Device for studying interactions of biological specimen

Abstract

The present invention relates to a device (10) for culturing and / or investigating biological specimen. The device comprises a plurality of wells (11), wherein at least two wells arranged next to one another each comprise an open upper section (12) and a lower section (13) wherein the upper section and the lower section are in fluid communication. At least two lower sections (13) of the at least two wells (11) are connected to one another by a conduit (15).

Description

[0001] Device for studying interactions of biological specimen

[0002] FIELD OF THE INVENTION

[0003] The present application relates to a device for studying interactions of biological specimen.

[0004] BACKGROUND

[0005] Biological specimen play an important role in biological sciences and applications. 3D tissue cultures (so-called microtissues) are more and more used in the testing or screening of compounds for their toxicity, immunogenity or therapeutic effect.

[0006] Furthermore, with the advent of immunooncological approaches, the demand for being able to study interactions between different biological specimen (e.g., a tumor model and an immune cell repertoire) has increased. It has turned out that in order to study interactions between different biological specimen, the current culturing devices are not sufficient, and do not offer enough degrees of freedom. The present invention solves this and other problems.

[0007] BRIEF DESCRIPTION OF THE FIGURES

[0008] Note that the Figures are not necessarily drawn to scale. In particular the relationship between the biological specimen and the device, and the height of the conduit, is not necessarily drawn to scale. Further note that in all figures the liquid content in the wells (water or aqueous media) are not shown for clarity.

[0009] Figure 1 gives a general overview of an embodiment of the device 10 for culturing and / or investigating biological specimen 16. Figure 1A shows a cross section of such device. The device comprises a plurality of wells 11, wherein at least two wells 11 arranged next to one another each comprise an open upper section 12, and a lower section 13. The upper section and the lower section are in fluid communication. In this embodiment, the lower section 13 has a smaller cross-sectional area than the open upper section 12. The wells comprise an intermediate section disposed between the open upper section 12, and the lower section 13, said intermediate section comprising a pipetting stage 17 having an inclination. Further, the wells comprise a chute 18 chute being provided between the pipetting stage 17 and the lower section 13.

[0010] In the illustrated embodiment, a base layer 14 is provided that forms the bottom of at least one well 11 and at least one conduit 15. At least two lower sections 13 of at least two wells 11 are connected to one another by a conduit 15. The conduit is formed by longitudinal grooves 24a in the basal parts of some wells, and the upper side of the base layer 14.

[0011] In one embodiment shown, as top view, in Figure IB, at least one set of at least two wells is arranged relative to at least one other set of at least two wells in such way that the conduits 15a, 15b are arranged in essentially rectangular fashion relative to one another.

[0012] Figure 2 shows two other embodiments of the device 10 for culturing and / or investigating biological specimen 16. In Figure 2A, the conduit 15 is formed by longitudinal grooves 25 in the upper side of the base layer 14, which create a distance between the nasal parts of the wells and the base layer 14, thus determining the height of the conduit 15.

[0013] Depending on purpose, the height of the conduit 15 can be chosen to be as high or even higher than the diameter of the biological specimen 16, or can be chosen to be smaller than the diameter of the biological specimen 16. In this regard, Figures 1 and 2 are not necessarily drawn to scale. In Figure 2 A, the conduit 15 connects two lower sections 13 of two wells to one another. As described in the specification, even more wells can be connected by the conduit. As an example, in Figure 2B, the conduit 15 four lower sections 13 of four wells to one another

[0014] Figure 3 shows other embodiments of the device 10 for culturing and / or investigating biological specimen 16, comprising a separation layer 22 arranged between the lower section 13 of the well and the conduit 15. Said separation layer 22 may comprise a mesh or a semipermeable membrane.

[0015] In one embodiment shown in Figures 3 and 3B, a cell monolayer 23 is grown on the separation layer.

[0016] In one embodiment shown in Figure 3C, the base layer 14 is connected to the device 10 by a series of layers comprising adhesive layers 20a, 20b and 20c, as well as a spacer layer 21 that mainly determines the height of the conduit 15. This embodiment can also be put into practice without the separation layer 22.

[0017] Depending on purpose, the height of the conduit 15 can be chosen to be as high or even higher than the diameter of the biological specimen 16, or can be chosen to be smaller than the diameter of the biological specimen 16. In this regard, Figure 3 is not necessarily drawn to scale.

[0018] Figure 4 shows other embodiments of the device 10 for culturing and / or investigating biological specimen 16, with different well shapes. In Figure 4A, the wells are essentially funnel-shaped while in Figure 4B, the intermediate section disposed between the open upper section 12 and the lower section 13 is hemispherical.

