Synthetic basement membrane and methods for its production

Abstract

The present invention relates to methods for producing an artificial basement membrane comprising a fiber mesh, artificial basement membranes comprising a fiber mesh, in particular produced by means of the method according to the invention, as well as in vitro test systems, cell culture systems, filtration systems and organ systems comprising at least one artificial basement membrane of the present invention.

Description

[0001] The present invention relates to methods for producing an artificial basement membrane comprising a fiber mesh, artificial basement membranes comprising a fiber mesh, in particular produced by means of the method according to the invention, as well as in vitro test systems, cell culture systems, filtration systems and organ systems comprising at least one artificial basement membrane of the present invention.

[0002] The basement membrane is a cell-free tissue, 1 to 3 µm thick, composed of fibrillar and reticular structural proteins. The fibrillar structures provide the membrane's basic stability and are primarily composed of type III collagen. Towards the stromal tissue, this structure is anchored to the adjacent tissue via other collagens, such as type VII. On the other side, the basement membrane consists of reticular structural proteins, mainly type IV collagen and laminins, which form a dense network. This structure primarily serves to provide specific adhesion sequences at high density for epithelial and endothelial cells. Generally, this membrane is permeable to most molecules and macromolecules and can also be penetrated by immune cells such as macrophages for defense against pathogens.The basement membrane therefore represents a boundary tissue which, together with the cellular component, creates the barrier in every epithelium and endothelium.

[0003] In the development of new pharmaceutical agents, the ability to cross these barriers is of essential importance, as bioavailability in the body is crucial for the efficacy of increasingly complex drugs. Therefore, there is a steadily growing demand for human physiological tissue models that replicate the barrier for transport studies. Barrier models are also being developed that represent pathological properties such as infections, genetic diseases, or tumors and are likewise used in the development of pharmaceutical agents. At the same time, the goal is also to reduce animal testing and offer more potent alternatives (based on the 3Rs principle). The 3Rs principle stands for Reduce, Refine, and Replace, and thus for the reduction, improvement, and replacement of animal testing.

[0004] To produce these tissues, endothelial or epithelial cells require the defined, dense structure of the basement membrane as well as the provision of adhesion sequences by type IV collagen and / or various laminins. In the standard procedure for these models, tracked-etched membranes are used as cell culture surfaces. These membranes provide a dense surface via their smooth plastic surface and allow the permeability of culture medium and active substances through pores of defined sizes ranging from a few hundred nm to several micrometers (depending on the application). However, to provide the cell adhesion points, these surfaces must first be coated with proteins or peptides / peptide sequences from the basement membrane, particularly type IV collagen, laminins, and fibronectin. This results in several disadvantages, primarily concerning the substrate material and its protein coating.

[0005] The smooth substrate material with incorporated pores has the disadvantage that molecules, after passing through the cellular barrier, have only a small number of penetrations or a small surface area available. This significantly slows down, and potentially prevents or delays, the transport of active substances through the barrier construct, leading to an underestimation of the drug's bioavailability (incorrect simulation of drug uptake). In some cases, the hydrophobic surface even makes transport studies impossible due to nonspecific molecular adsorption to the membrane material. In specific applications with additional cells below the barrier model, the small number and size of the pores also reduce cellular interactions between barrier cells and adjacent stromal cells or immune cells below.

[0006] The coating used for this purpose consists of two main types: firstly, defined protein solutions made from collagen type 4 and / or laminins and / or fibronectin, which functionalize the material surface via non-specific adsorption. A disadvantage of defined protein solutions is primarily their complex production and purification, and the associated considerable costs. Secondly, functionalization is achieved using undefined protein solutions: these solutions are produced from animal or human tissues that have a high proportion of basement membrane proteins. The most widely used material is Matrigel. TMThis method involves isolating artificial tumors in animal experiments. Artificial tumors are induced in mice, and once they have grown, the tumors are removed. The material is then liquefied, purified, and finally offered as a coating solution. Disadvantages include high batch variance, relatively high costs due to the complex manufacturing process, and the annual number of animals used, approximately 300,000 mice worldwide. As an alternative, advanced developments are currently underway that replace the isolation of basement membrane proteins in mice with direct human sources. Placental tissue, which is produced at every birth and would be available as a byproduct for processing and isolating ECM (extracellular matrix) proteins, is used for this purpose. However, batch variance due to genetic or pathological differences in the respective human proteins is also a disadvantage.

[0007] Another disadvantage of using coating solutions for the basement membrane is that only the qualitative and quantitative provision of adhesion sequences can be achieved. The simple principle of non-specific protein adsorption thus produces a randomly generated supply of the necessary peptide sequences, but not a physiological structural arrangement of these proteins to structurally mimic the basement membrane.

[0008] The technical problem underlying the present invention is to provide methods for producing basement membranes and basement membranes produced therein which do not have the aforementioned disadvantages of known basement membranes, in particular those which make it possible to provide functionally improved in vitro test systems, cell culture systems, filtration systems and organ systems.

[0009] This technical problem is solved by providing the teachings of independent and dependent claims and the associated description.

[0010] This technical problem is solved in particular by a method for producing an artificial basement membrane comprising a fiber mesh embedded in a matrix composed of at least one basement membrane protein, comprising the following process steps: a) Manufacturing and applying a fiber fabric comprising fibers with a fiber diameter of 100 to 2000 nm, by electrospinning from a fiber material onto the surface of a carrier element arranged in an electrospinner, wherein the fiber fabric has a fiber count of 1000 to 30000 fibers / mm² 2 exhibits b) Applying a stabilizing frame made of at least one polymer to the fiber fabric arranged on the support element under conditions that allow the stabilizing frame to associate with the fiber fabric, c) Cultivating the fiber web associated with the stabilizing frame with cells of at least one cell type introduced into the fiber web, which are capable of expressing at least one basement membrane protein, under conditions that allow the expression of the at least one basement membrane protein and while maintaining a matrix composed of the at least one basement membrane protein in which the fiber web is embedded and d) Removal of cells of at least one cell type while preserving the artificial basement membrane.

[0011] The present invention accordingly provides that in a first process step a) a fiber fabric is produced by electron spinning, wherein this fiber fabric comprises fibers with a fiber diameter of 100 to 2000 nm. Process step a) is carried out such that the fiber material is electrospun onto the surface of a carrier element arranged in an electrospinner. The carrier element is a detachably arranged, in particular planar, element on a collector of an electrospinner, which serves to collect and support the spun fibers. The surface of the carrier element is a defined surface, that is, a surface of a fixed size and geometry. Process step a) is further carried out such that a fiber fabric with a fiber count (here also referred to as fiber density) of 1000 to 30000 fibers / mm² is produced on the defined surface of the carrier element. 2The process step a) thus produces the fiber fabric and simultaneously applies it to the surface of a support element. The invention provides for an electrospinning process with a defined material onto a defined surface in such a way that a precisely defined, particularly low fiber density is achieved. In a particularly advantageous manner, a very thin fiber fabric with low fiber density is thus provided, which, in the subsequent process steps, is contacted with basement membrane protein-secreting cells that deposit the basement membrane proteins in and onto the fiber fabric and create a matrix embedding the fiber fabric, the thickness of which is on the order of natural basement membranes.

