Apparatus for culturing biological cellular material and method for its manufacture

Abstract

A culture device 100 for culturing biological cellular material has a base body 10 having an upper surface 11, a lower surface 12, at least one receptacle cavity 20 having a bottom wall 21 and a surrounding side wall 22, and at least one fluid passage 30 communicating with the at least one receptacle cavity 20, the at least one receptacle cavity 20 having an opening 23 toward the upper surface 11 of the base body 10 and arranged to accommodate biological cellular material, the inner surface of the side wall 22 being provided with an anchor notch 24 arranged to hold the biological cellular material in the at least one receptacle cavity 20, the upper surface 11 of the base body 10 having a chamfered portion 13 around the opening 23 of the at least one receptacle cavity 20, the chamfered portion 13 being adapted to accommodate a cavity lid sheet 40 covering the opening 23 of the at least one receptacle cavity 20. Additionally, methods of manufacturing the incubation device 100 and applications of the incubation device 100 are described.

Description

[Technical field]

[0001] The present invention relates to a culture device configured for culturing biological cellular material, such as, for example, tissue model material. Furthermore, the present invention relates to a method for manufacturing the culture device and a method for using the culture device. Applications of the present invention can be used in the fields of biotechnology, biochemistry and medicine, for example, for producing and / or investigating cellular complexes. [Background technology]

[0002] Herein, reference is made to the following prior art which illustrates the technical background of the present invention: [1] A. van den Berg et al., “Personalised organs-on-chips: functional testing for precision medicine”, Lab Chip, Vol. 19, p. 198 (2019) [2] G. Trujillo-de Santiago et al., “The Tumor-an-Chip: Recent Advances in the Development of Microfluidic Systems to Recapitulate the Physiology of Solid Tumors,” Materials, Vol. 12, p. 2945 (2019) [3] J. Hagenbuchner et al., “3D bioprinting: novel approaches for engineering complex human tissue equivalents and drug testing,” Essays Biochem, Vol. 65, No. 3, pp. 417-427 (2021) [4] HW Kang et al., “A 3D bioprinting system to produce human-scale tissue constructs with structural integrity,” Nature Biotechnology, Vol. 34, No. 3, pp. 312-319 (2016) [5] S. Ji et al., “Complex bioprinting methods,” APL Bioeng. 5, 011508 (2021) [6] WO 2020 / 120341 A1 [7] US 2018 / 223251 A1

[0003] In recent years, in vitro tissue models mimicking the individual microenvironment of specific human tissues have become increasingly important for patient-tailored therapy, especially in the field of cancer therapy or immunotherapy (see, for example, [1]). Modeling the tumor environment holds enormous potential for a better understanding of the mechanisms of how tumors communicate with nearby non-transformed cells. Due to individual gene expression patterns and mutations, the modes of interaction between tumor cells and the tissue environment differ between tumor types and patients, therefore personalized tumor tissue models may serve as drug testing platforms to identify and optimize individual therapeutic regimens and provide novel insights into signaling between specific cancer types and their microenvironment.

[0004] Another important application of in vitro tissue models is the attempt to replace live animals, e.g. rodents, during drug testing in medical research and development procedures. Besides the practical moral aspects, species-specific differences in drug transport mechanisms, digestive behavior, and especially in liver metabolism, can be quite significant from the equivalent procedures in the human body. Furthermore, the "living nature" of live animals introduces additional uncertainties during a series of measurements, so larger sample sizes are needed to separate these effects from the actual measurements of interest.

[0005] Currently, there are two main methods for utilizing suitable in vitro models, which include two-dimensional (2D) and three-dimensional (3D) cell culture. Current strategies for 2D culture of living tissue models include one of the following approaches. As a first approach, it is known to culture one to two layers of tissue models (e.g., skin equivalents) in so-called Boyden chamber inserts on a semipermeable membrane. These are relatively easy tissue models to prepare, but have limitations in terms of lack of substructure (e.g., no vascular imprinting), complex manual processes, static culture conditions, difficulties in handling for upper / lower microscopes, and difficulties in standardization.

[0006] As a second approach, organ-chip systems can be implemented in microfluidic devices, where fluidic chips with fine channels are fabricated by photolithographic or stereolithographic printing. While the channels offer advantages for moderate flows (oxygen and nutrient / growth factor supply, removal of metabolites), limitations arise from the low thickness of the tissue (e.g., 50-150 μm) and the limited access to samples embedded in the chip for further analysis. In particular, tissue models obtained in such 2D cultures lack the complexity of physiological, branched and interconnected microvascular networks and capillary geometries formed by cells in their natural environment.

[0007] A third known approach, organs on membranes, represents a combination of the above techniques (see, e.g., [2]), in which a chip with a semipermeable membrane is flushed with culture medium and a cell layer is applied to the membrane. A drawback of this concept is that true 3D structures cannot be obtained, since the tissues are monolayered or consist of only a very few cell layers.

[0008] On the other hand, 3D cultures can be obtained by 3D bioprinting of cellular materials, which allows the assembly of multi-layered, millimeter-thick cell-laden hydrogels that resemble natural human tissues much better than 2D cultures (see, for example, [3] to [7]). 3D printing of living human or animal tissues has been rapidly developed in recent years, and it is now already possible to print living heart and lung models. Tissue equivalents are usually printed in receptacle cavities in substrate bodies, like small containers in fluidic chips, in order to further culture them or to perfuse them, like organs, with nutrient medium before transplantation.

[0009] A major challenge in 3D culture is to induce blood vessel formation and angiogenesis in the culture volume. To this end, additively manufactured cell complexes containing passageways have been proposed, which are produced by printing 3D cultures in combination with multiple filaments of removable additive substances (e.g. Pluronic, see for example [3] and [5]). The additive substances are degraded after printing, thus creating elongated voids to provide blood vessel-like passageways. Despite this vascularization advantage for nutrient supply, 3D cultures still suffer from substantial limitations regarding the stability of the culture conditions.

[0010] Actual tests have shown unstable connections between cells and materials, including blood vessel-like passages and external supply tubes, such as passages in chips housing 3D cultures. This limitation is also influenced by the shortcomings of conventionally used hydrogel components, which typically include natural polymers, such as collagen and fibrin. These natural polymers are applied due to their high similarity to physiological extracellular matrix composition, favorable adhesive incentives for cell adhesion, and the characteristic of natural materials to promote angiogenesis. However, they have poor mechanical stability and durability during long-term culture.

[0011] Due to the limited stability, durability, and reproducibility of the culture conditions, the practical application of current 3D bioprinted models is limited to basic research using relatively small cell complex samples and specialized cell types. For routine applications in medicine and biology, there is a need for a reproducible and easy-to-use platform technology for parallelized long-term culture under physiological flow conditions in a perfusion system.

[0012] As a further general limitation of the prior art, and due to the low stability of cell culture, it is not possible to link 3D bioprinted models of different tissue types in a sequential manner, which would make it possible, for example, to test the effect of a drug on a specific organ (e.g., the lung) if the drug is first transformed by the digestive process, e.g., by the liver, or to test the possibly toxic effects of metabolic degradation products of the drug on other organs. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Publication No. 2020 / 120341 [Patent Document 2] US Patent Application Publication No. 2018 / 223251 [Non-patent literature]

[0014] [Non-Patent Document 1] Lab Chip, 19(2), 198-205(2019) [Non-Patent Document 2] Materials,12(18),2945(2019) [Non-Patent Document 3] Essays Biochem,65(3),417-427(2021) [Non-Patent Document 4] Nature Biotechnology,34(3),312-319(2016) [Non-Patent Document 5] APL Bioeng,5,011508(2021) Summary of the Invention [Problem to be solved by the invention]

[0015] It is an object of the present invention to provide an improved culture device configured for culturing three-dimensional (3D) cell cultures and capable of avoiding the limitations of the prior art. In particular, it is an object of the present invention to provide a culture device with improved stability, durability and / or reproducibility of the culture conditions and / or with the capability as a reproducible and easy-to-use platform technology for parallelized long-term culture in physiological conditions, especially flow conditions in perfusion systems, and / or with the capability of using natural polymers as cell matrix materials. Furthermore, the culture device is capable of processing multiple cell cultures in a sequential manner. Furthermore, the culture device is capable of culturing larger samples. Furthermore, it is an object of the present invention to provide an improved method of manufacturing the culture device capable of avoiding the limitations of the prior art. [Means for solving the problem]

[0016] The above object is solved by an incubation device and a method for its manufacture which respectively comprise the features of the independent claims. Preferred embodiments and applications of the invention result from the dependent claims.

