3D perfusable tissue construct with crosslinking initiator

Abstract

41 ABSTRACT A method of genera ng a perfusable structure for cell and / or ssue cul va on is provided, the method comprising the steps of: providing a crosslinkable composi on (20), providing a sacrificial scaffold (10) separately from the crosslinkable composi on (20), wherein the sacrificial scaffold (10) comprises a matrix polymer and a crosslinking ini ator for ini a ng crosslinking of the crosslinkable composi on (20), wherein the matrix polymer is configured for releasing the crosslinking ini ator when the sacrificial scaffold (10) is in contact with the crosslinkable composi on (20), a er separately providing the sacrificial scaffold (10), embedding the sacrificial scaffold (10) in the crosslinkable composi on (20) such that the sacrificial scaffold (10) comes into contact with the crosslinkable composi on (20), and releasing the crosslinking ini ator from the matrix polymer into the crosslinkable composi on (20) to crosslink a layer (22) of the crosslinkable composi on around the sacrificial scaffold (10), the crosslinked layer (22) forming at least one channel (28). Figure 3A

Description

[0001] 3D perfusable tissue construct with crosslinking initiator

[0002] Field of the invention

[0003] The present invention relates to a method of generating a perfusable network structure for cell and / or tissue cultivation. More particularly, the present invention relates to a method, which makes it possible to purposefully fabricate interconnected vascular-like and perfusable channel networks that can be used for cell and / or tissue cultivation. The present invention further relates to a perfusable network structure provided by such a method.

[0004] Background of the invention

[0005] The fabrication of perfusable network structures is an important technology in the fields of basic research, medicine, and pharmacy. Perfusable network structures can mimic natural vascularization and can provide valuable insights into cell biology, tissue engineering and organ development.

[0006] The creation of a complex vascular network structure is still a key challenge. Various techniques are known to provide vascular network structures, such as vessel sprouting, bioprinting and templating. All of these techniques, however, are very expensive, nonstandardized and time-consuming.

[0007] One approach for generating a perfusable three-dimensional network structure is provided in US 2023 / 0 398 803 Al. The authors propose to use a printable composition including sacrificial ink and a crosslinking agent. The printable composition is injected into a surrounding hydrogel containing crosslinkable polymers. When the printable composition is injected into the hydrogel, the crosslinking agent diffuses into the hydrogel and forms a diffusion layer around the sacrificial ink. Activation of the crosslinking reaction, e.g. activation of a photocrosslinking reaction using UV irradiation and Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) as crosslinking agent, leads to a selective crosslinking of the hydrogel around the sacrificial ink. The sacrificial ink is then removed, for example by melting the ink at temperatures from about 25°C to about 37°C. As a result, a hollow perfusable network structure is generated.

[0008] Although US 2023 / 0 398 803 Al provides a method for fabricating a three-dimensional perfusable network structure, the method has one main drawback. For example, a penetration depth of the crosslinking agent into the surrounding hydrogel and therewith a wall thickness of the crosslinked shell around the sacrificial ink varies depending on the length and / or complexity of the strands forming the network structure. Especially large and complex structures may suffer from this effect. Therefore, the high complexity and small sizes needed for micro-vascularization may not be achievable using the technique proposed in US 2023 / 0 398 803 Al.

[0009] It is thus an object of the present invention to provide an improved method for fabricating / generating a perfusable network structure for cell and / or tissue cultivation. In particular, it is an object of the present invention to solve or ameliorate at least some of the drawbacks encountered in the prior art.

[0010] Solution to the problem

[0011] These and other objects, which become apparent upon reading the description, are solved by the subject-matter of the independent claims. Further embodiments and developments are provided in the dependent claims.

[0012] According to a first aspect of the present invention, a method of generating a perfusable structure for cell and / or tissue cultivation is provided. The perfusable structure is suitable for mimicking natural vascularization. The method comprises: providing a crosslinkable composition; providing a sacrificial scaffold separately from the crosslinkable composition, wherein the sacrificial scaffold comprises a matrix polymer and a crosslinking initiator for initiating crosslinking of the crosslinkable composition, and wherein the matrix polymer is configured for releasing the crosslinking initiator when the sacrificial scaffold is in contact with the crosslinkable composition; after separately providing the sacrificial scaffold, embedding the sacrificial scaffold in the crosslinkable composition such that the sacrificial scaffold comes into contact with the crosslinkable composition; and releasing the crosslinking initiator from the matrix polymer into the crosslinkable composition to crosslink a layer of the crosslinkable composition around the sacrificial scaffold, the crosslinked layer forming at least one channel.

[0013] The present invention is at least partially based on the idea that the sacrificial scaffold is provided separately from the crosslinkable composition. In other words, the sacrificial scaffold may be fabricated, formed, and / or given its intended shape outside the crosslinkable composition. This may include printing (e.g., 3D printing, such as by fused filament deposition), molding (e.g., injection molding), drying, and / or cooling the sacrificial scaffold outside the crosslinkable composition.

[0014] The present invention is further based on the idea that only after separately providing the sacrificial scaffold, the sacrificial scaffold as a whole is embedded into the crosslinkable composition. In this way a more uniform penetration depth of the crosslinking initiator into the crosslinkable composition may be achieved by bringing the separately provided sacrificial scaffold into contact with the crosslinkable composition. Without wanting to be bound by theory, it is believed that this effect is achieved because, when the separately provided sacrificial scaffold as a whole is embedded into the crosslinkable composition, the crosslinking initiator is released from the matrix polymer along the entire length and / or structure of the sacrificial scaffold at the same time. In other words, upon contact of the sacrificial scaffold with the crosslinkable composition, the crosslinking initiator may be released into the crosslinkable composition. This is believed to provide for a more uniform penetration depth of the crosslinking initiator into the crosslinkable composition and thus a more uniform crosslinked layer around the sacrificial scaffold. The method presented in US 2023 / 0 398 803 Al, on the other hand, uses an aqueous solution of a sacrificial ink that is printed into a hydrogel using an injection nozzle. The injection nozzle is moved in a desired three-dimensional shape to provide the three-dimensional network structure. Due to the printing process of US 2023 / 0 398 803 Al, the crosslinking agent (crosslinking initiator) is released from the ink as soon as the ink comes into contact with the hydrogel. As a result, diffusion of the crosslinking agent into the hydrogel is started as soon as the strands of the network structure are generated (printed). These different onsets of diffusion, however, have been found to lead to non-uniform penetration depth of the crosslinking agent and thus to non-uniform wall thicknesses which are of disadvantage when it comes to the generation of complex micro-vascularized network structures. In addition, as the printing process proposed in US 2023 / 0 398 803 Al requires an aqueous solution of sacrificial ink, there are several drawbacks, which come with the printing process. For example, the printing process is relatively slow and / or only specific aqueous solutions which are printable can be used. The proposed method according to the present invention, however, does not use a printable ink and the matrix polymer is not a printable ink. The sacrificial scaffold as proposed is not necessarily printed. The sacrificial scaffold can be prepared outside and separately from the crosslinkable composition. This speeds up the fabrication process. The separately fabricated sacrificial scaffold may even be shelved and used later on.

[0015] As noted above, the sacrificial scaffold comprises a matrix polymer and a crosslinking initiator. In other words, the sacrificial scaffold preferably does not only comprise the matrix polymer but also the crosslinking initiator. In still other words, both the crosslinking initiator and the matrix polymer preferably are included in the sacrificial scaffold. This sacrificial scaffold (including the matrix polymer and the crosslinking initiator) preferably is separately provided from the crosslinkable composition.

[0016] The matrix polymer and the crosslinking initiator may be formed into the sacrificial scaffold by any technique known to those skilled in the art. For example, the matrix polymer and the crosslinking initiator may be provided as a blend (e.g., by mixing respective pellets, powders, or liquids), which may then be processed by known techniques (such as, e.g., casting, extrusion, injection molding, 3D printing, or melt electro-writing). Alternatively or additionally, the matrix polymer and the crosslinking initiator may be separately provided into the screw of an extruder or injection molding machine and blended therein.

[0017] For example, the matrix polymerand / orthe crosslinking initiator may be dissolved in a solvent. Preferably, a solvent in which both the polymer and the crosslinking initiator are soluble is used. However, also a dispersion of one of the matrix polymer and the crosslinking agent in the other one of the matrix polymer and the crosslinking agent is envisaged as a possibility. Preferably, the solvent is subsequently removed such that a dry mixture (e.g., as a powder or pellets) is obtained, which can then be processed into the sacrificial scaffold via the well- known techniques mentioned above.