[0012] In process step b), after the fiber fabric has been applied according to process step a), a stabilizing frame is applied to the fiber fabric located on the carrier element. Process step b) is preferably carried out in or with a polymer application device, in particular in a polymer application device separate from the electrospinner. In one embodiment, process step b) can also be carried out in the electrospinner with a polymer application device. The stabilizing frame also serves to enable the fiber fabric according to the invention to be removed from the surface of the carrier element without damage and while maintaining its shape, thus ensuring mechanical stabilization, particularly during removal.

[0013] The stabilizing frames are applied to the fiber fabric under conditions that allow the stabilizing frame to associate with the fiber fabric. In the context of the present invention, "association" refers in particular to a bonding, preferably reversible, of the stabilizing frame to the fiber fabric, which is achieved especially by adhesion and is strong enough to allow the fiber fabric associated with the stabilizing frame to detach from the surface of the support element, preferably as provided for in a process step v). In one embodiment of the invention, the bonding of the stabilizing frame to the fiber fabric can also be irreversible.

[0014] In a subsequent process step c), the fiber mesh is cultured with cells of at least one cell type introduced into the fiber mesh, wherein this cell type is capable of expressing at least one basement membrane protein, in particular of expressing and secreting at least one basement membrane protein into the cell environment. The invention thus provides in process step c) to apply cells of the at least one cell type to and within the fiber mesh, i.e., to bring the fiber mesh associated with the stabilizing frame into contact with cells of the at least one cell type and to culture the cells in the fiber mesh.In one embodiment, the structure of fiber fabric and stabilizing frame connected to the support element can also be brought into contact with cells of at least one cell type, the cells in the fiber fabric can be cultivated and subjected to further process steps, optionally with subsequent removal of the support element.

[0015] Procedure step c) is carried out under conditions that allow the expression of this at least one basement membrane protein in the cell culture vessel, in particular conditions that allow the expression and secretion of the basement membrane protein from the cell into its environment and thus into the fiber matrix, so that in procedure step c) a fiber matrix is ​​obtained in whose intercellular spaces and on whose fiber surfaces basement membrane proteins secreted from the cells are embedded and deposited. These basement membrane proteins thus form an extracellular matrix with the fiber matrix embedded within it.

[0016] According to the invention, it is therefore advantageously possible to provide a protein composition and structure that is as natural and physiological as possible, largely corresponding to that of natural basement membranes. According to the invention, this naturally produced protein component of the basement membrane is located, in particular, in the spaces between and on the surfaces of the electrospun material, i.e., in and on the fiber mesh, and thus forms an integral hybrid structure with this fiber mesh, consisting of synthetically produced fibers and the naturally produced basement membrane proteins surrounding them. The cells of the at least one cell type are therefore particularly preferably cultured within the open and thin fiber mesh structure obtained according to process steps a) and b), and thus secrete the basement membrane proteins within the fiber mesh and not solely on its surface.According to the invention, it is thereby possible to obtain a basement membrane whose protein components do not lie on the surface of a synthetic fiber mesh, but where the fiber mesh is an integral part of the basement membrane protein structure and the basement membrane proteins are therefore also present within the fiber mesh and thus have an overall low thickness.

[0017] The basement membrane proteins provided according to the invention can correspond exactly to the naturally occurring basement membrane proteins with respect to their modifications, such as glycosylation, just like the proteins found in natural basement membranes. In particular, cells that produce reticular basement membrane proteins such as collagen type 4, laminins, and fibronectins are preferably used according to the invention. Cultivating such cells on the fiber layers for a period sufficient to express and secrete the desired proteins allows proteins to be synthesized within the electrospun, especially thin, fiber layer that are entirely of biological and thus natural origin, and which can be produced without the use of animal materials and media.

[0018] In a subsequent process step d), the cells of the at least one cell type are removed, so that the biologically produced matrix and the synthetically produced fiber mesh embedded in it, and thus the basement membrane according to the invention, remain and are preserved.

[0019] The inventive method advantageously provides the inventive basement membrane directly using living cells for introducing naturally produced basement membrane proteins into the, preferably very thin, fiber mesh, so that the basement membrane is very similar to a naturally occurring basement membrane, particularly due to the in-vitro produced basement membrane proteins and its small thickness.

[0020] The inventive method advantageously allows the production of a particularly thin basement membrane, the minimization of the amount of artificial, i.e. synthetic, material used for production, and the obtaining of a particularly high proportion of natural basement membrane protein to synthetic material through the expression of natural basement membrane proteins provided for in the method.

[0021] The fiber material used in process step a) is an electrospinnable material, in particular a polymer, especially a synthetic or natural polymer. Advantageously and in a preferred embodiment, the fiber material is water-insoluble. In a particularly preferred embodiment, the fiber material is biocompatible, meaning in particular that it does not impair the growth and functionality of cells, especially their ability to express and secrete proteins.

[0022] In a particularly preferred embodiment, the fiber material is not biodegradable.

[0023] In a particularly preferred embodiment, the fiber material is biodegradable.

[0024] In a particularly preferred embodiment, the fiber material used in process step a) is selected from the group consisting of non-degradable polymers, in particular polyethers, polyamides, polyacrylonitrile, polystyrene, polyethylene terephthalate, polyoxazolines, chemically functionalized polysaccharides and (block) copolymers thereof; degradable polymers, in particular polyesters, poly-α-hydroxy acids, polyglycolic acid, polylactides, polybutyl acids, polyurethanes, polypeptides and (block) copolymers thereof; natural polymers, in particular polysaccharides, plant, animal and human proteins, lecithins; and inorganic materials, in particular condensed tetraethyl orthosilicate or condensed metal alcoholates, silica gel, SiO2, TiO2, bioglass, and carbon. In one embodiment, combinations of the aforementioned substances can also be spun.

[0025] In a particularly preferred embodiment, the fiber material is polyamide, poly-α-hydroxy acid or polyacrylonitrile.

[0026] In a particularly preferred embodiment, the fibers can have a diameter of 200 to 1500 nm, in particular 300 to 1000 nm, in particular 300 to 600 nm.

[0027] According to the invention, the individual fibers of the fiber fabric are preferably freely movable from one another and, in a particularly preferred embodiment, are not connected to one another.

[0028] The electrospinning of the at least one fiber material in process step a) can preferably be carried out with a voltage of 6 kV to 25 kV, in particular 8 to 20 kV. The relative humidity (at room temperature) during electrospinning can preferably be set to 10 to 90%, in particular 20 to 80%, in particular 25 to 50%.

[0029] In a preferred embodiment, additional polymers for electrospinning the at least one fiber material in process step a) can be electrospun simultaneously and / or alternately via separate nozzles or cannulas with suitable positioning. Furthermore, additional polymers can be dissolved directly in the solution of the at least one fiber material in process step a) and spun as a polymer mixture.

[0030] In a particularly preferred embodiment of the invention, it is provided that process step a), i.e., electrospinning, takes place over a period of 1 to 30, preferably 2 to 25, in particular 3 to 20 min with reference to a fiber source.

