Apparatus for culturing a tissue section

Abstract

The invention relates to an apparatus for culturing a tissue section (4), which apparatus comprises a frame (3) having a peripheral profile and a central open space formed by the peripheral profile, wherein the frame (3) comprises two frame elements (1, 2), each having a peripheral element profile (100, 110, 200, 210) and an element open space (101, 111, 201, 211) which is central in each case and is formed by the peripheral element profile, which frame elements (1, 2) can be assembled so as to cover one another to form the frame (3). The apparatus also comprises two support components (12, 22), the support components (12, 22) being designed to receive a tissue section (4). Each frame element (1, 2) comprises a frame-element main body (10, 20) and a frame-element insert (11, 21), which can be connected to form the frame element (1, 2). For each frame element (1, 2), the frame-element insert (11, 21) and the frame-element main body (10, 20) are designed to hold the support component (12, 22) such that the support component (12, 22) is clamped in the frame element so as to span the open space.

Description

[0001] Description

[0002] title

[0003] Device for cultivating a tissue section

[0004] The present invention relates to a device for culturing a tissue section, in particular a tumor tissue section, for example, for drug testing. Furthermore, the present invention relates to a cultivation system and a method for culturing a tissue section.

[0005] State of the art

[0006] Cancer patients with solid tumors unfortunately respond differently to drug therapy. The main reasons for this are the individual variability and heterogeneity of the tumors, as well as the complexity of the tumor microenvironment. There is a great need for increasing personalized cancer treatment, which, among other things, takes into account the cellular characteristics of the individual patient in order to derive an individually optimized therapy. Furthermore, there is a great need for a reliable in vitro tumor model that adequately reflects the in vivo situation and can be used for the development and preclinical testing of new antitumor agents.

[0007] During a biopsy or surgery, cancer tissue is removed from a patient. The cancer tissue is then cultured and used for in vitro testing of cancer drugs and to investigate individual drug effects. For in vitro testing, a suitable cell-based tumor model is required, which can be obtained from tissue samples from cancer patients and reflects key aspects of complex in vivo tumor biology. Thin tissue sections with a typical tissue thickness of approximately 150 μm to 400 μm are prepared from the removed tumor sample. These tumor tissue sections are particularly suitable for use as tumor models due to the preservation of in vivo tissue heterogeneity with various cell types and the preserved tumor microenvironment.

[0008] Various methods have been developed for culturing tumor tissue sections, but these only allow for a limited cultivation time. After a few days, the viability of the cells in the tissue section decreases significantly, and the cells die. This very limited cultivation time is due to the fact that removing the tumor inevitably leads to a complete cutoff of the blood supply, and supplying tumor tissue cultured ex vivo in medium with physiological oxygen concentrations is very difficult. This is due to the different oxygen uptake capacity of culture medium compared to blood.

[0009] According to the current state of the art, viable tumor tissue sections are cultured either floating in a suitable medium or lying on a filter. The most commonly used culture system is a tissue culture insert for multiwell plates (Millipore filters). In this system, the tissue sections are cultured at the air-liquid interface, thus achieving a higher oxygen supply.Although the known systems with cultivation devices for tissue at the air-liquid interface can partially remedy this deficiency by exposing one side of the tissue to air, which makes this type of cultivation of tissue sections clearly superior to liquid culture, this leads to the formation of an artificial gradient oriented from the air to the medium side, in such a way that only the upper cell layers facing the air correspond in their morphology and nature to the original tissue from which the tumor sections were obtained, while the cell layers facing away from the air show signs of hypoxia and necrosis.

[0010] WO 2019 / 029947 A1 discloses a cultivation approach in which, instead of static cultivation, a fluidic supply of culture medium to the tissue sections is implemented. A so-called Perfusion Air Culture (PAC) system with adjustable medium or drug supply is used for this purpose. The device comprises a frame, at least one support component, wherein the support component is configured to receive a tissue section, and at least one fluid-absorbing strip element. The frame is configured to hold the at least one support component and the at least one strip element.

