Alginate microbeads, methods for their preparation, and bioink formulations containing the same.

Abstract

Provided are methods for preparing alginate microbeads that are particularly suitable as porogens for pore-forming hydrogel containing bioink formulations. Further provided are alginate microbeads obtainable by saidmethods, bioink formulations containing the same, and a method for producing a porous three-dimensional object using said bioink formulation.

Description

ALGINATE MICROBEADS, METHODS FOR THEIR PREPARATION,AND BIOINK FORMULATIONS CONTAINING THE SAMEField of Invention

[0001] The present application claims the benefit of priority of U.S. provisional patent application no. 63 / 634,071, filed on April 15, 2024, the content of which is hereby incorporated in its entirety.

[0002] The present invention provides methods for preparing alginate microbeads that are particularly suitable as porogens for pore-forming hydrogel containing bioink formulations.

[0003] Said preparation methods allow a rapid and efficient sterile synthesis of alginate microbeads which can be easily separated from bath solution after synthesis, treated to remove excess ions / alcohols, and isolated by centrifugation. The alginate microbeads are compact, do not aggregate and have a defined spherical and narrow particle size distribution similar to cell size, making them particularly suitable for mixing as porogens in hydrogel bioink formulations. The microbeads allow the surrounding hydrogel matrix to show advantageous diffusion properties and enable a highly concentrated and uniform pore distribution when processing the bioink formulation.

[0004] Micropores can be formed in situ via degradation of the alginate microbeads, which thereby act as sacrificial porogens within the surrounding hydrogel matrix. These micropores create less constriction and increased surface area within the hydrogel matrix which allows cells encapsulated in the hydrogel matrix to exhibit three-dimensional (tissue) growth.

[0005] The present invention further provides alginate microbeads that are obtainable by said methods for their preparation and bioink formulations containing the same. The bioink formulations are biocompatible and biodegradable and they enable the production of three-dimensional objects that may be used as scaffolds for tissue engineering and other medical or non-medical applications. Hence, there is further provided a method for producing a porous three-dimensional object using saidbioink formulation.

[0006] Such three-dimensional objects can be used for various applications such as, for example, tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics and / or other biomedical applications.Background

[0007] Biofabrication is an interdisciplinary field that combines principles of engineering, biology, and material science and holds the potential to generate constructs that closely mimic the composition and hierarchical architecture of native tissues. Such constructs can be applied for various biomedical uses including tissue engineering, regenerative medicine, and drug discovery.

[0008] Among several fabrication methods, 3D bioprinting offers tremendous potential in the fabrication of functional tissue as it provides better control over the spatial arrangement of the matrix, architectural fidelity, and compositional reproducibility of the constructs. 3D bioprinting techniques allow precise placement of cells in spatially predefined locations within confined 3D structures.

[0009] The most common 3D bioprinting techniques include DLP-based bioprinting, droplet-based bioprinting, extrusion-based bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography-based bioprinting, magnetic bioprinting, and volumetric bioprinting. These 3D bioprinting techniques allow precise placement / positioning in spatially predefined locations within confined 3D structures.

[0010] One of the critical components of 3D bioprinting is the bioink used for creating tissue constructs. The bioink is an aqueous solution of one or more biomaterials in the form of a hydrogel, which forms a matrix and optionally contains one or more desired cell types. A bioink should therefore be able to transport cells during printing, allow for diffusion of nutrients to embedded cells, and support cell growth after printing. For this purpose, a bioink should meet certain materialproperties and biological requirements.

[0011] Typical material properties include, for example, printability, crosslinkability, matrix stiffness, protection against shear stresses during the printing process, and dimensional fidelity of printed structures during subsequent cell cultivation. In addition, the formation of pores in the processed bioink formulation plays a crucial role, as these pores can provide surface area where cells can grow in a non-constricted space during cell culturing. Biological requirements of bioinks mainly include, for example, degradability, cytocompatibility, and promotion of cell bioactivity during subsequent cell cultivation.

[0012] Among the different biomaterials, hydrophilic polymers are the most prominent materials which are used in bioink formulations. Hitherto, natural polymers, such as collagen, gelatin, hyaluronic acid, alginic acid, chitosan, have claimed central roles in bioink formulations due to their availability and ability to provide adapted scaffolding systems for the structural and functional organization of cells.

[0013] Various concepts for the formation of pores in hydrogel matrices are know from the prior art, as shown below.

[0014] C. Fan et al. reports on the potential use of alginate beads as a chondrocyte vehicle and stepwise dissolving porogen in a hydrogel scaffold for cartilage tissue engineering. Here, alginate beads fabricated by needle extrusion are used to prepare a chondroitin sulfate (CS)-alginate beads composite gel (see C. Fan et al., RSC Adv. 2015, 5, 80688-80697).

[0015] C.M. Hwang et al. reports on the fabrication of three-dimensional porous cell-laden hydrogel for tissue engineering. Here, gelatin beads of 150-300 pm diameter were used as a sacrificial porogen for generating pores within cell-laden hydrogels based on an alginate solution (see C.M. Hwang et al. Biofabrication 2, 2010, 035003, 1-12).

[0016] Y. Luo et al. describes 3D bioprinting scaffolds using alginate / poly vinyl alcohol bioinks, wherein micropores are formed by diffusion of polyvinyl alcohol solsin aqueous CaCh solution (see Y. Luo et al., Materials Letters, 2017, 189, 295-298).

[0017] F. Shi et al. reports a one-step method to construct hydroxyapatite scaffolds with 3D interconnected structure by a novel hydrogel bead porogen process. Alginate hydrogel beads were manufactured by dropwise extruding a 2.0% (w / v) alginate solution through gauge needles into a 1 M CaCh solution. Said beads were then used to prepare hydroxy apatite scaffolds, from which they were removed by sintering at 1200°C to produce porous hydroxyapatite scaffolds (see F. Shi et al., Materials Letters, 2017, 203, 13-16).

[0018] C.M. Hwang et al. assessed injectable alginate particle-embedded fibrin hydrogels for soft tissue reconstruction. Alginate beads were produced by mixing CaCh solution and 5% (w / v) alginate solution using a dual syringe system assembled with a screw mixer top. Said alginate beads were embedded into fibrin hydrogel to improve dimensional stability, long-term volume retention as well as high cellular mobility and proliferation (see C.M. Hwang et al., Biomed. Mater. 2013, 8, 014105, 1-9).

[0019] US 2015 / 0315353 Al relates to a process for producing polymer foams with use of hydrogel pearls as porosity generating template.

[0020] US 2022 / 0192986 Al relates to injectable, pore-forming hydrogels for materials-based cell therapies. Described are compositions and methods to form pores in situ within hydrogels, wherein said pores are formed via degradation of sacrificial porogens within the surrounding hydrogel.

[0021] However, the processes, pore-forming formulations and porogens known from the prior art have disadvantages, as described in the following.

[0022] The prior art porogens are not readily usable for bioink formulations, since they aggregate, are too large for high resolution printing, and exhibit a microfluidiclike behavior, making them difficult to resuspend in a hydrogel formulation. For this reason they usually contain surfactants, oils and antisolvents, all of which can be cytotoxic.

[0023] The prior art does not yield or involve compact porogens with a definedspherical and narrow particle size distribution, which can be easily and homogeneously mixed in high concentrations with a hydrogel matrix.

[0024] Furthermore, the prior art porogens cannot be easily separated from solution after synthesis and washing by centrifugation. This results in a large excess of washing solution remaining with the microparticles, which has a detrimental effect on the bioink to be produced. As a result, the concentration of prior art porogens in the bioinks is rather low.

[0025] Furthermore, the prior art porogens show poor diffusion properties and performance when formulated with a hydrogel matrix in bioinks. Three-dimensional objects made therefrom do not provide satisfactory pore properties for efficient cell cultivation.

[0026] For this reason, there is a continuous need to develop improved porogens and methods for their preparation and improved bioink formulations containing said improved porogens for the production of porous three-dimensional objects, which overcome the disadvantages from the prior art.

[0027] In particular, there is a need for improved porogens that are particularly suitable for pore-forming hydrogel containing bioink formulations, where the porogens are compact, have a defined spherical and narrow particle size distribution similar to cell size, do not aggregate, can be easily separated after synthesis and washing, can be formulated in high concentration to hydrogel containing bioink formulations, and once dissolved, show advantageous diffusion properties and performance when formulated into bioink formulations, enabling the production of three-dimensional objects with satisfactory pore properties for efficient cell cultivation.Objective

[0028] The present invention aims to overcome the disadvantages known from the prior art.

[0029] Hence, it is an object of the present invention to provide a method forpreparing alginate microbeads that are readily usable for bioink formulations.

[0030] It is an object of the present invention to provide a method for preparing alginate microbeads, which are compact, have a defined spherical and narrow particle size distribution similar to cell size in the range from 25 to 300 pm, do not aggregate, and can be easily separated after synthesis and washing.

[0031] It is a further object of the present invention to provide a method for preparing alginate microbeads, which can be formulated in high concentration to hydrogel containing bioink formulations and once dissolved, show advantageous diffusion properties and performance when formulated into bioink formulations, enabling the production of three-dimensional objects with satisfactory pore properties for efficient cell cultivation.

[0032] Yet another object of the present invention is to provide alginate microbeads showing the above-mentioned beneficial effects.