[0031] In a particularly preferred manner, it may also be provided to selectively adjust the amount of material applied per area of ​​the support element, in particular by varying the parameters area, spinning time, delivery rate and concentration of the polymer in the spinning solution.

[0032] In a preferred embodiment, the amount of fiber material applied to the surface of the support element is 10 to 150 µg / cm². 2 preferably 18 to 70 µg / cm³ 2 (each referring to the surface area of ​​the support element).

[0033] In a preferred embodiment, the fiber fabric has meshes with an average mesh area of ​​a maximum of 400 µm. 2 , in particular a maximum of 200 µm 2 , in particular a maximum of 150 µm 2 on.

[0034] In a further preferred embodiment, the average mesh area of ​​the fiber fabric is in a range of 10 to 400 µm. 2 preferably 10 to 200 µm 2, preferably 10 to 150 µm 2 preferably from 25 to 120 µm 2 .

[0035] In a preferred embodiment, the average mesh area is particularly 20 to 200 µm². 2 , especially 50 to 200 µm 2 , especially 100 to 200 µm 2 , especially 150 to 200 µm 2 , especially 10 to 150 µm 2 , especially 20 to 150 µm 2 , especially 50 to 150 µm 2 , especially 100 to 150 µm 2 , especially 10 to 100 µm 2 , especially 20 to 100 µm 2 , especially 50 to 100 µm 2 , especially 10 to 50 µm 2 , especially 20 to 50 µm 2 .

[0036] In a particularly preferred embodiment, the invention provides that the number of fibers per area is from 1000 to 30000 fibers per mm². 2 , in particular 1500 to 25000 fibers per mm 2 , in particular 2000 to 20000 fibers per mm 2, amounts.

[0037] In a particularly preferred embodiment, the fiber fabric is provided to have an average mesh area of ​​10 to 200 µm. 2 exhibits or the amount of fiber material applied to the surface of the carrier element is 10 to 150 µg / cm² 2 The surface area of ​​the support element is, or both.

[0038] In a particularly preferred embodiment, the fiber fabric is 1 to 20 µm thick, preferably 1.5 to 10 µm, more particularly 1 to 5 µm, more particularly 1 to 3 µm, more particularly 1.5 to 3 µm, more particularly up to 1 to 2 µm or 2 to 3 µm. In a particularly preferred embodiment, the fiber fabric is 1 to 3 µm thick.

[0039] In a particularly preferred embodiment, the fiber fabric is provided that, without considering any optional stabilizing fibers, it is 1 to 20 µm thick, preferably 1.5 to 10 µm thick, more particularly 1 to 5 µm thick, more particularly 1 to 3 µm thick, more particularly 1.5 to 3 µm thick, more particularly 1 to 2 µm thick, or 2 to 3 µm thick. In a particularly preferred embodiment, the fiber fabric is provided that, without considering any optional stabilizing fibers, it is 1 to 3 µm thick.

[0040] In a particularly preferred embodiment, the fiber fabric is provided that, taking into account optionally present stabilizing fibers, it is 1 to 20 µm thick, preferably 1.5 to 10 µm thick, more particularly 1 to 5 µm thick, more particularly 1 to 3 µm thick, more particularly 1.5 to 3 µm thick, more particularly 1 to 2 µm thick, or 2 to 3 µm thick. In a particularly preferred embodiment, the fiber fabric is provided that, taking into account optionally present stabilizing fibers, it is 1 to 3 µm thick.

[0041] In a particularly preferred embodiment, the support element is a planar metal structure, in particular a metal foil, especially an aluminum foil.

[0042] In a particularly preferred embodiment, the surface of the support element to be spun can be coated with a modification material, in particular a water-soluble material. This can advantageously prevent or reduce electrostatic adhesion of the electrospun fibers to the support element.

[0043] In a particularly preferred embodiment, it is provided that the surface of the support element is coated with a water-soluble modification material in a process step x) prior to process step a).

[0044] In a particularly preferred embodiment, the water-soluble modification material can be a water-soluble polymer, a water-soluble saccharide, or a water-soluble inorganic material.

[0045] In a particularly preferred embodiment, the water-soluble modification material can be a coating, a film, or a particulate coating. In particular, the water-soluble modification material can be a water-soluble polymer, especially polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, polyacrylamide, polyacrylic acid, or a mixture thereof. In a particularly preferred embodiment, the water-soluble modification material can also be a water-soluble saccharide, for example, an oligosaccharide. In a further preferred embodiment, the water-soluble modification material can also be an inorganic material, for example, a water-soluble salt, especially sodium chloride or other sodium, potassium, and nitrate salts.

[0046] In a particularly preferred embodiment, it is provided that following process step a), the carrier element having the fiber fabric is removed from the electro-spinner in a process step z) and transferred to a device for carrying out process step b).

[0047] In a particularly preferred embodiment, the stabilization frame is configured as a cell culture container in process step b). In this preferred embodiment, a stabilization frame is applied in process step b) that is designed, configured, and suitable to allow the cultivation of cells together with the fiber mat. In this embodiment, the stabilization frame serves in the subsequent process step c) for cultivating the cells of the at least one cell type.

[0048] In a particularly preferred embodiment, in process step b) the stabilization frame is designed as a cell culture container and the cultivation of the cells in the stabilization frame designed as a cell culture container, as provided for in process step c), is carried out.

[0049] Preferably, the device for carrying out process step b) is a polymer application device, in particular a printer or sprayer.

[0050] The stabilizing frame is preferably composed of one or more polymers. In a particularly preferred embodiment, the polymer is a biocompatible polymer, in particular a thermoplastic, especially polycaprolactone (PCL), polyetheretherketone (PEEK), polylactide (PLA), polyetherimide (PEI), PE (polyethylene), PP (polypropylene), PC (polycarbonate), PS (polystyrene), PET (polyethylene terephthalate), PA (polyamide), POM (polyoxymethylene), PGA (polyglycolic acid), PHB (polyhydroxybutyric acid), or poly(methyl methacrylate) (PMMA), or a combination thereof.

[0051] In a particularly preferred embodiment, it is provided that the stabilizing frame is applied to the fiber fabric in process step b) by means of a printing process, in particular to its upper side, i.e. the side facing away from the surface of the support element.

[0052] In a particularly preferred embodiment, the printing process can be carried out using a 3D printer.

[0053] In a particularly preferred embodiment, the material of the stabilizing frame can be applied to the fiber fabric by means of a fused deposition modeling (FDM) process, preferably using thermoplastics, in particular biocompatible materials, for three-dimensional printing.

[0054] According to the invention, it is preferred that the stabilization frame applied in process step b) is individually adapted to the size and shape of the application intended for the produced artificial basement membrane. In a preferred embodiment, the stabilization frame can have the shape and size of a Transwell system, a bioreactor, or an organ / human-on-a-chip system. In a preferred embodiment, the stabilization frame can be annular, i.e., for example, a stabilization ring, or angular, in particular rectangular.

