Use of polymer-based microcarriers in the generation of tissue scaffolds with complex geometries

Abstract

The present invention relates generally to regenerative medicine, and specifically to tissue engineering. The present invention provides an improved manufacturing methodology that allows for the generation of complex three-dimensional tissue scaffolds. The scaffolds, when implanted in a subject, are essentially non-immunogenic, mimic the natural extracellular matrix, and can be repopulated and regulated by host cells, thereby becoming living tissue. In general, the present invention involves the generation of engineered tissue scaffolds, developed primarily by in vitro culture of a first population of cells that adhere to biocompatible hydrogel beads and secrete extracellular matrix.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates generally to regenerative medicine, and in particular to tissue engineering.The present invention provides an improved manufacturing methodology that allows the generation of complex three-dimensional tissue scaffolds.The scaffolds, when implanted in a subject, are essentially non-immunogenic, mimic natural extracellular matrix, and can be repopulated and regulated by host cells, thereby becoming living tissue. [Background technology]

[0002] 2. Background of the Invention Transplants from human donors are currently the primary source for replacing damaged or dysfunctional organs and tissues in the clinic. Although transplantation has become a successful practice worldwide, this approach is greatly limited by a severe shortage of donors, the complexity of the procedure, and the potential risk of transmission of infectious agents. Thus, the use of animal tissues or synthetic materials to generate grafts has emerged as a possible alternative to human tissues. However, the drawbacks of such procedures are many, one of the most important being the immunological barrier. Thus, there is a need for innovative approaches in regenerative medicine to increase the number of patients who can undergo clinical transplantation and have a higher success rate.

[0003] Tissue engineering is a continuously growing interdisciplinary field that applies principles of engineering and life sciences to generate functional replacement tissues for clinical use. The field seeks to develop biological substitutes to restore, maintain or improve the biological function of whole tissues or organs. Furthermore, such tissue engineered constructs can also serve as in vivo systems for the delivery of cell-secreted molecules or therapeutic agents, or as in vitro models of tissue function to test the effects of various treatments.

[0004] In general, tissue engineering involves the cultivation of cells on a support matrix (scaffold) to form new tissue of intended shape and function. Thus, the support matrix must be processable to form a scaffold of the desired shape for the tissue of interest, and the cells can be obtained from the recipient to reduce the chance of immune system rejection or other biocompatible sources such as established human cell lines. However, to date, successful implantation of such engineered tissues in the clinic has been limited, and innovative approaches are needed to improve the methodology and increase the transitional value of the product.

[0005] Tissue scaffolds, which serve as intermediates in the generation of new functional tissues, may present a risk of immunological and / or inflammatory responses when implanted. Therefore, methods to develop human decellularized extracellular matrix tissues as support matrices for tissue culture are currently being investigated with the goal of generating more biocompatible scaffolds. Human-derived extracellular matrix proteins are expected to be essentially non-immunogenic when implanted in humans and mimic native extracellular matrix composition to achieve the required functional tissue properties. Furthermore, human decellularized extracellular matrix tissues can be repopulated and regulated by host cells, thereby becoming living tissues. This makes human extracellular matrix proteins preferable to proteins derived from animal sources and synthetic degradable or non-degradable polymers as tissue scaffolds.

[0006] To date, such tissue engineered decellularized extracellular matrix scaffolds have been produced by culturing human cells on a tubular biodegradable polyglycolic acid (PGA) substrate that degrades as the cells produce extracellular matrix proteins (WO 2012 / 094611). The tubular biodegradable PGA constructs are produced by wrapping a biodegradable PGA sheet around a mandrel (a gas permeable silicone tube) such that opposing edges of the biodegradable PGA sheet meet at an interface. At the interface, PGA fibers are pulled from each opposing edge of the sheet and intertwine to form a seam that creates a tubular construct of uniform density. As extracellular matrix proteins have been secreted onto the tubular PGA construct, the construct is decellularized, leaving the construct substantially acellular, extracellular matrix construct.

[0007] This approach has several drawbacks. First, the procedure using tubular PGA scaffolds is primarily tailored to form grafts of relatively simple geometric shapes, such as vascular grafts, limiting the potential use of this technique for replacement of other more complex dysfunctional tissues or organs. Second, the methodology requires complex and time-consuming manual procedures to proceed from PGA sheets to vascular grafts. The complex procedures often make it difficult to avoid product-to-product variability, and the seams connecting the sheets represent weak links in the construct. Third, the procedure relies on treating the tubular PGA construct to increase the rate of degradation and treating the cultured cells to degrade and remove the supporting PGA matrix. The residual fraction of the PGA matrix may exhibit immunogenic and / or inflammatory responses when implanted. Fourth, the use of macroporous PGA membranes limits the thickness of the final decellularized extracellular matrix construct. This again limits its potential use in the replacement of various dysfunctional tissues and organs. As a result, there is a need in the art to develop techniques to improve tissue scaffolds for use in tissue engineering.

[0008] Although WO 0214480 discloses that such decellularized tissue engineered constructs produced in WO 2012 / 09461 can be configured to assume any desired three-dimensional shape, it does not disclose how such three-dimensional shapes can be created from the original biodegradable PGA sheet other than in a tubular shape. Thus, there is a need in the art to enable the manufacture of complex three-dimensional tissue scaffolds for use in tissue engineering.

[0009] WO 2020 / 045162 discloses a drug delivery system (DDS) in the form of a sustained release composition comprising a drug encapsulated in alginate microbeads with a diameter of about 80 μm. To investigate how the DDS affects cell viability, the microbeads are mixed with cells and then subjected to an alginate lyase treatment to dissolve the microbeads. However, alginate without grafted cell adhesion ligands does not support cell attachment. WO 2017 / 223529 discloses a method for producing a tissue matrix scaffold (TMS) that can be used as a three-dimensional cell culture environment for culturing cells or tissues. TMS hydrogels are produced by extracting extracellular matrix (ECM) from homogenized animal tissues and concentrating the ECM extract. The concentrated ECM extract can be dehydrated to obtain porous TMS. A similar methodology is also described in WO 2018 / 213375. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2012 / 094611 [Patent Document 2] WO 02 / 14480 [Patent Document 3] International Publication No. 2012 / 09461 [Patent Document 4] International Publication No. 2020 / 045162 [Patent Document 5] International Publication No. 2017 / 223529 [Patent Document 6] International Publication No. 2018 / 213375 Summary of the Invention

[0011] Summary of the Invention The present inventors have addressed the above needs by providing an improved manufacturing methodology that allows for the production of complex three-dimensional tissue scaffolds that, when implanted in humans, are essentially non-immunogenic, mimic the natural extracellular matrix, and can be repopulated and regulated by host cells, thereby becoming living tissue.

[0012] A first aspect of the present invention is a method of producing a tissue scaffold, comprising: a) providing a first population of cells and biocompatible hydrogel beads; b) combining the first population of cells with biocompatible hydrogel beads to obtain a first cell-bead mixture; c) culturing the first cell-bead mixture to produce an extracellular matrix, thereby obtaining a first tissue scaffold; d) optionally subjecting the first tissue scaffold to mechanical conditioning, thereby obtaining a mechanically conditioned first tissue scaffold, - the first cell population is anchorage dependent cells capable of producing and secreting extracellular matrix, - the biocompatible hydrogel beads support the attachment of a first cell population; and - combining the first cell population and the biocompatible hydrogel beads of step b) in a mold to obtain a first cell-bead mixture; or Incubating the first cell-bead mixture of step b) to allow for the formation of bead-associated cells and then transferring to a mold; or incubating the first cell population of step b) and the biocompatible hydrogel beads to allow for the formation of bead-associated cells, and then transferring the bead-associated cells to a mold to obtain a first cell-bead mixture; The present invention relates to a method comprising the steps of:

[0013] In one embodiment, the first cell population of step b) and the biocompatible hydrogel beads are combined in a mold to obtain a first cell-bead mixture, or the first cell-bead mixture of step b) is incubated to allow for the formation of bead-associated cells and then transferred to a mold.

[0014] In another embodiment, the first group of cells of step b) and the biocompatible hydrogel beads are combined in a mold to obtain the first cell-bead mixture, or the first group of cells of step b) and the biocompatible hydrogel beads are incubated to allow for the formation of bead-associated cells, and then the bead-associated cells are transferred to a mold to obtain the first cell-bead mixture.

[0015] In one embodiment, the first cell-bead mixture of step c) is cultured at least until the first tissue scaffold is removed from the mold and still able to maintain its shape, i.e., until the first tissue scaffold is self-supporting. Example 2 provides a methodology that can be used to determine when a tissue scaffold is self-supporting.

[0016] Further steps In one embodiment, step d) is mandatory.

[0017] In one embodiment, the first tissue scaffold or the mechanically conditioned first tissue scaffold is subjected to decellularization, thereby obtaining a second tissue scaffold.

[0018] In one embodiment, the second tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned second tissue scaffold.

[0019] In one embodiment, the second tissue scaffold or the mechanically conditioned second tissue scaffold is incubated in the presence of a second cell population, thereby obtaining a second tissue scaffold regenerated with the second cell population.

[0020] In one embodiment, - subjecting the first tissue scaffold or said mechanically conditioned first tissue scaffold to a means for dissolving or degrading said biocompatible hydrogel beads, thereby obtaining a third tissue scaffold; or The biocompatible hydrogel beads are autodegradable or autodissolving, and the first tissue scaffold or the mechanically conditioned first tissue scaffold is cultured until the biocompatible hydrogel beads autodissolve or autodegrade, thereby obtaining a third tissue scaffold.

[0021] In one embodiment, the third tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned third tissue scaffold.

[0022] In one embodiment, the third tissue scaffold or the mechanically conditioned third tissue scaffold is incubated in the presence of the second cell population, thereby obtaining a third tissue scaffold regenerated with the second cell population.

[0023] In one embodiment, the third tissue scaffold or the mechanically conditioned third tissue scaffold is subjected to decellularization, thereby obtaining a fourth tissue scaffold.

[0024] In one embodiment, the fourth tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned fourth tissue scaffold.

[0025] In one embodiment, the fourth tissue scaffold or the mechanically conditioned fourth tissue scaffold is incubated in the presence of the second cell population, thereby obtaining a fourth tissue scaffold regenerated with the second cell population.

[0026] In a first preferred embodiment according to the first aspect of the invention, the method comprises the steps of: e) subjecting the first tissue scaffold or the mechanically conditioned first tissue scaffold to a means for dissolving or degrading the biocompatible hydrogel beads, thereby obtaining a third tissue scaffold; f) optionally subjecting the third tissue scaffold to mechanical conditioning, thereby obtaining a mechanically conditioned third tissue scaffold; g) subjecting the third tissue scaffold or the mechanically conditioned third tissue scaffold to decellularization, thereby obtaining a fourth tissue scaffold; h) optionally subjecting the fourth tissue scaffold to mechanical conditioning, thereby obtaining a mechanically conditioned fourth tissue scaffold; i) optionally incubating the fourth tissue scaffold or the mechanically conditioned fourth tissue scaffold in the presence of a second cell population, thereby obtaining a fourth tissue scaffold regenerated with the second cell population; Further includes:

[0027] In a preferred embodiment, at least one of steps f), h) and i) is mandatory. In a more preferred embodiment, at least two of steps f), h) and i) are mandatory. In an even more preferred embodiment, steps f), h) and i) are mandatory. In a most preferred embodiment, steps f) and i) are mandatory.

[0028] In one embodiment, step f) is mandatory.

[0029] In one embodiment, step h) is mandatory.

[0030] In one embodiment, step i) is mandatory.

[0031] In one embodiment, the third tissue scaffold comprises an extracellular matrix embedding the first cell population and hollow bodies previously occupied by biocompatible hydrogel beads.

