Basic element of multichamber biochip, manufacturing of multichamber biochip, establishment of organ and disease model, and method for using multichamber biochip for substance test

Abstract

To provide a multichamber biochip that can be used for a plurality of experiment applications.SOLUTION: The present invention relates to basic element 1 of a multichamber biochip that has a lowermost part 3 with a frame 4 positioned above the part, and is open on a side facing the opposite direction to the lowermost part, and an internal space 6 is surrounded by a frame. Further, at least one first vertical part 5 is present, preferably the first vertical part and a second vertical part 7 are present. Culture chambers 14 whose boundaries are defined with each other by using a film formed by the vertical part and the frame contact by a channel, and work so as to generate a biological model system. The basic element is in further detail formed as a single part (monolithic). The present invention also provides a method for manufacturing a multichamber biochip set, and the multichamber biochip, and furthermore, a method for using the multichamber biochip.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to basic elements that can be used to fabricate multi-chamber biochips for a range of innovative applications. [Background technology]

[0002] In terms of this application, the biochip serves to simulate a biological system, such as an organ or tissue, for example in an experimental setup, in that biological structures and / or situation / environmental conditions are reproduced artificially but as close as possible to reality in the reaction chamber. For example, the biochip can be formed from several components arranged one above the other, cooperating to form at least one culture chamber, but more often two culture chambers. These culture chambers can be separated from each other by membranes of selected properties and are filled with one or several culture media. Optionally, various substances, such as aerosols, cells, microorganisms and / or spheroids as well as nutrients, active substances and synthetic materials can be introduced in a targeted manner into the culture medium in order to simulate a biological system or a particular situation / environment under controlled conditions.

[0003] One feasible means for designing such a biochip is known from a publication by Raasch, M., et al. (Raasch, M., et al. "An integrative microfluidically supported in vitro model of an endothelial barrier combined with cortical spheroids simulates effects of neuroinflammation in neocortex development", 2016, Biomicrofluidics 10; doi 10.1063 / 1.4955184). The two basic elements each forming a preliminary culture chamber are each combined with one separation membrane, then linked together by adhesive bonding, then sealed with a closing film and partially sealed with an adhesive or connecting substance. The filling of the three culture chambers arranged one above the other and separated by two separation membranes is carried out via channels similarly formed in the two basic elements. In this way, at least two culture chambers can be created in a small space and can be operated and investigated under laboratory conditions, i.e., while monitoring, for example, the biological structures and composition used, the temperature and, optionally, the flow of the culture medium. The use of such a general biochip in a cell culture incubator further allows, for example, operation in ambient atmosphere at controlled temperature and composition.

[0004] Detection of processes inside the culture chamber can be performed, for example, using optical analysis (e.g., using transmitted light microscopy, live cell imaging) through a transparent window of the biochip. Furthermore, substances can be introduced through the separation membrane via the channel to estimate the barrier / seal / permeability of the biological structure or to evaluate transport processes on the biological structure and / or to analyze the reaction of proteins on and / or inside the biological structure. Furthermore, the biological structure and introduced substances such as culture medium (potentially rich in the aforementioned substances) can be removed for optical analysis, molecular biological analysis, and chemical analysis.

[0005] However, a biochip as described above must be constructed in layers from several different elements, each layer must be tightly connected to the corresponding neighboring elements, and in addition the requirements of the desired dimensions of the culture chambers and the flow and physiological conditions that are determined as a result must be ensured. This means in particular that the basic elements must be manufactured with high accuracy and dimensional precision, resulting in high production costs.

[0006] In our experiments we have further found that gluing the base element introduces stresses which cause deformation of the biochip, and that the adhesion effect makes the biochip leaky and subject to degradation processes which cause it to lose its functionality. In addition, it is disadvantageous that a separation membrane must be introduced before bonding the base element, which is associated with the risk of contamination of the membrane by the adhesive, for example. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Raasch, M., et al. "An integrative microfluidically supported in vitro model of an endothelial barrier combined with cortical spheroids simulates effects of neuroinflammation in neocortex development," 2016, Biomicrofluidics 10; doi 10.1063 / 1.4955184 [Non-Patent Document 2] Auner et al. “Chemical PDMS binding kinetics and implications for bioavailability in microfluidic devices,” 2019, Lab Chip 19:864-874 Summary of the Invention [Problem to be solved by the invention]

[0008] The aim of the present invention is to reduce or overcome the disadvantages known from the prior art and to propose an option that makes it possible to provide a multi-chamber biochip that can be used for several experimental applications. [Means for solving the problem]

[0009] This object is achieved not only by the independent claims but also by the subject matter of the dependent claims. Advantageous developments are the subject matter of the dependent claims.

[0010] The object of the invention is achieved with a basic element of a multi-chamber biochip, which has a bottom part with at least one frame located thereon and is open at least on the side facing away from the bottom part. In this case, the frame surrounds an internal space. The frame can be designed as a stepped section surrounding the internal space. In an advantageous option, since it is easier to manufacture, the frame can also be made with a flat, in particular rectangular, casting of the basic element, which has a correspondingly large thickness, in particular corresponding to the height of the internal space. Several frames that delimit several internal spaces can be placed on top of the bottom part. Alternatively, in a variant of the flat casting of the basic element, several internal spaces can also be surrounded by the flat casting.

