A device for studying the interactions of biological samples.

Abstract

The present invention relates to an apparatus (10) for culturing and / or investigating biological samples. The apparatus comprises a plurality of wells (11), wherein at least two wells arranged adjacent to each other each have an open upper (12) and lower (13), and the upper and lower are in fluid communication. At least two lower (13) of at least two wells (11) are connected to each other by conduits (15).

Description

Technical Field

[0001] The present application relates to an apparatus for studying interactions of biological samples.

Background Art

[0002] Biological samples play an important role in biological science and its applications. Three-dimensional tissue culture (so-called microtissues) is being increasingly utilized in tests and screening relating to the toxicity, immunogenicity, or therapeutic effect of compounds.

Summary of the Invention

[0003] Furthermore, with the advent of immuno-oncology approaches, there is a growing demand for the ability to study interactions between different biological samples (e.g., tumor models and immune cell repertoires). It has been found that current culture apparatuses are insufficient and do not provide sufficient degrees of freedom for studying interactions between different biological samples. The present invention solves this and other problems.

Brief Description of the Drawings

[0004] It should be noted that the drawings are not necessarily drawn to scale. In particular, the relationship between the biological sample and the apparatus, as well as the height of the conduits, are not necessarily drawn to scale.

[0005] Furthermore, it should be noted that in all the drawings, the liquid (water or aqueous medium) in the wells is not shown for the sake of clarity.