[0017] According to a first general aspect of the present invention, the above object is solved by a culture device configured for culturing biological cellular material and comprising a base body having an upper surface, a lower surface, at least one receptacle cavity having a bottom wall and a surrounding side wall, and at least one fluid passage communicating with the at least one receptacle cavity, the at least one receptacle cavity having an opening towards the upper surface of the base body and arranged to accommodate the biological cellular material.

[0018] According to the invention, the inner surface of the side wall is provided with an anchor notch arranged to hold biological cell material in at least one receptacle cavity, and the top surface of the base body has a chamfered portion around the opening of at least one receptacle, said chamfered portion adapted to receive a cavity lid sheet covering said opening of said at least one receptacle cavity. Optionally, at least one fluid passage may also be provided with a chamfered portion.

[0019] According to a second general aspect of the present invention, the above object is solved by a method for manufacturing a culture device according to the first general aspect of the present invention or an embodiment thereof, the method comprising a step of producing a base body with at least one receptacle cavity, at least one fluid passage, an anchor notch and a chamfered portion by subtractive manufacturing from a preformed base body workpiece and / or by additive manufacturing.

[0020] Advantageously, the present invention overcomes the drawbacks of the prior art by combining 3D bioprinting methods with fluidic chip technology to generate complex, preferably vascularized, tissue-on-a-chip models for medium and high throughput applications. The culture device of the present invention provides a platform that combines the standardized production and easy handling of fluidic chips with the complexity of millimeter-thick human vascularized soft tissue equivalents.

[0021] The term "biological cellular material" refers to a material comprising at least one biological cell and its cellular components. The biological cells may comprise any cell type of human or animal origin. The biological cellular material may be exclusively composed of biological cells and / or cellular components or it may comprise biological cells and / or cellular components and additionally natural and / or non-natural polymers, such as collagen, fibrin, and / or at least one hydrogel. The cells may comprise, for example, undifferentiated cells, such as stem cells, in particular induced pluripotent stem cells (iPSCs) and / or iPSC-derived cells and / or mesenchymal stem cells. Alternatively or additionally, the cells may comprise differentiated cells of any cell type present in human or animal tissue, such as fibroblasts, endothelial cells, mesothelial cells, smooth muscle cells, organ cells, such as lung cells or liver cells, and / or epithelial cells from human and / or animal origin. Alternatively or additionally, the cells may comprise spheroid cell bodies. The cells may comprise one single cell type or a mixture of at least two cell types. Depending on the particular application, the biological cellular material is also referred to below as tissue equivalent, tissue model, cell model, cell complex, cell culture, 3D culture or biological sample.

[0022] Preferably, the hydrogels comprising cells and / or cellular components of the biological cellular material comprise at least one natural hydrogel component, such as gelatin methacryloyl (GelMA), alginate, functionalized alginate-YISGR, chitosan, hyaluronic acid, hyaluronic acid methacrylate, collagen, nanocellulose, silk, synthetic hydrogels, such as PEG, PEGDA or PVA, synthetic extracellular proteins, and / or glycoproteins, such as fibrinogen and / or fibronectin. In particular, functionalized GelMA, as a semi-natural polymer, offers advantageous hydrogel properties, including tunable mechanical stability, intact adhesion triggers, protease degradation sites, and fast crosslinking capabilities.

[0023] Depending on the consideration of the stage of producing or using the culture device, the hydrogel and / or the biological cell material comprising the hydrogel is in a (particularly fluid) liquid or in a solidified, e.g. crosslinked, state. The biological cell material is introduced into the receptacle cavity in a fluid state. The biological cell material is then solidified. The solidified biological cell material, in particular the solidified hydrogel, has a permeable structure, i.e. it can be permeated (perfused) by a liquid, e.g. a nutrient solution.

[0024] Due to the thickness of the biological cellular material in the receptacle cavity (preferably more than 200 μm), the biological cellular material is a 3D material, i.e., the cells or cell components in the biological cellular material experience ambient conditions and interactions in a volume, for example in a natural environment, for example in a biological tissue. Introducing the biological cellular material includes feeding a liquid filament and / or the biological cellular material into the receptacle cavity. This process is also called printing the biological cellular material.

[0025] The substrate body (also indicated as fluidic chip or substrate chip) preferably comprises a solid part made of a rigid or slightly deformable (but precise in shape) material. The substrate body may be provided by a monolithic (integral) material or a multi-component, for example a multi-layer material, comprising at least one receptacle cavity and at least one fluid passage communicating with the at least one receptacle cavity. Preferably, the substrate body has a plate shape with a main extension, which is particularly preferably a substantially planar extension. For reference purposes, it is indicated that the upper and lower surfaces of the substrate body extend in a horizontal direction (xy direction), while the normal direction of the upper and lower surfaces is vertical (z direction). Preferably, the opening of at least one receptacle cavity faces vertically, i.e. at least one receptacle cavity is exposed in the z direction.

[0026] Preferably, the substrate is made of a biologically inert and non-conductive material. Particularly preferably, a plastic material such as polymethylmethacrylate (PMMA) is used. Alternatively, the substrate may be made of another solvent-free plastic, such as acrylonitrile butadiene styrene (ABS) polymer or polycaprolactone, or another inert material, such as PDMS, ceramic or glass. Advantageously, with a biologically inert and non-conductive material, the influence of the substrate material on the biological material is minimized or eliminated.

[0027] According to a further alternative, the material of the substrate body may be made of an electrically conductive material or may include electrically conductive components, such as electrodes. Advantageously, with an electrically conductive material, the culture device may fulfill additional functions. For example, biological material, such as cardiomyocytes, may be electrically stimulated or electrophysiological measurements may be performed with the biological material in the sample receptacle. The electrically conductive material is preferably a biologically inert metal, such as gold or platinum.

[0028] Furthermore, the material of the substrate body may be of biological origin, such as polylactic acid (PLA) or cellulose, which advantageously has a high compatibility with the biological material in the sample receptacle.

[0029] The substrate body may contain one single receptacle cavity (single sample embodiment) or an array of at least two receptacle cavities (multiple sample embodiment), preferably in a line or matrix arrangement. In the case of multiple receptacle cavities, at least two of the receptacle cavities may be fluidically coupled via at least one fluid passage, thus allowing multiple cell cultures to be processed in a sequential manner. In a number of cavities greater than one, the cavities may be fluidically coupled in a series or parallel configuration, or as a combination of both. Advantageously, this allows the simulation of the sequential implementation of metabolic processes in different cell types. Furthermore, multiple separate culture devices can be connected to a network via interconnecting tubes. For example, a drug may be first digested by liver cells before it hits the tumor, followed by testing the effect of the residual products on kidney cells.

[0030] The fluid passage generally comprises a part of the substrate body, for example a line or tube, which is capable of conducting a fluid (gas or liquid), in particular supplying a fluid to or withdrawing a fluid from the receptacle cavity. The fluid passage opens directly and / or via a ring passage into the receptacle cavity, which connects the receptacle cavity with a fluid system, for example a fluid reservoir for nutrient supply, which may be at least partially contained in the substrate body or may be arranged externally and separately from the substrate body.

[0031] Preferably, the at least one fluid passage is a radially closed passage extending in the substrate body, particularly preferably along its main extension. Alternatively or additionally, the at least one fluid passage can be provided as a closed or open passage on the surface of the substrate body, but optionally extending in a direction deviating from the main extension of the substrate body. Thus, the fluid passage can be located in the substrate body, on its upper and / or lower surface. Preferably, at least two fluid passages are provided per receptacle cavity, but it is also possible to associate a single fluid passage with a receptacle cavity.