[0011] Disclosure of the invention

[0012] A device for cultivating a tissue section is proposed. In particular, the tissue section is a tumor tissue section. The device has a frame with a circumferential profile and a central free space formed by the circumferential profile. The frame comprises two frame components, each with a circumferential component profile and a central component free space formed by the circumferential component profile. Both frame components can preferably be assembled to form the frame by means of a connecting element. Each frame component further comprises a frame-component base body and a frame-component insert, which can be plugged together to form the frame component. The profile of the frame-component insert preferably has a collar that can be inserted into the free space of the frame-component base body. In addition, the device has two support components, one support component each for a frame component.The two support components are particularly organotypic and designed to receive a tissue section. For each frame component, the frame component insert and the frame component base body are designed to hold a support component when assembled. For this purpose, the support component can be clamped by the frame component insert and the frame component base body. Each frame component is designed such that, after assembly, it holds one support component each in such a way that it is clamped to the frame component, spanning the free space of the frame component. The frame component insert and the frame component base body can hold the support component equally on all sides all the way around. Alternatively, it can also be provided that the support component is not held on all sides, but rather, for example, on two opposite sides.The other sides of the support component can then, for example, be pulled through the free space in the frame component base body. The profile of the frame component insert can have notches that allow the support component to be positioned more easily. For each frame component, a support component is inserted between the frame component base body and the frame component insert. By plugging them together, the support component is clamped between the frame component base body and the frame component insert inserted into it. This connection can be released again by separating the frame component base body and the frame component insert. The support component can then be removed and transported away, for example, for further examination of the tissue section. A new support component can then be easily inserted and clamped in again as described above.

[0013] The two frame components are then assembled to form the frame. Preferably, the two frame-component base bodies are aligned with each other. After the two frame components are assembled to form the frame, the tissue to be examined or cultured, for example, a thin tissue section, and in particular a tumor tissue section, can be held between the two support components. The two support components together can form a chamber into which the tissue section can be placed before the frame is assembled, so that it is held on both sides by the support components. This supplies the tissue section with optimal oxygen concentrations on both sides, enabling atmospheric cultivation of the tissue section.

[0014] The carrier components can be supplied directly with fluid. The fluid can be introduced between the carrier components, particularly into the chamber formed by the carrier components. This enables constant perfusion with a culture medium containing a desired concentration of nutrients. Furthermore, an active ingredient solution, such as a drug solution, can be introduced. This ensures a supply of nutrients and, if necessary, a uniform application of drugs. This eliminates the need for filter papers, as used in current delivery systems. The device thus allows the testing of active ingredients and drugs while maintaining the native individual tissue, for example, tumor tissue, under largely physiological conditions.The total amount of perfusate and thus the consumption of the active ingredient to be tested can also be kept low.

[0015] In the present case, a "circumferential profile" and a "central free space formed by the circumferential profile" are understood to mean any enclosure suitable for holding or stabilising one or more support components clamped or accommodated in the (circumferential) frame.

[0016] The frame, or the two frame components, is / are preferably essentially rectangular or narrow cuboid-shaped and accordingly has / have four sides.

[0017] Preferably, the at least one carrier component is made of a material selected from the group consisting of cotton fabric, nylon fabric, polycarbonate, cellulose hydrogel, animal intestine, in particular pig intestine, 3D-printed biomaterials, in particular 3D-printed alginate fabric or 3D-printed collagen fabric. Preferably, the at least one carrier component is or comprises a lattice-shaped and oxygen-permeable fabric.

[0018] The frame and its components can preferably be made of a solvent-resistant material. In particular, biocompatible polymers such as polycarbonate (PC), polypropylene (PP), acrylonitrile-butadiene-styrene copolymer (ABS), polyethylene terephthalate (PET), polylactic acid (PLA), polyetheretherketone (PEEK), and polyoxymethylene (POM) can be used as the material, as these can be easily cleaned and / or sterilized. 3D printing or injection molding, for example, can be used to manufacture the components. Alternatively, other materials, such as metals or ceramics, and corresponding manufacturing processes can also be used.

[0019] Preferably, a connecting element is provided by means of which the frame components can be assembled to form the frame. The connecting element is preferably designed as a frame-component insert. When assembling the frame, the connecting element can be connected to the other frame-component insert. The latter can additionally have a corresponding connection feature with which the connecting element interacts during the connection, for example a notch, a hook, an eyelet or the like. As described above, the frame-component inserts are preferably arranged on the outside of each frame component in the assembled device. This creates a secure and easily accessible connection concept that ultimately holds the entire device together. The connecting element is preferably designed as a click connection, guide rail, clamp connection or as a closure.

[0020] The frame advantageously has a guide for the liquid supply. This guide is preferably formed by connecting or joining the two frame components. Particularly preferably, the guide for the liquid supply is formed by connecting or joining the two frame component base bodies. Alternatively, the guide is located on one of the two frame components. A liquid, for example a cultivation medium or an active ingredient solution, in particular a medication solution, can be supplied to the device - and thus to the tissue section to be cultivated in the device - via the guide, and the tissue can thus be perfused with the liquid. The liquid is introduced into the free space within the frame and preferably between the support components, where the liquid comes into contact with the tissue held therein.