[0006] [Figure 1]Figure 1 shows a schematic of one embodiment of a apparatus 10 for culturing and / or investigating a biological sample 16. Figure 1A shows a cross-sectional view of the apparatus. The apparatus comprises a plurality of wells 11, and at least two adjacent wells 11 each have an open upper section 12 and a lower section 13. The upper and lower sections are in fluid communication. In this embodiment, the lower section 13 has a smaller cross-sectional area than the open upper section 12. The well includes an intermediate section located between the open upper section 12 and the lower section 13, which includes a pipetting stage 17 with an inclination. Furthermore, the well includes a chute 18 provided between the pipetting stage 17 and the lower section 13. In the illustrated embodiment, a base layer 14 forming the bottom surface of at least one well 11 and at least one conduit 15 are provided. At least two lower sections 13 of at least two wells 11 are connected to each other by the conduit 15. This conduit is formed by a longitudinal groove 24A at the base of some of the wells and the upper surface of the base layer 14. Figure 1B shows a top view. In one embodiment shown as a top view in Figure 1B, at least one set of at least two wells is arranged such that the conduits 15A and 15B are substantially rectangular relative to each other with respect to at least one other set of at least two wells. [Figure 2] Figure 2 shows two other embodiments of the apparatus 10 for culturing and / or investigating a biological sample 16. In Figure 2A, the conduit 15 is formed by a longitudinal groove 25 provided on the upper surface of the base layer 14, which creates a distance between the well tip and the base layer 14, and as a result determines the height of the conduit 15. Depending on the application, the height of the conduit 15 can be set to be equal to or greater than the diameter of the biological sample 16, or to be less than the diameter of the biological sample 16. In this regard, Figures 1 and 2 are not necessarily drawn to actual size. In Figure 2A, the conduit 15 connects the lower parts 13 of two wells to each other. It is also possible to connect more wells by conduit, as described in the specification. As an example, in Figure 2B, the conduit 15 connects the lower parts 13 of four wells to each other. [Figure 3] Figure 3 shows another embodiment of the apparatus 10 for culturing and / or investigating a biological sample 16, comprising a separation layer 22 positioned between the lower part 13 of the well and the conduit 15. The separation layer 22 may consist of a mesh or a semipermeable membrane. In one embodiment shown in Figures 3 and 3B, a cell monolayer 23 is formed on the separation layer. In one embodiment shown in Figure 3C, the base layer 14 is connected to the apparatus 10 by a series of layers consisting of adhesion layers 20a, 20b, 20c and a spacer layer 21 that primarily determines the height of the conduit 15. This embodiment can also be carried out without the separation layer 22. Depending on the application, the height of the conduit 15 can be set to be equal to or greater than the diameter of the biological sample 16, or to be less than the diameter of the biological sample 16. In this regard, Figure 3 is not necessarily drawn to actual size. [Figure 4] Figure 4 shows another embodiment of the apparatus 10 for culturing and / or investigating a biological sample 16, with a different well shape. In Figure 4A, the well is essentially funnel-shaped, while in Figure 4B, the intermediate section located between the open upper part 12 and lower part 13 is hemispherical. Depending on the application, the height of the conduit 15 can be set to be equal to or greater than the diameter of the biological sample 16, or to be less than the diameter of the biological sample 16. In this regard, Figure 3 is not necessarily drawn to actual size. [Figure 5]Figure 5 shows another embodiment of the apparatus 10 for culturing and / or examining biological samples 16. The dotted line indicates that the left and right halves belong to arbitrarily different embodiments. In Figure 5A, the base layer 14 is provided with undercuts 19a located at the edges of the conduits 15. These undercuts can be inclined (left half of Figure 5A) or vertical (right half of Figure 5A). In Figure 5B, the lower part 13 of the well is provided with only a chamber bevel 19b (right half of Figure 5B) or in combination with undercuts 19a located at the edges of the conduits 15 (left half of Figure 5B). All possible combinations of chamber bevels 19b and undercuts 19a, including inclined and vertical ones, are considered disclosed in this figure. These chamber bevels 19b and undercuts 19a facilitate the use of the apparatus 10 in light-sheet microscopy with the top of the apparatus open. In this method, two microscope lenses 26 (excitation lens and focusing lens) are used, tilted from below the apparatus 10, to image a biological sample. Light sheet microscopy allows the use of standard sample carriers, such as microtiter plate-shaped culture devices, as provided in embodiments of the present invention, while using a fluorescence microscope for, for example, live cell imaging at intracellular resolution. Such light sheet microscopes are offered, for example, under the brand name ZEISS LATTICE LIGHTSHEET 7. As illustrated, in these embodiments, the height of the undercut 19a or chamber bevel 19b is preferably smaller than the diameter of the biological sample, such as a microtissue. This also preferably applies to embodiments in which the undercut 19a and chamber bevel 19b are combined, as shown, for example, in the left half of Figure 5B. However, in Figure 5B, the relationship between (1) the combined height of the undercut 19a and chamber bevel 19b and (2) the diameter of the biological sample 16 is not depicted to scale. In further embodiments, the height of the undercut 19a and / or chamber bevel 19b is preferably smaller than the diameter of the biological sample, such as a microtissue.In further embodiments, the height of the undercut, the height of the chamber bevel, or a combination thereof (if applicable) is equal to or less than 3 / 4, 2 / 3, or 1 / 2 of the diameter of the biological sample, such as microtissue. [Figure 6] Figure 6 shows another embodiment of the apparatus 10 for culturing and / or investigating a biological sample 16, comprising a separation layer 22 positioned between the bottom 13 of the well and the conduit 15. In this embodiment, the biological sample 16 consists of cells or tissues with a specific gravity / density lower than that of an aqueous medium such as water or saline (e.g., adipocytes or adipocyte microtissues). The separation layer 22 prevents the sample 16 from floating and moving upward towards the liquid-air interface (not shown) in the well. All of this can be done on a microscope stage having single-cell resolution. In this way, it can be ensured, for example, that the sample remains within the focal range of the microscope lens 26. In this embodiment, the biological sample 16 containing cells or tissues with a specific gravity / density lower than that of an aqueous medium is introduced into the apparatus through a well lacking such a separation layer (Figure 6A) and reaches the conduit. Then, by slightly tilting the apparatus, the biological sample moves through the conduit towards an adjacent well having a separation layer 22. In this embodiment, the height of the conduit 15 is preferably greater than the diameter of the biological sample 16, in order to allow the biological sample 16 to pass through the conduit. [Figure 7] Figure 7 shows an embodiment of the apparatus 10 used in the Transwell migration assay for culturing and / or investigating a biological sample 16. The apparatus 10 comprises a separation layer 22 on which a monolayer of cell monolayers 23 is formed. This assay can be used to verify whether immune cells 27 migrate through the cell monolayer 23 and the mode of migration in response to the presence of a chemoattractant 28 (e.g., inflammatory cytokine). The monolayer (indicated by an asterisk) can be used to evaluate whether and how it migrates in response to the presence of a tumor sample or tumor microtissue 16a. All of these can be performed on a microscope stage with single-cell resolution. For further details of the Transwell migration assay, please refer to this specification. [Figure 8] Figure 8 shows one embodiment of a apparatus 10 for culturing and / or investigating biological samples 16 to investigate interactions between two biological samples 16a and 16b. Such interactions include, for example, the effects of a tumor sample or tumor microtissue 16a treated with a drug (examples shown are antibodies, small molecule drugs, viral vectors, or recombinant immune cells) 29 on a non-tumor microtissue (e.g., liver organoids) 16b. For example, it is possible to study whether cytokines 30, indicated by asterisks, released from the tumor sample or tumor microtissue in response to treatment, result in adverse off-target effects on the non-tumor microtissue, such as a cytokine storm. The possibility of abscopal effects can also be investigated. For example, it is possible to investigate whether systemic effects are observed on untreated tumor microtissue 16b when the initial tumor microtissue 16a is treated with drug 29. It is also possible to investigate the effects of the same anticancer drug on two different tumor samples or tumor microtissues. All of these can be performed on a microscope stage with single-cell resolution. Other interactions that could be studied include, but are not limited to, all kinds of inter-tissue interactions, such as liver-pancreas and liver-heart interactions. [Figure 9] Figure 9 shows one embodiment of the apparatus 10 for culturing and / or investigating a biological sample 16 similar to that shown in Figure 3C, and illustrates the exemplary thicknesses of the adhesive layers 20a, 20b, 20c, as well as the spacer layer 21, the separation layer 22, and the base layer 14. [Figure 10]Figure 10 shows one embodiment of a apparatus 10 for culturing and / or investigating a biological sample 16, which includes different groups of wells, some of which are connected to one another by conduits 15a. The conduits 15a and 15b are arranged substantially orthogonal to each other with respect to at least two other groups of wells. When installed on an inclined apparatus that performs one-dimensional tilting motion, this embodiment allows for the study of interactions between biological samples placed in a set of wells connected to each other by conduits 15b arranged orthogonal to the tilt axis 31. This is because the tilting motion facilitates the flow of liquid 32 and the movement of components contained therein in the conduits between the wells. Between wells connected to each other by conduits 15a arranged parallel to the tilt axis, there is little or no exchange of liquid and components contained therein. Therefore, there is little or no interaction between the biological samples placed therein, and these can serve as negative controls. [Figure 11] Figure 11 shows one embodiment of a apparatus 10 for culturing and / or investigating a biological sample 16, illustrating how the number of wells connected to each other by the same conduit varies. This apparatus consists of rows of 16 wells. In the first four rows, 8 x 2 wells are connected by one conduit each (doublet). In rows 5-8, 4 x 4 wells are connected by one conduit each (quadruplet). In rows 9-12, 2 x 8 wells are connected by each conduit (octet), in rows 17-20, 5 x 3 wells are connected by each conduit (triplet), and in rows 21-24, 1 x 14 wells are connected by one conduit. It is important to note that not all wells in a row or column are ever connected by the same conduit.

