymers (including but not limited to GelMA, HAMA, chitosan, gelatin, protein or polyethylene glycol microgel); the photo-crosslinking molecules are high polymers containing photo-reactive groups; the bioprinting substrate is capable of forming a hydrogel having a heterogeneous microstructure upon ultraviolet irradiation.

2. The bioprinting matrix of claim 1, wherein the microgel forms a composite hydrogel with photocrosslinking molecules and bioactive substances through two modes of physical encapsulation and chemical crosslinking under illumination, wherein the microgel forms a microsphere with a compact crosslinking structure inside, and the microgel forms chemical crosslinking with photocrosslinking molecules and forms physical encapsulation with bioactive substances.

3. The bioprinting substrate of any one of claims 1-2, said microgel having shear thinning properties with a yield stress of 10 to 200Pa; preferably, the microgel is a GelMA microgel, and the modification degree of the GelMA is 45-90%.

4. The bioprinting substrate of any one of claims 1-3, said microgel prepared by a complex coacervation process comprising: reducing the solubility of the biocompatible polymer in a solvent by utilizing a complex coacervate, so that the biocompatible polymer is precipitated to form microgel, wherein the complex coacervate is a polymer which is incompatible with the biocompatible polymer and can generate electrostatic action;

preferably, the microgel is a GelMA microgel and the complex coacervate is gum arabic;

preferably, the microgel is a GelMA microgel, and the solvent is an aqueous ethanol solution.

5. The bioprinting matrix of any one of claims 1-4, said GelMA microgel made by a process comprising the steps of:

step 1: dissolving GelMA and an optional surfactant in an aqueous ethanol solution to form a GelMA solution;

Step 2: adding complex coacervate into the GelMA solution, dissolving the complex coacervate, regulating and controlling the pH value of the solution to 6-7, and stirring to form GelMA microgel;

Step 3: stopping stirring to enable the GelMA microgel to settle; washing and collecting GelMA microgel;

preferably, the method has one or more of the following features:

(1) In the step 1, the mass concentration of GelMA in the solution is 2g/100 mL-5 g/100mL;

(2) In the step 1, the volume fraction of the ethanol in the ethanol water solution is 50% -80%;

(3) In the step 1, the surfactant is Pluronic F-127;

(4) The mass concentration of the surfactant in the solution is 0-0.5 g/100mL;

(5) Step 1 is carried out under heating and/or stirring conditions; preferably, the heating temperature is 45-55 ℃;

(6) The mass concentration of the complex coacervate in the solution is 0.02g/100 mL-0.1 g/100mL;

(7) The step 3 comprises the following steps: the stirring was stopped, the GelMA microgel was allowed to settle, the supernatant was removed, the GelMA microgel was washed with buffer solution and collected by centrifugation.

6. The bioprinting matrix of any one of claims 1-5, wherein the photocrosslinking molecule is a biocompatible polymer containing a photoreactive group;

Preferably, the biocompatible polymer containing a photoreactive group is selected from the group consisting of: sodium alginate, chitosan, silk fibroin, hyaluronic acid, fibrinogen, chondroitin sulfate, methacryloylated products of albumin (e.g., methacryloylated hyaluronic acid (HAMA), methacryloylated sodium alginate (AlgMA), methacrylated gelatin (GelMA)), polyethylene glycol diacrylate (PEGDA), tetra-arm polyethylene glycol acrylate (4-arm-PEG-AC), methacryloylated polyvinyl alcohol (PVAMA));

Preferably, the biocompatible polymer containing a photoreactive group is selected from the group consisting of: HAMA, PEGDA, gelMA.

7. The bioprinting matrix of any one of claims 1-6, said bioactive substance being a protein (e.g., collagen, fibrinogen, fibronectin) or an extracellular matrix (e.g., matrigel);

Preferably, the bioactive substance is protein, and the mass concentration of the bioactive substance is 0.2g/100 mL-0.5 g/100mL;

Preferably, the bioactive substance is Matrigel, the ratio of the volume of Matrigel to the mass of the biocompatible polymer forming the microgel is 6-8 mL/1 g, or the ratio of Matrigel to the microgel is 1:3-5;

Preferably, the bioactive agent is a cytokine capable of inducing differentiation of an undifferentiated cell into a smooth muscle cell or endothelial cell, such as TGF-alpha 1, PDGF-BB, VEGF or b-FGF.

8. The bioprinting substrate of any one of claims 1-7, said ultraviolet light having an illumination intensity of 2-10mW/cm ²; preferably, ultraviolet light irradiation with a wavelength of 365nm or more is used.