[0021] The guide for the liquid supply is preferably divided into several different sections within the frame profile. An outer section can be widened so that a conventional tubular injection cannula can be easily inserted. In a middle section, a constriction with a diameter of, for example, 0.3 mm to 1.2 mm can be provided, with which the cannula is held in particular straight and firmly. An inner section can in turn be widened and form a cavity. The cavity is connected to the two support components and improves the fluidic transfer of the liquid from the small cannula opening to the flat and closely spaced support components. For this purpose, the cavity can be filled with a fluid, for example with the cultivation medium and / or the drug solution. The result is a complete cannula connection.A conventional cannula can be inserted into the guide up to the chamber between the support components. The cannula, in turn, can be connected via a tube to a pumping mechanism, such as a syringe pump or a peristaltic pump. The cannula can be used to precisely supply the tissue section with the culture medium and, if necessary, with the drug solution—particularly with a flow rate in the range of pl / h to ml / h.

[0022] Advantageously, the frame component base body has a trapezoidal bulge on one side facing outwards. The trapezoidal bulge widens the profile of the frame component base body in the plane. The narrow side of the trapezoidal bulge points outwards and the long side points towards the central component free space. The trapezoidal bulge therefore widens towards the central component free space. Preferably, the associated support component on this side is also trapezoidal in shape and adapted to the bulge. When plugged together, the support component rests on the frame component base body with the trapezoidal side on the side facing away from the component insert. For this purpose, the side of the support component can be pulled through the free space of the frame component base body.Advantageously, the fluid supply guide described above is located in the trapezoidal recess. The trapezoidal shape allows the fluid supplied from the outside to be better distributed inward, creating a homogeneous fluid flow into the preferably rectangular free space and to the tissue incision. This eliminates the need for filter papers, as used in current supply systems.

[0023] To further improve the distribution of the fluid flow, the frame component base body can have structures for distributing the fluid on the surface of the trapezoidal bulge. The structures are, for example, elevations that protrude from the frame component base body. The structures lie against the support component or penetrate into it. The structures therefore represent obstacles to the fluid flow, by which the fluid flow is deflected and widened. The structures can take on any shape in their cross-section, for example round, rectangular or oval, and preferably have a cross-sectional size between 0.1 mm and 1.0 mm and a height corresponding to the distance between the frame component base bodies. The structures can be arranged regularly or irregularly and can be arranged in a suitable number and spacing.In addition, elongated fluidic barriers can be provided in the trapezoidal bulge, in particular at its edges, in order to prevent leakage of the liquid in the trapezoidal bulge.

[0024] Furthermore, a cultivation system for cultivating tissue sections, in particular for testing active ingredients and / or medications, is proposed. The cultivation system comprises a device according to the invention as described above, a holding frame and a receiving vessel. The device is inserted into the holding frame and held thereby. For this purpose, the holding frame can preferably have a holding and positioning structure with a lower stop on both sides. The holding structure can also prevent unintentional opening of the frame, the frame components and / or the chamber. In this case, the connecting element on the frame described above can be omitted. The holding frame is then inserted into the receiving vessel. The holding frame is designed such that it can be inserted into the receiving vessel and can preferably be placed at a predetermined depth. This has the advantage that, for example,a conventional vessel, in particular a ventilated tube, for example an appropriately dimensioned centrifuge tube, can be used as the receiving vessel, in which the device according to the invention can be fastened by means of the holding frame. The receiving vessel can have a lid, in particular a screw cap, which enables sterile gas exchange and allows access to the guide for the liquid supply, for example through a septum slit. An injection cannula for guiding the liquid supply can be inserted through the access. Additionally or alternatively, a withdrawal cannula can be inserted into the receiving vessel through the access. The ability of the tube to be closed with the lid is not impaired. The holding frame can have a structure for holding and guiding the cannula, in particular the withdrawal cannula.

[0025] According to a preferred embodiment, the tube can be selected from a tube that holds between 10 and 100 ml, and in particular a 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ml tube. Preferred dimensions for the frame are between 18 mm to 28 mm x 21 mm to 31 mm, in particular 23 x 26 mm, and for the free space, 9 mm to 19 mm x 10 mm to 20 mm, in particular 14 x 15 mm.

[0026] With this system according to the invention, a vertical orientation of the device according to the invention in the receiving vessel is advantageously achieved, and thus also a vertical orientation of the tissue section in a closed, sterile, ventilated receiving vessel. The system can then be kept, for example, in an incubator under a controlled atmosphere. With a vertical orientation, the device is preferably accommodated in the receiving vessel in such a way that a guide for fluid supply, for example by means of a perfusion needle, is located on the upper side of the frame. This guide can optionally be connected to a perfusion tube. This can preferably be connected to a syringe and controlled by a pumping mechanism, e.g., a syringe pump or a peristaltic pump, via which controlled perfusion is possible.The liquid introduced via this system can be collected in the receiving vessel after passing through the carrier component and thus also the tissue at the bottom of the receiving vessel.

