EP3164484A1

EP3164484A1 - Tissue model, method for producing said tissue model, and use of said tissue model

Info

Publication number: EP3164484A1
Application number: EP15747391.9A
Authority: EP
Inventors: Andreas BLAESER; Benjamin THEEK; Twan LAMMERS; Fabian Kiessling; Horst Fischer
Original assignee: Rheinisch Westlische Technische Hochschuke RWTH
Current assignee: Rheinisch Westlische Technische Hochschuke RWTH
Priority date: 2014-07-04
Filing date: 2015-07-06
Publication date: 2017-05-10
Also published as: WO2016001442A1; US20170130193A1; DE102014109360A1

Abstract

In a first aspect, the present application relates to a method for producing a tissue model comprising supply structures. In a further aspect, the application relates to tissue models that can be obtained in this way and to the use of said tissue models as models for tissue genesis, in particular tumorigenesis, including angiogenesis, in the tissue model. Finally, a method for testing and identifying active ingredient candidates, including treatment strategies, is provided.

Description

Tissue Model and Method of Making the Same and Use Thereof In a first aspect, the present application is directed to a method of making a tissue model having utility structures. In a further aspect, the application is directed to these tissue models thus obtainable as well as their use as models for tissue genesis, in particular tumorigenesis, including angiogenesis and the tissue model. Finally, a method for testing and identifying drug candidates including treatment strategies is provided.

PRIOR ART For the development of new active substances, for example in the field of tumor treatment, potential pharmacological substances over a multistage process are first investigated in cell culture and in the case of promising results in vivo in animal experiments. It turns out again and again, however, that the results of / n-V7fro experiments are not readily transferable to in vivo experiments, many potential active substances fail in these experimental phases. More effective screening for V7fro screening, in particular for subsequent / nV / Vo experiments, is desirable in order to reduce the cost of developing active agents and, at the same time, reduce the numbers of experimental animals. With the aim to establish an improved screening platform between cell culture and animal studies are currently different 3D / nv / ^'fro-tissue engineering (TE) pursues concepts. A first A step towards a 3D tumor model was made with the study of statically cultured cell-loaded microparticles, eg, Horning JL, et al. Mol. Pharm. 2008 5: 849-62. With the help of important key technologies such as microengineering (eg photolithography) and microfluidics, more complex hybrid systems of synthetic microchannels, membranes and cells embedded in biomaterials could be developed in the following years Huh, D. et al., 201 1, Trends Cell Biol. 21: 745-54. Systems prepared in this way are already being used to study angiogenesis and tumor cell extravasation, eg, described in Zheng, Y., et al., Proc. Natl. Acad. Be USA, 2012; 109: 934-7 or Jeon, JS et al. Plos 1 2013; 8: e5691 0. It has also already been possible to develop a microfluidic TE model based on exclusively biological material, Sekine, Het al. Nat Commun 2013, 4: 1 399. A capillary tissue from a donor animal connected to an artery is used as a vascular bed for the dynamic cultivation of in vitro expanded cells.

Bioprinting is a special tissue engineering process that uses the principle of generative manufacturing techniques - also known as rapid prototyping - to generate three-dimensional living tissue. In contrast to industrially applied generative manufacturing processes, such as e.g. In stereophotography, selective laser melting or fused deposition modeling, bioprinting uses cell-loaded hydrogels as a building material. According to a 3D model, this layer is applied layer by layer. Hydrogels mimic the extracellular matrix of natural tissue, providing a cell-friendly environment for 3D tissue engineering, see e.g. Lee, K.Y., Chem Ref 2001; 101: 1869-80.

In unpolymerized form, hydrogels are easy to handle and dispense. By a chemical, thermal or light-induced polymerization, the gel solidifies, so that successive layers can be applied successively. On various parameters, such as the gel concentration, the crosslinker concentration, the pH value and the temperature Elasticity and polymerization time and polymerization rate can be adjusted. Bioprinting can be used to construct complex 3D tissue constructs of different materials with different cell types, as described in various ways in the literature, eg Shuurman W., et al., Biofabrication 201 1; 3: 021001.

Many methods of forming models for angiogenesis describe methods in which the various cell types are simultaneously introduced into the matrix.

