Detergent-free decellularized extracellular matrix preparation method and bio-ink for 3D printing

Abstract

The present invention relates to a method for preparing a detergent-free decellularized ECM, a detergent-free decellularized ECM in powder form and liquid form, a method for preparing a primary bio-ink, a primary bio-ink, a method for preparing a vascular bio-ink, a vascular bio-ink, a three-dimensional structure comprising the primary bio-ink and / or the vascular bio-ink, and a method for preparing the three-dimensional structure.

Description

Technical Field

[0001] The present invention relates to a method for preparing a detergent-free decellularized ECM, a detergent-free decellularized ECM in powder form and liquid form, a method for preparing a primary bio-ink, a primary bio-ink, a method for preparing a vascular bio-ink, a vascular bio-ink, a three-dimensional structure comprising the primary bio-ink and / or the vascular bio-ink, and a method for preparing the three-dimensional structure. Background Art

[0002] Bioprinting enables the automated deposition of living cells and other components to form three-dimensional (3D) tissue constructs. Bioink formulations are produced from different sources, including synthetic and natural polymers such as collagen, gelatin, alginate, hyaluronic acid, fibrin, and polyethylene glycol. It is well known that the matrix materials used for bioprinting do not represent the complexity of the natural extracellular matrix (ECM), which constitutes the microenvironment of cells and can regulate cellular processes including migration, differentiation, and other functions. Therefore, the presence of ECM in bioink is believed to be beneficial for recreating a microenvironment with cell-cell connections.

[0003] International patent application WO2017014582 discloses a bio-ink composition comprising 0.05 to 60×10 6 / mL cells, 0.1 to 10 w / v% cell carrier material, 0.01 to 1 w / v% viscosity enhancer, 1 to 30 v / v% lubricant and 0.1 to 10 w / v% structural material. The bio-ink composition may further include tissue-derived component materials. Preferably, the cell carrier material is gelatin or collagen, the viscosity enhancer is hyaluronic acid or dextran, the lubricant is glycerol, and the structural material is fibrinogen or methacrylated gelatin (GelMa).

[0004] The literature includes many publications on the problem of selecting appropriate bio-ink compositions with optimal properties for tissue engineering applications. Mohamed Ali et al. conducted work on the production of bio-inks based on decellularized ECM (dECM) derived from kidney [1]. Relatively low concentration (1% to 3%) dECM hydrogels were obtained by dissolution method using 0.5 M acetic acid and 0.1 mg / mL pepsin. In addition, the dECM was methacrylated by adding a photoinitiator (Irgacure).

[0005] Subsequent research groups have attempted to obtain bioinks using ECMs supplemented with gelatin methacrylate (GelMa) and a photoinitiator (i.e., LAP) (lithium phenyl-2,4,6-trimethylbenzoylphosphite) [2]. Others have used dECM hydrogels obtained with relatively high concentrations of pepsin, adding polycaprolactone (PCL) as a preservative synthesis agent [3].

[0006] Patent specification KR20180125776 describes a bio-ink composition comprising dECM powder and a hydrogel. The dECM powder may be selected from liver tissue, heart tissue, cartilage tissue, bone tissue, adipose tissue, muscle tissue, skin tissue, mucosal epithelial tissue, amniotic tissue or corneal tissue. Preferably, the particle size of the dECM powder is 0.05 μm to 100 μm. The hydrogel may include one or more selected from the group consisting of gelatin, hyaluronic acid, dextran and collagen.

[0007] Falguni et al. (2014) developed tissue-specific dECM bioinks containing adipose, cartilage, and cardiac tissues that provide key cues for cell engraftment, survival, and long-term function. The bioprinting approach was able to recreate intrinsic cell morphology and function. Higher-order assembly of printed cell constructs was observed through organized spatial patterns and tissue-specific gene expression. The key advantage of the methodology is the application of tissue-specific ECM that provides key cues for cell engraftment, survival, and long-term function [3].

[0008] Many research groups have conducted studies involving organ decellularization to obtain dECM as a bio-ink component [4, 5, 7]. Various substances are used for decellularization, mainly Triton X-100 and / or dodecyl sulfate (SDS) detergents. KR1020180011607A describes a liver decellularization method in which liver tissue is treated with a desaturated solution containing a surfactant and a superactive solution. 0.5% Triton X-100 (Triton X-100) can be used as a surfactant.

[0009] Mohamed Ali and colleagues constructed a photocross-linked kidney containing ECM-derived bioink[1]. Porcine whole kidneys were decellularized by perfusion, dissolved in an acidic solution, and chemically modified with methacrylic acid. The results showed that the bioprinted human kidney cells had a high survival rate and matured over time. In addition, the bioprinted kidney constructs displayed structural and functional characteristics of native kidney tissue. Tissue-specific ECM-derived bioinks enhanced cell maturation and ultimately enhanced tissue formation.

[0010] Mirmalek-Sani et al. (2013) described the decellularization of porcine pancreas to create a scaffold for human stem cells and porcine islets. Cellular material was effectively removed while retaining ECM proteins and native vasculature. Furthermore, the study showed that the decellularized pancreas supported cell adhesion and maintenance of cell function [6]. Summary of the invention

[0011] The object of the present invention is to provide a detergent-free dECM that can be used for bioprinting. The literature data do not provide results on the method for determining the amount of detergent residue or its content in the ECM obtained by decellularization. In various tissue decellularization procedures previously published, the detergent removal stage is relatively short. It is believed that the absence of detergent in the dECM will substantially affect the quality of the obtained dECM. The process developed by the applicant allows the removal of almost all detergents without the addition of other chemicals. A second object of the present invention is to obtain a bio-ink with appropriate consistency and viscosity without the addition of viscosity enhancers.

[0012] In a first aspect, a method for preparing a detergent-free decellularized extracellular matrix (dECM) is provided, the method comprising the following steps:

[0013] - mechanically disrupting, preferably mechanically squeezing, an organ of animal origin selected from the group consisting of pancreas, liver, kidney, heart, skin, lung, large intestine, small intestine, arteries and veins, adipose tissue and placenta, wherein the organ is separated from the body of the animal;

[0014] - incubating the fragmented organs in a buffered detergent solution, preferably comprising 1× phosphate buffered saline (PBS), wherein the buffered detergent solution comprises 0.5% to 1.5%, preferably 1% (v / v) octoxynol-9, wherein the detergent solution is supplemented with an antimicrobial agent, preferably streptomycin, preferably at a concentration of 0.01% (w / v), and the incubation is carried out under stirring at a temperature below room temperature, preferably 4° C., for at least 72 hours, wherein the fragmented organs are transferred to fresh detergent solution every 4 to 12 hours;

[0015] - incubating the organ fragments in a first buffered washing solution, preferably comprising 1×PBS, wherein the first buffered washing solution comprises an antimicrobial agent, preferably streptomycin, preferably at a concentration of 0.01% (w / v), the incubation being carried out under stirring at a temperature below room temperature, preferably 4° C., for at least 72 hours, wherein the organ fragments are transferred to fresh washing solution every 4 to 12 hours;

[0016] - incubating the organ fragments in a DNase solution comprising DNase, preferably at a concentration of 0.0001% to 0.0003% (w / v), most preferably at a concentration of 0.0002% (w / v), preferably at a temperature suitable for DNase performance for at least 8 hours;

[0017] - incubating the organ fragments in a second buffered wash solution, preferably comprising 1×PBS, wherein the second buffered wash solution comprises an antimicrobial agent, preferably streptomycin, preferably at a concentration of 0.01% (w / v), the incubation being carried out under stirring at a temperature below room temperature, preferably 4° C., for at least 72 hours, wherein the organ fragments are transferred to fresh wash solution every 4 to 12 hours;

[0018] - Freezing the organ fragments and crushing the frozen organ fragments into pieces;

[0019] - freeze drying the frozen organ fragments preferably at -32°C, preferably at a pressure of 0.31 mbar (31 Pa);

[0020] - optional final drying at 0.0010 mbar (0.1 Pa) and -76°C for 5 to 15 minutes;

[0021] - grinding the crushed and dried product into dECM powder of 25 μm to 500 μm;

[0022] - Optional sterilization of the product, preferably by radiation and / or ethylene oxide.

[0023] Mechanical disruption of organs enhances the removal of detergents from the organs and produces a product with lower fat content, which improves the properties of the final product, namely increased viscosity and improved printability. The addition of DNase is essential for the removal of DNA from animal-derived organs. If the resulting printed 3D structures contain dECM with DNA, they cannot be further used for transplantation experiments.

[0024] Preferably, the grinding step is followed by a step of examining the dECM powder for the amount of octoxynol-9, wherein the powder is preferably treated with collagenase, preferably at a concentration of at least 43.953 PZ / g dECM, prior to examining the dECM powder for the presence of octoxynol-9.

[0025] Preferably, the grinding step is followed by the following steps:

[0026] - Dissolve dECM powder in a hydrochloric acid solution, preferably 0.01 M, supplemented with 0 to 10 mg / ml of pepsin;

[0027] - Mixing at room temperature for 48 hours to 72 hours, preferably 72 hours;

[0028] - Neutralize on ice, preferably using 0.1 M sodium base and PBS solution.

[0029] In a second aspect, a detergent-free decellularized ECM in powder form is provided, which can be obtained by a method for preparing a detergent-free decellularized extracellular matrix (dECM). Preferably, the dECM powder is sterile. If necessary, the powder can be sterilized by radiation sterilization or ethylene oxide sterilization.