[0032] In a preferred embodiment, the third tissue scaffold is cultured to i) generate extracellular matrix in the hollow bodies previously occupied by the biocompatible hydrogel beads, and / or ii) promote the migration of the first cell population into the hollow bodies, and / or iii) allow the contractile forces of the first cell population to close the hollow bodies. It is preferred that the hollow bodies are filled with extracellular matrix and / or the first cell population. It is particularly preferred that the hollow bodies are filled with extracellular matrix and / or the first cell population before step f) and / or step g).

[0033] In a second preferred embodiment according to the first aspect of the invention, the method comprises the steps of: e) the biocompatible hydrogel beads are autodegradable or autodissolving, and the first tissue scaffold or the mechanically conditioned first tissue scaffold is cultured until the biocompatible hydrogel beads autodissolve or autodegrade, thereby obtaining a third tissue scaffold; f) optionally subjecting the third tissue scaffold to mechanical conditioning, thereby obtaining a mechanically conditioned third tissue scaffold; g) subjecting the third tissue scaffold or the mechanically conditioned third tissue scaffold to decellularization, thereby obtaining a fourth tissue scaffold; h) optionally subjecting the fourth tissue scaffold to mechanical conditioning, thereby obtaining a mechanically conditioned fourth tissue scaffold; i) optionally incubating the fourth tissue scaffold or the mechanically conditioned fourth tissue scaffold in the presence of a second cell population, thereby obtaining a fourth tissue scaffold regenerated with the second cell population; Further includes.

[0034] In a preferred embodiment, at least one of steps f), h) and i) is mandatory. In a more preferred embodiment, at least two of steps f), h) and i) are mandatory. In an even more preferred embodiment, steps f), h) and i) are mandatory. In a most preferred embodiment, steps f) and i) are mandatory.

[0035] In one embodiment, step f) is mandatory.

[0036] In one embodiment, step h) is mandatory.

[0037] In one embodiment, step i) is mandatory.

[0038] In one embodiment, the third tissue scaffold comprises an extracellular matrix embedding the first cell population and hollow bodies previously occupied by biocompatible hydrogel beads.

[0039] In a preferred embodiment, the third tissue scaffold is cultured to i) generate extracellular matrix in the hollow bodies previously occupied by the biocompatible hydrogel beads, and / or ii) promote the migration of the first cell population into the hollow bodies, and / or iii) allow the contractile forces of the first cell population to close the hollow bodies. It is preferred that the hollow bodies are filled with extracellular matrix and / or the first cell population. It is particularly preferred that the hollow bodies are filled with extracellular matrix and / or the first cell population before step f) and / or step g).

[0040] cell In one embodiment, the first population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0041] The second population of cells may be the same as or different from the first population of cells.

[0042] In one embodiment, the second population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0043] In one embodiment, the second population of cells is different from the first population of cells and is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0044] The second population of cells is preferably anchorage-dependent cells.

[0045] In one embodiment according to the invention, the second population of cells are progenitor cells or adult stem cells (ASCs), which are preferably capable of differentiating and developing into a desired tissue or organ within the tissue scaffold in question.

[0046] Biocompatible Hydrogel Beads According to a first embodiment, a first population of cells is combined with biocompatible hydrogel beads to obtain a first cell-bead mixture (see step (b) of the method above).

[0047] In one embodiment according to the present invention, the biocompatible hydrogel beads are degradable or dissolvable.

[0048] In one embodiment according to the present invention, the biocompatible hydrogel beads are autodegradable or autodissolving.

[0049] In one embodiment according to the present invention, the biocompatible hydrogel beads have one or more cell adhesion ligands on their outer surface. The one or more cell adhesion ligands can be, for example, i) peptides containing cell adhesive amino acid sequences such as RGD, YIGSR, GFOGER or PHSRN, or ii) charged polymers such as poly-L-Lys. The one or more cell adhesion ligands are preferably incorporated into the biocompatible hydrogel beads via covalent tethering, electrostatic interactions, and / or interpenetrating networks.

[0050] In one embodiment, the one or more cell adhesion ligands are a peptide comprising the amino acid sequence RGD, for example, a peptide comprising the amino acid sequence GRGDSP.

[0051] In one embodiment, the biocompatible hydrogel beads comprise alginate, fibrin, gelatin, agarose, modified or unmodified polyethylene glycol, carrageenan, pectin, or any combination thereof.

[0052] In one embodiment, the biocompatible hydrogel beads are made from alginate, fibrin, gelatin, agarose, modified or unmodified polyethylene glycol, carrageenan, pectin, or any combination thereof.

[0053] In a preferred embodiment, the biocompatible hydrogel beads are biocompatible alginate beads. The biocompatible alginate beads are preferably produced by competitive ligand exchange crosslinking, for example as described in Example 1 of WO2017153947.

[0054] In another preferred embodiment, the biocompatible hydrogel beads are biocompatible hydrogel beads comprising alginate. The biocompatible hydrogel beads comprising alginate are preferably produced by competitive ligand exchange crosslinking, for example as described in Example 3 of WO2017153947.

[0055] In one embodiment, the biocompatible hydrogel beads are biocompatible alginate beads, and the alginate is an RGD-conjugated alginate or a GRGDSP-conjugated alginate.

[0056] In one embodiment, the biocompatible hydrogel beads are biocompatible alginate beads and the means for dissolving or degrading the biocompatible hydrogel alginate beads is selected from the group consisting of alginate lyase treatment, treatment with a divalent cation chelator, and any combination thereof.

[0057] In one embodiment, the biocompatible hydrogel beads are biocompatible hydrogel microbeads, preferably with an average size of less than 500 μm in their largest dimension, more preferably in the range of 10-200 μm, such as an average size of 10-150 μm or 30-150 μm, even more preferably in the range of 30 μm-90 μm, such as an average size of 30 μm-80 μm, and most preferably in the range of 50-80 μm in their largest dimension. The size of the beads is measured at their swelling equilibrium size in a liquid environment of physiological ion concentrations, such as cell culture medium (see Example 1, section 1.2).

[0058] In one embodiment, at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, such as a size in the range of 10-150 μm in their largest dimension, a size in the range of 30-150 μm in their largest dimension, or a size in the range of 30-90 μm in their largest dimension. Thus, one skilled in the art will understand that if there are 100 000 biocompatible hydrogel beads and 60% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, then in reality there are 60 000 biocompatible hydrogel beads having a size in the range of 10-200 μm in their largest dimension.

[0059] Mold In one embodiment, the mold is made of a bioinert material.

[0060] In one embodiment, the mold has one or more of the following characteristics: -Allows control of geometric shapes; - prevent cell attachment or cell invasion; and -Allows exchange of nutrients and gases between the exterior and interior of the mold.

[0061] In one embodiment, the mold is designed to prevent cell attachment or infiltration.

[0062] In one embodiment, the mold allows for the exchange of nutrients and / or gases between the exterior and interior of the mold.

[0063] In one embodiment, the mold is fully or partially immersed in the cell culture medium.

[0064] In a more preferred embodiment, the mold is designed to allow nutrient and gas exchange between the exterior and interior of the mold, preventing cell attachment or infiltration, and is fully or partially immersed in cell culture medium.

[0065] In one embodiment, the mold and the first tissue scaffold have substantially the same shape as the first tissue scaffold.

[0066] In one embodiment, the mold has the shape of a circular well.

[0067] In one embodiment, the mold comprises a container and at least one element disposed inside the container. The container and the at least one element can have any geometric shape. The space in the container that is not occupied by the at least one element is suitable for culturing the first cell-bead mixture to generate an extracellular matrix, thereby obtaining a first tissue scaffold. The at least one element may be included in the container before the first cell group and the biocompatible hydrogel beads are combined in the mold, or the first cell group and the biocompatible hydrogel beads are combined inside the container, and then the at least one element is included inside the container. In the latter case, the at least one element should be included in the container before culturing to generate an extracellular matrix (step c). The at least one element may be a prefabricated element attached to the container, or may be generated by 3D printing on the surface of the container. Those skilled in the art are familiar with suitable 3D printing techniques, including suspended layer additive manufacturing (Adv. Funct. Mater. 2019, 29, 1904845; Lee et al., Science 365, 482-487, 2019).

[0068] A second aspect of the invention relates to a tissue scaffold obtainable by the method according to the first aspect of the invention.

[0069] In one embodiment according to the second aspect of the invention, the tissue scaffold is a first tissue scaffold, a mechanically conditioned first tissue scaffold, a second tissue scaffold, a mechanically conditioned second tissue scaffold, or a second tissue scaffold regenerated with a second population of cells.

[0070] In another embodiment according to the second aspect of the invention, the tissue scaffold is a third tissue scaffold, a mechanically conditioned third tissue scaffold, a fourth tissue scaffold, a mechanically conditioned fourth tissue scaffold, or a fourth tissue scaffold regenerated with a second population of cells.

[0071] In preferred embodiments according to the second aspect of the invention, the tissue scaffold is a fourth tissue scaffold, a mechanically conditioned fourth tissue scaffold, or a fourth tissue scaffold regenerated with a second population of cells.

[0072] In one embodiment according to the second aspect of the invention, the tissue scaffold is suitable for regenerating a functional organ by introducing progenitor cells or adult stem cells (ASCs) and allowing them to differentiate within the tissue scaffold and develop into a desired tissue.

[0073] In one embodiment according to the second aspect of the invention, the tissue scaffold is suitable for being implanted into a subject, such as a human subject.

[0074] A third aspect of the present invention is a first population of cells; and - biocompatible hydrogel beads that support the attachment of a first cell population; - the extracellular matrix produced by the first group of cells; Including, - the first cell population is anchorage-dependent cells capable of producing and secreting extracellular matrix; - a first population of cells is attached to an exterior surface of the biocompatible hydrogel beads; and - a first tissue scaffold, comprising a first cell population and biocompatible hydrogel beads embedded in an extracellular matrix.

[0075] In one embodiment according to the third aspect of the invention, adjacent biocompatible hydrogel beads are connected to each other by a network of a first population of cells and extracellular matrix.

[0076] In one embodiment according to the third aspect of the invention, the first tissue scaffold has a tubular shape, such as the shape of a circular tube.

[0077] In one embodiment according to the third aspect of the invention, the first tissue scaffold is obtained by the method according to the first aspect of the invention.

[0078] In one embodiment according to the third aspect of the invention, the first tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned first tissue scaffold.

[0079] In one embodiment according to the third aspect of the invention, the first tissue scaffold or the mechanically conditioned first tissue scaffold is repopulated with a second population of cells.

[0080] In one embodiment according to the third aspect of the invention, the first tissue scaffold or the mechanically conditioned first tissue scaffold may be used to regenerate a functional organ by repopulating the scaffold with a second population of cells that can differentiate and develop into a desired tissue within the first tissue scaffold.

[0081] In one embodiment according to the third aspect of the invention, the first tissue scaffold or the mechanically conditioned first tissue scaffold is suitable for implantation into a subject, such as a human subject.

[0082] cell In one embodiment according to the third aspect of the invention, the first population of cells are anchorage-dependent cells, more preferably human anchorage-dependent cells.

[0083] In one embodiment according to the third aspect of the invention, the first population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably human primary mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue derived mesenchymal stem cells, or any combination thereof.

[0084] The second population of cells may be the same as or different from the first population of cells.

[0085] In one embodiment, the second population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0086] In one embodiment, the second population of cells is different from the first population of cells and is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0087] The second population of cells is preferably anchorage-dependent cells.

[0088] In one embodiment according to the invention, the second population of cells are progenitor cells or adult stem cells (ASCs), which are preferably capable of differentiating and developing into the desired tissue or organ within the tissue scaffold in question.

[0089] Biocompatible Hydrogel Beads In one embodiment according to the third aspect of the present invention, the biocompatible hydrogel beads have one or more cell adhesion ligands on their outer surface, and the first population of cells are attached to the outer surface of the biocompatible hydrogel beads via interaction with the one or more cell adhesion ligands.