[0055] In a particularly preferred embodiment, the stabilizing frame has a width of 100 to 1000 µm, preferably 200 to 800 µm, and a thickness of 50 to 600 µm, in particular 100 to 500 µm.

[0056] In a particularly preferred embodiment, the fiber fabric associated with the stabilizing frame is detached from the support element in a process step v) after process step b), in particular before carrying out process step c), after carrying out process step c), before carrying out process step d) or after carrying out process step d).

[0057] The detachment of the stabilizing frame with the attached fiber fabric from the support element, preferably provided for in process step v), can be carried out in particular by incubation in aqueous solution, especially water, for example for 0.5 to 5 minutes, in particular 0.5 to 1 min.

[0058] In a preferred embodiment, in process step v) it is provided that, prior to cultivating the fiber fabric associated with the stabilizing frame in process step c), the fiber fabric associated with the stabilizing frame is first detached from the surface of the support element.

[0059] In a particularly preferred further embodiment, in a subsequent process step following process step b), also referred to here as process step w), the fiber fabric associated with the stabilizing frame is transferred into a cell culture container. Depending on the device in which the stabilizing frame was applied, the transfer takes place either from the electrospinner or from the polymer application device.

[0060] In a preferred embodiment, in a process step v) it is provided that, prior to the transfer of the fiber fabric associated with the stabilizing frame into the cell culture container as provided in process step w), the fiber fabric associated with the stabilizing frame is first detached from the surface of the support element, so that only the fiber fabric fixed to the stabilizing frame is transferred, without the support element.

[0061] Preferably, following the detachment of the fiber mesh connected to the stabilizing frame from the support element as provided in process step v), this construct is transferred to a cell culture container in process step w) and cultured in the cell culture container with cells of at least one cell type introduced into the fiber mesh in the subsequent process step c), wherein this cell type is capable of expressing at least one basement membrane protein, in particular of expressing and secreting at least one basement membrane protein into the cell environment. In one embodiment, the construct consisting of the fiber mesh and stabilizing frame connected to the support element can also be transferred to the cell culture container and subjected to the further process steps, optionally with subsequent detachment of the support element.

[0062] In a particularly preferred embodiment, following process step b), the fiber fabric associated with the stabilizing frame is transferred in process step w) to a separate cell culture container and process step c) is carried out in the separate cell culture container.

[0063] In a further preferred embodiment, it may be provided that the fiber fabric is stabilized on one of its sides, in particular the top or bottom.

[0064] In a particularly preferred embodiment, it is provided that, prior to process step c), stabilizing fibers with a fiber diameter of 1 to 500 µm made of a stabilizing material are applied to at least one side, in particular the top or bottom or both sides of the fiber lay-up, in a process step y).

[0065] In a further preferred embodiment, it may be provided that the fiber fabric is stabilized on its underside by applying stabilizing fibers, i.e. on the side on which the stabilizing frame does not rest.

[0066] In a further preferred embodiment, it may be provided that the fiber fabric is stabilized on its upper side by applying stabilizing fibers, that is, on the side on which the stabilizing frame rests.

[0067] In a particularly preferred embodiment, the fiber diameter of the stabilizing fibers can be in a range of 1 to 500 µm, in particular 5 to 200 µm, in particular 5 to 20 µm.

[0068] In a particularly preferred embodiment, the stabilizing fibers may consist of or comprise polyamide, polycaprolactone or polyester.

[0069] In a particularly preferred embodiment, commercially available nets, especially made of polyamide or polyester, can be applied.

[0070] In a particularly preferred embodiment, the stabilizing fibers can be applied by directly applying the fibers using microfiber spinning processes, for example melt electrowriting, pressure spinning, melt blow spinning or fiber drawing.

[0071] In a particularly preferred embodiment, it is provided that the stabilizing fibers are applied to the top of the fiber fabric located on the support element in a process step y1) following process step a) and before process step b).

[0072] In a particularly preferred embodiment, it is provided that the stabilizing fibers are applied to the underside of the fiber fabric in a process step y2) following the detachment of the fiber fabric associated with the stabilizing frame from the support element in process step v) and before the transfer to a cell culture container preferably provided in process step w).

[0073] In a particularly preferred embodiment, the cells of at least one cell type cultivated in process step c) are cells that are able to express and secrete proteins of the basement membrane, in particular structural proteins, in particular proteoglycans, in particular collagen type 4, laminins, fibronectin, or a combination thereof.

[0074] In a particularly preferred embodiment, the cells of at least one cell type are cells that are capable of expressing and secreting fibrillar structural proteins such as collagen type 1, 3 or type 7.

[0075] In a particularly preferred embodiment, the human cell lines providing fibrillar collagens can be MCF-8, HeLa, HUVEC, HEK203, L929, A549, Caco-2 or MDCK.

[0076] In a particularly preferred embodiment, the fibrillar collagen-providing cells can be human stromal cell lines, in particular HFF-1, MRC-5, HS-27A, HS-5 or NGH-3T3.

[0077] In a particularly preferred embodiment, the fibrillar collagen-providing cells can be induced pluripotent stem cells (iPS), in particular IMR90-4, UKBi005 or UKKi011.

[0078] Furthermore, primary human cell lines capable of expressing and secreting Col3 (type 3 collagen) can be used. Additionally, primary human stromal cell lines, iPS-derived stromal cells, and animal stromal cells, in particular, can be used for this purpose.

[0079] In a particularly preferred embodiment, the cells of at least one cell type are cells that are able to express and secrete reticular structural proteins such as collagen type 4, fibronectins or laminins.

[0080] In a particularly preferred embodiment, endothelial cells, epithelial cells or tumor cells can be used.

[0081] In a particularly preferred embodiment, the cells can be cells from human cell lines, primary human cells, iPS-derived cells, or animal cells.

[0082] In a particularly preferred embodiment, the human cell lines can be cell lines from gigablastomas or cell lines from other carcinomas, in particular from breast, lung, prostate, gastric cancer, or invasive or metastatic melanomas. In a particularly preferred embodiment, the human cell line can be derived from the epithelium. In a particularly preferred embodiment, the cell line can be derived from the endothelium.

[0083] In a particularly preferred embodiment, the primary human cells can be tumor cells, epithelial cells, or endothelial cells. In a particularly preferred embodiment, the iPS-derived cells can be tumor cells, epithelial cells, and / or endothelial cells. In a particularly preferred embodiment, the animal cells can also be animal cell lines, in particular tumor cells, epithelial cells, and endothelial cells.

[0084] In a particularly preferred embodiment, the cell type is selected from the group consisting of human cell lines, animal cell lines, primary human cells, primary animal cells, induced pluripotent cell lines (IPS), IPS-derived cells and stromal cells.

[0085] In a particularly preferred embodiment, cultivation takes place for a period of 5 to 30, in particular 7 to 21 days.

[0086] In a particularly preferred embodiment, it is provided that process step c) is carried out at least twice sequentially with cells of different cell types.

[0087] In a particularly preferred embodiment, a method is provided in which a two-layered basement membrane is produced. In a particularly preferred embodiment, it may be provided in a process step c) that cells of at least one cell type are cultivated in a first phase and subsequently cells of at least one second cell type are cultivated in a second phase, such that different basement membrane products are deposited in the fiber matrix in the two different phases.