[0018] A preferred biocompatible solvent that may be used in the context of the present invention is water, but other solvents may be used as well. When water is used as a solvent, preferably, the matrix polymer and the crosslinking agent are water-soluble. One example of a preferred water-soluble matrix polymer is poly(2-cyclopropyl-oxazoline). One example of a preferred water-soluble crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (available, e.g., as LAP, Catalog #5269, from Advanced BioMatrix, Inc., Carlsbad, CA 92010, U.S.A). Preferably the water is removed to obtain a dry mixture of the matrix polymer and the crosslinking agent (e.g., a dry polymer / LAP mixture).

[0019] Preferably, the solvent (e.g. water) is removed by lyophilization to obtain the dry mixture of matrix polymer and crosslinking agent that can subsequently be processed further via the well- known techniques mentioned above (e.g., casting, extrusion, injection molding, 3D printing, or melt electro-writing).

[0020] Preferably, the matrix polymer is a thermoplastic matrix polymer, such as polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolic acid (PGA), Poly(N-isopropylacrylamide), poly(vinylcaprolactam), polyvinylmethylether, poly(oxazoline) (preferably poly(2-oxazoline), more preferably a poly(2-cyclopropyl-2-oxazoline)). By using a thermoplastic matrix polymer, self-supported and free-standing network structures may be separately provided. These structures may be prefabricated and may be shipped to a customer using these structures, e.g. for cell and / or tissue cultivation. The method of US 2023 / 0 398 803 Al, on the other hand, uses an aqueous solution of a sacrificial ink which by itself is unstable. The aqueous solution needs to be printed into the hydrogel for forming the structure. Shipping of prefabricated structures using the technique of US 2023 / 0 398 803 Al is not possible. Preferably, the crosslinkable composition comprises a first hydrogel. The first hydrogel may comprise at least one type of crosslinkable monomers or polymers, which may be selected in accordance with the chosen crosslinking mechanism. Examples of crosslinking mechanisms may be enzymatic crosslinking, small molecule crosslinking or photocrosslinking. Preferably, photocrosslinking may be used. A polymer suitable for photocrosslinking is, for example, methacrylated gelatin (GelMA).

[0021] Preferably, the method further comprises the step of removing the sacrificial scaffold, preferably wherein removing the sacrificial scaffold includes dissolving the matrix polymer. The step of removing or dissolving the sacrificial scaffold may occur separately from (e.g., after) or, at least partially, concurrently with the step of releasing the crosslinking initiator from the matrix polymer.

[0022] Preferably, the crosslinked layer forms a network of channels, preferably an interconnected network of channels, more preferably a three-dimensional network of channels.

[0023] The channels preferably have an inner diameter (i.e. a diameter of a lumen extending therethrough) of less than 1 mm, more preferably less than 500 micrometers, more preferably less than 300 micrometers, more preferably less than 200 micrometers, more preferably less than 100 micrometers, such as less than 50 micrometers or even smaller.

[0024] Alternatively or additionally, the channels preferably have an inner diameter of more than 10 micrometers, more preferably more than 25 micrometers, even more preferably more than 50 micrometers.

[0025] Preferably, the sacrificial scaffold is three-dimensional, and / or the sacrificial scaffold comprises one or more filaments, preferably wherein the sacrificial scaffold comprises a plurality of connected filaments forming a network, the filaments preferably extending in at least two nonparallel directions, and / or the sacrificial scaffold has at least one first node at which one filament branches into two or more filaments, and / or the sacrificial scaffold has at least one second node at which two or more filaments merge into one filament. The sacrificial scaffold may be configured for mimicking natural vascularization.

[0026] Preferably, the step of releasing the crosslinking initiator from the matrix polymer comprises releasing the crosslinking initiator by diffusion. For example, the crosslinking initiator may be released due to osmotic pressure.

[0027] Preferably, the step of releasing the crosslinking initiator from the matrix polymer may comprise swelling, preferably by uptake of water, and / or dissolution of the matrix polymer, preferably in an aqueous solution. "Swelling" preferably refers to an uptake of water molecules, such as hydration, without instant dissolution of the matrix polymer. For example, where a water-soluble polymer matrix is used, both diffusion and swelling may occur upon contact with the crosslinkable composition. Thereby the crosslinking initiator may be released into the crosslinkable composition.

[0028] Preferably, the matrix polymer is water soluble, and / or comprises or consists of polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolic acid (PGA), Poly(N-isopropylacrylamide), a carbohydrate-based material (such as isomalt), or a thermo-responsive polymer.

[0029] Preferably, the matrix polymer comprises, or consists of, a thermo-responsive polymer that is not soluble in aqueous solutions above a lower critical solution temperature and becomes soluble in aqueous solutions below the lower critical solution temperature (LCST).

[0030] Preferably, the thermo-responsive polymer is poly(vinylcaprolactam), polyvinylmethylether, or poly(oxazoline), preferably poly(2-oxazoline), more preferably a poly(2-cyclopropyl-2- oxazoline).

[0031] Preferably, the thermo-responsive polymer is not dissolvable (or substantially not dissolvable) in water at human body temperature and becomes dissolvable in water at a temperature below 30°C, preferably below 25°C. Human body temperature, as referred to herein, is preferably a temperature in the range of 35 °C to 40 °C, such that the polymer preferably is not dissolvable at a temperature falling within this range. "Not dissolvable" includes in this context that the polymer is a solid at this temperature.

[0032] The thermo-responsive polymer may dissolve in water within 30 minutes, preferably within 20 minutes, and more preferably within 15 minutes, at 25°C.

[0033] The thermo-responsive polymer may have a lower critical solution temperature (LCST) in water in a range of 4°C to 36°C, preferably 4°C to 30°C, more preferably in a range of 4 °C to 25 °C. The reduction of temperature for a short time period is a very cell friendly and biocompatible stimulus without the need for organic solvents. Furthermore, compared to channel creation via sugar-based fibers, no osmotic pressure is introduced. This may avoid a hypertonic environment and may thus, inter alia, reduce cell death (e.g., when cells are provided in the crosslinkable composition, as discussed in more detail below).

[0034] The thermo-responsive polymer may have a glass transition temperature (Tg) in a range of 30°C to 100°C, preferably 40°C to 100°C, and still more preferably 50°C to 100°C. The glass transition temperature may be determined via differential scanning calorimetry (DSC), e.g. with a heating rate of 20 °C / min. With a high glass transition temperature, sufficient stiffness at room temperature can be ensured. The sacrificial scaffold and / or the device can thus be handled, shipped and / or stored without the requirement of cooling or a special atmosphere.

[0035] The thermo-responsive polymer may be a poly(oxazoline).

[0036] The thermo-responsive polymer may comprise or consist of repeating units derived from 2- oxazoline or 2-substituted 2-oxazolines as monomers. Preferably, the thermo-responsive polymer is a poly(2-oxazoline) polymer, i.e. a polymer wherein all repeating units are derived from 2-oxazoline or 2-substituted 2-oxazolines as monomers. It is particularly preferred that the thermo-responsive polymer is a homo- or copolymer consisting of repeating units of the formula -N(C(O)R1)-CH2-CH2- which are provided by 2-substituted 2-oxazolines as monomers. R1is selected from C1-C6 alkyl and C3-C6 cycloalkyl. For example, it is possible to adjust the LCST of the thermo-responsive polymer via copolymerization of 2-oxazolines carrying different 2-substituents. It is preferably an n-propyl group or a cyclopropyl group, and most preferably a cyclopropyl group. Thus, preferred specific examples of a poly(2-oxazoline) for use as the thermo-responsive polymer in the context of the invention are poly(2-n-propyl-2-oxazoline) and / or poly(2-cyc / opropyl-2-oxazoline). More preferably, the thermo-responsive polymer comprises, and most preferably consists of, poly(2-cyc / opropyl-2-oxazoline). With these poly(2-oxazolines), the above-mentioned LCST may be provided. Moreover, a further advantageous feature of, for example, poly(2-cyc / opropyl-2-oxazoline) is swelling in aqueous solutions. After adding the scaffold to an aqueous solution, the filaments swell because the polymer chains get hydrated. This effect may be employed to release the crosslinking initiator from the polymer matrix into the first hydrogel.

[0037] The thermo-responsive polymer is preferred because it is possible to adjust the solubility and the material remains stable for storage for sufficient time. In particular, a low hygroscopicity may be helpful so that the filaments maintain their stability. Thermo-responsive polymers with a higher glass transition temperature (Tg) may provide for sufficient stiffness.

[0038] Poly(oxazolines) have a high bio-compatibility. Poly(2-cyc / opropyl-2oxazloine) or a copolymer comprising cyc / opropyl-oxazoline and another oxazoline are preferred since the material is relatively stiff (advantageous regarding the stability when filling the hydrogels), have a low hygroscopicity, and have a relatively high glass transition temperature (Tg) that is adequate for stable fixation, e.g. when filling the hydrogel into the chamber. The material can also be processed with various forming processes.

