Novel vertical incubation chamber for cell plates

Abstract

The present invention relates to a novel vertical incubation chamber for cell plates, comprising of a hollow body; means for vertical fixation of cell plates on two or more side walls; indentations at the side walls; and an indentation at the bottom wall of the body for introducing a stirring element. The invention specifically relates to a device which allows vertical particle-cell interaction experiments in a non-static system. The invention also relates to the use of said device, an array of said devices, a kit of parts comprising said device, a mounting plate for analysis, and a method for analyzing cell interactions using the device.

Description

[0001] NOVEL VERTICAL INCUBATION CHAMBER FOR CELL PLATES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a novel vertical incubation chamber for cell plates, comprising a hollow body and means for vertical fixation of cell plates on two or more side walls, indentations at the side walls, and an indentation of the bottom wall of the hollow body for a stirring element. The invention specifically relates to a device which allows vertical particle-cell interaction experiments in a non-static system. The invention also relates to the use of said device, an array of said devices, a kit of parts comprising said device, a mounting plate for analysis, a positioning plate for magnetic stirring, and a method for analyzing cell interactions using the device.

[0004] BACKGROUND OF THE INVENTION

[0005] Nano- and micro-sized vehicles are important carriers for diagnostic and therapeutic agents. Nanoparticles, microparticles, microcapsules as well as drug conjugates are used as versatile vehicles for drug delivery, which can be easily modified for selective tissue targeting.

[0006] The cellular dose describes the measured number of particles or drug formulation that get in contact with a cell layer, in other words, it measures the degree of particlecell or substance-cell interactions. The cellular dose depends highly on the shearing motion or shear stress and the physicochemical properties of particles such as size, shape, or surface characteristics, and is less influenced by the number of particles added. Thus, modelling interactions between particles and biological systems in vitro provides fundamental understanding for the development of drug delivery vehicles.

[0007] Horizontal cell monolayers are commonly used as in vitro particle-cell interaction testing systems; however, they often cannot model the effects of particle sedimentation and flotation, or dynamic flow conditions occurring in vivo. Recent studies have shown that particle suspensions containing large and dense particles, or porous particles, are not well dispersed despite Brownian motion (Feliu et al., 2017). Larger and denser particles show a higher cellular dose than smaller particles due to their higher sedimentation velocity, which increases contact probability with the cell layer. Highly porous particles or capsules with a low-density core float and their reduced cell contact leads to a lower cellular dose. As a result, the outcome of conventional, horizontal cell interaction experiments can be heavily influenced by sedimentation or flotation artefacts. Inverted particle-cell interaction testing systems characterized by inversion of cell monolayers have been described to overcome this problem (Cho et al., 2011), however, inversed cell monolayers do not have the capacity to model the three-dimensional structure of hollow organs in vivo.

[0008] CN112450067A and CN213819292U disclose a culture dish holder with brackets for vertical fixation of a petri dish which is connected to a turntable. A motor can continuously rotate the turntable. A plurality of petri dish holders can be arranged as a rack which can be placed in an incubator.

[0009] CN112505042B discloses an observation system to control cell-interaction and growth using a camera.

[0010] US2018057784A1 discloses a bioreactor for culturing cells, comprising a culture screen holder with grooves or slits on the internal surface of the sidewalls to horizontally insert culture screens.

[0011] US2021047595A1 discloses a cell culture device comprising a culture container and a rotating polymeric porous film housing part within the culture container, which is rotated with e.g., a magnetic stirrer.

[0012] EP3476489A1 discloses a centrifuge insert suited for at least two sample containers or carriers.

[0013] CN111548938A discloses a culture bottle for suspension and adherent cell culture, comprising a breathable film.

[0014] CN220597420U discloses a culture dish fixing frame for vertically placed culture dishes.

[0015] CN216670465U discloses a system for capturing an image of a culture dish.

[0016] CN1 16083216A discloses a culture dish with three separate culture areas, which allows to cultivate different microbial cells.

[0017] EP3395939A1 discloses a vessel which allows for isolation, culture, and proliferation of cells.

[0018] CN1 14085776A discloses a system for adherent cell culture.

[0019] CN1875093A discloses a tissue culture vessel for co-cultivating different types of cells and tissues.

[0020] CN217733139U discloses a cuboidal cell culture dish for multi-objective microscopy. CN104096265B describes a three-dimensional contraction model for constructing an artificial blood vessel model.

[0021] CN206843961 U describes a light barrier including a base plate and a column, wherein the base plate is fixed on the ground and the column is fixed vertically on the base plate.

[0022] Vertical particle-cell interaction testing systems allow a vertical positioning of cellcontaining plates to better represent the in vivo conditions of hollow organs (Cui et al., 2016). However, dynamic flow, an important parameter to model physiological substance or particle-cell interactions of human organs, has so far not been considered. Dynamic flow and shear stress may also alter the cell surface and consequently, impact internalization behavior of soluble substances.

[0023] A recently described system introduced dynamic flow to a horizontal particle-cell experiment by tilting the cell plate from side to side (Bjbrnmalm et al., 2016), however, tilting a horizontal plate was not sufficient to model the dynamic multidimensional flow or the influence of shear stress on cells of in vivo systems.

[0024] Therefore, there is still the need for a device which allows particle-cell interaction experimentation of vertically positioned cells under non-static conditions.

[0025] SUMMARY OF THE INVENTION

[0026] It is the objective of the present invention to provide a device which enables particle-cell interaction determination of cells under non-static conditions.

[0027] The problem is solved by the present invention.

[0028] It has been shown by the inventors that particle-cell interaction determination in a non-static system can be successfully performed in an incubation chamber which holds two or more cell plates vertically and accommodates for a stirring element. Said device was shown to be easy to handle, and also allows cheap and thus highly economic determination of cell-particle interaction.

[0029] According to the invention there is provided a device comprising an open-top hollow body (1), comprising at least 4 side walls (2), comprising a) slits or rails (3) for vertical fixation of a cell plate (4) at two or more side walls; b) an indentation (5) in the side wall behind the cell plate; and c) an indentation (6) at the bottom, specifically of the bottom wall of the hollow body. In an embodiment of the invention, the shape of the device is a cube, a cylinder, or a prism, specifically, of pentagonal, hexagonal, heptagonal, or octagonal prismatic shape.

[0030] According to a further embodiment, the device is made from plastic, glass, metal, or a polymer, specifically, the device is sterilizable or autoclavable.

[0031] According to an embodiment, the slits or rails (3) are parallel to the side walls (2), specifically, at an angle of 90 degrees + / - 5 degrees to the bottom wall of the device.

[0032] According to a further embodiment, the slits or rails (3) are size-fitting for the cell plate (4) and end above the indentation at the bottom wall (6).

[0033] In a further embodiment the indentation in the side wall (5) ends above the cell plate (4).

[0034] Provided in a further embodiment is an indentation at the bottom wall (6) of the device, specifically, said indentation is cylindrical or outwardly curved and allows for accommodation of a stirring element.

[0035] In a further embodiment the stirring element (7) is a magnetic stirrer bar, or a propeller.

[0036] According to a further embodiment, the device comprises a stirring element (7) in the bottom wall indentation (6), specifically, a magnetic stirring bar.

[0037] In an embodiment of the invention, the cell plate (4) is made from glass, specifically it is a coverslip, plastic, or a polymer, specifically it is a translucent material.

[0038] In a further embodiment, the cell plate (4) is a membrane, specifically, a bioprinted membrane, more specifically, a membrane which allows cells to invade.

[0039] In a further embodiment, the cell plate (4) is of square, polygonal, or round shape.

[0040] According to a further embodiment, the cell plate (4) is size-fitting for the slits or rails of the device.

[0041] According to a further embodiment, the cell plate (4) is untreated, or surface- treated with one or more cell attachment-facilitating substances, specifically fibronectin, laminin, collagen, gelatine, fetal calf serum, or poly-lysine.

[0042] In an embodiment of the invention, the device comprises of a connection element (13) at least at one side wall.

[0043] According to the invention there is also provided an array of devices comprising two or more devices connected to each other, or comprising two or more devices attached by one or more connection elements (13). Provided in a further embodiment is a lid (8; 16).

[0044] In a further embodiment, the lid (8; 16) is size-fitting for the device or the array of devices as described herein.

[0045] According to a further embodiment, the lid (8; 16) comprises a slit or rail (9) for horizontal fixation of a cell plate (4).

[0046] In a further embodiment, the lid (8; 16) is of the same material as the device, or of a different material, specifically it is plastic, glass, metal, or a polymer.

[0047] According to a further embodiment, the lid (8; 16) comprises a sealing ring.

[0048] In a further embodiment, the lid (8; 16) comprises nubs (17) on the inside of the lid for placing the lid on the device.

[0049] According to the invention there is further provided a kit comprising a) the device, or the array of devices described herein; b) a mounting plate (10) comprising i. one or more recesses (11 ) with beveled edges for placement of at least one cell plate, ii. a hole (12) at the bottom of each recess; and c) optionally, at least two cell plates; and d) optionally, tweezers for placement of cell plates.

[0050] According to a further embodiment, there is further provided a kit comprising a) the device, or the array of devices described herein; b) a positioning plate (14) comprising one or more recesses (15) for placement of the device; c) optionally, at least two cell plates; and d) optionally, tweezers for placement of cell plates.

[0051] According to a further embodiment, there is further provided a kit comprising a) the device, or the array of devices described herein; b) a mounting plate (10) comprising iii. one or more recesses (11 ) with beveled edges for placement of at least one cell plate, iv. a hole (12) at the bottom of each recess; and c) a positioning plate (14) comprising one or more recesses (15) for placement of the device or the array of devices; d) optionally, at least two cell plates; and e) optionally, tweezers for placement of cell plates.

[0052] In an embodiment of the invention, the kit comprises a mounting plate (10) or a positioning plate (14) made from plastic, metal, or a polymer, specifically, the mounting plate is sterilizable or autoclavable.

[0053] In a further embodiment, the kit comprises a mounting plate (10), wherein the size of the mounting plate is adapted for a plate reader.

[0054] According to a further embodiment, the kit comprises a cell plate, wherein the cell plate is made from glass, specifically, it is a coverslip, plastic, or a polymer,

[0055] In a further embodiment, the kit comprises a cell plate, wherein the cell plate is a membrane or a scaffold.

[0056] In a further embodiment, the kit comprises a cell plate wherein the cell plate is made from translucent material.

[0057] In a further embodiment, the kit comprises a cell plate, wherein the cell plate is of square, polygonal, or round shape.

[0058] According to a further embodiment, the kit comprises a cell plate, wherein the cell plate is size-fitting for the slits or rails.

[0059] In a further embodiment, the kit comprises a cell plate, wherein the cell plate is untreated, or surface-treated with one or more cell attachment-facilitating substances, specifically, fibronectin, laminin, collagen, gelatine, fetal calf serum, or poly-lysine.