[0011] Furthermore, within each internal space, at least one first vertical part is formed, fixed to the bottom and extending around the first surface, the height of which is less than the height of the frame. The first vertical part can in this case be designed to be freestanding or as a stepped part in the frame. The vertical part has a first side facing the internal space and a first support surface facing the bottom. The first support surface is designed to support a first separation membrane. The lower pre-incubation chamber (first chamber) is surrounded by the first side of the first vertical part and the surface surrounded by the first vertical part. The remaining internal space between the first support surface and the upper side of the frame forms the upper pre-incubation chamber (second chamber). The surface surrounded by the first vertical part can be polygonal, preferably quadrangular, in particular rectangular, but can also be circular. Advantageously, the support surface of the vertical part extends around the surrounded area with a uniform height relative to the bottom. The lower pre-incubation chamber and the upper pre-incubation chamber are each connected to the surrounding environment by at least one separate channel that is open on the outside of the base element.

[0012] In another embodiment of the basic element according to the invention, the lower pre-culture chamber and / or the upper pre-culture chamber are connected to the surrounding environment by at least two separation channels, which advantageously allow the supply / discharge of culture medium into / out of the culture channels, in the assembled state of the multi-chamber biochip.

[0013] For example, to be able to culture spheroids of approximately 1 mm size, at least one culture chamber and associated channels are designed with structures having a defined height or diameter greater than 1 mm, for example 1.1 mm or 1.2 mm.

[0014] The basic element according to the invention is formed from a single workpiece (monolithically) and preferably has the format of a microscope slide (76 mm x 26 mm ± 3 mm). The monolithic design allows easier handling than the solutions according to the prior art, since it is not necessary to connect two or more basic elements to each other, whose passages (channels, internal spaces, culture chambers, etc.) must be aligned and glued before connection. In addition, the basic element is more stable in the monolithic configuration, retains its initial shape (no stress-related bending of the body) and does not leak at the adhesion points, and is therefore more practical to handle and use. In particular, higher flow rates of supplied medium (perfusion rate) can be achieved without leakage. This is advantageous in particular when establishing organ models, since in such a case a perfusion rate matching the in vivo perfusion rate must be maintained for the longest possible period. The resulting omission of adhesion points also has the advantageous consequence of omitting an adhesive, which is absolutely necessary in the prior art and which may interact adversely with biological structures. In addition, the requirements for the dimensional accuracy of the basic elements according to the invention may be lower than those of a biochip consisting of several elements.

[0015] In a further advantageous embodiment of the basic element, a second vertical part is formed which is located at the bottom within the interior space and extends around the second surface. The height of the second vertical part is less than the height of the frame but greater than the height of the first vertical part. The second vertical part can also be designed to be freestanding or as a stepped part in the frame, with a support surface located opposite the bottom, hereafter referred to as the second support surface. The second support surface is designed to support a second separation membrane. Between the first and second support surfaces, the second vertical part forms a second side surface which faces towards the interior space. The second surface and the second side surface surrounded by the second vertical part define an intermediate preliminary incubation chamber (third chamber). The surface surrounded by the second vertical part can be polygonal, preferably quadrangular, in particular rectangular, but can also be circular. Preferably, the geometry of the surface surrounding the second vertical part corresponds to the geometry of the surface surrounded by the first vertical part. The intermediate pre-incubation chamber is also connected to the outside of the base element by at least one channel.

[0016] As will be explained further below, multi-chamber biochips having three or more culture chambers arranged one above the other can advantageously be manufactured using such embodiments of the basic element.

[0017] Advantageously, the material from which the basic element is manufactured comprises an injection molded biocompatible plastic. Examples of such plastics are polyurethane (PU), polyimide, styrene (SEBS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyethylene terephthalate (PET) and polyesters such as cyclic polyolefins (COP and COC). In a corresponding approach to material selection, the basic element is manufactured as a single part in an injection molding or casting process using a corresponding injection molding or casting tool.

[0018] Biochips are also often produced from polydimethylsiloxane (PDMS) in conventional techniques. However, in contrast to the aforementioned plastics, the use of chips with PDMS for complex cell, organ, and disease models is complicated, since PDMS must be activated to be suitable for biological structures, such as cell culture. In order to affect the biological structures and cultured systems to be investigated with as few biochips as possible, the biochip material should ideally be inert. However, the frequently used material PDMS is known, for example, to have a high bonding ability with some compounds (Auner et al. "Chemical PDMS binding kinetics and implications for bioavailability in microfluidic devices", 2019, Lab Chip 19:864-874). This bonding ability may have difficult-to-estimate effects on experiments (e.g., substance testing) performed with biochips made from PDMS. For example, substances with a logP greater than 1.8 and a small hydrogen donor count are strongly absorbed on PDMS, which makes active substance testing and the interpretation of the resulting data much more difficult. For example, the substance propiconazole has a logP of 3.72 and a hydrogen donor count of 0. Propiconazole is very hydrophobic and binds irreversibly to PDMS, so that it can only be detected in the medium after 24 hours in the PDMS chip at less than 30% of the starting concentration (Auner et al., 2019). This problem concerns, although not exclusively, the classical active substance group of small molecules. Many pharmaceutical active substances belong to this group of drugs, and as a result, biochips using PDMS are not suitable as test systems. In addition, one-piece building blocks for multi-chamber biochips made from only a single workpiece cannot be manufactured with PDMS, since with this substance the necessary separation membrane can only be fixed between the two individual components.

[0019] Thus, in an advantageous embodiment of the base element according to the invention, the base element is manufactured from polybutylene terephthalate (PBT).

[0020] PBT is a polymer that is usually used to manufacture products that are subjected to high mechanical loads and / or come into repeated contact with hot culture media. Typical uses of PBT are, for example, plain bearings, valve parts, screws, parts for household appliances such as coffee machines or hair dryers, connectors for pulse oximeters, tips for electrosurgical instruments, and parts for medical devices such as clips for respiratory masks. The production of suitable PBT-initiated polymers can even be achieved in a GMP-compliant manner. PBT is very suitable for injection molding due to its favorable cooling and processing behavior.