[0039] Preferably, when using a thermos-responsive polymer, removing the sacrificial scaffold comprises the step of reducing the temperature below the lower critical solution temperature such that the sacrificial scaffold dissolves. This preferred embodiment is based on the idea that by using a thermo-responsive polymer as matrix polymer, the diffusion of the crosslinking initiator into the crosslinkable composition can be separated from the removal of the sacrificial scaffold. In other words, by using the thermo-responsive polymer, the wall thickness of the perfusable structure can be adjusted independently from the removal of the sacrificial scaffold. In particular, the sacrificial scaffold may remain in place util the channel wall is advantageously stable for removal of the scaffold.

[0040] Preferably, the crosslinkable composition and the crosslinking initiator are suitable for photocrosslinking and the method further comprises the steps of: exposing the crosslinkable composition to radiation, such as ultraviolet light. This preferred embodiment is based on the idea that photocrosslinking offers the possibility to initiate the crosslinking process by exposing the diffusion layer around the sacrificial scaffold to light, preferably UV light. One example of a crosslinking initiator suitable for photocrosslinking is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP). One example of a polymer suitable for photocrosslinking is methacrylated gelatin (GelMA).

[0041] Preferably, the method comprises the step of: after embedding the sacrificial scaffold in the crosslinkable composition, waiting for a predetermined period of time before exposing the crosslinkable composition to radiation. This is based on the idea that a contact or diffusion time prior to exposing the sacrificial scaffold to UV light affects the thickness of the diffusion layer and thus the wall thickness of the tissue construct. Examples of diffusion times until UV irradiation are 10 minutes, preferably 5 minutes, more preferably 2 minutes, more preferably less than 2 minutes, such as 1-2 minutes.

[0042] Preferably, the step of exposing the crosslinkable composition to radiation is performed for a predetermined duration. The exposure time may affect the amount of crosslinking and thus may affect the rigidity / viscosity of the crosslinked layer surrounding the sacrificial scaffold. Examples for irradiation times are 10 minutes, preferably 5 minutes, more preferably 2 minutes, more preferably less than 2 minutes, more preferably 1 minute or less than one minute such as 20-60 seconds.

[0043] Preferably, the method comprises a step of removing crosslinkable composition that has not been crosslinked from around the crosslinked layer, preferably by washing. Hydrogels may be removed (washed) from the crosslinked layer. Preferably, the method further comprises freeze-drying and / or cryopreserving the perfusable structure (e.g., the channel network remaining after washing the crosslinkable composition that has not been crosslinked and / or dissolving the sacrificial scaffold). The stable freeze-dried and / or cryopreserved perfusable structure may be shipped to the customer for perfusion applications.

[0044] Preferably, the crosslinkable composition comprises at least one type of cells, preferably endothelial cells such as umbilical vein endothelial cells, smooth muscle cells, astrocytes, stellate cells, such as stellate macrophages cells (Kupffer cells), pericytes, macrophages, mesangial cells or othertypes of cells. Cells may be used for mimicking tunica media of a blood vessel.

[0045] Preferably, the cells and more preferably the endothelial cells may be encapsulated in the crosslinked layer. The cells may be provided within a hydrogel such as within a first hydrogel.

[0046] Preferably, the method further comprises the step of seeding tissue and / or cells around the at least one channel, preferably around the three-dimensional network of channels. Examples of cells are all cell types including and without limitation endothelial cells, pericytes, muscle cells including smooth muscle cells, cardiomyocytes, organ cells (such as hepatocytes), stem cells, fibroblasts, hematopoietic cells, stromal cells, plant cells, podocytes, etc.

[0047] Preferably, the step of seeding tissue and / or cells around the at least one channel comprises filling a second hydrogel into a cavity surrounding the at least one channel. Various cells or types of cells may be provided in different layers in or around the channels.

[0048] Preferably, the method further comprises the step of seeding tissue and / or cells within the at least one channel, preferably within a three-dimensional network of channels. For example, endothelial cells such as for mimicking a tunica intima of a blood vessel, and / or umbilical vein endothelial cells may be used. Cells may be seeded, e.g., by flowing liquid with these cells through the channels. Preferably, the method further comprises the steps of: cryopreserving the perfusable structure together with the seeded tissue and / or cells, and vitally thawing the cryopreserved perfusable structure such that the tissue and / or cells are vital and perfusable. This is based on the idea that a vascular network structure with cells may be preserved, such as cryopreserved, and may be shipped to a customer who vitally thaws the preserved vascular network structure such that the cells are vital and perfusable.

[0049] Preferably, the method further comprises the steps of: removing the crosslinkable composition that has not been crosslinked from around the crosslinked layer, preferably by washing, wherein the crosslinkable composition is a first crosslinkable composition; embedding the sacrificial scaffold in a second crosslinkable composition, allowing the crosslinking initiator to diffuse into the second crosslinkable composition and crosslinking a second layer of the second crosslinkable composition around the crosslinked first layer of the first crosslinkable composition. This preferred embodiment is based on the idea that different layers may be provided around the hollow channels. The different layers may include the same or different types of cells. Preferably, the sacrificial scaffold is only removed after all crosslinkable layers have been crosslinked.

[0050] Preferably, the second crosslinkable composition comprises at least one type of cells, preferably fibroblasts. This is based on the idea that fibroblasts may be used for mimicking tunica adventitia of a blood vessel. The second crosslinkable composition may comprise at least one third hydrogel in which the cells are provided. Examples of cells are all cell types including and without limitation endothelial cells, pericytes, muscle cells including smooth muscle cells, cardiomyocytes, organ cells (such as hepatocytes), stem cells, fibroblasts, hematopoietic cells, stromal cells, plant cells, podocytes, etc.

[0051] Preferably, the cells are encapsulated in the crosslinked second layer.

[0052] Preferably, the sacrificial scaffold is formed by one of 3D printing, preferably by Fused Deposition Modeling (FDM), injection molding, such as micro-injection molding, and melt electro-writing. Preferably, the step of providing a sacrificial scaffold separately from the crosslinkable composition, wherein the sacrificial scaffold includes a matrix polymer and a crosslinking initiator, includes the step of: dissolving the crosslinking initiator and the matrix polymer in a solvent. Preferably, the solvent is water. Preferably, the crosslinking initiator and the matrix polymer are dissolved simultaneously in the solvent. Alternatively, in a first step, one of the crosslinking initiator and the matrix polymer may be dissolved in the solvent, and in a subsequent second step, the other of the crosslinking initiator and the matrix polymer may be dissolved in the mixture of solvent and crosslinking initiator or matrix polymer. Preferably after dissolving the crosslinking initiator and matrix polymer, the dissolution is dried such by lyophilization.

[0053] Alternatively or additionally, the step of providing a sacrificial scaffold separately from the crosslinkable composition, wherein the sacrificial scaffold includes a matrix polymer and a crosslinking initiator, may include shaping a mixture and / or blend of the matrix polymer and the crosslinking agent (e.g., a mixture and / or blend of one or more pellets, one or more powders, and / or one or more liquids). The mixture may be a solution (e.g., a dried solution).

[0054] Shaping the mixture may include melting at least the matrix polymer within said mixture, e.g. for casting, injection molding, extruding, 3D printing (e.g., fused deposition modelling), melt electro-writing, or other micro-fabrication techniques.

[0055] According to a second aspect of the present invention, a perfusable structure for cell and / or tissue cultivation is provided. The perfusable structure is provided by the method according to the first aspect and / or preferred embodiments thereof.

[0056] Preferably, the perfusable structure comprises a crosslinked layer of crosslinkable composition surrounding at least one perfusable and / or hollow channel, preferably a three-dimensional network of perfusable and / or hollow channels.

[0057] Preferably, the perfusable structure comprises a first layer of cells, such as endothelial cells, arranged within the at least one channel. Preferably, the perfusable structure comprises a second layer of cells, such as smooth muscle cells, arranged within the crosslinked layer.

[0058] Advantageous embodiments of the first aspect are advantageous embodiments of the second aspect, and vice versa.

[0059] Brief description of the drawings

[0060] Figures 1A-1C are microscopic images of examples of sacrificial scaffolds used to provide perfusable network structures according to the present invention.

[0061] Figure 2A are schematic images for illustrating the process of fabricating a perfusable network structure according to an embodiment of the present invention.

[0062] Figure 2B shows a microscopic image of a fabricated perfusable network structure after freeze drying and a detailed view of a fluid tight connection to an apparatus for fabricating the perfusable network structure.