However, it is necessary to improve such three-dimensional tumor angiogenesis models, in particular to improve the production methods thereof, in order to provide corresponding tissue models for screening methods. These models should be suitable for examining tumor growth, tumor angiogenesis and tumor therapy effects. Both the tumor or the tissue structure and the supply structure should consist of essentially naturally obtained materials, preferably biological material, such as corresponding hydrogels and cells. In contrast to the prior art, the individualization of the tissue model should be possible in the present case.

Brief description of the invention

The present invention provides a method of making a tissue model having utility structures. This method comprises the steps of: a) applying a living cell composition to or around a preformed first structure to form a coating on and optionally completely encasing it around the preformed first structure, the composition being suitable as a coating Train supply structure; b) optionally embedding this coated first structure and / or cultivating the coated structure to form the utility structure;

c) applying a living cell-containing suspension to the coating obtained in the previous step or to the supply structure, to form a tissue structure on the coating or the supply structure and to obtain a tissue model;

d) optionally embedding the tissue model obtained in step c). The present invention further includes corresponding tissue models and the use of these tissue models for the analysis of tissue genesis, in particular tumorigenesis, including aniogenesis. In addition, the use of the tumor tissue model to test a possible therapy with a predetermined therapy or to stratify a selected therapy to treat the tumor as reflected by the tissue model is suitable.

In another aspect, the application is directed to methods for testing and identifying potential drug candidates using the tissue model of the present invention to identify those potential drug candidates that exhibit a pharmacological effect on the tissue model.

These potential drug candidates are, in particular, those which, in the tissue model, exhibit cytotoxic, cytostatic, tumor-affecting, ie in particular anti-angiogenic and anti-inflammatory properties.

The inventors were able to successfully generate three-dimensional biomimetic tumor angiogenesis models, also referred to as 3D-TAM, with the aid of a 3D bio-printer. With the help of the 3D bioprinting method, both the supply structure of the tissue model and the tissue structure of the tissue model are determined Fabric model produced. Thus, in one embodiment, the following invention relates to an artificially constructed tissue structure, such as a tumor, which is seated on a supply structure, usually a supply vessel, and which can be appropriately cultured and supplied via this supply structure. Angiogenesis is thereby induced in the tissue structure, such as the tumor, so that angiogenesis takes place on the basis of the tissue structure, such as the tumor, in order to enable the tissue structure / tumor to be connected to the supply structure during cultivation and thus to improve the supply ,

This tissue model with supply structure allows a reduction in the number of experimental animals, since this model can be used after the normal cell culture but before first / ^' nv / Vo experiments in the animal, for example, to test potential drug candidates.

However, the tissue model which can be produced according to the invention is also suitable for developing a strategy of tumor therapy tailored individually to the patient with the aid of preliminary investigations carried out in vitro. In this case, the tumor cells for the tissue model and possibly also the cells necessary for the supply structure are isolated from the individual. Subsequently, with the aid of the method according to the invention, the tissue structure having the supply structure is generated in order to test possible forms of therapy on this tissue model and thus to determine the most effective form of therapy for the treatment of the individual.

The biomimetic tissue model according to the invention is characterized in that both the structure and the composition of the supply structure and the structure and composition of the tissue structure and thus of the entire tissue model can be individually designed on account of the synthetic production process. That is to say, by using the 3D bioprinting technology according to the invention, the tumor compositions can be distinguished in their type and variety of printed cells as well as the structure and composition of the tumor cells. composition of the supply structure. Furthermore, by the individual supply of the tissue model on the supply structure, for example, a comprehensive and individual testing in relation to a therapy is possible.