[0019] Depending on purpose, the height of the conduit 15 can be chosen to be as high or even higher than the diameter of the biological specimen 16, or can be chosen to be smaller than the diameter of the biological specimen 16. In this regard, Figure 3 is not necessarily drawn to scale. Figure 5 shows other embodiments of the device 10 for culturing and / or investigating biological specimen 16. The dotted line symbolizes that the left and right half belong, optionally, to different embodiments.

[0020] In Figure 5A, the base layer 14 comprises undercuts 19a arranged at the edge of the conduit 15. These undercuts can be inclined (left half of Figure 5A) or perpendicular (right half of Figure 5 A). In Figure 5B, the lower sections 13 of the wells comprise chamber bevels 19b, alone (right half of Figure 5B) or in combination with the undercuts 19a arranged at the edge of the conduit 15 (left half of Figure 5B). All possible combinations of chamber bevels 19b and undercuts 19a, both inclined and perpendicular, shall be deemed disclosed by this figure.

[0021] These chamber bevels 19b and undercuts 19a facilitate the use of such device 10 in open top light sheet microscopy, wherein two microscope lenses 26 (excitation lens and collection lens) are being used in inclined fashion from underneath the device 10 to image the biological specimen.

[0022] Light sheet microscopy uses a fluorescence microscope e.g. for live cell imaging at subcellular resolution, while allowing to use standard sample carriers, like microtiter plate-shaped culturing devices, as e.g. provided by embodiments of the present invention. Such light sheet microscopes are e.g. provided under the brand ZEISS Lattice Lightsheet 7.

[0023] As can be seen, in these embodiments, the height of the undercuts 19a, or the chamber bevels 19b is preferably smaller that the diameter of the biological specimen, like e.g. a microtissue.

[0024] Note that this also applies, preferably, for the embodiment where undercuts 19a and chamber bevels 19b are combined, as e.g. shown in left half of Figure 5B, in which however the relation between (1) combined height of undercut 19a and chamber bevel 19b and (2) diameter of the biological specimen 16 is not drawn to scale.

[0025] In further embodiments, the height of the undercuts 19a and / or the chamber bevels 19b is preferably smaller than the diameter of the biological specimen, like e.g. a microtissue. In further embodiments, the height of the undercuts, the chamber bevels or the combination of the two, if applicable, is equal to, or smaller than,3 / 4,2A or / i of the diameter of the biological specimen, like e.g. a microtissue.

[0026] Figure 6 shows another embodiment of the device 10 for culturing and / or investigating biological specimen 16, comprising a separation layer 22 arranged between the lower section 13 of the well and the conduit 15.

[0027] In this case, the biological specimen 16 comprises cells or tissue with lower specific weight / density than aqueous media like water or saline (like e.g. adipocytes or an adipocyte microtissue). The separation layer 22 avoids floating of the specimen 16, and moving thereof upwards to the liquid air interface (not shown) in the well. All this can be done on a microscopic stage with single cell resolution. In such way, it can for example be ensured that the specimen stays within the focal range of a microscopic lens 26.

[0028] In this embodiment, a biological specimen 16 comprising cells or tissue with lower specific weight / density than aqueous media can be introduced into the device through the well that lacks such separation layer (Figure 6A), and reaches the conduit. If the device is then tilted slightly, the biological specimen will travel through the conduit into the direction of the neighbouring well comprising the separation layer 22.

[0029] In such embodiment, the height of the conduit 15 is preferably greater than the diameter of the biological specimen 16, in order to allow the latter to travel through the conduit.

[0030] Figure 7 shows embodiments of the device 10 for culturing and / or investigating biological specimen 16 when used in a transwell migration assay. The device 10 comprises a separation layer 22 on which a monolayer of endothelial cells 23 is grown. The assay can be used to test if and how immune cells 27 migrate through the endothelial monolayer 23 in response to the presence of a chemoattractant 28 (e.g., an inflammatory cytokine) symbolized by the star symbol, monolayer in response to the presence of a tumor sample or tumor microtissue tissue 16a. All this can be done on a microscopic stage with single cell resolution. For more details of the transwell migration assay see the specification herein. Figure 8 shows an embodiment of the device 10 for culturing and / or investigating biological specimen 16 in which the interaction between two biological specimen 16a, 16b is investigated.

[0031] Such interaction, for example, can be the effect a tumor sample or a tumor microtissue 16a being treated with a pharmaceutical entity 29 (being shown by the example of an antibody, a small molecular drug, a viral vector, or a recombinant immune cell) has on a non-tumor microtissue (e.g., a liver organoid) 16b. One can, for example, study whether cytokines 30, symbolized by the star symbol released by the tumor sample or a tumor microtissue in response to the therapy may have a detrimental off-target effect on the non-tumor microtissue, e.g. caused by a cytokine storm.