[0060] According to the invention there is provided a mounting plate (10) comprising a) one or more recesses (11 ) with beveled edges for placement of at least one cell plate, and b) a hole (12) at the bottom of each recess.

[0061] According to an embodiment of the invention, there is further provided a combination of a device or an array of devices described herein and a mounting plate (10) comprising a) one or more recesses (11 ) with beveled edges for placement of at least one cell plate, and b) a hole (12) at the bottom of each recess.

[0062] In an embodiment of the invention, the mounting plate is made of plastic or metal, or a polymer, specifically, a sterilizable or autoclavable material.

[0063] According to a further embodiment, the size of the mounting plate is adapted for a plate reader. According to the invention there is further provided a positioning plate (14) comprising one or more recesses (15) for placement of at least one device in the one or more recesses.

[0064] According to an embodiment of the invention, there is further provided a combination of a device or an array of devices described herein and a positioning plate (14) comprising one or more recesses (15) for placement of at least one device in the one or more recesses.

[0065] In an embodiment of the invention, the positioning plate is made of plastic, metal, or a polymer, specifically a sterilizable, autoclavable and / or 3D-printable material.

[0066] In a further embodiment, the size and shape of the positioning plate is appropriate for use with a magnetic agitator or stirrer.

[0067] According to the invention, the device, the array of devices, the kit, the combination comprising the device and the mounting plate, or the combination comprising the device and the positioning plate described herein is used for analyzing cell interaction, specifically, for analyzing protein-cell interactions, cell-cell interactions, cell adhesion to a surface, the effect of shear stress on cells, microbiome formation, or particle-cell interactions, specifically, microparticles, nanoparticles and microcapsules.

[0068] According to the invention there is also provided a method for analyzing cell interactions using the device, the array of devices, or the combination comprising the device and the mounting plate, or the combination comprising the device and the positioning plate or the kit described herein, comprising a) adhering cells to the cell plate (4); b) placing the cell plate in a slit or rail (3) with adherent cells facing inwards; c) optionally, washing the cells with a buffer solution; d) introducing a test solution or suspension into the hollow body; e) placing a magnetic stirrer (7) at the indentation at the bottom wall (6); f) optionally, placing the device on a positioning plate (14) and the positioning plate on a magnetic agitator; g) incubating the cells with the test solution or suspension under stirring conditions; h) optionally, washing the cells with a buffer solution; i) optionally, placing the cell plates on a mounting plate (10); j) analyzing of test substance-cell interactions. According to an embodiment of the invention, the method for analyzing cell interactions comprises adherent cells, specifically, epithelial cells, endothelial cells, or fibroblasts.

[0069] DESCRIPTION OF DRAWINGS

[0070] Figure 1 : A. Incubation chamber. A device comprising an open-top hollow body (1 ), comprising at least 4 side walls (2), comprising slits or rails (3) for vertical fixation of a cell plate (4) at two or more side walls, an indentation (5) in the side wall behind the cell plate, and an indentation (6) at the bottom wall of the hollow body for accommodation of a stirring element (7). The incubation chamber is shown from three different angles (top, middle, bottom). The spiral pattern displayed on a side wall is a graphical element without any technical function. B. Lid for incubation chamber with fixation means for cell plate. A lid (8) comprising a slit or rail (9) for horizontal fixation of a cell plate is shown from two different angles (left, right). C. Lid for incubation chamber without fixation means for cell plate. A lid (8) without slits or rails for fixation of a cell plate (left). The spiral pattern displayed on the outside of the lid is a graphical element without any technical function (right). D. Mounting plate for cell plates. A mounting plate (10) comprising one or more recesses (11) with beveled edges for placement of at least one cell plate, and a hole (12) at the bottom of each recess. One recess on top left is highlighted by a circle. Holes on the right side of the plate facilitate use with a plate reader.

[0071] Figure 2: Cyto-adhesive potential of microparticles. The cyto-adhesive potential of microparticles is shown in % of cell interaction for cancerous urothelial cell line 5637 and non-cancerous SV-HUC-1. Experiments were performed in the incubation chamber (‘CellCube’) and on monolayers with non-functionalized (blank-MP) and wheat germ agglutinin (WGA)-functionalized (WGA-MP) microparticles (p < 0.05, **p < 0.01). Particle-cell interaction was measured using the mounting plate of Figure 1 D in a plate reader.

[0072] Figure 3: Cyto-adhesive potential of microcapsules. The cyto-adhesive potential of microcapsules is shown in % of cell interaction for cancerous urothelial cell line 5637 and non-cancerous SV-HUC-1. Experiments were performed in the incubation chamber (‘CellCube’) and on monolayers with non-functionalized (blank-MC) and WGA functionalized (WGA-MC) microcapsules (p < 0.05, **p < 0.01). Particle-cell interaction was measured using the mounting plate of Figure 1 D in a plate reader.

[0073] Figure 4: Cyto-adhesive potential of nanoparticles. The cyto-adhesive potential of nanoparticles is shown in % of cell interaction for cancerous urothelial cell line 5637 and non-cancerous SV-HUC-1. Experiments were performed in the incubation chamber (‘CellCube’) and on monolayers with non-functionalized (blank-NP) and WGA functionalized (WGA-NP) nanoparticles (p < 0.05, **p < 0.01). Particle-cell interaction was measured using the mounting plate of Figure 1 D in a plate reader.

[0074] Figure 5: Comparison of particle-cell interactions in monolayer experiments and cuboidal incubation chamber. Interactions of nanoparticles (NP), microparticles (MP), microcapsules (MC), and soluble ligands (fluorescent wheat germ agglutinin, fWGA) with 5637 cells attached to cell plates without prior cell plate coating in an incubation chamber were compared to cell monolayers. Cells were incubated for 8 hours at 37°C under stirring and non-stirring conditions (indicated by stripes; ‘static’). Particle-cell interactions were analyzed following detaching of cells from the cell plates and measuring interactions by flow cytometry. Interactions with monolayers were normalized to 100% to form a baseline, and interactions with cells in the incubation chamber are shown as percentage relative to this baseline.

[0075] Figure 6: A. Incubation chamber with pentagonal prismatic shape. Incubation chamber comprising an open-top hollow body (1), side walls (2), slits or rails (3) for vertical fixation of a cell plate, an indentation (5) in the side wall behind the cell plate, and an indentation (6) at the bottom wall of the hollow body for accommodation of a stirring element. B. Incubation chamber with hexagonal prismatic shape. Incubation chamber comprising an open-top hollow body (1), side walls (2), slits or rails (3) for vertical fixation of a cell plate, an indentation (5) in the side wall behind the cell plate, and an indentation (6) at the bottom wall of the hollow body for accommodation of a stirring element. C. Incubation chamber with heptagonal prismatic shape. Incubation chamber comprising an open-top hollow body (1), side walls (2), slits or rails (3) for vertical fixation of a cell plate, an indentation (5) in the side wall behind the cell plate, and an indentation (6) at the bottom wall of the hollow body for accommodation of a stirring element. D. Incubation chamber with octagonal prismatic shape. Incubation chamber comprising an open-top hollow body (1), side walls (2), slits or rails (3) for vertical fixation of a cell plate, an indentation (5) in the side wall behind the cell plate, and an indentation (6) at the bottom wall of the hollow body for accommodation of a stirring element.

[0076] Figure 7: A. Array of devices. Device comprising an open-top hollow body (1), comprising side walls (2), slits or rails (3) for vertical fixation of a cell plate, an indentation (5) in the side wall behind the cell plate, and an indentation (6) at the bottom wall of the hollow body for accommodation of a stirring element, connected or attached with another device through a connection element (13) to form an array. B. Cross-section of device shown in Figure 7A. A device as shown in Fig. 7A comprising a connection element (13) can connect or attach to another device to form an array. C. Lid for array of devices. A lid (16) for an array of devices (1 ) as shown in Fig. 7A. D. Bottom-up view of the lid (16) as described in Fig. 7C comprising knobs or nubs (17) for fixing the lid on the device and to allow for gas exchange while avoiding tight sealing.

[0077] Figure 8: A. Positioning plate for incubation chambers. One or more incubation chambers can be placed on a positioning plate (14) which comprises one or more recesses (15) with straight edges. The positioning plate can be placed onto a magnetic agitator to induce stirring of a magnetic stirrer in the incubation chambers. Upwards- facing protrusions are at the corners of the positioning plate. The spiral pattern displayed within the recesses and the pattern on the corner protrusions are a graphical element without any technical function. B. Side-view of positioning plate for incubation chambers.

[0078] Figure 9: Cell adhesion and growth on cell plates with different coatings. Cell plates were coated with coating materials poly-L-lysine, laminin, collagen, fibronectin and fetal calf serum (FCS). HEK or CaCo-2 cells were seeded onto coated and uncoated cell plates and growth curves for each cell line were monitored using the PHIO Cell Watcher. Confluency tracked over a period of around 300 hours is shown in %.

[0079] Figure 10: Cell interactions in cuboidal and hexagonal incubation chamber. Interactions of nanoparticles incubated with HeLa, Caco-2 and 5637 cells cultured under stirring conditions as monolayers (‘wellplate’), or in a device (‘FlowCube’) comprising 4 cell plates (slides; coverslips) or 6 cell plates were measured. Cell plates coated with poly-L-lysine before cell attachment are indicated by stripes. Incubation was conducted for 2 hours at 37°C with stirring at 350 rpm. Particle-cell interactions were analyzed following detaching of cells from the cell plates and measuring interactions by flow cytometry. Cell interaction is indicated in % (**p < 0.01). DETAILED DESCRIPTION

[0080] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017), and Murphy & Weaver.

[0081] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0082] The term “about” or “around” as used herein refers to the same value or a value differing by + / - 5 % of the given value.

[0083] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

[0084] The herein referred to “device” refers to a device comprising an open-top hollow body for placing cell plates, specifically for experimentation and testing, comprising at least 4 side walls.

[0085] The term “incubation chamber” may be used interchangeably with the term “device”.

[0086] Specifically, the device comprises an open-top hollow body comprising at least 4 side walls; slits or rails for vertical fixation of a cell plate at two or more side walls; an indentation in the side wall behind the cell plate; and an indentation at the bottom wall of the hollow body for a stirring element.

[0087] Specifically, the device is made from plastic, glass, metal, or a polymer, specifically, but not limited to, clear resin, synthetic resin, polyether ether ketone (PEEK), polypropylene (PP), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polystyrene (PS), or nylon. Specifically, the device is made from a biocompatible material and / or a sterilizable or autoclavable material. Specifically, the device is made from a 3D-printable material, or it is produced by cast molding, fused deposition modeling (FDM), stereolithography (SLA), selective laser sintering (SLS), poly jet, direct metal laser sintering (DMLS), or multi jet fusion (MJF). The shape of the device can be, but is not limited to, a cube, a cylinder, or a polygonal prism, e.g., it can be, but is not limited to, having a pentagonal, hexagonal, heptagonal or octagonal prismatic shape. The device can have a flat bottom wall, or can have means to provide a horizontal position of the bottom wall, e.g., through attached legs.