Brief description of the illustrations

FIG. 1 shows schematically the various steps of the method according to the invention. In FIG. 1A, the first structure is provided on a carrier. As shown in FIG. 1B, the composition having living cells is applied to this first structure in accordance with the invention in order to form a supply structure as a coating. FIG. 1C shows the tissue model according to the invention with the supply structure and the tissue structure constructed thereon. FIG. 1 D shows a section through the fabric model according to the invention. The inner channel of the supply structure, referred to herein as a flow channel, allows a passage of fluids. The delivery channel has a two-layered structure, an inner layer with endothelial cells and an outer layer with fibroblasts to correspond to a vascular vessel. The tumor tissue of a hydrogel cell suspension with tumor cells and fibroblasts has printed structures with endothelial cells to study the angiogenesis of these structures. FIG. 1E shows the tissue model according to the invention as an artificial tumor with a supply structure, the vessel canal, embedded in an embedding compound. The vessel channel is connected for the passage of fluids, with connections to a medium flow. Thereby, e.g. to be examined molecules are entered with the medium in the tissue model.

Detailed Description of the Invention In a first aspect, the present invention is directed to a method of making a tissue model having utility structures, comprising the steps of: a) applying a living cell composition to or about a preformed first structure to form a coating on and optionally completely encasing it around the preformed first structure, the composition being adapted to form a coating as a supply structure;

b) optionally embedding this coated first structure and / or cultivating the coated structure to form the utility structure;

c) applying a living cell-containing suspension to the coating or the supply structure obtained in the previous step, to form a tissue structure on the coating or the supply structure and to obtain a tissue model;

d) optionally embedding the tissue model obtained in step c).

The method according to the invention is characterized in that, in a first step, the supply structure is produced, usually with a 3D bioprinting method that can be done by printing, spraying or extruding. In this first step, a coating is applied to a preformed first structure. This coating contains living cells to form the supply structure. If desired, this coating may represent a complete sheathing, so that a supply vessel-like structure is obtained. The resulting coated first structure may then be embedded in a suitable medium. Suitable media are discussed below. This coated first structure may either be embedded or further cultured in culture medium to form the supply structure, e.g. in the form of a supply vessel.

The living cell composition for coating the preformed first structure is one which comprises cells and is suitable for use Formation of vessel-forming cells is. In particular, this composition includes endothelial cells, fibroblasts and / or smooth muscle cells or progenitor cells thereof. The term precursor cells includes multipotent cells as well as direct progenitor cells of the above-mentioned endothelial cells, fibroblasts and / or smooth muscle cells.

The term "multipotent cells" refers to those cells that can still differentiate into two or more cell types: multipotent cells include, for example, totipotent and pluripotent stem cells, induced pluripotent stem cells and embryonic stem cells, precursor cells include endothelial cell direct progenitor cells, fibroblasts or smooth Muscle cells, including human umbilical vein endothelial cells (HUVECs), in one embodiment, human embryonic stem cells are excluded.

The application to the preformed first structure can take place in one or more steps or layers. The goal is the formation of the supply structure, such as a vascular structure. In the vascular structures, usually the inner layer is an endothelial cell layer, while the outer layer is formed by fibroblasts. In one embodiment, therefore, a composition having endothelial cells or progenitor cells thereof is applied to the preformed first structure in a first step, and a composition comprising fibroblasts or precursor cells in a further step. Alternatively, these cell types can also be applied in one step.

The preformed first structure is in one embodiment a removable support material. In another embodiment, this preformed structure may be a solid structure. In yet another embodiment, this preformed first structure is formed on a surface. This is done with a removable material, eg biomaterials such as polyethylene glycol, Gelatin, fats and waxes or other easily removable materials. This preformed first structure, also referred to herein as a channel core, is formed on a surface, for example by a printing process. This structure is one that can be removed at a later time, eg, by dissolution, rinsing, etc. Typically, the first structure is elongated. The first structure may further be branched.

In one embodiment, further, after removal of the preformed first structure, the resulting utility structure can be further functionalized. This includes in the interior and exterior of the supply structure, an introduction of other cells but also an inner or outer coating with functional ingredients.

