METHOD FOR PRODUCING VASCULARIZED BIOLOGICAL TISSUE

Description

[0001] The invention relates to a method for producing biological tissue, in particular tissue from heart muscle cells, liver cells, kidney cells, nerve cells and / or pancreatic cells. Applications of the invention include, for example, biomedicine, especially in so-called tissue engineering.

[0002] It is well known that biological tissue, comprising a variety of differentiated biological cells with a common function, can be recreated for research or implantation purposes using biotechnological methods. For example, cardiac muscle cells can be derived from pluripotent stem cells through differentiation and multiplied by cultivation in an incubator. However, conventional bioreplicated biological tissues (tissue products), which consist of an accumulation of differentiated cells, have the following disadvantages, resulting in limitations on the application of tissue products and their associated costs.

[0003] First, natural biological tissue contains not only differentiated cells but also blood vessels and an extracellular matrix. Only in combination with these other tissue components does the accumulation of differentiated cells provide an adaptation to the properties of natural biological tissue. Therefore, replicas of biological tissue can currently only fulfill the functions of natural tissue to a limited extent. Furthermore, the size of conventional replicas of biological tissue is limited because cells within a cell culture may not be optimally supplied with nutrients and oxygen and may die. Particularly when cultivated in the adherent state, conventional replicas of biological tissue often have a layered structure that is not optimally adapted to the natural spatial form of tissue.When conventional tissue products are implanted into a biological organism, undesirable immune reactions or even rejection reactions can occur, as the tissue products are recognized as foreign materials due to their composition differing from that of natural tissues.

[0004] WO 2019 / 226710 A1 discloses a method for producing vascularized biological tissue, comprising the steps of: producing a network structure from a multitude of interconnected filaments of a support polymer; coating the network structure with a protein material; seeding the coated network structure with endothelial cells and tissue-forming biological cells; and dissolving the filaments of the network structure to form the vascularized tissue. The production of the network structure includes deposition of the filaments onto a support substrate coated with a degradable matrix material and subsequent detachment of the network structure from the support substrate.

[0005] The object of the invention is to provide an improved method for the production of biological tissue, avoiding the limitations of conventional techniques.

[0006] The process for producing biological tissue should be characterized in particular by the creation of a tissue whose properties are better adapted to the properties of natural tissue, which can be produced in larger dimensions, which can be produced with a freely selectable spatial shape, which is characterized by reduced immune reactions, which is more cost-effective to produce, and / or which enables new or expanded applications of tissue products.

[0007] This problem is solved by a method for producing biological tissue that has the features of claim 1. Preferred embodiments and applications of the invention are set forth in the dependent claims.

[0008] According to a general aspect of the invention, the above problem is solved by a method for producing vascularized biological tissue according to claim 1. The method comprises the steps of producing a network structure from a plurality of interconnected filaments of a support polymer, coating the network structure with a protein material, seeding the coated network structure with endothelial cells and with tissue-forming biological cells, and dissolving the filaments of the network structure, so that the vascularized tissue is formed.

[0009] According to the invention, the coated network structure provides a single- or multi-layered bulk substrate formed by the filaments of the supporting polymer. The filaments, which can also be described as threads, fibers, or elongated polymer segments, form, for example, a disordered or regular distribution in space, in which the filaments are in point or section contact with each other or spaced apart. This creates an inner surface of the network structure, which enables the coating of the network structure with the protein material and the subsequent seeding with vascular cells (e.g., endothelial cells) and tissue-forming biological cells, optionally with the addition of extracellular matrix (ECM). The filaments are preferably made of a polysaccharide, such as alginate or gelatin.The coating of the network structure specifically involves coating the filaments within the network structure with the protein material. The protein material advantageously promotes adherent cell growth on the filaments.

[0010] To populate the coated network structure with endothelial cells and tissue-forming biological cells, the network structure is incubated with a cell suspension so that the cells adhere to the filaments, and the adherent cells are then cultured, preferably with the addition of culture medium (nutrient medium). Preferably, the endothelial cells are introduced into the network structure first, followed by the formation of a layer (multiple layer, monolayer, or sub-monolayer) of endothelial cells and then the tissue-forming biological cells.