[0088] In a particularly preferred embodiment, in a first phase of process step c), it may be provided to cultivate cells of a first cell type that enable the provision of fibrillar proteins, in particular collagen type 3 or collagen type 1, and in a second phase to cultivate cells of a second cell type that enable the provision of reticular proteins, in particular collagen type 4 or laminins. Due to the temporal sequence, a reticular protein layer is formed in the second phase above the fibrillar proteins introduced during the first phase.

[0089] In a particularly preferred embodiment, the removal of the cells of the at least one cell type provided for in process step d) while preserving the artificial basement membrane can be carried out by incubation in decellularization solutions, in particular in solutions containing the agents sodium dodecyl sulfate, sodium deoxycholate, Triton X-100, tri-n-butyl phosphate (TnBP), CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) or hypertonic or hypotonic solutions.

[0090] Preferably, decellularizing solutions are used, wherein the decellularizing agents are present in a concentration of 0.1 to 3 vol.%.

[0091] In a particularly preferred embodiment, the incubation period for decellularization is from 0.5 to 5 hours (h).

[0092] In a particularly preferred embodiment, it may be provided that the cell fragments possibly present in the product produced according to the invention after decellularization are washed out by one or more rinsing, in particular with physiological saline solution, and that any remaining DNA is degraded by incubation in DNase solution for in particular 0.5 to two hours, in particular one hour.

[0093] In one embodiment of the invention, the stabilizing frame can be removed following process step c), in particular following process step d).

[0094] In a particularly preferred embodiment, the artificial basement membrane is provided to be 1 to 20 µm, preferably 1.5 to 10 µm, in particular 1 to 5 µm, in particular 1 to 3 µm, in particular 1.5 to 3 µm, in particular 1 to 2 µm or 2 to 3 µm thick.

[0095] In a particularly preferred embodiment, the artificial basement membrane is provided to be 1 to 3 µm thick.

[0096] In a particularly preferred embodiment, the artificial basement membrane is provided to be 1 to 20 µm thick, preferably 1.5 to 10 µm, in particular 1 to 5 µm, in particular 1 to 3 µm, in particular 1.5 to 3 µm, in particular 1 to 2 µm or 2 to 3 µm, without taking into account any optional stabilizing fibers.

[0097] In a particularly preferred embodiment, the artificial basement membrane is provided to be 1 to 3 µm thick, without taking into account any optional stabilizing fibers.

[0098] In a particularly preferred embodiment, the artificial basement membrane is provided to be 1 to 20 µm, preferably 1.5 to 10 µm, in particular 1 to 5 µm, in particular 1 to 3 µm, in particular 1.5 to 3 µm, in particular 1 to 2 µm or 2 to 3 µm thick, taking into account optionally present stabilizing fibers.

[0099] In a particularly preferred embodiment, the artificial basement membrane is provided to be 1 to 3 µm thick, taking into account optionally present stabilizing fibers.

[0100] The present invention also provides an artificial basement membrane comprising a fiber mesh of electrospun fibers with a fiber diameter of 100 to 2000 nm, wherein the fiber mesh is embedded in a matrix produced by cells of at least one cell type capable of expressing at least one basement membrane protein, in particular produced by a method according to the invention.

[0101] The present invention also provides an in vitro test system, cell culture system, artificial tissue, implant, insert system, filtration system and human-on-a-chip / organ system, comprising at least one basement membrane according to the invention.

[0102] The present invention provides in particular an in vitro test system, especially a barrier model, comprising at least one basement membrane according to the invention.

[0103] The basement membrane according to the invention can be used cell-free or cell-containing.

[0104] According to the invention, the basement membrane can preferably be used in a cell-free or cell-containing version as an insert system or as an organ- or human-on-a-chip system, particularly in applications in the field of: Blood-brain barrier / neurovascular unit, intestine / gastrointestinal tract, endothelium, lungs, airways, skin, oral mucosa, cornea, retinal pigmented epithelium, pathological variants of the aforementioned models, liver, kidney, all other internal organs.

[0105] The basement membrane according to the invention can be used in vitro or in vivo, for example as a cell-free or cell-containing implant.

[0106] The basement membrane according to the invention can also be used as a filtration system, in particular as a filter or filter material, especially for sterile filtration.

[0107] The basement membrane according to the invention can also be used for stem cell culture or for the culture and application of spheroids, organoids and assembloids.

[0108] In a particularly preferred embodiment, the artificial basement membrane comprises cells of at least one target cell type applied in or onto the artificial basement membrane.

[0109] Cells of a target cell type are cells that are placed on or into a basement membrane according to the invention and cultured in order to provide a desired cell-based applicability using cultured cells, for example as a test system, in particular an in vitro test system, in particular a barrier model, or as an artificial tissue or organ system.

[0110] In a preferred embodiment, cells of at least one target cell type can be small intestinal cells, epithelial cells, endothelial cells or tumor cells.

[0111] In a preferred embodiment of the invention, the at least one target cell type is selected from the group consisting of fibroblasts, in particular cancer-associated fibroblasts, keratinocytes, mesenchymal stem cells, iPS cells (induced pluripotent stem cells), tumor cells, endothelial cells and combinations thereof.

[0112] The invention also relates to the use of a basement membrane according to the invention for the cultivation or differentiation of cells.

[0113] The invention also relates to an artificial tissue comprising at least one basement membrane according to the invention and at least one cell of at least one target cell type.

[0114] In a preferred embodiment, the artificial tissue has several, in particular at least two, in particular at least three, basement membranes according to the invention.

[0115] The invention also relates to an artificial tissue comprising a basement membrane according to the invention and cells of at least one target cell type, wherein the artificial tissue comprises 1 to 50 wt.% basement membrane and 50 to 99 wt.% cells, in particular 80 to 99 wt.% cells (in each case based on the total weight of the artificial tissue).

[0116] In a preferred embodiment, the artificial tissue is a small intestine model.

[0117] In a preferred embodiment, the artificial tissue is a model of a blood-brain barrier / neurovascular unit.

[0118] In a preferred embodiment, the artificial tissue is a skin model, in particular comprising a dermis comprising at least one basement membrane according to the invention. In a preferred embodiment, the artificial tissue is a skin model, preferably comprising a subcutis comprising at least one basement membrane according to the invention.

[0119] In a preferred embodiment, the artificial tissue is a skin model, in particular comprising a dermis, comprising at least one basement membrane according to the invention, and a subcutis comprising at least one basement membrane according to the invention, preferably a different basement membrane.

[0120] Preferably, the at least two basement membranes used in the skin model according to the invention are specifically designed, in particular they each have at least one cell of a specific cell type, in particular of different cell types.

[0121] In a preferred embodiment, the cells are homogeneously distributed within the basement membrane according to the invention.

[0122] The present invention also relates to therapeutic applications of the basement membranes according to the invention and artificial tissues comprising them.

[0123] The present invention also relates to methods for the therapy of, in particular, sick, humans or animals, and products for use in such methods.