[0032] One can also study a possible abscopal effect, e.g., whether, when a first tumor microtissue 16a is treated with pharmaceutical entity 29, a systemic effect on non-treated tumor microtissue 16b can be observed.

[0033] One can also investigate effects of the same anti-cancer therapeutic on two different tumor samples or tumor microtissue. All this can be done on a microscopic stage with single cell resolution. Other types of interaction that can be studied encompass, yet are not limited to, any kind of tissue-tissue interactions, like e.g. liver-pancreas, liver-heart, etc.

[0034] Figure 9 shows an embodiment of the device 10 for culturing and / or investigating biological specimen 16 similar to the one shown in Figure 3C, with adhesive layers 20a, 20b and 20c, as well as exemplary thicknesses of the spacer layer 21, the separation layer 22 and the base layer 14.

[0035] Figure 10 shows an embodiment of the device 10 for culturing and / or investigating biological specimen 16, which comprises different sets of wells, some of which are connected to one another by a conduit 15a which is arranged relative to other sets of at least two wells in such way that the conduits 15a, 15b are arranged in essentially rectangular fashion relative to one another.

[0036] If placed on a tilting device that performs a one-dimensional tilting movement, this embodiment allows to study the interaction between biological specimen that are placed within a set of wells connected to one another by conduits 15b that are arranged rectangular to the tilting axis 31, as the tilting movement facilitates the liquid flow 32 in the conduits between the wells, and the components comprised therein.

[0037] Wells connected to one another by conduits 15a that are arranged in parallel to the tilting axis will experience less or even no exchange of liquids, and of the components comprised therein. Biological specimen placed therein will hence experience less or no interaction, and can thus serve as a negative control.

[0038] Figure 11 shows an embodiment of the device 10 for culturing and / or investigating biological specimen 16, which shows different numbers of wells connected to one another by the same conduit. The device comprises rows with 16 wells. In the first four rows, 8 x 2 wells are connected by a conduit each (doublets). In rows 5 - 8, 4 x 4 wells are connected by a conduit each (quadruplets). In rows 9 - 12, 2 x 8 wells are connected by a conduit each (octets), in rows 17 - 20, 5 x 3 wells are connected by a conduit each (triplets), and in rows 21 - 24, l x 14 wells are connected by a conduit. It is important to mention that never all wells of one row or column are connected by the same conduit.

[0039] A device according to the invention can e.g. have doublets, triplets, quadruplets, octets or other combinations only, or, as exemplarily shown in Figure 11, can comprise a mixture of e.g. doublets, triplets, quadruplets, octets and 14ets, e.g., in different rows or columns, or even in the same rows or columns.

[0040] As a general rule, if the device comprises e.g. n wells in a row or column, a maximum of n-2 wells are connected to one another by the same conduit.

[0041] DETAILED DESCRIPTION

[0042] According to a first aspect of the invention, a device (10) for culturing and / or investigating biological specimen (16) is provided. The device comprises a plurality of wells wherein at least two wells (11) arranged next to one another each comprise an open upper section (12), a lower section (13), wherein the upper section and the lower section are in fluid communication. Further at least two lower sections (13) of the at least two wells (11) are connected to one another by a conduit (15). In several embodiments, the device culturing and / or investigating biological specimen adopts the format of a microwell plate or microtiter plate, with different wells arranged in rows and columns.

[0043] In several 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 a conduit.

[0044] As a general rule, the device comprises e.g. n wells in a row or column, a maximum of n-2 wells are connected to one another by the same conduit. If for example, the device comprises rows with 16 wells, 8 x 2 wells can be connected by a conduit each, 4 x 4 wells can be connected by a conduit each, 2 x 8 wells can be connected by a conduit each, 5 x 3 wells can be connected by a conduit each, or 1 x 14 wells can be connected by a conduit, yet never all 16 wells are connected by the same conduit.

[0045] However, in no embodiment of the device according to the present invention, all wells of one row or column are connected to one another by the same conduit, as e.g. disclosed in W02019008189A1.

[0046] In several embodiments, the maximum number of wells connected to one another by the same conduit equals half of the number of wells in a given row or column of a given device for culturing and / or investigating biological specimen.

[0047] In other embodiments, the maximum number of wells connected to one another by the same conduit equals n-2, wherein n is the number of wells in a given row or column of a given device for culturing and / or investigating biological specimen.

[0048] In table 1, some exemplary dimensions of devices for culturing and / or investigating biological specimen are shown, and the maximum number of doublets, triplets, quadruplets, or octets connected by one conduit each is shown.