[0030] In a third aspect, a detergent-free decellularized ECM in a solution form is provided, which can be obtained by a method for preparing a detergent-free decellularized extracellular matrix (dECM).

[0031] In a fourth aspect, a method for preparing a primary bio-ink is provided, the method comprising the following steps:

[0032] - preparing a paste comprising 5% to 50% (w / v), preferably 15% to 25% (w / v), of the dECM powder according to the first aspect of the invention and 1% to 10% (w / v), preferably 8% to 10% (w / v), of the dECM solution according to the third aspect of the invention by mixing;

[0033] - Incubate the paste at a temperature of 7°C to 10°C for at least 24 hours;

[0034] - Add 1.46% to 7.32% (w / v) of gelatin methacrylate, 0.15% to 1.10% (w / v) of hyaluronic acid methacrylate, 5% to 10% (w / v) of glycerol, and a photoinitiator, preferably 0.03% to 0.17% (w / v) of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and mix gently.

[0035] Since the dECM powder is initially prepared by freeze drying and does not dissolve afterwards, it retains the entire quaternary structure of the ECM. Therefore, the use of dECM in paste form (including both dECM powder and dECM solution) allows the primary bio-ink to have an appropriate consistency, and since the dECM powder is not dissolved in the primary bio-ink, the entire quaternary structure of the ECM is retained.

[0036] In a fifth aspect, a primary bio-ink is provided, comprising a dECM paste and 1.46% to 7.32% (w / v) methacrylated gelatin, 0.15% to 1.10% (w / v) methacrylated hyaluronic acid, 5% to 10% (w / v) glycerol, and a photoinitiator, preferably 0.03% to 0.17% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphite, wherein the dECM paste comprises 5% to 50% (w / v), preferably 15% to 25% (w / v) of dECM powder according to the second aspect of the invention, and 1% to 10% (w / v), preferably 8% to 10% (w / v) of dECM solution according to the third aspect of the invention, and wherein the viscosity of the primary bio-ink is at least 5 Pa·s measured in a cone and plate system at a constant shear rate of 2 1 / s and a temperature of 37°C.

[0037] The use of dECM is able to replicate the extracellular conditions of the body, thus providing bioprinting with native tissue characteristics, stimulating cell differentiation and improving their survival. In addition, the extracellular matrix is ​​necessary to obtain appropriate bioink viscosity and maintain a stable three-dimensional structure of the printed construct through the additional possibility of thermal cross-linking in the temperature range of 33°C to 37°C.

[0038] Compared to chemical cross-linking using chemicals that are toxic to cells in the primary bio-ink, the use of photoinitiators allows for cross-linking that is non-toxic to cells. Compared to thermal cross-linking, cross-linking using photoinitiators and visible light minimizes cellular DNA damage. Both temperature and light have negative effects on cells, causing DNA damage. However, when visible light cross-linking is used, these changes are kept to a minimum.

[0039] Gelatin methacrylate (GelMa) is used for the shaping of printed structures. In addition, it holds the microfilaments together to prevent leaflet delamination and improves cell and islet viability. Compared to gelatin, GelMa is stable at higher temperatures, which is beneficial during thermal cross-linking.

[0040] Methacrylated hyaluronic acid (HAMA) helps maintain the three-dimensional structure through cross-linking. In addition, HAMA provides smoothness, silkiness, uniformity of printed filaments, and supports cell culture. These characteristics cannot be obtained by adding unmethacrylated hyaluronic acid.

[0041] The use of glycerol improved the functionality of cells and islets. It also increased the lubricity of the bio-ink, enabled the formation of continuous filaments, improved the mixing of bio-ink components in syringes or mixers, and reduced pressure consumption during printing.

[0042] Preferably, the primary bio-ink comprises at least one additive selected from the following: hyaluronic acid at a concentration of 0.001 to 0.100 mg / mL bio-ink, preferably 0.007 mg / mL bio-ink, laminin at a concentration of 0.005 to 0.100 mg / mL bio-ink, preferably 0.084 mg / mL bio-ink, collagen I at a concentration of 0.001 to 0.100 mg / mL bio-ink, preferably 0.041 mg / mL bio-ink, 5mg / mL bio-ink, preferably 0.122mg / mL collagen IV, 3 to 300μg / mL, preferably 100μg / mL fibronectin, 10 to 100mg / mL human plasminogen, 1 to 2EPU / mL aprotinin, 0.05 to 2mg / mL polysorbate, 5 to 55mg / mL human thrombin, 20 to 60mM / mL bio-ink Calcium chloride in water; vitamins that promote angiogenesis: vitamin A at a concentration of 1nM to 500μM, preferably 100μM, vitamin B1 at a concentration of 50μM to 100μM, preferably 100μM, vitamin B3 at a concentration of 1μM to 10μM, preferably 10μM, vitamin B12 at a concentration of 10 to 100mg / mL bio-ink, vitamin D3 at a concentration of 0.1nM to 10nM, preferably 10nM; growth factors that support angiogenesis: concentration of 10 to 30ng / mL bio-ink Water, preferably 30ng / mL of VEGF in bio-ink, 10 to 20ng / mL of FGF in bio-ink, preferably 20ng / mL of FGF in bio-ink, 1 to 10ng / mL of TGF-β in bio-ink, optionally 20ng / mL of TGF-β in bio-ink, 0 to 100ng / mL of interleukin (IL)-8 in bio-ink, preferably 10ng / mL of interleukin (IL)-17A in bio-ink, 20 to 50ng / mL of IL-17A in bio-ink, preferably 20ng / mL of IL-17A.

[0043] Commercially available additives, such as hyaluronic acid, collagen types I and IV, and laminin, further improve the functionality of printed 3D structures.

[0044] Vitamin A-ATRA (all-trans retinoic acid), as one of the metabolites of vitamin A, has a pro-angiogenic effect - it can improve the expression of factors behind angiogenesis (such as cyclooxygenase-2 (COX-2), hypoxia-inducible factor (HIF)-1, CXC, chemokine receptor (CXCR)-4, vascular endothelial growth factor (VEGF), angiotensin (Ang)-2, angiotensin-4). In addition, it has been shown that ATRA reduces the activity of pro-MMP2 (pro-matrix metalloproteinase-2-type IV collagenase).

[0045] Vitamin B1-benfotiamine (a thiamine derivative) inhibits apoptosis on the protein-dependent B kinase pathway (PKB / Akt) and is responsible for inducing proliferation of endothelial progenitor cells.

[0046] Vitamin B3-niacin, through its receptor, hydroxycarboxylic acid receptor 2 (GPR109A), enhances and promotes endothelial cell function that supports angiogenesis. In addition, vitamin B3 is a precursor of NAD(+), which induces and supports angiogenesis through its reaction with sirtuin mediators (SIRT).

[0047] Vitamin B12 (cobalamin) induces the production of prostaglandin E1, prostacyclin, and nitric oxide (NO), all of which have a favorable effect on the initiation of angiogenesis.

[0048] Vitamin D3 is intended to stimulate angiogenesis in vitro. It induces increased expression of VEGF and pro-MMP2 activity. It also affects the function of ECFC (endothelial colony forming cells).

[0049] VEGF induces proliferation, migration, spore formation and junction formation between endothelial cells and, in addition, affects the degradation of the extracellular matrix (ECM) and activates cell surface integrins of endothelial cells by inducing the production of various proteases.

[0050] Fibroblast growth factor (FGF) increases endothelial cell migration and promotes capillary morphogenesis. It also increases endogenous VEGF production.

[0051] Transforming growth factor (TGF-β) promotes the formation of ECM (proteoglycans, fibronectin, collagen), regulates the proliferation of endothelial cells, their migration and the formation of blood vessels. TGF-β mediates the interaction between endothelial cells and pericytes.

[0052] Interleukin (IL)-8 has a potent pro-angiogenic effect on endothelial cells by interacting with CXCR1 and CXCR2 receptors. It stimulates the formation of microvascular networks.

[0053] IL-17A-induced angiogenesis, cell migration, and cytoskeletal rearrangement.

[0054] Preferably, the primary bio-ink comprises one or more animal-derived or human-derived additives selected from the following: endothelial cells at a density of 0.1 to 10×10 5 / mL bio-ink, primary microvascular endothelial cells, concentrations ranging from 0.1 to 10×10 5 / mL bio-ink, animal-derived or human-derived α cells, with a concentration of 3 to 9×10 6 / mL bio-ink, animal-derived or human-derived β cells, with a concentration of 1.1 to 3.4×10 7 / mL bio-ink, animal-derived or human-derived pancreatic islets, preferably in an amount of 20,000iEq / mL bio-ink.

[0055] Pancreatic islets are responsible for the production of insulin. Endothelial cells are added to form vascular networks more quickly in printed 3D structures. Primary microvascular endothelial cells are used to support the formation and growth of microvasculature in bioprinted 3D structures.