[0088] The device can be of any size appropriate for the cell plates and particle-cell determination.

[0089] In a specific embodiment, the side length of a cube-shaped or rectangular device is in the range of 15 to 25 mm, specifically 16-20 mm, more specifically 18 mm. Specifically, the side length of a rectangular device can be about 20 mm. The height cube-shaped or rectangular device can be in the range of 15-25 mm, specifically 16-20 mm, more specifically 18 mm or 19 mm.

[0090] In another specific embodiment, the side length of a device with a polygonal shape e.g., with pentagonal, hexagonal, heptagonal or octagonal prismatic shape, is in the range of 10 to 30 mm, specifically, in the range of 10 to 20 mm, more specifically, 13 to 20 mm. Specifically, the side length of a pentagonal device can be about 17 mm. Specifically, the side length of a hexagonal device can be about 15 mm. Specifically, the side length of a heptagonal device can be about 14 mm. Specifically, the side length of an octagonal device can be about 13 mm. The height of a polygonal device can be in the range of 15-25 mm, specifically 16-20 mm, more specifically about 18 mm or 19 mm.

[0091] Specifically, a rectangular device has a side length of 20 mm and a height of 19 mm. Specifically, a pentagonal device has a side length of 16.96 mm and a height of 19 mm. Specifically, a hexagonal device has a side length of 15.33 mm and a height of 19 mm. Specifically, a heptagonal device has a side length of 14.28 mm and a height of 19 mm. Specifically, an octagonal device has a side length of 13.55 mm and a height of 19 mm.

[0092] Specifically, the hollow body of the device can hold around 0.5 to 10 ml, specifically, 1 to 5 ml of liquid, e.g., cell culture medium or test solution or test suspension. Specifically, a cube-shaped or rectangular device can hold around 1 to 1.5 ml of liquid. Specifically, a heptagonal device can hold around 2 to 3 ml of liquid. Specifically, an octagonal device can hold around 3 to 4 ml of liquid. Specifically, a cubeshaped or rectangular device can hold 1 .2 ml. Specifically, a hexagonal device can hold 2.9 ml. Specifically, an octagonal device can hold 3.6 ml. As used herein the term “open-top hollow body” refers to a structure or object comprising at least 4 side walls, and a bottom wall.

[0093] As used herein the term “cell plate” refers to a carrier for isolated cells, appropriate for adhering said cells to the plate. The cell plate can be of any shape, thickness, or size applicable for use in the device described herein. Specifically, the shape of said cell plate can be round, square, or polygonal. Specifically, the size and thickness of said cell plate is appropriate to fit into the slits, rails, or means for fixation on the side wall and / or on the lid of the device. Specifically, the cell plate is optically transparent such that the contents of the plate can be viewed. Specifically, the cell plates can be made from glass, plastic, or a polymer specifically it is a coverslip. Specifically, the cell plate has a plain surface. Specifically, the cell plate can be made from any biocompatible material, or materials used as cell culture vessels (e.g., polystyrene, polycarbonate, stiff resins, glass), or appropriate material for cell culture, or other materials as can be envisioned by one of ordinary skill in the art.

[0094] The term ‘cell plate’ also encompasses a cell plate which comprises one or more cell attachment-facilitating substances on its surface, specifically compounds representing extracellular matrices, which can be, but are not limited to, collagen, fibronectin, laminin, gelatine, poly-lysine or fetal calf serum (FCS). Specifically, the cell plate can be pre-treated by etching, which can be, but is not limited to, etching by sulfuric acid and hydrogen peroxide.

[0095] Specifically, the cell plate is not a petri-dish, microplate, a microtiter plate, a PCR plate or a deep well plate.

[0096] Specifically, the cell plate has a thickness of 0.10 to 1.50 mm, more specifically 0.10 to 1.00 mm.

[0097] Specifically, the cell plate has a side length of 8 to 25 mm, specifically 18, 20, or 22 mm.

[0098] Specifically, the cell plate can be a coverslip. Specifically, said coverslip may have a thickness in the range of about 0.10 to 0.50 mm, specifically, 0.10 to 0.20 mm, more specifically a thickness of 0.13 to 0.17 mm, more specifically 0.13 to 0.16 mm.

[0099] Alternatively, the cell plate can be a membrane or a scaffold, specifically, a bioprinted membrane which allows cells to invade and form tissue-like structures. Specifically, the cell plate can comprise a hydrogel. Said membrane cell plates may be bio-printed. Specifically, said membrane cell plates have a side length of 8 to 25 mm. □

[0100] -14-

[0101] Specifically, said membrane cell plates have a thickness in the range of about 0.1 to 1.5 mm, more specifically, a thickness in the range of 0.5 to 1 mm. Specifically, said bioprinted membrane cell plates have a thickness in the range of about 0.1 to 1 .5 mm, more specifically, 0.5 to 1 mm. Specifically, said membrane cell plates have a pore size in the range of about 0.1 to 100 pm, more specifically, a pore size of 0.1 to 0.4 pm. Specifically, said membrane cell plates have a pore size of about 0.4 pm. Specifically, said membrane cell plates have a frame around the membrane or scaffold. Specifically, said membrane cell plates are size-fitting for slits or rails of the device. Cells may adhere and grow on and / or into the membrane or scaffold of said membrane cell plates. Specifically, cells are bio-printed together with bioink while printing of the membrane cell plates and may proliferate within the membrane. Non-limiting examples for bioinks for bioprinting are GelMA (Gelatine-Methacryloyl) and derivatives thereof. Optionally, bioinks can comprise gelatine, fibrine, collagen, hyaluronic acid but also synthetic polymers such as PLGA / PLA / PCL, PEG, or Pluronic, and optionally, may be optimized or customized depending on the cell type. Alternatively, cells attach or adhere to membrane cell plates within the device after seeding, optionally, cells invade membrane cell plates.

[0102] “Slits or rails” as used herein refer to means for the vertical fixation of cell plates in the device. Means for fixation can be slits, and rails, but may also be indentations, or fixation clips. Specifically, the slits or rails are size-fitted to accommodate the cell plates, and to maintain the cell plates in position. Specifically, the cell plates can be inserted into the device through the slits or rails or removed from the slits and rails without removing the adherent cells on the plates, optionally using tweezers. Slits or rails are located on at least two side walls, but can be on more side walls depending on the total amount of side walls. Exemplarily, in a device containing four side walls there can be two, three, or four slits or rails. Specifically, the slits or rails can be parallel to the side walls, but can also be at an angle of 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 degrees relative to the horizontal bottom wall of the device to allow the cell plate to be attached at an angle slightly different than 90°. More specifically, the slits or rails are at an angle of 90 degrees + / - 5 degrees to the bottom wall of the device. Specifically, if the indentation at the bottom wall extends beyond the edges of the cell plates, the slits or rails end above the indentation at the bottom wall of the device, to place and maintain them above the indentation accommodating the stirring element. The slits or the rails can be of any shape that is suitable for receiving, holding, or guiding the cell plate. They can be continuous or non-continuous.

[0103] “Indentation” as used herein refers to an indentation at the bottom wall of the device, and an indentation at a side wall, specifically, a recess of the side walls.

[0104] Specifically, the indentation at the side wall is directed outwards, and the shape of the indentation can be e.g. round, square, or polygonal. Specifically, the size of the indentation at the side wall is appropriate for a tool for placing and / or removing cell plates in the device, specifically tweezers or tweezer-like tools. Specifically, each side wall with slits or rails for placing cell plates has an indentation. Specifically, the indentation at the side wall ends above the bottom end of the inserted cell plate.

[0105] Specifically, the indentation at the bottom wall of the device is directed outwards, and its bottom can be of any shape, e.g., round, square, or polygonal, forming a cylindrical or curved outwardly-shaped hollow space. Specifically, the indentation at the bottom wall of the device allows for accommodation of a stirring element. Specifically, the size of the indentation at the bottom wall of the device is appropriate for accommodating a stirring element. Specifically, the indentation at the bottom wall of the device accounts for 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the area of the bottom of the device and is size-fitting to accommodate said stirring element.

[0106] Specifically, the device comprises a stirring element placed in the bottom indentation. Specifically, the device comprises a magnetic stirring bar placed in the bottom indentation.

[0107] The term “stirring element” as used herein refers to means for inducing dynamic flow in the incubation chamber, specifically, can be a magnetic stirring element, or a propeller or propeller-like element. The stirring element can be placed through the opentop hollow body of the device, e.g., a magnetic stirrer, or, the stirring element can consist of multiple parts, with one part inserted through the bottom of the device which is tightly sealed, and one part inserted through the hollow body, e.g., a rotor located inside the indentation at the bottom wall of the device, with an externally located motor.

[0108] The term “lid” as used herein refers to an additional part separate of the device or connected to the device, e.g. via a tab, or loop. The lid can be made of the same material as the device, but can also be of a different material. The lid can be composed of plastic, glass, metal, or a polymer. Specifically, the material can be, but is not limited to, clear resin, synthetic resin, polyether ether ketone (PEEK), polypropylene (PP), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polystyrene (PS) or nylon. Specifically, the lid is made from a biocompatible material and / or a sterilizable or autoclavable material. Specifically, the lid is made from a 3D-printable material, or it is produced by cast molding, fused deposition modeling (FDM), stereolithography (SLA), selective laser sintering (SLS), poly jet, direct metal laser sintering (DMLS), or multi jet fusion (MJF).

[0109] The lid can comprise a slit or rail or any other mean for fixation of a cell plate. Specifically, the lid can comprise means for horizontal fixation of a cell plate, which allow insertion and removal of the cell plate on the lid without interfering the adherent cells on the plate.

[0110] The size of the lid is appropriate for the lid to be placed on the open top of the device, or on top of the array of devices, specifically, to add and remove the lid without interfering with the cell plate on the lid or the cell plates on the side walls. Specifically, the lid can be the same size as the top opening of the device, or array of devices, or bigger, specifically, 100%, 110%, 120%, 130%, 140%, or 150% the size of the top opening of the device to allow easier grip and handling. Specifically, the lid can be of round, square or polygonal shape. Specifically, the lid can comprise a square bulge the same size of the open top to fit onto the device, or the lid can be of a different size than the open top of the device and can comprise means for fixation such as connection elements for reversible placement.

[0111] Specifically, the lid comprises nubs or knobs on the inside of the lid for placing the lid on the device, which thereby allows air exchange.

[0112] Specifically, the lid can comprise an indentation to place a sealing ring. Specifically, the lid comprises a sealing ring.