[0007] The apparatus according to the present invention may have, for example, only doublets, triplets, quadruplets, octets, or other combinations thereof, or, as illustrated in Figure 11, may include, for example, doublets, triplets, quadruplets, octets, and 14 mixtures in different rows or columns, or even in the same row or column.

[0008] Generally, if the device contains, for example, N wells in a row or column, then up to N-2 wells are connected to each other by the same conduit. [Modes for carrying out the invention]

[0009] According to a first aspect of the present invention, an apparatus (10) for culturing and / or investigating a biological sample (16) is provided. The apparatus includes a plurality of wells, at least two wells (11) arranged adjacent to each other, each having an open upper (12) and a lower (13), the upper and lower being in fluid communication. Furthermore, at least two lowers (13) of the at least two wells (11) are connected to each other by conduits (15).

[0010] In some embodiments, the apparatus for culturing and / or investigating biological samples employs the form of a microwell plate or microtiter plate, in which different wells are arranged in rows and columns.

[0011] In some embodiments, two wells (doublets), three wells (triplets), four wells (quadruplets), five wells, six wells, seven wells, eight wells (octets), nine wells, ten wells, eleven wells, or twelve wells are connected to one another by conduits.

[0012] Generally, this device contains, for example, N wells in a row or column, and at most N-2 wells are connected to each other by the same conduit. For example, if the device contains a row of 16 wells, it is possible that 8 x 2 wells are connected by one conduit each, 4 x 4 wells are connected by one conduit each, 2 x 8 wells are connected by one conduit each, 5 x 3 wells are connected by one conduit each, or 1 x 14 wells are connected by one conduit, but it is never the case that all 16 wells are connected by the same conduit.

[0013] However, in any embodiment of the apparatus according to the present invention, all wells in a row or column are not connected to one another by the same conduit, as disclosed, for example, in WO2019008189A1.

[0014] In some embodiments, the maximum number of wells connected to one another by the same conduit is equal to half the number of wells in a given row or column in a particular apparatus for culturing and / or investigating biological samples.

[0015] In other embodiments, the maximum number of wells connected to one another by the same conduit is equal to N-2, where N is the number of wells in a given row or column in a particular apparatus for culturing and / or investigating biological samples.

[0016] Table 1 shows exemplary dimensions of apparatus for culturing and / or testing biological samples, and indicates the maximum number of doublets, triplets, quadruplets, or octets connected by each conduit.

[0017] Furthermore, in some embodiments, the apparatus according to the present invention does not include a reservoir containing a culture medium, washing solution, or test solution that is fluidly connected to one or more wells via a conduit, as disclosed in, for example, WO2019008189A1.

[0018] It should be emphasized in this context that embodiments without a reservoir can be combined with embodiments in which not all wells in the same row or the same column are connected to each other by the same conduit, particularly with regard to the various possibilities of the latter disclosed herein.

[0019]

Table 1

[0020] In some embodiments, the lower portion (13) has a smaller cross-sectional area than the open upper portion (12).

[0021] According to one embodiment, the device according to the present invention is of a unitary structure, that is, without a separate base layer. Such a unitary structure can be manufactured, for example, by 3D printing.