[0063] Figure 3A is an annotated microscopic image of a section of a perfusable network structure that includes a crosslinked layer around a network of perfusable channels.

[0064] Figure 3B shows annotated microscopic images obtained during fabricating the perfusable network structure.

[0065] Figure 4A is schematic representation of how a thickness of a crosslinked layer may be adjusted.

[0066] Figure 4B is a collage of microscopic images of a filament of the sacrificial scaffold highlighting the effect of increased diffusion times on the thickness of the crosslinked layer. Figure 5 is a photograph of one example of a vascularized network.

[0067] Figure 6 is a microscopic image of one example of a channel, wherein cells are seeded in the channel and wherein cells are encapsulated in a crosslinked layer around the channel.

[0068] Figure 7 are further microscopic images (with staining) of another example of a channel, wherein cells are seeded in the channel and wherein cells are encapsulated in a crosslinked layer around the channel; before cryopreservation and thawing (left) and after cryopreservation and thawing (right).

[0069] Figure 8 is a schematic representation of a channel having multiple layers.

[0070] Figure 9 shows schematic images of fabricating a perfusable network structure with multiple cell layers and / or cell types.

[0071] Figure 10 are microscopic images of a channel with seeded cells obtained by the methods and structures according to the invention.

[0072] Detailed description

[0073] Within the figures, same components are referenced by the same reference numerals. The figures only show exemplary embodiments for facilitating the full understanding of the invention. The figures shall not be construed to limit the scope and spirit of the present invention.

[0074] Figures 1A-1C show three examples of sacrificial scaffolds 10 used to provide perfusable network structures according to an aspect of the present invention.

[0075] In Figure 1A, a first example of a sacrificial scaffold 10 is shown. The sacrificial scaffold 10 includes multiple filaments 12. The filaments 12 are connected to one another forming a network of filaments. One filament 12 branches at a first node 14 forming two further filaments 12. Each of these filaments 12 then branch at another node 14 into another two filaments 12. The filaments 12 merge into one filament at second nodes 16.

[0076] Figure IB shows another example of a sacrificial scaffold 10. Compared to the example shown in Figure 1A, the example shown in Figure IB is a more complex sacrificial scaffold 10 with more filaments 12 that branch and merge at nodes 14, 16.

[0077] Figure 1C shows another example of a sacrificial scaffold 10. In the example of Figure 1C, the filaments 12 branch off and merge at nodes 14, 16, and even cross each other, e.g. at nodes 18. At some of the nodes 14, 16, more than two filaments 12 branch off and / or merge.

[0078] Figures 1A-1C show only some of many examples of how sacrificial scaffolds 10 may look like. The sacrificial scaffolds 10 may have various shapes and may mimic natural vascularization. The sacrificial scaffolds 10 may be two-dimensional or three-dimensional. The sacrificial scaffolds 10 may have any suitable shape. The sacrificial scaffolds 10 may be used to provide a perfusable network structure such as a perfusable network structure suitable for cell and / or tissue cultivation.

[0079] The scaffolds 10 may be manufactured via, for example, a 3D printing processes, Fused Deposition Modeling (FDM), injection molding, such as micro-injection molding, melt electrowriting, freeform printing or other suitable microfabrication processes. Preferably, melt electro-writing is used, which allows to create scaffolds 10 with very thin filaments. Such filaments may emulate a micro-vascular network.

[0080] The sacrificial scaffold 10 comprises a matrix polymer. The matrix polymer may be thermoplastic matrix polymer. Thermoplastic matrix polymers may be self-supporting and / or free-standing. Thus, the sacrificial scaffold 10 generated using the methods described above may be self-supporting and / or stable. "Self-supporting" may refer to the fact that the sacrificial scaffold is able to maintain its shape under the influence of gravity, for example at a temperature of 25 °C or 37 °C. The prefabricated sacrificial scaffold includes the crosslinking initiator incorporated or embedded in the matrix polymer. The matrix polymer may be water soluble.

[0081] The matrix polymer may be polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolic acid (PGA), Poly(N-isopropylacrylamide), a carbohydrate-based material (such as isomalt), or a thermo-responsive polymer, such as poly(vinylcaprolactam) or polyvinylmethylether.

[0082] Preferably, a thermo-responsive polymer that becomes soluble at temperatures below a critical solution temperature, the so-called lower critical solution temperature (LCST), in an aqueous solution may be employed. The LCST of the sacrificial scaffold 10 may be configured to be within the physiological temperature range of 4 to 37°C to avoid temperature-related damages to the cells during dissolution of the scaffold 10. Preferably, the thermo-responsive polymer becomes soluble in an aqueous solution at temperatures below 30°C, for example at temperatures below 25°C. For example, the scaffold 10 may be made from a poly(2-oxazoline) or a poly-N-isopropylacrylamide.

[0083] Referring to Figure 2A, schematic images for illustrating a process of fabricating a perfusable network structure according to the present invention are shown.

[0084] On the left-hand side of Figure 2A, one example of a sacrificial scaffold 10 is shown. The sacrificial scaffold 10 may have been produced by any of the methods described above, such as 3D printing, injection molding or melt electro-writing. The sacrificial scaffold 10 includes various strands 12 and nodes 14 as explained in connection with Figure 1A-1C. The sacrificial scaffold 10 includes a matrix polymer mixed with a crosslinking initiator. The sacrificial scaffold 10 is self-supporting and freestanding. In other words, the sacrificial scaffold 10 can be prepared separately and / or may be shelfed for later use.

[0085] In the middle of Figure 2A, the separately provided sacrificial scaffold 10 is embedded in a crosslinkable composition 20. The crosslinkable composition 20 includes crosslinkable monomers and / or polymers. The crosslinkable molecules are configured for crosslinking based on a specific crosslinking reaction. The crosslinking reaction is initiated using the crosslinking initiator provided in the sacrificial scaffold 10 and / or mixed with the matrix polymer. The matrix polymer is configured to release the crosslinking initiator into the crosslinkable composition 20 once the sacrificial scaffold 10 comes into contact with the crosslinkable composition 20. The crosslinkable composition 20 may comprise a hydrogel including the crosslinkable monomers and / or polymers.

[0086] Possible crosslinking reactions may be, without limitation, photocrosslinking, crosslinking with small molecules, and / or enzymatic crosslinking.

[0087] Photocrosslinking mechanisms may utilize photocrosslinking polymers. Any photocrosslinking polymers deemed useful to the fabrication of the perfusable network may be used. For instance, the crosslinkable composition may include photocrosslinkable polymers including, without limitation, gelatin methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyethylene glycol) diacrylate (PEGDA), etc. Thus, when photocrosslinking is desired, the sacrificial scaffold 10 may include the matrix polymer together with a crosslinking initiator specific for the photocrosslinking reaction. The crosslinking initiator may be, for example, lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP).

[0088] Crosslinking with small molecules may utilize small molecule crosslinkable monomers or polymers. Any small molecule crosslinkable monomer or polymer deemed useful to the fabrication of the perfusable network may be used. For instance, small molecule crosslinkable monomers include, without limitation, acrylamide, and small molecule crosslinkable polymers include, without limitation, polyacrylamide (PAAm), alginate, etc. Thus, when crosslinking with small molecules is desired, the sacrificial scaffold 10 may include the matrix polymer together with a specific crosslinking initiator. When the small molecule crosslinkable polymer in the crosslinkable composition 20 is alginate, e.g., the crosslinking initiator is CaC . In some embodiments, when the small molecule crosslinkable monomer is acrylamide, the polymerization initiator is ammonium persulfate (APS). When the small molecule crosslinkable monomer is acrylamide, the matrix polymer and / or the crosslinkable composition may comprise a co-initiator. In some embodiments, the co-initiator is tetramethylethylenediamine (TEMED) and the crosslinker is bis acrylamide.

[0089] Enzymatic crosslinking may utilize enzymatic crosslinkable polymers. Any enzymatic crosslinkable polymer useful in the fabrication of the perfusable network may be used. For instance, enzymatic crosslinkable polymers include, without limitation, fibrinogen, gelatin, etc. Thus, when enzymatic crosslinking is desired, the sacrificial scaffold 10 may include the matrix polymer together with a specific crosslinking initiator. When the enzymatic crosslinkable polymer is fibrinogen, the crosslinking initiator may be thrombin. When the enzymatic crosslinkable polymer is gelatin, the crosslinking initiator may be transglutaminase.

[0090] Preferably, but without limitation, a photocrosslinking mechanism is used. The crosslinkable composition may be, for example, a hydrogel including gelatin methacryloyl (GelMA), and the crosslinking initiator may be, for example, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP).