[0090] In one embodiment according to the third aspect of the invention, the biocompatible hydrogel beads are degradable or dissolvable.

[0091] In one embodiment according to the third aspect of the invention, the biocompatible hydrogel beads are autodegradable or autodissolving.

[0092] In one embodiment according to the third aspect of the invention, the biocompatible hydrogel beads have one or more cell adhesion ligands on their outer surface. The one or more cell adhesion ligands can be, for example, i) peptides containing cell adhesive amino acid sequences such as RGD, YIGSR, GFOGER or PHSRN, or ii) charged polymers such as poly-L-Lys. The one or more cell adhesion ligands are preferably incorporated into the biocompatible hydrogel beads via covalent tethering, electrostatic interactions, and / or interpenetrating networks.

[0093] In one embodiment according to the third aspect of the invention, the one or more cell adhesion ligands are a peptide comprising the amino acid sequence RGD, for example a peptide comprising the amino acid sequence GRGDSP.

[0094] In one embodiment according to the third aspect of the present invention, the biocompatible hydrogel beads comprise alginate, fibrin, gelatin, agarose, modified or unmodified polyethylene glycol, carrageenan, pectin or any combination thereof.

[0095] In one embodiment according to the third aspect of the present invention, the biocompatible hydrogel beads are made from alginate, fibrin, gelatin, agarose, modified or unmodified polyethylene glycol, carrageenan, pectin or any combination thereof.

[0096] In one embodiment according to the third aspect of the present invention, the biocompatible hydrogel beads are biocompatible alginate beads. The biocompatible alginate beads are preferably produced by competitive ligand exchange crosslinking, for example as described in Example 1 of WO2017153947. In another preferred embodiment, the biocompatible hydrogel beads are biocompatible hydrogel beads comprising alginate. The biocompatible hydrogel beads comprising alginate are preferably produced by competitive ligand exchange crosslinking, for example as described in Example 3 of WO2017153947.

[0097] In one embodiment according to the third aspect of the invention, the biocompatible hydrogel beads are biocompatible alginate beads and the alginate is an RGD-conjugated alginate or a GRGDSP-conjugated alginate.

[0098] In one embodiment of the third aspect, the biocompatible hydrogel beads are biocompatible alginate beads and the means for dissolving or degrading the biocompatible hydrogel alginate beads is selected from the group consisting of alginate lyase treatment, treatment with a divalent cation chelator, and any combination thereof.

[0099] In one embodiment according to the third aspect of the invention, the biocompatible hydrogel beads are biocompatible hydrogel microbeads, preferably with an average size of less than 500 μm in their largest dimension, more preferably in the range of 10-200 μm, such as an average size of 10-150 μm or 30-150 μm, even more preferably in the range of 30 μm-90 μm, such as an average size of 30 μm-80 μm, and most preferably in the range of 50-80 μm in their largest dimension. The size of the beads is measured at their swelling equilibrium size in a liquid environment of physiological ion concentrations such as cell culture medium (see section 1.2 of Example 1).

[0100] In one embodiment, at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, such as a size in the range of 10-150 μm in their largest dimension, a size in the range of 30-150 μm in their largest dimension, or a size in the range of 30-90 μm in their largest dimension. Thus, one skilled in the art will understand that if there are 100 000 biocompatible hydrogel beads and 60% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, then in reality there are 60 000 biocompatible hydrogel beads having a size in the range of 10-200 μm in their largest dimension.

[0101] A fourth aspect of the present invention is - biocompatible hydrogel beads; -Extracellular matrix, Including, - a second tissue scaffold in which biocompatible hydrogel beads are embedded in the extracellular matrix.

[0102] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold has a tubular shape, such as the shape of a circular tube.

[0103] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold is obtained by a method according to the first aspect of the invention.

[0104] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned second tissue scaffold.

[0105] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold or the mechanically conditioned second tissue scaffold is repopulated with a second population of cells.

[0106] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold or the mechanically conditioned second tissue scaffold may be used to regenerate a functional organ by repopulating the scaffold with a second population of cells that can differentiate and develop into a desired tissue within the second tissue scaffold.

[0107] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold or the mechanically conditioned second tissue scaffold is suitable for implantation into a subject, such as a human subject.

[0108] cell In one embodiment, the second population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0109] The second population of cells is preferably anchorage-dependent cells.

[0110] In one embodiment according to the invention, the second population of cells are progenitor cells or adult stem cells (ASCs), which are preferably capable of differentiating and developing into the desired tissue or organ within the tissue scaffold in question.

[0111] Biocompatible Hydrogel Beads In one embodiment according to the fourth aspect of the invention, the biocompatible hydrogel beads are degradable or dissolvable.

[0112] In one embodiment according to the fourth aspect of the invention, the biocompatible hydrogel beads are autodegradable or autodissolving.

[0113] In one embodiment according to the fourth aspect of the invention, the biocompatible hydrogel beads have one or more cell adhesion ligands on their outer surface. The one or more cell adhesion ligands can be, for example, i) peptides containing cell adhesive amino acid sequences such as RGD, YIGSR, GFOGER or PHSRN, or ii) charged polymers such as poly-L-Lys. The one or more cell adhesion ligands are preferably incorporated into the biocompatible hydrogel beads via covalent tethering, electrostatic interactions, and / or interpenetrating networks.

[0114] In one embodiment according to the fourth aspect of the invention, the one or more cell adhesion ligands are a peptide comprising the amino acid sequence RGD, for example a peptide comprising the amino acid sequence GRGDSP.

[0115] In one embodiment according to the fourth aspect of the present invention, the biocompatible hydrogel beads comprise alginate, fibrin, gelatin, agarose, modified or unmodified polyethylene glycol, carrageenan, pectin or any combination thereof.

[0116] In one embodiment according to the fourth aspect of the present invention, the biocompatible hydrogel beads are made from alginate, fibrin, gelatin, agarose, modified or unmodified polyethylene glycol, carrageenan, pectin or any combination thereof.

[0117] In one embodiment according to the fourth aspect of the present invention, the biocompatible hydrogel beads are biocompatible alginate beads. The biocompatible alginate beads are preferably produced by competitive ligand exchange crosslinking, for example as described in Example 1 of WO2017153947. In another preferred embodiment, the biocompatible hydrogel beads are biocompatible hydrogel beads comprising alginate. The biocompatible hydrogel beads comprising alginate are preferably produced by competitive ligand exchange crosslinking, for example as described in Example 3 of WO2017153947.

[0118] In one embodiment according to the fourth aspect of the invention, the biocompatible hydrogel beads are biocompatible alginate beads and the alginate is an RGD-conjugated alginate or a GRGDSP-conjugated alginate.

[0119] In one embodiment of the fourth aspect, the biocompatible hydrogel beads are biocompatible alginate beads and the means for dissolving or degrading the biocompatible hydrogel alginate beads is selected from the group consisting of alginate lyase treatment, treatment with a divalent cation chelator, and any combination thereof.

[0120] In one embodiment according to the fourth aspect of the invention, the biocompatible hydrogel beads are biocompatible hydrogel microbeads, preferably with an average size of less than 500 μm in their largest dimension, more preferably with an average size in the range of 10-200 μm, such as 10-150 μm or 30-150 μm, even more preferably with an average size in the range of 30 μm-90 μm, such as 30 μm-80 μm, and most preferably with an average size in the range of 50-80 μm in their largest dimension. The size of the beads is measured at their swelling equilibrium size in a liquid environment of physiological ion concentration such as cell culture medium (see Example 1, section 1.2).

[0121] In one embodiment, at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, such as a size in the range of 10-150 μm in their largest dimension, a size in the range of 30-150 μm in their largest dimension, or a size in the range of 30-90 μm in their largest dimension. Thus, one skilled in the art will understand that if there are 100,000 biocompatible hydrogel beads and 60% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, then in reality there are 60,000 biocompatible hydrogel beads having a size in the range of 10-200 μm in their largest dimension.

[0122] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold or the mechanically conditioned second tissue scaffold may be used to regenerate a functional organ by repopulating the scaffold with a second population of cells that can differentiate and develop into a desired tissue within the second tissue scaffold.

[0123] In one embodiment according to the fourth aspect of the invention, the second tissue scaffold or the mechanically conditioned second tissue scaffold is suitable for implantation into a subject, such as a human subject.

[0124] A fifth aspect of the present invention is a first population of cells; and -Extracellular matrix, Including, - a third tissue scaffold, wherein the first population of cells is embedded in an extracellular matrix.

[0125] In one embodiment according to the fifth aspect of the invention, the third tissue scaffold comprises the first population of cells and an extracellular matrix embedding the hollow bodies.

[0126] In one embodiment according to the fifth aspect of the invention, the third tissue scaffold is obtained by a method according to the first aspect of the invention.

[0127] In one embodiment according to the fifth aspect of the invention, the third tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned third tissue scaffold.

[0128] In one embodiment according to the fifth aspect of the invention, the third tissue scaffold or the mechanically conditioned third tissue scaffold is repopulated with a second population of cells.

[0129] In one embodiment according to the fifth aspect of the invention, the third tissue scaffold or the mechanically conditioned third tissue scaffold may be used to regenerate a functional organ by repopulating the scaffold with a second population of cells that can differentiate and develop into a desired tissue within the third tissue scaffold.

[0130] In one embodiment according to the third aspect of the invention, the first tissue scaffold or the mechanically conditioned first tissue scaffold is suitable for implantation into a subject, such as a human subject.

[0131] In one embodiment according to the fifth aspect of the invention, the third tissue scaffold has a tubular shape, such as the shape of a circular tube.

[0132] cell In one embodiment according to the fifth aspect of the invention, the first population of cells are anchorage-dependent cells, more preferably human anchorage-dependent cells.

[0133] In one embodiment according to the fifth aspect of the invention, the first population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably human primary mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue derived mesenchymal stem cells, or any combination thereof.

[0134] The second population of cells may be the same as or different from the first population of cells.

[0135] In one embodiment, the second population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0136] In one embodiment, the second population of cells is different from the first population of cells and is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0137] The second population of cells is preferably anchorage-dependent cells.

[0138] In one embodiment according to the invention, the second population of cells are progenitor cells or adult stem cells (ASCs), which are preferably capable of differentiating and developing into the desired tissue or organ within the tissue scaffold in question.

[0139] A sixth aspect of the invention relates to a fourth tissue scaffold comprising an extracellular matrix.

[0140] In one embodiment according to the sixth aspect of the invention, the fourth tissue scaffold is obtained by a method according to the first aspect of the invention.

[0141] In one embodiment according to the sixth aspect of the invention, the fourth tissue scaffold is subjected to mechanical conditioning, thereby obtaining a mechanically conditioned fourth tissue scaffold.

[0142] In one embodiment according to the sixth aspect of the invention, the fourth tissue scaffold or the mechanically conditioned fourth tissue scaffold is repopulated with a second population of cells.

[0143] In one embodiment according to the sixth aspect of the invention, the fourth tissue scaffold has a tubular shape, such as the shape of a circular tube.

[0144] In one embodiment according to the sixth aspect of the invention, the fourth tissue scaffold or the mechanically conditioned fourth tissue scaffold may be used to regenerate a functional organ by repopulating the scaffold with a second population of cells that can differentiate and develop into a desired tissue within the fourth tissue scaffold.

[0145] In one embodiment according to the sixth aspect of the invention, the fourth tissue scaffold or the mechanically conditioned fourth tissue scaffold is suitable for implantation into a subject, such as a human subject.

[0146] cell In one embodiment, the second population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof.

[0147] The second population of cells is preferably anchorage-dependent cells.

