Cell culture unit, system, cell culturing arrangement, and method for preparing a membrane in a cell culture unit

Abstract

Cell culture unit (100) for enabling a preparation of a membrane (5), comprising a well arrangement (1) with a membrane support structure (2), wherein said membrane support structure (2) comprises an opening (3), at least one support face (4) laterally extending from said opening (3) and at least one confining outer edge (6) for confining a curable liquid matrix material (7), which can be provided on said at least one support face (4) spanning across said opening (3), wherein said at least one confining outer edge (6) extends circumferentially around said opening (3) and said at least one support face (4). Furthermore, the invention relates to a system, a method for preparing a membrane in a cell culture unit (100), a cell culturing arrangement an a method for manufacturing a cell culture unit (100).

Description

[0001] Cell culture unit, system, cell culturing arrangement, and method for preparing a membrane in a cell culture unit

[0002] The present invention relates to a cell culture unit for enabling a preparation of a membrane, comprising a well arrangement with a membrane support structure.

[0003] Further, the present invention relates to a system comprising a cell culture unit and a curable liquid matrix material and to a cell culturing arrangement comprising at least one cell culture unit.

[0004] In addition, the present invention relates to a method for preparing a membrane in a cell culture unit and to a method for manufacturing a cell culture unit.

[0005] Cell culturing is a widely used laboratory technique for growing and maintaining living cells in environments with controlled conditions, allowing to study the cellular behaviour outside of an organism. In particular, cell culturing may be used for the investigation of biological processes, for example in the context of drug development and the investigation of disease mechanisms. By providing controlled conditions, cell culturing enables a precise manipulation of different growth factors, like temperature, growth medium or growth substrate.

[0006] In pharmaceutical research, cell culture inserts are widely used for the development and study of in vitro barrier tissues and, for example, for the prediction of drug permeability, metabolism and transepithelial I -endothelial transport (Eltanameli et al.

[0007] 2024; Youhanna et al. 2021). Using permeable cell culture inserts, gastrointestinal permeability has been extensively studied in monocultures of immortalized cell lines such as Caco-2 epithelial cells (Breemen et al. 2005; Hubatsch et al. 2007; Yamashita et al. 2000; Artursson et al. 2001), and in more complex multicellular models (Marzorati et al. 2014; Reale et al. 2021 ; Araujo et al. 2013; Lozoya-Agullo et al. 2017; Antunes et al. 2013; Zhang et al. 2024). Similarly, pulmonary permeability has been investigated in both monoculture and coculture models (Foster et al. 2000; Mathias et al. 2002; Grainger et al. 2006; Forbes et al. 2003; Salomon et al. 2014; Dekali et al. 2014; Costa et al. 2019; Bluhmki et al. 2020; Hittinger et al. 2016). Furthermore, insert cultures have been used to study the permeability of the brain endothelium (Poller et al. 2011 ; Hellinger et al. 2012, Weksler et al. 2005, Feng et al. 2019,Nakagawa et al. 2009, Abbott et al. 2006, Hatherell et al. 2011 , Nakagawa et al. 2007, Bell et al. 2010, Stone et al. 2019).

[0008] For studying such barrier tissue models in conditions comparable with an in vivo environment, it has become known to use static insert cultures, wherein on either side of a porous membrane, which may be associated with a basal and an apical chamber, cells may be cultured. However, these static systems lack physiological tissue complexity and cellular interactions with the surrounding fibrous extracellular environment. Furthermore, the cells can only be cultured on stiff, two-dimensional substrates used as membranes with relatively high elastic moduli (Eltanameli et al.

[0009] 2024; Jain et al. 2022).

[0010] To address these limitations, different improvement strategies have been proposed. Altay et al. 2019 demonstrated intestinal monolayer growth from mouse organoid-derived crypts on micrometer-thin Matrigel-coated permeable inserts. In order to replicate the biochemical gradient along the small intestinal crypt-villus axis, or the colonic crypt axis respectively, in vitro, micropatterned collagen hydrogels were fabricated on top of porous insert membranes using a PDMS stamp for micromolding (Wang et al. 2017; Wang et al. 2018). Kim et al. 2018 substituted the porous membrane with a micropatterned, impermeable photoresist film, covered by a crosslinked and dehydrated collagen hydrogel. Since barrier tissues interact closely with their underlying lamina propria, it may be beneficial to incorporate an apical and a basal chamber or a stromal chamber to achieve models for more accurate testing of drug permeability and also for the metabolization of compounds (Szabo et al. 2023; Szabo et al. 2024; Asal et al. 2024; Darling et al. 2020).

[0011] Hinman et al. 2022 modified hanging baskets with polyester struts that support a suspended collagen hydrogel simultaneously serving as matrix for top-seeded epithelial cells and as connective tissue supporting immune cell migration from the basal chamber towards the barrier upon challenge with Clostridium difficile Toxin A. For an improved control over the mechanical material properties, Vila et al. 2020 generated semi-synthetic hydrogel co-networks of gelatine methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) that were photo-crosslinked on PET membranes prior to attachment to the frame of a cell culture insert, in order tocircumvent meniscus formation inside the commercially available tissue culture-treated transwell with its permeable polyester membrane.

[0012] Others described insert modifications that avoid the use of any plastic materials to support hydrogel-based membranes that separate an apical from a basal chamber. Kreuder et al. 2020 presented a 3D bioprinted membrane system modeling the placental barrier by directly bioprinting a fibroblast-laden GelMA bioink onto an insert frame. However, this approach requires users to have access to a bioprinter and limits the material choice to shear-thinning hydrogels. Dogan et al. 2022 developed a 3D-printed insert platform library ranging from 24- to 96-well plate format, which can be filled with bioactive gelatin membranes with a dip-casting process. However, high failure rates of 25-30% of this system are reported, which makes this system unsuitable for high-throughput screenings performed at early-stage compound testing in pharmaceutical development.

[0013] The above mentioned cell culture inserts are either cumbersome, manual, work only with specific biomaterials, or even require special devices such as bioprinters, thereby limiting versatility, throughput and scalability.

[0014] One of the objectives of the present invention is to improve and further develop a cell culture unit, a system, a cell culturing arrangement, a method for preparing a membrane in a cell culture unit and a method for manufacturing a cell culture unit for enabling the formation of a membrane enabling to provide cell culturing conditions comparable to an in vivo environment with easy means.

[0015] In a first aspect, the present invention provides a cell culture unit for enabling a preparation of a membrane, comprising a well arrangement with a membrane support structure, wherein said membrane support structure comprises an opening, at least one support face laterally extending from said opening and at least one confining outer edge for confining a curable liquid matrix material, which can be provided on said at least one support face spanning across said opening, wherein said at least one confining outer edge extends circumferentially around said opening and said at least one support face.In a second aspect, the present invention provides a system comprising a cell culture unit according to the first aspect of the invention and a curable liquid matrix material.