[0021] This material, which is unusual for use in cell culture chambers, shows a very low bond-forming capacity in tests compared to the set of components of the culture medium used, so that the influence of the material of the multi-chamber biochip, in particular the (pre)culture chambers, on the tests carried out in such cell culture chambers can be advantageously reduced. For example, in tests on active substance absorption with propiconazole and troglitazone, the inventors found that PBT is very suitable for active substance testing of substances with a logP of up to 3.72 (logP for propiconazole: 3.72; logP for troglitazone: 3.60). After a 24-hour culture period, at least 80% of the starting concentration of propiconazole or troglitazone is detectable in the culture medium.

[0022] In other embodiments, the basic element according to the invention can be designed such that a portion of the channel is formed in the side facing away from the bottom frame and / or in the side facing the bottom of the basic element. Preferably, the channel portion in one or more sides can be formed completely or partially as a spare channel portion due to the formation of circumferential channel boundary verticals. These channel boundary verticals are created in the basic element, adjacent to the basic element or projecting outward from the basic element. The channel boundary verticals have an inward facing side that bounds the channel space and thus acts as a channel wall. The support surface provided for supporting the (closure) membrane or the channel cover extends on the upper side (end face) of the channel boundary vertical.

[0023] The channels are connected to connectors (for example standard Luer type) for supplying or removing the medium, respectively, these connectors being advantageously formed opposite each other at the bottom.

[0024] For example, a window may be present at the bottom to allow visual detection of a process in at least one of the culture chambers.

[0025] In order to obtain a multi-chamber biochip according to the invention, basic elements according to the invention are provided, which serve as the basis for the multi-chamber biochip and advantageously allow both an efficient production and a flexible adaptation to the respective requirements.

[0026] In the case of a fully assembled and ready-to-use multi-chamber biochip, a first separation membrane is placed on and connected to a first support surface of the first vertical section. A lower culture chamber is provided by the first vertical section and the first separation membrane. Depending on the embodiment of the base element, a second separation membrane is optionally placed on and connected to a second support surface of the second vertical section. In this case, an intermediate culture chamber is provided between the first and second separation membranes. A closing membrane is placed on and connected to the frame. This closing membrane delimits an upper culture chamber, which is provided between the first and closing membranes or between the second and closing membranes depending on the design of the base element.

[0027] Optionally, an additional closure membrane is provided that is located on and connected to the bottom, side facing away from the frame.

[0028] The separation membrane used is preferably a film that can be fixedly integrated to a prespecified extent and can be permeable but also impermeable (semi-permeable) to gases, fluids, particles and / or more complex molecules depending on the material, thickness and manufacture of the separation membrane. Preferred but non-limiting materials for the separation membrane are polyethylene terephthalate or polycarbonate. The membrane preferably has pores with a size between 0.4 μm and 8 μm and has a thickness between 5 μm and 50 μm, preferably between 10 μm and 20 μm, particularly preferably 12 μm. The separation membrane can be at least semi-transparent, preferably transparent, for at least one selected wavelength range to allow improved optical detection of processes in at least one of the culture chambers. The closing membrane (sometimes also called a bonding film) can also be integrated in a preferably flexible manner and can be selected accordingly to block or be (semi)permeable to certain substance classes. The closing membrane can be transparent for at least one selected wavelength range to allow optical detection of processes in at least one of the culture chambers. In some embodiments, the closure membrane can be designed as a transparent closure film, for example as a polycarbonate film or a polyethylene terephthalate film. Glass or polystyrene or COC / COP are also conceivable materials from which the closure membrane can be made. The closure membrane (or closure film) can also function as a channel cover, since it optionally extends beyond the existing channel boundary vertical and rests on the supporting surface of the existing channel boundary vertical and is connected to the supporting surface in an air-tight and liquid-tight manner.

[0029] In certain embodiments of the multi-chamber biochip, at least one of the separation membranes may furthermore comprise depressions, also called (micro)cavities, in which cells, cell complexes, spheroids and / or organoids can be colonized and / or cultured.

[0030] In this case, an example of an application is the improvement / expansion of spheroid and organoid culture over periods of up to 4 days compared to static cell culture.

[0031] In the design of a multi-chamber biochip with microcavities, the spheroids and organoids can be cultured in an immobilized manner under (microfluidic) cell culture conditions with a flow-through fraction usually composed of proteins of the extracellular matrix in individual or mixed form, without further embedding in a (hydro)gel. The diameter of the microcavities can be between 500 μm and 1,500 μm, preferably 800 μm. Gel-free culture allows good optical analysis during the culture period. In addition, gel-free culture allows a gentler and non-destructive recovery of the intact cellular organization, in particular the spheroids and organoids from the biochip for other analyses, such as tissue sections, fluorescent immunostaining, flow cytometry, ELISA-based assays, or tissue lysis for DNA / RNA and Western blot analysis.

[0032] The gel-free immobilized culture of spheroids and organoids further allows easier co-culture with vascular tissue arrays (vascularization) on different isolation membranes (indirect vascularization) or on the same (micro)cavity or flat isolation membranes (direct vascularization). In addition, the immobilized vascularized spheroids and organoids with immune cells can be washed directly in the medium. The gel-free culture of spheroids and organoids makes it much easier for immune cells to migrate into the spheroids and organoids.

[0033] Cultivation under cell culture conditions without flow-through and / or gel allows a better maintenance of different biological structures than under comparable static cell culture conditions, in particular for spheroids, due in particular to improved nutrient and oxygen conditions, which allows the maintenance of the functionality of such biological structures for longer times for testing purposes.