[0027] Furthermore, a method for culturing a tissue section is proposed. This method utilizes the device or cultivation system described above.

[0028] First, the frame components are prepared. Two frame component base bodies and two frame component inserts, one for each frame component, as well as two support components are provided. A support component is placed on each frame component insert in such a way that the support component spans the component free space and extends into the profile of the frame component insert. All sides can extend equally into the frame component insert profile all the way around. Alternatively, sides of the support component can be left free. The frame component insert is then inserted into the frame component base body, thus forming a frame component. The support component is now held by the sides that extend into the frame component insert profile and are covered by the frame component base body profile.It may be possible to extend the sides of the support component through the free space of the frame component base body to the other side. This can improve the holding effect and leak resistance, and the sides can be used for additional functions, such as better distribution of the supplied fluid. The frame components can then be sterilized.

[0029] A tissue section is placed on a support component in one of the frame components. The other frame component is then placed on top, with the two frame component base bodies lying on top of each other and the support component of the other frame component resting on the tissue section. The support components thus form a chamber in which the tissue section is stored. The frame components are then connected to one another to form the frame. For this purpose, a connecting element, which is preferably arranged on one of the frame component inserts, is preferably connected to the other frame insert component. The tissue section is clamped and held between the support components.

[0030] Preferably, the frame containing the tissue section is inserted into a holding rack. The holding rack, including the frame, is then inserted into a receiving vessel, such as a vented tube. The receiving vessel can then be sealed with a lid. Additionally, the receiving vessel can be placed in an incubator.

[0031] For cultivation, the cultivation system can be fluidically connected to a supply system that provides a cultivation medium, and to a collection system that collects the fluid from the cultivation system. A fluid, in particular a cultivation medium, is fed into the free space surrounded by the support components within the frame, i.e. the chamber formed by the support components. A defined volume flow and / or a desired time duration can be set for the feed. The fluid can penetrate the support components and, in particular, act on the tissue section on both sides in the receiving vessel. In the process, metabolic products from the tissue section are transferred into the fluid. The fluid containing the metabolic products can then be drained away, in particular outside the frame.

[0032] Guides for cannulas can be provided in the lid for the supply and removal of fluids. To supply fluids, an injection cannula is guided through the lid to the frame. If a guide for fluid supply is present in the frame, the injection cannula can be inserted into it. To remove fluids, an extraction cannula can also be guided through the lid into the receiving vessel. It is preferably inserted outside the frame and advantageously extends to the bottom of the receiving vessel to drain the fluid collected there.

[0033] For drug testing and analysis, the supply system to which the cultivation system is connected can additionally provide a drug solution, which is fed into the free space within the frame surrounded by the carrier components. A defined dose and a desired time duration can be set for the delivery of the drug solution. The drug solution can penetrate the carrier components and, particularly in the receiving vessel, act on the tissue section on both sides. In addition, the collection system can have an analysis system or be fluidically connected to it, which performs an analysis of the metabolites. After drug testing, the frame can be removed from the receiving vessel. The frame components can be detached from one another, and the tissue section can be removed for further examination.

[0034] Short description of the drawings

[0035] Embodiments of the invention are illustrated in the drawings and explained in more detail in the following description.

[0036] Figures 1 AH show isometric views of basic components of the device according to the invention.

[0037] Figures 2A, B show isometric views of the device according to the invention before (Figure 2A) and after assembly (Figure 2B).

[0038] Figure 3A shows a side view of the device according to the invention from Figure 2 with a cannula.

[0039] Figure 3B shows an enlarged sectional view of a section of Figure 3A, rotated by 90°. Figure 4A shows a plan view of a frame component base body with a carrier component according to another embodiment.

[0040] Figures 4B-D show three enlarged sections from Figure 4A.

[0041] Figures 5A-D show isometric views and a top view (Figure 5C) of basic components of the cultivation system according to the invention.

[0042] Figure 6 shows an isometric view of the cultivation system according to the invention.

[0043] Figure 7 shows a flow diagram of an embodiment of the cultivation method according to the invention.

[0044] Figure 8 shows a flowchart of a drug testing and analysis procedure.