[0016] In a third aspect, the present invention provides a method for preparing a membrane in a cell culture unit according to the first aspect of the invention, in particular with a system according to the second aspect of the invention, comprising the steps of:

[0017] - Applying said curable liquid matrix material on said at least one support face in a way that said curable liquid matrix material spans across said opening; and

[0018] - Curing said curable liquid matrix material to form said membrane.

[0019] In a fourth aspect, the present invention provides a cell culturing arrangement comprising at least one cell culture unit according to the first aspect of the invention, preferably a plurality of such cell culture units.

[0020] In a fifth aspect, the present invention provides a method for manufacturing a cell culture unit according to the first aspect of the invention and / or a cell culturing arrangement according to the fourth aspect of the invention, comprising the steps of:

[0021] - Providing multiple material layers, preferably comprising a first material layer forming said at least one confining outer edge and / or a second material layer forming said at least one support face and / or an inner edge face of said opening; and

[0022] - Connecting said multiple material layers to each other, preferably by means of adhesive bonding, in particular with a pressure-sensitive adhesive.

[0023] It has been recognized, that the at least one confining outer edge enables the curable liquid matrix material, which is applied on the at least one support face and spanning across the opening, to form a meniscus with a convex form and maintain a freestanding coherent structure. This effect can be ascribed to cohesive intermolecular forces within the liquid matrix material exceeding adhesive forces between the liquid matrix material and the at least one confining outer edge. The cohesive forces are present at the surface of the liquid matrix material in the form of the surface tension, which causes the liquid matrix material to curve outward, creating a higher central elevation compared to the lateral elevation. Consequently, the liquid matrix material is prevented from spilling over the at least one confining outer edge.By subsequently curing the liquid matrix material, the free-standing membrane spanning across the opening can be formed in situ. The retaining of the liquid matrix material by the at least one support face and the at least one confining outer facilitates the control of the amount of liquid matrix material that can be provided. Therefore, the membrane thickness of the cured membrane, which depends on the amount of liquid matrix material provided, can be easily controlled. Hence, a good reproducibility of the membrane thickness can be achieved. In particular, the invention enables an in situ formation of membranes with particularly low thicknesses.

[0024] Preferably, the cell culture unit comprises a single confining outer edge, which extends circumferentially around the opening and the at least one support face. Such a single confining outer edge may thus form a continuous support ring without an interruption. However, it is also conceivable that a plurality of confining outer edges is provided, wherein these confining outer edges form one support ring with interruptions. These interruptions may be small enough to enable the formation of a convex meniscus of the liquid matrix material.

[0025] It is noted, that the at least one confining outer edge may preferably be sharp. However, it is also conceivable that the at least one confining outer edge may be rounded. The term “laterally extending from said opening” with regard to the at least one support face is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to an extension direction, which comprises a lateral component and may additionally comprise an axial component. The terms “lateral” and “axial” may relate to a longitudinal axis, along which the well arrangement may extend and / or which may extend perpendicular to a plane defined by the opening. The longitudinal axis may be a central axis of the well arrangement and extend through a centre point of the opening.

[0026] The term “confining outer edge” with regard to the at least one support face is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a transition section of one surface to another surface. The transition section may comprise a low curvature radius, preferably a curvature radius approaching zero forming a discrete edge line. In particular, the curvature radius ofthe discrete transition may be substantially lower than the curvature radii of the two surfaces.

[0027] The term “curable liquid matrix material” is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a fluid in a liquid or a semi-liquid state, which solidifies upon curing. Preferably, the curable liquid matrix material may serve as a support and / or a growth substrate for cell cultures. With respect to initiating and maintaining the curing process, the curing of the liquid matrix material may comprise at least one of the following: thermal curing, UV / photo curing, chemical curing, moisture curing, pressure curing, microwave or radiofrequency curing. The cured liquid matrix material may comprise mechanical properties, such as a bending stiffness or a hardness, comparable to mechanical properties of tissue to closely simulate conditions of an in vivo environment.

[0028] The formed membrane may be suitable for cultivating a wide range of different cell cultures comprising mammalian and / or animal cells, such as caco-2 intestinal monolayers, mouse organoid-derived intestinal epithelium, cocultures of mouse intestinal epithelium and fibroblasts, human organoid-derived intestinal epithelium and / or cocultures of human intestinal epithelium and mast cells.

[0029] Furthermore, it has been recognized that the assembly of the cell culture unit and / or the cell culture arrangement can be facilitated by stacking multiple material layers on one another and connecting them to each other. A first material layer may form the at least one confining outer edge. The geometry of the at least one confining outer edge and / or of the at least one support face and / or an inner edge face of the opening may be directly formed by cutting the respective first and / or second material layer in the desired shape. The material layers may be individually cut, for example laser-cut, from the same or different materials.

[0030] Prior to adhesively bonding the material layers to each other, the material layers may be washed with an ultrapure water, being substantially free from ions, organics, particulates and biological contaminants like bacteria or endotoxins, and blown dry with pressurized air. Additionally or alternatively, the material layers may be cleaned by means of plasma activation, gamma radiation and / or using ethylene oxide and / or other suitable gases. Subsequently, the material layers may be aligned by means ofpins and matching alignment holes prior to applying the adhesive and / or during the curing. When using a pressure-sensitive adhesive, a pressure may be applied for activating the curing process of the adhesive. For doing so, a 2 kg hand roller may be used to firmly attach the material layers to each other to ensure the cell culture unit and / or the cell culture arrangement is leak-proof. Additionally or alternatively, a pressure may be applied to the cell culture unit and / or the cell culture arrangement by means of a hydraulic press, wherein a pressure force of 15 kN may be applied for 15 min. After curing of the adhesive, the cell culture unit and / or the cell culture arrangement may be washed again with ultrapure water, fully dried and UV-sterilized for 60 min. Finally, the cell culture unit and / or the cell culture arrangement may be covered with the lid.

[0031] Further features, advantages and preferred embodiments are disclosed or may become apparent in the following.

[0032] According to a preferred embodiment of the invention, said at least one confining outer edge is stepped back from an inner edge face of said opening and / or that said at least one confining outer edge is offset inwardly from an inner side wall face of said well arrangement. Such a design of the cell culture unit can be easily manufactured, for example by assembling material layers on each other or by machining the membrane support structure. Additionally or alternatively, at least parts of the cell culture unit, in particular the membrane support structure, may be manufactured by means of injection moulding or overmoulding. As a result of the at least one confining outer edge being stepped back, the at least one confining outer edge may form the outer edge of a step arising from the at least one support face. Furthermore, a transition from this step to the at least one support face may comprise the form of an inner edge with a square angle.

[0033] According to a further preferred embodiment of the invention, said opening and said at least one confining outer edge both define a same type of shape, preferably a circular shape, an elliptical shape, a square shape, preferably with rounded comers, a rhombus shape, preferably with rounded comers, or a rectangular shapes, preferably with rounded comers. Such shapes are particularly uniform and comprise minimal discontinuities, thereby facilitating the formation of a stable meniscus of liquid matrix material during the preparation of the membrane.According to a further preferred embodiment of the invention, said opening comprises a width d, in particular a diameter d, wherein said width d ranges from 1 to 15 mm, preferably from 2 to 7 mm. Such dimensions allow for the formation of a stable meniscus and a free-standing coherent structure of the liquid matrix material spanning across the opening.