[0056] In a sixth aspect, a method for preparing a vascular bio-ink is provided, the method comprising the following steps:

[0057] a) optionally preparing a microbial gelatin solution supplemented with CMC, comprising preparing a 1% to 2% (w / v) microbial gelatin solution in a buffer solution, preferably PBS, by suspending the microbial gelatin in the buffer solution under stirring at a temperature between 50° C. and 65° C., preferably 60° C., adding a 2% to 5% (v / v) carboxymethyl cellulose (CMC) aqueous solution to obtain a final concentration of CMC in the bio-ink of 0.2% to 1% (v / v), and cooling the solution to a temperature equal to or lower than 40° C.;

[0058] b) preparing a 5% to 10% (w / v) dECM solution by adding the dECM powder according to the second aspect of the invention, preferably sterilized by radiation, to (i) the microbial gelatin solution supplemented with CMC obtained in step a) or (ii) a buffer solution or (iii) a cell culture medium solution under gentle stirring;

[0059] c) subjecting the resulting solution to ultrasonic treatment at a temperature not exceeding 37° C. for 0.5 to 2.0 hours;

[0060] d) optionally adding at least one animal-derived or human-derived additive selected from the following: fibronectin at a concentration of 3 to 300 μg / mL, preferably 100 μg / mL, VEGF at a concentration of 10 to 30 ng / mL, preferably 30 ng / mL, FGF at a concentration of 10 to 20 ng / mL, preferably 20 ng / mL, PGE2 at a concentration between 100 and 300 nM, preferably 100 nM, PGE2 at a density of 0.1 to 10×10 7 Endothelial cells with a density between 0.1 and 10 × 10 cells / mL bio-ink 6 Fibroblasts between cells / mL bio-ink.

[0061] In a seventh aspect, a method for preparing a vascular bio-ink is provided, the method comprising the following steps:

[0062] a) optionally preparing a microbial gelatin solution supplemented with CMC, comprising preparing a 1% to 5% (w / v) microbial gelatin solution in a buffer solution, preferably PBS, by suspending the microbial gelatin in the buffer solution under stirring at a temperature between 50° C. and 65° C., preferably 60° C., adding a 2% to 5% (v / v) carboxymethyl cellulose (CMC) aqueous solution to obtain a final concentration of 0.2% to 2% (v / v) CMC in the bio-ink, and cooling the solution to a temperature equal to or lower than 40° C.;

[0063] b) preparing a 2% to 10% (w / v) dECM solution by adding the dECM powder according to the second aspect of the invention, preferably sterilized by radiation, to (i) the microbial gelatin solution supplemented with CMC obtained in step a) or (ii) a buffer solution or (iii) a cell culture medium solution under gentle stirring;

[0064] c) cooking the mixture at 100° C. for 15 to 30 minutes;

[0065] d) optionally adding at least one animal-derived or human-derived additive selected from the following: fibronectin at a concentration of 3 to 300 μg / mL, preferably 100 μg / mL, VEGF at a concentration of 10 to 30 ng / mL, preferably 30 ng / mL, FGF at a concentration of 10 to 20 ng / mL, preferably 20 ng / mL, PGE2 at a concentration between 100 and 300 nM, preferably 100 nM, PGE2 at a density of 0.1 to 10×10 7 Endothelial cells with a density between 0.1 and 10 × 10 cells / mL bio-ink 6 Fibroblasts between cells / mL bio-ink.

[0066] In an eighth aspect, a vascular bio-ink is provided, the ink comprising the above-mentioned ultrasonically treated or boiled dECM solution according to the third aspect of the present invention, having a concentration of 2% to 10% (w / v), preferably supplemented with microbial gelatin having a concentration of 1% to 5% (w / v) and / or CMC having a concentration of 0.2% to 2% (v / v).

[0067] The sonicated or boiled dECM changes its physical and chemical properties with temperature. This composition is designed to ensure that the bio-ink has the appropriate viscosity when printed at relatively low temperatures (15°C to 20°C) and to preserve the printed conduits until cell infiltration and slow liquefaction at a culture temperature of 37°C.

[0068] Microbial gelatin provides an ideal consistency and improves cell survival. CMC increases viscosity and stabilizes the consistency of the bioink. Fibronectin promotes angiogenesis and, depending on the dose, stimulates the elongation of the formed tubes without affecting the proliferation rate.

[0069] Preferably, the vascular bio-ink comprises at least one animal-derived or human-derived additive selected from the following: fibronectin at a concentration of 3 to 300 μg / mL, preferably 100 μg / mL, VEGF at a concentration of 10 to 30 ng / mL, preferably 30 ng / mL, FGF at a concentration of 10 to 20 ng / mL, preferably 20 ng / mL, PGE2 at a concentration between 100 and 300 nM, preferably 100 nM, and a density of 0.1 to 10×10 7 Endothelial cells with a density between 0.1 and 10 × 10 cells / mL bio-ink 6 Fibroblasts between cells / mL bio-ink.

[0070] Endothelial cells give rise to blood vessels. Fibroblasts produce angiogenesis-inducing factors. VEGF induces proliferation, migration, spore formation and junction formation between endothelial cells. In addition, by inducing the production of various proteases, VEGF affects the degradation of the ECM and activates cell surface integrins of endothelial cells. FGF increases endothelial cell migration and promotes capillary morphogenesis. It also increases endogenous VEGF production. PGE2 - prostaglandin E2, induces migration, proliferation and formation of new tubes by activating (phosphorylated) FGF at the (R)-1 receptor.

[0071] In a ninth aspect, a three-dimensional structure is provided, comprising at least three adjacent bio-ink layers, wherein a layer of vascular bio-ink according to the eighth aspect of the invention is arranged between two layers of primary bio-ink according to the fifth aspect of the invention.

[0072] In a tenth aspect, a method for preparing a three-dimensional structure is provided, wherein the primary bio-ink according to the fifth aspect of the invention and the vascular bio-ink according to the eighth aspect of the invention are deposited layer by layer in a 3D bioprinting process at a printing speed of 5 mm / s to 50 mm / s, a pressure of 4 kPa to 300 kPa and a temperature of 4° C. to 37° C., and wherein during or after deposition, the primary bio-ink is exposed to ultraviolet and / or visible light for at least 5 seconds, preferably at a wavelength of 365 nm to 405 nm, more preferably at a wavelength of 405 nm. Crosslinking at 405 nm is preferred because it is non-toxic to cells contained in the three-dimensional structure.

[0073] The present invention enables to obtain models of leaflets with a size of 27×17×2.5 mm. Leaflets consisting of 5 layers were printed in 3 to 10 minutes. In addition, a three-dimensional model of a functional organ prototype with a size of 30×40×20 mm was obtained. The model consists of 30 layers and is printed in 20 to 60 minutes. Equally important, this is the first report of the use of dECM treated by boiling or ultrasound. Since the printing speed is appropriately related to the viscosity of the bio-ink (up to 30 mm / s), the present invention enables to obtain structures in a short time. Stable three-dimensional porous structures (30 layers) can be obtained, which can be stored at a temperature of 37°C for 20 days. In a preferred embodiment, the primary bio-ink is based on the use of a less toxic photoinitiator, namely LAP, instead of a relatively low concentration of Irgacure. In addition, a smaller amount of pepsin than found in the literature is used to obtain a dECM solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 .Effect of pepsin concentration on the properties of dECM hydrogels.

[0075] Figure 2 A-2F. Relationship between dECM solution concentration and viscosity of the following solutions: A) 5% (w / v) dECM solution supplemented with 15% (w / v) dECM powder, B) 5% (w / v) dECM solution supplemented with 25% (w / v) dECM powder, C) 8% (w / v) dECM solution supplemented with 15% (w / v) dECM powder, D) 8% (w / v) dECM solution supplemented with 25% (w / v) dECM powder, E) 10% (w / v) dECM solution supplemented with 15% (w / v) dECM powder, F) 10% (w / v) dECM solution supplemented with 25% (w / v) dECM powder.

[0076] Figure 3 A-3F. Kinetic analysis of 5% (w / v) dECM solution (A, B), 8% (w / v) dECM solution (C, D), and 10% (w / v) dECM solution (E, F) at predetermined temperatures.

[0077] Figure 4 .Printed leaflets: absorbance analysis of GelMa, HAMA, and Mix.

[0078] Figure 5 A-5B. Viscosity of A) cooked and B) sonicated dECM(r).

[0079] Figure 6 A-6B. A) 3D model of vascularized leaflet and B) photograph of printed structure.

[0080] Figure 7A-7D. Visualization of the vasculature of the pancreas 3D model (A, B) and prototype (C, D).

[0081] Figure 8 A-8B. Effects of glycerol on islet function A) at the beginning of the experiment and B) after 24 h of incubation.

[0082] Fig. 9 A-9C. Effects of commercial additives on islet function and viability A) at the beginning of the experiment, B) after 24 hours of incubation, C) after 48 hours of incubation.

[0083] Fig.10 A-10D. Effect of the addition of methacrylated gelatin on islet viability A) at the beginning of the experiment, B) after 24 h incubation, C) after 48 h incubation, D) after 72 h incubation.

[0084] Fig.11 A-11C. Effect of addition of methacrylated hyaluronic acid on islet viability A) at the beginning of the experiment, B) after 24 h of incubation, C) after 48 h of incubation.

[0085] Fig.12 A-12D. Effects of adding a mixture of GelMa and HAMA in different ratios on islet viability at A) the beginning of the experiment, B) after 24 hours of incubation, C) after 48 hours of incubation, and D) after 72 hours of incubation.

[0086] Fig.13 A-13D. Effect of adding cut or ground ECM powder on islet activity A) at the beginning of the experiment, B) after 24 hours of incubation, C) after 48 hours of incubation, D) after 72 hours of incubation.

[0087] Fig.14 A-14B. Effect of adding GelMa and HAMA to the primary bio-ink on the viability of printed islets A) at the beginning of the experiment, B) after 24 hours of incubation.

[0088] Fig.15 A-15F. Electron microscopy visualization showing the protein structure at various stages of dECM preparation in order to use it as a feedstock for bioprinting.

[0089] A)-C) are SEM (scanning electron microscope) images: A) native tissue before decellularization; B) tissue after decellularization; C) structure printed from primary bio-ink.