[0148] In one embodiment according to the invention, the second population of cells are progenitor cells or adult stem cells (ASCs), which are preferably capable of differentiating and developing into the desired tissue or organ within the tissue scaffold in question. [Brief description of the drawings]

[0149] [Figure 1a-c] Figure 1a-c are schematic diagrams of the approach used to generate bead-associated cells. RGD-functionalized alginate microbeads are generated in a microfluidic process (a) and mixed with human anchorage-dependent cells (b). The human anchorage-dependent cells attach to the outer surface of the RGD-functionalized alginate microbeads to obtain bead-associated cells (c). [Figure 2a-d] Figure 2a-g show the bead-associated cells of Figure 1c utilized to generate extracellular matrix and thereby obtain various tissue scaffolds. Panels a-e and f-g are schematics (lower panels) and corresponding microscopic images (upper panels) of the static culture and mechanical conditioning stages of the various tissue scaffolds described herein. Human anchorage-dependent cells attach to RGD-functionalized alginate microbeads and form a 3D lattice structure with each other (a), on which the cells can grow and proliferate (b). Over time, the cells generate their own supporting extracellular matrix (c) that can support the structure when the microbeads are selectively removed (d). The cells continue to deposit extracellular matrix after the microbeads are removed (e). Subjecting the sample to mechanical conditioning increases the deposition of extracellular matrix and generates aligned fibers (f). Finally, the cells can be removed leaving a tissue scaffold generated solely from human extracellular matrix (g). Black arrow = microbead; white arrow = cell; black arrowhead = cell secreting extracellular matrix; white arrowhead = extracellular matrix with aligned fibers upon mechanical conditioning. [Figure 2e-g]Figure 2a-g show the bead-associated cells of Figure 1c utilized to generate extracellular matrix and thereby obtain various tissue scaffolds. Panels a-e and f-g are schematics (lower panels) and corresponding microscopic images (upper panels) of the static culture and mechanical conditioning stages of the various tissue scaffolds described herein. Human anchorage-dependent cells attach to RGD-functionalized alginate microbeads and form a 3D lattice structure with each other (a), on which the cells can grow and proliferate (b). Over time, the cells generate their own supporting extracellular matrix (c) that can support the structure when the microbeads are selectively removed (d). The cells continue to deposit extracellular matrix after the microbeads are removed (e). Subjecting the sample to mechanical conditioning increases the deposition of extracellular matrix and generates aligned fibers (f). Finally, the cells can be removed leaving a tissue scaffold generated solely from human extracellular matrix (g). Black arrow = microbead; white arrow = cell; black arrowhead = cell secreting extracellular matrix; white arrowhead = extracellular matrix with aligned fibers upon mechanical conditioning. [Figure 3a-d] Figure 3a-d are schematic illustrations of the tissue scaffold fabrication process, in which the tissue scaffold has the shape of a circular tube. The bead-associated cells in Figure 1c (a) are cast into an agarose mold, viewed from the side (b) and top (c), forming a cohesive ring as the cells grow and secrete extracellular matrix. Upon removal from the mold, the tissue scaffolds maintain their shape and high viability despite the large size of the scaffold (d). [Figure 4a] Figure 4a is a photograph of a tissue scaffold with the shape of a circular tube. When cast into a mold, the bead-associated cells in Figure 1c form a cohesive ring, and the cells proliferate and grow as they deposit extracellular matrix into the ring-like structure. Despite a ring edge thickness approaching 1 mm, the tissue scaffold remains viable throughout the entire construct. [Figure 4b]Figure 4b is a photograph and microscopic image of the ring-shaped tissue scaffolds. Upon removal from the mold shown in Figure 4a, the constructs retain their shape and high viability despite the large size of the constructs (two separate representative samples). [Diagram 5] FIG. 5 is a schematic diagram of four different tissue scaffolds referred to herein showing the presence or absence of biocompatible hydrogel beads and a first population of cells. [Figure 6] FIG. 6 shows a self-supporting tissue scaffold having the shape of a ring. [Figure 7] Figures 7a-g are photographs of T1 tissue rings at various time points after molding (7a, 1 hour after molding; 7b, 6 hours after molding; 7c, 24 hours after molding; 7d, 48 hours after molding; 7e, 72 hours after molding; 7f, 96 hours after molding; 7g, 120 hours after molding). T1 tissue rings have been generated by using annular wells with an inner diameter of 3 mm, a well thickness of 1 mm, and a depth of 3 mm (Example 2, section 2.4). T1 tissue rings are thinner than T2 tissue rings. The distance between the outer surface of the tissue ring and the surface of the mold is indicated by the black arrow, and the greater the distance, the greater the degree of contraction of the tissue ring. [Figure 8] Figures 8a-g are photographs of T2 tissue rings at various time points after molding (8a, 1 hour after molding; 8b, 6 hours after molding; 8c, 24 hours after molding; 8d, 48 hours after molding; 8e, 72 hours after molding; 8f, 96 hours after molding; 8g, 120 hours after molding). T2 tissue rings have been generated by using annular wells with an inner diameter of 3 mm, a well thickness of 2 mm, and a depth of 4 mm (Example 2, section 2.4). T2 tissue rings are thicker than T1 tissue rings. The distance between the outer surface of the tissue ring and the surface of the mold is indicated by the black arrow, and the greater the distance, the greater the degree of contraction of the tissue ring. [Figure 9a-b]Figures 9a-b show the shrinkage of T1 and T2 tissue rings over time, respectively. The x-axis shows the time after molding (hours) and the y-axis shows the degree of shrinkage (tissue ring width:mold width ratio). Marked lines represent the average ring width:mold width ratio at different time points, while unmarked lines show ± standard deviation. A high degree of shrinkage, i.e., a low value on the y-axis, indicates that the tissue rings are self-supporting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0150] definition As used herein, the term "anchorage-dependent cells", also known as substrate-dependent cells, are any type of cell that can grow, survive, maintain function, or differentiate when attached to a surface.

[0151] As used herein, the term "regenerating" or "regeneration" encompasses the process of seeding any of the tissue scaffolds referred to herein in which a first population of cells has been removed and replaced with a second population of cells, thus allowing this new population of cells (second population of cells) to grow, survive, maintain function or differentiate on the decellularized tissue scaffold.

[0152] As used herein, the term "hydrogel" is a crosslinked hydrophilic polymer that is highly absorbent while still maintaining a well-defined structure. Hydrogels may be synthetic or naturally derived.

[0153] As used herein, the expression "hydrogel beads" refers to a three-dimensional crosslinked network of hydrophilic polymers formed into a spherical shape.

[0154] The terms "extracellular matrix proteins" or "extracellular matrix" are used interchangeably herein to describe an intricate network composed of an array of multidomain macromolecules such as collagen, elastin, enzymes, glycoproteins and hydroxyapatite, organized in a cell / tissue specific manner.

[0155] As referred to herein, the expression "bead-associated cells" is used to describe anchorage-dependent cells that are linked to hydrogel beads through a variety of interactions.

[0156] "Cell attachment to hydrogel beads" and "cell adhesion" are used interchangeably herein to describe the attachment of a first cell population to biocompatible hydrogel beads, thus obtaining bead-associated cells.

[0157] The term "hollow body" as referred to herein refers to the empty space that occurs within the tissue scaffold when the biocompatible hydrogel beads degrade or dissolve, i.e., the space previously occupied by the hydrogel beads.

[0158] As used herein, the terms "biologically inactive" or "inactive" are used interchangeably to describe the property of lacking the property of activity, i.e., lacking the usual or expected chemical or biological action.

[0159] As used herein, the terms "biocompatible" or "compatible" are used interchangeably to describe the characteristic of being compatible with living tissue or systems by not being toxic, physiologically reactive, or causing immune rejection.

[0160] As used herein, the term "microbeads" refers to beads that are less than 1000 μm in their largest dimension.

[0161] Detailed Description of the Invention Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of genetics, biochemistry, and molecular biology.

[0162] All methods and materials similar or equivalent to those described herein can be used to carry out or test the present invention, and suitable methods and materials are described herein.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of discrepancy, the present specification, including definitions, will take precedence.

[0163] Where numerical limits or ranges are stated herein, the endpoints are included, and all values ​​and subranges within the numerical limit or range are specifically included, as if expressly written.

[0164] Tissue engineering as a field seeks to develop biological substitutes to restore biological function of tissues or entire organs, and includes methods of culturing cells on a support matrix to form new tissues of intended shape and function. However, this method has several drawbacks, and translation of such engineered tissues to the clinic is poor. Tissue scaffolds, which act as intermediates in the generation of new functional tissues, may present a risk of immunological and / or inflammatory responses when implanted. Furthermore, tissue scaffolds must be processable to form tissue scaffolds of the desired shape for the tissue of interest. As a result, there is a need in the art to develop techniques to improve tissue scaffolds for use in tissue engineering.

[0165] The present inventors have addressed the above needs by providing an improved manufacturing methodology that allows for the production of complex three-dimensional tissue scaffolds that, when implanted in humans, are essentially non-immunogenic, mimic the natural extracellular matrix, and can be repopulated and regulated by host cells, thereby becoming living tissues.

[0166] In general, the invention involves the generation of engineered tissue scaffolds developed primarily by in vitro culture of a first population of cells that adhere to biocompatible hydrogel beads and secrete extracellular matrix. Removal of the biocompatible hydrogel beads and decellularization of the first population of cells provides a tissue scaffold containing an extracellular matrix of a desired shape. The decellularized tissue scaffold may be further repopulated and in vitro cultured with a second population of cells characteristic of the intended tissue or organ to be replaced or augmented. However, also within the scope of the invention, the decellularized tissue scaffold may be implanted into a human or animal, where the recipient's cells may infiltrate and repopulate the decellularized tissue scaffold in vivo. Thus, engineered tissue scaffolds that are primarily cultured in vitro, and in certain embodiments, in vivo, are included within the scope of the invention.

[0167] Generally, for a tissue scaffold product to be safe for implantation in humans, the reagents used throughout the method, such as hydrogel beads, are preferably biocompatible, degradable or soluble, and / or capable of being removed, e.g., washed away, before the implantable product is ready for use.

[0168] Thus, according to one aspect, the present invention provides a method of producing a tissue scaffold, comprising: a) providing a first population of cells and biocompatible hydrogel beads; b) combining the first population of cells with biocompatible hydrogel beads to obtain a first cell-bead mixture; c) culturing the first cell-bead mixture to produce an extracellular matrix, thereby obtaining a first tissue scaffold; d) optionally subjecting the first tissue scaffold to mechanical conditioning, thereby obtaining a mechanically conditioned first tissue scaffold, - the first cell population is anchorage-dependent cells capable of producing and secreting extracellular matrix; - the biocompatible hydrogel beads support the attachment of a first cell population; and - combining the first cell population and the biocompatible hydrogel beads of step b) in a mold to obtain a first cell-bead mixture; or incubating the first cell-bead mixture of step b) to allow for the formation of bead-associated cells and then transferring to a mold; The present invention provides a method comprising:

[0169] First cell population The first step of the method refers to a first cell population, which are anchorage-dependent cells used to seed the hydrogel beads and preferably secrete as much extracellular matrix as possible to construct a first tissue scaffold that retains its shape without the support of an additional substrate.

[0170] The first cell population may be derived from an animal or human donor, or an established cell line. Preferably, the cells are of the same species as the intended recipient, for example to reduce immunological reactions and increase compatibility between the tissue scaffold and the recipient. Rigorous screening of the cells for transmissible diseases (e.g., HIV or Hepatitis) further reduces the risk of infection associated with the tissue scaffold product. The cells may further have desirable properties, such as the ability to grow well in culture, or the cells are genetically modified to alter secretion of extracellular matrix components.

[0171] The first cell population can in principle be any type of cell that grows, survives, maintains function or differentiates when attached to a surface, also known as anchorage-dependent cells. Moreover, according to the method, the first cell population is capable of producing and secreting extracellular matrix proteins. Preferably, the first cell population produces as much extracellular matrix as possible. Thus, in one embodiment according to the present invention, the first cell population is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells, or any combination thereof. In a preferred embodiment, the first cell population is adipose tissue-derived mesenchymal stem cells. In one embodiment, the first cell population is genetically modified to increase secretion of extracellular matrix.