[0034] According to a further preferred embodiment of the invention, said at least one confining outer edge comprises a width D, in particular a diameter D, wherein said width D ranges from 2 to 20 mm, preferably from 6 to 10 mm. Such dimensions allow for the formation of a stable meniscus and a free-standing coherent structure of the liquid matrix material spanning across the opening.

[0035] According to a further preferred embodiment of the invention, said opening comprises a width d, in particular a diameter d, and that said at least one confining outer edge comprises a width D, in particular a diameter D, wherein a ratio of said widths d / D is less than or equal to 1 , preferably less than or equal to 0.7, more preferably less than or equal to 0.5. Such dimensions allow for the formation of a stable meniscus and a free-standing coherent structure of the liquid matrix material spanning across the opening.

[0036] According to a further preferred embodiment of the invention, said at least one confining outer edge comprises a first material, wherein said first material comprises a liquid-repellent material, in particular a hydrophobic material, and / or a polymer, for example polyethylene terephthalate and / or cyclic olefin copolymer and / or polymethyl methacrylate, preferably polyethylene terephthalate and / or cyclic olefin copolymer. It is conceivable that the first material layer is coated with a liquid-repellent material, in particular a hydrophobic material. A liquid-repellent material comprises a low surface energy resulting in low adhesive forces between the liquid matrix material and the at least one confining outer edge. Therefore, the formation of a meniscus by means of the cohesive intermolecular forces or the surface tension, respectively, can be promoted. Polymers, in particular polyethylene terephthalate and / or cyclic olefin copolymer, may comprise hydrophobic properties. Furthermore, polymers can be easily processed and can provide a neutral environment for cell culturing.According to a further preferred embodiment of the invention, said at least one support face and / or an inner edge face of said opening comprises a second material, wherein said second material comprises a liquid-attracting material, in particular a hydrophilic material, and / or a polymer, for example polyethylene terephthalate and / or cyclic olefin copolymer and / or polymethyl methacrylate, preferably polymethyl methacrylate and / or polyethylene terephthalate. A liquid-attracting material comprises a high surface energy resulting in high adhesive forces between the liquid matrix material and the at least one support face and / or the inner edge face of the opening. In other words, the liquid matrix material can “hold on” to the at least one support face and / or the inner edge face of the opening, which can prevent it from collapsing through the opening. Consequently, the formation of a free-standing coherent structure of the liquid matrix material spanning across the opening can be promoted. Furthermore, polymers can be easily processed and can provide a neutral environment for cell culturing. The inner edge face of the opening may comprise a height ranging from 5 pm to 500 pm, in particular from 5 pm to 250 pm.

[0037] The term “liquid-repellent material” with regard to the at least one support face is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a material with a surface contact angle greater than 90° for a definable liquid, in particular the liquid matrix composition. The term “liquid-attracting material” with regard to the at least one support face is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a material with a surface contact angle of less than 90° for a definable liquid, in particular the liquid matrix composition. Since the liquid matrix material may comprise an aqueous solution, the hydrophobicity / hydrophilicity may be a relevant characteristic of the first and / or second material. For determining the surface contact angle, the sessile drop contact angle test may be performed, preferably using a Young-Laplace fit to determine the surface contact angle in a drop shape analysis. In particular, an amount of 4 pl of distilled water may be used per test to assess the hydrophobicity / hydrophilicity.

[0038] According to a further preferred embodiment of the invention, said at least one confining outer edge forms part of a first material layer and / or that said at least one support face and / or an inner edge face of said opening forms part of a second material layer. Hence, the first and second material layers can be arranged on one anotherand connected to each other, thereby facilitating the assembly of the cell culture unit and / or the cell culture arrangement. Furthermore, such a design of the cell culture unit and / or the cell culture arrangement facilitates the use of different materials for first and the second material layers.

[0039] According to a further preferred embodiment of the invention, said first and second material layers are connected to each other by means of adhesive bonding, preferably with a pressure-sensitive adhesive. It is also conceivable that the first and second material layers are connected to each other by means of photobonding, laser welding, solvent bonding and / or other suitable bonding techniques. An adhesive bond between the first and second material layer can ensure a leak-proof connection. By using a pressure-sensitive adhesive, wherein a certain amount of pressure is applied during the curing process, a bond line with a particularly uniform thickness may be achieved. This can result in a cell culture unit with a high dimensional accuracy.

[0040] According to a further preferred embodiment of the invention, said membrane support structure comprises at least one lateral face extending from said at least one confining outer edge, preferably wherein said at least one lateral face extends substantially vertically, and / or that said at least one support face extends substantially horizontally. The lateral face, in particular a vertically extending lateral face, can influence the angle, at which the meniscus meets the confining outer edge, in a way that the formation of a meniscus with a curvature extending over the opening is promoted. Hence, the process of applying the liquid matrix material on the membrane support structure can be facilitated. A horizontally extending at least one support face can prevent the liquid matrix material from running off to the sides, i.e. in a lateral direction, further facilitating the process of preparing the membrane. The term “lateral face” is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a surface with a normal, wherein the normal comprises a laterally inward directed component and may additionally comprise an axial component. In other words, the lateral face faces the opening. The terms “lateral” and “axial” may relate to a longitudinal axis, along which the well arrangement may extend and / or which may extend perpendicular to a plane defined by the opening. The longitudinal axis may be a central axis of the well arrangement and extend through a centre point of the opening. The term “vertical” is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to anorientation parallel to the gravity direction when the cell culture unit is arranged in a position of its intended use. The term “horizontal” is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to an orientation perpendicular to the gravity direction when the cell culture unit is arranged in a position of its intended use. Additionally or alternatively, the at least one lateral face may extend substantially parallel to the longitudinal axis, along which the well arrangement may extend and / or which may extend perpendicular to a plane defined by the opening, and / or the at least one support face may extend substantially perpendicular to the longitudinal axis.

[0041] According to a further preferred embodiment of the invention, said at least one lateral face comprises a height of at least 10 pm and a maximum of 1000 pm, preferably of at least 25 pm and a maximum of 500 pm, more preferably of at least 50 pm and a maximum of 200 pm, most preferably of 100 pm. Such dimensions of the lateral face can facilitate the formation of a meniscus of the liquid matrix material. Furthermore, such heights enable to provide an amount of liquid matrix material sufficient for it to span across the opening of the membrane support structure.

[0042] According to a further preferred embodiment of the invention, said at least one lateral face extends from said at least one support face forming at least one inner edge, preferably wherein said at least one inner edge comprises an angle of less than or equal to 135°, more preferably of less than or equal to 90°. This can promote the formation of a well-defined and stable meniscus of the liquid matrix material by creating a sharp boundary for the liquid matrix material interface. This configuration can enhance the control over the meniscus shape, minimizing irregularities and aiding in precise liquid placement or containment.