[0034] In order to allow the user to have a wide range of usability options, a set for a multi-chamber biochip according to the present invention can be provided, comprising the basic elements as described above. Furthermore, at least one first separation membrane is present in such a set, which is placed on the first vertical part and serves to close the lower culture chamber. Optionally, at least one second separation membrane is included in the set, which is placed on the second vertical part and serves to close the middle culture chamber, if the basic element included in the set has a second vertical part. In addition, a closing membrane is included in the set, which is placed on the frame and serves to close the remaining internal space as the upper culture chamber.

[0035] In addition, it may include an additional closure membrane that is intended to be placed over the bottom, side facing away from the frame.

[0036] A set according to the invention can, for example, be provided directly to a user, or can be given to a service provider who, for example, performs the assembly of the components of the set on behalf of the user and according to the user's specifications.

[0037] Advantageously, providing such a set also allows the introduction of biological material, such as larger organs or cell masses or tissue pieces or multicellular organisms, such as parasites, which due to their size cannot be flushed into the chamber by the existing channels, such that such material can be applied in a sterile environment directly into the still open pre-culture chamber, which is subsequently closed with a separating or closing membrane, for example using an adhesive method.

[0038] The multi-chamber biochip is manufactured by providing a base element and placing a separation membrane on the first vertical section. The multi-chamber biochip is connected to the first vertical section to form a seal. If necessary, biological material can be introduced into the lower pre-culture chamber before applying the first separation membrane, as further described above.

[0039] In view of this description, a sealed connection is understood to mean, in particular, that a liquid-tight and gas-tight flat or straight connection is made, so that the membrane-bounded culture chamber or the membrane-bounded channel reliably withstands the flow of culture medium under certain static or dynamic operating pressures.

[0040] The connection is advantageously, but not exclusively, made by a guided beam of high energy radiation that is directed and controlled along the produced interlocking seam or connecting surface, the high energy radiation being in particular laser radiation of a wavelength and intensity matched to the materials to be joined.

[0041] Once the first separation membrane is attached to the first vertical section, a second separation membrane is optionally placed on top of the second vertical section and connected to the second vertical section to form a seal. Optionally, biological material can also be introduced into the intermediate pre-culture chamber before applying the second separation membrane. Correspondingly, a closure membrane is placed on the frame and the closure membrane and frame are connected to form a seal. Optionally, an additional closure membrane is placed on the bottom side facing away from the frame and connected to the side to form a seal.

[0042] At least one channel contacts each pre-incubation chamber, and each of the resulting culture chambers, so that culture medium can be supplied and / or discharged into each of the culture chambers of the multi-chamber biochip.

[0043] The above-mentioned specific embodiments of the multi-chamber biochip according to the invention advantageously allow a considerable number of possible uses of such a multi-chamber biochip. All of the intended uses comprise at least the following steps: First, a multi-chamber biochip according to the invention is selected and provided. The selection can be made, for example, with respect to the number of existing culture chambers and / or with respect to the choice and / or combination of one or more separation membranes. During use, the multi-chamber biochip can then be sterilized to avoid undesired interactions and contaminations. The assembly of sterile components under conditions of increased purity is also equivalent to sterilization. Then, for example, culture medium, cells, microorganisms, spheroids and / or organoids and / or cell clusters and / or tissue fragments or multicellular organs can be introduced into the existing culture chambers.

[0044] In a specific embodiment of the method of use of the multi-chamber biochip, or in one of the provided configurations of that embodiment, a hydrogel, preferably composed of components of the extracellular matrix such as collagen, fibronectin, laminin, etc., can be introduced into at least one of the culture chambers.

[0045] The multi-chamber biochip according to the invention can be used to generate and / or culture spheroids and / or organoids in at least one of the culture chambers of the multi-chamber biochip, for example by colonizing the spheroids and / or organoids in one (micro)cavity of the membrane.

[0046] The multi-chamber biochip according to the present invention can also be used to test existing cells, cell cultures, organoids or spheroids with various substances, active substances, nanomaterials, microorganisms, mediators, antibodies and the like.

[0047] Due to the structure of the invention based on the basic elements according to the invention, the invention is easy for the user to use. The assembly of the multi-chamber biochip is considerably simplified and significantly less error-prone compared to the prior art. Furthermore, due to the integral design of the basic elements, it allows efficient production and universal combination with a wide range of separation and / or closure membranes. For example, by using a laser joining method to connect the membranes and form a seal, it is possible to dispense with glue or other adhesives.

[0048] The multi-chamber biochip according to the invention can be used, for example, for active substance testing, or for the establishment and characterization of organ or organoid models as well as disease and infection models, depending on the specific embodiment. For active substance testing, it is conceivable, for example, to investigate the immune response of cultured cells to the administration of a substance. In this case, there are various possibilities, for example, the substance can be placed in the chamber in which the cells to be tested grow. The effect of the substance on the cells can then be determined, for example, by microscopic observation of the cells or by investigating the cell culture medium for, for example, messenger substances, markers, etc., released by the cells into the culture medium. Alternatively, a substance can also be added to the chamber facing the cells to be tested, for example, in order to investigate the effect of the cells growing in this chamber, placed opposite the cells to be tested, on the effect of the substance in the cells to be tested of the other chambers, which may, for example, weaken or strengthen the effect of the substance (gradient formation). Furthermore, the spheroids and organoids with and without immune cell populations can be washed / perfused. The immune cells can be washed / perfused into the chamber with the spheroids or organoids, or can be preferably flushed / perfused via the vasculature in one of the adjacent chambers. In addition, immune cells can be permanently integrated into the vascular structures and / or spheroids / organoids. In addition, the spheroids or organoids can be vascularized by introducing vascular cells, such as endothelial cells alone or in combination with pericytes and smooth muscle cells, without limitation. For example, to stimulate cells traversed by blood vessels, the upper and lower walls of the middle culture chamber can be lined with endothelial cells alone or in combination with pericytes and smooth muscle cells introduced through the inlet channel, while organ-specific epithelial cells are introduced into the lower and upper culture chambers. However, in other embodiments, endothelial cells can also be introduced on the upper or lower walls of the upper or lower chambers, alone or in combination with pericytes and smooth muscle cells, through a given inlet channel, and in the middle chamber, the endothelial cells can be integrated in the form of a layer of stratified cells or in the form of spheroids and organoids.