This preformed first structure is then coated with the living cell composition, e.g. with a printing process by printing, spraying or extruding. The composition is one which, at the time of application, is a liquid-viscous composition and which is then e.g. cured by polymerization. Materials suitable for this purpose are known hydrogels including alginate, agarose, hyaluronan, fibrinogen, extracellular matrix components, gelatin, collagen, peptide hydrogels, etc. In one embodiment, these are natural materials, in another embodiment synthetic materials. The gels may be hydrophilic or hydrophobic gels. Suitable hydrogels include those derived from collagen, hyaluronate, fibrin, alginate, agarose, hydrolyzates, and combinations thereof. Other suitable hydrogels which are not of the natural origin but are obtained synthetically include those derived from polyacrylic acid and derivatives thereof, polyethylene oxides and copolymers thereof, polyvinyl alcohols, polylactic acid, etc. Suitable hydrogels include:

Natural hydrogels:

Collagen, fibrinogen, alginate, hyaluronan, gelatin, agarose, chitosan, matri- gel®, hydrogels based on heparin and elastin, including chemically, biologically or physically modified forms of said hydrogels.

Synthetic hydrogels:

Polyacrylic acid and its derivatives, polyethylene oxide and its co-polymers, polyvinyl alcohol, polyphosphazenes and polypeptides, including chemically, biologically or physically modified forms of said hydrogels. In one embodiment, the materials used are biomaterials. For the first structure, which forms a channel core, a dissolvable or removable material is used, which in particular has non-cytotoxic properties or can be removed under non-cytotoxic conditions. Suitable for this were gelatin and polyethylene glycols. Hydrogels which are particularly suitable for the living cell compositions include alginate, agarose, chitosan, fibrinogen, collagen, etc., such as a combination of agarose and gelatin.

In various specific embodiments, the gel is selected from hydrogel, novogel ™, agarose, alginate, gelatin, Matrigel ™, hyaluronan, poloxamer, peptide hydrogels, polyethylene glycol, silicone gels, polylactic acid gels, or combinations thereof.

In some embodiments, the hydrogel is a thermo-reversible gel, i.e., it is not liquid at room temperature, but has a gel-forming temperature at> 25 degrees, such as> 30 degrees, such as> 35 degrees, e.g. > 40 degrees.

The present preformed first structure with coating, for example formed in a plurality of layers, suitable for forming the supply structure is embedded in one embodiment of the invention. As a suitable embedding medium, possible embedding media, such as those used in 3D bioprinting, can be used. In particular, this includes compositions consisting of agarose, gelatin, polyacrylamide, polyacrylic acid and their derivatives, Polyethylene oxide and its co-polymers, polyvinyl alcohols, polyphosphazenes, polypeptides, polydimethylsiloxane, polymethyl methacrylate, silicones, as well as chemically, biologically or physically modified variants of said materials. Suitable compositions include combinations of agarose and gelatin.

In one embodiment, this structure, possibly embedded therein, is then cultured for a predetermined period of time to facilitate the formation of the supply structure, e.g. to allow the formation of vascular vascular structures. Such cultivation takes place under known conditions. For example, the duration of such cultivation is at least one week, e.g. a maximum of two weeks, to form the supply structure to obtain a vascular vessel similar to a blood vessel. This supply structure has an inner channel and is usually elongated. If appropriate, the structure can be branched.

The method according to the invention further comprises applying a suspension containing living cells to the coating or the postculturing of the present supply structure. The cell suspension, after further cultivation, forms a tissue structure on the coating or supply structure to obtain a tissue model.

In one embodiment, the cells are already suspended in the gel to be applied. Alternatively, the gel can first be applied to the coating or the formed supply structure and then the cells are introduced site-specifically, for example injected. In one embodiment, these steps may also be combined. That is, additional cells, such as tumor cells, are introduced into the present tissue structure of gel and cells.

The term "tissue model" is understood here to mean: a synthetically produced, cell-containing 3D tissue with at least two partial structures. tures, supply structure and tissue structure.

The term "supply structure" is understood in the present case as follows: A synthetically produced, cell-containing 3D cell structure having an inner region which can form a cavity and has no cells, this inner region having a first access and at least one second access, wherein These fluid additions can be delivered and removed, and an outer region with cells that form a cell structure, such as a cell structure similar to a vascular cell structure The supply structure is typically elongate and may be branched.