[0043] According to a further preferred embodiment of the invention, said at least one confining outer edge comprises an angle of less than or equal to 135°, more preferably of less than or equal to 90°. Such dimensions can promote the formation of a well-defined and stable meniscus of the liquid matrix material by creating a sharp boundary for the liquid matrix material interface. Furthermore, this configuration can enhance the control over the meniscus shape, minimizing irregularities and aiding in precise liquid placement or containment.According to a further preferred embodiment of the invention, said well arrangement comprises a main well with a first and a second chamber, wherein said opening is interposed between said first and second chambers, preferably wherein said second chamber is fluidly connected to at least one auxiliary well of said well arrangement. This design enables a selective exchange of media, nutrients or signaling molecules between the chambers, while maintaining a spatial separation. Hence, coculture systems or a gradient formation can be realized, for example enabling to study cell migration and interaction. The first chamber may serve as an apical chamber and the second chamber may serve as a basal chamber. This facilitates physiologically relevant cell culturing by mimicking the polarity of epithelial or endothelial cells. A width, preferably a diameter, of the first chamber may range from 8 to 10 mm. A height of the first chamber may range from 8 to 12 mm. A height of the second chamber, in particular a space between the formed membrane and a bottom of the second chamber, may range from 0.2 to 1 mm. The first chamber may in particular be arranged substantially above the second chamber, when the cell culture unit is used for its intended purpose. In other words, the first chamber may be a top chamber and the second chamber may be a bottom chamber.

[0044] According to a further preferred embodiment of the invention, said at least one auxiliary well is at least partially arranged circumferentially around said main well. Such an arrangement can enable an efficient use of space while maintaining a separation between the first and second chambers.

[0045] According to a further preferred embodiment of the invention, said curable liquid matrix material comprises a hydrogel. Hydrogel can provide a biocompatible substrate for cell attachment and growth, closely mimicking an extracellular matrix. Furthermore, hydrogel can be configured to mimic specific physiologically relevant cell culturing conditions, for example for tissue engineering and drug testing. The hydrogel may comprise a polymer network with water as the dispersion medium. It is noted that the hydrogel is in a liquid or semi-liquid state when applying it on the support face and is subsequently solidified by curing for forming the membrane.

[0046] According to a further preferred embodiment of the invention, said hydrogel comprises a natural hydrogel, for example comprising a solubilized extracellular matrix (ECM), such as Matrigel® and / or VitroGel® RGD, and / or collagen, and / or comprises asynthetic hydrogel, for example comprising polyethylene glycol. A natural hydrogel may comprise a polymer network derived from biological sources, such as polysaccharides (e.g., alginate, hyaluronic acid) or proteins (e.g., collagen, gelatin). Such natural hydrogels may be biocompatible and biodegradable and can be well suited for mimicking the extracellular matrix. Hence, natural hydrogels may for example be used for applications in tissue engineering, wound healing or drug delivery. The natural hydrogel may in particular comprise a bovine collagen solution and Matrigel®. A synthetic hydrogel may comprise a polymer network produced from synthetic materials, such as polyacrylamide (PAM), polyethylene glycol (PEG), in particular 8-arm PEG, or polyvinyl alcohol (PVA). Synthetic hydrogels can enable a precise control over their mechanical, chemical and / or physical properties. A hydrogel comprising 8-arm PEG shows a particularly low swelling upon being crosslinked as a suspended membrane, thereby ensuring to maintain its shape upon crosslinking.

[0047] According to a further preferred embodiment of the invention, preferably said opening comprises a width d, in particular a diameter d, and said at least one confining outer edge comprises a width D, in particular a diameter D, and said curable liquid matrix material comprises a viscosity of: at least 5 mPa*s, preferably wherein a ratio of said widths d / D is less than or equal to 0.5; preferably of at least 25 mPa*s, preferably wherein a ratio of said widths d / D is less than or equal to 0.67; more preferably of at least 150 mPa*s, preferably wherein a ratio of said widths d / D is less than or equal to 0.7. Such viscosities are particularly suitable for forming a free-standing coherent structure of the liquid matrix material spanning across the opening, in particular in combination with the specified ratios of the widths d / D.

[0048] The formed membrane may comprise a trans-epithelial electrical resistance (TEER) of a maximum of 50 Q*cm2, in particular of a maximum of 30 Q*cm2. Such low TEER values indicate that the membrane comprises a low permeability to ions and molecules, allowing better diffusion of nutrients, oxygen, and waste products and therefore promoting cell growth..

[0049] Furthermore, the formed membrane may comprise a baseline resistance value of at least 200 Q, in particular of at least 400 Q. Such high baseline resistance value can indicate a strong barrier integrity, which is beneficial for accurately modelling in vivo epithelial or endothelial barriers.Additionally or alternatively, the cell culture unit may comprise an apical and a basal chamber, wherein, for closely simulating conditions of an in vivo environment, the formed membrane may be configured in a way that a CB concentration in the basal chamber after 3h of CB diffusion across the membrane comprises a maximum of 0.95 pM, in particular a maximum of 0.7 pM. This way, a controlled molecular transport can be modelled, which is comparable to in vivo conditions.

[0050] Additionally or alternatively, for closely simulating conditions of an in vivo environment, the formed membrane may comprise an apparent paracellular permeability (Papp) of at least 200 nm / s, in particular of at least 550 nm / s. Such values can ensure an efficient solute diffusion through the membrane, closely simulating physiological paracellular transport conditions.

[0051] According to a further preferred embodiment of the invention, prior to said curing, a surplus amount of said curable liquid matrix material is removed, preferably wherein said surplus amount depends on a desired membrane thickness. Hence, the membrane thickness can be easily controlled.

[0052] According to a further preferred embodiment of the invention, said applying and / or removing of curable liquid matrix material is performed by means of a pipetter, preferably with a circular motion of a pipette tip of said pipetter. This technique allows for a precise and uniform application of a specific amount of the liquid matrix material. The circular motion facilitates an even distribution of the liquid matrix material on the at least one support face and helps to span the fluid across the opening. In particular, the applying and / or removing of curable liquid matrix material may be integrated in an automated process.

[0053] According to a further preferred embodiment of the invention, said formed membrane comprises a thickness of a maximum of 500 pm, in particular of at least 150 pm and a maximum of 350 pm. Such membrane thicknesses may enable a mechanical support and / or a nutrient diffusion for the cultured cells, which are comparable to conditions in an in vivo environment.According to a further preferred embodiment of the invention, said cell culturing arrangement comprises at least one of the following: a single well plate; a multi well plate; a cell culture insert. Single well plates can offer controlled conditions for specific experiments. Multi well plates can enable a high-throughput screening. Cell culture inserts can facilitate the study of cellular interactions across different chambers, making them versatile for a range of cell culture applications, from drug testing to coculture studies.