[0032] The receptacle cavity is a hollow space in the base body and can be designated as a bowl, compartment, vessel or culture space. The bottom wall of the receptacle cavity is located opposite the opening of the receptacle cavity. The bottom wall preferably extends parallel to the main extension, i.e. in a horizontal direction. The peripheral side wall of the receptacle cavity is an inner receptacle wall provided between the bottom wall and the opening. Preferably, the receptacle cavity has a cylindrical shape, such that the peripheral side wall is a cylindrical surface. The vertical direction of the receptacle cavity is also called the axial direction, in which the peripheral side wall limits the receptacle cavity in a radial direction relative to the axial direction. Preferably, all side walls of the receptacle cavity are provided with anchor notches.

[0033] Preferably, the receptacle cavity has an axial depth of at least 5 mm, particularly preferably at least 10 mm to 15 mm, and / or at most 30 mm to 40 mm, particularly preferably at most 50 mm. The cross-sectional dimension of the receptacle cavity, for example its diameter, is at least 5 mm, particularly preferably at least 10 mm to 15 mm, and / or at most 30 mm to 40 mm, particularly preferably at most 50 mm. Advantageously, the receptacle cavity provides sufficient space for cell culture for near-lifelike testing thereon. It is emphasized that the invention is not limited to these exemplary ranges. The advantages of the anchor notch in particular can also be obtained with larger receptacle cavities, whereby larger receptacle cavities are able to accommodate larger tissue constructs, for example for transplantation purposes, such as for skin or organ transplants. For adaptation to available laboratory equipment, the culture device with the receptacle cavity may be of similar dimensions to a standard microscope slide (76 mm x 26 mm).

[0034] The anchor notch (or recessed structure, recess) is recessed in the radial direction from the inner space of at least one receptacle cavity to the surrounding substrate body material. Preferably, the anchor notch has a radially extending retention profile formed such that the material in the sample receptacle is captured in the anchor notch, in particular mechanically fixed. The anchor notch on the side wall of the receptacle allows the hydrogel material including cells to be safely locked in the receptacle cavity, thus allowing the material to be cultured in the receptacle cavity for a long period of time without detachment from the wall, thus ensuring a permanent supply of nutrients and oxygen, as well as the evacuation of cell degradation products. Furthermore, the anchor notch allows the stable culture of larger samples of cellular material. Furthermore, cellular material including natural polymers with inherently low mechanical stability can be stabilized in the receptacle cavity during culture.

[0035] The beveled portion is generally a step, where the surface of the base body is deeper than the remaining surface. The beveled portion extends along the edge of the opening of at least one receptacle cavity, and optionally along at least one of the side surfaces, preferably along both sides of at least one fluid passage. The width of the beveled portion is preferably selected in the range between 1 mm and 5 mm. Depending on the design of the culture device, smaller or larger widths can be provided.

[0036] The inventors have found that the limitations of the culture stability of conventional 3D cultures and changes in culture conditions are caused by changes in biological samples during the course of culture. The 3D cellular material is deformed during culture due to the influence of internal cellular forces. During the course of tissue culture and maturation, the tissue shrinks over days or weeks, it detaches from the fluid passages and can no longer be perfused. Deformations occur when biological cells (particularly fibroblasts, myofibroblasts, and muscle cells, but also other cell types) produce extracellular matrix and / or exert mechanical forces, displacing the cellular material against or even detaching from the walls of the receptacle cavity. Thus, for example, during perfusion, nutrient medium can no longer pass through the printed tissue and the system becomes prone to leaking.

[0037] Furthermore, the inventors have found that displacement of the biological sample relative to the chamber wall can be reduced or even eliminated by the anchor notch in the receptacle cavity and the beveled portion on the top surface of the base body. Advantageously, both the anchor notch and the beveled portion improve stable fixation of the biological cell material in the receptacle cavity without compromising perfusion culture of the biological cell material.

[0038] Advantageously, the beveled portion can fulfill several functions. First, it facilitates the placement of a cavity lid sheet that can be provided to close at least one receptacle cavity. The lid can be manufactured as a breathable cover or as a sealing foil. Second, the cellular material can protrude from the receptacle cavity and extend to the beveled portion. Thus, the biological sample can be further stabilized in the receptacle cavity. Furthermore, any material in the beveled portion, e.g. the cellular material and / or part of the cavity lid, can also cover part of the fluid passage. The beveled portion facilitates the sealing of the receptacle cavity and / or the fluid passage connected to the receptacle cavity.

[0039] By providing both the anchor notch and the chamfered portion, certain further important qualitative and quantitative advantages of the present invention are provided:

[0040] The combination of the microfluidic substrate body with 3D bioprinting and the direct connection of the fluid passages in the substrate body with the cellular material, particularly the bioprinted passages therein, allows for the integration of a reproducible, standardized, and medium-throughput production of printed fluidic chips for multicellular tissue equivalents. The anchor notch, particularly its preferred tooth-like design, e.g. its triangular, rectangular, or trapezoidal cross-sectional design, and the resulting chamber structure allow the printed biological tissue in the receptacle cavity to be "interlocked" with the substrate body portion during hydrogel polymerization and integrated into the substrate body. Peeling / splitting adhesion of the tissue equivalent to the surface is prevented by the chamfered portion provided adjacent to the receptacle cavity to form the maximum adhesion area.

[0041] With the present invention, a stabilized fixation of the connection is obtained, thus facilitating efficient perfusion and more stable fixation of the tissue in the receptacle cavity over long culture periods. The culture device allows efficient and standardized production in medium / high throughput processes and efficient handling for measurement purposes (e.g. microscopy, etc.), since the substrate body does not deform or, preferably, does not detach from the provided glass substrate and can be further cultured in a corresponding automated culture system.

[0042] The culture device of the present invention allows the deposition of cell material in at least one receptacle cavity using a liquid cell-material complex (bioink), thus inducing the spontaneous formation of blood vessels. For this purpose, for example, iPS cells can be differentiated into a mesenchymal stem cell population, thus promoting the formation of vascular structures by endothelial cells.

[0043] According to a preferred embodiment of the invention, the anchor notch has an angular shape providing a narrowing of the anchor notch towards the inner surface of the side wall. According to another preferred embodiment of the invention, the anchor notch has a stepped shape providing an undercut slit opening of the anchor notch towards the inner surface of the side wall. Thus, the anchor notch of the side wall preferably comprises an undercut recess in the inner surface of at least one receptacle cavity. Advantageously, when material is added to the receptacle cavity and adjacent notches, the material is held in place by the notches, as by barbs. If the cellular material in the receptacle cavity changes over time due to deformation and / or biological self-assembly processes, the angled and / or stepped anchor notch secures the cellular material and prevents it from peeling off from the side wall. The angled shape with narrowing towards the inner surface of the receptacle cavity prevents the material in the anchor notch from being pulled towards the center of the receptacle cavity, preventing the cellular material from peeling off from the wall.

[0044] Advantageously, various configurations of the anchor notches are possible. Preferably, the anchor notches are distributed in the azimuth direction, i.e. they are located continuously along the circumferential direction of the inner side wall of the receptacle cavity. Alternatively, the anchor notches may be distributed axially, e.g. a series of ring-shaped anchor notches are located continuously along the axial direction of the receptacle cavity. According to further preferred embodiments of the invention, the anchor notches may be distributed evenly or unevenly along the side wall. In an even distribution, the anchor notches are evenly spaced along the side wall, while in the case of an uneven distribution, irregular spacing is provided.

[0045] The uneven distribution has particular advantages when the strength of the tensile forces in the cellular material toward the center of the cavity through contraction of the material is not axially symmetric about the center of the cavity, as may be the case for muscle cells, for example. This ensures that the cultured cellular material is held in place and that fluid passageways, such as supply tubes, are not compromised by shear stresses, even when the strength of the forces is unevenly distributed. Furthermore, all of the anchor notches may have the same shape and size, or they may be different sizes and / or shapes.