[0011] Endothelial cells are differentiated cells that form the inner lining of blood vessels in natural tissue. Preferably, endothelial cells from the organism whose tissue is to be replicated are used, e.g., from a mammal, particularly a human. The tissue-forming biological cells are differentiated cells of at least one cell type from which the tissue to be replicated is to be built. The differentiation of the tissue-forming biological cells preferably occurs before the network structure is populated.

[0012] The network structure is disintegrated after it has been populated with cells. Disintegration preferably comprises a chemical and / or thermal conversion of the filaments into a liquid state and a flushing of the converted material from the populated cell cluster. Upon disintegration, interconnected cavities remain within the cell cluster, the inner surfaces of which are formed by endothelial cells. The inventors have observed that these cavities are distributed geometrically like blood vessels in natural tissue and thus form blood vessels in the vascularized tissue, which preferably constitutes the finished tissue product.

[0013] The network structure is preferably dissolved after colonization is complete and the internal volume of the network structure is densely filled with cells. Alternatively, dissolution can be initiated when the internal volume of the network structure is only partially filled, thus facilitating the growth of further vessels.

[0014] Advantageously, the following advantages are achieved with the vascularized tissue produced according to the invention.

[0015] The properties of vascularized tissue are better adapted to the properties of the corresponding natural tissue than is the case with conventional tissue products. Vascularized tissue can even be indistinguishable from natural tissue. Accordingly, a reduction in immune reactions is also possible when the tissue product is implanted into a living organism.

[0016] Furthermore, the size limitations of conventional tissue products are overcome by allowing the differentiated cells in the tissues to be supplied via the vessels in the vascularized tissue. The vascularized tissue is not limited to a layered shape but can be produced with a freely selectable spatial form that is adapted to the shape of natural tissue or a desired implant.

[0017] The inventive method has a positive effect on the cost of the vascularized tissue, which can be produced with larger volumes and quantities with increased effectiveness than conventional tissue products.

[0018] The vascularized tissue produced according to the invention also offers new applications for tissue products, particularly in research and in implant medicine, e.g. in the creation of model tissues that do not differ from natural tissue or only differ negligibly.

[0019] The invention is applicable with various tissue-forming cells. According to a preferred application of the invention, the tissue-forming cells comprise cardiac muscle cells, and the vascularized tissue comprises a cardiac muscle tissue product. Alternatively, the tissue-forming cells comprise liver cells, kidney cells, nerve cells, or pancreatic cells, wherein the vascularized tissue is, accordingly, a liver tissue product, kidney tissue product, nerve tissue product, or pancreatic tissue product. The vascularized tissue can alternatively contain several cell types, such as cardiac muscle and nerve cells, or other cells in combination with nerve cells.

[0020] Advantageously, various embodiments of the invention are available in which the network structure is produced on a solid support substrate or without being bound to a solid support substrate.

[0021] According to a preferred embodiment of the invention, the fabrication of the network structure comprises deposition of the filaments onto a support substrate coated with a degradable matrix material, such as a polysaccharide, in particular dextran, and subsequent detachment of the network structure from the support substrate. Advantageously, the network structure is supported by the support substrate.

[0022] The deposition of the filaments comprises an arrangement of a multitude of pre-fabricated, e.g., extruded, filaments of the support polymer and / or a deposition of the support polymer using a 3D deposition process such that the filaments are built up on the support substrate. A 3D deposition process includes, for example, a 3D printing process, in particular 3D freeze-printing, in which the filaments of the support polymer are formed in the frozen state.

[0023] In a preparatory step immediately before the filaments are depositiond, the support substrate is coated with the matrix material. Preferably, the base of a vessel is used as the support substrate. This vessel then receives solutions or suspensions of the respective components during the subsequent steps of coating the filaments and seeding them with cells, thus forming an incubator. The vessel is, for example, a reaction vessel of a reaction plate, such as a microtiter plate.

[0024] The matrix material forms an insulating layer. Detaching the network structure from the support substrate involves dissolving the matrix material. The matrix material preferably differs in its chemical solubility from that of the filaments. Particularly preferably, the filaments and the matrix material are made of different polysaccharides. Advantageously, this allows the filaments to initially remain intact when the network structure detaches from the support substrate.

[0025] In this embodiment of the invention, a two-stage dissolution of the matrix material and the support polymer, particularly preferably made of polysaccharides, is preferably provided, in which first the network structure is detached from the support substrate and then, after colonization with the cells, the network structure is dissolved.