[0054] There are several ways how to design and further develop the teaching of the present invention in an advantageous way. To this end, it is to be referred to the patent claims subordinate to the patent claims directed to the first, second and third aspect of the invention on the one hand and to the following explanation of preferred examples of embodiments of the invention, illustrated by the drawing on the other hand. In connection with the explanation of the preferred embodiments of the invention by the aid of the drawing, generally preferred embodiments and further developments of the teaching will be explained.

[0055] In the drawing

[0056] Fig. 1a shows a cell culture unit according to an embodiment of the present invention in a cross-sectional top view,

[0057] Fig. 1b shows the cell culture unit according to the embodiment of Fig. 1a in a cross-sectional side view,

[0058] Fig. 2a-d show shapes of the opening and the confining outer edge of the cell culture unit according to different embodiments of the present invention in a top view,

[0059] Fig. 3a-c show a visualization of steps of a method according to the embodiment of the invention for preparing a membrane in a cell culture unit according to the embodiment of Fig. 1a-b,Fig. 4 shows steps of a method according to the embodiment of the invention for manufacturing a cell culture unit according to the embodiment of Fig.

[0060] 1a-b,

[0061] Fig. 5 shows: (a) shows schematic representations of a Hydrogel Membrane Plate (HMP) in a 3D view with the apical and basal chamber in each main well as shown in across-sectional view (b), and (c) the geometry of the membrane support structure in a schematic cross-sectional view, (d) Suspended hydrogel membranes fabricated in the HMP from PEG 3 % and MG / COL (1:3) (Scale bars = 1 mm), (e) results of measurements of the thickness [pm] and (f) vertical deviation [pm] of the suspended PEG 3 % and MG / COL (1:3) membranes, (g) results of Baseline resistance value measurements [Q], (h) TEER values [Q*cm2], (i) basal concentration of the small molecule CB [pM] after addition to the apical chamber and 3h incubation and the resulting (j) Papp values [nm / s] compared between acellular Costar Transwells® (black dots) and HMPs (white dots), both in empty state and with a hydrogel membrane,

[0062] Fig. 6 shows: (a) the results of an experiment testing the shear ratedependency of the viscosities [mPa*s] of H2O, PEG 2 %, COL 5.5 mg / mL and VitroGel® RGD. (b) the results of surface contact angle measurements with 4 pL-droplets of distilled water (using Young- Laplace fit) on COC, PMMA with a thickness of 50 pm, PMMA with a thickness of 250 pm, PET and hydrophilic film, (c) shows a schematic of the geometry for hydrogel confining with the opening comprising a diameter d and the confining outer edge comprising a diameter D,

[0063] Fig. 7-9 show tabular summaries of the results for VitroGel® RGD, COL 5.5 mg / mL and PEG 2 % achieved from counting the hydrogel loading successes out of n = 2 attempts per condition for different diameters D, diameters d and ratios of the diameters d / D, and for the materials COC, PMMA with a thickness of 50 pm, PMMA with a thickness of 250 pm and the hydrophilic film,Fig. 10 shows mass-based swelling ratios after crosslinking Q’ of 8-arm photocrosslinked PEG hydrogels with concentrations from 2-5 (w / v)% swollen in PBS overnight at room temperature,

[0064] Fig. 11 shows: (a) Bright field images (taken at 20x magnification) of Caco-2 intestinal epithelial cells cultured in Costar Transwells® and on MG / COL (1:3) hydrogel membranes in HMPs on days 1, 8 and 18 (Scale bars = 250 pm), (b) Papp values [nm / s] calculated from the diffusion of CB (MW 596.44 Da, 3h incubation) across Caco-2 barriers cultured in Costar Transwells® and HMPs and acellular controls over time for a total of 23 days,

[0065] Fig. 12 shows: (a) Bright field images (taken at 20x magnification) of mouse intestinal organoid-derived monolayers cultured in Costar Transwells® and on MG / COL (1:3) membranes in HMPs on days 0, 3 and 7 (Scale bars = 250 pm), (b) IF-stained mouse intestinal epithelium with E- Cadherin in red and DAPI in blue (Scale bar = 100 pm), (c) Papp values [nm / s] calculated from the diffusion of CB (MW 596.44 Da, 3h incubation) across mouse organoid-derived intestinal epithelium cultured in Costar Transwells® and HMPs and acellular controls for 7 days, (d) H&E-stained section of mouse organoid-derived intestinal epithelium on day 4 cultured in a Costar Transwell® and on MG / COL (1:3) membranes in HMPs (Scale bar = 100 pm),

[0066] Fig. 13 shows: (a) Bright field images (taken at 20x magnification) of mouse embryonic fibroblasts, mouse intestinal organoid-derived monolayers and their coculture in / on free-standing MG / COL (1:3) membranes in HMPs on days 0, 4 and 7 (Scale bars = 250 pm), (b) Papp values [nm / s] over time calculated from the diffusion of CB (MW 596.44 Da, 3h incubation) across mono- and cocultures of mouse fibroblasts and mouse intestinal barriers cultured in HMPs over time for a total of 7 days, (c) the results of Fig.13b at a different scale for the y-axis for a better distinction of Papp values [nm / s] between the monoculture of mouse intestinal epithelium and the coculture of mouse intestinal epithelium with fibroblasts, (d) TEER values [Q*cm2] (normalized to acellularcontrols) of mouse embryonic fibroblasts, mouse intestinal organoid- derived monolayers and their coculture in HMPs for 7 days, (e) H&E- stained sections of mouse intestinal epithelium and the coculture of mouse intestinal epithelium with fibroblasts on day 9 of culture inside HMPs,

[0067] Fig. 14 shows: (a) H&E and IF-stained sections (imaged at 25x magnification) of human intestinal organoid-derived monolayers cultured on MG / COL (1:3) membranes in HMPs on day 9 (Scale bars = 100 pm), after 48h pro-inflammatory cytokine treatment with (b) 20 ng / mL TNFa or (c) TNFa + IFNy (20 ng / mL, each). E-Cadherin staining in green, DAPI staining in blue and Ki67, Fabpl, Muc2, ZO1 and Casp3, respectively, in red. (d) Papp values [nm / s] calculated from the diffusion of CB (MW 596.44 Da, 3h incubation) across human organoid-derived intestinal epithelium cultured in Costar Transwells® and HMPs and acellular controls for 9 days, and

[0068] Fig. 15 shows: (a) Bright field images (taken at 20x magnification) of LAD2 human mast cells, human intestinal organoid-derived monolayers and their coculture in / on MG / COL (1:3) membranes in HMPs on days 1, 4 and 7 (Scale bars = 250 pm), (b) Papp values [nm / s] calculated from the diffusion of CB (MW 596.44 Da, 3h incubation) across mono- and cocultures of human mast cells and human intestinal barriers cultured in HMPs for 9 days, (c) TEER values [Q*cm2] (normalized to acellular controls) of human mast cells, human intestinal organoid-derived monolayers and their coculture in HMPs for 9 days.