[0124] In particular, the basement membrane according to the invention can be used as a wound dressing, especially for the treatment of preferably chronic wounds or burns, in particular by insertion, in particular by placing, into wounds, colonization with tissue cells from the wound edges, closure of the wound over the basement membrane according to the invention by cells, in particular keratinocytes.

[0125] The basement membrane according to the invention can also be used as a porous tissue which enables the ingrowth of cells in vivo during regeneration after, for example, surgical procedures or resections in (cancer) therapy.

[0126] In particular, the present invention also relates to methods for the therapy of, especially diseased, humans or animals using the artificial tissue or basement membrane according to the invention, and the basement membrane or the artificial tissue according to the invention for use in such methods. In particular, the artificial tissue according to the invention can be used as an ATMP (Advanced Therapy Medicinal Product), preferably seeded with tissue-specific cells of a target cell type, wherein its use in or as stromal tissue / organ(s) is preferred, in particular in or as skin, cartilage, arteries and / or veins, heart, kidney, liver, urogenital tract, respiratory tract, bones, intestine or cornea.

[0127] The present invention also relates to non-therapeutic applications of the basement membrane according to the invention and artificial tissues comprising it, for example cosmetic applications.

[0128] In connection with the present invention, the term "fiber count / mm" is used. 2 “the number of fibers per mm 2 Fiber density, also referred to here as fiber layup, refers to the entire surface area of ​​the top or bottom surface of the fiber layup in its planar dimensions, i.e., length and width. Only the top or bottom surface is considered for area calculation, not both, nor are the edge surfaces. Fiber density is preferably determined using light or scanning electron microscopy.

[0129] The directions of extension of spatial structures, such as fiber mats, support elements, or basement membranes of the present invention, are designated as length, width, and thickness with reference to a Cartesian three-dimensional coordinate system, wherein the length corresponds to the x-axis, the width to the y-axis lying in the same plane, and the thickness to the z-axis perpendicular, in particular vertical, to this plane spanned by the x- and y-axes. Planar directions of extension of the aforementioned spatial structures, in particular length and width, are directions in which the extension is significantly greater, in particular many times greater, in particular at least 5 times greater, than in non-planar directions of extension, in particular thickness.

[0130] In connection with the present invention, the “amount of material applied per area of ​​the support element” is calculated according to Example 1.

[0131] In connection with the present invention, the "mesh area" of the fiber layup is determined using microscopy methods, particularly without a medium or in water, and especially without mechanical stress. The most suitable methods are transmitted light microscopy or 3D methods such as laser scanning microscopy (LSM) or the similar method of convocal reflection microscopy. The individual images of a nanofiber layer are then superimposed from the resulting 3D image to form a 2D image. Subsequently, the area of ​​the spaces between the nanofibers is determined. In particular, the mesh size is determined according to the procedure described in Example 4.

[0132] In connection with the present invention, the term "fiber mat", also referred to here as fiber fleece, is understood to mean a sheet structure consisting of disordered or specifically aligned fibers, wherein in a preferred embodiment the fibers are not connected, interwoven or entangled with each other.

[0133] In connection with the present invention, the term "side" is understood to mean the planar side of a fiber fabric or a basal membrane, in particular the top or bottom side, wherein the edge surfaces or end faces of the membrane or fiber fabric are not covered by the term "side" due to their small thickness.

[0134] In the context of the present invention, the term "electrospinner" refers to a device used in electrospinning to produce very small diameter fibers from a polymer solution or melt, in particular by applying a high voltage to draw a polymer liquid droplet into a thin stream, which is then transformed into a nanofiber structure by evaporation or solidification. An electrospinner comprises, in particular, a high-voltage source, a syringe pump for conveying the polymer solution at a controlled rate, a nozzle, for example a needle or capillary, for the exit of the polymer solution, and a collector, for example a metal plate or drum, for collecting and forming the fibers.

[0135] In the context of the present invention, the term "biocompatible" means that a material or substance does not impair the growth and function of cells, in particular their ability to express and secrete proteins, and especially that the material or substance is accepted by the human or animal body, particularly without triggering a harmful immune response or toxic effect upon contact. Biocompatible materials are preferably non-toxic, non-immunogenic, non-carcinogenic, and / or, depending on the application, stable or biodegradable.

[0136] In the context of the present invention, the term “biodegradable” means that the element in question, in particular substance, in particular polymer, can be degraded by biological processes, in particular naturally occurring biological processes, in particular biological processes occurring naturally in a human or animal body, and thus loses its structural and / or material integrity.

[0137] In connection with the present invention, the term "cell culture container" preferably refers to a cell culture plate, microplate, Petri dish, bioreactor, microfluidic chip, multi-chamber system, roller bottle or cell culture flask.

[0138] The methods according to the invention are characterized by a preferred sequence of process steps x) (optional coating with modification material), a), y1) (optional application of stabilizing fibers to the top of the fiber fabric), z) (optional removal from electrospinner), b), v) (optional detachment of the carrier element), y2) (optional application of stabilizing fibers to the underside of the fiber fabric after carrying out process step v)), c) and d).

[0139] The methods according to the invention are characterized by a preferred sequence of process steps x) (optional coating with modification material), a), y1) (optional application of stabilizing fibers to the top of the fiber fabric), z) (optional removal from electrospinner), b), v) (optional detachment of the carrier element), y2) (optional application of stabilizing fibers to the underside of the fiber fabric after carrying out process step v)), w) (transfer to cell culture container), c) and d).

[0140] In a particularly preferred embodiment, the process steps take place in the order specified in the present disclosure, unless otherwise stated or as is evident to a person skilled in the art.

[0141] In a particularly preferred embodiment of the present invention, it is provided that no further process steps are carried out between the individual explicitly specified process steps.

[0142] In the context of the present invention, the term "and / or" is understood to mean that all members of a group connected by the term "and / or" are represented both cumulatively in any combination and alternatively to one another. For example, the expression "A, B and / or C" is understood to have the following disclosure content: i) (A or B or C), or ii) (A and B), or iii) (A and C), or iv) (B and C), or v) (A and B and C), or vi) (A and B or C), or vii) (A or B and C), or viii) (A and C or B).

[0143] In connection with the present invention, individual components or constituents of a whole, in particular the basement membrane according to the invention or one of its components, which are quantitatively determined in relative form, in particular in percentages, unless otherwise specified, preferably add up to 100 wt.% of the whole referred to or of the basement membrane or, if referred to, of a component thereof.

[0144] In connection with the present invention, unless otherwise stated, any unspecified decimal places are to be understood as 0.

[0145] Further advantageous embodiments of the invention are set out in the dependent claims.

[0146] The following examples and the accompanying figures illustrate the present invention without limiting it.