[0046] Preferably, the anchor notch extends in a direction perpendicular to the upper and lower surfaces of the base body, i.e. parallel to the axial direction of the receptacle cavity. This configuration has advantages for manufacturing the culture device. Alternatively, the anchor notch may have an angle other than 90° to the upper and lower surfaces, allowing additional fixing forces to be formed in an advantageous manner.

[0047] According to a further preferred embodiment of the invention, the chamfered portion has a rough finish. Advantageously, this rough finish improves adhesion of the cellular material and / or the cavity lid sheet on the chamfered portion. In particular, when the chamfered portion is used to stretch the cavity lid sheet over the receptacle cavity and, optionally, over the fluid passageway, the cavity lid sheet material can adhere better to the rough surface, thereby creating a more stable condition for the culture device.

[0048] According to another advantageous embodiment of the invention, the beveled portion may have a profile with a recessed groove extending around the opening of the at least one receptacle cavity and opening towards the anchor notch, advantageously the groove providing additional retention of cellular material in the area covered by the lid.

[0049] Preferably, the peripheral side wall may have a peripheral bottom recess arranged adjacent to the bottom wall and extending radially in the substrate body. Advantageously, the peripheral bottom recess provides an additional fixation effect for the cellular material. Particularly preferably, the peripheral bottom recess may have, for example, a contour, for example a step shape, which is formed such that the cellular material in the sample receptacle is captured in the peripheral bottom recess.

[0050] According to a particularly preferred embodiment of the present invention, advantages for the cultivation of biological cellular material can be obtained if a crosslinked hydrogel is disposed in at least one receptacle cavity, with a portion of the hydrogel being contained in the anchor notch. The crosslinked hydrogel may be disposed as a coating on the inner surface of the receptacle cavity. Preferably, biological cells or cellular components or cellular material or tissue components in a particular portion of the biological cellular material to be cultivated, such as extracellular matrix proteins, e.g. collagen, laminin, and fibronectin, may be contained in the hydrogel. Optionally, the inner surface of at least one fluid passage may also be dressed with a coating of hydrogel, while preserving the flow capacity of the at least one fluid passage. As an example, the inner wall of at least one fluid passage may be coated with an extracellular matrix protein that promotes adhesion of endothelial cells.

[0051] Thus, at least one receptacle cavity including the anchor notch can be pre-filled with a hydrogel, optionally including cells or cellular components. Pre-filling may include coating the inner surface of the receptacle cavity including the anchor notch or at least partially filling the receptacle cavity including the anchor notch. In the case of coating, growth of cellular material is supported in the receptacle cavity and the anchor notch during application of the culture device. In the case of partial or complete pre-filling, the culture device is provided in a pre-filled state, for example as a kit for investigating biological cell complexes.

[0052] A preferred embodiment of the method for producing the culture device preferably comprises the steps of depositing a liquid hydrogel in at least one receptacle cavity such that the anchor notch is filled with the liquid hydrogel (optionally containing biological cell material) and crosslinking the hydrogel by at least one of irradiation, temperature setting (heating or cooling) and chemical crosslinking. Crosslinking the hydrogel preferably comprises irradiation with light having a wavelength in the range of 365 nm to 700 nm. Advantageously, in this wavelength range from UVA to red, suitable photoinitiators are available that can be used for the polymerization of biological hydrogels.

[0053] According to a further advantageous embodiment of the invention, the culture device comprises a membrane layer which provides a cavity cover sheet and is combined with the beveled portion to cover the opening of at least one receptacle cavity. With respect to the method of the invention, the membrane layer is preferably combined with the beveled portion during or after the manufacture of the culture device. With the membrane layer, further stabilization of the cellular material in the receptacle cavity is obtained in an advantageous manner.

[0054] The membrane layer can be fixed around the entire periphery of the beveled portion, such that the receptacle cavity is closed. This variant can be used with pre-filled receptacle cavities. If hydrogel is present in the cavity, the lid sheet material covers it. Alternatively, the membrane layer can be fixed around a portion of the periphery of the beveled portion, such that the receptacle cavity is partially closed but still accessible for the supply of cellular material. In the latter case, the membrane layer is preferably fixed completely after the supply of cellular material, such that the membrane layer adheres to the entire periphery of the beveled portion during the culture of the cellular material.

[0055] Preferably, one single membrane layer is provided per receptacle cavity. In case of multiple receptacle cavities, a membrane layer can be provided for at least one of the receptacle cavities, preferably for all receptacle cavities. Alternatively, one common single membrane layer covers all receptacle cavities in the substrate body. According to a preferred variant, the cavity lid sheet provided by the membrane layer comprises a web of filaments or a tight material, for example a film or foil. When the membrane layer comprises a web of filaments, advantages are obtained for gas supply to the cell material, for example for adjusting the CO2-containing environment and for improving cell adhesion.

[0056] Regarding the method of manufacturing the culture device, the membrane layer is preferably a spun web of filaments. The membrane layer is preferably produced by electrospinning (producing filaments by spinning and randomly depositing them on a substrate under the effect of an electric field). According to a preferred embodiment of the invention, the membrane layer is bonded to the beveled area by the effect of a chemical solvent and / or a melting process.

[0057] The membrane layer may comprise a synthetic or semi-synthetic layer, but preferably has a thickness in the range of 100 nm to 50 μm. The membrane may comprise fibers with a diameter in the range of 50 nm to 300 nm, which are referred to below as "nano-membranes". In a preferred example, the membrane layer is made of polycaprolactone and / or gelatin, or another biocompatible, spinnable material.

[0058] Preferably, the membrane layer is fabricated using an electrospinning apparatus configured to pull threads of membrane material across a substrate body and deposit them onto the surface of the substrate body exposing at least one receptacle cavity. This semi-permeable, non-contact spun "nano-patch" bonds the surface of the biological tissue to the surface of the substrate body and prevents it from peeling off from the edges even during shrinkage.

[0059] According to another modification of the invention, further biological cellular material may be deposited on the membrane layer, which advantageously allows, for example, to study the interaction of the cellular material in the receptacle cavity with the cellular material on the membrane layer, to better mimic the basement membrane in human tissues that include thin membrane layers, for example human skin or lung tissue.

[0060] According to a further preferred embodiment of the invention, the substrate body can be bonded via the underside to a carrier substrate, in particular comprising glass and / or plastic (e.g. PMMA) or a composite material with similar rigidity to glass. From the point of view of the manufacturing method, it is preferred to bond the substrate body to the carrier substrate and to subject the at least one receptacle cavity and the at least one fluid passage (including the anchor notch and the beveled portion) to a plasma treatment. The bonding can be achieved by means of glue or a biocompatible adhesive tape (e.g. ARcare 90106NB from Adhesive Research Inc.). Advantageously, the mechanical stability and the operating efficiency of the culture device are improved by the carrier substrate. Furthermore, the fluid passage can be exclusively or additionally formed in the carrier substrate. As an example, it is possible to generate the fluid passage in a carrier substrate, for example made of plastic, by stereolithography 3D printing. The plasma treatment further improves the adhesion of biological cellular material, such as tissue cells or muscle cells, and fixes the cells responsible for the contraction of the biological sample to the inner walls and the anchor notch.

[0061] The subtractive manufacturing method includes providing a preformed substrate body workpiece and creating at least one receptacle cavity with associated anchor notch and facet recess and at least one fluid passage by material removal. The material removal preferably includes laser beam-based cutting, especially engraving. Laser beam-based engraving has the advantage of creating nanostructured surfaces with improved adhesion properties for biological cell materials. The laser intensity, laser pulse frequency, and the moving speed of the laser relative to the substrate body can be selected depending on the desired structure to be created. Increasing the laser pulse frequency results in a smooth surface. In particular, a low laser pulse frequency performed with a pulsed laser having a repetition frequency of less than 400 Hz, preferably between 250 Hz and 400 Hz, is preferred to create a rough surface finish.