[0113] “Sealing ring” as used herein refers to a mechanical gasket in the shape of a torus; it is a loop of elastomer with a round cross-section, designed to be seated in a groove and compressed during assembly between two or more parts, forming a seal at the interface. Specifically, the ring is made from flexible material, which allows waterproof sealing of the lid onto the device. Specifically, it is made from biocompatible material, specifically, silicone or elastomeric materials.

[0114] “Connection element” as used herein refers to one or more elements on one or more outer side walls of the device described herein to connect two or more of the devices. Specifically, connection elements can be, but are not limited to, mortise and tenon joints, bridle joints, or hook-and-loop joints. Specifically, connection elements can be located on an outer side wall and connect two devices through fastening means, such as, but not limited to screws, nails or rivets. Specifically, connection elements on an outer side wall can be formed by the side wall, or can be separate objects which are attached or adhered to the side wall, such as through joints, hooks, rivets, or other means to attach connection elements to a side wall. Alternatively, the devices can be interconnected via holes in the side walls, wherein the devices may further contain indentations for accommodating sealing elements such as sealing rings for connecting the devices water-tight. Specifically, devices can be interconnected via holes in the side walls comprising a membrane, more specifically, a semi-permeable or permeable membrane. Specifically, the membrane can be bio-printed. Specifically, a removable membrane comprising a cell layer may be used. Specifically, said membrane can have a pore size of about 0.4 pm to 10 pm. One non-limiting example is a modified transwell culture insert such as a commercially available snapwell insert. A snapwell insert may be of polyester, or polycarbonate. The snapwell insert may comprise a tissue culture- treated membrane supported by a detachable ring. Adherent cells may be grown on said membrane while the membrane is within the device, or outside of the device. Said removable membrane can be placed within the device once adherent cells are grown to confluency. Specifically interconnected devices are used for permeability assays.

[0115] “Array” or “array of devices” as used herein refers to two or more devices which can be handled together. An array of devices can be connected through connection elements. Connection elements can connect two or more devices to each other, specifically, two, three, four, five, six, seven, eight, nine, ten, or more devices to form an array of devices. The array of devices may form a consecutive line, a square or rectangle, but can be of any shape appropriate for handling of more than one single device, such as a star-like arrangement. Specifically, each device of an array comprises a magnetic stirrer placed in the bottom indentation.

[0116] “Tweezers” as used herein include devices comprising of two connected handle members each consisting of a handle part held by a finger, and a pressing part. Specifically, tweezers can be made from metal, or polymers. Specifically, tweezers can be made from a biocompatible material, and / or a sterilizable or autoclavable material.

[0117] A “mounting plate” as used herein refers to a plate for analysis, separate from said device. The mounting plate can be made from, but is not limited to plastic, metal, or a polymer, specifically a sterilizable and / or autoclavable and / or 3D-printable material. Specifically, the size and shape of the mounting plate is appropriate for use with a plate reader, e.g., this can be, but is not limited to, Multiskan or Varioskan models by Thermo Fisher Scientific, BioTek® or Accuris models by Sigma Aldrich, or Infinite® models by Tecan, or other plate readers, microplate photometers or other instruments which allow for e.g., measuring fluorescence, absorbance, or luminescence.

[0118] Specifically, the mounting plate comprises one or more recesses for placement of at least one cell plate for analysis, with a hole at the bottom of each recess to facilitate photometric measurements. Edges of said recess can be beveled for easier insertion and removal of cell plates. Specifically, the size of said recesses and the hole of the bottom of said recess are appropriate for holding cell plates. Specifically, mounting plates can comprise one or more additional holes or recesses for placement and / or fixation inside the plate reader, depending on the used plate reader.

[0119] The mounting plate can be of any size appropriate for analysis systems. In a specific embodiment, the side lengths are in the range of 60 to 130 mm, specifically in the range of 80 to 120 mm. In a specific embodiment, the mounting plate is rectangular, having one side length in the range of 80-90 mm, specifically of about 85 to 86 mm, more specifically about 85.5 mm, and the other side length is in the range of 110-140 mm, specifically 115-130 mm, specifically 127-128 mm, more specifically 127. 8mm.

[0120] The plate may further have a thickness in the range of 1 to 5 mm.

[0121] A “positioning plate” as used herein refers to a plate, separate from the device described herein. One or more devices comprising a magnetic stirrer can be placed on the positioning plate, and the positioning plate can be placed on a magnetic agitator. Stirring can be induced for multiple devices at the same time by one magnetic agitator.

[0122] The positioning plate can be made from, but is not limited to plastic, metal, or a polymer, specifically a sterilizable, autoclavable and / or 3D-printable material. Specifically, the size and shape of the positioning plate is appropriate for use with a magnetic agitator or stirrer, e.g., this can be, but is not limited to, Cimarec™ Mono Direct or Maxi Direct stirrers from Thermo Fisher Scientific, or inductive magnetic stirrers from 2mag, or other magnetic agitators or magnetic stirring inductor devices.

[0123] Specifically, the positioning plate comprises one or more recesses for placement of at least one device. Edges of said recesses can be straight or can be beveled for easier insertion and removal of devices. Specifically, the size of said recesses is appropriate for holding devices.

[0124] The positioning plate can be of any size appropriate for magnetic agitators. In a specific embodiment, the side lengths are in the range of 60 to 130 mm, specifically in the range of 80 to 120 mm. In a specific embodiment, the positioning plate is rectangular, having one side length in the range of 80-90 mm, specifically of about 85 to 86 mm, more specifically about 85.5 mm, and the other side length is in the range of 110-140 mm, specifically 115-130 mm, specifically 127-128 mm, more specifically 127.8 mm. The positioning plate may further have a thickness in the range of 1 to 5 mm.

[0125] The term “combination” as used herein refers to a combination of the device or the array of devices as described herein and a mounting plate, or a combination of the device or the array of devices as described herein and a positioning plate.

[0126] The term “in vitro” refers to activities that take place outside an organism, such as experimentation, treatment, or measurements done in or on living tissue or cells, in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions. In contrast, the term “in vivo” refers to activities that take place inside an organism, such as experimentation, treatment, or measurements done in or on living mammals, specifically humans or animals.

[0127] The herein referred term “isolated cell” refers to an isolated, eukaryotic cell, specifically extracted from an organism or obtained through propagation or differentiation from a progenitor cell. Specifically, an “isolated cell” is a cell that exists in vitro and is separate from the organism from which it was originally derived. Specifically, an isolated cell can be derived from a eukaryotic organism, specifically from plants, animals, or fungi, specifically the isolated cell can be derived from, but not limited to, Homo sapiens, Mus musculus, or Rattus rattus.

[0128] The herein referred to “adherent cell” refers to a cell that attaches to its environment through specialized proteins on the cell surface. Specifically, an “adherent cell” is attached to its surrounding extracellular matrix, a gel-like structure released by cells into spaces between them. For example, adherent cells may be derived from adipose tissue, bone marrow, umbilical cord, Wharton’s Jelly, placenta, chorionic plate, synovial membrane, or dental pulp. Specifically, adherent cells are human or animal cells, or plant cells. Specifically, an adherent cell can be, but is not limited to, an epithelial cell, endothelial cell, or a fibroblast, specifically, Caco-2 colon cancer cells, 5637 bladder cancer cells, T-24 bladder cancer cells, RT4 bladder cancer cells, HT-1376 bladder cancer cells, KB cells, or SV-HUC-1 uroepithelial cells, or HeLa cells, MSC / TERT, HEK293 cells, Vero cells, and induced pluripotent cells (iPSCs), MDCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, WI-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, PerC6 cells, COR cells, HUVEC cells, or QOR cells. It is within the knowledge of one skilled in the art to select an adherent cell suitable for use in the present invention.

[0129] In an alternative embodiment, the device described herein can also be used for non-adherent cells, e.g. for testing adherence of such cells to surface coating materials. As an example, such cells may be, but are not limited to, Raji-cells.

[0130] “Adhering cells to the cell plate” as used herein refers to the attachment of adherent cells onto the cell plates, while providing an appropriate environment including culture media, pH, temperature, and incubation time appropriate for said cells, which is well known to persons skilled in the art.

[0131] The term “cell monolayer” as used herein refers to adherent cells cultured as a monolayer on a cell carrier, specifically a cell plate. It contrasts with three-dimensional culturing methods such as cell aggregates or organoids.

[0132] “Horizontal cell monolayers” used herein refer to cell monolayers cultured on cell carriers, specifically a cell plate, which are maintained in a horizontal position.

[0133] “Vertical cell monolayers” used herein refer to cell monolayers cultured on cell carriers, specifically a cell plate, which are maintained in a vertical position, or cell monolayers cultured on cell carriers, specifically a cell plate, which are maintained in a horizontal position and placed in a vertical position for experimentation.

[0134] “Inverted cell monolayers” used herein refer to cell monolayers cultured on cell carriers, specifically a cell plate, which are maintained in an inverted position compared to horizontal cell monolayers, e.g., as described by Cho etal., 2011 for experimentation.

[0135] “Cellular dose” used herein is defined as the measured number of particles that gets in contact with a cell monolayer. The cellular dose depends highly on the physicochemical properties of particles such as size, shape or surface characteristics and is less influenced by the number of particles added.

[0136] The term “sedimentation” as used herein refers to the process by which particles in suspension settle out of the fluid in which they are entrained and come to rest against a barrier. Sedimentation occurs due to the particle’s motion through the fluid in response to the forces acting on them, which can be, but are not limited to, gravity, Brownian motion, or centrifugal acceleration. Particles in a vessel that experience gravity will tend to move in a uniform manner in the direction exerted by that force and tend to move to the bottom of the vessel. In a system which is only influenced by gravity and Brownian motion, the sedimentation velocity depends on the size, weight, and density of particles, with larger and denser particles showing a higher sedimentation velocity compared to smaller particles.

[0137] The term “flotation” as used herein refers to phenomena related to the relative buoyancy of objects. Buoyancy or upthrust is an upward force exerted by a fluid that opposes the weight of a partially or fully immersed object. In a column of fluid, pressure increases with depth because of the weight of the overlying fluid. Thus, the pressure at the bottom of a column of fluid is greater than at the top of the column. Similarly, the pressure at the bottom of an object submerged in a fluid is greater than at the top of the object. The pressure difference results in a net upward force on the object. The magnitude of the force is proportional to the pressure difference and is equivalent to the weight of the fluid that would otherwise occupy the submerged volume of the object, i.e. the displaced fluid. For this reason, an object whose average density is greater than that of the fluid in which it is submerged tends to sink. If the object is less dense than the liquid, the force can keep the object afloat.

[0138] The term “cyto-adhesive potential” as used herein refers to the ability of particles to demonstrate particle-cell interactions in experimentation.