9. The bioprinting substrate of any one of claims 1-8, further comprising a photoinitiator;

Preferably, the photoinitiator is selected from the group consisting of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (I2959), phenyl (2, 4, 6-trimethylbenzoyl) phosphate lithium salt (LAP), 2-hydroxy-2-methyl-1-phenylpropanone (1173), 1-hydroxycyclohexylphenyl ketone (184), 2-methyl-2- (4-morpholino) -1- [4- (methylthio) phenyl ] -1-propanone (907), 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide (TPO), ethyl 2,4, 6-trimethylbenzoyl phenylphosphonate (TPO-L), 2-dimethylamino-2-benzyl-1- [4- (4-morpholino) phenyl ] -1-butanone (IHT-PI 910), 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (695), methyl Benzoate (MBF), or any combination thereof;

Preferably, the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (I2959) or phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP);

Preferably, the photoinitiator concentration is between 0.01g/100mL and 0.1g/100mL.

10. The bioprinting matrix of any one of claims 1-9, further comprising cells;

Preferably, the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a unicellular eukaryotic organism, a protozoan cell, a plant cell, an animal cell, an algal cell, a fungal cell, an artificial cell, or any combination thereof; or the cell is selected from stem cells, somatic cells, germ cells, or any combination thereof;

Preferably, the stem cells are selected from Embryonic Stem (ES) cells, induced Pluripotent Stem (iPS) cells, mesenchymal stem cells, neural stem cells, muscle stem cells, hematopoietic stem cells, epithelial stem cells, breast stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, or any combination thereof;

Preferably, the somatic cells are selected from the group consisting of epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, myocytes, osteocytes, hepatocytes, pancreatic cells, exogenous cells, endogenous cells, cardiomyocytes, skeletal cells, cardiac myoblasts, skeletal myoblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, or any combination thereof;

preferably, the unit volume of bioprinting matrix contains 10 ⁵～10⁷ cells/mL.

11. The bioprinting matrix of any one of claims 1-10, wherein the mass concentration of the microgel is 2.5g/100mL or more, the bioprinting matrix being an embedded printing matrix or an extruded printing matrix; or the mass concentration of the microgel is lower than 2.5g/100mL, and the biological printing matrix is an extrusion printing matrix;

Preferably, the biological printing matrix is an embedded printing matrix, the mass concentration of the photo-crosslinking molecules is 0.2g/100 mL-0.6 g/100mL, and the mass concentration of the microgel is 2.5g/100 mL-4 g/100mL;

Preferably, the biological printing matrix is an extrusion printing matrix, the mass concentration of the photo-crosslinking molecules is 2g/100 mL-4 g/100mL, and the mass concentration of the microgel is 0.2g/100 mL-0.6 g/100mL.

12. A method of preparing the bioprinting substrate of any one of claims 1-11, the method comprising: the ingredients contained in the bioprinting matrix are mixed uniformly, optionally the method further includes irradiating the bioprinting matrix under ultraviolet light to produce cross-links.

13. A kit comprising the bioprinting substrate of any one of claims 1-11.

14. Use of the bioprinting matrix of any one of claims 1-11 in the preparation of a kit for in vitro 3D bioprinting or in vitro biomimetic structure construction.

15. Use of the bioprinting matrix of any one of claims 1-11 for in vitro 3D bioprinting or in vitro biomimetic structure construction, preferably for non-diagnostic or therapeutic purposes.

16. A method of bioprinting comprising using the bioprinting matrix of any one of claims 1-11 or using the kit of claim 13;

preferably, the bioprinting method comprises extrusion printing and/or embedded printing.

17. The bioprinting method of claim 16, comprising: forming a predetermined structure by extrusion printing using the bioprinting substrate of any one of claims 1-11 as a first printing ink and a sacrificial ink as a second printing ink; irradiating the predetermined structure with ultraviolet light to crosslink the bioprinting substrate; the sacrificial ink is removed to obtain a bioprinting product.

18. The bioprinting method of claim 16, comprising: extruding the sacrificial ink into the bioprinting matrix of any one of claims 1-11; irradiating the bioprinting substrate with ultraviolet light to produce cross-links; the sacrificial ink is removed to obtain a bioprinting product.

19. A product obtained by the bioprinting method of any one of claims 16-18;

preferably, the product is an organoid;

Preferably, the product is a angioid.

20. Use of the bioprinting matrix of any one of claims 1-11, the kit of claim 13, or the product of claim 19 for repair of diseased tissue or organs, drug development and screening, or preparation of a pathology study model; preferably, the use is for non-diagnostic or therapeutic purposes.

Preferably, the tissue or organ is selected from the group consisting of heart, liver, kidney, pancreas, brain or tissue constituting any of the above organs.