[0062] Alternatively or additionally, the material to be removed from the pre-formed substrate workpiece can be removed by mechanical and / or chemical processing methods, such as milling, etching, grinding, etc. A combination of mechanical and / or chemical processing methods with additional laser processing is also possible, particularly to obtain a desired surface structure.

[0063] Additive manufacturing includes, for example, injection molding, die casting, deep drawing, stereolithography methods, or holographic volume 3D printing (where structures are generated in polymers by focusing photons, for example by two-photon polymerization, rather than layer by layer), or layer by layer 3D printing. Subsequent treatment of the surface with a pulsed laser can additionally modify the surface texture. In particular, a rough finish can be produced in the fall-off area.

[0064] According to another particularly preferred embodiment of the present invention, at least one hollow vascular structure is prepared in the hydrogel by depositing a vessel-shaped sacrificial material in the hydrogel, which is combined with at least one fluid passageway, and the sacrificial material is removed after crosslinking the hydrogel to form at least one hollow vascular structure. The sacrificial material may, for example, comprise the polymer Pluronic F127. Alternatively, other unstable substances may be used, which act as a support material and can be removed, for example, by temperature change (gelatin, low melting agarose).

[0065] Alternatively, the at least one hollow vascular structure may be printed in the biological material by additive printing techniques, such as, for example, stereolithography or holographic printing methods. Advantageously, the at least one hollow vascular structure can be formed in this case without the use of a sacrificial support material.

[0066] Preferably, the inner surface of the at least one fluid passage and the at least one hollow vascular structure is coated with at least one of biological cells, collagen, laminin, and fibronectin. The sacrificial material is deposited in a liquid state by a deposition technique that allows for localized placement of the sacrificial material on or in the hydrogel. The deposition technique may include, for example, direct dispensing, extrusion printing, droplet dispensing, or spraying.

[0067] Preferred applications of the present invention include at least one of the following: culture of biological cells, organ chip processes, self-organization of complexes of biological cells, maturation of biological cells, study of degeneration processes of biological cells, testing of pharmacologically active substances (e.g. drug testing), creation of multi-tissue / organ models, study of wound healing processes, creation of biosensors, biohazard detection, parasitology studies, creation of barrier models (e.g. blood-brain barrier, skin, intestine, lung), in vitro vaccination processes by implementation of antigen presenting cells, antigen and vaccine screening, mechanobiology studies, study of biotechnical circulatory systems, formation of tumor environment, study of tumor angiogenesis, metastasis and adhesion of tumor metastases, study of aging processes, and study of tissue specific metabolism, as well as methods of disease modeling.

[0068] The above preferred examples show that the application of the present invention is diverse and in particular coincides with that of all other organ chip technologies.The advantage of the present invention is the possibility of long-term culture of perfused cellular materials, such as tissue fragments, so that self-organization, maturation processes, as well as slow degeneration processes can be studied.In addition, mechanobiological phenomena can be studied in 3D tissue equivalents, which correspond much better to living tissue than, for example, cell monolayers on semipermeable membranes.

[0069] The features disclosed in the context of the culture device and embodiments thereof also represent preferred features of the inventive method of producing a culture device and embodiments of said method. The above-mentioned aspects and preferred features of the invention, in particular with regard to the configuration of the culture device and the dimensions and configuration of the individual components described in relation to the culture device, also apply to the method. The above-mentioned preferred embodiments, variants and features of the invention can be combined with each other if desired.

[0070] Further details and advantages of the invention are described below with reference to the accompanying drawings, which are shown diagrammatically below. [Brief description of the drawings]

[0071] [Figure 1]FIG. 1 is a perspective view overviewing the features of a preferred embodiment of an incubation device according to the present invention. [Diagram 2] 1 illustrates features of a preferred embodiment of a method for manufacturing an incubation device according to the present invention. [Diagram 3] The preparation of a cavity lid sheet is illustrated. [Figure 4] The preparation of a cavity lid sheet is illustrated. [Diagram 5] FIG. 2 is a perspective view of a further embodiment of an incubation device according to the present invention. [Figure 6] FIG. 2 is a perspective view of a further embodiment of an incubation device according to the present invention. [Figure 7] FIG. 13 is a top view of a further embodiment of an incubation device according to the present invention comprising multiple receptacle cavities. [Figure 8] FIG. 13 is a top view of a further embodiment of an incubation device according to the present invention comprising multiple receptacle cavities. [Figure 9] The preparation of a cavity lid sheet is illustrated. [Figure 10] FIG. 2 is a perspective view of a further embodiment of an incubation device according to the present invention. [Figure 11] FIG. 2 is a perspective view of a further embodiment of an incubation device according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Features of preferred embodiments of the invention are described below with particular reference to the construction of the culture device and its manufacture, in particular with regard to the provision of a receptacle cavity with an anchor notch and a chamfered portion. Further features of the culture device, in particular with regard to its implementation as a fluidic chip and its coupling with further fluidic components, such as fluidic reservoirs and / or pumps, valves, sensors and / or monitoring devices, are not described, since they are known as such from the prior art. Furthermore, features of culturing biological cells and adjusting the culture and / or perfusion conditions, such as the supply of nutrients and / or differentiation factors, are not described, since they are also known as such from the prior art.

[0073] FIG. 1 illustrates a first embodiment of a culture device 100 according to the present invention, including a base body 10, a receptacle cavity 20, a fluid passageway 30, and a cavity lid sheet 40. The receptacle cavity 20 is adapted to accommodate biological cellular material (not shown in FIG. 1, see FIG. 2B). FIG. 1 illustrates an embodiment of a culture device having a single receptacle cavity 20. The present invention is not limited to this configuration, but rather can be implemented with multiple receptacle cavities, as shown in FIGS. 7 and 8. In the illustrated example, the fluid passageway 30 includes four fluid lines 31, each in fluid communication with the receptacle cavity 20 and having a port section 32 (see FIG. 1) for coupling the receptacle cavity 20 to other components (not shown) of a fluid system via a tube 33 (see FIG. 2C). In alternative embodiments, another number of fluid lines, including a single fluid line, may be provided per receptacle cavity 20 .

[0074] The substrate body 10 is a rectangular parallelepiped-shaped plate having an upper surface 11 and a lower surface 12 extending parallel to the horizontal direction (xy plane). The receptacle cavity 20 is made as a through hole passing through the substrate body 10. On the upper surface 11, the receptacle cavity 20 has an opening 23, and the internal space of the receptacle cavity 20 is exposed to the surroundings or covered with a cavity lid sheet 40. On the lower surface 12, a carrier substrate 14 is bonded to the substrate body 10. The carrier substrate 14 is made of, for example, glass. The carrier substrate 14 closes the receptacle cavity 20 toward the lower surface 12 of the substrate body 10.

[0075] The receptacle cavity 20 is defined by a bottom wall 21 and a side wall 22 that includes an anchor notch 24 of the present invention. The bottom wall 21 is provided by an exposed portion of the carrier substrate 14. The side wall 22 is provided by the material of the base body 10. The vertical direction (z-direction) defines the axial direction of the receptacle cavity 20.

[0076] The anchor notches 24 comprise recesses in the inner sidewall 22 of the receptacle cavity 20. Each of the anchor notches 24 extends radially in the xy plane. Additionally, each of the anchor notches 24 extends axially (z-direction) through the entire thickness of the substrate body 10. The anchor notches 24 are evenly distributed at equiangular intervals around the circumference of the sidewall 22. Alternatively, the anchor notches 24 can be distributed non-uniformly and / or with varying shapes and sizes (not shown).

[0077] As shown in FIG. 1, the anchor notch 24 is preferably an angular shape forming the inner profile of the anchor notch 24. The anchor notch 24 is shaped such that its azimuthal width narrows in the radial direction toward the center of the receptacle cavity 20, in particular toward the inner sidewall 22. According to the illustrated example, the anchor notch 24 has a cross-sectional shape resembling a thick arrow, i.e., the inner profile has a narrowing portion and a portion with a constant cross section. The invention is not limited to this illustrated shape. Alternatively, as a further variation, the profile of the anchor notch 24 can be formed such that the narrowing portion extends over the entire depth of the anchor notch 24, or the profile of the anchor notch 24 can be formed like a sawtooth profile or with a step shape (see FIGS. 9, 10).