[0049] The invention is explained in more detail below with reference to exemplary embodiments and figures. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 is a schematic perspective view of an exemplary embodiment of a basic element according to the invention; [Diagram 2] 2 is a schematic diagram of an exemplary embodiment of a channel in the bottom part of a base element according to the present invention; [Diagram 3] FIG. 1 is a schematic diagram of a representative embodiment of a set according to the invention for providing a multi-chamber biochip (exploded view). [Figure 4] 1 shows a cross-section through a multi-chamber biochip according to the present invention with three culture chambers and a schematic diagram of an apparatus for operating the multi-chamber biochip. [Diagram 5] 1 illustrates a possible use of a multi-chamber biochip according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] In the following, the invention is described by a representative embodiment in which two internal spaces 6 are each bounded by a rectangular flat casting of a frame 4 on the bottom 3 of the basic element 1. As will be explained in more detail below, the internal spaces 6 are divided in a stepped manner in such a way that they each form a (pre) multi-chamber cavity 2.1. As a result, a multi-chamber biochip 2 can be provided by the basic elements of Fig. 1, Fig. 2 and Fig. 3, or a multi-chamber biochip 2 with two multi-chamber cavities 2.1 is provided (both multi-chamber cavities 2.1 functionally forming a complete multi-chamber biochip). To improve the clarity of the figures, both existing multi-chamber cavities 2.1 are used in Fig. 1, Fig. 2 and Fig. 3 to show elements of a single multi-chamber cavity 2.1 or elements of a multi-chamber biochip 2 with only one multi-chamber cavity 2.1. In this case, the description only concerns one multi-chamber cavity 2.1.

[0052] The basic element 1 according to the invention is formed in a single piece from a biocompatible material, and in particular from a biocompatible injection-molded plastic (Figure 1). Starting from the bottom part 3 (see also Figure 2) a frame 4 is formed which is open on the side facing away from the bottom part. The frame 4 is flat and encloses an interior space 6.

[0053] Inside the internal space 6, a first vertical section 5 extending around the periphery of the first surface is made in the form of a stepped section present in the material of the frame 4. The height of the first vertical section 5 is lower than the height of the frame 4. The vertical section 5 has a first side 5.2 facing towards the internal space 6 and a first support surface 5.1 facing the bottom part 3. The first support surface 5.1 is designed to support a first separation membrane 11 (see Fig. 3, Fig. 4). The lower preliminary incubation chamber 8 is delimited by the first side 5.2 and by the surface enclosed by the first vertical section 5. In the representative embodiment of Figs. 1 to 3, the surface enclosed by the first vertical section 5 is rectangular.

[0054] In addition, a second vertical part 7 is provided, which is placed inside the internal space 6 on the bottom part 3 and surrounds a second surface, which is also designed as a stepped part made from the material of the basic element 1 (FIG. 1). The second surface surrounded by the second vertical part 7 is larger than the first surface surrounded by the first vertical part 5 and is also rectangular. The height of the second vertical part 7 is lower than the height of the frame 4 but higher than the height of the first vertical part 5. A second support surface 7.1 is formed on the second vertical part 7. The second support surface 7.1 is designed to support a second separation membrane 12 (see FIG. 3). In the part between the first support surface 5.1 of the first vertical part 5 and the second support surface 7.1 of the second vertical part 7, the second vertical part 7 forms a second side surface 7.2 facing towards the internal space 6. The intermediate pre-incubation chamber 9 is bounded by a second surface surrounded by the second vertical portion 7 and by a second side surface 7.2. The remaining internal space 6 between the second support surface 7.1 and the upper side 1.1 of the basic element 1 forms an upper pre-incubation chamber 10. The upper side 1.1 is formed by the side of the basic element 1 facing the lowermost part 3.

[0055] In order to be able to supply the culture chambers 8, 9 and 10, which are obtained from the preliminary culture chambers 8, 9, 10 in the assembled state of the multi-chamber biochip 2, with a culture medium 18 (see FIG. 4), two channels 14 are formed between the culture chambers 8, 9 and 10 and the upper side 1.1 of the basic element 1. In FIG. 1, the channels 14 can be seen as round passages 10.1, 16.1 and as a passage 9.1 with a rectangular cross section. The channels 14 lead from the culture chambers 8, 9 and 10 in the direction of the bottom part 3, where the distribution takes place to the connector 16 (see FIGS. 1, 3, 4).

[0056] In the illustrated exemplary embodiment, the connectors 16 are located on the upper side 1.1 of the base element 1. In the exemplary embodiment, each of the connectors 16 is configured to supply and discharge the culture medium 18 through each of the culture chambers 8, 9, 10. Thus, each of the culture chambers 8, 9, 10 of the multi-chamber biochip 2 that is ready for operation allows the culture medium 18 to pass therethrough independently of the other culture chambers 8, 9, 10. In particular, each culture medium 18 can be individually selected for the corresponding culture chamber 8, 9, 10 and applied to the corresponding culture chamber 8, 9, 10 by an individual, controllable volumetric flow.

[0057] To allow optical detection of processes at least in the lower culture chamber 8 during operation of the multi-chamber biochip 2, a window 17 is formed in the bottom part 3 (FIG. 4). The surface of the window 17 coincides with the surface enclosed by the first vertical part 5.