[0022] According to one embodiment, the device according to the present invention comprises a base layer (14) that forms the bottom surface of at least one well, thereby forming the bottom surface of at least one well and at least one conduit, and thus has a two-part configuration (no adhesive layer or separation layer is included). In certain embodiments, such a base layer consists of a transparent film attached from the underside of the well via an adhesive or an adhesive layer.

[0023] In this embodiment, the well does not have its own bottom surface, and the bottom surface is formed by the base layer (14) attached thereto. The base layer (14) can be attached from the underside of the well in a releasable or non-releasable manner.

[0024] Such a two-part configuration can be manufactured by die casting, molding, embossing, or 3D printing methods using a suitable material such as a thermoplastic resin including the materials discussed below, for example.

[0025] Preferably, at least one conduit (15) consists of a longitudinal groove defined by two side walls. As described in the drawings, these grooves may be formed at the bottom of some wells (the base layer (14) forming its bottom surface), or they may be formed on the upper surface of the base layer (14) such that the bottom of the well forms its top surface.

[0026] According to one embodiment of the apparatus according to the present invention, the base layer (14) is made of an optically transparent material.

[0027] According to one embodiment, the base layer comprises a cycloolefin polymer (COP). COP possesses distinctive physical and chemical properties because it combines optimal transparency, thermal stability, biocompatibility, low autofluorescence, flatness, and chemical resistance in an excellent manner.

[0028] According to another embodiment, the base layer comprises polystyrene. According to another embodiment, the base layer comprises PMMA (polymethyl methacrylate). According to another embodiment, the base layer comprises polypropylene. According to another embodiment, the base layer comprises polydimethylsiloxane (PDMS).

[0029] According to one embodiment of the apparatus according to the present invention, the biological sample (16) is one or more selected from the following group. • Tissue sample • Microtissue, • Monolayer of adherent cells, and / or • Floating cells.

[0030] As used herein, the term “microtissue” is intended to encompass 3D cell culture models, including organoids, 3D tissue spheroids, embryoid bodies, pancreatic islets, and precision sectioned tissue slides. Microtissues may be derived from stem cells, naturally occurring cells, or artificially created from isolated cells, or passively or actively reconstructed. Microtissues may contain, or consist solely of, primary cells obtained from organs, tumor cells obtained from tumors, or cells obtained from cell cultures. In other embodiments, such microtissues may contain two or more different cell types.

[0031] In many cases, microtissues are prepared from isolated cells that, when held under appropriate conditions, aggregate to form such microtissues. Details of methods for preparing microtissues are well known to those skilled in the art, for example, from WO2015158777A1 (liver microtissue) and KELM et al., 2003 (tumor microtissue). The contents of these documents are incorporated herein by reference for the purpose of enabling implementation.

[0032] As used herein, the term “tissue sample” refers to a sample from a liquid (blood, lymph, cerebrospinal fluid) or solid tissue (connective tissue, organs, muscles, skin, tumors, etc.). Tissue samples are provided in the form of sections, aspirates, or other appropriate forms.

[0033] The term "adherent cell monolayer" refers to a type of cell culture in which cells adhere to or are bound to a specific surface in order for proliferation to occur. Most vertebrate-derived cells (with the exception of hematopoietic cells) are culturable and require a two-dimensional monolayer to facilitate cell adhesion and diffusion.

[0034] The term "suspension cells" refers to cells that are not attached to a given surface, or have never attached to one, and cells that do not form or have never formed a microtissue. Such suspension cells include immune cells such as PBMCs, macrophages, T cells, and NK cells, as well as circulating tumor cells (CTCs).

[0035] In one embodiment, the height of at least one conduit is set to be smaller than the diameter of the biological sample contained in the well, thereby preventing the biological sample from moving between wells. Typically, the diameter of the microtissue in the above description is between 100 μm and 500 μm. In this case, the height of the conduit is smaller than its diameter.

[0036] In one embodiment, the height of at least one conduit is greater than the diameter of the biological sample contained in the well, thereby allowing the biological sample to move into the conduit. Typically, the diameter of the microtissue in the above description is between 100 μm and 500 μm. In this case, the height of the conduit is greater than the diameter.

[0037] According to other embodiments, the height of at least one conduit is 1 mm or less and 0.03 mm or more, and specifically, preferred heights are 0.06, 0.09, 0.10, 0.12, 0.15, 0.18, 0.21, 0.24, 0.27, 0.3, 0.33, 0.36, 0.39, 0.42, 0.45, 0.48, 0.51, 0.54, 0.57, 0.6, 0.63, 0.66, 0.69, 0.72, 0.75, 0.78, 0.81, 0.84, 0.87, 0.9, 0.93, 0.96, or 0.99 mm.

[0038] The height of the conduit can be determined, for example, by the thickness of the spacer layer that separates the well from the base layer.

[0039] According to one embodiment of the apparatus according to the present invention, at least one well comprises a separation layer (22) positioned between the lower part (13) of the well and the conduit (15).