[0078] On the top surface 11, the base body 10 has a chamfered portion 13. The chamfered portion 13 surrounds the opening 23 of the receptacle cavity 20 and includes the exposed contour of the anchor notch 24. In accordance with the cylindrical shape of the receptacle cavity 20, the chamfered portion 13 has the shape of a ring circle having a width greater than the depth of the anchor notch 24. In an alternative embodiment, the chamfered portion 13 can have a contour as illustrated in Figures 9 and 10.

[0079] Preferably, the chamfered portion 13 also extends along the fluid passageway 30, with the upper surface 11 being chamfered on either side of the fluid line 31 and around the port 32, as shown in FIG.

[0080] In a practical example, the substrate body 10 has a thickness of 3 mm and an area of ​​21 mm x 26 mm. The inner diameter of the receptacle cavity 20 is, for example, 10 mm, while about 10 to 20 anchor notches are provided, each having a radial depth of 1.5 mm and an azimuthal width of about 1.5 mm. The fluid passage 30 has a depth of 2 mm and a length between the receptacle cavity 20 and the port section 32 of 4 mm. The depth of the chamfered portion 13 is, for example, 400 μm.

[0081] FIG. 1 illustrates a cavity lid sheet 40 as a preferred feature of the culture device 100 according to the present invention. For illustrative purposes, the cavity lid sheet 40 is shown apart from the top surface 11. In practical applications, the cavity lid sheet 40 is placed directly on the culture device 100, in particular on the biological cell material in the receptacle cavity 20. Depending on the application of the culture device 100, the cavity lid sheet 40 is provided on the culture device 100 pre-filled (with the receptacle cavity 20 containing the biological cell material) or it is fabricated after printing the biological cell material in the receptacle cavity 20. Fabricating the cavity lid sheet 40 is described in an exemplary manner with reference to FIG. 3 and FIG. 4 below.

[0082] Figure 2 illustrates diagrammatically the steps of a method for manufacturing an incubation device according to the invention, such as the incubation device 100 of Figure 1. In the illustrated example, reference is made to manufacturing the incubation device 100 by a subtractive process.

[0083] First, the substrate body 10 is formed from a plate-shaped substrate body workpiece 10A, for example, an acrylic plate, having the size of the resulting substrate body 10. The receptacle cavity 20 with the anchor notch 24 (not shown in detail) and the fluid passage 30, as well as the beveled portion 13, are produced by laser processing, in particular laser cutting and / or laser engraving (see FIG. 2A), using a laser tool 200. The laser tool 200 includes, for example, a Laser System 8000 Laser System Mini (manufacturer: Epilog Laser Systems, Golden, Colorado, USA). The operating conditions of the laser tool 200 are selected depending on the material to be processed and the structure to be produced. To provide a rough finish of the beveled portion 13, the laser tool 200 operates at a repetition rate of about 300 Hz. FIG. 2A shows the situation after the completion of the receptacle cavity 20 and the fluid passage 30.

[0084] Subsequently, the carrier substrate 14 is adhered to the lower surface 12 of the substrate body 10. As an example, the carrier substrate 14 comprises a glass sheet having a size equal to the size of the lower surface 12 of the substrate body 10 and a thickness of, for example, 100 μm. Alternatively, the carrier substrate 14 may comprise a film attached to the lower surface 12.

[0085] After the substrate body 10 has been attached to the carrier substrate 14, the inner surface of the substrate body 10 is coated with a hydrogel 2, for example GelMA. For this purpose, a liquid volume of hydrogel 2 can be provided by a liquid pumping device 310 and the liquid hydrogel 2 can be distributed on the inner surface of the substrate body 10 by spraying the substrate body 10 directly from the print head into the anchor notch during the printing process and / or by moving the substrate body 10, in particular by swirling it on an orbital shaker. After contacting the inner surface of the substrate body 10, the hydrogel 2 dries and / or crosslinks, so that a coating having a thickness of, for example, 100 μm is formed. This lowest hydrogel layer preferably contains biological cell material and particularly preferably has the same composition as the remaining material in the sample receptacle. The biological cell material 1 to be cultured is then added to the coated substrate body 10 (FIG. 2B).

[0086] The biological cell material 1, which comprises the same or modified hydrogel with embedded biological cells, is applied by using another liquid pumping device and / or a droplet dispensing device 320. For example, a 3D Discovery BioSafety bioprinter (manufacturer: Regenhu, Switzerland) is used for printing the biological cell material. Alternative deposition techniques include microjet printing, spraying by ultrasonic effect, or printing droplets by needle printer. The biological cell material 1, e.g. stem cells in alginate, is filled into the interior space of the receptacle cavity 20 and into the anchor notch. The hydrogel contained in the biological cell material can then be crosslinked by irradiation, temperature setting, e.g. heating or chemical crosslinking, depending on the composition of the hydrogel. This process polymerizes the hydrogel and fixes it in the anchor notch together with the biological cells. The exact hydrogel composition is selected depending on the specific requirements, e.g. the desired mechanical strength of the crosslinked material, based on previous tests.

[0087] Simultaneously with the introduction of the biological cell material, a vessel-shaped sacrificial material 3 is deposited in the biological cell material, as shown diagrammatically in FIG. 2C. The sacrificial material 3 can also be printed in the fluid lines 31 and, optionally, introduced in the tubes 33. The sacrificial material includes, for example, Pluronic F-127, as described in [3]. The sacrificial material 3 provides a network of filaments, as shown diagrammatically, with cross-sectional dimensions like the blood vessels and vasculature in the biological cell material 1 to be cultured. Furthermore, the sacrificial material 3 is bonded to the fluid lines 31 of the fluid passage. After cross-linking the hydrogel, the sacrificial material 3 is removed by dissolution, resulting in hollow blood vessels and vasculature.

[0088] 3 and 4 illustrate the formation of the cavity-lid sheet 40 as a mesh-like membrane layer 41 by electrospinning. The provision of the mesh-like membrane layer 41 has particular advantages in terms of providing an increased surface for efficient cell attachment and cell migration. The spinning apparatus 330 includes a syringe driver 331 containing the polymer solution, a spinning nozzle 332, a high voltage source 333, and a conductive platform 334. The conductive platform 334 is movable in the x and y directions, the movement in the x direction being preferably a stepwise movement and slower than the movement in the y direction. Additionally or alternatively, the syringe driver 331 may be movable in the x and y directions. As a further alternative to the illustrated configuration, instead of the conductive platform 334, two elongated electrodes can be placed along the culture device and the high voltage can be switched between these two electrodes. Since no speed limitations are imposed in this embodiment, advantages in terms of even better parallel alignment of the filaments can be achieved. The polymer solution includes biomolecules, such as gelatin, collagen, etc., dissolved in an organic solvent. Cell adhesion for cells added to membrane layer 41 can be improved by adding peptides or proteins, such as ECM proteins, to the polymer solution.

[0089] Using a syringe driver 331, the polymer solution is supplied under pressure to the spinning nozzle 332. A high voltage, for example 8.5 kV, is applied between the spinning nozzle 332 and a conductive substrate 334 together with a high voltage source 333. Due to the electric field created by the high voltage source, a jet of the polymer solution is formed at the spinning nozzle 332. The pressure of the polymer solution and the high voltage are preferably adjusted such that a Taylor cone is formed at the nozzle opening with the jet at the tip of the cone.