[0058] A representative course of the channels 14 and their connection to the corresponding connectors 16 is shown in FIG. 2 with a side view of the bottom part 3 pointing outwards. To supply and discharge the medium 18 through the lower culture chamber 8, there are two lower channels 14.2 which lead into the lower culture chamber 8 through two supply openings 8.1 arranged diagonally opposite each other. To supply the intermediate culture chamber 9 with the other medium 18, two intermediate channels 14.3 are provided which are formed in the part like opposed rectangular supply passages 9.1 arranged between the first vertical part 5 and the second vertical part 7 (see also FIG. 1). The upper culture chamber 10 is supplied with the other medium 18 by the two upper channels 14.4 in that each of the two upper channels 14.4 contacts the upper culture chamber 10 through the outermost round supply passages 10.1.

[0059] The connector passage 16.1 shown in each case with a round cross section creates in this case the respective connection leading to the connector 16.

[0060] In the exemplary embodiment of FIG. 2, the portion 14.1 of the channel 14 between the supply opening 8.1 and the supply passages 9.1, 10.1 on the one hand is present as an associated connector passage 16.1 shaped as a reserve channel portion 14.1 on the other hand. The reserve channel portion 14.1 is formed by a recess incorporated in the basic element 1. In the exemplary embodiment, the recess is arranged in the form of a groove with a rectangular cross section. Naturally, other types of recesses with other cross sections are also possible, such as a semicircular cross section. The recess has an internal channel wall 14.5 surrounding the connector passage 16.1 and the supply passages 9.1, 10.1, respectively, or in the case of the lower culture chamber 8, a window 17 with two associated connector passages 16.1 and two diagonally opposite supply passages 8.1. The channel wall 14.5 is flush with the side surface and points in the opposite direction to the frame 4 of the bottom part 3. By applying the channel cover, the preliminary channel parts 14.1 can be closed, so that the functional state of these channel parts 14.1 can be created. In the exemplary embodiment of Fig. 3 and Fig. 4, the lower closing membrane 15 is applied to the side of the bottom part 3 pointing away from the frame 4 and is connected in a liquid-tight manner to the bottom part 3 along the channel wall 14.5 in the manner of a seam. The connection is preferably made by means of laser welding along the connection seam created, but can also be achieved, for example, by gluing or solvent welding. Due to the liquid-tight connection of the closing membrane 15, the preliminary channel parts 14.1 achieve their functional state.

[0061] In FIG. 3 a set for a multi-chamber biochip 2 is shown by way of example, the illustration can also be considered as an exploded view of the components of a representative embodiment of the multi-chamber biochip 2. A basic element 1 according to the invention is present as a central element. A first flexible separation membrane 11 is provided for placing on the first support surface 5.1 of the first vertical part 5. The first separation membrane 11 closes the lower culture chamber 8 when it is attached there. The first separation membrane 11 comprises, for example, microcavities 19 in which, for example, spheroids or organoids can be introduced and / or cultured therein (see FIG. 5). A second flexible separation membrane 12 serves for placing on the second support surface 7.1 of the second vertical part 7 and closes the intermediate culture chamber 9. A transparent closing membrane 13 is also provided which can be placed on the frame 4, which membrane, in the assembled state, serves to close the remaining internal space 6 between the second separation membrane 12 and the closing membrane 13 as the upper culture chamber 10.

[0062] Furthermore, in order to close not only the channel portion 14.1 formed in the bottom portion 3 but also the window 17, there is an additional transparent closing film 15 applied on top of the bottom portion 3 which seals the corresponding channel portion 14.1 and window 17, as already explained above.

[0063] A multi-chamber biochip 2 according to the invention is shown in a side cross-sectional view in Figure 4, with a lower culture chamber 8, a middle culture chamber 9 and an upper culture chamber 10. The drawing plane of Figure 4 corresponds to the line aa in Figure 3, and for clarity only the left half of the multi-chamber biochip of Figure 3 is shown in Figure 4.

[0064] The monolithic structure of the basic element 1 of the multi-chamber biochip 2 can be clearly seen in FIG. 4. Starting from the bottom part 3, a flat frame 4 is formed, which is open on the side facing away from the bottom part 3. Inside the frame 4, a first vertical part 5, which extends around the first surface, is made in the form of a stepped part present in the material of the frame 4. The height of the first vertical part 5 is lower than the height of the frame 4. The first vertical part 5 has a first side 5.2 and a first support surface 5.1 facing the bottom part 3. In the area of ​​the surface surrounding the first vertical part 5, the bottom part has a window 17. Furthermore, there is a second vertical part 7, which is placed on the bottom part 3 and surrounds the second surface, and is similarly designed as a stepped part made from the material of the basic element 1. The height of the second vertical part 7 is lower than the height of the frame 4, but higher than the height of the first vertical part 5. A second support surface 7.1 is formed on the second vertical part 7. Between the first support surface 5.1 of the first vertical portion 5 and the second support surface 7.1 of the second vertical portion 7, the second vertical portion 7 forms a second side surface 7.2 facing towards the interior space 6.

[0065] A first separation membrane 11 is applied to the first support surface 5.1 and a second separation membrane 12 is applied to the second support surface 7.1, in each case in a liquid-tight manner. The first separation membrane 11 has microcavities 19. On the side of the bottom part 3 facing away from the frame 4, a lower closing membrane 15 is applied, in each case in a liquid-tight manner, and an upper closing membrane 13 is applied to the upper side of the frame. The lower culture chamber 8 is delimited by the first side 5.2, the first separation membrane 11 and the lower closing membrane 15. The middle culture chamber 9 is delimited by the second side 7.2, the first separation membrane 11 and the second separation membrane 12, and the upper culture chamber 10 is delimited by the remaining frame 4, the second separation membrane 12 and the upper closing membrane 13.