[0026] According to the invention, a network structure-cell composite is formed by coating the network structure with the protein material and seeding the coated network structure with endothelial cells and tissue-forming biological cells before the network structure is detached from the support substrate, wherein side sections of the filaments in the network structure-cell composite (i.e., a lateral surface of the network structure-cell composite) touch the support substrate, wherein the following steps are then provided: detachment of the network structure-cell composite from the support substrate, folding of the network structure-cell composite into a multilayer such that the side sections of the filaments in the network structure-cell composite at least partially touch, and fixation of the folded network structure-cell composite with subsequent unraveling of the network structure.

[0027] By coating the network structure with the protein material and seeding the coated network structure before detaching the network structure from the support substrate, the support substrate advantageously fulfills its supporting function until the cells are arranged in the cluster, thus preserving the arrangement of the filaments and, consequently, the subsequently formed blood vessels. Folding the network-cell cluster into a multilayer advantageously simplifies the formation of a three-dimensional tissue product.

[0028] Advantageously, various folding methods for the network-cell assembly are available, which can be implemented individually or in combination. According to one method, the network-cell assembly is suspended over an elongated holding element, such as a holding thread or rod, in such a way that the surfaces of the network-cell assembly, where the side sections of the filaments are exposed, touch each other. The network-cell assembly is folded over the elongated holding element so that two sections of a surface of the network-cell assembly, separated by the elongated holding element, lie against each other. Advantageously, the surface sections adhere to each other, forming a closed cell assembly from which the elongated holding element can be easily separated.

[0029] According to a second variant (the "origami" variant), the network-cell assembly is placed on a folding substrate such that the side sections of the filaments are exposed, and the folding substrate is deformed such that the surfaces of the network-cell assembly where the side sections of the filaments are exposed touch each other. Advantageously, the deformation of the folding substrate provides a force under which the sections of a lateral surface of the network-cell assembly touch, thereby supporting the adhesive connection of the parts of the network-cell assembly.

[0030] According to a third variant, the network structure-cell assembly is placed on a folding tool such that the side sections of the filaments are exposed, and the folding tool is actuated in such a way that the surfaces of the network structure-cell assembly where the side sections of the filaments are exposed touch each other. In this case as well, the folding tool advantageously exerts a force under which the cell assembly stabilizes itself in the folded state.

[0031] Particularly preferred is a network structure that is mirror-symmetrical with respect to a predetermined reference plane perpendicular to the extent of the network structure, with the folding of the network-cell assembly occurring along the reference line. Accordingly, after folding, filament segments with identical shapes touch, resulting in larger vessel diameters after the filaments dissolve.

[0032] According to an alternative embodiment of the invention, the coating of the network structure with the protein material and the seeding of the coated network structure with the endothelial cells and the tissue-forming biological cells are provided after the network structure has been detached from the support substrate. In this case, advantages can be gained from a simplified delivery of the protein material, the endothelial cells, and the tissue-forming biological cells into the network structure from multiple sides.

[0033] According to a further preferred embodiment of the invention, the fabrication of the network structure preferably comprises a 3D deposition of the filaments without bonding to a solid support substrate. The 3D deposition can be carried out, for example, in a highly viscous support fluid or a soft polymer, in particular a substrate block, or on a layer of prefabricated filaments.

[0034] The term "3D deposition" generally refers to processes by which a three-dimensional structure is produced from filaments by building up the support polymer, with or without substrate binding. The application of 3D deposition has the particular advantage that the distribution of the filaments, and thus the blood vessels, in vascularized tissue can be precisely controlled by spatially resolved monitoring of the support polymer deposition.

[0035] Advantageously, the 3D deposition of the filaments can include 3D freeze-printing of the support polymer, in which a solution of the support polymer is built up and cross-linked layer by layer at a temperature below the freezing point of the support polymer, so that the arrangement of the filaments with an internal volume is formed.

[0036] Alternatively, the 3D deposition of the filaments can include extruding the support polymer into a substrate using a cannula device. Particularly preferably, the cannula device comprises a coaxial cannula with which the support polymer and the endothelial cells are simultaneously introduced into the substrate.