[0049] Furthermore, in several embodiments, the device according to the present invention does not provide reservoirs comprising culture media, washing media or test media, which reservoirs would be fluidically connected to one or more wells by means of a conduit, as e.g. disclosed in W02019008189A1.

[0050] It is important to stress, in this context, that embodiments which do not provide reservoirs and the embodiments in which not all wells of one row or column are connected to one another by the same conduit can be combined, in particular with regard to the different possibilities of the latter, as disclosed herein.

[0051] Table 1: some exemplary dimensions of devices according to the present invention

[0052] In several embodiments, said lower section (13) has a smaller cross-sectional area than the open upper section (12).

[0053] According to one embodiment, the device according to the invention is a one-part structure, i.e., without a separate base layer. Such one-part structure can e.g. be produced by 3D printing.

[0054] According to one embodiment, the device according to the invention comprises a base layer (14) that forms the bottom of at least one well and at least one conduit, hence forming a two- part structure (not counting adhesive layers or separation layers in). In one embodiment, such base layer comprises a transparent foil that is attached from underneath to the wells by means of an adhesive or an adhesive layer.

[0055] In this embodiment, the wells have no bottom per se, as the bottom is formed by the base layer (14) attached thereto. The base layer (14) can be releasably or irreleasably attached from underneath to the wells. Such two-part structure can be produced, e.g., by die-casting, molding, embossing or 3D printing methods of suitable material, e.g., thermoplasts including the materials discussed hereinbelow.

[0056] Preferably, at least one conduit consists (15), essentially, of a longitudinal groove defined by two lateral walls. As discussed in the figure section, these grooves can be formed in the basal parts of some wells (with the base layer (14) forming the bottom thereof) , or in the upper side of the base layer (14), with the basal parts of the wells forming the ceiling thereof.

[0057] According to one embodiment of the device according to the invention, the base layer (14) comprises an optically transparent material.

[0058] According to one embodiment, said base layer comprises a cyclo-olefin polymer (COP). COP has distinctive physical and chemical properties, as it creates a superior combination of optimal clarity, thermal stability, biocompatibility, low auto-fluorescence, flatness and chemical resistance.

[0059] According to another embodiment, said base layer comprises polystyrene. According to another embodiment, said base layer comprises PMMA (polymethyl methacrylate). According to another embodiment, said base layer comprises polypropylene. According to another embodiment, said base layer comprises Polydimethylsiloxane (PDMS).

[0060] According to one embodiment of the device according to the invention, the biological specimen (16) is one or more selected from the group consisting of

[0061] • tissue sample

[0062] • microtissue,

[0063] • adherent cell monolayer, and / or

[0064] • suspended cells.

[0065] The term “microtissue” as used herein, is meant to encompass 3D cell culture models including organoids, 3D tissue spheroids, embryoid bodies, islets, precision cut tissue slides, and the like. The microtissues can derive from stem cells or can be of natural origin, or can be engineered or passively or actively reassembled from isolated cells. Microtissues can comprise, or even consist, of primary cells obtained from organs, as well as tumor cells obtained from tumors or cells obtained from cell culture. In other embodiments, such microtissues comprise two or more different cell types.

[0066] Oftentimes, microtissues are created from isolated cells which aggregate when held under suitable conditions to form such microtissue. Details of how microtissues are produced are known to the skilled artisan e.g. from WO2015158777A1 (liver microtissues) or Keim et al., 2003 (tumor microtissues). The content of these documents is incorporated by reference herein for enablement purposes.

[0067] The term “tissue sample”, as used herein, refers to sample from liquid (blood, lymph, liquor) or solid tissues (connective tissue, organs, muscle, skin, or tumor, to name a few). Tissue samples can be provided as slices, aspirations and other suitable formats.

[0068] The term “adherent cell monolayer” relates to a type of cell culture in which cells are attached or adhered to a given surface in order for growth to occur. Most vertebrate derived cells (with the exception of hematopoietic cells) can be cultured and require a 2 dimensional monolayer to facilitate cell adhesion and spreading.

[0069] The term “suspended cells” relates to cells that do not attach, or have not attached, to a given surface, nor do they form, or have they formed, a microtissue. Such suspended cells are for example immune cells, like PBMC, Macrophages, T cells, NK cells or the like, yet also circulating tumor cells (CTC).

[0070] In one embodiment, the height of at least one conduit is smaller than the diameter of the biological specimen comprised in the well, so as to avoid that the biological specimen migrate between the wells. Typically, a microtissue in the meaning of the above description has a diameter between 100 pm and 500 pm. In such case the conduit has a height smaller than such diameter.

[0071] In one embodiment, the height of at least one conduit is greater than the diameter of the biological specimen comprised in the well, so as to let the biological specimen migrate into the conduit. Typically, a microtissue in the meaning of the above description has a diameter between 100 pm and 500 pm. In such case the conduit has a height greater than such diameter.