[0139] The term “analyzing of test substance interactions” as used herein refers to methods for analysis of cell interactions, specifically, particle-cell interactions, cellular dose and / or cyto-adhesive potential. Non-limiting examples are analyzing of protein-cell interactions, cell-cell interactions, cell adhesion to a surface, the effect of shear stress on cells, microbiome formation, or particle-cell interactions. Specifically, analysis methods include, but are not limited to, flow cytometry, microscopy, cell lysis and sequencing of cells, or the use of a plate reader to measure the relative fluorescence intensity (RFI) of particles interacting with cells following the particle-cell interaction experiments, which are all methods well known to a person skilled in the art. Cells may be detached or dissociated from cell plates prior to analysis, e.g., for flow cytometry. Dissociated adherent cells can be collected using methods from the group consisting of, but not limited to, centrifugation, filtration and tangential flow filtration (TFF).

[0140] Cells can also remain attached to cell plates for analysis, e.g., using a plate reader, specifically, placing cell plates on the mounting plate for use with a plate reader. Specifically, the device or the array of devices described herein may be used for analyzing test substance interactions, optionally, with the mounting plate and / or the positioning plate as described.

[0141] Specifically, a method for analyzing test substance-cell interactions using the device, or the array of devices, or the combination, or the kit as described, comprises following steps: a) adhering cells, specifically adherent cells, more specifically, epithelial cells, endothelial cells, or fibroblasts, to the cell plate; b) placing the cell plate in a slit or rail on the side wall of the device with adherent cells facing inwards; c) optionally, washing the cells with a buffer solution; d) introducing a test substance solution or suspension into the hollow body; e) placing a magnetic stirrer at the indentation at the bottom wall; f) optionally, placing the device on the positioning plate and the positioning plate on a magnetic agitator; g) incubating the cells with the test solution or suspension under stirring conditions; h) optionally, washing the cells with a buffer solution; i) optionally, placing the cell plates on the mounting plate; j) analyzing of test substance interactions.

[0142] The term “dynamic flow” as used herein refers to the multi-dimensional, dynamic movement of fluids occurring in fluid-containing mammalian organs. It contrasts with static flow conditions of culture media occurring in conventional cell monolayer in vitro culture, where particle movement is mostly limited to Brownian motion.

[0143] The herein referred to “non-static system” refers to a system under continuous dynamic flow. The herein referred to “cells under non-static conditions” refer to cells which are exposed to dynamic flow introduced by movement of the culture media through movement of a stirring element in the bottom indentation of the incubation chamber, specifically a magnetic stirrer or a propeller. Specifically, non-static conditions are stirring conditions.

[0144] The herein referred to “particles” can be, but are not limited to, nanoparticles, microparticles, or microcapsules. The device or the array of devices as described herein can be used to analyze particle-cell interactions, optionally, using the mounting plate and / or the positioning plate according to the invention.

[0145] The term “nanoparticles” as used herein refers to a particle of matter of 1 to 500 nanometers in diameter. Nanoparticles in the context of drug delivery can be, but are not limited to, polymeric nanoparticles, dendrimers, inorganic nanoparticles, organic nanoparticles, liposomes, or solid lipid nanoparticles.

[0146] The term “microparticles” as used herein refers to a particle of matter of 0.5 to 100 pm in diameter. Microparticles in the context of drug delivery can be, but are not limited to, polymeric microparticles, inorganic microparticles, or organic microparticles.

[0147] The term “microcapsules” as used herein refers to a spherical particle of 0.1 to 100 pm in diameter, consisting of a core particle or droplet surrounded by a coating or shell. Microcapsule coatings in the context of drug delivery can be, but are not limited to, proteins, ethyl cellulose, polyvinyl alcohol, gelatin, or sodium alginate. The core material can be, but is not limited to, an adsorbent particle, an emulsion, or a suspension.

[0148] “Functionalization” of particles and capsules refers to the addition of molecules to the surface of particles to modulate particle-cell interactions. Specifically, modulation can promote particle-cell interaction by using formulations comprising a targeting ligand, specifically, a bio-recognitive targeting ligand. Specifically, modulation can also promote reduced interactions with the surrounding, e.g., for stealth particles to reduce opsonization. Specifically, functionalization can be, but is not limited to, treatment with wheat germ agglutinin (WGA), human or bovine serum albumin (HSA or BSA), antibodies or fragments thereof, peptides, e.g., Arginylglycylaspartic acid (RGD), carbohydrate ligands, e.g., N-Acetylglucosamine (GIcNAc), or folic acid.

[0149] The herein used term “substances” refers to soluble substances and solutions. Substances differ from particles in that particles are non-soluble in cell culture media, buffer, or water and form a dispersion. Substances are dissolved in the medium and therefore form a solution whereas particles are dispersed in the medium and therefore form a dispersion, e.g., an emulsion or suspension. Substance-cell interactions can be assessed, e.g. through fluorescence labelling and following analysis with a plate reader or flow cytometry. Substance-cell interactions can further be assessed through expression of markers, knock-ins, or knock-outs, such as GFP or RFP proteins. Nonlimiting examples for soluble substances are soluble ligands, e.g., lectins such as fluorescent wheat germ agglutinin (fWGA). Further non-limiting examples for substances are fluorescence labelled antibodies; or fluorescence labelled receptor substrates e.g. fluorescence labelled carbohydrate moieties. Specifically, substances may be mRNA or plasmids that induce a knock-in or knock-out of the expression of a fluorescence protein in the cell.

[0150] The term “kit” as used herein refers to a kit comprising a device according to the invention, or the array of devices according to the invention.

[0151] Specifically, the kit comprises a) the device or the array according to the invention; b) a mounting plate comprising: i.one or more recesses with beveled edges for placement of at least one cell plate; ii. a hole at the bottom of each recess; c) optionally at least two cell plates; and d) optionally tweezers for placement of cell plates.

[0152] Specifically, the kit comprises a) the device or the array according to the invention; b) a positioning plate comprising: i. one or more recesses for placement of at least one device according to the invention in one or more recesses; c) optionally at least two cell plates; and d) optionally tweezers for placement of cell plates.

[0153] Specifically, the kit comprises a) the device or the array according to the invention; b) a mounting plate comprising: i.one or more recesses with beveled edges for placement of at least one cell plate; ii. a hole at the bottom of each recess; c) a positioning plate comprising: i. one or more recesses for placement of at least one device according to the invention in one or more recesses; d) optionally at least two cell plates; and e) optionally tweezers for placement of cell plates.

[0154] Specifically, the kit comprises a mounting plate made from plastic, metal, or a polymer, specifically it is sterilizable or autoclavable. Specifically, the size of the mounting plate is adapted for a plate reader. Specifically, the kit comprises a cell plate made from glass, specifically it is a coverslip, plastic, or a polymer, specifically a translucent material. Specifically, the cell plate is of square, polygonal, or round shape, and the cell plate is size-fitting for the slits or rails of the device. Specifically, the cell plate is untreated, or surface-treated with one or more cell attachment-facilitating substances, specifically fibronectin, laminin, collagen, gelatine, fetal calf serum, or polylysine.

[0155] The present invention also encompasses the following embodiments:

[0156] 1. A device comprising an open-top hollow body (1 ), comprising at least 4 side walls (2), comprising: a) slits or rails (3) for vertical fixation of a cell plate (4) at two or more side walls; b) an indentation (5) in the side wall behind the cell plate; and c) an indentation (6) at the bottom of the hollow body.

[0157] 2. The device of embodiment 1 , wherein the shape of the device is a cube, a cylinder, or a prism, specifically of pentagonal, hexagonal, or octagonal prismatic shape.

[0158] 3. The device of embodiment 1 or 2, wherein the device is made from plastic, glass, metal, or a polymer, specifically the device is sterilizable or autoclavable.

[0159] 4. The device of any one of embodiments 1 to 3, wherein the slits or rails (3) are parallel to the side walls, specifically the slits or rails are at an angle of 90 degrees + / - 5 degrees to the bottom of the device.

[0160] 5. The device of any one of embodiments 1 to 4, wherein the slits or rails (3) are sizefitting for the cell plate and end above the indentation at the bottom.

[0161] 6. The device of any one of embodiments 1 to 5, wherein the indentation at the side wall (5) ends above the cell plate. The device of any one of embodiments 1 to 6, wherein the indentation at the bottom (6) is cylindrical or outwardly curved and allows for accommodation of a stirring element (7). The device of any one of embodiments 1 to 7, wherein the stirring element (7) is a magnetic stirrer bar, or a propeller. The device of any one of embodiments 1 to 8, further comprising a lid (8). The device of any one of embodiments 1 to 9, wherein the lid (8) comprises a slit or rail (9) for horizontal fixation of a cell plate (4). The device of any one of embodiments 1 to 10, wherein the lid (8) is of the same material as the device, or of a different material, specifically it is plastic, glass, metal, or a polymer. The device of any one of embodiments 1 to 11 , wherein the lid (8) comprises a sealing ring. The device of any one of embodiments 1 to 12, wherein the cell plate (4) is made from glass, specifically it is a coverslip, plastic, or a polymer, specifically it is a translucent material. The device of any one of embodiments 1 to 13, wherein the cell plate (4) is of square, polygonal, or round shape. The device of any one of embodiments 1 to 14, wherein the cell plate (4) is sizefitting for the slits or rails of the device. The device of any one of embodiments 1 to 15, wherein the cell plate (4) is untreated or surface-treated with one or more cell attachment-facilitating substances, specifically fibronectin, laminin, collagen, gelatine or poly-lysine. The device of any one of embodiments 1 to 16, wherein the device comprises of a connection element at least at one outer side wall. An array of devices comprising two or more devices of any one of embodiments 1 to 16 connected to each other, or comprising two or more devices of claim 17 attached by one or more connection elements. A kit comprising: a) the device of any one of embodiments 1 to 17, or the array of claim 18; b) a mounting plate (10) comprising: i. one or more recesses (11) with beveled edges for placement of at least one cell plate; □

[0162] -27- ii. a hole (12) at the bottom of each recess; c) optionally at least two cell plates; and d) optionally tweezers for placement of cell plates.

[0163] 20. The kit of embodiment 19, wherein the mounting plate (10) is made from plastic, metal, or a polymer, specifically it is sterilizable or autoclavable.

[0164] 21. The kit of embodiment 19 or 20, wherein the size of the mounting plate (10) is adapted for a plate reader.

[0165] 22. The kit of any one of embodiments 19 to 21 , wherein the cell plate is made from glass, specifically it is a coverslip, plastic, or a polymer, specifically a translucent material.

[0166] 23. The kit of any one of embodiments 19 to 22, wherein the cell plate is of square, polygonal, or round shape.

[0167] 24. The kit of any one of embodiments 19 to 23, wherein the cell plate is size-fitting for the slits or rails.

[0168] 25. The kit of any of embodiments 19 to 24, wherein the cell plate is untreated, or surface-treated with one or more cell attachment-facilitating substances.

[0169] 26. A mounting plate comprising: a) one or more recesses with beveled edges for placement of at least one cell plate; and b) a hole at the bottom of each recess.