[0045] Embodiments of the invention

[0046] Figures 1A-H show the basic components of the device according to the invention. Figures 1A-D show a first frame component base body 10 and a first frame component insert 11, which, as shown in Figure 1D, are assembled to form a first frame component 1, as well as a first organotypic support component 12. The first frame component base body 10 shown in Figure 1A has a profile 100 that surrounds a central free space 101. The frame component base body profile 100 and the free space 101 are square, with the exception of one side, on which the profile 100 has a trapezoidal bulge 102 in the plane. The trapezoidal bulge 102 is isosceles, the short side points outwards and the long side merges into the rest of the profile 100, so that the trapezoidal bulge 102 widens inwards.On the short side, one half 103 of a guide, or in other words a cannula connection 33, is arranged, which will be discussed in detail below. The first frame component insert 11 shown in Figure 1B also has a profile 110 that is rectangular and has the same dimensions as the profile 100 of the associated first frame component base body 10. The frame component insert profile 110 surrounds a likewise rectangular, centrally arranged free space 111. On the edge of the frame component insert profile 110, a collar 112 is formed that projects perpendicular to the profile 110 and adjoins the profile 110 inwards. In the frame component insert profile 110, incisions 113 are machined on two opposite sides, which serve to position the first carrier component 12.

[0047] In Figure 1C, the first support component 12 is placed on the first frame component insert 11. The support component 12 is, for example, a grid-like, oxygen-permeable cotton fabric. One side 122 of the support component 12 is also trapezoidal and corresponds in shape and dimensions to the trapezoidal bulge 102 of the frame component base body 10. Two other sides 120 of the support component 12 are held in the notches 113 of the profile 110.

[0048] In Figure 1D, the first frame component base body 10 is placed onto the first frame component insert 11. The first frame component base body 10, the first frame component insert 11, and the first carrier component 12 are aligned with one another such that the profile 100 of the first frame component base body 10 lies flush over the profile 110 of the first frame component insert 11, the collar 112 of the first frame component insert 11 engages in the free space 101 of the first frame component base body 10, and the trapezoidal side 122 rests on the trapezoidal bulge 102 of the frame component base body 10. For this purpose, the trapezoidal side 122 is guided upwards from below through the free space 101 of the frame profile base body 10 and then folded outward along the line 123. The opposite side of the first support component 12 is processed in the same way.The first support component 12 is held on the sides 120 by the first frame component base body 10 and the first frame component insert 11. Thus, the first frame component 1 is formed.

[0049] Figures 1 EH show a second frame component base body 20 and a second frame component insert 21, which, as shown in Figure 1 H, are assembled to form a second frame component 2, as well as a second organotypic support component 22. The second frame component base body 20 shown in Figure 1 E is designed with the same shape and the same dimensions as the first frame component base body 10. The second frame component base body 20 has a profile 200 that surrounds a central free space 201. The frame component base body profile 200 and the free space 201 are square, with the exception of one side, on which the profile 200 has a trapezoidal bulge 202 in the plane. The trapezoidal bulge 202 is isosceles, the short side points outwards and the long side merges into the remaining profile 200, so that the trapezoidal bulge 202 widens inwards.The other half 203 of the cannula connection 33 is arranged on the short side and will be discussed in more detail below. The second frame component insert 21 shown in Figure 1F also has a profile 210 which is rectangular and has the same dimensions as the profile 200 of the associated second frame component base body 20. The frame component insert profile 210 surrounds a likewise rectangular, centrally arranged free space 211. On the edge of the frame component insert profile 210, a collar 212 is formed which projects perpendicularly to the profile 210 and adjoins the profile 210 inwards. In the frame component insert profile 210, incisions 213 are machined on two opposite sides, which serve to position the second carrier component 22.In contrast to the first frame component insert 11, the second frame component insert 21 has four connecting elements 214 in the form of click elements arranged on the outside at the corners of the frame component insert profile 210. Their function is described in connection with Figure 2.

[0050] In Figure 1G, the second support component 22 is placed on the second frame component insert 21. The support component 22 is also a grid-shaped and oxygen-permeable cotton fabric. One side 222 of the support component 22 is also trapezoidal and corresponds in shape and dimensions to the trapezoidal bulge 202 of the frame component base body 20. Two other sides 220 of the support component 22 are held in the notches 213 of the profile 210. In Figure 1H, the second frame component base body 20 is placed on the second frame component insert 21.The second frame component base body 20, the second frame component insert 21 and the second carrier component 22 are aligned with one another such that the profile 200 of the second frame component base body 20 lies flush over the profile 210 of the second frame component insert 21, the collar 212 of the second frame component insert 21 engages in the free space 201 of the first frame component base body 20 and the trapezoidal side 222 rests on the trapezoidal bulge 202 of the frame component base body 20. For this purpose, the trapezoidal side 222 is guided upwards from below through the free space 201 of the frame profile base body 20 and then folded outwards along the line 223. The same procedure is followed with the opposite side of the second carrier component 22. The second support component 22 is held on the sides 220 by the second frame component base body 20 and the second frame component insert 21.Thus, the second frame component 2 is formed.