[0090] D)-F) are TEM (transmission electron microscopy) images: D) tissue after decellularization; E), F) structures printed by primary bio-ink with preserved collagen quaternary structure (collagen fibers are visible). DETAILED DESCRIPTION

[0091] Example 1: Preparation of detergent-free dECM

[0092] A. Pancreatic Decellularization Process

[0093] A 1% (v / v) Triton X-100 solution with 0.1% (v / v) ammonia in a 1× concentrated PBS solution with 0.01% (w / v) streptomycin was prepared to remove the cell structure of the pancreatic organ while retaining the extracellular matrix (scaffold). After collection, the tissue material was frozen at -80°C. Then, after thawing, the outer layer of adipose tissue and the surrounding membrane were removed from the organ. The prepared pancreas was processed in two ways: cut into small pieces (about 1 cm to 1.5 cm) and mechanically ground (using extrusion grinding).

[0094] The fragmented tissue is placed in a bottle and suspended in the previously prepared Triton X-100 solution. The specimen is placed in a 4°C incubator and maintained at a constant stirring of 150 rpm. The detergent is replaced every 4 to 12 hours until the cell part is completely removed (3 to 5 days). The detergent is then washed out of the obtained scaffold. For this purpose, a 1× PBS solution containing 0.01% (w / v) streptomycin is used. The washing process is carried out at 4°C for 72 hours with continuous stirring at 150 rpm.

[0095] The next stage - decellularization - mainly consisted of applying a deoxyribonuclease solution (0.0002% (w / v) DNase in 1× PBS, supplemented with 0.12 mM calcium and magnesium ions). The scaffolds were incubated in the above solution at 37°C with stirring at 150 rpm for 8 hours. The last step involved another wash with a 1× PBS solution containing 0.01% (w / v) streptomycin under standard conditions (4°C; 150 rpm; 72 hours). In addition, the use of ammonia with a concentration of 0.1% (v / v) in 1× concentrated PBS solution to wash out the detergent was also tested. In addition, the effect of increasing the temperature to 20°C to 24°C on the washing steps was studied.

[0096] After the decellularization process, the obtained scaffolds were frozen in liquid nitrogen and crushed into pieces of about 0.5 cm in size. The material was freeze-dried at a temperature of -32°C and a pressure of 0.31 mbar (31 Pa) for 26 hours. The final drying process lasted for 10 minutes at a pressure of 0.0010 mbar (0.1 Pa) and a temperature of -76°C. The crushed and dried scaffolds were ground into powder using a cryogenic mill. The grinding process consisted of 3 cycles, each lasting 1 minute at 15 impacts per second.

[0097] In order to characterize the obtained product, i.e. dECM powder abbreviated as "dECM(p)", the powder particle size distribution in the flow gradient was measured using a laser diffraction spectrometer Spraytec (Malvern, UK) equipped with an auxiliary inhalation chamber for studying inhalation sprays. In all cases studied, the values ​​of the parameters describing the analyzed powders after atomization were comparable, indicating that no additional energy in the form of increased airflow was required to break the powder into individual particles. Table 1 gives the values ​​of the parameters describing the powder particle diameters, where:

[0098] Dv(50) - median of the volume particle size distribution: the particle diameter that bisects the cumulative volume distribution, in other words, all particles smaller or larger than the median have the same volume (particles below this diameter account for 50% of the sample volume).

[0099] Dv(10) - Particles below this diameter account for 10% of the sample volume.

[0100] Dv(90) - Particles below this diameter account for 90% of the sample volume.

[0101] D[3][2] - The Sauter diameter is the diameter of a particle whose volume to surface ratio is the same as the ratio of the volume of all analyzed particles to the total surface of all such particles.

[0102] D[4][3] - diameter defined as the ratio of the sum of the fourth powers of the particle diameters to the sum of the third powers of the particle diameters.

[0103] Table 1. Parameters describing powder particle diameter

[0104]

[0105] The results of the measurements of the dECM powder after aerosolization showed that the powder was polydisperse. The median value of the volumetric particle diameter distribution in the nominal air flow of the Cyclohaler type inhaler - Dv(50) was equal to 148.43±10.14μm. At the same time, the diameter of the smallest particles whose total volume did not exceed 10% of the total volume of the sample was less than 28.23±1.48μm (Dv(10)), while the diameter of the particles whose total volume was less than 90% of the total volume of the sample was 410.10±29.41μm. The air flow rate delivered to the inhaler was increased to 200dm 3 / min and 270dm 3 / min did not significantly affect the volume particle size distribution median or Dv(10) value. Only a slight increase in the size of the largest particles (Dv(90)) from about 410.1±29.41 μm to 498.3±62.7 μm was observed. This resulted in a distribution range of 2.57±0.04 (for 100 dm 3 / min airflow) increased to 3.3±0.4 (for 270dm 3 / min air flow).

[0106] B. Efficacy of the Decellularization Method

[0107] -Protein properties of the product:

[0108] Using mass spectrometry, the protein composition of the ECM of decellularized porcine pancreas was determined. The results obtained clearly showed that the highest percentage of collagen was found in the samples tested, with type 1 collagen (COL1) of the alpha (A)-1 chain, the so-called COL1A1, showing the highest values ​​compared to the other collagen types detected.

[0109] Table 2. Percentage of COL1A1 in the tested samples. M18, M22, M23, M24s, M24, M25 represent the sample numbers.

[0110]

[0111] Additionally, significant amounts of collagen types IV and VI were found. This suggests that the decellularization protocol used allowed for the retention of the collagen types with the highest degree of integration with the islets and beta cells. Collagen types I and IV are the most effective in supporting islet function and viability, and they are often used as supplements in biomedical applications based on islet cell function. Notably, collagen types VI and IV are present on the secretory outer surface of the islets and in the basement membrane, where they regulate fibronectin activity. The percentage content of any of the other collagen types analyzed (COL1A2, COL3A1, COL4A2, COL6A1, COL6A2, COL6A3, COL14A1) did not exceed 3.5% in any of the samples examined.

[0112] - Final DNA concentration

[0113] To determine the residual DNA concentration, three analyses were performed:

[0114] (a) PicoGreen was used to determine the residual DNA concentration

[0115] (b) Agarose gel electrophoresis is used to determine the particle size of the remaining genetic material

[0116] (c) Microscope imaging-hematoxylin & eosin (H&E) staining.

[0117] The decellularization process was successfully completed when the concentration of residual DNA did not exceed 50 ng double-stranded DNA (dsDNA) / mg dry weight ECM and the molecules of the remaining DNA did not exceed 200 base pairs (bp). In addition, the microscopic images of the scaffolds obtained were evaluated for the presence of cell nuclei (hematoxylin & eosin staining).

[0118] The concentration of residual DNA in the dry matter averaged 0.077 ng / mg. In all samples tested, the residual DNA content was below 0.15 ng / mg. The analysis was performed using the DNeasy Blood & Tissue Kit for the isolation of residual DNA and the Quant iT PicoGreen dsDNA reagent and kit for the determination of the concentration of the isolated genetic material.

[0119] No signal was found using agarose gel electrophoresis. All samples were below the detection level, which clearly indicated that there was no residual DNA in the ECM in the form of particles larger than 200 bp.

[0120] Microscopic examination showed that after the decellularization process, no genetic material and no cell nuclei were visible.

[0121] -Triton X-100 residues and effective methods for their detection and removal

[0122] For comparison, decellularization was performed using 0.5% (w / v) SDS. However, the results were not satisfactory due to the large amount of detergent remaining in the final product. The ECM powder showed high levels of foaming when trying to dissolve it. This was not observed when TritonX-100 was used.

[0123] To verify the remaining Triton X-100 concentration after the decellularization process, its residual amount in the final product was determined.

[0124] Prepare samples for analysis:

[0125] Given the white color of dissolved dECM, samples were treated with three concentrations of collagenase: 4.3953 PZ activity units / g dECM(p), 43.953 PZ / g dECM(p), and 87.906 PZ / g dECM(p). Collagenase was prepared in a special solution containing 150 mL of Ringer's solution with a pH of 7.2 to 7.4, 2.72 mL of Hepes (1 M), 1.125 mL of sodium bicarbonate (7.5%), and 1.05 mL of calcium chloride (1 M).

[0126] The samples were stirred at 37°C for 24 hours under constant shaking at 1000 rpm. The obtained solutions were analyzed for residual concentrations of non-ionic detergent Triton X-100. Sample A is the result of treating dECM with a single collagenase concentration. Sample B was treated with 10-fold collagenase concentration. Sample C was treated with 20-fold collagenase concentration (A = 6.977 μg Triton X-100 / g dECM (p), B = 40.475 μg Triton X-100 / g dECM (p), C = 39.325 μg Triton X-100 / g dECM (p)).

[0127] Importantly, the highest concentration of residual Triton X-100 was found in the dECM solution treated with collagenase at a concentration of 43.953 PZ / g dECM. Increasing the collagenase concentration did not result in obtaining a greater amount of Triton X-100, indicating that the 43.953 PZ / g concentration of dECM was sufficient to extract all of the remaining Triton X-100 from the sample.

[0128] Previously published attempts to evaluate this detergent have not yielded any practical results for the following reasons:

[0129] - Evaluation of Triton X-100 powder was not possible, as this form of ECM absorbed the dye, thus distorting the results. This correlation was demonstrated by analyzing ECM powder after decellularization using SDS detergent, which indicates the presence of TritonX-100, which was not possible since the detergent used was not Triton X-100.

[0130] - dECM dissolved in pepsin and neutralized affects the concentration reading due to its white color.