[0033] Yet another object of the present invention is to provide bioink formulations comprising a crosslinkable hydrogel matrix and alginate microbeads showing the above-mentioned beneficial effects.

[0034] Yet another object of the present invention is to provide a method for producing a porous three-dimensional object from bioink formulations comprising a crosslinkable hydrogel matrix and alginate microbeads showing the above-mentioned beneficial effects.Summary

[0035] The embodiments of the present invention described hereinafter provide a technical solution to the aforementioned objectives.

[0036] In a first embodiment, the present invention provides a method for preparing alginate microbeads, wherein the method comprises the following steps: (a) providing a first solution comprising alginic acid or a salt thereof; and (b) processing said first solution through a coaxial spray nozzle into a second solution comprising a divalentmetal halide and an alcohol to obtain alginate microbeads.

[0037] In a second embodiment, the present invention provides alginate microbeads, which are obtainable or obtained by the method for preparing alginate microbeads according to the first embodiment of the present invention.

[0038] In a third embodiment, the present invention provides a bioink formulation comprising a crosslinkable hydrogel matrix, and alginate microbeads according to the second embodiment of the present invention.

[0039] In a fourth embodiment, the present invention provides a method for producing a porous three-dimensional object, wherein the method comprises the following steps: (i) 3D bioprinting the bioink formulation according to the third embodiment of the present invention to obtain a three-dimensional object; (ii) crosslinking said three-dimensional object to obtain a crosslinked three-dimensional object; and (iii) treating said crosslinked three-dimensional object with a solution comprising a chelating agent to obtain a porous three-dimensional object.

[0040] The present invention is further described in the embodiments following hereinafter in the detailed description.Brief Description of the Figures

[0041] Fig. 1: Particle size distribution measured by DLS of alginate microbeads obtained from Example 1-1, Example 1-2 and Example 1-3.

[0042] Fig. 2a: Microscope image of alginate microbeads obtained from Example 1-1 (no ethanol in bath solution, heat treated, lOx magnification).

[0043] Fig. 2b: Microscope image of alginate microbeads obtained from Example 1-2 (5% v / v ethanol in bath solution, heat treated, lOx magnification).

[0044] Fig. 2c: Microscope image of alginate microbeads obtained from Example 1-3 (20% v / v ethanol in bath solution, heat treated, lOx magnification).

[0045] Fig. 3: Particle size distribution measured by DLS of alginate microbeadsobtained from Example 1-2 and Example 1-4.

[0046] Fig. 4: Particle size distribution measured by DLS of alginate microbeads obtained from Example 1-2, Example 1-4 and Example 1-5.

[0047] Fig. 5: Microscope images of alginate microbeads obtained from Example 2; prior art (1), (2) and (3) (lOx magnification).

[0048] Fig. 6a: Cell viability testing according to Example 4 (black: untreated porous bioink; gray: porous bioink).

[0049] Fig. 6b: Metabolic activity testing according to Example 4 (black: untreated porous bioink; gray: porous bioink).

[0050] Fig. 7: Cell viability images of hMSCs cultured on bioprinted three- dimensional objects after 1 day, 3 days, 7 days and 14 days according to Example 4 (4x magnification).

[0051] Fig. 8: Cell elongation and structure stability of hMSCs cultured on bioprinted three-dimensional object after 14 days according to Example 4 (lOx magnification).

[0052] Fig. 9: Cell elongation and structure stability of hMSCs cultured on bioprinted three-dimensional object after 14 days according to Example 4 (2x magnification).

[0053] Fig. 10: Effect of the dissolution solution with different application times on hMSC culture, which was cultivated on a bioprinted three-dimensional object for 14 days according to Example 4. A: No treatment; B: 20 min treatment; C: 45 min treatment; D: 45 min treatment with cell overlay (gray to light gray) (bright-field microscopy).Detailed DescriptionMethod for Preparing Alginate Microbeads

[0054] In a first embodiment of the present invention, a method for preparing alginate microbeads is provided, wherein the method comprises the following steps:(a) providing a first solution comprising alginic acid or a salt thereof; and (b) processing said first solution through a coaxial spray nozzle into a second solution comprising a divalent metal halide and an alcohol to obtain alginate microbeads.

[0055] The method for preparing alginate microbeads as described herein provides an optimized way of preparing alginate microbeads in bulk. The procedure in step b) enables the particles to sink and crosslink immediately in the second solution without aggregating.

[0056] Preferably, the mass concentration of the alginic acid or salt thereof in the first solution is from 0.01% (w / v) to 5.0% (w / v), preferably from 0.05% (w / v) to 2.0% (w / v), more preferably from 0.1% (w / v) to 1.0% (w / v), and most preferably from 0.4% (w / v) to 0.6% (w / v), based on the total volume of the first solution.

[0057] Preferably, the weight average molecular weight (Mw) of the alginic acid or salt thereof in the first solution is in the range from 1,000 Da (1 kDa) to 10,000,000 Da (10 MDa), preferably from 2,000 Da (2 kDa) to 1,000,000 Da (1 MDa), more preferably from 3,000 Da (3 kDa) to 700,000 Da (700 kDa), and most preferably from 4,000 Da (4 kDa) to 500,000 Da (500 kDa). The weight average molecular weight (Mw) can be determined by any standard method known to those skilled in the art, such as GPC.

[0058] Preferably, the first solution further comprises one or more polymers that are miscible with the alginic acid or salt thereof. Preferred polymers are selected from polysaccharides such as, for example, chitosan, hyaluronic acid, fucoidan, poly(guluronate), etc., or polypeptides such as, for example, silk, collagen, RGD (H- Arg-Gly-Asp-Nth), etc.. After formation of the micropores by degradation of the alginate microbeads, the polymers remain in the micropores and provide a coating on the inner surface of the micropores in the hydrogel matrix. This allows for an improved cell growth in the hydrogel matrix.

[0059] Preferably, the mass concentration of the divalent metal halide in the second solution is from 0.01% (w / v) to 5.0% (w / v), preferably from 0.05% (w / v) to 2.0% (w / v), more preferably from 0.1% (w / v) to 1.0% (w / v), and most preferably from0.4% (w / v) to 0.6% (w / v), based on the total volume of the second solution.

[0060] Preferably, the divalent metal halide in the second solution is an alkaline earth metal halide. Preferred alkaline earth metal halides are alkaline earth metal chlorides such as, for example, MgCh, CaCh, SrCh and BaCh, and alkaline earth metal bromides such as, for example, MgBr2, CaBr2, SrB 12 and BaBr2.

[0061] More preferably, the divalent metal halide in the second solution is an alkaline earth metal chloride, preferably selected from the list consisting of MgCh, CaCh, SrCh and BaCl2.

[0062] Most preferably, the divalent metal halide in the second solution is CaCh.

[0063] It is preferred in the first embodiment of the present invention that the mass concentration of the alginic acid or salt thereof in the first solution is from 0.01% (w / v) to 5.0% (w / v), based on the total volume of the first solution, and that the weight average molecular weight (Mw) of the alginic acid or salt thereof in the first solution is in the range from 1,000 Da (1 kDa) to 10,000,000 Da (10 MDa). The weight average molecular weight (Mw) can be determined by any standard method known to those skilled in the art, such as GPC.

[0064] It is more preferred in the first embodiment of the present invention that the mass concentration of the alginic acid or salt thereof in the first solution is from 0.05% (w / v) to 2.0% (w / v), based on the total volume of the first solution, and that the weight average molecular weight (Mw) of the alginic acid or salt thereof in the first solution is in the range from 2,000 Da (2 kDa) to 1,000,000 Da (1 MDa). The weight average molecular weight (Mw) can be determined by any standard method known to those skilled in the art, such as GPC.

[0065] It is even more preferred in the first embodiment of the present invention that the mass concentration of the alginic acid or salt thereof in the first solution is from 0.1% (w / v) to 1.0% (w / v), based on the total volume of the first solution, and that the weight average molecular weight (Mw) of the alginic acid or salt thereof in the first solution is in the range from 3,000 Da (3 kDa) to 700,000 Da (700 kDa). The weight average molecular weight (Mw) can be determined by any standard methodknown to those skilled in the art, such as GPC.

[0066] It is most preferred in the first embodiment of the present invention that the mass concentration of the alginic acid or salt thereof in the first solution is from 0.4% (w / v) to 0.6% (w / v), based on the total volume of the first solution, and that the weight average molecular weight (Mw) of the alginic acid or salt thereof in the first solution is in the range from 4,000 Da (4 kDa) to 500,000 Da (500 kDa). The weight average molecular weight (Mw) can be determined by any standard method known to those skilled in the art, such as GPC.

[0067] Preferably, the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, a cyclic alkyl alcohol having 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms, or an aromatic alcohol having 6 to 10 carbon atoms, preferably 6 carbon atoms, wherein the alcohol has 1 to 3 hydroxy groups, preferably 1 hydroxy group.

[0068] More preferably, the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 4 carbon atoms, a cyclic alkyl alcohol having 3 to 6 carbon atoms, or an aromatic alcohol having 6 carbon atoms, wherein the alcohol has 1 hydroxy group.

[0069] Even more preferably, the alcohol in the second solution is selected from the list consisting of methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, 2-butanol, 2- methyl-propan-l-ol, and 2-methyl-propan-2-ol, cyclopropanol, cyclobutanol, cyclopentanol, cyclohexanol, and phenol.