[0037] Preferably, the support polymer comprises alginate, with the dissolution of the support polymer being carried out using alginate lyase. The use of alginate offers particular advantages due to its biocompatibility and its suitability for residue-free dissolution for the formation of vessels within the cell network. Alternatively or additionally, other uronic acid-based polysaccharides, such as galacturonic acid, or protein-based support polymers, such as gelatin, can be used. The dissolution of the support polymer is preferably carried out using alginate lyase, dextranase, and / or pectinase. Furthermore, a complexing agent, such as EDTA (ethylenediaminetetraacetic acid), can be used for dissolving the support structure.

[0038] A particular advantage of the invention is that the vascularized tissue can be produced in freely selectable sizes. Advantageously, a connection of at least two layers of vascularized tissue to form a tissue block can be provided.

[0039] Advantageously, the method according to the invention allows for modification of the cell assembly for the production of vascularized tissue, for example, to improve its supply. Preferably, according to one embodiment of the invention, at least one perfusion line is embedded in the cell assembly for the production of vascularized tissue, in the vascularized tissue itself, and / or in the tissue block. The perfusion line is made of a removable material and is arranged for the supply of a culture medium. For example, in a first culture phase of the vascularized tissue, the perfusion line allows for an increased supply of the culture medium to support cell proliferation in the vascularized tissue. The perfusion line can be dissolved in a later culture phase of the vascularized tissue or when the internal vessels are sufficiently developed.

[0040] Further details and advantages of the invention are described below with reference to the accompanying drawings. The drawings schematically show: Figures 1 to 5: Features of the method for producing vascularized tissue according to one embodiment of the invention, Figures 6 to 9: Features of the method for producing vascularized tissue according to further embodiments of the invention, Figure 10: the production of vascularized tissue with embedded perfusion lines, and Figure 11: the production of vascularized tissue in the form of a tissue block.

[0041] Features of embodiments of the invention are described below with exemplary reference to the production of vascularized cardiac muscle tissue. It is emphasized that the practical implementation of the invention is not limited to applications with cardiac muscle cells, but is also possible with other cell types, such as liver cells, kidney cells, nerve cells, and / or pancreatic cells. Details of the specific cell types used, such as endothelial cells and cardiac muscle cells, their provision (e.g., by differentiation from pluripotent stem cells), and their cultivation are not described, as these are known from the prior art.

[0042] The figures illustrate embodiments of the invention by means of enlarged sectional views, each showing a single-layer or double-layer section of a network structure or vascularized tissue in an area with fewer filaments or vessels. In practice, the invention is realized with extensive network structures that can comprise considerably more filaments or vessels and / or more layers or a spatial configuration of the network structure.

[0043] The production of vascularized biological tissue is carried out using techniques known from biotechnological tasks in the laboratory or industrial production per se, such as the use of incubators with facilities for supplying solutions and / or suspensions.

[0044] The Figures 1 to 5Illustrate a first embodiment of the inventive method for producing vascularized tissue, in which the production of a network structure and its seeding with endothelial cells and cardiac muscle cells on a solid carrier substrate take place.

[0045] The Figures 1A and 1B show the production of the network structure 10 from a multitude of interconnected filaments 11 of a support polymer, such as alginate, and the coating of the network structure 10 with a protein material 12, such as fibronectin or laminin.

[0046] The solid support substrate 20, preferably with a flat surface, is made, for example, of PMMA or glass. The support substrate 20 is formed, for example, by the base of a well in a microtiter plate (microwell) and coated with a matrix material 21, such as dextran. The thickness of the preferably closed layer of the matrix material 21 is, for example, 10 µm.

[0047] The filaments 11, which are shown in the figures in sectional view and can touch and / or cross each other outside the plane of the drawing, are according to Figure 1A For example, as a prefabricated network of precipitated alginate filaments or by 3D deposition on the support substrate 10, arranged randomly or with a regular distribution. Side sections 13 of the filaments 11 contact the coated support substrate 20, and gaps 14 remain between the filaments 11. The diameters of the alginate filaments 11 are preferably selected in the range of 50 µm to 500 µm or even above 500 µm. The gaps 14 have a size in the range of 100 µm to 500 µm.

[0048] The protein material 12 is according to Figure 1B e.g. from an ambient solution above the coated support substrate 20 or by targeted droplet deposition on the filaments 11 and the exposed areas of the matrix material 21.