[0131] Therefore, treatment of dECM with collagenase is the only method to date to assess detergent residues in biomaterials after decellularization. This has important implications for the use of such materials (dECM) in bioprinting processes using living cells. This is crucial if such materials are to be implanted in the human body.

[0132] C. Results

[0133] In the first step, the fat composition of the decellularized matrix was analyzed for differences in the functionality of the pancreas decellularized preparations. In the next step, the residual DNA content, collagen content, and residual detergent Triton X-100 were analyzed according to the pancreas preparation method.

[0134] The use of mechanical extrusion grinding method allowed to significantly reduce the fat content in the obtained extracellular matrix. In the mechanical extrusion grinding method, the fat content was 6.24 + / - 0.07% (w / w), while in the cutting method, the fat content was 21.47 + / - 0.07% (w / w). The difference was statistically significant (p < 0.001). The low fat content of the obtained dECM significantly improved the viability of cells and islets.

[0135] When the mechanical extrusion grinding method was used, the residual DNA content tested using Picogreen was significantly lower, ie 0.07 + / - 0.07 ng / mg tissue compared to 0.13 + / - 0.06 ng / mg tissue (p = 0.027). In both cases this is well below the allowed value of 50 ng / mg.

[0136] The use of mechanical extrusion grinding method allows to significantly reduce the Triton X-100 content in the extracellular matrix obtained. In the mechanical extrusion grinding method, the detergent content is 3.79 + / - 2.33 μg / g, while in the cutting method it is 6.53 + / - 2.34 μg / g. The difference is statistically significant (p = 0.008).

[0137] There were no differences in the collagen content of the tested materials resulting from the preparation methods, depending on the use of cutting and mechanical extrusion grinding.

[0138] Using ammonia to alkalinize the environment for better washing out of Triton did not improve the washing out of Triton. However, it caused a change in the composition of the collagen obtained. Likewise, washing at 24°C did not improve the washing out of Triton, while increasing the damage to the collagen structure, leading to higher results for DNA content, which may indicate a risk of infection in the material. Therefore, the best approach is to wash the decellularized material in PBS at a temperature of 4°C for 72 hours.

[0139] Example 2: Preparation of bio-ink

[0140] A. Dissolution of dECM (p)

[0141] In order to obtain dECM solution (dECM(r)), a dissolution process of dECM powder (dECM(p)) using pepsin and hydrochloric acid (HCl) was established. The process of obtaining dECM solution is divided into two parts:

[0142] (a) Dissolution of dECM

[0143] Pepsin (at a concentration of 0 mg / mL to 10 mg / mL, preferably 1 mg / mL) was dissolved in 50 mL of 0.01 M HCl, and then dECM (p) (0.5 g to 5 g) was added. The concentration of dECM (r) produced by this method ranged from 1% to 10% (w / v). The prepared solution was placed on a magnetic stirrer and the following stirring conditions were used: ambient temperature was about 25°C, dissolution time was 72 hours, and the solution was stirred every hour for the first 8 hours of stirring.

[0144] (b) Neutralization of dECM (r)

[0145] Neutralize 50 mL of dECM(r) on ice (ideal temperature of dECM solution is 4°C to 4.5°C) to a pH of 7.2 to 7.4 using the following:

[0146] -5 ml of 0.1 M NaOH (the volume of 0.1 M NaOH used for neutralization is equal to 1 / 10 of the volume of dECM(r);

[0147] - 5.56 mL of 10×PBS (the volume of 10×PBS used for neutralization is equal to 1 / 9 of the volume of dECM(r));

[0148] - Dilute the dECM solution with an appropriate volume of 1× PBS (1 ml to 10 ml).

[0149] To determine a suitable process for preparing dECM(r), solutions with a relatively high concentration of dECM(p) of 10% (w / v) were analyzed after irradiation sterilization and grinding with different pepsin contents.

[0150] dECM solutions with different pepsin contents were prepared. Solutions containing 1 mg / mL pepsin had a relatively high homogeneity: a smaller range of viscosity values. Turbidity varied slightly with temperature. All analyzed dissolution methods with different pepsin contents were used for the preparation of dECM(r), however, the amount of 1 mg / mL used proved to be optimal.

[0151] B. Preparation of Bio-ink

[0152] (a) Conditions for obtaining primary bio-ink:

[0153] - Neutralized dECM solution, dECM(p) ground, cut, sterilized with or without radiation or ethylene oxide

[0154] -dECM(p) powder, ground, cut, sterilized with or without radiation or ethylene oxide

[0155] - Sterile GelMa 10% to 20% (w / v) containing 0.2% to 0.5% (w / v) LAP

[0156] - Sterile HAMA 1% to 3% (w / v) containing 0.2% to 0.5% (w / v) LAP

[0157] -Sterile glycerol

[0158] - Medium 1: 5-7 v / v, pancreatic islets 20,000 iEq / mL and cell line: endothelial cells 1×10 5 / mL, primary microvascular endothelial cells 1×10 5 / mL, vitamins: A is 100μM, B1 is 100μM, B3 is 10μM, D3 is 10nM, growth factors: VEGF is 30ng / mL, FGF is 20ng / mL, tumor necrosis factor (TNF)-α is 10ng / mL, IL-8 is 10ng / mL, and IL-17A is 20ng / mL.

[0159] First, a paste containing an appropriate amount of neutralized dECM (r) and dECM (p) was prepared by thorough mixing using a sterile metal spatula. Since dECM (p) is prepared by freeze drying and does not dissolve subsequently, it retains the quaternary structure of ECM. The obtained paste was placed at a temperature of 7°C to 10°C for at least 24 hours. Before the paste was directly used for bio-ink production, it was placed in a sterile syringe and mixed between syringes. At the same time, GelMa (10% to 20% (w / v)) and HAMA (1% to 3% (w / v)) solutions were prepared using LAP according to the common process. The syringe containing the paste was connected to another syringe without a piston through a connector, which was moved upside down and stably placed vertically. Glycerol, culture medium, growth factors, vitamins, GelMa and HAMA solutions were added in sequence. The piston was then gently inserted to mix the paste with the other reagents. After mixing, the prepared bio-ink was placed in an incubator for 5 minutes, islets and cells were added, and then mixed again and introduced into the cartridge. In the next step, the filled cartridges were centrifuged at 1500 rpm for 2 min and then reintroduced into the incubator, after which they were printed for approximately 5 min.

[0160] The composition of the obtained primary bio-ink was as follows: 40% to 50% (v / v) dECM (r), 2.763% to 27.692% (w / v) dECM (p), 1.464% to 7.320% (w / v) GelMa, 0.146% to 1.098% (w / v) HAMA, 5.0% to 10.0% (w / v) glycerol, 0.03% to 0.17% (w / v) w / v) LAP, VEGF 30ng / mL, FGF 20ng / mL, TGF-β 10ng / mL, IL-8 10ng / mL, IL-17A 20ng / mL, vitamin A 100μM, vitamin B1 100μM, vitamin B3 10μM, vitamin D3 10nM, pancreatic islets 20000iEq / mL, endothelial cells 1×10 5 / mL, primary microvascular endothelial cells were 1×10 5 / mL.

[0161] (b) Vascular bio-ink

[0162] The process of producing vascular bioink using ultrasound treatment is a two-step process:

[0163] - Preliminary dissolution - An appropriate amount of microbial gelatin is suspended in PBS (1% to 2% (w / v)) and stirred at 60°C with a magnetic stirrer for about 10 minutes. Then, under continuous stirring, the temperature is lowered and 5% to 10% (w / v) of dECM (p) (ground and cut, after or without radiation sterilization or ethylene oxide sterilization) is added in batches, stirring the solution additionally every 2 minutes. According to a variant, a previously prepared PBS-based carboxymethylcellulose (CMC) solution (2% to 5% (v / v)) is added to the mixture.

[0164] -Ultrasonic treatment: Place the bottle with the prepared ECM solution in a beaker with ice, then place an ultrasonic probe and a temperature sensor in the beaker, and then perform ultrasonic treatment according to the developed process using a 3s pulse with an amplitude of 45%, and stop working when the temperature exceeds 30°C. The ultrasonic treatment time is 0.5 hours to 2.0 hours.

[0165] - Alternatively, the preliminary dissolution step is omitted and a 5% to 10% (w / v) dECM solution is prepared by adding the dECM powder to a buffer solution or cell culture medium solution with gentle stirring. Next, a sonication step is performed as described above.

[0166] The process of producing vascular bioink through cooking is a two-step process:

[0167] - Preliminary dissolution - An appropriate amount of microbial gelatin is suspended in PBS (1% to 5% (w / v)) and stirred with a magnetic stirrer at 60°C for about 10 minutes. Then, under continuous stirring, the temperature is lowered and 2% to 10% (w / v) of dECM (p) (ground and cut, after or without radiation sterilization or ethylene oxide sterilization) is added in batches, stirring the solution additionally every 2 minutes. According to a variant, a previously prepared PBS-based CMC solution (2% to 5% (v / v)) is added to the mixture.

[0168] - Cooking treatment: The bottle containing the prepared ECM solution or ECM powder dissolved in PBS solution (5% to 10% (w / v)) was placed on a magnetic stirrer equipped with a hot plate heated to 100°C, where the mixture was cooked for 15 to 30 minutes.

[0169] - Alternatively, the preliminary dissolution step is omitted and a 5% to 10% (w / v) dECM solution is prepared by adding the dECM powder to a buffer solution or cell culture medium solution with gentle stirring. Next, a sonication step is performed as described above.