[0070] Most preferably, the alcohol in the second solution is ethanol.

[0071] Preferably, the second solution further comprises water.

[0072] More preferably, the second solution comprises ethanol and water.

[0073] It is preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is an alkaline earth metal halide and that the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 8 carbon atoms, a cyclic alkyl alcohol having 3 to 8 carbon atoms, or an aromatic alcohol having 6 to 10 carbon atoms, wherein the alcohol has 1 to 3 hydroxy groups.It is preferred that the second solution further comprises water.

[0074] It is more preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is an alkaline earth metal chloride such as, for example, MgCh, CaCh, SrCh and BaCh, or an alkaline earth metal bromide such as, for example, MgBn, CaBri, SrB 12 and BaBri, and that the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 4 carbon atoms, a cyclic alkyl alcohol having 3 to 6 carbon atoms, or an aromatic alcohol having 6 carbon atoms, wherein the alcohol has 1 hydroxy group. It is preferred that the second solution further comprises water.

[0075] It is even more preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is selected from the list consisting of MgCh, CaCh, SrCh and BaCh and that the alcohol in the second solution is selected from the list consisting of methanol, ethanol, 1 -propanol, 2-propanol, 1- butanol, 2-butanol, 2-methyl-propan-l-ol, and 2-methyl-propan-2-ol, cyclopropanol, cyclobutanol, cyclopentanol, cyclohexanol, and phenol. It is preferred that the second solution further comprises water.

[0076] It is most preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is CaCh and that the alcohol in the second solution is ethanol. It is preferred that the second solution further comprises water.

[0077] Preferably, the volume ratio of alcohol in the second solution is from 0.01% (v / v) to 20% (v / v), preferably from 1% (v / v) to 10% (v / v), more preferably from 2% (v / v) to 8% (v / v), and most preferably from 4% (v / v) to 6% (v / v), based on the total volume of the second solution.

[0078] More preferably, the volume ratio of ethanol in the second solution is from 0.01% (v / v) to 20% (v / v), preferably from 1% (v / v) to 10% (v / v), more preferably from 2% (v / v) to 8% (v / v), and most preferably from 4% (v / v) to 6% (v / v), based on the total volume of the second solution.

[0079] It is preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is an alkaline earth metal halide and that the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1to 8 carbon atoms, a cyclic alkyl alcohol having 3 to 8 carbon atoms, or an aromatic alcohol having 6 to 10 carbon atoms, wherein the alcohol has 1 to 3 hydroxy groups, wherein the second solution further comprises water, and wherein the volume ratio of alcohol in the second solution is from 0.01% (v / v) to 20% (v / v), based on the total volume of the second solution.

[0080] It is more preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is an alkaline earth metal chloride such as, for example, MgCh, CaCh, SrCh and BaCh, or an alkaline earth metal bromide such as, for example, MgBn, CaBri, SrB and BaB . and that the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 4 carbon atoms, a cyclic alkyl alcohol having 3 to 6 carbon atoms, or an aromatic alcohol having 6 carbon atoms, wherein the alcohol has 1 hydroxy group, wherein the second solution further comprises water, and wherein the volume ratio of alcohol in the second solution is from 1% (v / v) to 10% (v / v), based on the total volume of the second solution.

[0081] It is even more preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is selected from the list consisting of MgCh, CaCh, SrCh and BaCh and that the alcohol in the second solution is selected from the list consisting of methanol, ethanol, 1 -propanol, 2-propanol, 1- butanol, 2-butanol, 2-methyl-propan-l-ol, and 2-methyl-propan-2-ol, cyclopropanol, cyclobutanol, cyclopentanol, cyclohexanol, and phenol, wherein the second solution further comprises water, and wherein the volume ratio of alcohol in the second solution is from 2% (v / v) to 8% (v / v), based on the total volume of the second solution.

[0082] It is most preferred in the first embodiment of the present invention that the divalent metal halide in the second solution is CaCh and that the alcohol in the second solution is ethanol, wherein the second solution further comprises water, and wherein the volume ratio of alcohol in the second solution is from 4% (v / v) to 6% (v / v), based on the total volume of the second solution.

[0083] It is preferred in the first embodiment of the present invention that in step (b) the first solution is processed through the interior of the coaxial spray nozzle andthat air is processed through the exterior of the coaxial spray nozzle. This allows good control of particle density and size distribution of the alginate microbeads.

[0084] It is preferred in the first embodiment of the present invention that the first solution is processed through interior of the coaxial spray nozzle in step (b) at a flow rate in the range from 0.01 to 10 mL / min, preferably from 0.1 mL / min to 5.0 mL / min, more preferably from 0.1 mL / min to 2.0 mL / min, and most preferably at a flow rate of 0.2 mL / min, 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, 1.0 mL / min, 1.2 mL / min, 1.4 mL / min, 1.6 mL / min or 1.8 mL / min. The person skilled in the art is aware that the flow rate of the first solution depends on various parameters such as, for example, the interior diameter of the coaxial spray nozzle, and can therefore vary. The flow rates stated above are particularly suitable for coaxial spray nozzles with an interior diameter in the range from 0.20 mm to 2.00 mm, preferably from 0.50 mm to 1.50 mm, more preferably from 0.80 mm to 1.00 mm, and most preferably with an interior diameter of 0.838 mm. Depending on the dimension of the coaxial spray nozzle, a person skilled in the art is able to determine appropriate flow rates of processing the first solution in step (b).

[0085] It is preferred in the first embodiment of the present invention that air is processed through a 0.22 pm filter and the exterior of the coaxial spray nozzle in step (b), wherein the air pressure is in the range from 1.65 bar to 3.45 bar, preferably from 1.72 bar to 2.76 bar, more preferably from 1.86 bar to 2.55 bar, and most preferably from 2.07 bar to 2.41 bar. The person skilled in the art is aware that the air pressure depends on various parameters such as, for example, the exterior diameter of the coaxial spray nozzle, and can therefore vary. The air pressures stated above are particularly suitable for coaxial spray nozzles with an exterior diameter in the range from 1.00 mm to 3.00 mm, preferably from 1.20 mm to 2.40 mm, more preferably from 1.40 mm to 1.80 mm, and most preferably with an exterior diameter of 1.600 mm. Depending on the dimension of the coaxial spray nozzle, a person skilled in the art is able to determine appropriate air pressures.

[0086] In a preferred embodiment of the first embodiment of the present invention, in step (b) the first solution is processed through the interior of the coaxial spray nozzle and air is processed through the exterior of the coaxial spray nozzle, wherein the flow rate of the first solution is in the range from 0.01 mL / min to 10 mL / min andthe air pressure is in the range from 1.65 bar to 3.45 bar, wherein the interior diameter of the coaxial spray nozzle is in the range from 0.20 mm to 2.00 mm and the exterior diameter of the coaxial spray nozzle is in the range from 1.00 mm to 3.00 mm.

[0087] In a more preferred embodiment of the first embodiment of the present invention, in step (b) the first solution is processed through the interior of the coaxial spray nozzle and air is processed through the exterior of the coaxial spray nozzle, wherein the flow rate of the first solution is in the range from 0.1 mL / min to 5.0 mL / min and the air pressure is in the range from 1.72 bar to 2.76 bar, wherein the interior diameter of the coaxial spray nozzle is in the range from 0.50 mm to 1.50 mm and the exterior diameter of the coaxial spray nozzle is in the range from 1.20 mm to 2.40 mm.

[0088] In an even more preferred embodiment of the first embodiment of the present invention, in step (b) the first solution is processed through the interior of the coaxial spray nozzle and air is processed through the exterior of the coaxial spray nozzle, wherein the flow rate of the first solution is in the range from 0.1 mL / min to 2.0 mL / min and the air pressure is in the range from 1.86 bar to 2.55 bar, wherein the interior diameter of the coaxial spray nozzle is in the range from 0.80 mm to 1.00 mm and the exterior diameter of the coaxial spray nozzle is in the range from 1.40 mm to 1.80 mm.

[0089] In a most preferred embodiment of the first embodiment of the present invention, in step (b) the first solution is processed through the interior of the coaxial spray nozzle and air is processed through the exterior of the coaxial spray nozzle, wherein the flow rate of the first solution is 0.2 mL / min, 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, 1.0 mL / min, 1.2 mL / min, 1.4 mL / min, 1.6 mL / min or 1.8 mL / min and the air pressure is in the range from 2.07 bar to 2.41 bar, wherein the interior diameter of the coaxial spray nozzle is 0.838 mm and the exterior diameter of the coaxial spray nozzle is 1.600 mm.

[0090] In the first embodiment of the present invention the volume of the first solution to be processed in step (b) is usually equal to or less than the volume of thesecond solution.

[0091] It is preferred that the ratio of the volume of the first solution to the volume of the second solution is in the range from 1:1 to 1:10. It is more preferred that the ratio of the volume of the first solution to the volume of the second solution is in the range from 1:2 to 1:8. It is even more preferred that the ratio of the volume of the first solution to the volume of the second solution is in the range from 1:4 to 1:6. It is most preferred that the ratio of the volume of the first solution to the volume of the second solution is 1:5.