[0072] According to other embodiments, at least one conduit has a height of between < 1 mm and > 0,03 mm, with 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 as preferred heights.

[0073] The height of the conduit can for example be determined by the thickness of a spacer layer that separates the wells and the base layer.

[0074] According to one embodiment of the device according to the invention, at least one well comprises a separation layer (22) arranged between the lower section (13) of the well and the conduit (15).

[0075] Such separation layer can be used for different applications. In one application, wherein the biological specimen comprises cells or tissue with lower specific weight / density than aqueous media like water or saline (like e.g. adipocytes or an adipocyte microtissue), the separation layer avoids floating of the specimen.

[0076] In another application, a cell monolayer can be grown on the separation layer, and its interaction with another biological specimen below the separation layer can then be investigated, or its effect in a transwell migration assay can be investigated.

[0077] According to one embodiment thereof, said separation layer (22) comprises a mesh or a semipermeable membrane.

[0078] According to one embodiment thereof, the mesh or semipermeable membrane comprises an average pore size of between > 0.1 pm and < 100 pm.

[0079] Note that in porous materials, like mesh and the like, the pore size is oftentimes subject to a gaussian distribution. The peak value of said distribution then indicates the average pore size. Typically the mesh size is a catalogue value provided by the respective manufacturers. In order to control the pore size or a given material, a sample can is placed under the microscope, and the pore sizes are measured directly from the magnified image. After obtaining high-resolution images of the mesh, digital image analysi s can be used to measure and average the pore sizes. Software tools can automate this process and provide distributions of pore sizes.

[0080] The use of semipermeable or porous membranes, and examples of such membranes, in tissue barrier and co-culture models is for example disclosed in Chung et al (2018), the content of which is incorporated by reference herein in its entirety for enablement purposes. Materials include, but are not limited to, PC (polycarbonate), PCL (poly(s-caprolactone)), PDMS (polydimethylsiloxane), PE (polyester), PET (Polyethylene terephthalate), PTFE (Polytetrafluoroethylene), Cellulose acetate, SiN (silicon nitride), or SiO2 (silicon dioxide)

[0081] One example for such semipermeable membrane is a Permeable Polycarbonate Membrane as for example being used in Corning™ Transwell™ Multiple Well Plates with Permeable Polycarbonate Membrane Inserts, as supplied by Fisher Scientific.

[0082] Another example for such semipermeable membrane is a hydrophilic polycarbonate (PCTE) filter membrane with an average pore size of e.g. 12 pm, as for example offered by Sterlitech (USA) (catalogue number: 1270170).

[0083] Examples for such meshes are shown in table 2, provided by NBC Meshtec (JP)

[0084] Table 2: Examples of meshes suitable as separation layer in the meaning of the present invention

[0085] According to different embodiments, the separation layer (22) is coated with at least one of a) a hydrophilic layer, b) one or more ECM peptides or proteins, and / or c) a coating comprising poly-amino acids.

[0086] Techniques to equip a given substrate with a hydrophilic layer are for example disclosed in Akther et al (2020), the content of which is incorporated by reference herein in its entirety for enablement purposes.

[0087] Extracellular matrix (ECM) peptides or proteins promote cell adhesion to a surface coated therewith. See for example Junker et al (1987), the content of which is incorporated by reference herein in its entirety for enablement purposes.

[0088] Such coatings can comprise e.g. collagen or collagen derivatives, or other matrix components (e.g. fibronectins or laminins), yet can also be cell-type specific, as e.g. disclosed in Zhang et al (2009), the content of which is incorporated by reference herein in its entirety for enablement purposes.

[0089] 14

[0090] SUBSTITUTE SHEET (RULE 26) A coating comprising poly-amino acids may for example comprise Poly-L-Lysine or Poly-L- Omithine. Respective coating protocols are published in the scientific literature as well as by laboratory suppliers.

[0091] In one embodiment, there can be different coatings on the two sides of the separation layer (22).

[0092] According to one embodiment, the device according to the invention comprises at least two sets of at least two wells, wherein the two lower sections of the at least two wells of each set are connected to one another by a conduit (15), wherein further at least one set of at least two wells is arranged relative to at least one other set of at least two wells in such way that the conduits (15a, 15b) are arranged in essentially rectangular fashion relative to one another.

[0093] If placed on a tilting device that performs a one-dimensional tilting movement, this embodiment allows to study the interaction between biological specimen that are placed with a set of wells connected to one another by conduits that are arranged rectangular to the tilting axis, as the tilting movement facilitates the liquid flow in the conduits between the wells, and the components comprised therein.