The term "tissue structure" as used herein means: a synthetically produced 3D cell structure which has cells at least in some regions, for example present in the entire structure The tissue structure is one that comprises tumor cells in one embodiment and cells such as progenitor cells In the context of angiogenesis, vessels forming blood vessel-like structures can be applied directly to the living cell-containing suspension applied to the coating or the supply structure according to the invention For example, a bioresorbable separation layer may be present between the suspension to form the fabric structure and the coating or utility structure, such a release layer may be one based on polylactide, polycaprolactone, or other gels finally the gel for embedding the coated first structure. Such a spacing can then be useful in order to prevent premature proliferation of the cells present in the tissue structure or the suspension into the supply structure and thus possibly to prevent clogging thereof. If the supply structure is already completely formed, for example in the form of a vascular vessel, then the suspension containing living cells can be applied directly to this supply structure. The living cells contained in the suspension have tumor cells in one embodiment. To form a tumor tissue model, these tumor cells are applied to the supply structure as tumor tissue structures. These tumor cells are living tumor cells, eg primary tumor cells isolated from an individual whose therapy is to be stratified or in which the therapy form is to be determined on the basis of this tissue model.

This suspension may further comprise other types of cells, in particular fibroblasts or endothelial cells but also smooth muscle cells. These cells are suitable for neovascularization (angiogenesis). The neovascularization can take place both within the tissue structure and between tissue structure and supply channel. By the latter, a connection of the optionally formed in the fabric structure vessels to the supply channel can be ensured.

In one embodiment, the tissue structures may be formed such that the entire tissue structure is formed with the same cell suspension. Alternatively, the tissue structure can be formed using more than one suspension of different composition. This makes it possible to form structured fabric structures, e.g. those in which in certain areas only tumor cells are present or in which in certain areas other cell types, such as fibroblasts, smooth muscle cells or endothelial cells are present to represent different tissue structures.

The suspension is one which comprises a medium suitable for 3D printing. This medium is one such as the above-mentioned medium for printing or coating the first structure. In particular, this medium is a gel of the type mentioned above. For example, a hydrogel is used, such as collagen, fibrinogen, alginate, hyaluronan, gelatin, agarose, chitosan, Matrigel ™, hydrogels based on heparin and elastin, polyacrylic acid and its Derivatives, polyethylene oxide and its co-polymers, polyvinyl alcohol, polyphosphazenes and polypeptides, including chemically, biologically or physically modified forms of said hydrogels. Suitable gels include a combination of agarose and gelatin. Thus, in a preferred embodiment, the tissue model is a tumor tissue model with a tissue structure containing tumor cells, wherein the suspension containing living cell is one containing tumor cells, such as primary tumor cells. In one embodiment, the suspension contains live tumor cells and stromal cells such as endothelial cells, fibroblasts, myofibroblasts, immune cells including macrophages and leukocytes. These are, in particular, primary cells, including stem cells and progenitor cells (progenitor cells) of these cell types.

The suspension is in the form of a hydrogel or a biocompatible polymer.

The tissue model with the tissue structure and the supply structure which is thus available is subsequently embedded in one embodiment. Suitable embedding media are the abovementioned media, namely gels, such as agarose, gelatin, polyacrylamide, polyacrylic acid and its derivatives, polyethylene oxide and its co-polymers, polyvinyl alcohols, polyphosphazenes, polypeptides, polydimethylsiloxane, polymethyl methacrylate, silicones, and also chemically, biologically or physically Modified variants of the materials mentioned, are used. Alternatively, the tissue model can be used in culture media as are well known to those skilled in the art. The investment material or the embedding medium, these terms are used interchangeably herein, is one that is cytocompatible. The term cytocompatible means that the embedding medium or the embedding mass exhibits cell and tissue compatibility.

The application of the suspension with living cells and / or the application of the composition with the living cells is carried out in one embodiment by the so-called bioprinting. The term bioprinting refers to the application of cell-loaded hydrogels. These hydrogels mimic the extracellular matrix of natural tissue and thus provide a cell-friendly environment for the present manufacturing process. In unpolymerized form, the hydrogels can be handled appropriately well and by induction, e.g. chemically, thermally or by light, solidify by polymerization.

The application can be done by means of printing, spraying or extrusion. In one embodiment, the application is carried out by printing e.g. through a 3D printer.