[0040] Such separation layers can be used for a variety of purposes. One application is when a biological sample contains cells or tissues with a lower specific gravity / density than an aqueous medium such as water or saline (e.g., adipocytes or adipocyte microtissues), in which case the separation layer prevents the sample from floating up.

[0041] Another application is to culture a cell monolayer on a separation layer to investigate its interaction with another biological sample below the separation layer, or to investigate its effect in a Transwell transfer assay.

[0042] According to one embodiment, the separation layer (22) comprises a mesh or a semipermeable membrane.

[0043] According to one embodiment, the mesh or semipermeable membrane has an average pore size of 0.1 μm or more and 100 μm or less.

[0044] It should be noted that in porous materials such as mesh, the pore size often follows a Gaussian distribution. The peak value of this distribution represents the average pore size.

[0045] Typically, the mesh opening size is the catalog value provided by each manufacturer. To control the pore size of a specific material, the sample can be placed under a microscope and the pore size can be measured directly from the magnified image. After obtaining a high-resolution image of the mesh, the pore size can be measured using digital image analysis and the average value can be calculated. Using software tools, this process can be automated, and the pore size distribution can be obtained.

[0046] The use of semipermeable or porous membranes in tissue barriers and co-culture models, and examples of such membranes, are disclosed, for example, in CHUNG et al. (2018), the entirety of which is incorporated herein by reference for the purpose of enabling implementation. Examples of materials include, but are not limited to, PC (polycarbonate), PCL (poly(E-caprolactone)), PDMS (polydimethylsiloxane), PE (polyester), PET (polyethylene terephthalate), PTFE (polytetrafluoroethylene), cellulose acetate, SIN (silicon nitride), or SiO2 (silicon dioxide).

[0047] An example of such a semipermeable membrane is a permeable polycarbonate membrane, such as those used in Corning®, Transwell®, and multiwell plates with permeable polycarbonate membrane inserts supplied by Fischer Scientific.

[0048] Another example of such a semipermeable membrane is a hydrophilic polycarbonate (PCTE) filter membrane with an average pore size of, for example, 12 μm, offered by Sterlitech (USA) (catalog number: 1270170).

[0049] Examples of such meshes are shown in Table 2, provided by NBC MESHTEC (Japan).

[0050] [Table 2] Table 2: Examples of meshes suitable as separation layers in the present invention

[0051] According to various embodiments, the separation layer (22) is coated with at least one of the following: a) hydrophilic layer, b) One or more ECM peptides or proteins, and / or c) Coating containing polyamino acids.

[0052] A technique for forming a hydrophilic layer on a given substrate is disclosed, for example, in Akther et al. (2020), and its contents are incorporated herein by reference in their entirety for the purpose of enabling implementation.

[0053] Extracellular matrix (ECM) peptides or proteins promote cell adhesion to surfaces coated with them. See, for example, Junker et al. (1987), the contents of which are incorporated herein by reference in their entirety for the purpose of enabling implementation.

[0054] Such coatings may include, for example, collagen or collagen derivatives, or other matrix components (e.g., fibronectin or laminin), but can also be cell-type specific, as disclosed by Zhang et al. (2009). The contents of that document are incorporated herein by reference in their entirety for the purpose of enabling implementation.

[0055] Polyamino acid-containing coatings may include, for example, poly-L-lysine or poly-L-ornithine. The respective coating protocols are published in scientific literature and by laboratory reagent manufacturers.

[0056] In one embodiment, different coatings can be applied to both sides of the separation layer (22).

[0057] According to one embodiment, the apparatus according to the present invention includes at least two sets of at least two wells, the lower two locations of at least two wells in each set being connected to each other by conduits (15), and furthermore, at least one set of at least two wells is arranged such that the conduits (15A, 15B) are substantially orthogonal to each other with respect to at least one other set of at least two wells.

[0058] When installed on an inclined device that performs unidirectional tilting motion, this embodiment makes it possible to study the interactions between biological samples placed in a set of wells connected to each other by conduits arranged perpendicular to the tilt axis. This is because the tilting motion promotes the flow of liquid in the conduits between the wells, thereby promoting the interactions of the components contained in the wells.

[0059] Between wells connected to each other by conduits positioned parallel to the inclined axis, little to no exchange of liquid and its components occurs.

[0060] According to one embodiment of the apparatus according to the present invention, the lower part (13) of at least one well is positioned eccentrically with respect to the vertical axis of its open upper part (12).

[0061] According to one embodiment of the apparatus according to the present invention, at least one well further comprises an intermediate section located between an open upper (12) and lower (13), the intermediate section comprising a pipetting stage (17) having an inclination.

[0062] The pipetting stage is configured as a platform within a well, and the platform has a slope such that any fluid applied to the platform is discharged downwards. The slope angle of the pipetting platform is at least 5°.