[0091] The matrix polymer of the sacrificial scaffold 10 is configured for releasing the crosslinking initiator after the sacrificial scaffold 10 comes into contact with the crosslinkable composition. In other words, as long as the sacrificial scaffold 10 is not in contact with the crosslinkable composition, the crosslinking initiator preferably is not released from the matrix polymer.

[0092] When the sacrificial scaffold 10 is embedded in the crosslinkable composition 20, as shown in the middle of Figure 2A, the sacrificial scaffold 10 comes into contact with the crosslinkable composition 20 and, as a result, the crosslinking initiator is released from the matrix polymer into the crosslinkable composition.

[0093] Release of the crosslinking initiator from the matrix polymer may be due to osmotic pressure and / or diffusion. Release of the crosslinking initiator from the matrix polymer may comprise swelling of the matrix polymer. Swelling may be an uptake of water without instant dissolution of the matrix polymer. When the crosslinking initiator is released from the matrix polymer, the crosslinking initiator diffuses into the crosslinkable composition and forms a diffusion layer 22 around the sacrificial scaffold 10. Activation of the crosslinking reaction then leads to a selective crosslinking of the crosslinkable monomers and / or polymers in the diffusion layer 22 around the sacrificial scaffold 10. As a result, a crosslinked layer 23 around the sacrificial scaffold 10 can be created, as indicated in the middle of Figure 2A.

[0094] Once the crosslinked layer 23 is formed around the sacrificial scaffold 10, the sacrificial scaffold 10 may be removed. This is indicated on the right-hand side of Figure 2A. Removing the sacrificial scaffold 10 may be done by any suitable method. Examples of such methods are explained later on. Removing the sacrificial scaffold 10 leaves behind a hollow network of prefusable channels.

[0095] Referring to Figure 2B, on the left-hand side of Figure 2B, a microscopic image of a network of hollow channels after freeze-drying is shown. The channel network was obtained by removing the sacrificial scaffold as explained in connection with Figure 2A. In the specific example, the sacrificial scaffold is fixated within a dedicated apparatus. For example, the sacrificial scaffold may be fixated in a device as disclosed, for example, in WO 2022 / 214 496 Al, which is incorporated by reference herein, but not necessarily. The sacrificial scaffold may have been made from a thermoplastic material and / or may be self-supported and / or freestanding. Once the sacrificial scaffold is removed, the hollow channel network as shown one the left-hand side of Figure 2B is obtained. The right-hand side of Figure 2B shows a detailed view of the connection between the sacrificial scaffold or hollow channel (once the sacrificial scaffold is removed) and the apparatus of WO 2022 / 214496 Al. More details on the apparatus and the fluid tight connection are provided in WO 2022 / 214496 Al, which is herein incorporated by reference in its entirety. It was found that a leak-free fluid connection can be provided even after freeze-drying and rehydrating the network of hollow channels in the apparatus.

[0096] Referring to Figure 3A, a detailed view of a section of a sacrificial scaffold 10 embedded in the crosslinkable composition 20 is shown. Figure 3A shows the basic principle of the underlying mechanism. Figure 3A shall not be understood as limiting the scope of the disclosure. Figure 3A is one of many possible examples of sacrificial scaffolds 10 embedded in the crosslinkable composition 20. As can be seen in Figure 3A, once the crosslinking initiator is released from the matrix polymer into the crosslinkable composition 20, a diffusion layer 22 (located between the sacrificial scaffold 10 and the dashed-dotted line) is formed around the filaments 12 of the sacrificial scaffold 10. Because the sacrificial scaffold 10 as a whole is embedded into the crosslinkable composition 20, diffusion of the crosslinking initiator into the crosslinkable composition 20 starts at every position along the sacrificial scaffold 10 at substantially the same time. As a result, a penetration depth of the crosslinking initiator into the crosslinkable composition 20, and thus a thickness of the diffusion layer 22 around the sacrificial scaffold 10 is more uniform. In regions where the sacrificial scaffold 10 comprises a node, such as node 14 indicated in Figure 3A, the crosslinking initiator may have different penetration depths due to multiple, or in this case two, neighboring filaments 12 releasing the crosslinking initiator. But in general, the diffusion layer 22 may be substantially uniform along a strand of the sacrificial scaffold 10, as indicated in Figure 3A.

[0097] Activation of the crosslinking reaction such as activation of the photocrosslinking reaction using UV light, selectively crosslinks the crosslinkable monomers and / or polymers contained in the diffusion layer 22 and forms a crosslinked layer 23 around the filaments 12 of the sacrificial scaffold 10. As can be seen in Figure 3A, the crosslinked layer 23 surrounding the filaments 12 forms a network, preferably a three-dimensional network, of interconnected channels or tubular structures. These channels may still be filled with the filaments 12 of the sacrificial scaffold 10. However, once the sacrificial scaffold 10 is removed, an interconnected network of hollow channels that are surrounded by the crosslinked layer 23 of crosslinkable polymers is provided. This interconnected network of hollow channels may mimic a natural vascularization and is suitable for perfusion.

[0098] Removal of the sacrificial scaffold 10 may include a full dissolution of the matrix polymer and / or may comprise a dissociation of the matrix polymer to such a degree that the matrix polymer may be washed out from the channels, e.g. by flowing a liquid therethrough. Dissolution and / or dissociation may occur separately from (e.g., after), or at least partially concurrently with the step of releasing the crosslinking initiator from the matrix polymer. Preferably, but without limitation, the sacrificial scaffold 10 may comprise or consist of a thermo-responsive polymer. The thermo-responsive polymer is not dissolvable in water or an aqueous solution above a lower critical solution temperature (LCST) but becomes dissolvable in water or the aqueous solution below the LCST. "Not dissolvable" includes in this context that the polymer is a solid above the LCST. In case the matrix polymer comprises or consists of a thermo-responsive polymer, removal of the sacrificial scaffold 10 may include reducing the temperature below the LCST. As a result, the sacrificial scaffold 10 dissolves and leaves behind hollow channels surround by a crosslinked layer of the crosslinkable composition 20. As mentioned earlier, thermo-responsive polymers with a LCST within a range 4°C to 37°C may be provided to avoid temperature-related damages to the cells during dissolution of the sacrificial scaffold 10. Examples of thermo-responsive polymers are, without limitation, poly(2- oxazoline), poly-N-isopropylacrylamide, poly(vinylcaprolactam) or polyvinylmethylether. Further examples are provided, for example, in WO 2022 / 214 496 Al incorporated by reference herein.

[0099] Once the hollow perfusable network structure is created, the network structure may be preserved, e.g. freeze-dried or cryo-preserved. The preserved perfusable network structure may then be shipped to a customer who may use the preserved network structure for perfusion purposes.

[0100] Figure 3B shows microscopic images that were obtained during the fabrication process according to the methods and structures presented herein.

[0101] On the top left, the sacrificial scaffold 10 is shown. In the specific example, the sacrificial scaffold is fixated in the apparatus explained in WO 2022 / / 214496 Al. As already mentioned, the sacrificial scaffold 10 is provided separately from the crosslinkable composition, and may be self-supporting and free-standing, unlike a printable ink.

[0102] On the top right, the sacrificial ink 10 is embedded in the crosslinkable composition 20. The crosslinking initiator diffuses into the crosslinkable composition 20 forming the diffusion layer 22. Crosslinking within the diffusion layer 22 by any suitable means, such as photocrosslinking, forms the crosslinked layer 23.

[0103] On the bottom left, a microscopic image during removal of the sacrificial scaffold 10 is shown.

[0104] On the bottom right, a microscopic image after removal of the sacrificial scaffold 10 is shown. Once the sacrificial scaffold 10 is removed, a network of hollow channels that are surrounded by the crosslinked layer 23 are provided.

[0105] In Figure 4A, an experimentally obtained correlation of how to adjust and / or tailor a wall thickness surrounding the hollow channels is shown. In this example, the polymer matrix was Poly(2-cyclopropyl-oxazoline), the crosslinking initiator was LAP, the crosslinkable composition was GelMA. Crosslinking was achieved by UV irradiation with a 60W 405 nm lamp for 30 seconds after waiting for the specified timeframes. Figure 4A highlights the underlying principle and shall not be understood as limiting the scope of the disclosure.

[0106] Figure 4A depicts one example of a relation between a wall thickness 24 (y-axis) of the crosslinked layer 23 surrounding the sacrificial scaffold 10 and a diffusion time (x-axis) of the crosslinking initiator into the crosslinkable composition 20. As can be seen, by increasing the diffusion time of the crosslinking initiator into the crosslinkable composition 20, the thickness 24 of the crosslinked layer 23 and thus the thickness 24 of the wall surrounding the filaments 12 of the sacrificial scaffold 10 (or the hollow channels once the sacrificial scaffold 10 is removed) can be increased. It should be noted that Figure 4A is for illustration purposes only. Hence, in other embodiments, the relation shown in Figure 4A may be different.