[0147] The figures show: Fig. 1. Handling of the thin fiber fabric. (A) After production of the thin fiber fabric, PCL rings were pressed directly onto the fibers. (B) Due to the water-soluble intermediate layer of modification material, the fiber fabric with the stabilizing frame can be detached and (C) removed after a short incubation in water. Fig. 2 Extension of the thin fiber layup (polyamide 6) with MEW reinforcing fibers (PCL, polycaprolactone). Fig. 3 Characterization of the thin fiber lay-ups by measuring the mesh areas. Fig. 4. Characterization of the cellularly modified and decellularized fiber network. In cross-section, the fibers can be identified by their characteristic shape. The immunohistochemically stained collagen IV is clearly visible around and between the fibers. Fig. Five small intestinal epithelial cells form a monolayer on a synthetic basement membrane according to the invention. The DAPI stain (A) shows the characteristic round nuclei with a diameter of approximately 10 µm. The phalloidin stain (B) describes the cell body via the cytoskeleton and stains the rest of the cell except for the nucleus. (C) shows a superimposition of both stains. Fig. Six HiPS cell-derived endothelial cells of the blood-brain barrier form monolayers on the biomodified synthetic basement membrane according to the invention. DAPI staining shows the characteristic round nuclei with a diameter of approximately 10 µm in all images. The cytoplasmic tight jugtion protein ZO-1 (A) is present around each cell body in varying concentrations. Another tight jugtion protein, occludin (B), connects the cells and is therefore visible as a line around each individual cell. The membrane transporter protein Glut-1 (C) is located on the cell surface and is therefore visible on all cells in varying concentrations. Examples: Example 1 - Basic framework made of electrospun fibers and stabilizing frame

[0148] The fiber fabrics were produced in an electric spinner with a rotating roller of 10.5 cm diameter. The surface was wrapped with an aluminum foil as a carrier element, and adhesive tape was used to create a defined surface area of ​​396 cm². 2The surface was then coated with the modification material NaCl powder to facilitate later fiber removal (process step x)). For the spinning process (process step a)), a polyamide 6 solution with a concentration of 12% w / v in 1,1,1,3,3,3-hexafluoro-2-propanol was prepared and placed in a syringe with a distance of 15 cm between the needle tip and the roller surface. During the spinning process, the pump was set to a delivery rate of 0.6 mL / h, a potential difference of 11 kV, a rotation speed of 100 rpm, and a parallel movement of the needle relative to the roller of 10 mm / s. The spinning duration was varied in this example over 6 min, 10 min, and 16 min.

[0149] The theoretical fiber content per area is determined from these parameters as follows: Mass promoted / surface area=(0.12*0.6 mL / h*1.14 g / cm3*spinning time) / 396 cm2.

[0150] The fiber content per area was: 20.7 µg / cm² 2 (6 min), 34.5 µg / cm³ 2 (10 min), 55.3 µg / cm² 2 (16 min).

[0151] Subsequently, the fiber layup associated with the stabilizing frame was removed from the electrospinner in process step z) and placed in an FDM 3D printer (Fused Deposition Modeling (FDM)). The printing (process step b)) of the stabilizing rings, i.e., the stabilizing frame, was carried out directly onto the fibers using polycaprolactone with an inner diameter of 9 mm and an outer diameter of 10 mm ( Fig. 1A). Finally, the construct, i.e., the fiber fabric associated with the stabilizing frame, can be detached from the support element by short incubation in water and removed (process step v)) ( Fig. 1B, C). Example 2 - Additional stabilization of the fiber layup via µm fibers

[0152] For additional stabilization, extra fibers can either be applied directly to the electrospun fibers (process step y1)) followed by printing on the stabilizing frame, or, as described below, to the underside of the construct after printing on the stabilizing frame (process step y2)). For this, the electrospun fiber and frame construct was detached from the aluminum surface with water (process step v)), dried, and placed upside down in a MEW (melt-electrowriting (MEW)) printer. Subsequently, the electrospun fibers were printed with polycaprolactone in a checkerboard pattern with fiber-to-fiber spacing of 300 µm and a fiber thickness of approximately 10 µm (process step y2)). Fig. Figure 2 shows the resulting structure using different microscopy methods and magnifications. Fig. Figure 2 shows light microscopy images (top) and SEM (scanning electron microscopy) images (bottom) with different electrospun fiber densities, produced with (A) 6 min, (B) 10 min and (C) 16 min spinning times. The reinforcing fibers, which have a significantly larger diameter than the fibers of the fiber layup also shown, were applied to the back of the fiber layup via melt electrowriting.

[0153] This approach allows great freedom in the structure and arrangement of these MEW fibers, especially with regard to fiber thicknesses, fiber spacings, angular orientation of the fibers, and non-linearly laid fibers. Example 3 - Biologization process of the synthetic scaffold

[0154] The synthetic scaffold, i.e., the fiber matrix, was first placed in a transwell device (clamped in a cell crown, two plastic cylinders of different diameters nested inside one another), positioned in a 24-well plate (process step w), transferred to cell culture containers, and covered with a total of 1.5 ml of culture medium, both inside and out. Subsequently, 100,000 cells per sample were cultured (process step c)) for 14 days (medium changes 3 times per week). To remove the cells, the medium was first removed, the sample was covered with a sodium deoxycholate solution (0.33 g / 10 ml deionized water; DE: fully demineralized), and incubated for 30 min at room temperature on a shaker (process step d)). To remove cell residues, 4 washing steps were performed with PBS (phosphate-buffered saline) for 20 min at room temperature on a shaker, followed by overnight incubation in fresh PBS at 4°C.In the final step, the DNA was removed by incubation in a DNase solution at 37°C for 1 hour. After three further washing steps in PBS, the synthetic, bio-modified scaffold was stored at 4°C until further use. All steps were performed under sterile conditions to avoid final, tissue-damaging sterilization methods. Example 4 - Stitches

[0155] To characterize the fiber densities, the number of fibers / area and the mesh areas were determined from scanning electron microscopic or light microscopic images of the fiber layups from Example 1. Fig. Figure 3 AC (above) shows, as examples, the fiber densities of the three different spinning times. The representation of the mesh areas describes Fig. 3A-C, below. Fig. Figure 3 shows a light microscopic image of the fiber layups (top) with the corresponding histogram of the mesh areas and number of meshes (bottom). The densities of the fiber layups were adjusted via the spinning times of (A) 6 min, (B) 10 min, and (C) 16 min. The fiber density was determined manually, and the mesh area was calculated using ImageJ and is shown in the size range of 1 µm. 2 and 1000 µm 2 . Example 5 - Biologically modified basement membrane

[0156] To investigate the structure and composition of the finished product of the synthetically based and bio-engineered basement membrane, immunofluorescence staining against collagen type 4 of paraffin sections was performed. Fig. Figure 4A shows the fiber layup with a 6-minute spinning time and Fig. 4B with a spinning time of 16 minutes. Secondly, the samples were processed according to... Fig. 4B (16 min spinning time) via critical spot drying ( Fig. 4C, E: for critical spot drying with 2 and 1 µm scale) or lyophilization ( Fig. 4D, F: for lyophilization with 10 and 1 µm scale) dried and its dense surface was checked by scanning electron microscopy. The presence of collagen IV (red staining) is visible across the entire surface and thickness of the fiber matrix by immunofluorescence of paraffin sections; exemplified in the sample with (A) 6 min spinning time and (B) 16 min spinning time. Complete filling of the interfiber spaces with ECM matrix can be confirmed after both (C) critical spot drying and (D) lyophilization (for 16 min spinning time: Fig. 4C, D, E and F). Example 6 - Small intestine