[0094] Wells connected to one another by conduits that are arranged in parallel to the tilting axis will experience less or even no exchange of liquids and the components comprised therein.

[0095] According to one embodiment of the device according to the present invention, the lower section (13) of at least one well is eccentrically arranged with respect to the longitudinal axis of the open upper (12) section thereof.

[0096] According to one embodiment of the device according to the present invention, at least one well further comprises an intermediate section disposed between the open upper section (12), and the lower section (13), said intermediate section comprising a pipetting stage (17) having an inclination.

[0097] The pipetting stage is configured as a platform within the well, said platform has a slope such that any fluid applied to the platform can drain off into the lower section. The slope of the pipetting platform has an angle of at least 5°. The slope of the pipetting stage (17) has an angle of not more than 45°, preferably of not more than 35°, more preferably of not more than 25°, and most preferably of not more than 15°. Most preferably, the slope of the pipetting platform has an angle of 10°.

[0098] The angle of the slope is to be understood as the angle between the plane or the pipetting stage and the plane of the horizontal cross-sectional area, which is parallel to the plane of the multiwell plate where the pipetting stage is in contact with the wall of the upper section of the well.

[0099] According to one embodiment of the device according to the present invention, the inclination of the pipetting stage (17) has an angle of at least 5°, and of not more than 45°, preferably of not more than 35°, more preferably of not more than 25°, and most preferably of not more than 15°.

[0100] According to one embodiment of the device according to the present invention, the intermediate section of at least one well further comprises a chute (18), said chute being provided between the pipetting stage and the lower section.

[0101] The pipetting stage (17) provides a slope such that any fluid applied thereto can run off. The chute (18) provides a transition between the lower edge of the pipetting stage and the lower section of the well. The chute (18) has a larger inclination than the slope of the pipetting stage (17).

[0102] Sometimes, such “pipetting stage” is also called “ledge”, like e,g, in the microtiter plates produced by InSphero (CH) applying Insphero’s SureXchange technology.

[0103] For adding or aspirating medium from the well, a pipette tip is inserted into the well at the side of the pipetting stage (17) such that any fluid to be supplied to the well hits the pipetting stage first, before flowing smoothly downwards the pipetting stage and along the chute (18) into the lower section (13) of the well. Thereby, the biological specimen is not directly present within the flow path of any fluid supplied to or being aspirated from the well. The pipetting stage (17) provides a slope upon which the vertical positioning of the pipette tip onto this slope forms a gap between the orifice of the pipette tip and the pipetting stage (17). At optimal inclination of the slope, the gap is small enough to prevent a microtissue to be aspirated inadvertently, while assuring efficient aspiration of fluid. This effect is supported by the surface and adhesion tension, so that a small volume remains in the lower section of the well. The chute has a slope, wherein the slope of the chute is steeper than the slope of the pipetting stage within the same well. The pipetting stage and / or the chute may be present in a well on only one inner side of the well, on two adjacent inner sides of the well, on three inner sides of the well, or on all four or all inner sides of the well.

[0104] Further optional details of the shape of the wells as such are for example disclosed in W02017001680A1, the content of which is incorporated herein by reference.

[0105] According to one embodiment, the device according to the present invention comprises a row of wells or an array of wells. According to embodiments, said device 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 embodiments, the multiwell plate is a standard SBS / ANSI format multiwell plate.

[0106] According to embodiments of the device according to the present invention, at least a) the lower section (13) of at least one well, and / or b) at least one section of the base layer (14) is provided with an ultra-low attachment coating.

[0107] Culturing cells in a well which is provided with an ultra-low adherence (ULA) surface maintains the cells in a suspended, unattached state. Under particular circumstances, self assembly of the cells to form a microtissue is thus promoted. Propagating a spheroid or microtissue in a well that is provided with an ultra-low attachment surface maintains integrity of the spheroid or microtissue, and prevents the spheroid or microtissue from undesired attachment-mediated de-differentiation or activation, including an undesired alteration in cell division properties.

[0108] Providing a well of a multiwell plate with an ultra-low attachment surface can be done in various different ways. An example of providing a well with an ultra-low attachment surface is covalently binding a hydrogel layer to the substrate such as polystyrene, said hydrogel being hydrophilic and neutrally charged. Multiwell plates comprising such an ultra-low attachment surface are commercially available from Corning Inc. Another example of an ultra-low attachment surface is a surface made of a polymer consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC). Such ULA coatings are for example present in the Akura™ Spheroid Microplates manufactured by Insphero (CH).

[0109] According to one embodiment of the device according to the present invention, the separation layer (22) is attached to the lower section (13) of at least one well by means of an adhesive layer (20a).