[0069] Fig. 1a and 1b show a cell culture unit according to an embodiment of the present invention in a cross-sectional top view (Fig. 1a) and in a cross-sectional side view (Fig. 1b).

[0070] The cell culture unit 100 comprises a well arrangement 1 with a main well 1a and two auxiliary wells 1b, which are arranged circumferentially around the main well 1a. The well arrangement 1, in particular the main well 1a, comprises a membrane supportstructure 2, wherein the membrane support structure 2 comprises an opening 3 and a support face 4 that laterally, in particular radially, extends from the opening 3. Furthermore, the membrane support structure 2 comprises a confining outer edge 6 for confining a curable liquid matrix material, which can be provided on the support face 4 spanning across the opening 3. The confining outer edge 6 extends circumferentially around the opening 3 and the support face 4 and comprises an angle 8 of 90°. Furthermore, the confining outer edge 6 is offset inwardly from an inner side wall face 1 c of the well arrangement 1 , in particular the main well 1 a.

[0071] The opening 3 and the confining outer edge 6 each define a circular shape. Therein, the opening 3 comprises a diameter d. The confining outer edge 6 comprises a diameter D.

[0072] The confining outer edge 6 forms part of a first material layer 9a and the support face 4 and an inner edge face 10 of the opening 3 form part of a second material layer 9b. The first material layer 9a, hence the confining outer edge 6, comprises a first material 11a, wherein the first material 11a comprises a polymer, for example polyethylene terephthalate and / or cyclic olefin copolymer. The second material layer 9b, hence the support face 4 and the inner edge face 10 of the opening 3, comprises a second material 11b, wherein the second material 11b comprises a polymer, for example polymethyl methacrylate and / or polyethylene terephthalate. The first and second material layers 9a, b are connected to each other by means of adhesive bonding with a pressure-sensitive adhesive 9c.

[0073] The cross-sectional side view of Fig. 1b is associated with a cross-sectional plane 12 shown in Fig. 1a. From Fig. 1b, it can be seen that the confining outer edge 6 is stepped back from the inner edge face 10 of the opening 3. This results in the membrane support structure 2 comprising a lateral face 13 extending from the confining outer edge 6. Therein, the lateral face 13 extends vertically and the support face 4 extends horizontally. Hence, the lateral face 13 and the support face 4 form an inner edge 14 with an angle 15 of 90°. Furthermore, the lateral face 13 extends parallel to a longitudinal axis 16, along which the well arrangement 1 , in particular the main well 1b, extends and which extends perpendicular to a plane (not shown) defined by the opening 3. Additionally, the support face 4 extends perpendicular tothe longitudinal axis 16. The longitudinal axis 16 is a central axis of the well arrangement 1 and extends through a centre point 17 of the opening 3.

[0074] The main well 1 comprises a first and a second chamber 18a, b, wherein the opening 3 is interposed between the first and second chambers 18a, b. The second chamber 18b is fluidly connected to the auxiliary wells 1b of the well arrangement 1. Additionally, the first chamber 18a forms an apical chamber and the second chamber 18b forms a basal chamber.

[0075] Fig. 2a-d show shapes of the opening and the confining outer edge of the cell culture unit according to different embodiments of the present invention in a top view.

[0076] Fig. 2a shows an opening 3 and a confining outer edge 6, each having a circular shape. The opening 3 comprises a diameter d and the confining outer edge 6 comprises a diameter D.

[0077] Three openings 3 and confining outer edges 6, each comprising a square shape with rounded comers 19, are shown in Fig. 2b. The square shapes differ from each other in the radius of the rounded comers 19. The openings 3 each comprise a width d and the confining outer edges 6 each comprise a width D.

[0078] Fig. 2c shows an opening 3 and a confining outer edge 6, each comprising an elliptical shape. The opening 3 comprises a diameter d, and the confining outer edge 6 comprises a diameter D, wherein the diameters d and D are associated with the minor axes of the elliptical shapes.

[0079] Furthermore, two openings 3 and confining outer edges 6, each comprising a rectangular shape with rounded comers 19, are shown in Fig. 2d. The rectangular shapes differ from each other in the radius of the rounded comers 19. The openings 3 each comprise a width d and the confining outer edges 6 each comprise a width D, wherein the widths d and D are associated with the narrow dimensions of the rectangular shapes.Fig. 3a-c show a visualization of steps of a method according to the embodiment of the invention for preparing a membrane in a cell culture unit according to the embodiment of Fig. 1a-b.

[0080] In a first step S1 shown in Fig. 3a, the curable liquid matrix material 7 is applied on the support face 4 in a way that the curable liquid matrix material 7 spans across the opening 3. For applying the curable liquid matrix material 7, a pipetter is used, wherein a pipette tip 20 of the pipetter is moved across the support face 4 in a circular motion. The confining outer edge 6 may serve as a lateral guidance for the pipette tip 20. For example, for an opening 3 with a diameter d of 3 mm, liquid matrix material 7 in an amount of 30-35 pL may be applied (S1 ).

[0081] In the next step S2, a surplus amount of the curable liquid matrix material 7 is removed (Fig. 3b). For example, when an amount of 30-35 pL has been applied in step S1, a surplus amount may be removed to reduce the remaining amount of liquid matrix material 7 to 12 pL, which still ensures that the liquid matrix material 7 spans across the opening 3.

[0082] Subsequently, the curable liquid matrix material 7 is cured to form the membrane 5 (step S3, Fig. 3c). The curing (S3) may for example be achieved by exposing the liquid matrix material 7 to UV radiation and / or heat for a specific exposure time.

[0083] Fig. 4 shows steps of a method according to the embodiment of the invention for manufacturing a cell culture unit according to the embodiment of Fig. 1a-b.

[0084] In a first step S101, multiple material layers 9, 9a, 9b comprising the first material layer 9a and the second material layer 9b are provided. The material layers 9, 9a, 9b are washed (step S102) with an ultrapure water, for example MilliQ, being substantially free from ions, organics, particulates and biological contaminants like bacteria or endotoxins, and blown dry (step S103) with pressurized air. Subsequently, for connecting the material layers 9, 9a, 9b to each other, a pressure-sensitive adhesive 9c is applied on the material layers 9, 9a, 9b in step S104 and the material layers 9, 9a, 9b are aligned by means of pins and matching alignment holes in step S105. For activating the curing process of the adhesive 9c, a pressure is applied (step S106). This step comprises the use of a 2 kg hand roller to firmly attach the materiallayers to each other and additionally comprises applying a pressure force of 15 kN for 15 min by means of a hydraulic press. After curing (step S107) of the adhesive 9c, the cell culture unit 100 is washed again with ultrapure water (step S108), fully dried (step S109) and UV-sterilized (step S110) for 60 min.