[0092] Preferably, the method for preparing alginate microbeads according to the first embodiment of the present invention further comprises the following steps: (c) separating the alginate microbeads obtained from step (b); and (d) washing the alginate microbeads obtained from step (c).

[0093] More preferably, the method for preparing alginate microbeads according to the first embodiment of the present invention further comprises the following steps: (c) separating the alginate microbeads obtained from step (b); (d) washing the alginate microbeads obtained from step (c); (e) heating the alginate microbeads obtained from step (d); and (f) washing the alginate microbeads obtained from step (e).

[0094] It is preferred in step (c) that separating the alginate microbeads is carried out by sieving.

[0095] It is preferred in step (d) that washing the alginate microbeads is carried out with sterile water.

[0096] It is preferred in step (e) that the alginate microbeads are heated to 70°C to 90°C. It is more preferred in step (e) that the alginate microbeads are heated to 80°C. It is preferred that the heating in step (e) is carried out for 0.5 h to 2 h. It is more preferred that the heating in step (e) is carried out for 1 h.

[0097] It is preferred in step (f) that washing the alginate microbeads is carried out with sterile water. It is more preferred in step (f) that washing the alginate microbeads is carried out with sterile water, wherein the volume of sterile water that is about fourtimes the volume of alginate microbeads.

[0098] Hence, it is a preferred embodiment of the present invention that the method for preparing alginate microbeads further comprises the following steps: (c) separating the alginate microbeads obtained from step (b) by sieving; and (d) washing the alginate microbeads obtained from step (c) with sterile water.

[0099] Hence, it is a more preferred embodiment of the present invention that the method for preparing alginate microbeads further comprises the following steps: (c) separating the alginate microbeads obtained from step (b) by sieving; (d) washing the alginate microbeads obtained from step (c) with sterile water; (e) heating the alginate microbeads obtained from step (d) to 70°C to 90°C for 0.5 h to 2 h; and (f) washing the alginate microbeads obtained from step (e) with sterile water.

[0100] Hence, it is a most preferred embodiment of the present invention that the method for preparing alginate microbeads further comprises the following steps: (c) separating the alginate microbeads obtained from step (b) by sieving; (d) washing the alginate microbeads obtained from step (c) with sterile water; (e) heating the alginate microbeads obtained from step (d) to 80°C for 1 h; and (f) washing the alginate microbeads obtained from step (e) with sterile water, wherein the volume of sterile water that is about four times the volume of alginate microbeads.Alginate Microbeads

[0101] In a second embodiment of the present invention, alginate microbeads are provided, wherein the alginate microbeads are obtainable or obtained by the method for preparing alginate microbeads according to the first embodiment of the present invention.

[0102] Preferably, the alginate microbeads obtained in step (b) of the method for preparing alginate microbeads according to the first embodiment of the present invention have a diameter in the range from 10 pm to 700 pm. More preferably, the alginate microbeads have a diameter in the range from 20 pm to 300 pm. Most preferably, the alginate microbeads have a diameter in the range from 25 pm to 200 pm. The diameter of the alginate microbeads can be determined by any standardmethod known to those skilled in the art, such as dynamic light scattering (DLS).

[0103] The diameter of the alginate microbeads can be determined by any standard method known to those skilled in the art, such as dynamic light scattering (DLS).

[0104] The alginate microbeads according to the second embodiment of the present invention have a compact and spherical shape and a narrow particle size distribution, which distinguishes them from the prior art.Bioink Formulation

[0105] In a third embodiment of the present invention, a bioink formulation is provided comprising a crosslinkable hydrogel matrix, and alginate microbeads according to the second embodiment of the present invention.

[0106] Various biodegradable polymers can be used as crosslinkable hydrogel matrix in the bioink formulation according to the third embodiment of the present invention.

[0107] It is preferred in the third embodiment of the present invention that the crosslinkable hydrogel matrix is selected from the list consisting of alginates and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA) polymers, gelatin, gelatin methacrylate (GelMa), collagen, fibrin, hyaluronic acid, natural and synthetic polysaccharides such as agarose and chitosan, polyamino acids such as polypeptides, particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon-caprolactone, polyanhydrides, polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides), particularly poly(ethylene oxides), poly(allylamines) (PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly (vinylpyrrolidone), and copolymers of the above, including graft copolymers.

[0108] It is more preferred in the third embodiment of the present invention that the crosslinkable hydrogel matrix is gelatin methacrylate (GelMa).

[0109] It is preferred in the third embodiment of the present invention that the volume ratio of the crosslinkable hydrogel matrix in the bioink formulation is from1% (v / v) to 70% (v / v), preferably from 10% (v / v) to 50% (v / v), more preferably from 30% (v / v) to 40% (v / v), based on the total volume of the bioink formulation.Depending on the desired properties of the bioink formulation, a person skilled in the art is able to determine suitable volume ratios of the crosslinkable hydrogel matrix in the bioink formulation.

[0110] It is preferred that the crosslinkable hydrogel matrix component in the bioink formulation has a composition that enables good diffusion properties, which is important for the dissolution of the alginate microbeads. For example, if gelatin methacrylate (GelMa) is used as crosslinkable hydrogel matrix in the bioink formulation, it is preferred that the mass ratio of the polymer(s) in the crosslinkable hydrogel matrix component is from 0.1% (w / w) to 20% (w / w), preferably from 1% (w / w) to 10% (w / w), more preferably from 5% (w / w) to 7% (w / w), based on the total mass of the crosslinkable hydrogel matrix component. Depending on the crosslinkable hydrogel matrix component to be used in the bioink formulation, a person skilled in the art is able to determine suitable mass ratios of the polymer(s).

[0111] It is preferred in the third embodiment of the present invention that the volume ratio of the alginate microbeads in the bioink formulation is from 30% (v / v) to 99% (v / v), preferably from 50% (v / v) to 90% (v / v), more preferably from 60% (v / v) to 70% (v / v), based on the total volume of the bioink formulation. Depending on the desired properties of the bioink formulation, a person skilled in the art is able to determine suitable volume ratios of the alginate microbeads in the bioink formulation.

[0112] It is preferred in the third embodiment of the present invention that the bioink formulation further comprises one or more crosslinking agents. The crosslinking agent serves the purpose to crosslink the polymer(s) in the crosslinkable hydrogel matrix of the bioink formulation to form a crosslinked three-dimensional hydrogel.

[0113] Preferred crosslinking agents are selected from the list consisting of photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes mediating oxidative crosslinking, and agents mediating redox crosslinking.

[0114] Preferred photoinitiators are selected from the list consisting of acetophenone, p-anisil, benzil, benzoin, benzophenone, 2-benzoylbenzoic acid, 4,4’-bis(diethylamino)benzophenone, 4,4’ -bis(dimethylamino)benzophenone, benzoin methyl ether, benzoin isopropyl ether, benzoin isobutyl ether, benzoin ethyl ether, 4- benzoylbenzoic acid, 2, 2’-bis(2-chlorophenyl)-4, 4’, 5, 5 ’-tetraphenyl- 1,2’ -biimidazole, methyl 2-benzoylbenzoate, 2-(l,3-benzodioxol-5-yl)-4,6-bis(trichloromethyl)- 1,3,5- triazine, 2-benzyl-2-(dimethylamino)-4’ -morpholinobutyrophenone, (+)- camphorquinone, 2-chlorothioxanthone, 4,4’ -dichlorobenzophenone, 2,2- diethoxyacetophenone, 2,2-dimethoxy-2-phenylacetophenone, 2,4- diethylthioxanthen-9-one, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 1,4- dibenzoylbenzene, eosin Y, 2-ethylanthraquinone, 1 -hydroxycyclohexyl phenyl ketone, 2-hydroxy-2-methylpropiophenone, 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone, 2-isopropylthioxanthone, lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate, 2-methyl-4’-(methylthio)-2-morpholino- propiophenone, 2-isonitrosopropiophenone, 2-phenyl-2-(p-toluenesulfonyl- oxy)acetophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, tris(2,2’- bipyridyl)dichlororuthenium(II) hexahydrate, and a combination of tris(2,2’- bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate.

[0115] More preferred photoinitiators are selected from the list consisting of eosin Y, 2-hydroxy-4’(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl(2,4,6- trimethylbenzoyl)phosphinate, tris(2,2’ -bipyridyl)dichlororuthenium(II) hexahydrate, and a combination of tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate.

[0116] Preferred thermal initiators are selected from the list consisting of tert- amyl peroxybenzoate, 4,4-azobis(4-cyanovaleric acid), 1,1’- azobis(cyclohexanecarbonitrile), 2,2’-azobisisobutyronitrile (AIBN), 2,2’-azobis(2- methylpropionamidine)dihydrochloride, benzoyl peroxide, 2,2-bis(tert- butylperoxy)butane, l,l-bis(tert-butylperoxy)cyclohexane, 2,5-bis(tert-butylperoxy)- 2,5-dimethylhexane, 2,5-bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne, bis(l-(tert- butylperoxy)-l-methylethyl)benzene, l,l-bis(tert-butylperoxy)-3,3,5- trimethylcyclohexane, tert-butyl hydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butyl peroxybenzoate, tert-butylperoxy isopropyl carbonate, cumene hydroperoxide, cyclohexanone peroxide, dicumyl peroxide, lauroyl peroxide, 2,4- pentanedione peroxide, peracetic acid, ammonium persulfate, potassium persulfate,and sodium persulfate.