[0090] The concentration of the polymer solution is adjusted so that the material leaving the spinning nozzle 332 crosslinks and dries during the flight towards the substrate. Thus, nanofibers are created, which form a nanomesh on the substrate. The nanomesh can include a random distribution of nanofibers or an at least partially oriented arrangement of nanofibers. Following the movement of the conductive substrate 334, the nanofibers 4 are not only deposited on the conductive platform 334, but are drawn directly onto the hydrogel in the culture device 100 and the sample receptacle 20. The high speed movement of the conductive substrate 334 in the y direction results in a preferential orientation of the nanofibers in the y direction. The diameter of the fibers can be set, for example, in the range of 50 nm to 150 nm in the case of gelatin fibers. The thickness of the membrane layer 41 depends on the spinning time, which can reach, for example, 30 μm. After completing the spinning, the culture device 100 with the coated membrane layer 41 is transferred from the conductive substrate 334 to the application site.

[0091] After spinning membrane layer 41 to create cavity-lid sheet 40, biological cell material can be applied to the top exposed surface of cavity-lid sheet 40. As shown in the exemplary electron microscope image of Figure 4, single cells, e.g., keratinocytes, attach to the surface of membrane layer 41 due to cell-filament interactions.

[0092] 5 and 6 illustrate a further alternative embodiment of the culture device 100 (without the optional cavity lid sheet shown), which is similarly constructed as the embodiment of FIG. 1, in particular with the base body 10, the receptacle cavity 20 and the fluid passageway 30. Deviating from FIG. 1, the embodiment of FIG. 5 has a wider chamfered portion 13. The radial width of the chamfered portion 13 is about twice or more the radial depth of the anchor notch 24. With the wider chamfered portion 13, improved adhesion of the cavity lid sheet and / or biological cell material is obtained.

[0093] In the embodiment of Fig. 6, a ring passage 34 is additionally provided in the fluid line 31. The ring passage 34 is made in the substrate body. The fluid line 31 and the anchor notch 24 are fluidly coupled with the ring passage 34. The anchor notch 24 is therefore open towards the interior space of the receptacle cavity 20 and towards the ring passage 34. In the embodiment of Fig. 6, certain advantages are obtained with regard to an even perfusion of biological cell material in the receptacle cavity 20.

[0094] 7 and 8 illustrate a further embodiment of the culture device 100, in which a plurality of receptacle cavities 20 with anchor notches are provided in the base body 10. Each of the receptacle cavities may have a separate fluid line, as shown in Figs. 1, 5 and 6. Alternatively, the receptacle cavities 20 may be fluidly coupled via a shared fluid line 31 of the fluid passage 30. As shown in Fig. 7, a group of receptacle cavities 20 are coupled in parallel via a common fluid line 31 with a common perfusion fluid reservoir (not shown). According to Fig. 8, a group of receptacle cavities 20 are coupled in a serial manner via a fluid line 31, and perfusion fluid flowing through one of the receptacle cavities is supplied to the subsequent receptacle cavity 20. According to a further alternative, a network of fluid lines including the receptacle cavities can be formed.

[0095] 9 and 10 illustrate further advantageous features of the embodiment of the culture device of the present invention. The top view of the base body 10 in FIG. 9 shows modified shapes of anchor notches 24, each extending radially with a retention profile for capturing material in the receptacle cavity 20. The retention profile is formed with a stepped shape 25 that provides an undercut slit opening 26 of the anchor notch 24. The slit opening 26 opens toward the inner surface of the side wall 22. The cellular material is captured by the undercut slit opening 26, similar to the profile of the anchor notch 24 shown in FIG. 1. In this embodiment, the anchor notch 24 preferably has an angular shape narrowing in the radial direction, as shown in FIG. 9, or a linear shape, as shown in FIG. 10 (cross-sectional view).

[0096] As a further optional feature, the beveled portion 13 may be contoured in the xy plane with a recessed groove 15 extending azimuthally around the opening 23 of the receptacle cavity 20, as shown in Figures 9 and 10. The recessed groove 15 is in direct communication with the anchor notch 24, and a ring of material, such as cellular material and / or biomatrix material, may be polymerized in direct connection with the anchor notch 24 and the material in the receptacle cavity 20. This ring of material provides an additional retention function to stabilize the cellular material in the receptacle cavity 20.

[0097] Additionally or alternatively, the peripheral sidewall 22 may have a peripheral bottom recess 27 disposed adjacent the bottom wall 21 and extending radially into the substrate body 10. Preferably, the peripheral bottom recess 27 has a stepped profile 28 such that a structure similar to a recessed trench 15 is formed. The peripheral bottom recess 27 traps cellular material in the receptacle cavity 20 in the axial direction (z direction), while the optional stepped profile 28 additionally traps cellular material in the radial direction (xy direction).

[0098] The recessed face 13 contoured with grooves 15 and the peripheral bottom recess 27 with step-shaped contour 28 form annular retention structures at the top and bottom, with anchor extensions directly embedded in the cellular material. The retention structures can be formed, for example, by laser engraving. In particular, the top and bottom annular retention structures, where the anchors do not reach the bottom, allow the degradable material to create cavities below the cellular material. Thus, the cellular material is lowered into the chip (3D anchors) and can be supplied with medium over the entire surface, including from below. Also, pressure changes (e.g. pulsating flow of medium or different air pressures in a skin equivalent) can move the cellular material and thus mechanically stimulate it.

[0099] 11 shows an example of a workflow for making a vessel-shaped sacrificial material by additive printing technology. In step S1, a substrate body 20 is prepared by laser engraving and / or cutting receptacles into a PMMA plate from the top and bottom. After oxygen plasma treatment to charge the plate surface (step S2) and coating the parts of the plate surface that form the receptacles with a biopolymer, such as collagen (step S3), the substrate body plate is rotated (step S4) and a degradable vessel-shaped sacrificial polymer material 3, such as water-soluble PVA (polyvinyl alcohol), is printed into the engraved recessed areas on the bottom of the substrate body plate (step S5). A transparent PMMA foil is then fixed underneath (step S6) and the substrate plate is cut, for example by laser cutting, into single slices each with one receptacle cavity (step S7), followed by an oxygen plasma treatment (step S8) to charge the surface of the PMMA foil, improve wettability, remove the solvent and sterilize the finished slides.

[0100] In summary, the present invention provides a method for medium throughput production of 3D bioprinted tissue equivalents, preferably embedded in laser-fabricated acrylic / glass chips, where the engraved passages are directly connected to conduits in the tissue equivalent during the printing process. Anchor notches provide specific macro- and microstructures in the fluidic chip design that integrate the bioprinted tissue into the substrate body and prevent detachment of the biological tissue from the walls of the plastic chip during post-processing. The tissue is connected and stabilized within the plastic part of the substrate body, preferably by a combination of laser-based surface treatment and electrospun semi-synthetic nanomembranes. With these on-chip tissue models, the inventors were able to show that drug testing can be performed directly and that tests on physiology, cell biology, mechanobiology, and morphogenesis can be performed serially or in parallel.

[0101] In fact, the culture device of the present invention has been used to investigate neuroblastoma, an extracranial solid tumor that occurs in childhood and still has a poor prognosis. One of our strategies to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interactions between the patient's cancer cells and the tumor environment. The culture device of the present invention has been used to develop a perfused and microvascularized tumor environment model directly bioprinted into the receptacle cavity. A gelatin methacrylate / fibrin-based matrix containing multiple cell types mimics the tumor microenvironment that encourages spontaneous microvessel formation by embedded endothelial cells. The fluid passages are coated with endothelial cells after printing to form a blood vessel-tissue barrier. The tissue model thereby mimics the structure and function of human soft tissue with larger blood vessels for perfusion coated with endothelial cells and a microvascular network within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process. We have demonstrated that microvessels are attracted and grow into tumor spheroids, and neuroblastoma cells invade the tumor microenvironment as soon as the spheroids break down. These studies have resulted in long-term stability of cell cultures, for example over 28 days. Using this 3D bioprinted microvascularized neuroblastoma microenvironment model printed directly into the culture device, we have described a novel medium-throughput platform suitable for reproducibly and stably testing drug response and metastasis for precision medicine approaches.

[0102] The features of the invention disclosed in the above specification, in the drawings and in the claims may be useful individually, in combination or in subcombination for the implementation of the invention in its different embodiments.