[0051] Figures 2A and B show the assembly of the two frame components 1, 2 from Figure 1 into a frame 3 in which a fabric section 4 is accommodated between the support components 12 and 22. Figure 2A shows the frame components 1, 2 and the fabric section 4 separated from one another. The first frame component 1 is rotated with the first frame component base body 10 in the direction of the second frame component base body 20 of the second frame component 2. The first frame component 1 and the second frame component 2 are aligned such that the profiles 100 and 200, the free spaces 101 and 201, and the trapezoidal bulges 102 and 202 of the base bodies 10, 20 lie one above the other. The fabric section 4 is placed on the support component 22 of the second frame component 2 within the free space 22. The first frame component 1 is then placed from above onto the second frame component 2. Figure 2B shows the frame 3 in its assembled state.The frame components 1, 2 surround a central free space 31 which corresponds to the free spaces 111 and 211. The fabric section 4 is arranged between the base bodies 10, 20 of the first frame component 1 and the second frame component 2 and within the free space 31 and is located in a chamber formed by the two support components 12 and 22 in the free spaces 101, 111, 201, 211. The connecting elements 214 of the second frame component insert 21 enclose the second frame component 2 and the first frame component 1 and are clipped into the first frame component insert 21 of the first frame component 1. The first frame component insert 21 can have corresponding recesses (not shown) for this purpose. Alternatively, other types of connecting elements 214 can be selected. This creates a firm connection and clamps the tissue section 214 firmly and securely between the support components 21 and 22.The connection can be released by loosening the connecting elements 214. The two cannula connection halves 103, 203 lie on top of each other and together form the cannula connection 33, which is described below.

[0052] Figure 3A shows the device according to the invention in an upright position, so that the cannula connection 33 points upwards. An injection cannula 5 is inserted into the cannula connection 33. This has a Luer connection 51 on the opposite side for fluidic connection to a supply system. Figure 3B shows an enlarged sectional view of section A from Figure 3A, rotated by 90° compared to Figure 3A. The cannula connection 33 has a widening 330 in the upper area, which acts as an injection aid. In the middle area, a cannula channel is provided which is narrower than the widening 330 and encloses the cannula 5 and thus holds it securely. The lower area widens out to form a cavity 332 in which the fluid to be introduced is held.From the cavity 332, the fluid introduced through the cannula 5 can penetrate between the two carrier components 12, 22 into the chamber and thus to the tissue section 4.

[0053] Figure 4A shows a top view of the first frame component base body 10 with the first support component 12. A plurality of structures 124 are formed in the trapezoidal bulge 122, which rest against or penetrate the support component 12. The structures 124 serve to distribute the fluid introduced via the cannula connection 33, of which the cannula connection half 103 is shown here. Elongated fluidic barriers 125 are formed at the edges of the trapezoidal bulge 122, which additionally prevent fluid leakage.

[0054] Figure 4B shows a top view of section B of Figure 4A, depicting a cannula connection half 103. Here, too, the cannula connection half 103 has a widened portion 330 in the upper region as an injection aid, a narrower cannula channel, also referred to as a constriction 331, in the middle region, and a cavity 332 in the lower region. Reference is made to the description of Figure 3B.

[0055] Figures 4C and 4D show two different embodiments of the structures 124 for distributing the fluid. In Figure 4C, the structures 124 have a triangular shape, with one tip pointing toward the cannula connection 33 or the cannula connection half 103. In Figure 4D, the structures 124 are round. They can be circular or oval, as shown here. The structures 124 form obstacles to the fluid flow, so that it is deflected and thus distributed.

[0056] Figure 5A shows a tube 6 that serves as a receptacle for the device according to the invention and in which the tissue section can be cultivated. The tube is cylindrical and has a conical tip at the lower end. The tube 6 has a lid 7 that can be placed fluid-tight onto the tube 6. The lid 7 has a septum 71 in the center of the top side and several filter openings 72 around the outside. Figures 5B and 5C show a holding frame 8 that can accommodate and hold the device according to the invention. The holding frame 8 has an elongated, cylindrical frame structure 80 that is adapted in shape and size to the tube 6. Two guide rails 81 are arranged inwardly on the frame structure 80 and run from the upper edge to across the center of the frame structure 80.The frame 3 of the device, including the support components 12, 22 and the tissue section 4, is inserted into the guide rails 81 from above and is then held centrally in the frame structure 80 by the guide rails 81. The frame 3 is inserted so that the cannula connection 33 faces upward. Furthermore, the holding frame has a holding and guiding structure 82 for a cannula, which is formed by an opening in a projection on the upper side of the frame structure 80. In Figure 5D, the holding frame 8 is inserted into the tube 6.

[0057] The frame structure 80 rests against the inner wall of the tube 6 and rests on the conical tip. The frame 3 of the device according to the invention is held centrally in the tube 6.