[0066] The function of membranes 11, 12, 13 and 15 is clearly visible and is to delimit culture chambers 8, 9 and 10 from each other and from the surrounding environment, and to provide desired options for the exchange of molecules and / or cells between culture chambers 8, 9 and 10.

[0067] In the example of Figures 3 and 4, the multi-chamber biochip 2 has three chambers. The lower chamber 8 is approximately 42 mm 2 60 mm including the usable base area of ​​10 mm, the height of 0.5 mm, and the volume of the inlet and outlet channels 14. 3 In the region of the feed opening 8.1, the inlet and outlet channels 14 have a diameter of approximately 0.5 mm. The intermediate chamber 9 has a volume of 160 mm. 2 The usable base area of ​​the channel 14 is just 200 mm, including the height of 1.1 mm and the volume of the inlet and outlet channels 14. 3 The inlet and outlet channels 14 are rectangular in the area of ​​the feed passage 9.1 and have a width of 2 mm. The upper chamber 10 has a volume of 216 mm. 2 The usable base area of ​​the 150 mm 2 tube is approximately 150 mm 2 , which includes a height of 0.7 mm and a volume of the inlet and outlet channels 14 . 3 The inlet and outlet channels 14 have a diameter of 0.8 mm in the area of ​​the supply passage 10.1. All single dimensional specifications mentioned are subject to variation, for example ±0.5 mm, resulting in changes in surface and volume sizes.

[0068] The various culture media 18 (indicated by arrows) can flow along the associated channels 14 into and out of the corresponding culture chambers 8, 9, 10, the supply of the culture media 18 into the culture chambers 8, 9, 10 being possible independently of one another (FIG. 4). The connection principle is illustrated in FIG. 4 using the example of a lower culture chamber 8, in which in each case the associated connector 16 with the supply lines 23 and the discharge lines 24 are arranged leads into the lower culture chamber 8 via a supply opening 8.1 and is connected to a lower channel 14.2 which supplies the lower chamber 8 with the culture media 18. The lower channel 14.2 extends partly offset relative to the drawing plane and is therefore partly drawn with dashed lines.

[0069] In a similar manner, the middle culture chamber 9 can be supplied with the medium 18 via the middle channel 14.3 and the upper culture chamber 10 can be supplied with the medium 18 via the upper channel 14.4. The multi-chamber biochip 2 can be operated with an instrument, such as a reading instrument or a microscope, having a lens 21 facing the window 17 and capable of monitoring and optionally detecting, storing and evaluating the process of the multi-chamber biochip 2. For this purpose, a light source 22 may also be present to illuminate the multi-chamber biochip 2 in a desired manner. In addition, a pump 25 can be arranged connected to a supply line 23 and an outlet line 24, which in turn are attached to corresponding connectors 16. The pump 25 and optionally the light source 22 can be controlled with the controller 20, for example, so that the perfusion of the culture chambers 8, 9, 10 can be performed in a controlled manner and can be optically monitored. The controller 20, implemented for example by a computer, can also optionally store and / or evaluate the optically detected data in addition to generating the control commands. For example, the controller 20 can be used to control pump flow rates for the individual culture chambers 8, 9, 10 as a function of the optically detected data.

[0070] The present invention advantageously allows the construction of complex biological models, for example microfluidic cultures of spheroids 26 and / or organoids 26 with integrated vascular and immune cell circulation.

[0071] As a result, a model for studying pancreatic cancer (PDAC, pancreatic ductal adenocarcinoma) can be created (Figure 5). For this purpose, a lower culture chamber 8 is provided with a well-defined height of 0.5 mm between the lower closure membrane 15 and the first separation membrane 11. The first separation membrane 11 is porous and comprises microcavities 19 on the side of the first separation membrane 11 that faces towards the intermediate culture chamber 9, in which cavities the spheroids 26 can be colonized and cultured. The intermediate culture chamber 9 has a well-defined height of 1.1 mm, so that the spheroids 26 can be introduced in a non-destructive manner up to a size of approximately 1 mm. The channels 14 are also made to specific dimensions so that they are correspondingly large.

[0072] In the exemplary embodiment of Fig. 5, the second separation membrane 12 is designed as a porous PET film and covers the intermediate culture chamber 9. A cell layer 27 composed of microvascular pancreatic endothelial cells 28, accompanied by macrophages 29, is formed in the upper culture chamber 10. To improve clarity, the cell layer 27 is shown at a distance from the second separation membrane 12. In reality, the cell layer 27 attaches and grows on top of the second separation membrane 12. Through the second separation membrane 12, the perfused monocytes 30 and T cells 31 can pass, for example, into the intermediate culture chamber 9.

[0073] The upper culture chamber 10 has a clear height between the second separating membrane 12 and the closing membrane 13 of 0.7 mm. [Explanation of symbols]

[0074] 1 Basic elements 1.1 Upper side 2. Multi-chamber biochip 2.1 Multi-chamber cavity 3. Bottom 4 Frames 5 First vertical section 5.1 First Support Surface (First End Face) 5.2 First aspect 6. Interior Space 7 Second vertical section 7.1 Second Support Surface (First End Face) 7.2 The second aspect 8 Lower (spare) culture chamber 8.1 Supply opening 9 Intermediate (spare) incubation chamber 9.1 Supply passage 10 Upper (spare) culture chamber 10.1 Supply passage 11 First separation membrane 12 Second separation membrane 13 Upper closing film (or upper joining film) 14 Channels 14.1 Spare Channel Part 14.2 Bottom Channel 14.3 Middle Channel 14.4 Top Channel 14.5 Channel Walls 15 Additional (lower) closure film (or lower bonding film) 16 Connectors 16.1 Connector Passage 17. Windows 18 Culture solution 19 Microcavity 20 Controller 21 Lens 22 Light source 23 Entrance 24 Release 25 Pump 26 Spheroid / organoid 27 cell layers 28 (Pancreatic) Endothelial cells 29 Macrophages 30 Monocytes 31 T cells