[0117] More preferred thermal initiators are selected from the list consisting of 4,4- azobis(4-cyanovaleric acid), 2,2'-azobisisobutyronitrile (AIBN), 2,2’-azobis(2- methylpropionamidine)dihydrochloride, benzoyl peroxide, tert-butyl peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0118] Preferred basic initiators are selected from the list consisting of hydroxides, amines and amides. More preferred basic initiators are selected from the list consisting of MOH, MOR, NH3 , RNH2, and R2NM, wherein M is an alkali metal, preferably Li, Na, K; and R is a carbyl moiety, preferably Ci-Ce alkyl. Most preferred basic initiators are selected from the list consisting of OPP, NH3 and (z‘Pr)2NLi (LDA).

[0119] Preferred oxidative initiators are selected from the list consisting of hydroxyl (HO") and peroxyl (ROO"), peroxynitrous acid / peroxynitrite (ONOOH / ONOO ), nitrogen dioxide (NO2*), nitrosoperoxycarbonate (ONOOCO2 ), carbonate (CO3" ), and lipid hydroperoxides(LOOH), wherein R is a carbyl moiety, preferably Ci-Ce alkyl, and L is a lipid moiety.

[0120] Preferred enzymes mediating oxidative crosslinking are selected from the peroxidase families, including horseradish peroxidase, myeloperoxidase, laccase, etc..

[0121] Preferred agents mediating redox crosslinking are selected from the list consisting of peroxides, hypochlorous acid, chloramines, hypobromous acid, bromamines, hypothiocyanous acid, nitroxyl, peroxynitrous acid, and other nitrating species.

[0122] Preferably, the bioink formulation comprises two crosslinking agents selected from the lists described above.

[0123] It is particularly preferred in the third embodiment of the present invention that the bioink formulation comprises lithium phenyl(2,4,6-trimethyl- benzoyl)pho sphinate .

[0124] In a preferred embodiment of the present invention, the concentration of the crosslinking agents in the bioink formulation is from 0.1 mM to 100 mM, preferably from 0.2 mM to 50 mM, more preferably from 0.5 mM to 40 mM, particularlypreferably from 1 mM to 30 mM, even particularly preferably from 2 mM to 25 mM, and most preferably from 5 mM to 15 mM.

[0125] It is preferred in the third embodiment of the present invention that the bioink formulation comprises one or more diluents. Such diluents typically act as solvents and / or dispersants diluting the crosslinkable hydrogel matrix and alginate microbeads in the bioink formulation. Preferred diluents are compounds that are liquid at room temperature. Preferred diluents are water, cell culture media such as, for example, DMEM, RPMI and MEM, or water-based buffer systems such as, for example, phosphate buffered saline (PBS), Hank’s buffer, Earle’s balanced salt solution, Tyrode buffer, HEPES buffer, etc..

[0126] The bioink formulations of the present invention are useful for different 3D bioprinting techniques including DLP-based bioprinting, droplet-based bioprinting, extrusion-based bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography -based bioprinting, magnetic bioprinting, and volumetric bioprinting.

[0127] In addition, the bioink formulations of the present invention are biocompatible with several human and non-human cell types and are therefore useful to print various tissue contracts.

[0128] In conclusion, the embodiments described herein overcome the disadvantages known from the prior art. In particular, the method for preparing alginate microbeads allows the preparation of alginate microbeads that are particularly suitable as porogens for hydrogel containing bioink formulations, where the porogens are compact and have a defined spherical and narrow particle size distribution similar to cell size, do not aggregate, can be easily separated after synthesis and washing, can be formulated in high concentration, and once dissolved, show advantageous diffusion properties and performance when formulated into bioink formulations, enabling the production of three-dimensional objects with satisfactory pore properties for efficient cell cultivation.

[0129] To produce alginate microbeads of similar density without using the second solution as described in the first embodiment of the present invention, one would have to work with higher concentrations and molecular weights of alginic acid, whichresults in slower processing due to the higher viscosity. Beyond that, one would require higher concentrated dissolution solutions to degrade the alginate microspheres.Method For Producing Porous Object

[0130] In a fourth embodiment of the present invention, a method for producing a porous three-dimensional object is provided, wherein the method comprises the following steps: (i) 3D bioprinting the bioink formulation according to the third embodiment of the present invention to obtain a three-dimensional object; (ii) crosslinking said three-dimensional object to obtain a crosslinked three-dimensional object; and (iii) treating said crosslinked three-dimensional object with a solution comprising a chelating agent to obtain a porous three-dimensional object.

[0131] Similar to thermoplastics commonly used in traditional 3D printing, bioinks can be extruded through printing nozzles or needles into filaments that retain their shape once applied. However, bioinks are sensitive to the processing conditions of conventional 3D printing. Compared to traditional 3D printing materials, bioinks typically require lower printing temperatures (typically 37 °C or less) and milder curing conditions to avoid compromising cytocompatibility and bioactivity.

[0132] Extrusion-based bioprinting and digital light processing (DLP)-based bioprinting are both 3D bioprinting techniques commonly used to produce biofabricated three-dimensional objects. In extrusion-based bioprinting, a bioink in solution or semi-solution form is loaded into a cartridge and a mechanical force, usually air pressure or a motor-drive piston or screw, pushes the bioink through a nozzle to form a filament that can be deposited layer by layer to that the desired three- dimensional object is produced. DLP-based bioprinting is also a layer-by-layer process. However, instead of extruding the material through a nozzle, a source of illumination treats each layer with a still image. This image is projected into a vat of light-sensitive liquid, triggering a chemical reaction that causes the liquid to cure in the illuminated area. The printed three-dimensional object is obtained by stacking these cured layers on a build platform.

[0133] Traditional bioprinting techniques involve depositing material layer-by-layer to create the end structure, but recently a new method called volumetric bioprintingwas reported (see: P.N. Bernal, P. Delrot, D. Loterie, Y. Li, J. Maida, C. Moser and R. Levato, Volumetric Bioprinting of Complex Living-Tissue Constructs within Seconds, Adv. Mater. 2019, 31, 1904209). Volumetric bioprinting occurs when a bioink is placed in a liquid cell and is selectively irradiated by an energy source. This method will actively polymerize the irradiated material and that will comprise the final structure. Manufacturing biomaterials using volumetric bioprinting of bioinks can greatly decrease the manufacturing time. In materials science, this is a breakthrough that allows personalized biomaterials to be quickly generated.

[0134] Unlike traditional 3D printing materials such as thermoplastics that are essentially fixed once they are printed, bioinks are a dynamic system because of their high-water content and often non-crystalline structure. The shape fidelity of the bioink after filament deposition must also be taken into account. Finally, the printing pressure and nozzle diameter must be taken into account to minimize the shear stresses placed on the bioink and on any cells within the bioink during the printing process. Too high shear forces may damage or lyse cells, adversely affecting cell viability.

[0135] It is preferred in the fourth embodiment of the present invention that 3D bioprinting in step (i) is performed using one of the following 3D bioprinting techniques: DLP-based bioprinting, droplet-based bioprinting, extrusion-based bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography-based bioprinting, magnetic bioprinting, and volumetric bioprinting. These 3D bioprinting techniques allow precise placement / positioning in spatially predefined locations within confined 3D structures.

[0136] It is preferred in the fourth embodiment of the present invention that the three-dimensional object is crosslinked in step (ii) by exposure to light. Such exposure to light is typically performed after the bioink formulation has been applied to a 3D bioprinting technique and has been deposited to form a three-dimensional object. The exposure to light for crosslinking the polymer(s) in the crosslinkable hydrogel matrix to form a three-dimensional object can be optionally carried out during or after the 3D bioprinting depending on the 3D bioprinting technique used such as, for example, extrusion-based bioprinting or DLP-based bioprinting or any one of the otherbioprinting techniques mentioned above.

[0137] It is preferred in the fourth embodiment of the present invention that the solution comprising a chelating agent in step (iii) comprises citrate or EDTA (ethylenediaminetetraacetate). It is more preferred in the fourth embodiment of the present invention that the solution in step (iii) comprises alkali metal citrate or alkali metal EDTA. Preferred alkali metal citrate is sodium citrate or potassium citrate. Preferred alkali metal EDTA is disodium EDTA or dipotassium EDTA. It is most preferred in the fourth embodiment of the present invention that the solution in step (iii) comprises sodium citrate, potassium citrate, disodium EDTA or dipotassium EDTA, preferably sodium citrate or potassium citrate, more preferably sodium citrate.

[0138] It is preferred in the fourth embodiment of the present invention that the concentration of the chelating agent is > 10 mM, preferably > 30 mM, more preferably > 40 mM, and most preferably > 50 mM. It is preferred in the fourth embodiment of the present invention that the concentration of the chelating agent is < 200 mM. Preferred concentrations of the chelating agent are in the range from 10 mM to 200 mM, preferably from 30 mM to 200 mM, more preferably from 40 mM to 200 mM, and most preferably from 50 mM to 200 mM. The chelating agent complexes the metal ions in the alginate microbeads. Hence, the concentrations of the chelating agent mentioned enable a clean and efficient pore formation via degradation of the alginate microbeads in the crosslinked hydrogel matrix while maintaining cell viability.