[0170] The vascular bio-ink substrate thus prepared was supplemented with fibronectin, growth factors and endothelial cells.

[0171] The composition of the obtained ultrasonically treated vascular bio-ink is as follows: 5% to 10% (w / v), preferably 7.5% (w / v) dECM (p), 0.2% to 1% (v / v) CMC, 1% to 2% (w / v), preferably 1% (w / v) microbial gelatin, 100 μg / mL fibronectin, 30 ng / mL VEGF, 20 ng / mL FGF, 100 nM PGE2, 1.5×10 7 / mL of endothelial cells and 3×10 6 / mL of fibroblasts.

[0172] The composition of the boiled vascular bio-ink is as follows: 2% to 10% (w / v), preferably 5% (w / v) dECM (p), 0.2% to 2% (v / v) CMC, 1% to 5% (w / v), preferably 1% (w / v) microbial gelatin, 100 μg / mL fibronectin, 30 ng / mL VEGF, 20 ng / mL FGF, 100 nM PGE2, 1.5×10 7 / mL of endothelial cells and 3×10 6 / mL of fibroblasts.

[0173] Alternatively, the vascular bio-ink consists of 5% to 10% (w / v), preferably 5% (w / v), dECM(p) in a buffer solution or cell culture medium.

[0174] Example 3: Characteristics of primary bio-ink

[0175] A. Rheology

[0176] The tests performed served as a basis for determining the values ​​of characteristic parameters constituting factors limiting the possibility of printing pancreatic lobule models using a specific system - viscosity values ​​exceeding 5 Pa·s. The effect of pepsin concentration on the properties of dECM hydrogels is described below.

[0177] Table 3. Effect of temperature (25°C to 37°C) on dECM(r) turbidity

[0178]

[0179] Table 4. Effect of exposure time at 37°C on dECM(r) turbidity

[0180]

[0181] Table 5. Effect of pepsin concentration on dECM(r) viscosity, measured at a constant shear rate (21 / s) for 50 min

[0182] Pepsin concentration [mg / mL] η[mPa·s] 0 2109.7<η<5611.9 1 3026.9<η<4040.7 10 3287.6<η<4691.3

[0183] To determine the composition of the bioink with the best performance, the viscosity of the dECM solutions and pastes was tested using an MCR 72 rheometer (Anton Paar) following a specially developed procedure that represents the conditions present during the bioprinting process: cone and plate system, with a constant shear rate of 21 / s and a test temperature of 37°C. Taking into account the sample differences in the type of powder used (MS-milled sterile, CS-cut sterile, MNS-milled non-sterile, CNS-cut non-sterile) and the concentrations of the components used, the system rheological test results are shown in Figure 2. Figure 2 shown.

[0184] The increase in dECM solution concentration resulted in an increase in viscosity [ Figure 2 ]. The viscosity values ​​obtained seem too low to use dECM(r) as an agent to impart appropriate consistency to bioinks. In each case considered, the viscosity values ​​of the solutions obtained with sterilization of dECM(p) were lower than those of the non-sterile powder solutions. Slight differences in solution consistency were observed when using milled and cut powders at lower dECM(r) concentrations. For 10% (w / v), the viscosity of the milled powder dECM(r) was slightly higher than that of dECM(r) prepared from cut dECM(p).

[0185] The results summarized showed that the use of dECM pastes was necessary to obtain a bioink substrate with an appropriate consistency. The viscosity of all systems summarized was within the acceptable range for use during printing. In addition, the use of sterile powders for bioprinting of the mixtures of the components with cells and islets after sterilization appeared to be advantageous.

[0186] The addition of glycerol to the primary bio-ink (paste) resulted in a slight decrease in the viscosity of the primary bio-ink, which is contrary to literature data, which reported an increase in the viscosity of the bio-ink after the addition of glycerol. Each reagent added to the paste induces a change in viscosity. The addition of substances that support the maintenance of cell and islet structure or viability results in changes in paste fluidity that are minimal or even negligible. Pastes from dECM form the basis for producing primary bio-inks and determining whether a particular bio-ink can be used for printing.

[0187] B. Curing Method of Bio-ink

[0188] - Cross-linking printouts using cross-linking agents

[0189] The following table shows the differences in the cross-linker composition used in the primary bio-inks [Table 6].

[0190] Table 6. Various cross-linkers used in primary bio-inks

[0191]

[0192] The above system has been tested for crosslinking using light in the wavelength range of 365nm to 405nm with positive results [Table 7].

[0193] Table 7. % damage to DNA exposed to light of 365 nm and 405 nm wavelengths

[0194]

[0195] Analysis of cross-linking results after the process or during bioprinting showed that the use of light with a wavelength of 365nm and 405nm achieved the desired effect, i.e., the hydrogel form changed from liquid to solid. However, since the bio-ink contains cells and microorganisms, only visible light can be used. Therefore, the most preferred cross-linking method is to use light with a wavelength of 405nm.

[0196] Adding supplementary chemicals to the dECM paste smoothed the topography of the filament surface. In addition, increased aeration of the bioink was confirmed when GelMa and HAMA were added, with the strongest effect being seen with HAMA.

[0197] -Thermogel

[0198] The strength of the gelling process is tested by identifying the turbidity of the solution over a wide range of temperatures and exposure times to its effect using specialized equipment. Figure 3 Examples of results from cross-linking experiments with dECM solutions are shown. For 5% (w / v), a slight increase in absorbance was observed for all tested systems produced by increasing temperatures in the range of 25°C to 37°C. The use of milled sterile powders reduced the turbidity of the dECM solutions. For cut powder solutions, the same sterility correlation was obtained at higher concentrations of dECM(r) (8% and 10% (w / v)), while the opposite was true for milled powders. The turbidity of both 8% and 10% (w / v) dECM(r) increased slightly with increasing temperature. The 10% dECM(r) showed a relatively high turbidity and was stable over the tested temperature range.

[0199] According to the gelation kinetics at 37° C., no significant changes in turbidity were observed when increasing the exposure time at 37° C. The dECM(r) from the milled sterile powder had the lowest absorbance value, while the cut non-sterile powder solution had the highest turbidity for all dECM(r) concentrations.

[0200] C. Permeability of bio-ink components using glucose diffusion as an example

[0201] As the so-called driving force (i.e., glucose concentration) increases, the delay time and the time to reach equilibrium state decrease, and the diffusion rate increases accordingly. The data in Table 8 show that the diffusion rate of the membranes obtained using the primary bio-ink is comparable to that obtained using 4% (w / v) alginate (Alg4).

[0202] Table 8. Diffusion rate of membranes with primary bioinks with different additives. 4% alginate was added for comparison.

[0203]

[0204] D.Absorbance

[0205] To evaluate the usability of the obtained bio-ink, the printed leaflets were analyzed by absorbance using a specially prepared buffer that simulates the conditions inside the body. During the first 15 minutes, a slight increase in the weight of the printed structure was observed, which then decreased and stabilized at a specific level. In the next step, the weight of the printed leaflets was observed over time to study the degradation phenomena in the SBF buffer environment. Figure 4 ].

[0206] Example 4: Characteristics of vascular bio-ink

[0207] A. Rheology

[0208] The boiled dECM(r) had higher viscosity values ​​than the sonicated dECM(r). However, due to the appropriate stability of the vascular bio-ink after sonication, this method was determined to be more preferred for the printing of tubular catheters.

[0209] B. Gelation

[0210] Increased temperatures in the range of 25°C to 37°C and longer exposure times at 37°C resulted in slightly decreased dECM concentrations for both boiled and sonicated treatments.

[0211] Table 9. Effect of temperature on dECM concentration used to produce vascular bio-inks

[0212]

[0213] Table 10. Effect of exposure time at 37°C on dECM concentration for producing vascular bio-ink

[0214]

[0215] Example 5: Effect of pressure used in bioprinting on cell and micro-organ viability

[0216] Viability tests were performed on fibroblasts (cell lines 3T3-L1 and HFF-1) and pancreatic islets. For this purpose, pressures ranging from 15 kPa to 100 kPa were applied to the pancreatic cells / islets using needles with diameters of 0.2 mm and 0.6 mm. The results of the tests performed showed that the shear forces induced during 3D bioprinting using the extrusion method can lead to significant changes in cell and micro-organ viability.

[0217] Table 11. Percentage of surviving and dead 3T3-L1 cells after application of predetermined stresses

[0218]

[0219] Table 12. Percentage of HFF-1 cells that survive and die after application of predetermined pressures

[0220]

[0221] Table 13. Viability of islets subjected to specific pressures using a 0.6 mm needle. For islets, it is preferred not to use a smaller needle diameter, as the diameter of islets varies between 50 μm and 500 μm.

[0222]

[0223] In order to obtain viable functional biological 3D structures, the pressure and diameter of the needle need to be matched to the specific cell type. However, it is preferred to apply a pressure of no more than 30 kPa.

[0224] Example 5: Printability

[0225] Printouts using primary bio-inks were performed using the following parameters: pressure: 4 kPa to 100 kPa, print speed: 5 mm / s to 40 mm / s, temperature: print head: 10°C to 37°C; print bed: 4°C to 37°C, needle diameter: 100 nm to 1 mm. Printouts using vascular bio-inks were performed using the following parameters: pressure: 5 kPa to 100 kPa, print speed: 5 mm / s to 40 mm / s, temperature: print head: 10°C to 37°C; print bed: 4°C to 37°C, needle diameter: 100 nm to 1 mm.