[0157] To confirm the applicability of the bio-modified basement membrane according to the invention, human primary small intestine organoids, cultured in Matrigel® (3D environment), were dissociated into single cells using TrypLE Express, and 400,000 single cells were applied apically to the bio-modified basement membrane. For the culture of the primary organoids, a medium cocktail consisting of a variety of growth factors that support proliferation, suppress apoptosis, and prevent differentiation was used for a period of 5 days until a closed epithelial cell layer was formed. For a further 5 days, another medium cocktail is applied (daily medium changes) which specifically induces differentiation of the intestinal epithelium (Schweinlin et al., 2016, DOI: 10.1089 / ten.TEC.2016.0101, Däullary et al., 2023, https: / / doi.org / 10.1080 / 19490976.2023.2186109).The formation of a closed, single-layered cell structure was confirmed by immunofluorescence staining. The nuclei were stained with DAPI (blue) and the cell membrane with phalloidin (red) (see figure). Fig. 5). Fig. Figure 5 shows representative images of an immunofluorescence stain. The markers (A) DAPI (blue, nuclei) and (B) phalloidin (red, cell membrane) show the formation of a single-layered, closed epithelial cell layer on the decellularized basement membrane. (C) shows a merger of the two stained cell structures. Example 7 - Blood-Brain Barrier

[0158] Another application example of the bio-modified basement membrane according to the invention is the construction of in vitro models / test systems of the human blood-brain barrier / neurovascular unit. For this purpose, endothelial cells of the blood-brain barrier, derived from hiPS cells (human iPS cells) and differentiated on day 8 of differentiation, were seeded as single cells at a defined density of 1 x 10 6 cells / cm² 2 The culture surface was applied to the biologically modified basement membrane. The cells were treated with cell-specific medium for 2 days to ensure the formation of a dense, barrier-forming monolayer. To characterize the differentiation success of the hiPS cell-derived endothelial cells of the blood-brain barrier, immunofluorescence staining for tissue-specific tight junction proteins and transporters was performed. Fig. 6A-C). Fig.Figure 6 shows representative images of immunofluorescence staining for the markers (A) ZO-1 (Zona Occludens), (B) Occludin and (C) Glut-1 (glucose transporter), which show the formation of a monolayer on the biosynthesized basement membrane and the expression of characteristic blood-brain barrier markers.

Claims

[1] A method for producing an artificial basement membrane comprising a fiber matrix embedded in a matrix composed of at least one basement membrane protein, comprising the following process steps: a) Manufacturing and applying a fiber fabric comprising fibers with a fiber diameter of 100 to 2000 nm, by electrospinning from a fiber material onto the surface of a carrier element arranged in an electrospinner, wherein the fiber fabric has a fiber count of 1000 to 30000 fibers / mm² 2 exhibits b) Applying a stabilizing frame made of at least one polymer to the fiber fabric arranged on the support element under conditions that allow the stabilizing frame to associate with the fiber fabric, c) Cultivating the fiber web associated with the stabilizing frame with cells of at least one cell type introduced into the fiber web, which are capable of expressing at least one basement membrane protein, under conditions that allow the expression of the at least one basement membrane protein and while maintaining a matrix composed of the at least one basement membrane protein in which the fiber web is embedded and d) Removal of cells of at least one cell type while preserving the artificial basement membrane. [2] Method according to claim 1, wherein in process step b) the stabilization frame is designed as a cell culture container and the cultivation of the cells provided for in process step c) is carried out in the stabilization frame designed as a cell culture container. [3] Method according to claim 1, wherein following process step b) the fiber fabric associated with the stabilizing frame is transferred in a process step w) into a separate cell culture container and process step c) is carried out in the separate cell culture container. [4] Method according to one of the preceding claims, wherein, after process step b), in a process step v), the fiber fabric associated with the stabilizing frame is detached from the support element. [5] Method according to any of the preceding claims, wherein the fiber fabric has an average mesh area of ​​10 to 200 µm 2 exhibits or the amount of fiber material applied to the surface of the carrier element is 10 to 150 µg / cm² 2 The surface area of ​​the support element is, or both. [6] Method according to one of the preceding claims, wherein the surface of the support element is coated with a water-soluble modification material in a process step x) prior to process step a). [7] Method according to one of the preceding claims, wherein the stabilizing frame is applied to the fiber fabric in process step b) by means of a printing process. [8] Method according to one of the preceding claims, wherein following process step a) the carrier element having the fiber lay-up is removed from the electro-spinner in a process step z) and transferred to a device for carrying out process step b). [9] Method according to one of the preceding claims, wherein in a process step y) prior to process step c) stabilizing fibers with a fiber diameter of 1 to 500 µm made of a stabilizing material are applied to at least one side of the fiber lay-up. [10] Method according to claim 9, wherein the stabilizing fibers are applied to the top of the fiber lay-up on the support element in a process step y1) following process step a) and before process step b). [11] Method according to claim 9, wherein the stabilizing fibers are applied to the underside of the fiber fabric in a process step y2) following the detachment of the fiber fabric associated with the stabilizing frame from the support element as provided in process step v) and prior to the cultivation provided in process step c). [12] Method according to any of the preceding claims, wherein process step c) is carried out at least twice sequentially with cells of different cell types. [13] Method according to any of the preceding claims, wherein the fiber material is selected from the group consisting of non-degradable polymers, in particular polyethers, polyamides, polyacrylonitrile, polystyrene, polyethylene terephthalate, polyoxazolines, chemically functionalized polysaccharides and (block) copolymers thereof, degradable polymers, in particular polyesters, poly-α-hydroxy acids, polyglycolic acid, polylactides, polybutyl acids, polyurethanes, polypeptides and (block) copolymers thereof, natural polymers, in particular polysaccharides, plant, animal and human proteins, lecithins, and inorganic materials, in particular condensed tetraethyl orthosilicate or condensed metal alcoholates, silica gel, SiO2, TiO2, bioglass and carbon. [14] Method according to any of the preceding claims, wherein the cell type is selected from the group consisting of human cell lines, animal cell lines, primary human cells, primary animal cells, induced pluripotent cell lines (IPS), IPS-derived cells and stromal cells. [15] Method according to any of the preceding claims, wherein the artificial basement membrane is 1 to 20 µm thick. [16] Artificial basement membrane comprising a fiber mesh of electrospun fibers with a fiber diameter of 100 to 2000 nm, wherein the fiber mesh is embedded in a matrix produced by cells of at least one cell type capable of expressing at least one basement membrane protein, in particular produced by a method according to any one of claims 1 to 15. [17] Artificial basement membrane according to claim 16, comprising cells of at least one target cell type applied in or onto the artificial basement membrane according to claim 16. [18] In vitro test system, cell culture system, artificial tissue, implant, insert system, filtration system or human-on-a-chip / organ system comprising at least one basement membrane according to claim 16 or 17.

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