[0049] Then, according to Figure 2 the formation of a network structure-cell complex 4. For this purpose, a first type of endothelial cells 2 is first adherently settled on the filaments 11 and the exposed areas of the matrix material 21 (see Figure 2A The cells are supplied from a suspension that covers the coated support substrate 20 and the filaments 11. Preferably, a closed layer (mono- or multi-layer) of endothelial cells 2 is formed. Optionally, a second type of cell 2A, such as smooth muscle cells, is additionally seeded onto the filaments 11 and / or the areas between the filaments 11 (see Figure 2BBy using two or more types of endothelial cells, the composition of vessel walls can advantageously be formed similarly to that in natural tissues. Optionally, the endothelial cells 2, 2A can be cultivated in an adherent state on the support substrate 20 by supplying a culture medium, whereby the endothelial cells 2, 2A proliferate.

[0050] The tissue-forming cells 3, such as cardiac muscle cells, are subsequently arranged on the endothelial cells 2, 2A (see Figure 2C The cardiac muscle cells are again supplied from a suspension that covers the coated carrier substrate 20 and the filaments 11 equipped with the endothelial cells 2, 2A. The tissue-forming cells 3 form a continuous layer that extends over the filaments 11 and the areas between them. The total thickness of the resulting network-cell assembly 4 is, for example, 5 mm.

[0051] The network structure-cell assembly 4 is detached from the support substrate 20. For this purpose, the matrix material 21 is dissolved by the addition of a solvent that dissolves the matrix material 21 but not the support polymer of the filaments, such as dextranase, so that the network structure-cell assembly 4 separates from the support substrate 20 (see Figure 3A ).

[0052] Preferably, the network structure 10 is shaped in a mirror-symmetric form with respect to a predetermined reference plane 6 perpendicular to the extent of the network structure 10, as shown in Figure 3A This is shown schematically. By folding the network structure-cell assembly 4 at the reference plane (see arrow in Figure 3A ) the filaments with the same positions relative to fold line 7 and the same sizes fit together (see Figure 3BFrom the filaments 11, which were initially laterally flattened due to contact with the support substrate 20, rounded filaments are formed whose cross-sectional shape is adapted to the cross-sectional shape of the vessels to be formed. In the folded state, contacting surfaces of the network structure-cell assembly 4 are connected to one another, thereby fixing the network structure-cell assembly 4.

[0053] Symmetry with respect to the reference plane 6 is not a mandatory feature of the invention. Even in non-symmetrical distributions, folding of the network structure-cell assembly 4 through self-organization processes results in a distribution of touching filaments 11, which are intended for subsequent vascularization.

[0054] Subsequently, the network structure 10 is dissolved, so that the vascularized tissue 1 is formed (see Figure 3CThe network structure 10 is dissolved, for example, by the supply of alginate lyase. As a result, the vascularized tissue 1 consists of tissue-forming cells 3, within which cavities remain after the filaments dissolve. These cavities are interconnected, forming vessels 8 within the vascularized tissue 1. The inner walls of the vessels 8 are formed by the endothelial cells 2 and 2A.

[0055] The Figures 4 and 5 show variants of the folding of the network structure-cell assembly 4 by suspension on an elongated holding element 30 ( Figure 4 ), using a folding substrate 40 ( Figure 5A ) or using a folding tool 50 ( Figure 5B ).

[0056] The elongated retaining element 30 is, for example, a retaining thread over which the network structure-cell assembly 4 is placed along the intersection line of the reference plane 6 with the network structure-cell assembly 4 (see Figure 4A). Surfaces of the network structure-cell assembly 4, where the side sections 13 of the filaments 11 are exposed, pivot on both sides of the holding element 30 under the influence of gravity until they touch and connect by forming cell-cell contacts (see Figure 4B ). Subsequently, the retaining element 30 can be removed and the filaments 11 dissolved to form the vascularized tissue 1.