[0172] Hydrogel beads and bead-associated cells According to the second step of the method, the first cell population is combined with biocompatible hydrogel beads to promote the attachment of the first cell population to the biocompatible hydrogel beads, thereby obtaining bead-associated cells. Thus, the biocompatible hydrogel beads are the initial substrate for the growth of the first cell population when generating a tissue scaffold of a desired shape for a target tissue or organ.

[0173] Those skilled in the art will readily understand the conditions necessary to achieve cell attachment and / or growth on the hydrogel beads. The first cell population and the biocompatible hydrogel beads can be incubated or cultured in a still / static, shaking or rotating incubator in a tissue culture flask or any other form of sterile container. See further Example 1, section 1.3.

[0174] In order for the first cell population to attach to the biocompatible hydrogel beads, the biocompatible hydrogel beads must support the attachment of the first cell population. If the biocompatible hydrogel beads do not support the attachment of the first cell population, one or more cell adhesion ligands can be incorporated into the biocompatible hydrogel beads or coated on the surface of the biocompatible hydrogel beads to achieve the appropriate conditions for attachment, also referred to herein as functionalization of the biocompatible hydrogel beads. For example, biocompatible hydrogel beads covalently conjugated with heparin-binding peptides or peptide sequences found in extracellular matrix proteins can be used to tailor the type and extent of interaction with the cells. The interaction of cells with biocompatible hydrogel beads containing cell adhesion ligands is often mediated through cell receptors on the surface of the cells that recognize the cell adhesion molecules on the surface of the hydrogel.

[0175] Thus, in one embodiment according to the present invention, the biocompatible hydrogel beads have one or more cell adhesion ligands on their outer surface.

[0176] Cells, especially anchorage-dependent cells, do not readily attach and grow on or within alginate hydrogels. However, free hydroxyl and carboxyl groups are distributed along the backbone of alginate, making it suitable for chemical modification, such as covalently conjugating alginate with cell adhesion ligands.

[0177] According to one embodiment of the present invention, the biocompatible hydrogel beads are alginate beads and include one or more cell adhesion ligands on the outer surface of the alginate beads. The one or more cell adhesion ligands can be, for example, i) peptides containing cell adhesive amino acid sequences such as RGD, YIGSR, GFOGER or PHSRN, or ii) charged polymers such as poly-L-Lys. The one or more cell adhesion ligands are preferably incorporated into the biocompatible hydrogel beads via covalent tethering, electrostatic interactions, and / or interpenetrating networks. Peptide-linked alginates are available, for example, from Novamatrix® (https: / / novamatrix.biz / ).

[0178] The functionalization of biocompatible hydrogel beads allows not only the attachment of one cell to one bead, but also various attachments of one or more cells to one or more beads, cell-to-cell attachments, and / or attachments between cells, beads and extracellular matrix, forming a three-dimensional lattice structure. Thus, in one embodiment according to the invention, adjacent biocompatible hydrogel beads are connected to each other by a network of a first group of cells and extracellular matrix.

[0179] Hydrogels such as alginate are ideal substrates for biomedical applications because they are cross-linked hydrophilic polymers that are insoluble in water and are highly absorbent while maintaining a well-defined structure, a beneficial feature when acting as a substrate for culturing cells, as the hydrogel itself can act as a reservoir of nutrients deep within the tissue scaffold.

[0180] Furthermore, the spacing between the biocompatible hydrogel beads creates a porous structure that allows for diffusion of nutrients throughout the tissue scaffold as cells grow and secrete extracellular matrix. This provides an advantage over the bulk gels used in the prior art in that thicker tissue scaffolds can also be produced without reseeding the biocompatible hydrogel beads to obtain or maintain a viable cell population that produces extracellular matrix. Thus, the expanded ability of nutrient diffusion by using the biocompatible hydrogel beads according to the present invention provides the advantage of enabling the production of complex three-dimensional tissue scaffolds.

[0181] Those skilled in the art will recognize that the size of the biocompatible hydrogel beads is likely to affect i) the porosity of the structure, i.e., the spacing between the biocompatible hydrogel beads, and ii) the surface area to volume ratio of the beads. Increasing the bead size is expected to increase the volume of the spacing between the beads, but is expected to decrease the surface area to volume ratio of the beads. Thus, increasing the size of the beads is likely to improve the diffusion of nutrients throughout the structure, but at the same time decrease the efficiency of extracellular matrix production. Therefore, the size of the biocompatible hydrogel beads should be selected for optimal nutrient diffusion and cell number per volume.

[0182] In one embodiment the biocompatible hydrogel beads are biocompatible hydrogel microbeads, preferably having an average size of less than 500 μm in their largest dimension, more preferably an average size in the range of 10-500 μm, such as 10-200 μm, e.g. 10-150 μm or 30-150 μm, even more preferably an average size in the range of 30 μm-90 μm, such as 30 μm-80 μm, and most preferably an average size in the range of 50-80 μm in their largest dimension.

[0183] In one embodiment, at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, such as a size in the range of 10-150 μm in their largest dimension, a size in the range of 30-150 μm in their largest dimension, or a size in the range of 30-90 μm in their largest dimension. Thus, one skilled in the art will understand that if there are 100 000 biocompatible hydrogel beads and 60% of the biocompatible hydrogel beads have a size in the range of 10-200 μm in their largest dimension, then in reality there are 60 000 biocompatible hydrogel beads having a size in the range of 10-200 μm in their largest dimension.

[0184] As previously mentioned, hydrogels are highly absorbent, therefore, bead size is measured at swelling equilibrium size in a liquid environment of physiological ion concentrations, such as cell culture medium. Section 1.2 of Example 1 provides a technique for measuring the diameter of hydrogel beads.

[0185] Biocompatible hydrogels can be produced by any method of crosslinking polymers, such as free radical polymerization, chemical reaction, or ionizing radiation. In the case of divalent ion crosslinked hydrogels, the biocompatible hydrogels are preferably prepared by competitive ligand exchange as described in WO2017153947, particularly in Example 1 or Example 3 thereof.

[0186] In one embodiment, the biocompatible hydrogel beads of the present invention are made of or comprise alginate; fibrin; gelatin; agarose; modified or unmodified polyethylene glycol; carrageenan; pectin; hyaluronic acid; collagen; cellulose derivatives, such as hydroxymethylpropylcellulose (HPMC) or methylpropylcellulose (MPC); or any combination thereof.Preferably, the polymers used to manufacture the biocompatible hydrogel beads are degradable or soluble, and preferably have inherently low immunogenicity.Preferably, the hydrogel beads are prepared from natural polymers.

[0187] According to one embodiment, the biocompatible hydrogel beads are either alginate beads or beads that include alginate.

[0188] Alginate is a promising candidate due to its biocompatibility, low toxicity, and the ability to withstand e.g. Ca 2+ Alginate hydrogels have been used in many biomedical applications due to the ease of gelation by the addition of divalent cations such as . Alginate hydrogels can be prepared from a variety of commercially available alginates, typically obtained from brown algae such as Laminaria hyperborean, Laminaria digitatat, Laminaria japonica, Ascopyllum nodosum or Macrocystis pyrifera, by a variety of cross-linking methods well known to those skilled in the art, including competitive ligand exchange as described above.

[0189] Alginate is a naturally occurring anionic polymer containing blocks of (1,4) linked β-D-mannuronate (M) and α-L-guluronic acid (G) residues. The blocks consist of consecutive G residues (GGGGGG), consecutive M residues (MMMMMM), and alternating M and G residues (GMGMGM). Alginates extracted from different sources vary in their M and G content and the length of each block, with over 200 different alginates currently being produced.

[0190] Only the G-blocks of alginate are capable of absorbing divalent cations (e.g., Ca 2+ ) to form hydrogels. Thus, the composition (i.e., M / G ratio), sequence, G-block length and molecular weight are factors that influence the physical properties of alginates and the resulting hydrogels.

[0191] The mechanical properties of alginate gels are typically enhanced by increasing the length and molecular weight of the G-blocks. However, alginate solutions formed from high molecular weight polymers also become highly viscous. Manipulation of the molecular weight and its distribution can independently control the solution viscosity before gelation and the stiffness after gelation. The elastic modulus of the gel can be significantly increased, while the viscosity of the solution is minimally increased by using a combination of high and low molecular weight alginate polymers.

[0192] Those skilled in the art will recognize that the percentage of M and G residues will vary depending on the species of alginate from which the alginate is derived. Those skilled in the art will also recognize that some alginates having a high percentage of G residues may be commonly referred to as G-rich alginates, while alginates having a low percentage of G residues may be commonly referred to as M-rich alginates.

[0193] In one embodiment, the biocompatible hydrogel beads used in the method are made of alginate with a high percentage of G residues, i.e. G-rich alginate. According to one embodiment, the biocompatible hydrogel beads used in the method are made of alginate with 30% or more G residues, such as 35% or more G residues, 40% or more G residues, 45% or more G residues, 50% or more G residues, 55% or more G residues, 60% or more G residues, 65% or more G residues or 70% or more G residues monomer units). In a preferred embodiment, the biocompatible hydrogel beads used in the method are made of alginate with 40% or more G residues, such as 45% or more G residues (monomer units).

[0194] In one embodiment, the biocompatible hydrogel beads used in the method are made from a mixture of at least two different alginates, each alginate independently comprising 30% or more G residues, e.g., 35% or more G residues, 40% or more G residues, 45% or more G residues, 50% or more G residues, 55% or more G residues, 60% or more G residues, 65% or more G residues or 70% or more G residues (monomer units).

[0195] In another embodiment, the biocompatible hydrogel beads used in the method are made from alginate containing 30-90% G residues, e.g., 30-80% G residues, 40-80% G residues or 40-75% G residues (monomer units).

[0196] In one embodiment, the biocompatible hydrogel beads used in the method are made from a mixture of at least two different alginates, each alginate independently comprising 30-90% G residues, e.g., 30-80% G residues, 40-80% G residues or 40-75% G residues (monomer units).

[0197] In another embodiment, the biocompatible hydrogel beads used in the method are made of alginate having a G:M ratio > 0.4, e.g., a G:M ratio > 0.5, a G:M ratio > 0.7, a G:M ratio > 0.9, a G:M ratio > 1, a G:M ratio > 1.2, a G:M ratio > 1.4 or a G:M ratio > 1.5.

[0198] In one embodiment, the biocompatible hydrogel beads used in the method are made from a mixture of at least two different alginates, each alginate independently having a G:M ratio > 0.4, e.g., a G:M ratio > 0.5, a G:M ratio > 0.7, a G:M ratio > 0.9, a G:M ratio > 1, a G:M ratio > 1.2, a G:M ratio > 1.4 or a G:M ratio > 1.5.

[0199] In one embodiment, the biocompatible hydrogel beads used in the method are made of alginate having a molecular weight in the range of 10-300 kDa, e.g., 10-250 kDa, 10-200 kDa, 10-150 kDa, 10-100 kDa, 50-300 kDa, 50-250 kDa, 50-200 kDa, 50-150 kDa or 50-100 kDa.

[0200] In one embodiment, the biocompatible hydrogel beads used in the method are made from a mixture of at least two different alginates, each alginate independently having a molecular weight in the range of 10-300 kDa, e.g., 10-250 kDa, 10-200 kDa, 10-150 kDa, 10-100 kDa, 50-300 kDa, 50-250 kDa, 50-200 kDa, 50-150 kDa or 50-100 kDa.

[0201] Mold According to the first aspect of the present invention, the first cell population of step b) and the biocompatible hydrogel beads are combined in a mold to obtain a first cell-bead mixture, or the first cell-bead mixture of step b) is incubated to allow the formation of bead-associated cells and then transferred to a mold.

[0202] Transfer of bead-associated cells by the present method can be any procedure that physically moves the bead-associated cells from one container to the next, such as injection or pipetting.