As noted above, the preformed material forming the first structure may be removed after coating and, if necessary, forming the supply structure. To use the tissue model in which the supply structure serves as a supply path for the cells present in the tissue model, this first structure is removed, for example by dissolving it. The person skilled in suitable methods for removing, for example, dissolution of this first structure formed, for example, from gels known. These gels may be gelatin, waxes, fats or readily soluble polymers such as polyethylene glycol. A suitable gel is a combination of agarose and gelatin. By appropriate thermal or chemical conditions, these gels can then be dissolved so that a channel is formed internally in the supply structure. Through this channel can then the necessary, the supply of Be fed to tissue models serving food etc. Furthermore, the supply structure can be used to supply the potential active substance candidates to be tested as well as chemical substances, biochemical / biological factors (eg cytokines, hormones, growth factors) or other cell types (eg macrophages) in order to test their effects on the tissue model.

The carrier on which the first structure is applied or which is integrated in the first structure can be designed such that it can be tempered, e.g. can be cooled or heated. This makes it possible to remove the materials forming the first structure after coating and curing. In the case of PEG or gelatin, heating to 37 ° C may result in liquefaction thereof while the coating-forming gel remains in its shape after cure and thus a channel is formed in that supply structure which provides for both the cells in the supply structure as well as in the tissue structure.

In contrast to the prior art in which the different cell types are introduced simultaneously, the method according to the invention is based on the idea of providing an improved tissue model by sequentially forming the structures.

In a further aspect, the present invention is directed to a fabric model so available. The fabric model obtainable in accordance with the invention is characterized in that it has a supply structure and a fabric structure. Due to the supply structure, a complete supply is possible. The supply structure supplies both the cells of the supply structure and the cells of the tissue structure. For this purpose, the angiogenesis in the tissue structure / tumor and thus the connection of this tissue structure / tumor is promoted to the supply structure. Depending on the requirements, different conditions can be simulated. In this way, hypoxic environments can be provided in the appropriate atmosphere. About the supply structure and connected to supply vessels can a long-term cultivation of the tissue model done. In this case, additives can be supplied to the tissue model at temporally limited times, for example potential drug candidates, in order to test their effect on the tissue model. In addition, various physical or chemical as well as cellular parameters can be altered on this tissue model. These include parameters such as pH values, CO2 level, pressure, flow rate and temperature, but also the provision of additives such as growth factors and cytokines as well as cells, etc. The tissue model according to the invention can be used in a variety of ways. As stated, studies on the influence of potential drug candidates on the tissue model can be performed. The tissue model can show both tissue structures of healthy origin and tissue structures with tumor cells or other cells to be treated. The tissue model thus allows the assessment of an effect of these potential drug candidates on healthy and altered tissue. It allows a systematic analysis of the various parameters. As a result, a reproducible model can be provided, which can be used, for example, in the pharmaceutical industry, for example as an intermediate step between the investigation of potential drug candidates in the cell culture and the subsequent in vivo animal experiments. This significantly reduces the number of animal experiments.

The tissue model according to the invention can be used as a model for tissue genesis, in particular tumorigenesis. In particular, angiogenesis can be induced and examined in tissue.

Another use is one in which the tumor tissue model is used for testing in a therapy of a predetermined form of therapy or for stratifying the therapy for treating a tumor. As a result, for example, the development of an individualized form of therapy of an individual to be treated is possible. The tissue model is thereby established from cells of the individual to be treated in order subsequently to test the therapeutic options in vitro. This has the advantage that the already weakened patient Only the optimal form of therapy for him, instead of having to be consulted beforehand to test a range of therapies, including their side effects. Both anti-angiogenesis and antiinflammatory as well as cytostatic and cytotoxic effects can be investigated on the tissue model. In this case, this tissue model can be constructed according to individual requirements, for example, the tissue structure can show different areas with different compositions of cells and cell types. The same applies to the supply structure.

Another significant advantage of the tissue model according to the invention is the possibility of studying angiogenesis, tissue genesis and tumor therapy effects with the aid of imaging methods. These imaging techniques include US (ultrasound), MRI, CT, PET, single photon emission computed tomography, optical imaging including microscopy. Depending on the particular imaging procedure, different contrast agents may be administered via the delivery structure, such as e.g. CT contrast agents, DS microbubbles, etc. The contrast agents can be used to better visualize and contrast the 3D TAM model, the vascular structure in the 3D TAM model, and to detect and localize specific cell types within the 3D TAM model. Furthermore, the 3D-TAM model can also be used to develop, study and establish new contrast agents for the aforementioned imaging techniques.