[0057] The folding substrate 40 is a foldable support element with two flat wings, which can be folded between a spread-out, flat state (in Figure 5A (shown dashed) into a folded state (in Figure 5A(shown with solid lines) is pivotable. The folding substrate 40 consists, for example, of an elastically resilient, bistable material that can be pivoted between the two states by internal mechanical stresses. Two parts of a network structure-cell assembly 4 can be laid flat against each other and contacted using the folding substrate 40. The network structure-cell assembly 4 is placed on the folding substrate 40 in its unfolded state, with the intersection line of the reference plane 6 and the network structure-cell assembly 4 (see Figure 3) coincides with the pivot axis between the two wings of the folding substrate 40. The transition to the folded state (see arrows) deforms the folding substrate 40 such that surfaces of the network-cell assembly 4, where the side segments of the filaments are exposed, touch and connect with each other by forming cell-cell contacts. Subsequently, the folding substrate 40 can be removed and the filaments unwound to form the vascularized tissue.

[0058] The folding tool 50 according to Figure 5B has a similar function to the folding substrate 40, in that it transitions between a spread-out, flat state (in Figure 5B (shown dashed) into a folded state (in Figure 5B(shown with solid lines) is pivotable. Contrary to the internal tension of the folding substrate 40, the folding tool 50 is actuated at at least one actuator 51 to fold the network structure-cell assembly 4.

[0059] The Figures 6 to 8 Figure 1 illustrates a second embodiment of the inventive method for producing vascularized tissue, in which only the network structure is produced on a solid support substrate. Subsequently, the network structure is detached from the support substrate, coated with a protein material, and seeded with endothelial cells and cardiac muscle cells.

[0060] Figure 6A Figure 1 shows the fabrication of the network structure 10 from a multitude of interconnected filaments 11 of a support polymer, such as alginate, with mutual distances 14 on the support substrate 20 coated with a matrix material 21, as shown in Figure 21. Figure 1AThe network structure 10 is then detached from the support substrate 20 and coated on all sides with the protein material 12 ( Figure 6B and 7A ).

[0061] The network structure 10, detached from the carrier substrate 20 and coated with the protein material 12, is seeded with endothelial cells 2 ( Figure 7B For this purpose, the network structure 10 is incubated in an incubator with a suspension of the endothelial cells 2.

[0062] By incubating with a cardiac muscle cell suspension, cardiac muscle cells 3 are seeded onto the endothelial cells 2, which are arranged on and between the filaments 11, thereby forming the network-cell complex 4 ( Figure 8A ). Subsequently, the supporting polymer of the filaments 11 is dissolved, thereby forming the vascularized tissue 1 inside the network structure-cell complex 4.

[0063] Figure 9Figure 1 illustrates a third embodiment of the inventive method for producing vascularized tissue, in which the production of the network structure and the seeding with endothelial cells and cardiac muscle cells take place without binding to a solid support substrate, but rather within a substrate block, e.g., made of a soft, incompletely cross-linked alginate. The alginate is formulated to be so soft that cells can align themselves within it and are simultaneously separated from their surroundings. Subsequently, the network structure is removed from the substrate block, and the supporting polymer of the filaments is dissolved.

[0064] According to Figure 9AThe filaments 11 of the support polymer, such as alginate or galacturonic acid, are embedded using a cannula. Preferably, a coaxial cannula is used, through whose inner channel the support polymer, in particular weakly cross-linked alginate and optionally extracellular matrix (ECM) for cell polarization, and through whose outer channel the endothelial cells 2 together with ECM and optionally also an addition of ethylenediaminetetraacetic acid (EDTA) are introduced. The coaxial cannula is positioned in the substrate block 22 according to a predetermined program and moved, e.g., withdrawn along predetermined paths, while releasing the support polymer and the endothelial cells 2 with the additives. Subsequently, cardiac muscle cells 3 with additives of ECM and optionally also EDTA are introduced using a cannula and placed on the endothelial cells 2, so that the network structure-cell complex 4 is formed in the substrate block 22.

[0065] In the next step, the network structure-cell assembly 4 is separated from the substrate block 22 by dissolving the substrate block 22 with alginate lyase ( Figure 9B ). Subsequently, the filaments 11 are dissolved, e.g., with pectinase, thereby forming the free vessels 8 of the vascularized tissue 1 ( Figure 9C ).

[0066] Figure 10 illustrates a modification of the procedure according to Figure 9, wherein, in addition, four perfusion lines 60 are embedded in the network-cell assembly to provide the cells with culture medium, particularly during culture in the substrate block 22. The perfusion lines 60 serve specifically to supply the cells during the maturation and organization of the network-cell assembly 4, and they are preferably made of a degradable material, such as dextran. For example, dialysis tubing can be used as perfusion lines 60. When the network-cell assembly 4 is separated from the substrate block 22, the perfusion lines 60 can be dissolved, for example, with dextranase.