[0203] In the context of the present invention, the mold may be made of a bioinert material and serves as a support for the first cell-bead mixture until the tissue scaffold can hold its shape by itself.Thus, in one embodiment, the mold has one or more of the following properties:(1) allows control of the geometric shape;(2) prevents cell attachment or infiltration;(3) allows exchange of nutrients and gases between the exterior and interior of the mold.

[0204] The mold may be made of any inert and biocompatible material having the above characteristics. For example, the mold material should be selected to allow precise control over the shape and dimensions of the construct, and can be removed mechanically (e.g., by pulling out) or chemically (through degradation) from the tissue scaffold. According to one embodiment, the mold may be made of copolymers of lactic and glycolic acid (PLGA or PDLG), polycaprolactone, carbohydrate glass, such as the inert Gore-Tex material typically used for vascular grafts, ePTFE (expanded polytetrafluoroethylene), PTFE (polytetrafluoroethylene), PDMS (polydimethylsiloxane), silicone, PEEK (polyetheretherketone) and / or agarose. According to one embodiment, the mold used in the method is agarose.

[0205] Agarose is widely used as a mold material for making self-assembled tissues due to its biocompatibility, permeability and non-cell adhesiveness. For example, an agarose mold can be obtained by 3D printing and formed into the desired shape of the tissue scaffold prepared according to the present method. For example, see Strobel et al. (2018), J. Vis. Exp., 134, 55618.

[0206] In one embodiment, the mold is fully or partially immersed in cell culture medium, hi one embodiment, the mold has the shape of a circular well.

[0207] The mold, in the context of the present invention, provides an external support for the first cell-bead mixture to form a lattice of a desired shape for the intended tissue or organ. Preferably, the mold allows for the generation of tissue scaffolds of any desired shape and / or complex geometry.

[0208] 1. Cultivation of the First Cell-Bead Mixture to Generate ECM According to step c) of the method according to the first aspect of the invention, the first cell-bead mixture is cultured in a mould to generate an extracellular matrix, thereby obtaining a first tissue scaffold, the formed extracellular matrix taking the shape of the mould used.

[0209] The conditions and culture medium suitable for culturing the first cell-bead mixture before or after transferring to the mold, and the cells present on the scaffold of the present invention are known to those skilled in the art. The suitable culture medium and supplements for culturing anchorage-dependent cells (e.g., the cells of the first cell group) applicable in the present method are available from various providers known to those skilled in the art, such as, for example, ThermoFisherScientific or PromoCell. In a preferred embodiment, the first cell group produces and secretes as many extracellular matrix proteins as possible as it grows and divides on the biocompatible hydrogel beads.

[0210] Additionally, various growth conditions can be selected to enhance the process of extracellular matrix production and / or stimulate the development of desired mechanical, physical, or biochemical properties. Such growth conditions may include the use of growth media supplemented with amino acids such as proline, alanine, glycine, and / or ascorbic acid to enhance extracellular matrix protein production. Additionally, the growth media may be supplemented with growth factors such as FGF2 (fibroblast growth factor) and / or any other growth factors that may be important in the regulation of extracellular matrix production and degradation and the interaction between cells and the extracellular matrix. In one embodiment, the medium is supplemented with amino acids as described in section 1.3 of Example 1 and / or growth factors as described in section 1.1 of Example 1. In one embodiment, the first cell-bead mixture is cultured in the mold in the presence of one or more agents that stimulate the cells of the first cell-bead mixture to produce and / or secrete extracellular matrix. The one or more agents that stimulate the cells to produce and / or secrete extracellular matrix are selected from the group consisting of proline, glycine, alanine, ascorbic acid, and any combination thereof.

[0211] The first cell-bead mixture is cultured in the mold to generate extracellular matrix until the first tissue scaffold is removed from the mold and still able to maintain its shape, i.e., the first tissue scaffold is self-supporting. Example 2 provides a methodology that can be used to determine when a tissue scaffold is self-supporting.

[0212] Culturing until the first tissue scaffold is removed from the mold and still able to maintain its shape typically ranges from 2 days to 2 weeks depending on the cell type, mold, and experimental conditions used. Example 2 provides a methodology that can be used to determine when a tissue scaffold is self-supporting.

[0213] Mechanical conditioning The first tissue scaffold, the second tissue scaffold, the third tissue scaffold and / or the fourth tissue scaffold can be subjected to mechanical conditioning to provide a more suitable tissue architecture and an extracellular matrix environment that more closely mimics native tissue. Clinical outcomes can be improved by mechanically conditioning the tissue scaffold to enhance properties such as expression of desired cell phenotypes, altered tensile strength, and fiber alignment under load that mimics loads naturally encountered in the body.

[0214] Mechanical conditioning of any tissue scaffold according to the present invention can be performed by various devices known in the art that can apply mechanical forces to cells (e.g., stretching) without damaging the tissue scaffold (e.g., Strobel, Tis Eng Part A 2018; J Tissue Eng Regen Med 10, E204, 2016). Such devices can include various types of bioreactor systems or thermoresponsive polymers that generate elastic substrates for cells, and the mechanical forces can be, but are not limited to, pneumatic or hydraulic or mechanical and / or thermal stretching.

[0215] When the tissue scaffold has the shape of a tube, the tissue scaffold can be subjected to a mechanical conditioning selected from the group consisting of, for example: - Internal cyclic strain and / or radial expansion of the scaffold, preferably applied via pressurized fluid or air flow; Internal cyclic circumferential strain and / or radial expansion of the scaffold, preferably applied via pressurized fluid or air flow, etc.; - Axial strain, preferably applied via extensions on opposing ends of the scaffold; - A compressive strain, preferably applied from one side of the scaffold; - Fluid shear stress on or through the scaffold; or -Any combination of them.

[0216] Removal of biocompatible hydrogel beads In one embodiment according to the present invention, the biocompatible hydrogel beads are subjected to a means to dissolve or degrade the biocompatible hydrogel beads, thereby obtaining a third tissue scaffold.

[0217] Those skilled in the art will recognize that the hydrogel beads may be removed by techniques well known to those of skill in the art. The method of dissolving or degrading the hydrogel will depend on the hydrogel in question.

[0218] In one embodiment, the biocompatible hydrogel beads are biocompatible alginate beads and the means for dissolving or degrading the biocompatible hydrogel alginate beads is selected from the group consisting of alginate lyase treatment, treatment with a divalent cation chelator, and any combination thereof.

[0219] In one embodiment according to the present invention, the biocompatible hydrogel beads are self-degrading or self-dissolving. The terms "self-degrading" and "self-dissolving" mean that the biocompatible hydrogel beads degrade or dissolve by themselves. Therefore, no agent needs to be added for the degradation or dissolution to occur. The time required for the degradation or dissolution to be complete typically decreases with increasing temperature.

[0220] Immediately after the biocompatible hydrogel beads are removed, there are hollow bodies previously occupied by the biocompatible hydrogel beads. In one embodiment according to the invention, the hollow bodies are reconstituted by i) secreting extracellular matrix in the hollow bodies, and / or ii) promoting the migration of the first cell population into the hollow bodies; and / or allowing the contractile forces of the first cell population to close the hollow bodies. The hollow bodies are preferably filled with extracellular matrix and / or the first cell population prior to mechanical conditioning and / or decellularization.

[0221] Decellularization in general The invention further includes subjecting the first or third tissue scaffold to decellularization, thereby obtaining a second or fourth tissue scaffold, respectively.

[0222] Decellularization is any technique that removes cellular components while leaving the extracellular matrix secreted by the first population of cells substantially intact in any step according to the invention.

[0223] In one embodiment according to the invention, the decellularization is selected from the group consisting of physical treatment, chemical treatment, enzymatic treatment, and any combination thereof.

[0224] Decellularization - Physical Treatment The most common physical treatments used to lyse, kill and remove cells from the extracellular matrix of tissue scaffolds are through the use of temperature, pressure and electrical disruption. Thus, in one embodiment, decellularization is achieved through the use of temperature, pressure and / or electrical disruption.

[0225] The rapid freeze-thaw mechanism often uses temperature methods. By rapidly freezing the tissue, fine ice crystals are formed around the cell membrane, lysing the cells. Thus, in one embodiment, the rapid freeze-thaw mechanism is used to achieve decellularization.

[0226] Pressure decellularization involves the controlled use of hydrostatic pressure applied to a tissue scaffold. This is best performed at elevated temperatures to avoid unmonitored ice crystal formation that can damage the scaffold. Thus, in one embodiment, decellularization is achieved by the use of hydrostatic pressure applied to the tissue scaffold.

[0227] Electrical disruption of the plasma membrane is another option to dissolve cells housed in the tissue scaffold. By exposing the tissue scaffold to an electric pulse, micropores are formed in the plasma membrane. The cells eventually die after the homeostatic electrical balance is disturbed by the applied stimulus. Thus, in one embodiment, decellularization is achieved by exposing the tissue scaffold to an electric pulse, thereby forming micropores in the plasma membrane of the cells.

[0228] In one embodiment, decellularization is achieved by a rapid freeze-thaw mechanism, the use of hydrostatic pressure applied to the tissue scaffold, exposing the tissue scaffold to electrical pulses, or any combination thereof.

[0229] After lysing the cells, the scaffold may preferably be further exposed to a liquefied chemical that breaks down and washes away undesirable components.

[0230] Decellularization - Chemical Treatment The appropriate combination of chemicals is selected for decellularization depending on the thickness, extracellular matrix composition, and intended use of the tissue scaffold. Chemicals typically used to kill and remove cells include acid, alkaline treatment, ionic surfactants, non-ionic surfactants, and zwitterionic surfactants. Thus, in one embodiment, decellularization is achieved by exposing the tissue scaffold to acid, alkaline, ionic surfactants, non-ionic surfactants, and / or zwitterionic surfactants.

[0231] Sodium dodecyl sulfate (SDS), an ionic detergent, is commonly used due to its high effectiveness in lysing cells without significant damage to the extracellular matrix. Thus, in one embodiment, decellularization is achieved by exposing the tissue scaffold to SDS.

[0232] The best known non-ionic detergent is Triton® X-100, which is popular due to its ability to disrupt lipid-lipid and lipid-protein interactions. Triton® X-100 does not disrupt protein-protein interactions, which is beneficial for keeping the ECM intact. Thus, in one embodiment, decellularization is achieved by exposing the tissue scaffold to Triton® X-100.

[0233] EDTA is a chelating agent that binds calcium, a necessary component for proteins to interact. By making calcium unavailable, EDTA prevents endogenous proteins between cells from binding to each other. EDTA is often used with trypsin, an enzyme that acts as a protease to break existing bonds between endogenous proteins of adjacent cells in tissue. Together, the EDTA-trypsin combination makes a good team for decellularizing tissue. Thus, in one embodiment, decellularization is achieved by exposing tissue scaffolds to EDTA, more preferably to EDTA-trypsin combination.

[0234] Thus, in one embodiment, decellularization is achieved by exposing the tissue scaffold to SDS, exposing the tissue scaffold to Triton® X-100, exposing the tissue scaffold to EDTA, exposing the tissue scaffold to an EDTA-trypsin combination, or any combination thereof.

[0235] After lysing the cells, the scaffold may preferably be further exposed to a liquefied chemical that breaks down and washes away undesirable components.

[0236] Decellularization by chemical treatment is the preferred decellularization option according to the present invention.

[0237] Decellularization - Enzyme Treatment Enzymes used in decellularization procedures are used to disrupt the bonds and interactions between nucleic acids, cells that interact through adjacent proteins, and other cellular components. Lipase, thermolysin, galactosidase, nuclease, and trypsin have all been used to remove cells. After cells are lysed with detergents, acids, physical pressure, etc., endonucleases and exonucleases can begin to degrade the genetic material.