Finally, the present invention relates to methods for testing potential drug candidates. The method according to the invention comprises the step of providing a tissue model according to the invention and introducing the drug candidates via the supply structure into the tissue structure and analyzing the effect of the drug candidates on the tissue model, wherein a drug candidate is identified as a potential drug, if this shows a desired effect on the tissue model , In one embodiment, this process is one wherein the potential drug candidates are those which have a cytostatic, cytotoxic on tumor cells or cells which contribute to the supply of the tumor, including endothelial cells, fibroblasts and smooth muscle cells, antiangiogenes and / or show anti-inflammatory effect. The method comprises the steps of providing a tissue model of the invention containing tumor cells, introducing the potential drug candidates via the supply structure into the tumor cell-containing tissue structure, and analyzing the effect of the potential drug candidates on the tissue model, identifying a potential drug as they reduce tumor growth, such as Tumor growth stopping effect or tumor growth-reducing effect, an anti-angiogenic or an anti-inflammatory effect.

Examples:

In the following, the invention will be explained in more detail by means of examples, without being limited thereto.

1 . Production of a predetermined first structure

On a support surface, e.g. a culture dish, a glass surface, a base layer (e.g., agarose) similar to the later potting material, or the surface of the bioreactor vessel, the preformed first structure is formed. This forming can be done by printing or extrusion. In the present case, a gelatin solution, with a heated to about 37 ° C printhead applied. In a second experiment, polyethylene glycol (PEG) is used as biomaterial. 2. Applying a coating to a preformed first structure

A) A first structure of gelatin or PEG is labeled with a living cell. len mixed, hydrogel-based coating, in this case alginate, applied. For this purpose, a suspension consisting of alginate (eg 2% by weight, sodium salt of alginic acid from brown algae, Sigma) and fibroblasts or HUVECs is applied to the preformed first structure at room temperature using a 3D printer and then by spraying a crosslinking solution consisting of calcium chloride (30 mg / ml) solidified.

B) A preformed first structure is made of gelatin or polyethylene glycol as described above.

Using a 3D printer, a composition of alginate (2% by weight, sodium salt of alginic acid from the brown algae, Sigma) with endothelial cells is applied as the first layer to this structure (10 million cells per milliliter). After this alginate layer has cured, a second composition containing fibroblasts (10 million cells per milliliter) is optionally applied as an outer layer.

3. embedding the coated first structure

The resulting coated structure is embedded in agarose (3% by weight).

4. Remove the first structure

PEG can be easily removed from the cured coating with the help of water. The gelatin is purified by heating e.g. liquefied to 37 ° C and e.g. sucked off with the help of a syringe. During and after removal of the PEG or gelatin, the cell-loaded alginate coating is retained and forms a stable channel wall.

It has been shown that the embedded hollow structure coated with alginate and cells prevents the escape of liquid so that liquids can easily be passed through the channel thus formed. After about two weeks of cultivation under usual cultivation conditions, a biofunctional tissue resembling a vascular vessel is formed.

5. Introduction of tumor tissue (completion of the 3D-TAM model)

The tumor tissue can be introduced into the 3D-TAM model in three different ways.

5.1 injection of tumor cells

The preparation of the supply channel, the embedding and the cultivation are carried out as described above (item 1 -4). After a the

Concerning the supply channel, homogeneous, densely grown endothelial layer has been formed, optionally with an additional layer containing fibroblasts, tumor cells are centrally injected by means of a syringe integrated in the SD printer and a few 100 μm are injected into the embedding compound above the supply channel.