[0170] 27. The mounting plate of embodiment 26, wherein said plate is made of plastic or metal, or a polymer, specifically it is sterilizable or autoclavable.

[0171] 28. The mounting plate of embodiment 26 or 27, wherein the size is adapted for a plate reader.

[0172] 29. Use of the device of any one of embodiments 1 to 17, the array of embodiment 18, the kit of any one of embodiments 19 to 25, or the mounting plate of any one of embodiments 26 to 28 for analyzing cell interaction, specifically for analyzing particle-cell interactions, specifically microparticles, nanoparticles and microcapsules; protein-cell interactions, cell-cell interactions, cell adhesion to a surface, the effect of shear stress on cells, or microbiome formation.

[0173] 30. A method for analyzing cell interactions using the device of any one of embodiments 1 to 17, the array of embodiment 18, the kit of any one of embodiments 19 to 25, or the mounting plate of any one of embodiments 26 to 28, comprising: □

[0174] -28- a) adhering cells, specifically adherent cells, more specifically, epithelial cells, endothelial cells, or fibroblasts, to the cell plate (4); b) placing the cell plate in a slit (3) with adherent cells facing inwards; c) optionally, washing the cells with a buffer solution; d) introducing a test solution or suspension into the hollow body; e) placing a magnetic stirrer (7) at the indentation at the bottom (6); f) incubating the cells with the test solution or suspension under stirring conditions; g) optionally, washing the cells with a buffer solution; h) optionally, placing the cell plates on a mounting plate (10); i) analyzing of test substance interactions, optionally using a plate reader.

[0175] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Different embodiments of the present invention have been described according to the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the invention.

[0176] EXAMPLES

[0177] Example 1 - Particle-cell interactions of suspensions with varying ph ysicochemical characteristics

[0178] Particle-cell interaction experiments were conducted with microparticles, microcapsules and nanoparticles. Two different cell lines were used: cell line 5637, originating from a bladder cancer tumor, and SV-HUC-1 , a non-cancerous urothelial cell line. Experiments were performed using a vertical, non-static device (‘incubation chamber’, Fig. 1A, with lid of Fig. 1 B) comprising a magnetic stirrer in the bottom indentation of the device and vertically fixed cell plates comprising attached cells, and compared to experiments performed with a conventional horizontal monolayer set-up. For analysis, cell-comprising cell plates were placed on a mounting plate (Fig. 1 D) and cell-associated relative fluorescence intensity (RFI) was determined at 485 / 525 nm (excitation / emission, ex / em).

[0179] First, microparticles (MPs) were tested (Fig. 2). Microparticles are hard particles consisting of a poly-lactic-co-glycolic acid (PLGA) matrix and are fluorescently dyed using BodiPy©. They are characterized by a mean diameter of 5.2 pm ± 0.84 pm, a sedimentation velocity of 4.47x1 O'6m / s and a density of 1 .3 g / cm3and were either non- functionalized (blank-MP) or treated with wheat germ agglutinin (WGA-MP). Fig. 2 shows the cyto-adhesive potential of WGA-functionalized and non-functionalized microparticles tested in the incubation chamber and on a conventional cell monolayer system. Particle-cell interaction was measured using the mounting plate of Figure 1 D in a plate reader. Particle-cell interactions are shown as percentage of interaction for cancer cell line 5637 and non-cancerous cell line SV-HUC-1. Particle-cell interaction of blank-MP in the incubation chamber was set to 100% and the other conditions were plotted relatively to that. Double asterisks indicate statistical significance (p<0.01).

[0180] MP-cell interactions were significantly reduced in the incubation chamber compared to the monolayer set-up. In the monolayer set-up, WGA-treated MPs showed elevated cell interactions, while in the incubation chamber WGA treatment had less influence on cell interaction.

[0181] Second, microcapsules (MCs) were tested (Fig. 3). Microcapsules consist of human serum albumin (HSA) as shell material and olive oil as a core. They are characterized by a mean diameter of 1.09 pm ± 0.03 pm, a sedimentation velocity of minus 1.15x1 O'8m / s, and a density of 0.98 g / cm3. They were either non-functionalized (blank-MC) or treated with wheat germ agglutinin (WGA-MC). Fig. 3 shows the cyto- adhesive potential of WGA-functionalized and non-functionalized microcapsules tested in the incubation chamber and on a conventional cell monolayer system. Particle-cell interactions are shown as percentage of interaction for cancer cell line 5637 and non- cancerous cell line SV-HUC-1. Particle-cell interaction of blank-MC in the incubation chamber was set to 100% and the other conditions were plotted relatively to that. Double asterisks indicate statistical significance (p<0.01 ).

[0182] MC-cell interactions were significantly, up to 3-fold, higher in the incubation chamber compared to the monolayer set-up. WGA treatment did not significantly influence MC-cell interactions.

[0183] Third, nanoparticles (NPs) were tested (Fig. 4). Similarly to microparticles, nanoparticles are hard particles consisting of a poly-lactic-co-glycolic acid (PLGA) matrix and are fluorescently dyed using BodiPy©. They are characterized by a mean diameter of 188 nm ± 6.8 nm, a sedimentation velocity of 2.60x1 O'9m / s and a density of 1 .3 g / cm3and were either non-functionalized (blank-NP) or treated with wheat germ agglutinin (WGA-NP). Fig. 4 shows the cyto-adhesive potential of WGA-functionalized and non- functionalized nanoparticles tested in the incubation chamber and on a conventional cell monolayer system. Particle-cell interactions are shown as percentage of interaction for cancer cell line 5637 and non-cancerous cell line SV-HUC-1. Particle-cell interaction of blank-NP in the incubation chamber was set to 100% and the other conditions were plotted relatively to that. Double asterisks indicate statistical significance (p<0.01).

[0184] NP-cell interactions were not significantly influenced by conditions in the incubation chamber compared to cell monolayer experiments. WGA-functionalization only slightly influenced particle-cell interactions.

[0185] Taken together, for dense microparticles, MP-cell interaction was significantly reduced in the incubation chamber, while for floating microcapsules, MC-cell interaction was significantly enhanced compared to the conventional horizontal monolayer set-up. Large and heavy particles are more likely to interact with horizontally aligned cells due to their sedimentation properties. In contrast, highly porous particles or capsules with a low-density float and their cell contact is reduced. In either case, Brownian motion in a conventional horizontal monolayer set-up is not sufficient to keep these particles dispersed. No difference could be observed for nanoparticles, indicating that these smaller particles remain well dispersed due to Brownian motion and are less influenced by sedimentation.

[0186] Example 2 - Cell internalization under shear stress

[0187] In the first example, particle-cell interaction was assessed by directly measuring cell-associated fluorescence intensity using the specified mounting plate in a plate reader. However, this method could not differentiate between particle internalization and surface adhesion. To address this limitation, in the following second example, flow cytometry was used to analyse cell-associated fluorescence intensity. During cell detachment from the growth surface, loosely attached particles on the cell surface are removed, ensuring that the measured fluorescence primarily reflected internalized particles.

[0188] Moreover, the effect of shear stress on particle-cell interactions was tested. Shear stress can further influence fluid dynamics and particle-cell interactions.

[0189] A comparison of particle-cell interactions obtained by monolayer experiments and cuboidal incubation chamber is shown in Fig. 5. Fig. 5 shows interactions of nanoparticles (NP, diameter: 100 nm), microparticles (MP, 5.2 pm), microcapsules (MC, 1.6 pm), and soluble ligands (fluorescent wheat germ agglutinin, fWGA) with 5637 cells incubated in the incubation chamber or as monolayers. Cells were attached to cell plates without prior cell plate coating. Cells were incubated for 8 hours at 37°C under stirring and non-stirring conditions (indicated by stripes, ‘static’). The positioning plate (Fig. 8A and B) was used for stirring. For analysis, cells were detached from the cell plates and cell-associated fluorescence measurement was performed by flow cytometry. In Fig. 5, interactions with monolayers were normalized to 100% to form a baseline, and interactions with cells in the incubation chamber are shown as percentage relative to this baseline.

[0190] For the soluble ligand fWGA, comparable interaction levels were observed between incubation chamber and monolayer setups, with slightly increased binding under shear stress.

[0191] For NPs, reduced adhesion was observed in incubation chamber experiments, suggesting that shear stress negatively impacts NP-cell interaction and especially particle internalisation.

[0192] For MPs, incubation with a monolayer of cells caused rapid sedimentation which led to increased cell adhesion. Incubation in the incubation chamber under stirring conditions increased interaction due to improved dispersion of the particle suspension compared to static conditions.

[0193] For MCs, incubation with cell monolayers limited cell contact due to the microcapsules’ buoyancy which resulted in lower adhesion. However, the vertical orientation of cell plates in the incubation chamber increased the interaction of MCs with cells.

[0194] Example 3 - Particle-cell interactions of cells on coated cell plates

[0195] Cell adhesion and growth was assessed for cell plates with different coatings or using uncoated cell plates (Fig. 9). Cell plates were coated with coating materials poly- L-lysine, laminin, collagen, fibronectin, or fetal calf serum (FCS). HEK or CaCo-2 cells were seeded onto coated and uncoated cell plates and growth curves for each cell line were monitored using the PHIO Cell Watcher. Confluency was tracked over a period of around 300 hours. Fig. 9 shows growth curves for HEK cells grown on uncoated cell plates and cell plates pre-coated with poly-L-lysine, laminin, fibronectin, or collagen. Pre-coating with laminin resulted in highest growth curves. Lysine and collagen also supported growth, whereas uncoated cell plates showed the lowest growth curves. CaCo-2 cells were grown on uncoated cell plates and cell plates pre-coated with collagen and fetal calf serum (FCS). Growth curves of CaCo-2 cells showed similar trends with collagen precoating resulting in higher growth curves compared to uncoated cell plates. FCS slightly supported growth compared to untreated cell plates.

[0196] As a next step, particle-cell interactions of cells grown on treated or non-treated cell plates were tested on monolayers, and in cuboidal and hexagonal incubation chambers (Fig. 10, FlowCube, used synonymously herein for“CellCube”). Fig. 10 shows interactions of nanoparticles (NPs) incubated with HeLa, CaCo-2 and 5637 cells cultured as monolayers, or in an incubation chamber device comprising 4 cell plates (‘slides’; ‘coverslips’, see device Fig. 1) or 6 cell plates (slides; see device Fig. 6B) under stirring conditions. Cell plates coated with poly-L-lysine before cell attachment are indicated by stripes. Incubation was conducted for 2 hours at 37°C with stirring at 350 rpm. The positioning plate (Fig. 8A and B) was used for stirring. For analysis, cells were detached from the cell plates and cell-associated fluorescence measurement was performed by flow cytometry.

[0197] A reduced adherence of NPs was observed for experiments performed with the incubation chambers compared to monolayer conditions across all tested cell lines. Cuboidal devices comprising 4 cell plates and hexagonal devices comprising 6 cell plates showed comparable results.