[0139] It is preferred in the fourth embodiment of the present invention that the solution in step (iii) further comprises an alkali metal halide, preferably potassium chloride or sodium chloride, more preferably sodium chloride. Preferably, the concentration of the alkali metal halide is in the range from 10 mM to 100 mM, preferably from 20 mM to 80 mM, more preferably from 40 mM to 70 mM, and most preferably from 50 mM to 60 mM. The alkali metal halide helps to maintain the osmotic pressure. Hence, the concentrations of the alkali metal halide mentioned enable an even and uniform pore formation while maintaining the structural integrity of the crosslinked hydrogel matrix while maintaining cell viability.

[0140] It is preferred in the fourth embodiment of the present invention that thetreatment of the crosslinked three-dimensional object with the solution in step (iii) is carried out at a temperature in the range from 20°C to 45°C, preferably from 30°C to 40°C, more preferably from 35°C to 40°C, and most preferably at a temperature of 37°C.

[0141] By treating the crosslinked three-dimensional object with the solution in step (iii) of the method for producing a porous three-dimensional object, the alginate microbeads are degraded, causing micropores in the crosslinked three-dimensional object. Complete degradation of the alginate microbeads provides increased porosity and surface area within the crosslinked bioink formulation, allowing for greater surface area for cell growth, better nutrient diffusion, easier matrix deposition, and increased cell-cell crosstalk.

[0142] However, in non-porous or insufficiently porous crosslinked bioink formulations, the cells are enclosed and take much longer to break down the highly cross-linked hydrogel matrix around them, causing them to slowly spread and elongate. Thick areas of highly cross-linked hydrogel matrices also inhibit the diffusion of essential nutrients and media, causing the cells in the innermost part of the bioink to die due to nutrient deficiency.Porous Object

[0143] The porous three-dimensional object, which is obtained by the method for producing a porous three-dimensional object according to the fourth embodiment of the present invention is preferably for use in tissue engineering, regenerative medicine, cell delivery, drug delivery, drug discovery, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics and / or other biomedical applications.

[0144] It is to be understood that a skilled person can freely combine the above- mentioned preferred, more preferred, even more preferred, particularly preferred and / or most preferred embodiments relating to the first embodiment, second embodiment, third embodiment, fourth embodiment and fifth embodiment of thepresent invention.Definitions

[0145] As used herein, the term “bioink formulation” means bioink formulations comprising at least one crosslinkable hydrogel matrix formed from a (biocompatible) polymer or (biocompatible) copolymer. Bioinks are formulations used to produce engineered artificial tissues or organs, disease models and organoids using 3D bioprinting. Bioinks must meet certain characteristics, including rheological, mechanical, biofunctional and biocompatible properties, among others. Such bioinks are considered as one of the most advanced tools for tissue engineering and regenerative medicine (TERM).

[0146] As used herein, the term “polymer” includes, but is not limited to, homopolymers, copolymers, for example, block, random, and alternating copolymers, terpolymers, quaterpolymers, etc., and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible configurational isomers of the molecule. These configurations include, but are not limited to isotactic, syndiotactic, and atactic symmetries. A polymer is a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units (i.e. repeating units) derived, actually or conceptually, from molecules of low relative mass (i.e. monomers).

[0147] As used herein, the term “monomer” refers to a molecule which can undergo polymerization thereby contributing constitutional units (repeating units) to the essential structure of a polymer or an oligomer.

[0148] As used herein, the term “copolymer” generally means any polymer derived from more than one species of monomer, wherein the polymer comprises more than one species of corresponding repeating unit. In one embodiment the copolymer is the reaction product of two or more species of monomer and thus comprises two or more species of corresponding repeating unit. It is preferred that the copolymer comprises two, three, four, five or six species of repeating unit. Copolymers that are obtained by copolymerization of three monomer species can also be referred to as terpolymers. Copolymers that are obtained by copolymerization of four monomer species can also be referred to as quaterpolymers. Copolymers may be present as block, random,and / or alternating copolymers.

[0149] As used herein, the term “block copolymer” refers to a copolymer, wherein adjacent blocks are constitutionally different, i.e. adjacent blocks comprise repeating units derived from different species of monomer or from the same species of monomer but with a different composition or sequence distribution of repeating units.

[0150] As used herein, the term “random copolymer” refers to a copolymer in which the probability of finding a given repeating unit at any given site in the chain is independent of the nature of the adjacent repeating units. Usually, in a random copolymer, the sequence distribution of repeating units follows Bemoullian statistics.

[0151] As used herein, the term “alternating copolymer” refers to a copolymer consisting of macromolecules comprising two species of repeating units in alternating sequence.

[0152] As used herein, the term “crosslinking agent” refers to a reagent that provides a reactive species capable of activating a reactive group to react with another reactive group in a crosslinking reaction. This enables the crosslinking of two or more polymer chains that carry reactive groups. Typical crosslinking agents in the context of the present invention are photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes mediating an oxidative crosslinking, and agents mediating a redox crosslinking. The crosslinking agents mentioned either directly provide a reactive species or must be activated for this purpose, for example by irradiation treatment, thermal treatment, etc..

[0153] In the context of the present invention, the term “diluent” as used herein, denotes one or more compounds serving as a solvent, suspending agent, carrier and / or matrix for the polymer and any other component comprised in the bioink formulation. Diluents are typically inert compounds that do not react with said polymers and said other components. Typical diluents are compounds that are liquid at room temperature.

[0154] As used herein, the term “alkyl” refers to a saturated hydrocarbon chain, such as, but not limited to, methyl, ethyl, propyl and butyl. The alkyl group may be straight-chain or branched-chain. For example, as used herein, propyl encompassesboth / -propyl and .so- ropyl; butyl encompasses n-butyl, sec -butyl, / .so-butyl and tertbutyl, and so forth. Divalent alkyl is also referred to as "alkylene" in the present application. Those skilled in the art are familiar with this nomenclature.

[0155] As used herein, the term “allyl” refers to a substituent with the structural formula R-CH2-CH=CH2, where R is any other group of atoms or H. It consists of a methylene bridge (-CH2-) attached to a vinyl group (-CH=CH2).

[0156] As used herein, the term “vinyl” refers to a substituent with the structural formula R-C(R’)=CH2, where R and R’ are independently from each other any other group of atoms or H. Preferably, it consists of a vinyl group (-CH=CH2).

[0157] The present invention is further illustrated by the examples following hereinafter which shall in no way be construed as limiting. A skilled person will acknowledge that various modifications, additions and alternations may be made to the invention without departing from the spirit and scope of the invention as defined in the appended claims.ExamplesMaterials Used

[0158] All chemicals and filters used in the following examples were obtained from Sigma- Aldrich. Ultrapure water was obtained from ultrapure lab water systems (Q- POD® Ultrapure Water Remote Dispenser).Example 1 - Preparation of Alginate Microbeads

[0159] An alginate microbead precursor solution was prepared by dissolving low endotoxin alginate (0.5% w / v) in ultrapure water (50 mL). The solution was filtered and sterilized using a 0.22 pm PES Steriflip filter.

[0160] A bath solution was prepared by mixing a certain volume ratio of ethanol (see Table 1 below) with ultrapure water at a total volume of 250 mL and dissolving calcium chloride (0.5 % w / v) in this mixture. The solution was filtered and sterilized using a 0.22 pm PES Stericup filter.

[0161] Sterile water was obtained by filtering ultrapure water through a 0.22 pmPES Stericup filter , which was then put into a sterile spray bottle.

[0162] Filtered air was obtained by attaching a 0.22 pm PVDF Millex GP filter to the tubing of an air pump.

[0163] All materials used were sterilely transferred into a biological safety cabinet by spraying / soaking the surfaces with 70% ethanol. Inside the safety cabinet, the sterile alginate microbead precursor solution was loaded into a 50 mL BD Syringe with a luer lock. A coaxial needle (Ramehart Instrument Co.) was custom made with stainless steel with an interior diameter of 18 G (0.838 mm) and an exterior diameter of 14 G (1.600 mm) with a needle length of 25 mm. A FEP tube (Dolomite Microfluidics) with an exterior diameter of 1 / 16” (1.59 mm) and a wall thickness of 0.25 mm was used to attach the 50 mL syringe and the interior channel of the coaxial needle. The exterior channel of the coaxial needle was attached to the luer lock end of the Millex GP filter attached to the tubing of the air pump. The coaxial needle was placed facing downward and orthogonal to a 8” (20.3 cm) pan containing the sterile bath solution placed on top of a shake plate (Benchmark). The coaxial needle was secured 15 cm above the surface of the sterile bath solution using a clamp centered to the pan. The 50 mL syringe was placed in a syringe pump (Harvard Apparatus) and secured.

[0164] The shake plate was turned on and set to 110 rpm. The air pump was set to a certain pressure (see Table 1 below). The syringe pump was set to a certain flow rate (see Table 1 below). The coaxial spraying was started and continued until the alginate microbead precursor solution (50 mL) was sprayed into the bath solution (250 mL). The obtained microbeads were separated using a mesh (25 pm) with the assistance of the sterile water spray bottle to dislodge the microbeads. The microbeads were transferred to an empty conical (50 mL) and sterile water was added in a volume ratio of 1:1 to soak the microbeads.