[0110] According to one embodiment of the device according to the present invention, a spacer layer (21) is attached to the lower side of the separation layer (22) by means of an adhesive layer (20b). In this embodiment, the height of the conduit is essentially determined by the thickness of the spacer layer (21).

[0111] According to one embodiment of the device according to the present invention, the base layer (14) that forms the bottom of at least one well and at least one conduit is attached to the lower side of the spacer layer (21) by means of an adhesive layer (20c).

[0112] According to one embodiment of the device according to the present invention, the

[0113] • at least one lower section (13) of at least one well comprises an undercut (19a) or chamber bevel (19b) and / or

[0114] • the base layer (14) comprises at least one chamber bevel (19a) arranged at the edge of the conduit (15).

[0115] These undercuts or chamber bevels facilitate the use of such device in open top light sheet microscopy, by avoiding blind angles. In open top light sheet microscopy, two microscope lenses (excitation lens and collection lens) are being used in inclined fashion from underneath the device to image the biological specimen.

[0116] In one embodiment, the height of at least one undercut or chamber bevel is smaller than the diameter of the biological specimen, like e.g. a microtissue. Note that this also applies, preferably, for an embodiment where undercuts and chamber bevels are combined, as e.g. shown in Figure 5B, left part.

[0117] Typically, a microtissue in the meaning of the above description has a diameter between 100 pm and 500 pm.

[0118] In further embodiments, the height of the undercuts, the chamber bevels or the combination of the two, if applicable, is equal to, or smaller than,3 / 4,2 / s or / i of the diameter of the biological specimen, like e.g. a microtissue.

[0119] These embodiments help to avoid that the biological specimen get trapped in the undercuts. In such way, the biological specimen can remain centered in the lower section of the well. Trapping in the space formed by the undercuts and / or chamber bevels would create light diffraction effects when observing the specimen e.g. by inverted microscopy.

[0120] According to another aspect according to the present invention, the device according to the above description is provided for use in the study of the interaction of two more biological specimen.

[0121] According to another aspect according to the present invention, use of the device according to the above description is provided for the study of the interaction of two more biological specimen.

[0122] According to another aspect according to the present invention, a method for studying the interaction of two more biological specimen is provided, in which method the device according to the above description is used.

[0123] In order to avoid repetitive text passages, it should be understood that embodiments disclosed above regarding a) the different types of biological specimen and b) the types of interaction between the biological specimen shall be deemed disclosed in the context of 1) the device for use, 2) the use of the device and 3) the method for studying as referred to above. Production of the device

[0124] As discussed above, the device can comprise a one-part structure, i.e., without a separate base layer. Such one-part structure can e.g. be produced by 3D printing.

[0125] The device can yet also comprise a two-part structure (not counting adhesive layers or separation layers in). In such embodiment, a base layer that forms the bottom of at least one well and at least one conduit is attached, releasably or irreleasably, from underneath to the wells, e.g., by means of an adhesive or an adhesive layer.

[0126] Such two-part structure can be produced, e.g., by die-casting, molding, embossing or 3D printing methods of suitable material, e.g., thermoplasts including the materials discussed herein..

[0127] Examples

[0128] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0129] Table 3: examples of biological specimen or combinations thereof that can be investigated

[0130] A transwell migration assay is a commonly used test to study, the migratory response of e.g. endothelial cells to angiogenic inducers or inhibitors, or of immune cells to immune cell engaging stimuli.

[0131] In such assay, endothelial cells are grown on a separation layer comprising a permeable membrane or mesh. Then, for example, a solution containing a test agent is placed below the cell permeable membrane. Following an incubation period, the cells that have migrated through the membrane are stained and counted. The membrane is usually coated with some extracellular matrix component (e.g. collagen) which facilitates both adherence and migration.

[0132] In another embodiment, immune cells are added to the well, and the test agent on the other side of the separation layer is for example an immune cell engager, like e.g. an inflammatory cytokine. In this approach, the immune cells have to pass the endothelial layer and the separation layer.

[0133] The main advantage of this assay is its detection sensitivity. Migration through the permeable filter can be caused by very low levels of e.g. angiogenic inducers or an inflammatory cytokines. The device according to the invention allows all this to be done on a microscopic stage with single cell resolution.

[0134] References

[0135] Keim J et al. Biotechnology and Bioengineering, 83(2) 173-180 (2003)

[0136] 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

[0137] Akther, F.; Yakob, S.B.; Nguyen, N.-T.; Ta, H.T. Surface Modification Techniques for Endothelial Cell Seeding in PDMS Microfluidic Devices. Biosensors 2020, 10, 182

[0138] 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

[0139] 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

[0140] WO2015158777A1

[0141] W02019008189A1

[0142] W02017001680A1 1 Reference Numbers

[0143] 10 device for culturing and / or investigating biological specimen

[0144] 11 well

[0145] 12 open upper section of well

[0146] 13 lower section of well

[0147] 14 base layer

[0148] 15, 15a, 15b conduit connecting lower sections of at least two wells

[0149] 16 biological specimen

[0150] 16a tumor sample or tumor microtissue

[0151] 17 pipetting stage within well

[0152] 18 chute within well portion.