[0198] For HeLa and 5637 cells, lysine coating of cell plates in devices had no impact on cell interaction, while CaCo-2 cells showed a slight increase in particle-cell interaction.

[0199] Taken together, particle-cell interaction was influenced by vertical fixation of cell plates in incubation chambers comprising 4 or more cell plates, with or without precoating of cell plates.

[0200] Data generated by the present invention demonstrates that particle sedimentation or flotation effects of particles with higher density or flotation properties significantly influence the outcome of in vitro particle-cell interaction experiments. A conventional horizontal monolayer set-up fails to model conditions occurring in vivo, e.g., in hollow human organs such as the urinary bladder. A vertical, non-static experimental set-up as demonstrated in the present invention is required to model physiological conditions, necessary for e.g., drug delivery studies in vitro.

[0201] Example 4 - Permeability studies of soluble substances in array of devices

[0202] To assess the permeability of test compounds across a polarized epithelial barrier, cells are cultured on semipermeable membranes and analysed using the device under stirred conditions. A cell line commonly used for permeability studies is the Caco- 2 cell line. Over an incubation period of 2 to 3 weeks, Caco-2 cells differentiate and form a tight barrier with a high transepithelial electrical resistance (TEER), similar in its characteristics to the epithelium of the small intestine.

[0203] A semipermeable membrane (e.g. Snapwell® membrane inserts) is equilibrated in cell culture medium at 37°C. Caco-2 cells are then seeded onto the apical side of the membrane, and medium is added to both apical and basolateral compartments. The cells are incubated under standard cell culture conditions (37°C, 5% CO2) for approximately 20 days. During this period, the medium is refreshed every 2-3 days. TEER measurements should be performed regularly to monitor barrier formation; a high TEER value confirms the development of a tight epithelial layer suitable for permeability studies.

[0204] Optional - Combination with binding studies: Cells are cultured on coverslips until a confluent monolayer forms, typically within 5-7 days. If necessary, coverslips can be coated with cell adhesion-promoting substances such as collagen, fibronectin, or poly- L-lysine before seeding.

[0205] Once the epithelial barrier is established, the semipermeable membrane (Snapwell membrane) is placed in the connection element between two devices forming an array. The devices are aligned and tightly sealed using the provided screws to ensure a leak-proof connection. Coverslips with cell layers may also be inserted into the remaining three side compartments of each of the device if binding studies are to be conducted simultaneously.

[0206] For the permeability assay, the donor phase (containing the test compound) is added to one device of the device array, while the acceptor phase (such as buffer or fresh culture medium) is added to the other device. Magnetic stir bars are placed in one or both chambers to simulate dynamic conditions, and the entire assembly is positioned on a magnetic stirring plate. Stirring should be homogenous, ensuring proper mixing. It is critical to confirm that the membrane and any coverslips are fully immersed in their respective media.

[0207] After a defined incubation period, samples are collected from the acceptor and donor device at regular time points. These samples are analysed to determine the amount of permeated compound, using techniques such as HPLC, photometry, or other appropriate analytical methods. The apparent permeability coefficient can then be calculated.

[0208] TEER values should be measured before, during, and after the experiment to ensure the integrity of the cell monolayer has not been compromised. Significant drops in TEER may indicate barrier disruption.

[0209] If binding studies are included, coverslips from both the donor and acceptor devices can be removed after the assay and analysed for compound-cell interactions. This can be done using flow cytometry, fluorescence microscopy, immunostaining, or analyte extraction followed by quantification or any other appropriate method.

[0210] The inventive array of devices allows for the simultaneous assessment of compound permeability and cell-associated interactions under dynamic conditions, closely mimicking physiological environments.

[0211] Methods

[0212] Nanoparticle preparation

[0213] Nanoparticles (NPs) were prepared by solvent-evaporation applying a single o / w nanoprecipitation system as described in Anzengruber et al., 2023. Briefly, 50 mg of poly-lactic-co-glycolic acid (PLGA) RG503H and 25 pg BodiPy® as a fluorescent marker for cell adhesion experiments were dissolved in 3 ml acetone. This solution was injected via syringe into 20 ml of an aqueous solution of PVA 0.5 % (w / v) and sonicated for two minutes at 40 % amplitude using a Bandelin HD70 Sonoplus sonifier. The mixture was then poured into another aqueous solution containing 0.1 % PVA (w / v) and stirred for one hour under continuous airflow to allow for evaporation of acetone. The nanoparticles were purified by several centrifugation steps at increasing g-forces. Each pellet was suspended in 0.1 % PVA (w / v). Apart from the first three centrifugation steps, the remaining seven pellets were pooled and filtered through a 5 pm polycarbonate filter (Whatman Nucleopore). For further use, 1 ml aliquots of the nanoparticle suspension were lyophilized and stored at 4°C. All preparation steps were carried out under light protection.

[0214] Microcapsule preparation

[0215] Microcapsules (MCs) were prepared as described in Skoll et al., 2021. 20 mg human serum albumin (HSA) were dissolved in 10 ml 100 mM phosphate buffer at pH 8 and overlaid with 1 ml olive oil. The probe micro tip of the sonifier (Sonoplus HD2070, Bandelin, Germany) was positioned at the interface of the two phases. The sample was sonicated with an acoustic power of =253 W cm-2 at 40% amplitude for 2 minutes. Subsequently, the capsules were washed four times by centrifugation (5204 x g, 40 min, 4 °C). For final separation, the capsules were left for 12 h in a separation flask at room temperature. The lower aqueous phase containing the capsules was used for further experiments. To produce WGA-targeted MCs, 3.6 mg WGA and 16.4 mg HSA were dissolved in 100 mM phosphate buffer pH 7.3 and treated as described above. Microcapsules used for cell-binding experiments were labelled with BodiPy®, a fluorescent dye. Therefore, 1 mg lipophilic dye was dissolved in 1 ml isopropyl alcohol, used as solubilizing agent and 25 pl of this stock solution were added to the oil phase with the isopropyl alcohol cleared off prior sonication.

[0216] Microparticle preparation

[0217] A solvent-evaporation protocol with a single o / w emulsion system was used for preparation of microparticles (MPs). The inner organic phase consisted of 50 mg PLGA RG503H and 25 pg BodiPy® dissolved in 3 ml of a 1 :1 mixture of dichloromethane and acetone. This solution was injected via syringe into 20 ml of an aqueous solution of PVA 0.5% (w / v) and emulsified for two minutes using an I KA UltraTurrax T8 at intensity level 4. The suspension was then poured in 80 ml of purified water and stirred for one hour under continuous airflow to allow for particle hardening and evaporation of dichloromethane and acetone. The particle suspension was then washed three times with purified water via centrifugation at 4275 x g for 15 minutes using a Sorvall Lynx 6000. The pellet was suspended in purified water and aliquots were lyophilized and stored at 4°C for further use.

[0218] Particle characterization □

[0219] -36-

[0220] Particle size distribution and polydispersity index (PDI) of the nanoparticle suspension and protein capsule suspension were determined in triplicates by dynamic light scattering (DLS) using a Zetasizer NanoZS, (Malvern Panalytical, Malvern, UK). Lyophilized nanoparticles were suspended in purified water prior to particle size characterization.

[0221] MCs were analyzed directly after production. Size distribution of microparticles was determined by laser diffraction using a Mastersizer 3000 with a Hydro SV dispersion unit (Malvern Panalytical, Malvern, UK).

[0222] Lyophilized MPs were suspended in purified water at a concentration of 1 mg / ml. A sufficient laser obscuration of 1 -10% was achieved using 100 pl of this suspension. The particle suspension was stirred at 700 rpm during the measurement.

[0223] Surface modification with Wheat Germ Agglutinin

[0224] The surface of PLGA-microparticles and PLGA-nanoparticles was modified with WGA to enable a specific interaction with certain sugar components in the glycocalyx of urothelial cells. The carboxylic groups of PLGA at the particle surface were linked to the amino groups of WGA via a carbodiimide method according to an adapted protocol previously described by Hermanson (2013). For activation of the carboxyl groups, 10 mg of freeze-dried microparticles or nanoparticles were suspended MES buffer 0.1 M, pH6.0 and mixed with 1 mg EDC and 2.2 mg sulfo-NHS dissolved in the same buffer. The activation proceeded under constant magnetic stirring for 10 minutes at room temperature. Subsequently, 4 mg of WGA were dissolved in MES buffer 0.1 M, pH8.0 and added to the reaction mix. The pH was adjusted to pH8.0 with 1 M NaOH solution. The reaction was let to proceed for further two hours at room temperature. Remaining activated carboxyl groups were then saturated by addition of glycine. To remove excessive cross-linking reagent, the nanoparticle suspension was transferred to a dialysis tube (Spectra / Por® cut-off 300 kDa) and extensively dialyzed against 0.1 M MES buffer, isotone, pH7.4. For further use, the WGA-nanoparticle-suspension was lyophilized and stored at 4°C. A final concentration of 0.1 % PVA was used as a cryoprotectant during lyophilization of nanoparticles. Microparticles were washed three times via centrifugation at 4275 x g for 15 minutes. The pellet was suspended in 0.1 M MES buffer, isotone, pH7.4 and subsequently freeze dried and stored at 4°C for further use. Cell culture

[0225] For cell interaction studies, two urothelial cell lines were used. The human urothelial cell line 5637 originates from a bladder carcinoma grade 2 and the SV-HUC-1 cell line was established from a donor without history of urothelial malignancy. Both cell lines were obtained from ATCC (Rockville, USA). Passages between 35 and 54 were used for this study. The 5637 cells were cultivated in RPMI-1640 nutrition medium fortified with 10% fetal calf serum, 0.5 mM L-glutamine and antibiotics. For the SV-HUC cell line Ham’s F- 12-K medium containing 10% fetal calf serum, 0.5mM L-glutamine and penicillin / streptomycin (10 U / ml / 10 pg / ml) was used for cultivation. Both cell lines were cultivated in a humidified 5% CO2 / 95% air atmosphere at 37°C. According to supplier’s instructions, both cell lines were sub-cultivated using a 0.25% (w / v) Trypsin / EDTA solution.

[0226] For binding studies on cell-monolayers, cells were seeded in 96-well plates at a density of 17,000 cells per well.

[0227] For binding studies using the incubation chamber, glass coverslips (8 mm x 8 mm) were pre-treated with freshly prepared peroxysulfuric acid for one hour. Subsequently, the glass coverslips were thoroughly washed with purified water, coated with fetal calf serum (FCS) and dried under laminar airflow. Dry glass slides were then placed in 24-well plates and 1 ml of cell suspension containing 120,000 cells was seeded in each well. Cells were cultivated to confluency within four days.