[0226] - Leaflet

[0227] Printing a pancreatic lobule with ducts takes about 3 minutes. Figure 6 A 3D model of the vascularized leaflet and a picture of the printed structure are shown. SEM was used to determine the morphology of the printed leaflet from the side and cross-section. Loose arrangement of bioink filaments was observed behind the substantial porosity of the leaflet. In addition, based on the cross-sectional analysis, the layering of the three-dimensional porous structure provided by the patented conduit of the mimic tube was determined.

[0228] - Vascularized three-dimensional structure

[0229] Printing of the biomimetic pancreas prototype with the patented ductal network took about 30 minutes. As in the case of the lobules, loose arrangement of bio-ink filaments was observed in the highly porous structure of the printed construct with the patented ductal network.

[0230] The printed vasculature was evaluated using magnetic resonance imaging. The resulting 3D reconstruction showed that the patented catheter had no tendency to collapse or dislodge.

[0231] Example 5: Cytotoxicity of printed leaflets

[0232] MTT assay was performed on fibroblast cell line (3T3) to evaluate the cytotoxicity of primary bio-inks. The results are expressed as % of control at the maximum extract concentration [Table 14]. The extract exposure time was 24 h, and the cell density was 1×10 5 / mL was plated on a plate. Both experiments showed no cytotoxicity to the tested cell lines.

[0233] Table 14. Results of MTT assay of primary bio-ink cytotoxicity. Tests were performed on fibroblast 3T3 cell line

[0234]

[0235] Example 6: Effects of individual bio-ink components on islet / cell function and viability

[0236] To evaluate the effects of individual components of the primary bioink on islet viability and function, a glucose stimulation assay was performed.

[0237] -glycerin

[0238] Due to its properties, the addition of 5% (w / v) and 10% (w / v) glycerol to the bio-ink improved the printability of the primary bio-ink. To evaluate its effect on islet function, glycerol was added to the culture medium at a concentration of 5% or 10%, and islets were incubated therein for 24 h. Figure 8 ]. In both cases, the islets functioned far better than islets grown in culture alone.

[0239] - Commercial protein supplements

[0240] The effect of the addition of extracellular matrix proteins on islet function and viability was tested. For this purpose, a solution consisting of 0.007 mg / mL hyaluronic acid, 0.041 mg / mL type I collagen, 0.122 mg / mL type IV collagen, and 0.084 mg / mL laminin was prepared and added to the culture medium. Two types of hyaluronic acid (high molecular weight or low molecular weight) were added to the culture medium for the experiment, and the islets were incubated in it for 48 hours. Fig. 9 In both high (H) and low (L) molecular weight hyaluronan variants, islets had functional levels comparable to islets not treated with any supplements.

[0241] -GelMA

[0242] We tested how islet viability might be affected by methacrylated gelatin, a component of the bio-ink used to ensure proper cross-linking of the prints. For this purpose, 7.8% v / v of GelMa was added to the culture medium and islets were incubated therein for 72 h. Fig.10 ]. Islets grown in medium supplemented with GelMa secreted similar or greater amounts of insulin, depending on the time of measurement, indicating that the compound has a favorable effect on the viability of islets at a given concentration.

[0243] -HAMA

[0244] We tested how islet viability might be affected by methacrylated hyaluronic acid, a component of the bio-ink used to ensure proper cross-linking of the prints. For this purpose, 0.78% v / v of HAMA was added to the culture medium and islets were incubated therein for 48 h. Fig.11Islets grown in medium containing HAMA secreted less insulin than control islets, which may indicate that the compounds at the tested concentrations have an adverse effect on islet viability.

[0245] -GelMA and HAMA

[0246] We tested how islet viability might be affected by a mixture of methacrylated gelatin and methacrylated hyaluronic acid as components of the bio-ink, which ensures proper cross-linking of the prints. For this purpose, 4.68% v / v of GelMa and 0.312% v / v of HAMA (G3:2H) or 3.12% v / v of GelMa and 0.468% v / v of HAMA (G2:3H) were added to the culture medium and islets were incubated therein for 72 h. Fig.12 ]. Islets grown in medium supplemented with the mixture in a G3:2H ratio secreted greater or lesser amounts of insulin compared to control islets, depending on the time point of measurement. This mixture variant appears to have a favorable effect on islet viability. Islets grown in medium supplemented with the mixture in a G2:3H ratio secreted significantly less insulin than control islets, suggesting that the GelMa and HAMA mixture at a given concentration has an adverse effect on islet viability.

[0247] -ECM powder

[0248] We investigated how the ECM obtained by decellularization affects the viability of islets. For this purpose, 3.33% v / v of the cut or ground ECM obtained during decellularization was added to the culture medium and islets were incubated therein for 72 h. Fig.13 Islets grown in medium supplemented with ground ECM had a favorable effect on islet insulin secretion over 24 hours. Medium supplemented with shredded ECM significantly reduced insulin secretion, which may indicate an adverse effect on islet viability.

[0249] Example 7: Viability of pancreatic islets after bioprinting

[0250] Three bioinks were selected to evaluate the viability of pancreatic islets after 3D bioprinting: methacrylated gelatin, methacrylated hyaluronic acid, and a mixture of methacrylated gelatin and methacrylated hyaluronic acid.

[0251] For this purpose, 7.8% v / v of GelMa or 0.78% v / v of HAMA or a mixture of 4.68% v / v of GelMa and 0.312% v / v of HAMA (MIX) was added to the primary bio-ink. After printing, the lobules with islets were incubated in culture medium for 24 h [ Fig.14 ].

[0252] Islets in lobules printed with primary bio-inks containing GelMa additions showed the highest levels of insulin production after the printing process, thus indicating that this component has a favorable effect on islet viability and function. Adding HAMA and a mixture of GelMa and HAMA to the bio-ink both caused a slight decrease in the levels of insulin produced by the islets compared to control islets grown in culture medium (without 3D bioprinting). While the results with the bio-ink with only GelMa additions showed the highest islet activity at a given glucose concentration, the printed structure was the least stable and decomposed the fastest in culture medium. Therefore, the best solution was to use a mixture of methacrylated gelatin and methacrylated hyaluronic acid for the bioprinting process. This combination allows the preservation of viable and functional islets while maintaining appropriate bioprinting parameters and printed model stability.

[0253] Example 8: Confirmation of dECM quaternary structure preserved in primary bio-ink

[0254] To visualize and confirm the preservation of the ECM quaternary structure in printed constructs containing the primary bioink, electron microscopy was used to visualize the protein structure at various stages of dECM preparation (in order to use it for bioprinting) ( Fig.15 ). The printed structures containing the primary bio-ink (E and F) showed collagen quaternary structure with visible collagen fibers.

[0255] References:

[0256] [1]Mohamed Ali, Anil Kumar PR, James J.Yoo, Faten Zahran, Anthony Atala, and Sang Jin Lee (2019). A Photo-Crosslinkable Kidney ECM-Derived BioinkAccelerates Renal Tissue Formation, Adv.Healthcare Mater, 1800992, DOI: 10.1002 / adhm.201800992

[0257] [2]Pengfei Chen,Lin Zheng,Yiyun Wang,Min Tao,Ziang Xie,Chen Xia,Chenhui Gu,Jiaxin Chen,Pengcheng Qiu,Sheng Mei,Lei Ning,Yiling Shi,Chen Fang,Shunwu Fan and Xianfeng Lin(2019).Desktop-stereolithography 3D printing of aradially oriented extracellular matrix / mesenchymal stem cell exosome bioinkfor osteochondral defect regeneration,Theranostics.9(9):2439-2459

[0258] [3]Falguni Pati,Jinah Jang,Dong-Heon Ha,Sung Won Kim,Jong-Won Rhie,Jin-Hyung Shim,Deok-Ho Kim&Dong-Woo Cho(2014).Printing three-dimensionaltissue analogues with decellularized extracellular matrix bioink,Naturecommunications,5:3935,DOI:DOI:10.1038 / ncomms4935 10.1038 / ncomms4935

[0259] [4]Lee,H.,Han,W.,Kim,H.,Ha,D.-H.,Jang,J.,Kim,B.S.,&Cho,D.-W.(2017).Development of Liver Decellularized Extracellular Matrix Bioink for Three-Dimensional Cell Printing-Based Liver Tissue Engineering.Biomacromolecules,18(4),1229-1237.doi:10.1021 / acs.biomac.6b01908

[0260] [5]Sackett,S.D.,Tremmel,D.M.,Ma,F.,Feeney,A.K.,Maguire,R.M.,Brown,M.E.,Odorico,J.S.(2018).Extracellularmatrix scaffold and hydrogel derivedfrom decellularized anddelipidized human pancreas.Scientific Reports,8(1).doi:10.1038 / s41598-018-28857-1

[0261] [6]Mirmalek-Sani,S.H.,Orlando,G.,McQuilling,J.P.,Pareta,R.,Mack,D.L.,Salvatori,M.,&Soker,S.(2013).Porcine pancreasextracellular matrix as aplatform for endocrine pancreasbioengineering.Biomaterials,34(22),5488-5495

[0262] [7]Brown,B.N.,Freund,J.M.,Han,L.,Rubin,J.P.,Reing,J.E.,Jeffries,E.M.,Badylak,S.F.(2011).Comparison of ThreeMethods for the Derivation of aBiologic Scaffold Composed ofAdipose Tissue Extracellular Matrix.TissueEngineering Part C:Methods,17(4),411-421.doi:10.1089 / ten.tec.2010.0342。