[0107] Using photocrosslinking as a crosslinking mechanism can help to gain control over the crosslinking kinetics. This is because the photocrosslinking reaction is only initiated upon exposure of the diffusion layer 22 to ultraviolet (UV) light. Thus, when the sacrificial scaffold 10 is embedded in the crosslinkable composition 20, diffusion of the crosslinking initiator into the crosslinkable composition 20 may start, but crosslinking within the diffusion layer 22 may only be initiated upon exposing the diffusion layer 22 to UV light. Hence, photocrosslinking offers the possibility to wait for a predetermined period of time before the crosslinking reaction is initiated. The predetermine time period may be the time the crosslinking initiator is allowed to diffuse into the crosslinkable composition 20. Examples of predetermined time periods and / or diffusion times are 10 minutes, preferably 5 minutes, more preferably 2 minutes, more preferably less than 2 minutes, such as 1-2 minutes.

[0108] Figure 4B shows a collage of microscopic images that have been taken of a filament 12 of the sacrificial scaffold 10 using the methods and structures herein. The images have been combined in the collage to highlight the effect of increased diffusion times on the thickness of the crosslinked layer 23. As can be seen in Figure 4B, in the specific example shown, a diffusion time of about 30 seconds may lead to a thickness of about 75 micrometers for the crosslinked layer 23. A diffusion time of about 45 seconds may lead to a thickness of about 100 micrometers and a diffusion time of about 90 second may lead to an even larger thickness of about 150 micrometers for the crosslinked layer 23. The skilled reader will understand that Figure 4B illustrates the underlying principle and shall thus not be understood as limiting the scope of this disclosure.

[0109] Additionally or alternatively to changing the diffusion time (as shown in Figure 4B), the duration of UV irradiation on the diffusion layer 22 may have an effect on the amount of crosslinking taking place within the diffusion layer 22. Longer durations may cause more crosslinking within the diffusion layer 22. Durations of UV irradiation may be 10 minutes, preferably 5 minutes, more preferably 2 minutes, more preferably less than 2 minutes, more preferably 1 minute or less than one minute such as 20-60 seconds.

[0110] Once enough crosslinking took place within the diffusion layer 22, the crosslinkable composition 20 around the diffusion layer 22 that has not been crosslinked can be removed, e.g. by washing. The sacrificial scaffold 10 may be removed. Thereby, a network of hollow channels delimited by the crosslinked composition in the diffusion layer 22 is formed. Such channel network is suitable for a great variety of cell cultivation and / or tissue engineering experiments. For example, cells and / or tissue may be provided around the perfusable channel network. The cells and / or tissue may be perfused via the channel network during a respective experiment. For example, the perfusable channel network may be arranged within a cavity and a second hydrogel containing cells and / or tissue (not shown) may be filled into said cavity. Examples of cells to be seeded around the channel network are all cell types including and without limitation endothelial cells, pericytes, muscle cells including smooth muscle cells, cardiomyocytes, organ cells (such as hepatocytes), stem cells, fibroblasts, hematopoietic cells, stromal cells, plant cells, podocytes, etc. Depending on the needs of the customer, the channel network could be freeze-dried and / or shipped as such or together with pre-seeded cells for experimental purposes (such as pre-seeded cells in a second hydrogel surrounding the channel network).

[0111] Referring to Figure 5, one example of a vascular network 26 that has been obtained using the technique as described herein is shown.

[0112] The example of Figure 5 shows a preservable and matured vascularized network of hollow channels suitable for cell and / or tissue engineering and suitable for perfusion purposes. The vascular network of Figure 5 may include one or more layers of cells and / or tissue and may be freeze-dried and / or cryo-preserved for shipping the vascular network to a customer. The customer may then thaw the preserved vascular network and may use the vascular network for perfusion purposes. If the vascularized network includes cells and / or tissue, the customer may thaw the preserved vascularized network until the cells and / or tissue are vital again and then use the thawed vascularized network for perfusion purposes.

[0113] Referring to Figure 6, a schematic detailed view of an example of a channel 28 is shown. Figure 6 is a brightfie Id image obtained from a section of the channel 28, which may also be referred to as a "vascularized" channel herein. The channel 28 is formed by a tubular wall 29 (resulting from the crosslinking of the diffusion layer) and an inner lumen 27 (resulting from dissolution of the sacrificial scaffold). In Figure 6, cells 30 of a first type are seeded into the channel 28. For example, endothelial cells such as for mimicking a tunica intima of a blood vessel, and / or umbilical vein endothelial cells may be seeded into the channel 28. Cells may be seeded, e.g. by flowing liquid with these cells through the channel 28. The cells 30 may adhere to the inner side of the crosslinked layer, may fully cover the inner wall of the channel 28 and may be perfusable by flowing perfusion liquid through the hollow channel 28.

[0114] As can be further seen in Figure 6, cells 32 of a second type are encapsulated in the tubular wall 29. The cells 32 may be of the same or a different type than the cells 30. Preferably, the cells 32 are pericytes or smooth muscle cells (SMCs).

[0115] Referring to Figure 7, microscopic images (with live-dead staining) of another example of a vascularized channel 28 are shown. The images have been obtained from a section of a vascularized channel 28. On the left-hand side of Figure 7, a microscopic image before cryopreservation and thawing is shown. On the right-hand side of Figure 7, a microscopic image after cryopreservation and thawing is shown.

[0116] As can be seen, cells 30 adhere to the inner wall of the channel 28 and cells 32 are encapsulated within the crosslinked layer 22 surrounding the channel 28. Cells 30 may be the same or different cells than cells 32. Preferably, cells 30, 32 may be endothelial cells such as umbilical vein endothelial cells or smooth muscle cells. Smooth muscle cells may be used for mimicking tunica media of a blood vessel. Other examples of cells 30, 32 are astrocytes, stellate cells, such as stellate macrophages cells (Kupffer cells), pericytes, macrophages, mesangial cells. The skilled reader will understand that cells 32 within an outer region of the diffusion layer 22 may grow faster than cells 32 within an inner region of the diffusion layer 22. As a result, the outer cells 32 may overgrow the inner cells 32 and thus the outer cells 32 are shown more dominantly in the stained images.

[0117] As noted above, cells 30 may be seeded into the channel 28 by flowing a liquid containing these cells 30 through the channel 28. As noted above, cells 32 may be encapsulated in the crosslinked layer 22. For example, cells

[0118] 32 may be provided within a first hydrogel comprised in the crosslinkable composition.

[0119] As the skilled person will appreciate, the technique of the present invention also allows for providing a channel having, along at least a portion thereof, a wall comprising two or more layers of crosslinked composition. These layers may be provided with different physical properties (such as for allowing a selective diffusion of nutrients) or with different cells (e.g., different cell-types) encapsulated therein. A schematic representation of such channel 28 with a layered channel wall 29 is shown in Figure 8.

[0120] The diffusion layer 22 may form a first layer 51 of the channel wall 29. The crosslinkable composition 20 that has not been crosslinked from around the first layer 51 may be removed, preferably by washing. The crosslinkable composition 20 may be a first crosslinkable composition comprising, e.g. a first hydrogel.

[0121] After removing the first crosslinkable composition that has not been crosslinked, the sacrificial scaffold together with the first layer 51 may further be embedded in a second crosslinkable composition 42 comprising, e.g. a further ("third") hydrogel. The crosslinking initiator may then diffuse through the crosslinked first layer 51 of the channel wall 29 into the second crosslinkable composition 42 to create a (second) diffusion layer 25 in said second crosslinkable composition 42. The diffusion layer 25 may be crosslinked to form a crosslinked second layer 52 of the channel wall 29. Crosslinking in the diffusion layer 25 may be achieved by one of enzymatic crosslinking, small molecule crosslinking or photocrosslinking.

[0122] If desired, the second crosslinkable composition 42 that has not been crosslinked to form the crosslinked second layer 52 may subsequently be removed from around the second layer 52, preferably by washing. As described above, the created channel could subsequently be surrounded with a further hydrogel (the above-mentioned "second" hydrogel) in which cells or tissue to be cultivated may be provided. As the skilled person will appreciate, cells (such as a third type of cells 33) may be encapsulated in the second layer 52. Examples of cells are all cell types including and without limitation endothelial cells, pericytes, muscle cells including smooth muscle cells, cardiomyocytes, organ cells (such as hepatocytes), stem cells, fibroblasts, hematopoietic cells, stromal cells, plant cells, podocytes, etc. Preferably, the cells are fibroblasts. Fibroblasts may be used for mimicking tunica adventitia of a blood vessel.