[0238] Thus, in a preferred embodiment, decellularization is accomplished following chemical or physical treatment or in combination with an enzyme treatment. In one embodiment, the enzyme treatment comprises exposing the tissue scaffold to an enzyme selected from the group consisting of lipase, thermolysin, galactosidase, nuclease, trypsin, and any combination thereof.

[0239] According to one embodiment, decellularization is achieved by exposing the tissue scaffold to at least one detergent and at least one nuclease, more preferably by exposing the tissue scaffold to a detergent selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), Triton® X and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), followed by exposing the tissue scaffold to a nuclease treatment, such as a DNase treatment.

[0240] The decellularized tissue scaffold from any of the steps of the present invention retains substantially the same shape as it had before decellularization. Therefore, a procedure that removes cells with less damage to the extracellular matrix is ​​preferred. In any of the steps according to the present invention, the decellularized tissue scaffold is thoroughly washed to remove residual decellularization solution that may reduce biocompatibility or inhibit subsequent growth of cells on or within the following tissue scaffold.

[0241] Second cell population The invention further includes repopulating the decellularized second or fourth tissue scaffold with a second population of cells.

[0242] The second cell population refers to cells that are used to repopulate the tissue scaffold, preferably after the first cell population has been removed by decellularization. The second cell population may be the same or different from the first cell population, or a cell population that has characteristics of the intended tissue or organ to be replaced or augmented.

[0243] The second cell population may be derived from an animal or human donor, or an established cell line. Preferably, the cells are of the same species as the intended recipient, for example to reduce immunological reactions and increase compatibility between the tissue scaffold and the recipient. Rigorous screening of the cells for transmissible diseases (e.g., HIV or Hepatitis) further reduces the risk of infection associated with the tissue scaffold product. The cells may further have desirable properties, such as the ability to grow well in culture, or the cells are genetically modified to alter secretion of extracellular matrix components.

[0244] The second cell group herein may be the same or different anchorage-dependent cells as the first cell group used to seed the biocompatible hydrogel beads (initial substrate). In one embodiment, the second cell group may be any type of cells suitable for regenerating a functional tissue or organ. For example, the cells used to seed the decellularized tissue scaffold, i.e., the second or fourth tissue scaffold, may be a different cell type from the cells used to generate the decellularized tissue scaffold.

[0245] The second population of cells preferably has the capacity to attach, grow and / or differentiate when subjected to a second or fourth tissue scaffold according to the invention.

[0246] In one embodiment, the second population of cells is selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated primary mesenchymal cells, such as fibroblasts and / or smooth muscle cells, preferably primary human mesenchymal cells, such as mesenchymal stem cells, in particular adipose tissue-derived mesenchymal stem cells. In one embodiment, the second population of cells is anchorage-dependent cells genetically modified to increase secretion of extracellular matrix.

[0247] In a preferred embodiment, the second cell population is a progenitor cell or adult stem cell (ASC) that differentiates within the tissue scaffold and develops into a desired tissue or organ. Cells applicable as the second cell population are available from various cell culture providers, see, for example, ThermoFisherScientific's 3D Cell Culture Handbook, https: / / assets.thermofisher.com / TFS-Assets / BID / Handbooks / 3d-cell-culture-handbook.pdf, page 18, or Lonza Bioscience Solutions.

[0248] Preferably, the second cell population is of the same species as the intended recipient, and most preferably, the second cell population is derived from the intended recipient. Preferably, the second cell population is a cell type characteristic of the intended quality / properties of the tissue or organ that the tissue scaffold is intended to replace or augment. For example, if the tissue scaffold is to be implanted in a human, the second cell population herein may preferably be human cells. Furthermore, for example, if the tissue scaffold is to be used to support vascular development, the second cell population herein preferably includes endothelial cells and / or smooth muscle cells.

[0249] In a most preferred embodiment, the second population of cells is endothelial cells.

[0250] According to one embodiment, the second tissue scaffold is regenerated with a second cell population. According to one embodiment, the fourth tissue scaffold is regenerated with a second cell population.

[0251] One of skill in the art will recognize that the culture time for repopulating a decellularized second or fourth tissue scaffold according to the present invention may depend on the size and density of the scaffold and the desired cellular structure obtained.

[0252] The tissue scaffold may be treated in a variety of ways either before or after seeding, for example, selected agents may be applied to the tissue scaffold to enhance cell attachment or growth.

[0253] Furthermore, with the goal of repopulating decellularized secondary or quaternary tissue scaffolds, various growth conditions can be selected to enhance the process.

[0254] Those skilled in the art will recognize that various growth conditions can be used and may vary depending on the selected cells used to repopulate the scaffold. Various growth conditions can be selected to stimulate the development of desired mechanical, physical or biochemical properties and / or to stimulate the migration of cells into the tissue scaffold. Such growth conditions may further include the use of supplemented growth media and / or the application of mechanical, electrical and / or chemical stimuli. Thus, in general, the decellularized tissue scaffold may be subjected to any of the tissue engineering steps according to the present invention in the manufacture of the tissue scaffold.

[0255] Having generally described the invention, a further understanding can be obtained by reference to specific embodiments that are provided herein for purposes of illustration only and are not intended to be limiting unless specifically stated. EXAMPLES

[0256] Example 1: Generation of ring-shaped tissue scaffolds 1.1 Tissue culture Adipose tissue-derived mesenchymal stem cells (AT-MSC, Lonza) were cultured at 37°C, 5% CO2 in a humidity-controlled incubator and supplemented with DMEM / F12 Glutamax growth medium (10% AB serum, 1% Pen-Strep, 5ng / ml FGF2). Cells were limited to less than 10 passages and subcultured prior to full cell confluency and use in tissue constructs.

[0257] 1.2 Preparation of hydrogel microbeads Alginate microbeads were generated using a two-stream flow-focus droplet chip (Wunderlichips, Switzerland) equipped with a 50 × 50 μm droplet-forming nozzle (Figure 1a). The continuous phase consisted of Novec 7500 fluorinated oil (3M, USA) containing 1% Pico-Surf (Sphere Fluidics, UK) as a surfactant.

[0258] Two dispersed phases were used: (1) 1.0% (wt) total alginate consisting of 0.5% NOVATACH™ VLVG 4GRGDSP (4GRGDSP-bound high G low MW alginate, G / M ratio ≥ 1.5, nominal viscosity 10-80 mPa.s, 69% guluronic acid monomer units, 33 kDa) and 0.5% PRONOVA UP LVG (20-200 mPa.s, 70% guluronic acid monomer units, 108 kDa) containing 80 mM CaEDTA and 20 mM HEPES at pH 7.0, and (2) 0.5% NOVATACH™ VLVG containing 80 mM ZnEDDA and 20 mM HEPES at pH 7.0. 1.0% (wt) total alginate consisting of 4GRGDSP (4GRGDSP-bound high G low MW alginate, G / M ratio ≥ 1.5, nominal viscosity 10-80 mPa.s, 69% guluronic acid monomer units, 33 kDa) and 0.5% PRONOVA UP LVG (20-200 mPa.s, 70% guluronic acid monomer units, 108 kDa).

[0259] The two aqueous phases meet in a parallel flow region within the microfluidic channel prior to droplet breakage. Flow rates were controlled by controlled injection using BD plastic syringes with PE plastic tubing (Scientific Commodities Inc.) attached to a syringe pump (Harvard Apparatus, PHD ULTRA) at 1000 μL h for the continuous phase and 1000 μL h for the continuous phase. -1 and 200 μL h for both aqueous phases. -1 was set to.

[0260] The microbeads were collected and the detergent was removed by adding perfluorooctanol (PFO, TCI Europe) at a 1:1 ratio to the generated bead volume. The beads were centrifuged at 1000 relative centrifugal force (rcf) for 1 min to remove the non-aqueous phase, then rinsed with 2:1 volume of complete growth medium and the collected beads were washed again. Any remaining non-aqueous residue was removed with a pipette. The final processed beads were stored at 4°C for at least 12 h in complete growth medium to swell to equilibrium size.

[0261] To measure bead size, an aliquot was taken, diluted 1:10 in complete growth medium, and applied to a glass slide. Transmitted light images were taken using an inverted optical microscope and a 4x objective. Beads were detected in the images using an algorithm based on the circular Hough transform. The diameter of the detected beads was then quantified from the images using the established pixel / micron ratio of the calibrated microscope, resulting in a size of 65.8 + / - 6.7 μm (SD).

[0262] 1.3 Preparation of bead-associated cells The cells from section 1.1 (Figure 1b) were mixed with the hydrogel beads from section 1.2 (Figure 1a) after a minimum of 12 hours of treatment with beads at a concentration of 10 million cells in complete growth medium / ml and 5.3 million beads / ml. The mixture was then incubated at 37 °C for 2 hours to promote cell attachment to the hydrogel beads (Figure 1c) and form bead-associated cells. To prevent cell settling, the mixture was gently mixed by tapping with a finger every 30 minutes.

[0263] 1.4 Formation of ring-shaped tissue scaffold Ring-shaped tissue scaffolds were formed by pouring into an agarose mold.

[0264] Similar to others (Strobel, Jove 2018), 2% agarose (w / v in base DMEM / F12 Glutamax growth medium) was poured into 6 cm culture dishes and a machined mold was used to create circular wells (3 mm inner diameter, 1 mm well thickness, 3 mm depth; Figure 3b). The agarose mold was sterilized under UV light for 2 h.

[0265] The mixture obtained in section 1.3 (Figure 3a) was centrifuged at 150 rcf for 5 min, excess medium was removed, and the bead-associated cells were transferred by micropipette into the agarose mold (Figure 3b) to a total volume of 30 μl per ring impression, filling the wells to a height of approximately 1 mm. The rings in the culture dish were cultured in a humidity-controlled incubator at 37 °C, 5% CO2, and a pO2 of 141.4 mmHg.

[0266] After 30 minutes in the agarose mold, 1 Tissue engineering medium (TE medium) was gently added to the dish (4 ml total) without disturbing the settled cell-bead mixture. Medium was changed at least twice a week. The tissue engineered rings were maintained for 6 weeks, at which point they were self-supporting.

[0267] 1 Tissue engineering medium (TE medium) consisted of complete growth medium (DMEM / F12 with supplements as described in 1.1) supplemented with proline (50 μg / ml) (Paz-Lugo 2018), glycine (50 μg / ml) (Paz-Lugo 2018), alanine (20 μg / ml) (ref) and ascorbic acid (50 μg / ml) (Murad 1981, Berg 1983) to enhance collagen production.

[0268] 1.5 Removal of hydrogel microbeads Alginate hydrogel beads were removed from the ring constructs obtained in section 1.4 by adding alginate lyase (Sigma, A1603) to a final concentration of 0.3 mg / ml to the dish containing the ring and incubated in TE medium for 30 min. The dish was then supplemented with fresh TE medium (Figure 2d). The tissue engineered rings were maintained for 6 weeks, with the medium being changed at least twice a week (Figure 2e).

[0269] 1.6 Decellularization The ring constructs obtained in section 1.5 were removed from the agarose mold and placed in a 15 ml conical tube containing 10 ml of a 1.5% solution of Tween® 20 in PBS. They were incubated for 18 hours at room temperature in the tube on a rocker (VWR rocker, speed 25, angle 10). The constructs were then transferred to another tube containing 10 ml of 40 U / ml DNase I (Sigma, D4513) diluted in PBS. They were incubated for 18 hours at room temperature in the tube on a rocker (VWR rocker, speed 25, angle 10). Decellularization was confirmed by imaging after staining with the nuclear binding dye Hoechst. The final product was free of cells as judged by the lack of intact nuclei and consisted only of cell-derived extracellular matrix.

[0270] Example 2: Self-supporting tissue scaffold 2.1 Tissue culture Adipose tissue-derived mesenchymal stem cells (AT-MSC, Lonza) were cultured at 37°C, 5% CO2 in a humidity-controlled incubator, supplemented with complete MEM alpha (Gibco catalog #32561-094) growth medium (supplemented with 10% FBS, 1% Pen-Strep, 10 μg / L hEGF, 720 mg / L hydrocortisone, 17.6 mg / L ascorbic acid, 10 mg / L insulin). Cells were limited to less than 10 passages and subcultured prior to full cell confluency and use in tissue constructs.