Claims

1. The basic element (1) of a multi-chamber biochip (2) formed (monolithically) from a single processed product, - The lowest part (3), accompanied by a frame (4) located above the lowest part (3) and open on the side facing the opposite direction from the lowest part (3), and at least one internal space (6) is bounded by the lowest part (3) by the frame (4), - At least one first vertical portion (5) located inside the internal space (6) above the lowest part (3), extending around a first surface having a first support surface (5,1) and a first side surface (5.2), and having a height lower than the height of the frame (4), The lower pre-culture chamber (8) is surrounded by a surface enclosed by the first side (5.2) and the first vertical portion (5), The remaining internal space between the first support surface (5.1) and the upper part of the frame (4) forms the upper pre-culture chamber. At least one first vertical section (5), - Open on the outside of the basic element, at least one channel (14) connects the lower preliminary culture chamber (8) to the surrounding environment, - Open on the outside of the basic element, and at least one other channel (14) connecting the upper pre-culture chamber (10) to the surrounding environment Basic element (1) having the following characteristics.

2. - A second vertical portion (7) extending inward into the internal space (6) around the second surface, having a height lower than the height of the frame (4) and higher than the height of the first vertical portion (5), The intermediate pre-culture chamber (9) is surrounded by the second side surface (7.2) of the second vertical section (7) and the surface surrounding the second vertical section (7). The second vertical section (7) and - Open on the outside of the basic element, and at least one other channel (14) connecting the intermediate pre-culture chamber (9) to the surrounding environment The basic element (1) according to claim 1, characterized by the above.

3. The basic element (1) according to claim 1 or 2, characterized in that the material from which the basic element (1) is manufactured comprises injection-molded biocompatible plastic, more particularly polybutylene terephthalate (PBT).

4. The basic element (1) according to claim 1 or 2, characterized in that the portion of the channel (14) is formed within the side surface of the lowest part (3) facing in the opposite direction from the frame (4), and is connected to a connector (16) of the channel (14) for supplying or releasing culture medium.

5. The basic element (1) according to claim 4, characterized in that the portion of the channel (14) is formed entirely or partially as a preliminary channel portion (14.1).

6. The basic element (1) according to claim 1 or 2, characterized in that the first vertical portion (5) and at least one other frame (4) having an optional second vertical portion (7) are formed on the lowest portion (3).

7. A multi-chamber biochip (2), - Having the basic element (1) described in claim 1 or 2, - The first separation membrane (11) is placed on the first vertical section (5) and connected to the first vertical section (5), The lower culture chamber (8) is provided by the first vertical section (5) and the first separation membrane (11). Yes, -Optionally, a second separation membrane (12) is placed on the second vertical section (7) and connected to the second vertical section (7), An intermediate culture chamber (9) is provided between the first separation membrane (11) and the second separation membrane (12). Yes, - The upper closing membrane (13) is placed on the upper side (1.1) of the frame (4) and connected to the frame (4), An upper culture chamber (10) is provided between the upper closing membrane (13) and the first separation membrane (11), or optionally between the upper closing membrane (13) and the second separation membrane (12). Yes, -Optionally, the lower closing membrane (15) is placed on the side surface of the lowest part (3) facing in the opposite direction from the frame (4), and connected to the side surface, a multi-chamber biochip (2).

8. The multi-chamber biochip (2) according to claim 7, characterized in that at least one of the separation membranes (11, 12) has a microcavity (19).

9. A method for manufacturing a multi-chamber biochip according to claim 7, - A step of providing the basic element (1) according to claim 1 or 2, - The steps include placing the first separation membrane (11) on the first vertical portion (5), connecting the first separation membrane (11) and the first vertical portion (5), and forming a seal. -Optionally, the second separation membrane (12) is placed on the second vertical portion (7), the second separation membrane (12) and the second vertical portion (7) are connected, and a seal is formed. - The steps include placing the upper closing membrane (13) on the frame (4), connecting the upper closing membrane (13) and the frame (4), and forming a seal. -Optionally, place the lower closing membrane (15) on the side of the lowest part (3) facing the opposite direction from the frame, connect the lowest part (3) and the lower closing membrane (15), and form a seal. A method for providing this.

10. The method according to claim 9, characterized in that the sealed connection is achieved using a bundle of induced and controlled high-energy radiation.

11. A set for the multi-chamber biochip (2) according to claim 7, - The basic element (1) described in claim 1 or 2, - At least one first separation membrane (11) placed on the first vertical section (5) to help close the lower culture chamber (8), -Optionally, at least one second separation membrane (12) placed on the second vertical section (7) to help close the intermediate culture chamber (9), - An upper closing membrane (13) that is placed on top of the frame (4) and helps to close the upper culture chamber (10) and A set that includes this item.

12. The set according to claim 11, characterized by an additional closing membrane (15) positioned on the bottommost (3) side facing the opposite direction from the frame.

13. A method for culturing cells, - The step of selecting and providing the multi-chamber biochip (2) described in claim 7, - The step of introducing culture medium (18) and cells (26, 28, 29, 30, 31) into existing culture chambers (8, 9, 10) A method for providing this.

14. The method according to claim 13, characterized in that a hydrogel is introduced into at least one of the culture chambers (8, 9, 10).

15. The method according to claim 13, characterized in that the spherical bodies (26) and / or organelles (26) are colonized in at least one of the culture chambers (8, 9, 10) by colonizing the spherical bodies (26) and / or organelles (26) in at least one of the culture chambers (8, 9, 10), and / or culturing them.

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