[0067] Several sections of the vascularized tissue 1, which is produced in particular by a method according to one of the described embodiments, can be joined to form a tissue block 9, as schematically shown in Figure 11The connection is shown. It occurs through mutual contact with the addition of genipin.

[0068] The features of the invention disclosed in the foregoing description, the drawings and the claims may be important for the realization of the invention in its various embodiments, both individually and in combination or sub-combination.

Claims

1. A method for manufacturing vascularized biological tissue (1), comprising the steps of - manufacturing a network structure (10) from a plurality of interconnected filaments (11) of a supporting polymer, wherein the manufacturing of the network structure (10) comprises a deposition of the filaments (11) on a carrier substrate (20) coated with a degradable matrix material (21) and a subsequent detachment of the network structure (10) from the carrier substrate (20), - coating the network structure (10) with a protein material (12), - colonization of the coated network structure (10) with endothelial cells (2, 2A) and with tissue-forming biological cells (3), and - dissolving the filaments (11) of the network structure (10) so that the vascularized tissue (1) is formed, wherein the method comprises the steps of: - forming a network structure-cell-composite (4) by coating the network structure (10) with the protein material (12) and colonizing the coated network structure (10) with the endothelial cells (2, 2A) and with the tissue-forming biological cells (3) before the network structure (10) is detached from the carrier substrate (20), wherein side portions (13) of the filaments (11) in the network structure-cell-composite (4) contact the carrier substrate (20), - detaching the network structure-cell-composite (4) from the carrier substrate (20), - folding the network structure-cell-composite (4) to form a multilayer (5) in such a way that the side sections (13) of the filaments (11) in the network structure-cell-composite (4) at least partially touch each other, and - fixing the folded network structure-cell-composite (4) with the subsequent dissolution of the network structure (10).

2. The method according to claim 1, wherein the folding of the network structure-cell-composite (4) comprises - suspending the network structure-cell-composite (4) over an elongate holding element (30) so that surfaces of the network structure-cell-composite (4) where the side portions (13) of the filaments (11) are exposed are in contact, - depositing the network structure-cell-composite (4) on a folding substrate (40) such that the side portions (13) of the filaments (11) are exposed, and deforming the folding substrate such that surfaces of the network structure-cell-composite (4) where the side portions (13) of the filaments (11) are exposed are in contact, or - depositing the network structure-cell-composite (4) on a folding tool (50) such that the side portions (13) of the filaments (11) are exposed, and actuating the folding tool such that surfaces of the network structure-cell-composite (4) at which the side portions (13) of the filaments (11) are exposed are in contact.

3. The method according to claim 1 or 2, wherein - the network structure (10) is shaped with mirror symmetry with respect to a predetermined reference plane (6) perpendicular to the extension of the network structure (10), and - the network structure-cell-composite (4) is folded along the reference plane (6).

4. The method according to claim 1, wherein - the manufacturing of the network structure (10) comprises a 3D deposition of the filaments (11) in such a way that the filaments (11) are built up on the solid carrier substrate (20).

5. The method according to claim 4, wherein - the 3D deposition of the filaments (11) comprises 3D freeze printing the supporting polymer.

6. The method according to any one of the preceding claims, having at least one of the features - the supporting polymer comprises alginate, another uronic acid-based polysaccharide, in particular galacturonic acid, and / or a protein-based supporting polymer, in particular gelatin, and - the supporting polymer is dissolved using alginate lyase, dextranase, pectinase and / or a complexing agent, in particular EDTA (ethylenediaminetetraacetic acid).

7. The method according to any one of the preceding claims, further comprising - joining at least two layers of vascularized tissue (1) to form a tissue block (9).

8. The method according to any one of the preceding claims, further comprising - an embedding of at least one perfusion pipe (60) in the vascularized tissue (1) or the tissue block (9), wherein the perfusion pipe (60) is made of a dissolvable material and is arranged for supplying a cultivation medium into the vascularized tissue (1) or the tissue block (9).

9. The method according to any one of the preceding claims, wherein - the tissue-forming biological cells (3) comprise heart muscle cells, liver cells, kidney cells, nerve cells and / or pancreatic cells.