Claims

1. A method for preparing a detergent-free decellularized extracellular matrix dECM, the method The following steps are involved: - mechanically disrupting an organ of animal origin selected from the group consisting of pancreas, liver, kidney, heart, skin, lung, large intestine, small intestine, arteries and veins, adipose tissue and placenta, wherein the organ is separated from the body of the animal; - incubating the organ fragments in a buffered detergent solution comprising 1× PBS, wherein the buffered detergent solution comprises 0.5% to 1.5% (v / v) octoxynol-9, wherein the detergent solution is supplemented with an antimicrobial agent, and the incubation is performed at a temperature below room temperature under agitation for at least 72 hours, wherein the organ fragments are transferred to fresh detergent solution every 4 to 12 hours; - incubating the organ fragments in a first buffered wash solution comprising 1×PBS, wherein the first buffered wash solution comprises an antimicrobial agent, the incubation being carried out at a temperature below room temperature under stirring for at least 72 hours, wherein the organ fragments are transferred to fresh wash solution every 4 to 12 hours; - incubating the fragmented organ in a DNase solution containing DNase at a temperature suitable for the performance of the DNase; - incubating the organ fragments in a second buffered wash solution comprising 1×PBS, wherein the second buffered wash solution comprises an antimicrobial agent, the incubation being performed at a temperature below room temperature under stirring for at least 72 hours, wherein the organ fragments are transferred to fresh wash solution every 4 to 12 hours; - freezing the organ fragments and crushing the frozen organ fragments into fragments; - Freeze drying of frozen organ fragments; - grinding the crushed and dried product into dECM powder of 25 μm to 500 μm, and treating the dECM powder with collagenase, after which the amount of octoxynol-9 remaining in the dECM powder was checked to confirm its complete removal; -Sterilized products.

2. The method according to claim 1, wherein the grinding step is followed by the following steps: - Dissolve dECM powder in hydrochloric acid solution supplemented with 0 to 10 mg / ml of pepsin; - Mix at room temperature for 48 to 72 hours; -Neutralize on ice.

3. A method for preparing a primary bio-ink, the method The following steps are involved: - preparing by mixing a paste comprising 5% to 50% (g / ml) of dECM powder obtained according to the method defined in claim 1 and 1% to 10% (g / ml) of dECM solution obtained according to the method defined in claim 2; - Incubate the paste at a temperature of 7°C to 10°C for at least 24 hours; - Add 1.46% to 7.32% (g / ml) of gelatin methacrylate, 0.15% to 1.10% (g / ml) of hyaluronic acid methacrylate and 5% to 10% (g / ml) of glycerol, as well as the photoinitiator, and mix gently.

4. A primary bioink, the ink comprising a dECM paste and 1.46% to 7.32% (g / ml) gelatin methacrylate, 0.15% to 1.10% (g / ml) hyaluronic acid methacrylate, and 5% to 10% (g / ml) glycerol, as well as a photoinitiator, wherein the dECM paste comprises 5% to 50% (g / ml) dECM powder obtained by the method defined in claim 1, and 1% to 10% (g / ml) dECM solution obtained by the method defined in claim 2, and wherein the viscosity of the primary bioink is measured to be at least 5 Pa·s in a cone-plate system at a constant shear rate of 21 / s and a temperature of 37°C.

5. The primary bioink according to claim 4, the ink comprising at least one additive selected from the following: hyaluronic acid at a concentration of 0.001 to 0.100 mg / mL of the primary bioink, laminin at a concentration of 0.005 to 0.100 mg / mL of the primary bioink, collagen I at a concentration of 0.001 to 0.100 mg / mL of the primary bioink, collagen IV at a concentration of 0.005 to 0.175 mg / mL of the primary bioink, fibronectin at a concentration of 3 to 300 μg / mL, human fibrinogen at a concentration of 10 to 100 mg / mL of the primary bioink, aprotinin at a concentration of 1 to 2 EPU / mL of the primary bioink, polysorbate at a concentration of 0.05 to 2 mg / mL of the primary bioink, human thrombin at a concentration of 5 to 55 mg / mL of the primary bioink, calcium chloride at a concentration of 20 to 60 mM / mL of the primary bioink; angiogenic vitamins: vitamin A at a concentration of 1 nM to 500 μM, vitamin B1 at a concentration of 50 μM to 100 μM, vitamin B3 at a concentration of 1 μM to 10 μM, vitamin B12 at a concentration of 10 to 100 mg / mL of the primary bioink, vitamin D3 at a concentration of 0.1 nM to 10 nM; angiogenesis-supporting growth factors: VEGF at a concentration of 10 to 30 ng / mL of the primary bioink, FGF at a concentration of 10 to 20 ng / mL of the primary bioink, TGF-β at a concentration of 1 to 10 ng / mL of the primary bioink, interleukin (IL)-8 at a concentration of 0 to 100 ng / mL of the primary bioink, IL-17A at a concentration of 20 to 50 ng / mL of the primary bioink.

6. The primary bio-ink according to claim 4 or 5, wherein the ink comprises at least one additive selected from the group consisting of: a density of 0.1 to 10×10 5 / mL of primary bio-ink with endothelial cells at a concentration of 0.1 to 10 × 10 5 / mL primary bio-ink with a concentration of 3 to 9 × 10 6 / mL of bio-ink with a concentration of 1.1 to 3.4×10 7 / mL bio-ink of β cells, pancreatic islets.

7. A method for preparing a vascular bioink, the method comprising the following steps: a) Preparation of a microbial gelatin solution supplemented with CMC, including preparing a 1% to 2% (g / ml) microbial gelatin solution in a buffer solution in the form of PBS. This preparation is carried out by suspending the microbial gelatin in the buffer solution at a temperature between 50 °C and 65 °C under stirring conditions, adding a 2% to 5% (v / v) aqueous solution of carboxymethyl cellulose CMC to obtain a final concentration of 0.2% to 1% (v / v) CMC in the vascular bioink, and cooling the solution to a temperature equal to or lower than 40 °C; b) Preparation of a 5% to 10% (g / ml) dECM solution, which is prepared by gently stirring and adding the dECM powder obtained by the method defined in claim 1 to (i) the microbial gelatin solution supplemented with CMC obtained in step a), or (ii) a buffer solution, or (iii) a cell culture medium solution; c) Ultrasonically treating the resulting solution for 0.5 hour to 2.0 hours at a temperature not exceeding 37 °C; d) adding at least one additive selected from the group consisting of: fibronectin at a concentration of 3 to 300 μg / mL, VEGF at a concentration of 10 to 30 ng / mL, FGF at a concentration of 10 to 20 ng / mL, PGE2 at a concentration between 100 and 300 nM, 7 Endothelial cells with a density between 0.1 and 10 × 10 cells / mL bio-ink 6 Fibroblasts between cells / mL bio-ink.

8. A method for preparing a vascular bioink, the method comprising the following steps: a) Preparation of a microbial gelatin solution supplemented with CMC, including preparing a 1% to 2% (g / ml) microbial gelatin solution in a buffer solution in the form of PBS. This preparation is carried out by suspending the microbial gelatin in the buffer solution at a temperature between 50 °C and 65 °C under stirring conditions, adding a 2% to 5% (v / v) aqueous solution of carboxymethyl cellulose CMC to obtain a final concentration of 0.2% to 1% (v / v) CMC in the vascular bioink, and cooling the solution to a temperature equal to or lower than 40 °C; b) Preparation of a 5% to 10% (g / ml) dECM solution, which is prepared by gently stirring and adding the dECM powder obtained by the method defined in claim 1 to (i) the microbial gelatin solution supplemented with CMC obtained in step a), or (ii) a buffer solution, or (iii) a cell culture medium solution; c) Boiling the mixture at 100 °C for 15 minutes to 30 minutes; d) adding at least one additive selected from the group consisting of: fibronectin at a concentration of 3 to 300 μg / mL, VEGF at a concentration of 10 to 30 ng / mL, FGF at a concentration of 10 to 20 ng / mL, PGE2 at a concentration between 100 and 300 nM, 7 Endothelial cells with a density between 0.1 and 10 × 10 cells / mL bio-ink 6 Fibroblasts between cells / mL bio-ink.

9. A vascular bioink, the ink comprising a dECM solution obtained by the method defined in claim 2, which has been ultrasonically treated or boiled, at a concentration of 2% to 10% (g / ml).

10. The vascular bioink according to claim 9, which is supplemented with microbial gelatin at a concentration of 1% to 5% (g / ml) and / or CMC at a concentration of 0.2% to 2% (v / v).

11. The vascular bio-ink according to claim 9 or 10, wherein the ink comprises at least one additive selected from the following: fibronectin at a concentration of 3 to 300 μg / mL, VEGF at a concentration of 10 to 30 ng / mL, FGF at a concentration of 10 to 20 ng / mL, PGE2 at a concentration between 100 and 300 nM, and a density of 0.1 to 10×10 7 Endothelial cells with a density between 0.1 and 10×10 cells / mL vascular bioink 6 cells / mL of fibroblasts between vascular bioinks.

12. A three-dimensional structure, the three-dimensional structure comprising at least three adjacent bioink layers, wherein the vascular bioink layer according to claim 9 is arranged between two layers of the primary bioink defined in claim 4.

13. A method for preparing a three-dimensional structure, wherein the primary bio-ink defined in claim 4 and the vascular bio-ink defined in claim 9 are deposited layer by layer during a 3D bioprinting process at a printing speed of 5 mm / s to 50 mm / s, a pressure of 4 kPa to 300 kPa and a temperature of 4°C to 37°C, and wherein during or after deposition, the primary bio-ink is exposed to ultraviolet light and / or visible light for at least 5 seconds.

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