[0063] The inclination angle of the pipetting stage (17) is 45° or less, preferably 35° or less, more preferably 25° or less, and most preferably 15° or less. Most preferably, the inclination angle of the pipetting platform is 10°.

[0064] This inclination angle refers to the angle between the plane of the pipetting stage and the horizontal cross-sectional plane parallel to the plane of the multiwell plate in which the pipetting stage is in contact with the upper wall of the wells.

[0065] According to one embodiment of the apparatus according to the present invention, the inclination angle of the pipetting stage (17) is at least 5°, 45° or less, preferably 35° or less, more preferably 25° or less, and most preferably 15° or less.

[0066] According to one embodiment of the apparatus according to the present invention, the middle section of at least one well further comprises a chute (18), the chute being located between the pipetting stage and the lower part.

[0067] The pipetting stage (17) has a slope that allows any liquid adhering to it to flow down. The chute (18) forms a transition between the lower end of the pipetting stage and the bottom of the well. The chute (18) has a greater slope than the pipetting stage (17).

[0068] These types of "pipette stages" are sometimes called "ledges." For example, microtiter plates manufactured by InSphero(CH) using its SureXchange technology are one such example.

[0069] When adding or aspirating culture medium into a well, the pipette tip is inserted into the well on the side of the pipetting stage (17). As a result, the liquid supplied to the well first hits the pipetting stage, then flows smoothly down the pipetting stage and along the chute (18) to the bottom of the well (13). Thus, the biological sample is not directly present in the flow path of the liquid supplied to or aspirated from the well. The pipetting stage (17) has a slope, and by positioning the pipette tip perpendicular to this slope, a gap is created between the opening of the pipette tip and the pipetting stage (17).

[0070] At the optimal angle of inclination, this gap is small enough to ensure efficient liquid aspiration while preventing unintentional aspiration of microtissue. This effect is supported by surface tension and adhesive tension, leaving a small amount of liquid at the bottom of the well. The chute has an inclination, which is steeper than the inclination of the pipetting stage within the same well. The pipetting stage and / or chute can be located on only one inner side of the well, two adjacent inner sides, three inner sides, all four inner sides, or all inner sides.

[0071] Any further details relating to the shape of the wells are disclosed, for example, in WO2017001680A1, which is incorporated herein by reference.

[0072] According to one embodiment, the apparatus according to the present invention comprises a row or array of wells. According to one embodiment, the apparatus is configured as a multiwell plate, preferably comprising an array of 96 wells, an array of 384 wells, or an array of 1536 wells. According to one embodiment, the multiwell plate is a standard SBS / ANSI format multiwell plate.

[0073] According to one embodiment of the apparatus according to the present invention, at least a) the bottom of at least one well (13), and / or b) At least one portion of the base layer (14) is coated with an ultra-low adhesion coating.

[0074] When cells are cultured in wells with ultra-low adhesion (ULA) surfaces, the cells maintain a suspended, non-adherent state. Under certain conditions, this promotes cell self-assembly and the formation of microtissues. Growing spheroids or microtissues in wells with ultra-low adhesion surfaces maintains the integrity of the spheroids or microtissues and prevents them from undergoing dedifferentiation or activation through undesirable adhesion, including undesirable changes in cell division characteristics.

[0075] There are various methods for providing an ultra-low adhesion surface to the wells of a multi-well plate. One example of this method involves covalently bonding a hydrophilic and neutrally charged hydrogel layer to a substrate such as polystyrene. Multi-well plates with such ultra-low adhesion surfaces are commercially available from Corning. Another example of an ultra-low adhesion surface is a surface formed from a polymer consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC). Such ULA coatings are used, for example, in Akura® Spheroid Microplates manufactured by Insphero(CH).

[0076] According to one embodiment of the apparatus according to the present invention, the separation layer (22) is attached to the bottom (13) of at least one well via an adhesive layer (20a).

[0077] According to one embodiment of the apparatus according to the present invention, the spacer layer (21) is attached to the underside of the separation layer (22) via an adhesive layer (20b). In this embodiment, the height of the conduit is substantially determined by the thickness of the spacer layer (21).

[0078] According to one embodiment of the apparatus according to the present invention, a base layer (14) that forms the bottom surface of at least one well and at least one conduit is bonded to the lower surface of a spacer layer (21) via an adhesive layer (20c).

[0079] According to one embodiment of the apparatus according to the present invention, • At least one lower section (13) of at least one well is provided with an undercut (19a) or a chamber bevel (19b), and / or The base layer (14) comprises at least one chamber bevel (19a) positioned on the edge of the conduit (15).

[0080] These undercuts or chamber bevels facilitate the use of this instrument in open-top light-sheet microscopy by avoiding blind spots. In open-top light-sheet microscopy, two microscope lenses (excitation lens and focusing lens) are used at an angle from the bottom of the instrument to image biological samples.