[0153] 19a undercut

[0154] 19b chamber bevels

[0155] 20a, 20b, 20c adhesive layers

[0156] 21 spacer layer

[0157] 22 separation layer arranged between the lower section of the well and the conduit

[0158] 23 cell monolayer

[0159] 24 basal parts of wells

[0160] 24a longitudinal grooves in the basal parts of wells

[0161] 25 longitudinal grooves in base layer

[0162] 26 microscope lens

[0163] 27 immune cells

[0164] 28 chemoattractant

[0165] 29 pharmaceutical entity

[0166] 30 cytokine

[0167] 31 tilting axis

[0168] 32 flow in conduits caused by tilting movement

Claims

What is claimed is:

1. A device (10) for culturing and / or investigating biological specimen (16), the device comprising a plurality of wells wherein at least two wells (11) arranged next to one another each comprise• an open upper section (12)• a lower section (13)• wherein the upper section and the lower section are in fluid communication, wherein further at least two lower sections (13) of the at least two wells (11) are connected to one another by a conduit (15).

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

3. The device according to claim 2, wherein the base layer (14) comprises an optically transparent material.

4. The device according to anyone of the aforementioned claims, wherein the biological specimen (16) is one or more selected from the group consisting of• tissue sample• microtissue,• adherent cell monolayer, and / or• suspended cells.

5. The device according to any one of the aforementioned claims, wherein at least one well comprises a separation layer (22) arranged between the lower section (13) of the well and the conduit (15).

6. The device according to any one of the aforementioned claims, wherein said separation layer (22) comprises a mesh or a semipermeable membrane.

7. The device according to claim 6, wherein the mesh or semipermeable membrane comprises an average pore size of between > 0.1 pm and < 100 pm.

8. The device according to any one of claims 5 - 7, wherein said separation layer (22) is coated with at least one of a) a hydrophilic layer, b) one or more ECM peptides or proteins, and / or c) a coating comprising poly-amino acids.

9. The device according to any one of the aforementioned claims, which comprises at least two sets of at least two wells, wherein the two lower sections of the at least two wells of each set are connected to one another by a conduit (15), wherein further at least one set of at least two wells is arranged relative to at least one other set of at least two wells in such way that the conduits (15a, 15b) are arranged in essentially rectangular fashion relative to one another.

10. The device according to any one of the aforementioned claims, wherein the lower section (13) of at least one well is eccentrically arranged with respect to the longitudinal axis of the open upper (12) section thereof.

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

12. The device according to claim 11, wherein the inclination of the pipetting stage (17) has an angle of at least 5°, and of not more than 45°, preferably of not more than 35°, more preferably of not more than 25°, and most preferably of not more than 15°13. The device according to any one of claims 11 - 12, wherein the intermediate section of at least one well further comprises a chute (18), said chute being provided between the pipetting stage and the lower section14. The device according to any one of the aforementioned claims, wherein the device comprises a row of wells or an array of wells.

15. The device according to any one of the aforementioned claims, wherein said device is configured as a multiwell plate, preferably comprising an array of 96 wells, an array of 384 wells or an array of 1536 wells.

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

17. The device according to any one of the aforementioned claims, wherein at least a) the lower section (13) of at least one well, and / or b) at least one section of the base layer (14) is provided with an ultra-low attachment coating.

18. The device according to any one of claims 5 - 17, wherein the separation layer (22) is attached to the lower section (13) of at least one well by means of an adhesive layer (20a).

19. The device according to claim 18, wherein a spacer layer (21) is attached to the lower side of the separation layer (22) by means of an adhesive layer (20b).

20. The device according to claim 19, wherein the base layer (14) that forms the bottom of at least one well and at least one conduit is attached to the lower side of the spacer layer (21) by means of an adhesive layer (20c).

21. The device according to any one of the aforementioned claims, wherein• at least one lower section (13) of at least one well comprises a chamber bevel (19b) and / or• the base layer (14) comprises at least one undercut (19a) arranged at the edge of the conduit (15)22. The device according to any one of the aforementioned claims for use in the study of the interaction of two more biological specimen.

23. Use of the device according to any one of the aforementioned claims for the study of the interaction of two more biological specimen.

24. A method for studying the interaction of two more biological specimen, in which method the device according to any one of the aforementioned claims is used.

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