[0085] Examples

[0086] Method 1: Assessing epithelial barrier integrity with TEER and Papp measurements

[0087] Trans-epithelial electrical resistance (TEER) values were measured with an EV0M2 Epithelial Voltohmmeter (189138 AC11A, World Precision Instruments) and a chopstick-style electrode by placing the electrode inside the HMP or Costar Transwell® with the intestinal monolayers and reading the measured resistance value [Q] from the device. TEER values were calculated by multiplying the measured resistance with the surface area of the Costar Transwell® using the following eguation: TEER = [Q] x [cm2]. To normalize TEER measurements, the values from acellular Costar Transwells® were subtracted from all other conditions. For each experimental condition, TEER values were measured in n = 3 biological replicates.

[0088] Furthermore, the flux of a small molecule across the intestinal barriers was measured to assess the apparent paracellular permeability (Papp), which is another measure of barrier integrity. First, the intestinal monolayers were gently washed with warm PBS. Then, 600 pL of cell culture medium were added to the basal chambers and 200 pL of cell culture medium containing 10 pM Cascade Blue™ (CB; C3239, Invitrogen) were added to the apical chambers. For Caco-2 cells, complete cell culture medium was used, while for the mouse and human intestinal organoid-derived monolayers Advanced DMEM / F12 medium + 1X Glutamax + 10 mM HEPES + 100 U / mL P / S was used instead of full medium. The samples were incubated for 3h at 37°C, 5% CO2 and 95% humidity. After the incubation period 100 pL were collected from each basal chamber in a black 96-well plate (655087, Greiner Bio-One), to measure the amount of CB that diffused across the intestinal epithelium. With a plate reader (FlexStation 3, FV06263, Molecular Devices), the fluorescence was measured with excitation at 380 nm and emission at 420 nm. A linear standard curve was fitted to themeasurements of the calibration samples to relate fluorescent intensity to CB concentration. The blank measurement was subtracted from the detected fluorescence values and using the following equation, Papp values were calculated: Papp = (Vrec x dCrec) / (A x dt x Cdon,t=o), with Vrec = medium volume in basal chamber in [mL], dCrec = CB concentration in [pM] at t = 3h in the basal chamber (calculated via calibration curve), A = diffusion area in [cm2], dt = incubation time in [s] and Cdon = CB concentration in [pM] at to in the apical chamber. For each experimental condition, Papp values were measured in n = 3 biological replicates. Acellular samples served as controls to the intestinal monolayers.

[0089] Method 2: Microtome sectioning & Hematoxylin and Eosin (H&E) staining

[0090] The fixed intestinal monolayers were carefully removed from the HMP with tweezers and embedded in HistoGel™ Specimen Processing Gel (12006679, Epredia). After histogel polymerization at 4°C, samples were transferred to biopsy cassettes and stored in 10% Formalin solution until processing. Samples were cut in half with a razor blade to make vertical sections, followed by overnight sample dehydration in the HistoCore PEARL tissue processor (Leica). Afterwards, the samples were embedded in liquid paraffin on the Medite embedding center and these formalin-fixed paraffin-embedded (FFPE) blocks were then cut into 5 pm-thick sections on a Microtome (Thermo Fisher) and transferred onto SuperFrost Ultra Plus™ GOLD Adhesion Slides (11976299, Epredia). The slides were kept at 40°C until they dried.

[0091] H&E staining was done manually with the Abeam H&E Staining Kit (ab245880, abeam) following the supplier’s instructions. Briefly, the sections were deparaffinized and hydrated in distilled water, followed by a 5 min incubation with Hematoxylin, Mayer’s (Lillie’s Modification) at room temperature. The slides were rinsed 2x in distilled water. Next, the tissues were stained for 10-15 seconds in Bluing Reagent and rinsed again 2x in distilled water. Slides were dipped in absolute alcohol, followed by a staining in Eosin Y Solution (Modified Alcoholic) for 2-3 min at room temperature. Slides were rinsed with absolute alcohol and dehydrated in three changes of absolute alcohol. After this, the slides were cleared and mounted in synthetic resin. H&E-stained sections were imaged with a bright field whole slide scanner (Hamamatsu, NanoZoomer S360) and visualized using the NDP.view2 Image viewing software.Method 3: 4-i

[0092]

[0093] immunofluorescence

[0094]

[0095] 4-plex IF staining of the FFPE sections was done with the Ventana Discovery Ultra automated tissue stainer (Roche Tissue Diagnostics, Tucson AZ USA). First, slides were baked at 60°C for 8 min, followed by deparaffinization over three cycles of 8 min at 69°C with Discovery Wash (950-510, Ventana). As a pretreatment, heat-induced antigen retrieval was done for 40 min with Discovery CC1 (950-500, Ventana) at 92°C, followed by a blocking pre-treatment with Discovery Goat Ig Block (760-6008, Ventana) for 32 min at 37°C. Then, samples were blocked for 16 min with Discovery Inhibitor (760-4840, Ventana) and subsequently neutralized. Primary antibodies were diluted in Discovery Ab Diluent (760-108, Ventana) in previously optimized concentrations and applied at 37°C for 40-60 min in consecutive staining sequences in two separate 4-plex panels. In panel 1 the order was as follows: Ki67 (MA5-14520, Thermo), Fabpl (PA5-28945, Thermo), Muc2 (MA5-12345, Thermo) and E-Cadherin (790-4497, Ventana). In panel 2 the order was Caspase3 (C10423, Thermo), Ki67, ZO1 (33-9100, Thermo) and E-Cadherin. Primaries were detected with species-matched secondary antibodies conjugated to horseradish peroxidase (HRP) (OmniMap anti-Mouse HRP, 760-4310, Ventana; OmniMap anti-Rabbit HRP, 760-4311, Ventana; OmniMap anti-Goat HRP, 760-4647, Ventana; OmniMap anti-Rat HRP, 760-4457, Ventana) for 8 min and subsequently visualized using Opal-driven fluorophores from Akoya (Opal 480 Reagent Pack, FP1500001 KT; Opal 520 Reagent Pack, FP1487001 KT; Opal 570 Reagent Pack, FP1488001 KT; Opal 620 Reagent Pack, FP1495001 KT). For multiplexed IF staining, after each sequence of primary and secondary antibody-HRP incubation and detection with Opal reagent, samples were prepared for the next staining sequence by antibody denaturation with ULTRA Cell Conditioning 2 (ULTRA CC2, 950-223, Ventana) for 4 min at 92°C and neutralization with Discovery Inhibitor (760-4840, Ventana) for 16 min at room temperature. Lastly, samples were counterstained with 4’,6-Diamidino-2-phenylindol (DAPI, Roche) for 32 min at room temperature. IF-stained samples were imaged with a Leica Stellaris 8 confocal microscope (Leica Microsystems) at 25x magnification with a water-immersion objective.

[0096]

[0097] 1.1 Aim of the Experiment

[0098] The aim of this experiment is to investigate the thickness of membranes formed from PEG and MG / COL hydrogels in cell culture units according to the invention.