[0081] In one embodiment, the height of at least one undercut or chamber bevel is smaller than the diameter of the biological sample, such as a microtissue. It should be noted that this also preferably applies to embodiments in which an undercut and a chamber bevel are combined, as shown on the left side of Figure 5B.

[0082] Typically, the diameter of the microstructure in the above description is between 100 μm and 500 μm.

[0083] In further embodiments, the height of the undercut, chamber bevel, or combination thereof (if applicable) is less than or equal to 3 / 4, 2 / 3, or 1 / 2 of the diameter of the biological specimen, such as microtissue.

[0084] These embodiments help prevent biological samples from becoming trapped within the undercut. In this way, the biological sample can remain in the center of the bottom of the well. Confinement in the space formed by the undercut and / or chamber bevel will cause a diffraction effect of light, for example, when observing the sample with an inverted microscope.

[0085] According to another aspect of the present invention, the apparatus described above is provided for use in studying the interaction of two or more biological samples.

[0086] According to another aspect of the present invention, the use of the apparatus described above is provided for studying the interaction of two or more biological samples.

[0087] According to another aspect of the present invention, a method is provided for studying the interaction of two or more biological samples, in which the apparatus described above is used.

[0088] To avoid redundancy, it should be understood that the above embodiments relating to a) various types of biological samples and b) types of interactions between biological samples are considered to be disclosed in the context of 1) the apparatus used, 2) the use of the apparatus, and 3) the research methods described above.

[0089] Manufacturing of equipment As described above, this device can have a one-piece structure without a separate base layer. Such a one-piece structure can be manufactured, for example, by 3D printing.

[0090] Furthermore, the apparatus may also have a two-part configuration (excluding adhesive and separation layers). In such an embodiment, the base layer forming the bottom of at least one well and at least one conduit are attached from the underside of the well, either in a removable or non-removable manner, using an adhesive or adhesive layer.

[0091] Such a two-part structure can be manufactured using a suitable material (e.g., a thermoplastic resin, including the materials discussed herein) by methods such as die casting, molding, embossing, or 3D printing.

[0092] Examples The present invention is illustrated and described in detail in the drawings and the foregoing description, but such illustrations and descriptions are illustrative or exemplary and not limiting. The present invention is not limited to the disclosed embodiments. Other modifications to the disclosed embodiments can be understood and implemented by a person skilled in the art by examining the drawings, the disclosure and the appended claims in practicing the invention described in the claims. In the claims, the word “equipped with” does not exclude other elements or steps, nor does one exclude more than one. The mere fact that certain measures are described in different dependent claims does not indicate that a combination of these measures cannot be used advantageously. No reference numeral in the claims should be construed as limiting the scope.

[0093] [Table 3] Table 3: Examples of biological samples or combinations thereof that can be investigated.

[0094] The Transwell migration assay is a commonly used test method to study, for example, the migration response of endothelial cells to angiogenesis-inducing or inhibitory factors, or the response of immune cells to immune cell binding stimuli.

[0095] In such assays, endothelial cells are cultured on a separation layer consisting of a permeable membrane or mesh. Next, a solution containing the test drug is placed, for example, beneath the cell-permeable membrane. After an incubation period, cells that have migrated across the membrane are stained and counted. The membrane is typically coated with some extracellular matrix component (e.g., collagen) that promotes both adhesion and migration.

[0096] In another embodiment, immune cells are added to the wells, and the test substance on the opposite side of the isolation layer is an immune cell-binding substance, such as an inflammatory cytokine. In this approach, the immune cells must pass through both the endothelial layer and the isolation layer.

[0097] The main advantage of this assay lies in its detection sensitivity. Movement through permeable filters can be triggered by extremely low concentrations of, for example, angiogenesis-inducing factors or inflammatory cytokines. The apparatus according to the present invention makes it possible to perform all of these operations on a microscope stage with single-cell resolution.

[0098] References Kelm J et al. Biotechnology and Bioengineering, 83(2)173‐180(2003) Chung HH, Mireles M, Kwarta BJ, Gaborski TR. Use of porous membranes in tissue barrier and co-culture models. Lab Chip. 2018 Jun 12;18(12):1671-1689 Akther,F.; Yakob,SB;Nguyen,N.-T.;Ta,HT Surface Modification Techniques for Endothelial Cell Seeding in PDMS Microfluidic Devices. Biosensors 2020,10,182 Zhang Y, He Y, Bharadwaj S, Hammam N, Carnagey K, Myers R, Atala A, Van Dyke M. Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype. Biomaterials. 2009 Aug;30(23-24):4021-8 Junker JL, Heine UI. Effect of adhesion factors fibronectin, laminin, and type IV collagen on spreading and growth of transformed and control rat liver epithelial cells. Cancer Res. 1987 Jul 15;47(14):3802-7 WO2015158777A1 WO2019008189A1 WO2017001680A1 [Explanation of Symbols]