5.2 Print the tumor on the supply channel

5.2.1 Print the tumor on a release liner

The supply channel is manufactured as described (pt. 1 -2). On the

Supply channel is applied to a biodegradable separation layer of, for example, polycaprolactone. The tumor tissue in the form of a tumor cell-containing hydrogel suspension is printed on the separating layer, for example tumor cells in Matrigel. Possibly. The tumor tissue can be structured with other cell types and other hydrogels. The construct thus prepared is further processed and cultured as described in pt. 3-5. The separation layer in this case prevents the proliferation of the tumor cells in the direction of the supply channel during the first weeks of cultivation. The separating layer is to be designed so that it has only dissolved when the endothelial layer in the wall of the channel is fully developed. After dissolving the separating layer, the tumor tissue is granted access to the supply channel and a connection by means of a new blood vessels.

5.2.2 Printing the tumor after previous cultivation of the supply channel

The production of the supply channel, the embedding and the cultivation are carried out as described (pt. 1 -4). After a homogenous dense endothelial layer lining the delivery channel is formed, the construct is removed from the culture and the investment is removed to within a few hundred microns above the supply channel (e.g., scalpel cutting). On the supply channel near the surface, the tumor tissue in the form of a tumor cell-containing hydrogel suspension is printed, e.g. Tumor cells in Matrigel. Possibly. The tumor tissue can be structured with other cell types and other hydrogels. Subsequently, the entire construct is embedded again and further cultivated.

Claims

claims:

1 . Method for producing a tissue model having care structures, comprising the steps of:

a) applying a living cell composition to or about a preformed first structure to form a coating on and optionally completely encasing it around the preformed first structure, the composition being adapted to form a coating as a supply structure;

b) optionally embedding this coated first structure

and / or cultivating the coated structure to form the supply structure;

c) applying a suspension containing live cells to the coating or the supply structure obtained in the previous step, to form a tissue structure on the coating or the supply structure and to obtain a tissue model;

d) optionally embedding the tissue model obtained in step c).

2. The method of claim 1, wherein the supply structure consists of or includes vessel-forming cells, in particular where this consists of or includes endothelial cells, fibroblasts and / or smooth muscle cells.

The method of claim 1 or 2, wherein the tissue model is a tumor tissue model with a tissue structure containing tumor cells, and the suspension containing living cells is one that contains live tumor cells, such as primary tumor cells.

The method of claim 3, wherein the suspension contains live tumor cells and stromal cells, such as endothelial cells, fibroblasts, myofibroblasts, immune cells, including macrophages and leukocytes, and especially primary cells, including stem cells and progenitor cells, of these cell types.

Method according to one of the preceding claims, wherein the suspension in the form of a hydrogel and / or a biocompatible polymer is applied.

Method according to one of the preceding claims, wherein the application of the suspension with living cells and / or the application of the composition with living cells by means of printing, spraying or extrusion takes place.

Method according to one of the preceding claims, wherein the tissue structure in the tissue model has different areas or structures.

Method according to one of the preceding claims, wherein the supply structure and / or the formed fabric model is fixed with a zyto-compatible investment material.

Fabric model obtainable by a process according to one of claims 1 to 8.

10. Use of a tissue model according to claim 9 as a model for tissue genesis, in particular tumorigenesis.

Use of a tissue model according to claim 9, wherein the tissue model is a tumor tissue model for testing a therapy with a predetermined therapy or for the stratification of therapy for the treatment of the tumor.

12. Use of a tissue model according to claim 9 in imaging including US, MRI, CT, PET, SPECT, optical imaging including microscopy.

13. A method for testing and identifying drug candidates, comprising the step of:

a) providing a tissue model according to claim 9;

b) introducing the drug candidate via the supply structure into the tissue structure and analyzing the effect of the drug candidate on the tissue model, wherein a drug candidate is identified as a potential drug, if this shows a desired effect on the tissue model.

14. A method for testing and identifying drug candidates according to claim 13, wherein said drug candidates are those which are cytostatic, cytotoxic to tumor cells or cells which contribute to the supply of the tumor, including endothelial cells, fibroblasts and smooth muscle cells which are anti-angiogenic and or anti-inflammatory, comprising the steps:

a) providing a tissue model according to claim 9 containing tumor cells;

b) introducing the drug candidates via the supply structure into the tumor cell-containing tissue structure and analyzing the effect of the drug candidate on the tissue model, identifying a potential drug when it has a tumor growth-reducing, such as tumor-stopping effect or a tumor-reducing effect, anti-angiogenic or shows anti-inflammatory effect.