[0165] The water-soaked microbeads were heated at 80°C for 1 h to remove excess ethanol and allow excess calcium ions to diffuse out. A sterile 1 mL disposable pipette was used to periodically homogenize the microbeads during the heating process. The microbeads were separated using a centrifuge set at 3,000 rpm for 5 min. The supernatant was removed and replaced by water in a volume ratio of 1 : 1. Themixture was homogenized and left to equilibrate for at least 24 h.

[0166] Alginate microbeads were prepared according to the method described above, where the alcohol content in the bath solution was varied as shown in Table 1.

[0167] Table 1: Preparation of alginate microbeads according to Example 1 using different volume ratios of ethanol in the bath solution.Characterization of Alginate Microbeads

[0168] The particle size distribution of the alginate microbeads from Example 1-1, Example 1-2 and Example 1-3 was measured by dynamic light scattering (DLS, Wyatt Technologies, Mobius Mobility Laser Photometer) (see Fig. 1). As can be seen in Fig. 1, the alginate microbeads from Example 1-2 have the narrowest particle size distribution, followed by the microbeads from Example 1-3, whereas the microbeads from Example 1-1 have the broadest particle size distribution. In conclusion, the presence of alcohol in the bath solution during preparation of the alginate microbeads provides a narrower particle size distribution and a more compact and spherical shape of the microbeads.

[0169] In addition, images of the alginate microbeads were captured using a microscope (see Fig. 2a for microbeads from Example 1-1; Fig. 2b for microbeads from Example 1-2; and Fig. 2c for microbeads from Example 1-3). As can be seenfrom Figs. 1-2 to 1-3, the presence of alcohol in the bath solution during preparation of the alginate microbeads provides finer particles with a more clearly defined spherical geometry that are not grown together, but are discrete.

[0170] Fig. 3 shows the particle size distribution of the alginate microbeads from Example 1-2 and Example 1-4 measured by dynamic light scattering (DLS, Wyatt Technologies, Mobius Mobility Laser Photometer). As can be seen in Fig. 3, the alginate microbeads from Example 1-2 have a narrower particle size distribution than the microbeads from Example 1-4.

[0171] Fig. 4 shows the particle size distribution of the alginate microbeads from Example 1-2, Example 1-5 and Example 1-6 measured by dynamic light scattering (DLS, Wyatt Technologies, Mobius Mobility Laser Photometer). As can be seen in Fig. 4, the alginate microbeads from Example 1-2 have the narrowest particle size distribution, followed by the microbeads from Example 1-5, whereas the microbeads from Example 1-6 have the broadest particle size distribution.Example 2 - Preparation of Reference Alginate Microbeads

[0172] Alginate microbeads were prepared according to the manufacturing processes known from the following publications:

[0173] (1) Potential use of alginate beads as a chondrocyte delivery vehicle and stepwise dissolving porogen in a hydrogel scaffold for cartilage tissue engineering (C. Fan, D.-A. Wang, RSC Adv., 2015, 5, 80688-80697). See section 2.3 “Preparation of alginate beads”.

[0174] (2) One-step method to construct hydroxyapatite scaffolds with 3-D interconnected structure by a novel hydrogel bead porogen process (F. Shi, W. Zhi, Y. Liu, T. Zhou, J. Weng, Materials Letters, 2017, 203, 13-16). See section 2.1 “Manufacture of alginate hydrogel beads”.

[0175] (3) Assessments of injectable alginate particle-embedded fibrin hydrogels for soft tissue reconstruction (C.M. Hwang, B. Ay, D.L. Kaplan, J.P. Rubin, K.G. Marra, A. Atala, J.J. Yoo, S J. Lee, Biomed. Mater. 2013, 8, 014105, 1-9). See section2.1 “Fabrication and characterization of alginate-fibrin hydrogels).

[0176] Particles recreated from the method in (1) had a bead size 1.2-3.3 mm, which is not usable in a 3D bioprinting application and workflow. Particles recreated from the method in (2) had a bead size 1.2-3.3 mm, which is not usable in a 3D bioprinting application and workflow. Particles recreated from the method in (3) had a fragment size of average diameter of 93.7 pm with a PDI (poly dispersity index, measured by DLS) of 0.6, but were not identifiable as spherical or somewhat spherical in shape and cannot be used as a spherical porogen in a 3D bioprinting application and workflow.

[0177] Images of the alginate beads / fragments of prior art (1), (2), and (3) are shown in Fig. 5. The alginate microbeads according to the present invention were fabricated with an average diameter of 178.9 pm with a PDI of 0.3 (Example 1-2), and are very spherical as shown in Figs 2b and 2c. The alginate microbeads according to the present invention can be used as spherical porogens in 3D bioprinting application and workflows, whereas the prior art particles are not suitable for such application.Example 3 - Preparation of Bioink Formulation

[0178] DBPS (Dulbecco’s phosphate buffered saline) (10 mL) was heated to 60°C and lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) (25 mg) was added and dissolved by stirring. Then discrete GelMA (1g) was added and dissolved by stirring under protection from light. The pH was adjusted to 7.0-7.5 using 3M NaOH and IM HC1. The GelMA solution was sterilized using a 0.22 pm PES Stericup filter.

[0179] 5 mL of the homogenized and equilibrated alginate microbead slurry fromExample 1 was taken up and centrifugated at 3,000 rpm for 5 min. The supernatant was removed and 2.5 mL microbeads were collected to which 1 mL of the GelMA solution was added. After homogenization the final sterile bioink formulation was obtained.

[0180] A 70% confluent plate of Human bone marrow-derived mesenchymal stem cells (hMSCs) was cultured in a T182 flask. The media was removed and the cells were gently swirled in DPBS (20 ml) at 37°C. DPBS was removed and a warmedtrypsin solution (5 mL) was added. It was incubated for 5 min to detach the cells. Warmed media (10 mL) was added and the cell solution was gently aspirated to homogenize and separate out cell clusters. A sample of the cell solution was taken and the cell concentration and quantity for 4 million cells was determined using a cell counter. 4 million cells in solution were centrifuged at 150 G for 5 min to obtain a cell pellet. The supernatant was removed and 2 mL of the sterile bioink formulation prepared before was added, followed by homogenization to evenly distribute the cells in the bioink formulation.Example 4 - Preparation of Porous Three-Dimensional Object

[0181] A dissolution solution was prepared by dissolving NaCl (0.146 g) and sodium citrate (0.809 g) in 50 mL ultrapure water. The solution was filtered and sterilized using a 0.22 pm PES Stericup filter.

[0182] The bioink formulation prepared Example 2 was loaded into a 3 mL syringe with a 22 G needle tip and a piston and air bubbles were removed. The bioink in the syringe was cooled to 4°C for 30 min to initiate gelation. A bioprinter (Cellink BioX6) with a square grid model (6x6x1 mm with 20% infill) was loaded. The temperature of the printhead was set to 14°C, temperature of the printbed was set to 10°C, the print speed was set to 10 mm / s, and the extrusion pressure was set to 45 kPa. The bioprinter was calibrated to a 24 well plate.

[0183] The syringe loaded with the bioink formulation from Example 2 was attached to the temperature controlled printhead and bioprinting was conducted. The bioprinted three-dimensional objects were crosslinked by irradiation at 405 nm (OmniCure / Exelitas) at 10 mW / cm2for 30 s.

[0184] The dissolution solution (2 mL) was applied at 37°C to each well containing a bioprinted crosslinked three-dimensional object. It was incubated at 37 °C for 45 min. Thereafter, the dissolution solution was removed and replaced with warm media. Cell culturing in the porous three-dimensional objects took place for at least 14 days.Cellular Cytocompatibility

[0185] Cellular cytocompatibility of the crosslinked porous bioink and dissolutiontime of the alginate microbeads in the crosslinked bioink were evaluated by a live / dead staining assay and a metabolic assay. Measurements were taken after 1 day, 3 days, 7 days and 14 days of the cell culture and compared to the performance of a non-treated version of the porous bioink.

[0186] Live / dead staining was conducted by applying 1 mL of a staining solution (1 pL of 2 mM ethidium homodimer-I solution in DMSO and I p L of I mM Calcein- AM in DMSO per 1 mL of media) to the bioprinted structures with subsequent incubation for at 37 °C for 15 min in a dark incubator.

[0187] Live-stained cell bodies (normally stained green fluorescent) and dead- stained nuclei (normally stained red fluorescent, cell membrane impermeable) were captured using a fluorescent microscope and counted using ImageJ software. The viability was determined by the percentage of live cells divided by the total number of cells with n = 3 images of cells. 1 mL of a metabolic solution (0.1 mL of PrestoBlue® per 1 mL of media) was applied to the bioprinted structures and a control blank well and it was incubated at 37°C for 4 h in a dark incubator. 80 pL of each sample was transferred onto a 96 well plate and read for fluorescence at an excitation wavelength of 560 nm and an emission wavelength of 590 nm from the bottom of the plate. All readings were decreased by the value of the reading of the blank well. The data was averaged and normalized to the first reading (after 1 day) of the respective study.

[0188] Cell viability testing is shown in Fig. 6a and metabolic activity testing is shown in Fig. 6b.

[0189] Cell viability images of hMSCs cultured on bioprinted three-dimensional objects after 1 day, 3 days, 7 days and 14 days are shown in Fig. 7. Cells were observed to have a circular shape on day 1, which gradually changes to grow and elongate in 3 dimensions as cell growth proceeds.