[0123] As the skilled person will further appreciate, further layers (not shown), such as a third layer could also be provided by repeating the steps described above.

[0124] The sacrificial scaffold 10 may be removed after the desired layers, such as the first layer 51 and the second layer 52, have been crosslinked.

[0125] Figure 9 shows schematic images of one example of fabricating a perfusable network structure with multiple cell layers and / or cell types.

[0126] As indicated on the left-hand side of Figure 9, the separately provided sacrificial scaffold may be embedded in a crosslinkable composition 20. The crosslinkable composition 20 may include a first type of cells (cells A, indicated by ovals). Crosslinking within the diffusion Iayer 22 results in cells A being encapsulated within the crosslinked layer 23. The sacrificial scaffold may then be removed or may be removed at a later stage to obtain a hollow perfusable channel network with cells A encapsulated within the channel walls.

[0127] As indicated in middle of Figure 9, crosslinkable composition that has not been crosslinked may be removed, e.g. by washing.

[0128] Before or after removal of the crosslinkable composition that has not been crosslinked, a second type of cells (cells B, indicated by hexagons) may be seeded through the hollow channels, e.g. by using a perfusion liquid containing cells B. Cells B may grow on an inside of the channel walls. A hollow perfusable channel network with cells A encapsulated within the channel walls and cells B growing and adhering to the inside of the channel walls may be obtained.

[0129] As indicated on the right-hand side of Figure 9, the hollow perfusable channel network with cells A and B may then be embedded in a further, e.g. second, crosslinkable composition 42. The second crosslinkable composition 42 may include a third type of cells (cells C, indicated by pentagons). A crosslinking within a second diffusion layer may occur, similarly to the process explained in connection with Figure 8, and cells C may be encapsulated within the second crosslinked layer.

[0130] Referring to Figure 10, microscopic images of a channel with seeded cells obtained by the methods and structures as proposed herein are shown. On the left-hand side a brightfield image and on the right-hand side a live-dead staining image are shown.

[0131] Experiments

[0132] This experimental section provides an in-depth description of how the crosslinking initiator may be incorporated into the matrix polymer and released from the matrix polymer. The basic principles have already been explained in connection with Figures 1-10. The following description of experiments is intended to provide a thorough understanding of the invention, but shall not be interpreted to limit the scope of the claims. a) Incorporation of the crosslinking initiator into the matrix polymer:

[0133] • 2% (w / v) of LAP (Advanced BioMatrix, Inc., Carlsbad, CA 92010, U.S.A) were dissolved in a suitable solvent, such as distilled water. For example, 0.2 g of LAP were dissolved in 10 ml of water.

[0134] • The matrix polymer was subsequently dissolved in the mixture of LAP and water. For example, 1,0 g poly(2-cyclopropyl-oxazoline) was dissolved in the water / LAP mixture, for example, over 24h at 4°C. As those of skill in the art will understand from the disclosure provided herein, other crosslinking initiators and matrix polymers may be used in conjunction with suitable solvents, as well known in the field.

[0135] The mixture of crosslinking initiator and matrix polymer was then frozen in liquid nitrogen and freeze-dried at -50°C.

[0136] The dried mixture may then be formed into the sacrificial scaffold via fabrication technologies already explained in connection with, for example, Figures 1A-1C and Figure 2A, such as 3D printing, Fused Deposition Modeling (FDM), injection molding (such as micro-injection molding), or melt electro-writing. For the experiments performed by the inventor, Fused Deposition Modeling (FDM) was found to be particularly useful. b) Formation of a crosslinked layer around the sacrificial scaffold by release of the crosslinking initiator from the matrix polymer of the sacrificial scaffold

[0137] • 10% (w / v) GelMA (Advanced BioMatrix, Inc., Carlsbad, CA 92010, U.S.A) was dissolved in phosphate-buffered saline (PBS). Dissolution was achieved at 37°C overnight on a shaker.

[0138] • A prefabricated sacrificial scaffold obtained in accordance with the experiment described above (see also Figures 1A-1C) was fixated in an apparatus (as shown in Figure 2B).

[0139] • 400 pl of dissolved 10% (w / v) GelMA (with or without cells, depending on the experiment being performed) were introduced into the hydrogel chamber of the apparatus, preferably such that the prefabricated sacrificial scaffold was completely covered.

[0140] • Upon contact of the sacrificial scaffold with the 10% (w / v) GelMA (with or without cells), the crosslinking initiator (in this example LAP) was released into the crosslinkable composition. • After waiting for a predetermined time period of about 5-10 seconds, crosslinking of the GelMA was initiated by shining a 405 nm lamp (60 W) with a distance of about 10 cm between the lamp and the GelMA for about 1 min onto the GelMA.

[0141] • Crosslinking of GelMA by LAP led to the generation of a crosslinked layer around the prefabricated sacrificial scaffold.

[0142] Once the crosslinked layer was formed, non-crosslinked GelMA was removed by washing (as described in connection with Figure 9 above). For example, non-crosslinked GelMA was removed by washing the sacrificial scaffold three times with 400 pl of warm PBS.

[0143] Subsequently, the sacrificial scaffold was removed by decreasing the temperature and washing (as described in connection with Figure 3A above) resulting in a self-supported and free-standing perfusable network structure, as shown, for example, in Figures 3A and 3B.

[0144] The following aspects are preferred embodiments of the invention:

[0145] 1. A method of generating a perfusable structure for cell and / or tissue cultivation, the method comprising the steps of: providing a crosslinkable composition (20), providing a sacrificial scaffold (10) separately from the crosslinkable composition (20), wherein the sacrificial scaffold (10) comprises a matrix polymer and a crosslinking initiator for initiating crosslinking of the crosslinkable composition (20), wherein the matrix polymer is configured for releasing the crosslinking initiator when the sacrificial scaffold (10) is in contact with the crosslinkable composition (20), after separately providing the sacrificial scaffold (10), embedding the sacrificial scaffold (10) in the crosslinkable composition (20) such that the sacrificial scaffold (10) comes into contact with the crosslinkable composition (20), and releasing the crosslinking initiator from the matrix polymer into the crosslinkable composition (20) to crosslink a layer (22) of the crosslinkable composition around the sacrificial scaffold (10), the crosslinked layer (22) forming at least one channel