[0058] Figure 6 shows the system according to the invention, which comprises the device according to the invention with the frame 3 and the support components 12, 22, the tube 6, the lid 7, the holding frame 8, the injection cannula 5, and a withdrawal cannula 9. The device according to the invention is inserted into the holding frame 8, and the holding frame is in turn inserted into the tube 6, as shown in Figure 5D and described in this context. The lid 7 is placed on the tube 6 and seals it fluid-tight. The injection cannula 5 and the withdrawal cannula 9 are guided through the septum 71 in the lid. On the outside, the cannulas 5, 9 each have a Luer connector 51, 91, with which the cannulas can be connected to a supply and / or drainage system. The injection cannula 5 is guided into the cannula connection 33 of the frame 3.Fluid can be introduced via the injection cannula 5 into the cannula connection 33 within the frame 3 and finally into the chamber between the support components 12, 22. The withdrawal cannula 9 is guided through the holding and guiding structure 82 of the holding frame 8 and runs past the frame 3 to the tip of the tube 6. Fluid that has escaped from the device through the support components 12, 22 can be removed from the tube 3 through the withdrawal cannula 9.

[0059] Figure 7 shows a flow diagram of an embodiment of the method according to the invention for cultivating a tissue section 4. With reference to Figures 1 AD, a first frame component base body 10 and a first frame component insert 11 are provided 1000, a first carrier component 12 is placed 1001 on the first frame component insert 11, the first frame component base body 10 is placed 1002 on the first frame component insert 11, wherein the collar 112 of the first frame component insert profile 110 is pushed into the free space 101 of the first frame component base body 10, and the first carrier component 12 is guided 1003 on the side of the trapezoidal bulge 102 and on the opposite side through the free space 101 of the first frame component base body 10, so that a first frame component 11 is formed.Accordingly, with reference to Figures 1 EH, a second frame component 12 is formed by providing 1010 a second frame component base body 20 and a second frame component insert 21, placing 1011 a second carrier component 22 on the second frame component insert 21, placing 1012 the second frame component base body 20 on the second frame component insert 21, wherein the collar 212 of the second frame component insert profile 210 is pushed into the free space 201 of the second frame component base body 20, and guiding 1013 the second carrier component 22 on the side of the trapezoidal bulge 202 and on the opposite side through the free space 201 of the second frame component base body 20.

[0060] The tissue section 4 is then placed 1022 on one of the support components 12, 22, for example the second support component 22, in the second frame component 2. Subsequently, the first frame component 1 is placed 1023 on the second frame component 2. The two frame components 1, 2 are then connected 1023 to one another, as shown in Figure 2B, and thus the frame 3 of the device according to the invention is produced. The tissue section 4 is placed between the support components 12, 22. The connecting elements 214 on the second frame component insert 21 are clipped into the first frame component insert 11 in order to create a fixed connection between the frame components 1, 2, which can, however, be removed again. The frame 3 with the support components 12, 22 and the tissue section 4 is then inserted 1030 into a holding frame 8 and the holding frame 8 as well as the frame 3 with the support components 12, 22 and the tissue section 4 are inserted 1031 into a tube 6, as shown in Figure 5.Subsequently, the tube 6 is closed 1032 with a lid 7 and placed 1033 in an incubator (not shown). Subsequently, the device according to the invention is fluidically contacted 1034 by, as shown in Figure 6, inserting the injection cannula 5 through the septum 71 in the lid 7 into the cannula connection 33 of the frame 3 and fluidically connecting it to a supply system via the Luer connection 51, and the withdrawal cannula 9 is also guided through the septum 71 in the lid 7 to the tip of the tube 6 and fluidically connecting it to a collection system via the Luer connection 91. Finally, a culture medium with a predeterminable composition of nutrients and a predeterminable oxygen concentration is fluidically supplied 1035 via the supply system and the injection cannula 5 over a predeterminable period of time.The culture medium which has escaped through the carrier components and has come into contact with the tissue section 4 is then removed via the withdrawal cannula 9 1036.

[0061] Figure 8 shows a flow diagram of a method for drug testing. Via the supply system and the injection cannula 5, a drug solution with a predeterminable dose and concentration is fluidically supplied 2000 to the device according to the invention and thus to the tissue section 4 over a predeterminable period of time. The supply of the culture medium can be maintained as described in step 1035 in Figure 7. The fluid returned in step 1036 is supplied to an analysis system and examined there 2001. Steps 2000 and 2001 can be repeated with different drug solutions. Finally, the fluid addition is stopped 2002, i.e., both the supply of the culture medium and the supply of drug solution.The tissue section 4 is finally removed by removing the holding frame 8 from the tube 6, pulling the frame 3 out of the holding frame 8 and separating the two frame components 1, 2 by loosening the connecting elements 214 2003.