[0099] 10 Apparatus for culturing and / or investigating biological samples 11 wells 12 wells open top 13 wells 14 Base Layer 15, 15a, 15b Conduits connecting the bottoms of at least two wells 16 Biological Samples 16a Tumor sample or tumor microtissue Pipetting stage in 17 wells 18 Shoots within the well section 19a Undercut 19b Chamber Bevel 20a, 20b, 20c adhesive layer 21 Spacer layer 22 Separation layer located between the bottom of the well and the conduit 23 Cell monolayer 24 well bottom 24a Longitudinal grooves at the bottom of the well 25. Longitudinal grooves in the base layer 26 Microscope lenses 27 Immune cells 28 Chemokines 29 Pharmaceutical ingredients 30 cytokines 31 Tilt axis 32 Flow in a conduit caused by tilting motion

Claims

1. An apparatus (10) for culturing and / or investigating a biological sample (16), wherein the apparatus comprises a plurality of wells, At least two wells (11) are arranged adjacent to each other, and each well is The open upper part (12) and The lower part (13) and the following are included: The upper and lower parts are in fluid communication, Furthermore, the apparatus wherein at least two lower parts (13) of the at least two wells (11) are connected to each other by conduits (15).

2. The apparatus according to claim 1, comprising a base layer (14) that forms the bottom surface of at least one conduit and at least one well.

3. The apparatus according to claim 2, wherein the base layer (14) is made of a light-transmitting material.

4. The aforementioned biological sample (16) is Tissue sample, microtissue, A monolayer of adherent cells, and / or The apparatus according to any one of claims 1 to 3, wherein one or more cells are selected from a group of suspended cells.

5. The apparatus according to any one of claims 1 to 4, wherein at least one well comprises a separation layer (22) disposed between the lower part (13) of the well and the conduit (15).

6. The apparatus according to any one of claims 1 to 5, wherein the separation layer (22) comprises a mesh or a semipermeable membrane.

7. The apparatus according to claim 6, wherein the average pore size of the mesh or semipermeable membrane is 0.1 μm or more and 100 μm or less.

8. The separation layer (22) is a) hydrophilic layer; b) One or more ECM peptides or proteins, and / or c) The apparatus according to any one of claims 5 to 7, wherein it is coated with at least one coating comprising a polyamino acid.

9. The apparatus according to any one of claims 1 to 8, comprising at least two sets of at least two wells, wherein the two lower parts of the at least two wells in each set are connected to one another by conduits (15), and further comprising at least one set of at least two wells, wherein the conduits (15a, 15b) of at least one set of at least two wells are arranged substantially perpendicular to each other with respect to at least one other set of at least two wells.

10. The apparatus according to any one of claims 1 to 9, wherein the lower part (13) of at least one well is positioned eccentrically with respect to the longitudinal axis of the open upper part (12).

11. The apparatus according to any one of claims 1 to 10, wherein at least one well further comprises an intermediate section located between the open upper (12) and lower (13) sections, the intermediate section comprising a pipetting stage (17) having an inclination.

12. The apparatus according to claim 11, wherein the inclination angle of the pipetting stage (17) is at least 5° and 45°, preferably 35° and 25°, most preferably 15° and 35°.

13. The apparatus according to any one of claims 11 to 12, wherein the intermediate portion of at least one well further comprises a chute (18), the chute being located between the pipetting stage and the lower part.

14. The apparatus according to any one of claims 1 to 13, wherein the apparatus comprises a row of wells or an arrangement of wells.

15. The apparatus according to any one of claims 1 to 14, wherein the apparatus is configured as a multiwell plate, and preferably comprises a 96-well arrangement, a 384-well arrangement, or a 1536-well arrangement.

16. The apparatus according to claim 15, wherein the multiwell plate is a standard SBS / ANSI standard multiwell plate.

17. at least, a) the lower part (13) of at least one well, and / or b) The apparatus according to any one of claims 1 to 16, wherein at least one section of the base layer (14) is coated with an ultra-low adhesion coating.

18. The apparatus according to any one of claims 5 to 17, wherein the separation layer (22) is attached to the lower part (13) of at least one well via an adhesive layer (20a).

19. The apparatus according to claim 18, wherein the spacer layer (21) is bonded to the lower surface of the separation layer (22) via the adhesive layer (20b).

20. The apparatus according to claim 19, wherein the base layer (14) forming the bottom surface of the at least one well and the at least one conduit is attached to the lower surface of the spacer layer (21) via an adhesive layer (20c).

21. At least one lower part (13) of at least one well is provided with a chamber bevel (19b), and / or The apparatus according to any one of claims 1 to 20, wherein the base layer (14) comprises at least one undercut (19a) positioned at the edge of the conduit (15).

22. The apparatus according to any one of claims 1 to 21, used for studying the interaction of two or more biological samples.

23. Use of the apparatus according to any one of claims 1 to 22 for studying the interaction of two or more biological samples.

24. A method for studying the interaction of two or more biological samples, comprising using the apparatus described in any one of claims 1 to 23.

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