[0228] Particle-cell interaction studies

[0229] The cell interaction of WGA-grafted particles (microparticles, nanoparticles and microcapsules) and non-modified particles was evaluated for 5637-cell monolayers and SV-HUC-1 -cell monolayers cultivated in 96-well plates as well as on cell monolayers cultivated on glass coverslips using the incubation chamber. All particle suspensions were adjusted to the same relative fluorescence intensity (RFI) by dilution with isotone 20mM HEPES pH7.4.

[0230] For cell interaction experiments in 96-well plates, cell layers were washed twice with 100 pl isotone HEPES, pH7.4 buffer to remove cell debris and nutrition media. Then 50 pl of the respective particle suspension were added to each well and cell-monolayers were incubated at 37°C for 60 minutes. Subsequently, cell layers were washed with 100 pl of isotone HEPES pH7.4 and cell associated RFI was determined at 485 / 525 nm (excitation / emission; ex / em) using a microplate reader (TECAN, Grading, Austria). □

[0231] -38-

[0232] To investigate the particle-cell interaction using the incubation chamber, four glass coverslips with adhered confluent cell monolayers were carefully removed from the 24- well plate and vertically placed in the incubation chamber with the cell monolayer facing inwards. The resulting cubical space surrounded by cell monolayers was filled with 1.2 ml of isotone HEPES pH 7.4. A magnetic stirrer was placed in the cylindrical cavity in the middle of the device and the device was then placed on a stirring plate for 30 seconds to remove nutrition media and cell debris. Then, the buffer was removed and replaced by 1 .2 ml of the respective particle suspension. The filled incubation chamber was again placed on the stirring plate and incubated at 37°C for 60 minutes.

[0233] As indicated, for several experiments, e.g., Fig. 2 to 4, incubation chambers were placed in recesses of a positioning plate and the positioning plate was placed on a magnetic stirrer for stirring.

[0234] Subsequently, cells were washed twice with isotone HEPES pH 7.4 as described previously. The glass coverslips with the adherent cell monolayer were then removed from the incubation chamber and placed on the specially designed mounting plate with the cell monolayer facing upwards. Each glass slide was covered with 20 pl of isotone HEPES pH7.4 to prevent cells from drying. Cell-associated RFI was determined at 485 / 525 nm (ex / em) using the microplate mounting plate.

[0235] For analysis of cell interaction via flow cytometry, cells first need to be detached from the cell plates. Therefore, following the incubation period, the cell plates with the adherent monolayer are removed from the device and placed in a 24-well plate. Cell layers are rinsed with isotone buffer. Subsequently, 100 pl of 0.05% trypsine-EDTA or Accutase (depending on the cell line) are added. Cells are incubated at 37°C for 15 minutes in case of 5637 cell line (other incubation times may be appropriate for different cells). Once cells are detached, buffer with 2% serum is added to neutralize the enzyme. Cells are thoroughly suspended to ensure single-cell suspension and then transferred to flow cytometric analysis. Cell associated fluorescence intensity was then determined. REFERENCES

[0236] Anzengruber M, Wimmer L, Szuchar R, Skoll K, Wirth M, Gabor F. LogP of N- acyl-gemcitabine and lectin-corona emerge as key parameters in nanoparticulate intravesical cancer therapy. Eur J Pharm Sci [Internet]. 180:106330 (2023)

[0237] Bjbrnmalm M, Faria M, Chen X, Cui J, Caruso F. Dynamic Flow Impacts Cell - Particle Interactions: Sedimentation and Particle Shape Effects. Langmuir. 32(42): 10995-1001 (2016).

[0238] Cho EC, Zhang Q, Xia Y. The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles. Nat Nanotechnol. 6(June):385-91 (2011).

[0239] Cui J, Faria M, Bjbrnmalm M, Ju Y, Suma T, Gunawan ST, Richardson J J , Heidari H, Bals S, Crampin EJ, Caruso F. A Framework to Account for Sedimentation and Diffusion in Particle - Cell Interactions. Langmuir. 32(47): 12394-12402 (2016).

[0240] Feliu N, Sun X, Puebla RAA, Parak WJ. Quantitative Particle-Cell Interaction: Some Basic Physicochemical Pitfalls. Langmuir. 33:6639-46 (2017).

[0241] Hermanson GT. Chapter 3 - The reactions of bioconjugation. Bioconjugate Techniques, Academic Press. Pages 229 - 258 (2013).

[0242] Skoll K, Ritschka M, Fuchs S, Wirth M, Gabor F. Characterization of sonochemically prepared human serum albumin nanocapsules using different plant oils as core component for targeted drug delivery. Ultrason Sonochem. 76:105617

[0243] (2021)

Claims

□-40-CLAIMS1. A device comprising an open-top hollow body (1 ), comprising at least 4 side walls (2), comprising: a) slits or rails (3) for vertical fixation of a cell plate (4) at two or more side walls; b) an indentation (5) in the side wall behind the cell plate; and c) an indentation (6) at the bottom wall of the hollow body.

2. The device of claim 1 , wherein the shape of the device is a cube, a cylinder, or a prism, specifically of pentagonal, hexagonal, heptagonal, or octagonal prismatic shape.

3. The device of claim 1 or 2, wherein the device is made from plastic, glass, metal, or a polymer, specifically the device is sterilizable or autoclavable.

4. The device of any one of claims 1 to 3, wherein the slits or rails (3) are parallel to the side walls, specifically the slits or rails are at an angle of 90 degrees + / - 5 degrees to the bottom wall of the device.

5. The device of any one of claims 1 to 4, wherein the slits or rails (3) are size-fitting for the cell plate and end above the indentation at the bottom wall.

6. The device of any one of claims 1 to 5, wherein the indentation at the side wall (5) ends above the cell plate.

7. The device of any one of claims 1 to 6, wherein the indentation at the bottom wall (6) is cylindrical or outwardly curved and allows for accommodation of a stirring element (7).

8. The device of any one of claims 1 to 7, wherein the stirring element (7) is a magnetic stirrer bar, or a propeller.

9. The device of any one of claims 1 to 8, further comprising a lid (8; 16).

10. The device of any one of claims 1 to 9, wherein the lid (8; 16) comprises a slit or rail (9) for horizontal fixation of a cell plate (4).11 . The device of any one of claims 1 to 10, wherein the lid (8; 16) is of the same material as the device, or of a different material, specifically it is plastic, glass, metal, or a polymer.

12. The device of any one of claims 1 to 11 , wherein the lid (8; 16) comprises a sealing ring.

13. The device of any one of claims 1 to 12, wherein the cell plate (4) is made from glass, specifically it is a coverslip, plastic, or a polymer, specifically it is a translucent material.

14. The device of any one of claims 1 to 13, wherein the cell plate (4) is a membrane, more specifically a bio-printed membrane.

15. The device of any one of claims 1 to 14, wherein the cell plate (4) is of square, polygonal, or round shape.

16. The device of any one of claims 1 to 15, wherein the cell plate (4) is size-fitting for the slits or rails of the device.

17. The device of any one of claims 1 to 16, wherein the cell plate (4) is untreated or surface-treated with one or more cell attachment-facilitating substances, specifically fibronectin, laminin, collagen, gelatine, fetal calf serum, or poly-lysine.

18. The device of any one of claims 1 to 17, wherein the device comprises of a connection element (13) at least at one outer side wall.

19. An array of devices comprising two or more devices of any one of claims 1 to 18 connected to each other, or comprising two or more devices of claim 17 attached by one or more connection elements (13).

20. A kit comprising: a) the device of any one of claims 1 to 18, or the array of claim 19; b) a mounting plate (10) comprising: i. one or more recesses (11) with beveled edges for placement of at least one cell plate; ii. a hole (12) at the bottom of each recess;□-42- c) optionally a positioning plate (14) comprising one or more recesses (15) for placement of the device; d) optionally at least two cell plates; and e) optionally tweezers for placement of cell plates.

21. A kit comprising: a) the device of any one of claims 1 to 18, or the array of claim 19; b) a positioning plate (14) comprising one or more recesses (15) for placement of the device; c) optionally a mounting plate (10) comprising: i. one or more recesses (11) with beveled edges for placement of at least one cell plate; ii. a hole (12) at the bottom of each recess; d) optionally at least two cell plates; and e) optionally tweezers for placement of cell plates.

22. The kit of claim 20 or 21 , wherein the mounting plate (10) or the positioning plate (14) is made from plastic, metal, or a polymer, specifically it is sterilizable or autoclavable.

23. The kit of any one of claims 20 to 22, wherein the size of the mounting plate (10) is adapted for a plate reader.

24. The kit of any one of claims 20 to 23, wherein the cell plate is made from glass, plastic, or a polymer, or is a membrane or a scaffold, specifically the cell plate is a coverslip, more specifically the cell plate is made from a translucent material.

25. The kit of any one of claims 20 to 24, wherein the cell plate is of square, polygonal, or round shape.

26. The kit of any one of claims 20 to 25, wherein the cell plate is size-fitting for the slits or rails.

27. The kit of any of claims 20 to 26, wherein the cell plate is untreated, or surface- treated with one or more cell attachment-facilitating substances, specifically fibronectin, laminin, collagen, gelatine, fetal calf serum, or poly-lysine.□-43-28. Combination of the device of any one of claims 1 to 18, or the array of claim 19 with a mounting plate (10) comprising: a) one or more recesses (11 ) with beveled edges for placement of at least one cell plate; and b) a hole (12) at the bottom of each recess.

29. The combination of claim 28, wherein said mounting plate is made of plastic or metal, or a polymer, specifically it is sterilizable or autoclavable.

30. The combination of claim 28 or 29, wherein the size of said mounting plate is adapted for a plate reader.31 . Combination of the device of any one of claims 1 to 18, or the array of claim 19 with a positioning plate (14) comprising one or more recesses (15) for placement of at least one device in a recess.

32. Use of the device of any one of claims 1 to 18, the array of claim 19, the combination of any one of claims 28 to 30, or the combination of claim 31 for analyzing cell interaction, specifically for analyzing particle-cell interactions, specifically microparticles, nanoparticles and microcapsules; protein-cell interactions, cell-cell interactions, cell adhesion to a surface, the effect of shear stress on cells, or microbiome formation.

33. A method for analyzing cell interactions using the device of any one of claims 1 to 18, the array of claim 19, the combination of any one of claims 28 to 30, or the combination of claim 31 comprising: a) adhering cells, specifically adherent cells, more specifically, epithelial cells, endothelial cells, or fibroblasts, to the cell plate (4); b) placing the cell plate in a slit (3) with adherent cells facing inwards; c) optionally, washing the cells with a buffer solution; d) introducing a test substance solution or suspension into the hollow body; e) placing a magnetic stirrer (7) at the indentation at the bottom wall (6); f) optionally, placing the device on the positioning plate (14) of claim 31 and the positioning plate on a magnetic agitator;□ g) incubating the cells with the test solution or suspension under stirring conditions; h) optionally, washing the cells with a buffer solution; i) optionally, placing the cell plates on a mounting plate of any one of claims 28 to 30; j) analyzing of test substance interactions.

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