[0271] 2.2 Preparation of hydrogel microbeads Alginate microbeads were generated using a two-stream flow-focus droplet chip (Wunderlichips, Switzerland) equipped with a 50 × 50 μm droplet-forming nozzle (Figure 1a). The continuous phase consisted of Novec 7500 fluorinated oil (3M, USA) containing 1% Pico-Surf (Sphere Fluidics, UK) as a surfactant.

[0272] Two dispersed phases were used: (1) 1.0% (wt) total alginate consisting of 0.5% NOVATACH™ VLVG 4GRGDSP (4GRGDSP-bound high G low MW alginate, G / M ratio ≥ 1.5, nominal viscosity 10-80 mPa.s, 69% guluronic acid monomer units, 33 kDa) and 0.5% PRONOVA UP LVG (20-200 mPa.s, 70% guluronic acid monomer units, 108 kDa) containing 80 mM CaEDTA and 20 mM HEPES at pH 7.0, and (2) 0.5% NOVATACH™ VLVG containing 80 mM ZnEDDA and 20 mM HEPES at pH 7.0. 1.0% (wt) total alginate consisting of 4GRGDSP (4GRGDSP-bound high G low MW alginate, G / M ratio ≥ 1.5, nominal viscosity 10-80 mPa.s, 69% guluronic acid monomer units, 33 kDa) and 0.5% PRONOVA UP LVG (20-200 mPa.s, 70% guluronic acid monomer units, 108 kDa).

[0273] The two aqueous phases meet in a parallel flow region within the microfluidic channel prior to droplet breakage. Flow rates were controlled by controlled injection using BD plastic syringes with PTFE plastic tubing (Bohlender) attached to syringe pumps (New Era, InfusionONE and SyringeTWO) at 1000 µL h for the continuous phase and 1000 µL h for the continuous phase. -1 and 250 μL h for both aqueous phases. -1 was set to.

[0274] The microbeads were collected in tubes containing PFO (TCI Europe) (volume equal to 20% of the total continuous phase volume, calculated from the total run time) and basal growth medium (volume 1:1 to the aqueous phase). The collected beads were centrifuged at 230 relative centrifugal force (rcf) for 2 min, the non-aqueous phase was removed, and TE medium was added to the collected beads in a volume of 1:2 and left to swell. The final processed beads were stored at 4°C for at least 12 h in the medium mixture to swell to equilibrium size.

[0275] To measure bead size, a 30ul aliquot was taken, diluted with 70ul complete growth medium and applied to a glass slide. Transmitted light images were taken using an inverted optical microscope and a 4x objective. Beads were detected in the images using an algorithm based on the circular Hough transform. The diameter of the detected beads was then quantified from the images using the established pixel / micron ratio of the calibrated microscope, resulting in a size of 58.2+ / -1.7μm (SD).

[0276] The bead concentration is determined by taking a 50 ul aliquot of beads and diluting with 450 ul of TE media. 10 ul of the bead dilution is transferred to a hemocytometer chamber and counted manually.

[0277] 2.3 Preparation of bead-associated cells The cells from section 2.1 (Figure 1b) were mixed with the hydrogel beads from section 2.2 (Figure 1a) at a concentration of 6.2 million cells / ml in TE medium and 2.9 million beads / ml in TE medium, a minimum of 12 hours after bead treatment. The mixture was then incubated at 37 °C for 2 hours to promote cell attachment to the hydrogel beads (Figure 1c) and form bead-associated cells. To prevent cell settling, the mixture was gently mixed by tapping with a finger every 30 minutes.

[0278] 2.4 Formation of ring-shaped tissue scaffold Ring-shaped tissue scaffolds were formed by pouring into an agarose mold.

[0279] Pour 2% agarose (w / v in base DMEM / F12 Glutamax growth medium) into the wells of a 6-well multiplate and use a machined mold to create circular wells of two different sizes: 3 mm inner diameter, 1 mm well thickness (T1), 3 mm depth; and 3 mm inner diameter, 2 mm well thickness (T2), 4 mm depth.

[0280] The mixture obtained in section 2.3 was centrifuged at 230 rcf for 8 min, excess medium was removed, and the bead-associated cells were transferred by micropipette into the agarose molds to a total volume of 30 μl per ring impression for T1 rings and 80 ul for T2 diameter rings, filling the wells to a height of approximately 1 mm and 2 mm, respectively. A total of 30 rings were filled for each mold size, allowing evaluation of 3 rings at 10 different time points. The rings in the multiwell plates were cultured in a humidity-controlled incubator at 37 °C, 5% CO2, and a pO2 of 141.4 mmHg.

[0281] After 60 min in the agarose mold, TE medium was gently added to the dish (4 ml total) without disturbing the settled cell / bead mixture. Medium was changed at least twice a week. Tissue engineered rings were maintained for 5 days to monitor self-supporting properties.

[0282] 2.5 Brightfield imaging and tissue shrinkage Brightfield images (Zeiss PrimoVert) of tissue rings were taken 1 hour after molding (immediately after adding medium) (T1: Fig. 7a; T2: Fig. 8a), as well as 2, 6 (T1: Fig. 7b; T2: Fig. 8b), 12, 18, 24 (T1: Fig. 7c; T2: Fig. 8c), 48 (T1: Fig. 7d; T2: Fig. 8d), 72 (T1: Fig. 7e; T2: Fig. 8e), 96 (T1: Fig. 7f; T2: Fig. 8f) and 120 (T1: Fig. 7g; T2: Fig. 8g) hours after molding.

[0283] Using a 4x objective, two images of different areas of the three rings were taken per time point, ensuring that each image included the inner and outer edges of both the formed tissue and the mold. Images were then analyzed to obtain measurements of ring width and mold width to measure tissue shrinkage over time (T1: Figure 9a; T2: Figure 9b).

[0284] [Table 1]

[0285] Average / time point Two images were taken of different areas of the three rings per time point, providing a total of six images. The width of the ring and the width of the mold were measured for each image. Based on these measurements, the ring width:mold width ratio was calculated for each image (two images per ring) and the average ratio for each ring was calculated. There were three rings per time point, and the "average / time point value" represents the average ring width:mold width ratio for these three rings.

[0286] [Table 2]

[0287] Average / time: Two images were taken of different areas of the three rings per time point, providing a total of six images. The width of the ring and the width of the mold were measured for each image. Based on these measurements, the ring width:mold width ratio was calculated for each image (two images per ring) and the average ratio for each ring was calculated. There were three rings per time point, and the "average / time point value" represents the average ring width:mold width ratio for these three rings.

[0288] 2.6 Self-supporting assessment: manual removal from agarose mold After imaging, demolding (removal from the mold in the dish) of three rings / time points was attempted using curved tweezers. Qualitative results detailing the self-supporting properties of the rings at each time point were recorded. Agarose "wells" containing rings that were not yet self-supporting were cut out of the mold, and both the contained ring material and the excised agarose were transferred together into a sample tube.

[0289] By 6 hours after molding, some tissue rings showed some self-supporting properties, such as bottoming out of the mold along the inner post, but did not survive handling and removal from the dish using tweezers. By 96 hours after molding, it was possible to remove the tissue rings from the dish and transport them while they maintained their ring shape (FIG. 6), and therefore these rings were considered self-supporting in the context of this application. The average shrinkage of T1 tissue rings at this time is 31.3% and 30% relative to T2 (68.7% and 70.0% of the mold width, respectively). Variation and error in these measurements occurs due to some rings in the mold moving up to the inner post, changing the focal length and relative width of the ring compared to the edge of the mold area.

Claims

1. A method for producing a tissue scaffold, wherein the method comprises the following steps: a) A step of providing a first group of cells and biocompatible hydrogel beads, b) A step of combining the first cell group with the biocompatible hydrogel beads to obtain a first cell-bead mixture, c) A step of culturing the first cell-bead mixture to generate an extracellular matrix and thereby obtain a first tissue scaffold, d) If necessary, subject the first tissue scaffold to mechanical conditioning to obtain a mechanically conditioned first tissue scaffold. Includes, - The first group of cells is a scaffold-dependent cell capable of generating and secreting extracellular matrix, - The biocompatible hydrogel beads support the attachment of the first group of cells, - The first cell group and the biocompatible hydrogel beads from step b) are brought together in a mold to obtain the first cell bead mixture, or A method comprising incubating the first cell population and the biocompatible hydrogel beads in step b) to enable the formation of bead-associated cells, and then transferring the bead-associated cells to a mold to obtain the first cell-bead mixture.

2. The method according to claim 1, wherein the first cell-bead mixture in step c) is cultured until at least the first tissue scaffold becomes self-supporting.

3. The method according to claim 1, wherein step d) is essential.

4. - The first tissue scaffold or the mechanically conditioned first tissue scaffold is subjected to means for dissolving or decomposing the biocompatible hydrogel beads, thereby obtaining a third tissue scaffold, or - The biocompatible hydrogel beads are self-degradable or self-soluble, and the first tissue scaffold or the mechanically conditioned first tissue scaffold is cultured until the biocompatible hydrogel beads self-dissolve or self-degrade, thereby obtaining a third tissue scaffold. The method according to claim 1.

5. The method according to claim 4, wherein the third tissue scaffold is subjected to mechanical conditioning to obtain a mechanically conditioned third tissue scaffold.

6. The method according to claim 4, wherein the third tissue scaffold is subjected to decellularization to obtain a fourth tissue scaffold.

7. The method according to claim 5, wherein the mechanically conditioned third tissue scaffold is subjected to decellularization to obtain a fourth tissue scaffold.

8. The method according to claim 6, wherein the fourth tissue scaffold is incubated in the presence of a second cell population to obtain a fourth tissue scaffold regenerated with the second cell population.

9. The method according to claim 7, wherein the fourth tissue scaffold is incubated in the presence of a second cell population to obtain a fourth tissue scaffold regenerated with the second cell population.

10. The aforementioned mold, - Made from bioinert materials, - Designed to allow the exchange of nutrients and gases between the outside and inside of the mold, - Completely or partially immersed in cell culture medium, The method according to claim 1.

11. The method according to claim 1, wherein the biocompatible hydrogel bead is a biocompatible alginate bead having one or more cell adhesion ligands on its outer surface.

12. The method according to claim 1, wherein the biocompatible hydrogel beads are biocompatible hydrogel microbeads.

13. The method according to claim 12, wherein the biocompatible hydrogel microbeads have an average size in the range of 10 to 200 μm in their maximum dimensions.

14. The method according to claim 1, wherein at least 60% of the biocompatible hydrogel beads have a size in the range of 10 to 200 μm in their maximum dimensions.

15. The first organization is a scaffold, - The first group of cells, - Biocompatible hydrogel beads that support the adhesion of the first group of cells, - The extracellular matrix generated by the first group of cells, Includes, - The first group of cells is a scaffold-dependent cell capable of generating and secreting extracellular matrix, - The first group of cells is attached to the outer surface of the biocompatible hydrogel beads, - The first group of cells and the biocompatible hydrogel beads are embedded in the extracellular matrix. The first organization is the scaffold.

16. The first tissue scaffold according to claim 15, wherein the biocompatible hydrogel beads are biocompatible alginate beads having one or more cell adhesion ligands on their outer surface.

17. The first tissue scaffold according to claim 15, wherein the biocompatible hydrogel beads are biocompatible hydrogel microbeads.

18. The first tissue scaffold according to claim 17, wherein the biocompatible hydrogel microbeads have an average size in the range of 10 to 200 μm in their maximum dimensions.

19. The first tissue scaffold according to claim 17, wherein at least 60% of the biocompatible hydrogel beads have a size in the range of 10 to 200 μm in their maximum dimensions.

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