[0062] Components 1, 2 themselves can be disassembled by detaching the frame-component insert 11 or 21 from the frame-component base body 10 or 20. The support component 12, 22 can then be removed and also examined.

Claims

Claims 1 . Device for cultivating a tissue section (4), comprising a frame (3) with a circumferential profile and a central free space formed by the circumferential profile, wherein the frame (3) comprises two frame components (1, 2), each with a circumferential component profile (100, 110, 200, 210) and a central component free space (101, 111, 201, 211) formed by the circumferential component profile, which frame components (1, 2) can be assembled to form the frame (3) in a manner that covers one another, and two carrier components (12, 22), wherein the carrier components (12, 22) are designed to receive a tissue section (4), characterized in that each frame component (1, 2) comprises a frame component base body (10, 20) and a frame component insert (11, 21) which can be plugged together to form the frame component (1, 2), wherein for each frame component (1, 2) the frame component insert (11, 21) and the frame component base body (10,20) are designed to hold the support component (12, 22) in such a way that the support component (12, 22) is clamped to the frame component spanning the free space, 2. Device according to claim 1, characterized in that a connecting element (214) is provided, by means of which the frame components (1, 2) can be assembled to form the frame (3), wherein the connecting element (214) is formed on one frame component insert (21) and can be connected to the other frame component insert (11).

3. Device according to claim 2, characterized in that the connecting element (214) is designed as a click connection, guide rail, clamp connection or as a closure.

4. Device according to one of the preceding claims, characterized in that the frame (3) and / or the two frame components (1, 2) have a guide (33) for the liquid supply.

5. Device according to claim 4, characterized in that the guide (33) for supplying liquid is formed in the assembled frame component base bodies (10, 20).

6. Device according to claim 4 or 5, characterized in that the guide (33) for supplying liquid within the frame profile (100, 200) has a widening (330) in an outer section, a constriction (331) in a middle section and forms a cavity (332) in an inner section.

7. Device according to one of the preceding claims, characterized in that the frame component base body (100, 200) has a trapezoidal bulge (102, 202) on one side, which widens towards the component free space (101, 201), that the carrier component (12, 22) is also trapezoidal on this side and that the trapezoidal side of the carrier component (12, 22) rests on the frame component base body (10, 20) on the side facing away from the frame component insert (11, 21).

8. Device according to claim 7, characterized in that the frame component base body (10, 20) has structures (124) for distributing the liquid on the surface of the trapezoidal bulge (102, 202), which structures rest on the carrier component (12, 22) or penetrate into it.

9. Cultivation system for cultivating tissue sections, characterized in that it comprises a device according to one of claims 1 to 8 and a receiving vessel, wherein the receiving vessel has a holding frame (8) which holds the device in the receiving vessel.

10. Cultivation system according to claim 9, characterized in that the receiving vessel is a ventilated tube (6).

11. Cultivation system according to one of claims 9 or 10, characterized in that the receiving vessel has a lid (7) through which an injection cannula (5) and / or a withdrawal cannula (9) can be guided.

12. A method for cultivating a tissue section (4) with a device according to one of claims 1 to 8 or a cultivation system according to one of claims 9 to 11, comprising the following steps: - Preparation (1000, 1010) of the frame components (1, 2) by placing a carrier component (12, 22) for each frame component (1, 2) on a frame component insert (11, 21), wherein the carrier component (12, 22) spans the component free space (111, 211) and extends into the profile (110, 210) of the frame component insert (11, 21), and the frame component insert (11, 21) is inserted into the frame component base body (10, 20) so that the carrier component (12, 22) is held; - placing (1022) a tissue section (4) on a support component (12, 22) in a frame component (1, 2); - placing (1023) the other frame component (1, 2), wherein the two frame component base bodies (10, 20) lie on one another and wherein the carrier component (12, 22) of the other frame component (1, 2) rests on the fabric section (4); - connecting (1024) the frame components to form a frame; - Supply (1035) of fluids into the free space (301) within the frame (3) surrounded by the support components (12, 22).

13. Method according to claim 12, characterized in that the frame components (1, 2) are connected by connecting a connecting element (214) formed on one frame component insert (21) to the frame component insert (11) of the other frame component (1).

14. Method according to claim 12 or 13, characterized in that the frame (3) is inserted into a holding frame (8) and the frame (3) and the holding frame (8) are inserted together into the receiving vessel be introduced.

15. The method according to claim 14, characterized in that the receiving vessel is closed with a lid (7), that for the supply (1036) of fluids an injection cannula (5) is guided through the lid (7) to the frame (3), and that for the removal (1036) of fluids a withdrawal cannula (9) is guided through the lid (7) into the receiving vessel.

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