[0190] Cell elongation and structure stability are shown in Fig. 8 (lOx magnification) and Fig. 9 (2x magnification) after 14 days hMSCs culturing. The structures in Figs. 8 and 9 were stained in the same manner as those portrayed in Fig. 7.

[0191] The effect of different application times of the dissolution solution on hMSCcultivated for 14 days on a bioprinted three-dimensional object according to Example 4 is shown in Fig. 10: A: No treatment; B: 20 min treatment; C: 45 min treatment; D: 45 min treatment with cell overlay (gray to light gray) (bright- field microscopy).

[0192] The examples provided herein are not intended in any way to limit the scope of the invention as set forth in the claims.

Claims

We claim:

1. Method for preparing alginate microbeads, wherein the method comprises the following steps:(a) providing a first solution comprising alginic acid or a salt thereof; and(b) processing said first solution through a coaxial spray nozzle into a second solution comprising a divalent metal halide and an alcohol to obtain alginate microbeads.

2. Method for preparing alginate microbeads according to claim 1, wherein the mass concentration of the alginic acid or salt thereof in the first solution is from 0.01% (w / v) to 5.0% (w / v), preferably from 0.05% (w / v) to 2.0% (w / v), more preferably from 0.1% (w / v) to 1.0% (w / v), and most preferably from 0.4% (w / v) to 0.6% (w / v), based on the total volume of the first solution.

3. Method for preparing alginate microbeads according to claim 1 or 2, wherein the weight average molecular weight (Mw) of the alginic acid or salt thereof in the first solution is in the range from 1,000 Da (1 kDa) to 10,000,000 Da (10 MDa), preferably from 2,000 Da (2 kDa) to 1,000,000 Da (1 MDa), more preferably from 3,000 Da (3 kDa) to 700,000 Da (700 kDa), most preferably from 4,000 Da (4 kDa) to 500,000 Da (500 kDa).

4. Method for preparing alginate microbeads according to any one of claims 1 to3, wherein the first solution further comprises one or more polymers that are miscible with the alginic acid or salt thereof, preferably wherein the polymer is selected from polysaccharides or polypeptides.

5. Method for preparing alginate microbeads according to any one of claims 1 to4, wherein the mass concentration of the divalent metal halide in the second solution is from 0.01% (w / v) to 5.0% (w / v), preferably from 0.05% (w / v) to 2.0% (w / v), more preferably from 0.1% (w / v) to 1.0% (w / v), and most preferably from 0.4% (w / v) to 0.6% (w / v), based on the total volume of the second solution.

6. Method for preparing alginate microbeads according to any one of claims 1 to5, wherein the divalent metal halide in the second solution is an alkaline earth metalhalide, preferably wherein the divalent metal halide in the second solution is an alkaline earth metal chloride, preferably selected from the list consisting of MgCh, CaCh, SrCh and BaCh, more preferably CaCh.

7. Method for preparing alginate microbeads according to any one of claims 1 to6, wherein the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 8 carbon atoms, a cyclic alkyl alcohol having 3 to 8 carbon atoms, or an aromatic alcohol having 6 to 10 carbon atoms, wherein the alcohol has 1 to 3 hydroxy groups, preferably wherein the alcohol in the second solution is a linear or branched chain alkyl alcohol having 1 to 4 carbon atoms, a cyclic alkyl alcohol having 3 to 6 carbon atoms, or an aromatic alcohol having 6 carbon atoms, wherein the alcohol has 1 hydroxy group, more preferably wherein the alcohol in the second solution is selected from the list consisting of methanol, ethanol, 1 -propanol, 2- propanol, 1 -butanol, 2-butanol, 2-methyl-propan-l-ol, and 2-methyl-propan-2-ol, cyclopropanol, cyclobutanol, cyclopentanol, cyclohexanol, and phenol, most preferably, wherein the alcohol in the second solution is ethanol.

8. Method for preparing alginate microbeads according to any one of claims 1 to7, wherein the second solution further comprises water, preferably wherein the second solution comprises ethanol and water.

9. Method for preparing alginate microbeads according to claim 8, wherein the volume ratio of alcohol in the second solution is from 0.01% (v / v) to 20% (v / v), preferably from 1% (v / v) to 10% (v / v), more preferably from 2% (v / v) to 8% (v / v), and most preferably from 4% (v / v) to 6% (v / v), based on the total volume of the second solution, preferably wherein the volume ratio of ethanol in the second solution is from 0.01% (v / v) to 20% (v / v), preferably from 1% (v / v) to 10% (v / v), more preferably from 2% (v / v) to 8% (v / v), and most preferably from 4% (v / v) to 6% (v / v), based on the total volume of the second solution.

10. Method for preparing alginate microbeads according to any one of claims 1 to9, wherein in step (b) the first solution is processed through the interior of the coaxial spray nozzle and air is processed through the exterior of the coaxial spray nozzle.

11. Method for preparing alginate microbeads according to any one of claims 1 to10, wherein the ratio of the volume of the first solution to the volume of the secondsolution is in the range from 1:1 to 1:10, preferably from 1:2 to 1:8, more preferably from 1:4 to 1:6, and most preferably 1:5.

12. Method for preparing alginate microbeads according to any one of claims 1 to 11, wherein the method further comprises the following steps:(c) separating the alginate microbeads obtained from step (b); and(d) washing the alginate microbeads obtained from step (c); preferably wherein the method further comprises the following steps:(e) heating the alginate microbeads obtained from step (d); and(f) washing the alginate microbeads obtained from step (e).

13. Alginate microbeads obtainable by the method for preparing alginate microbeads according to any one of claims 1 to 12.

14. Alginate microbeads according to claim 13, wherein the diameter of the alginate microbeads obtained in step (b) is in the range from 10 pm to 700 pm, preferably from 20 pm to 300 pm, more preferably from 25 pm to 200 pm.

15. Bioink formulation comprising a crosslinkable hydrogel matrix, and alginate microbeads according to claim 13 or 14.

16. Bioink formulation according to claim 15, wherein the crosslinkable hydrogel matrix is selected from the list consisting of alginates and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA) polymers, gelatin, gelatin methacrylate (GelMa), collagen, fibrin, hyaluronic acid, natural and synthetic polysaccharides such as agarose and chitosan, polyamino acids such as polypeptides, particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon-caprolactone, poly anhydrides, polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides), particularly poly(ethylene oxides), poly(allylamines) (PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly (vinylpyrrolidone), and copolymers of the above, including graft copolymers, preferably wherein the crosslinkable hydrogel matrix is gelatin methacrylate (GelMa).

17. Bioink formulation according to claim 15 or 16, wherein the volume ratio of the crosslinkable hydrogel matrix is from 1% (v / v) to 70% (v / v), preferably from 10% (v / v) to 50% (v / v), more preferably from 30% (v / v) to 40% (v / v), based on the total volume of the bioink formulation.

18. Bioink formulation according to one or more of claims 15 to 17, wherein the volume ratio of the alginate microbeads is from 30% (v / v) to 99% (v / v), preferably from 50% (v / v) to 90% (v / v), more preferably from 60% (v / v) to 70% (v / v), based on the total volume of the bioink formulation.

19. Bioink formulation according to one or more of claims 15 to 18, further comprising one or more crosslinking agents, preferably wherein the crosslinking agents are selected from the list consisting of photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes mediating oxidative crosslinking, and agents mediating redox crosslinking.

20. Method for producing a porous three-dimensional object, wherein the method comprises the following steps:(i) 3D bioprinting the bioink formulation according to any one of claims 15 to 19 to obtain a three-dimensional object;(ii) crosslinking said three-dimensional object to obtain a crosslinked three- dimensional object; and(iii) treating said crosslinked three-dimensional object with a solution comprising a chelating agent to obtain a porous three-dimensional object.

21. Method for producing a porous three-dimensional object according to claim 20, wherein the 3D bioprinting in step (i) is performed using one of the following 3D bioprinting techniques: DLP-based bioprinting, droplet-based bioprinting, extrusionbased bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography-based bioprinting, magnetic bioprinting, and volumetric bioprinting.

22. Method for producing a porous three-dimensional object according to claim 20 or 21, wherein the three-dimensional object is crosslinked in step (ii) by exposure to light.

23. Method for producing a porous three-dimensional object according to any one of claims 20 to 22, wherein the solution comprising a chelating agent in step (iii) comprises citrate or EDTA, preferably alkali metal citrate or alkali metal EDTA, more preferably sodium citrate, potassium citrate, disodium EDTA or dipotassium EDTA, even more preferably sodium citrate or potassium citrate, and most preferably sodium citrate.

24. Method for producing a porous three-dimensional object according to claim 23, wherein the concentration of the chelating agent is > 10 mM, preferably > 30 mM, more preferably > 40 mM, and most preferably > 50 mM.

25. Method for producing a porous three-dimensional object according to any one of claims 20 to 24, wherein the solution in step (iii) further comprises an alkali metal halide, preferably potassium chloride or sodium chloride, more preferably sodium chloride.

26. Method for producing a porous three-dimensional object according to claim 25, wherein the concentration of the alkali metal halide is in the range from 10 to 100 mM, preferably from 20 mM to 80 mM, more preferably from 40 mM to 70 mM, and most preferably from 50 mM to 60 mM.

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