[0146] (28). The method of aspect 1, wherein the matrix polymer is a thermoplastic matrix polymer. The method of aspect 1 or 2, wherein the crosslinkable composition (20) comprises a first hydrogel. The method of any one of the preceding aspects, further comprising: removing the sacrificial scaffold (10), preferably by dissolving the matrix polymer. The method of any one of the preceding aspects, wherein the crosslinked layer (22) forms a network of channels, preferably an interconnected network, more, more preferably a three-dimensional network of channels. The method of any one of the preceding aspects, wherein the sacrificial scaffold (10) is three-dimensional, and / or the sacrificial scaffold (10) comprises one or more filaments (12), preferably wherein the sacrificial scaffold (10) comprises a plurality of connected filaments (12) forming a network, the filaments (12) preferably extending in at least two nonparallel directions, and / or the sacrificial scaffold (10) has at least one first node (14) at which one filament (12) branches into two or more filaments (12), and / or the sacrificial scaffold (10) has at least one second node (16) at which two or more filaments (12) merge into one filament (12). The method of any one of the preceding aspects, wherein releasing the crosslinking initiator from the matrix polymer comprises releasing the crosslinking initiator by diffusion. The method of any one of the preceding aspects, wherein releasing the crosslinking initiator from the matrix polymer comprises swelling, preferably by uptake of water, and / or dissolution of the matrix polymer, preferably in an aqueous solution. The method of any one of the preceding aspects, wherein the matrix polymer is water soluble, and / or the matrix polymer comprises, or consist of, polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolic acid (PGA), Poly(N-isopropylacrylamide), a carbohydrate-based materials such as isomalt, or a thermo-responsive polymer, such as poly(vinylcaprolactam) or polyvinylmethylether. The method of any one of the preceding aspects, wherein the matrix polymer comprises, or consists of, a thermo-responsive polymer that is not soluble in aqueous solutions above a lower critical solution temperature and becomes soluble in aqueous solutions below the lower critical solution temperature. The method of aspect 10, wherein the thermo-responsive is poly(oxazoline), preferably poly(2-oxazoline), more preferably a poly(2-cyclopropyl-2-oxazoline). The method of aspect 10 or 11, wherein the lower critical solution temperature is within a range of 4°C to 37°C. The method of aspect 10, 11, or 12, when further dependent on aspect 4, wherein the removing the sacrificial scaffold comprises: reducing the temperature below the lower critical solution temperature such that the sacrificial scaffold (10) dissolves. The method of any one of the preceding aspects, wherein the crosslinkable composition (20) and the crosslinking initiator are suitable for photocrosslinking and the method further comprises the steps of: exposing the crosslinkable composition (20) to radiation, such as ultraviolet light. The method of aspect 14, further comprising: after embedding the sacrificial scaffold (10) in the crosslinkable composition (20), waiting for a predetermined period of time before exposing the crosslinkable composition (20) to radiation. The method of aspect 14 or 15, wherein the step of exposing the crosslinkable composition (20) to radiation is performed for a predetermined duration. The method of any one of the preceding aspects, further comprising: removing crosslinkable composition (20) that has not been crosslinked from around the crosslinked layer (22), preferably by washing. The method of any one of the preceding aspects, further comprising: freeze-drying and / or cryopreserving the perfusable structure. The method of any one of the preceding aspects, wherein the crosslinkable composition (20) comprises at least one type of cells, preferably endothelial cells or smooth muscle cells. The method of aspect 19, wherein the endothelial cells are encapsulated in the crosslinked layer (22). The method of any one of the preceding aspects, further comprising a step of: seeding tissue and / or cells around the at least one channel (28), preferably around the three-dimensional network of channels (28). The method of aspect 21, wherein the step of seeding tissue and / or cells around the at least one channel (28) comprises filling a second hydrogel into a cavity surrounding the at least one channel (28). The method of any one of the preceding aspects, further comprising a step of: seeding tissue and / or cells within the at least one channel (28), preferably within the three-dimensional network of channels (28). The method of any one of the preceding aspects, preferably of any one of aspects 19- 23, further comprising: cryopreserving and / or freeze-drying the perfusable structure together with the seed tissue and / or cells, and vitally thawing the cryopreserved and / or freeze-dried perfusable structure such that the tissue and / or cells are vital and perfusable. The method of any one of the preceding aspects, further comprising: removing the crosslinkable composition (20) that has not been crosslinked from around the crosslinked layer (22), preferably by washing, wherein the crosslinkable composition (20) is a first crosslinkable composition; embedding the sacrificial scaffold (10) in a second crosslinkable composition; allowing the crosslinking initiator to diffuse into the second crosslinkable composition and crosslinking a second layer of the second crosslinkable composition around the crosslinked first layer of the first crosslinkable composition. The method of aspect 25, wherein the second crosslinkable composition comprises at least one type of cells, preferably fibroblast. The method of aspect 26, wherein the cells are encapsulated in the crosslinked second layer. The method of any one of the preceding aspects, wherein the sacrificial scaffold (10) is formed by one of 3D printing, preferably by Fused Deposition Modeling (FDM), injection molding, such as micro-injection molding, and melt electro-writing. A perfusable structure for cell and / or tissue cultivation provided by the method of any one of the preceding aspects. The perfusable structure of aspect 29, wherein the perfusable structure comprises: a crosslinked layer (22) of crosslinkable composition (20) surrounding at least one perfusable and / or hollow channel (28), preferably a three-dimensional network of perfusable and / or hollow channels (28). The perfusable structure of aspect 29 or 30, wherein the perfusable structure comprises a first layer of cells, such as endothelial cells, arranged within the at least one channel (28). The perfusable structure of aspect 29, 30, or 31, wherein the perfusable structure comprises a second layer of cells, such as smooth muscle cells, arranged within the crosslinked layer.

Claims

CLAIMS1. A method of generating a perfusable structure for cell and / or tissue cultivation, the method comprising the steps of: providing a crosslinkable composition (20), providing a sacrificial scaffold (10) separately from the crosslinkable composition (20), wherein the sacrificial scaffold (10) comprises a matrix polymer and a crosslinking initiator for initiating crosslinking of the crosslinkable composition (20), wherein the matrix polymer is configured for releasing the crosslinking initiator when the sacrificial scaffold (10) is in contact with the crosslinkable composition (20), after separately providing the sacrificial scaffold (10), embedding the sacrificial scaffold (10) in the crosslinkable composition (20) such that the sacrificial scaffold (10) comes into contact with the crosslinkable composition (20), and releasing the crosslinking initiator from the matrix polymer into the crosslinkable composition (20) to crosslink a layer (22) of the crosslinkable composition around the sacrificial scaffold (10), the crosslinked layer (22) forming at least one channel (28).

2. The method of claim 1, wherein the matrix polymer is a thermoplastic matrix polymer.

3. The method of claims 1 or 2, further comprising: removing the sacrificial scaffold (10), preferably by dissolving the matrix polymer.

4. The method of any one of the preceding claims, wherein the sacrificial scaffold (10) is three-dimensional, and / or the sacrificial scaffold (10) comprises one or more filaments (12), preferably wherein the sacrificial scaffold (10) comprises a plurality of connected filaments (12) forming a network, the filaments (12) preferably extending in at least two nonparallel directions, and / orthe sacrificial scaffold (10) has at least one first node (14) at which one filament (12) branches into two or more filaments (12), and / or the sacrificial scaffold (10) has at least one second node (16) at which two or more filaments (12) merge into one filament (12).

5. The method of any one of the preceding claims, wherein the matrix polymer is water soluble, and / or the matrix polymer comprises, or consist of, polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolic acid (PGA), Poly(N-isopropylacrylamide), a carbohydrate-based materials such as isomalt, or a thermo-responsive polymer, such as poly(vinylcaprolactam) or polyvinylmethylether, and / or the matrix polymer comprises, or consists of, a thermo-responsive polymer that is not soluble in aqueous solutions above a lower critical solution temperature and becomes soluble in aqueous solutions below the lower critical solution temperature.

6. The method of claim 5, wherein the thermo-responsive is poly(oxazoline), preferably poly(2-oxazoline), more preferably a poly(2-cyclopropyl-2-oxazoline), and / or the lower critical solution temperature is within a range of 4°C to 37°C.

7. The method of claim 5, or 6, when further dependent on claim 3, wherein the removing the sacrificial scaffold comprises: reducing the temperature below the lower critical solution temperature such that the sacrificial scaffold (10) dissolves.

8. The method of any one of the preceding claims, wherein the crosslinkable composition (20) and the crosslinking initiator are suitable for photocrosslinking and the method further comprises the steps of: exposing the crosslinkable composition (20) to radiation, such as ultraviolet light, and preferablyafter embedding the sacrificial scaffold (10) in the crosslinkable composition (20), waiting for a predetermined period of time before exposing the crosslinkable composition (20) to radiation.

9. The method of any one of the preceding claims, further comprising: removing crosslinkable composition (20) that has not been crosslinked from around the crosslinked layer (22), preferably by washing.

10. The method of any one of the preceding claims, further comprising: freeze-drying and / or cryopreserving the perfusable structure.

11. The method of any one of the preceding claims, wherein the crosslinkable composition (20) comprises at least one type of cells, preferably endothelial cells or smooth muscle cells, preferably wherein the endothelial cells are encapsulated in the crosslinked layer (22).

12. The method of any one of the preceding claims, further comprising a step of: seeding tissue and / or cells around the at least one channel (28), preferably around the three-dimensional network of channels (28), preferably wherein the step of seeding tissue and / or cells around the at least one channel (28) comprises filling a second hydrogel into a cavity surrounding the at least one channel (28), and / or seeding tissue and / or cells within the at least one channel (28), preferably within the three-dimensional network of channels (28).

13. The method of any one of the preceding claims, preferably of claims 11 or 12, further comprising: cryopreserving and / or freeze-drying the perfusable structure together with the seed tissue and / or cells, and vitally thawing the cryopreserved and / or freeze-dried perfusable structure such that the tissue and / or cells are vital and perfusable.

14. The method of any one of the preceding claims, further comprising: removing the crosslinkable composition (20) that has not been crosslinked from around the crosslinked layer (22), preferably by washing, wherein the crosslinkable composition (20) is a first crosslinkable composition; embedding the sacrificial scaffold (10) in a second crosslinkable composition; allowing the crosslinking initiator to diffuse into the second crosslinkable composition and crosslinking a second layer of the second crosslinkable composition around the crosslinked first layer of the first crosslinkable composition, preferably wherein the second crosslinkable composition comprises at least one type of cells, preferably fibroblast, more preferably wherein the cells are encapsulated in the crosslinked second layer.

15. A perfusable structure for cell and / or tissue cultivation provided by the method of any one of the preceding claims, preferably wherein the perfusable structure comprises a crosslinked layer (22) of crosslinkable composition (20) surrounding at least one perfusable and / or hollow channel (28), preferably a three-dimensional network of perfusable and / or hollow channels (28) the perfusable structure comprises a first layer of cells, such as endothelial cells, arranged within the at least one channel (28) and preferably a second layer of cells, such as smooth muscle cells, arranged within the crosslinked layer.

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