Claims

1. A culture device (100) configured for culturing biological cellular material (1), comprising: An upper surface (11); A lower surface (12); At least one receptacle cavity (20) having a bottom wall (21) and a peripheral side wall (22); at least one fluid passageway (30) in communication with said at least one receptacle cavity (20); A substrate body (10) having the at least one receptacle cavity (20) has an opening (23) towards the top surface (11) of the substrate body (10) and is arranged to receive the biological cellular material (1); an inner surface of the side wall (22) having an anchor notch (24) arranged to retain the biological cellular material (1) in the at least one receptacle cavity (20); the anchor notch (24) has a radially extending retention feature such that material in the receptacle cavity (20) is captured in the anchor notch (24); The upper surface (11) of the base body (10) has a chamfered portion (13) around the opening (23) of the at least one receptacle cavity (20), and the chamfered portion (13) is adapted to accommodate a cavity lid sheet (40) that covers the opening (23) of the at least one receptacle cavity (20).

2. 2. The incubation device of claim 1, wherein the anchor notch (24) has an angular shape providing a narrowing of the anchor notch (24) toward the inner surface of the side wall (22).

3. The incubation device of claim 1 or 2, wherein the anchor notch (24) has a stepped shape (25) that provides an undercut slit opening (26) of the anchor notch (24) toward the inner surface of the side wall (22).

4. said anchor notches (24) being evenly distributed along said side wall (22); said anchor notches (24) being unevenly distributed along said side wall (22); The anchor notch (24) extends in a direction perpendicular to the upper and lower surfaces (11, 12) of the base body (10), The culture device according to any one of claims 1 to 3, comprising at least one.

5. The incubation device according to any one of claims 1 to 4, wherein the chamfered portion (13) has a rough finish.

6. The incubation device according to any one of claims 1 to 5, wherein the beveled portion (13) has an outer shape having a recessed groove (15) extending around the opening (23) of the at least one receptacle cavity (20) and opening toward the anchor notch (24).

7. The incubation device according to any one of claims 1 to 6, wherein the peripheral side wall (22) has a peripheral bottom recess arranged adjacent to the bottom wall (21) and extending radially within the base body (10).

8. 8. The incubation device of claim 7, wherein the peripheral bottom recess has a contour shaped such that material in the sample receptacle is trapped in the peripheral bottom recess.

9. The culture device of any one of claims 1 to 8, wherein a cross-linked hydrogel is disposed in the at least one receptacle cavity (20), and a portion of the hydrogel is contained within the anchor notch (24).

10. 10. The culture device according to claim 9, wherein the biological cells and / or cell components of the biological cellular material (1) are contained in the hydrogel.

11. The incubation device according to any one of claims 1 to 10, further comprising a membrane layer (41) bonded to the recessed portion (13) for providing the cavity lid sheet (40) and covering the opening (23) of the at least one receptacle cavity (20).

12. The incubation device of claim 11, wherein the membrane layer (41) comprises a web of filaments (42).

13. The culture device according to any one of claims 1 to 12, at least one of the at least one receptacle cavity (20) and the at least one fluid passageway (30) is coated with at least one of biological cells, collagen, laminin, and fibronectin; The biological cellular material (1) has the following characteristics: it comprises at least one of biological cells and cellular components; the substrate body (10) being bonded via the lower surface (12) to a carrier substrate (14), in particular comprising glass; the at least one fluid passage (30) is formed in the upper surface (11) of the substrate body (10); The at least one fluid passage (30) comprises features formed in the lower surface of the substrate body (10); The at least one fluid passage (30) is formed in the substrate body (10) by one or more of the following features: An incubation device comprising at least one

14. A method for manufacturing an incubation device (100) according to any one of claims 1 to 13, comprising the step of producing the base body (10) with the at least one receptacle cavity (20), the at least one fluid passage (30), the anchor notch (24), and the chamfered portion (13) by subtractive manufacturing and / or additive manufacturing from a preformed base body workpiece (10A).

15. Adhering the substrate body (10) to a carrier substrate; and subjecting the at least one receptacle cavity (20) and the at least one fluid passage (30), including the anchor notch (24) and the chamfered portion (13), to a plasma treatment; The method of claim 14 further comprising at least one.

16. 16. The method of claim 14 or 15, the substrate body (10) is fabricated by subtractive manufacturing, which involves removing material from a pre-formed substrate body (10) workpiece; and A method in which said material removal comprises laser beam based engraving, in particular performed with a pulsed laser having a repetition rate of less than 400 Hz.

17. The method according to any one of claims 14 to 16, depositing a liquid hydrogel (2) into said at least one receptacle cavity (20) such that said anchor notch (24) is filled with said liquid hydrogel (2); and The method further comprises crosslinking said hydrogel (2) by at least one of irradiation, temperature setting, and chemical crosslinking.

18. 18. The method according to claim 17, wherein the biological cells and / or cellular components of the biological cellular material (1) are contained in the hydrogel (2).

19. 19. The method of claim 17 or 18, The biological cellular material (1) is characterized in that it comprises at least one of fibroblasts, endothelial cells, mesothelial cells, smooth muscle cells, organ cells (such as hepatocytes), induced pluripotent stem cells (iPSCs) and iPSC-derived cells, mesenchymal stem cells and spheroid cell bodies, or at least one component thereof; Said hydrogel (2) comprises at least one natural hydrogel component, such as gelatin methacryloyl (GelMA), alginate, functionalized alginate-YISGR, chitosan, hyaluronic acid, hyaluronic acid methacrylate, collagen, nanocellulose, silk, synthetic hydrogels (such as PEG, PEGDA or PVA), synthetic extracellular proteins, and / or glycoproteins (such as fibrinogen and / or fibronectin), among other characteristics: The method includes at least one.

20. 20. The method according to any one of claims 17 to 19, wherein crosslinking the hydrogel (2) deposited in the at least one receptacle cavity (20) comprises irradiation with light having a wavelength in the range of 365 nm to 700 nm.

21. The method according to any one of claims 17 to 20, characterized in that at least one hollow vascular structure is formed in the hydrogel (2) deposited in the at least one receptacle cavity (20), by: depositing a vessel-shaped sacrificial material (3) in said hydrogel (2), bonding said sacrificial material (3) with said at least one fluid passage (30), and removing said sacrificial material (3) after crosslinking said hydrogel to form said at least one hollow vessel structure (4); or Additive printing technology allows the creation of hollow structures. The method further comprising preparing.

22. 22. The method of claim 21, further comprising coating the inner surface of the at least one fluid passage (30) and the at least one hollow vascular structure (4) with at least one of biological cells, collagen, laminin, and fibronectin.

23. 23. The method of any one of claims 14 to 22, further comprising bonding a membrane layer (41) to the recessed portion (13) to provide a cavity lid sheet (40) for covering the opening (23) of the at least one receptacle cavity (20).

24. 24. The method of claim 23, wherein further biological cellular material (1) is deposited on the membrane layer (41).

25. The membrane layer (41) is a spun web of filaments (42); and The membrane layer (41) is bonded to the recessed portion (13) by the effect of a chemical solvent and / or by a melting process, among other features:

25. The method of claim 23 or 24, comprising at least one.

26. A culture device according to any one of claims 1 to 13 or a method according to any one of claims 14 to 25, Cultivation of biological cells, Organ Chip Process, Self-organization of biological cell complexes, Study of maturation of biological cells, degeneration processes of biological cells, Testing for pharmacologically active substances (e.g. drug testing), Creation of multi-tissue / organ models, Research into the wound healing process, Fabrication of biosensors, Biohazard detection, parasitology research, Preparation of barrier models (e.g., blood-brain barrier, skin, intestine, lung), In vitro vaccination process by implementation of antigen-presenting cells, Antigen and vaccine screening, Mechanobiology research, Research on biotechnology circulation systems; Formation of the tumor environment, Research on tumor angiogenesis, metastasis, and adhesion of metastatic tumors, Research into the aging process, Research into tissue-specific metabolism, and Among the methods of disease modeling, The method used for at least one.

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