[0099] 1.2 Experimental Protocol

[0100] For testing, 24 cell culture units according to the embodiment of Fig. 1a-b were combined into an SBS standard well plate format to form a Hydrogel Membrane Plate (in the following “HMP”). The HMP represents a cell culture arrangement according to an embodiment of the invention. The opening had a diameter d = 3 mm. The confining outer edge had a diameter D = 6 mm. An inner edge face of the opening had a height of 250 pm. The lateral face extended vertically and had a height of 100 pm. Fig. 5 shows (a) the HMP with (b) the apical and basal chamber in each main well, and (c) the geometry of the membrane support structure.

[0101] HMPs were fabricated by individually laser-cutting different material layers, as indicated in Fig. 1b, using a Trotec Speedy 360. Before assembly, the laser-cut material layers were carefully washed with MilliQ water and blown dry with pressurized air. The HMP was manually assembled from top to bottom with pressuresensitive adhesive (PSA; 94119, Adhesives Research) layers in between the material layers for bonding. The material layers were aligned by means of alignment pins and matching alignment holes. A 2 kg hand roller (Ziegler) was used to firmly attach the layers to ensure the HMP was leak-proof. After assembly, the HMP was put into a hydraulic press at 15 kN for 15 min (Polystat 200 T, Servitec) for further activation of the adhesive, washed again in MilliQ water, fully dried and UV-sterilized for 60 min (PURITEC® HNS® UV-C lamp, OSRAM). Then, the HMP was covered with the lid from a commercially available 96-well tissue culture plate (CytoOne).

[0102] Generally, the workflow for forming the membranes corresponds to the method shown in Fig. 3a-c and described above. For example, for a device with an opening with a diameter d of 3 mm, hydrogel in an amount of 30-35 pL is applied on the support face by slow pipetting of the hydrogel in a circular motion, thereby closing the 3 mm-opening with hydrogel. Next, a surplus amount of hydrogel is slowly removed bypipetting, so that an amount of 12 pL of hydrogel remains. Subsequently, the hydrogel is cured, so that a free-standing hydrogel membrane is formed.

[0103] In the following the specific hydrogel compositions and adaptations to the procedure are described.

[0104] a) Matriqel / Collaqen (MG / COL)

[0105] An MG / COL (1:3) hydrogel solution (in the following “MG / COL (1:3)”) was prepared from neutralized TeloCol-6 Type I bovine collagen solution (COL; final concentration after neutralization = 5.38 mg / mL for 100% COL hydrogels; Catalog No.: 5225-50ML, Advanced BioMatrix), which was combined with Matrigel® Matrix Basement Membrane (MG; Catalog No.: 356231 , Coming, 7.5 mg / mL protein) in a 1 :3 volumetric ratio, e.g. 100 pL MG was added to 300 pL neutralized COL. Prior to applying the MG / COL (1:3) on the support face, the HMP was cooled to and stored at 4°C to prevent crosslinking. Upon 15 min crosslinking of the MG / COL (1 :3) at 37°C, medium was added to both basal and apical chambers to keep the membranes hydrated. Importantly, reverse pipetting technique was utilized to add medium, in order to prevent the introduction of air bubbles especially in the basal chamber.

[0106] b) Polyethylene glycol (PEG)

[0107] To produce synthetic hydrogel membranes, a 3 (w / v)% polyethylene glycol (PEG) hydrogel precursor solution (in the following “PEG 3 %”) was prepared from PEG-Norbornene (8-arm, 20 kDa, PSB-8210, Creative PEGWorks) and PEG-SH (8-arm, 20 kDa, PSB-854, Creative PEGWorks) with 3 mM Lithium-Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; 900889, Sigma) as a photoinitiator. The applied PEG 3 % was cured by photocrosslinking for 6 min under UV-C light (254 nm, 7 W, PURITEC® HNS® UV-C lamp, OSRAM) and immediately after crosslinking PBS (10010-015, gibco) was added.

[0108] To characterize the formed membranes, MG / COL (1 :3) membranes were stained with Recombinant Alexa Fluor® 647 Anti-Collagen I antibody (1 pg / mL, ab280968, abeam) and PEG 3 % membranes were stained with Alexa Fluor™ 546 C5 Maleimide (20 pM, A10258, Invitrogen) overnight at 4°C. Images were acquired with a Leica Stellaris 8 confocal microscope (Leica Microsystems) and membrane thickness and therespective vertical deviation were measured in Imaged with the lnteredgeDistance_lmageJMacro_2.0 with n = 5 replicates per condition.

[0109] 1.3 Results

[0110] As shown, in Fig. 5d-f, the formation of suspended hydrogel membranes works with both synthetic hydrogels, e.g. PEG 3 %, and natural hydrogels, e.g. MG / COL (1:3). PEG 3 % and MG / COL (1:3) membranes cross-linked and stained inside the HMPs are shown in a top and a side view in Fig. 5d. Image analysis and quantification revealed an average membrane thickness of 298.4 ± 7.7 pm for PEG 3 % membranes and 257.8 ± 23.7 pm for MG / COL (1:3) membranes (Fig. 5e), as well as a vertical deviation of -54.4 ± 4.6 pm for PEG 3 % membranes and -98.6 ± 38.1 pm for MG / COL (1:3) membranes (Fig. 5f).

[0111]

[0112] 2.1 Aim of the Experiment

[0113] The aim of this experiment is to characterize functional integrity, permeability, and selective transport properties of the membranes formed in a cell culture unit according to the invention, providing a comprehensive assessment of its ability to simulate physiological barrier functions. Specifically, the baseline resistance value, the trans-epithelial electrical resistance (TEER), the CB concentration diffusion and the apparent paracellular permeability (Papp) in both acellular HMPs and Costar Transwells® without and with membranes formed of hydrogel are determined.

[0114] 2.2 Experimental Protocol

[0115] For forming the membranes, HMPs with MG / COL (1:3) membranes according to the embodiment described in Example 1 were used. Furthermore, the trans-epithelial electrical resistance (TEER) and the apparent paracellular permeability (Papp) were measured. For details on the methods used for determining the TEER and Papp values, it is referred to “Method 1”.

[0116] 2.3 Results

[0117] The results are shown in Fig. 5g-j. In Costar Transwells® the baseline resistance measurements yielded 193.8 ± 14.1 0 for the empty Costar Transwell® and 181.8 ± 6.6 Q for the hydrogel-coated one. In HMPs, the baseline resistance values weregenerally increased compared to the empty Costar Transwells® with the wells having a baseline resistance of 389.0 ± 19.3 Q and the well with a suspended hydrogel membrane having a baseline resistance of 414.8 ± 17.2 Q (Fig. 5g). Baseline TEER values, which are the baseline resistance values that were normalized to the surface area, were shown to be 63.9 ± 4.7 0* cm2for empty Costar Transwells® and 60.0 ± 2.2 0* cm2for hydrogel-loaded Costar Transwells®, as well as 27.5 ± 1.40* cm2for empty HMPs and 29.3 ± 1.2 Q*cm2for hydrogel-loaded HMPs (Fig. 5h), indicating that the baseline TEER values of Costar Transwells® are already higher than the ones of HMPs. The measured CB concentrations in the basal chamber after 3h of CB diffusion across the apical-basal membrane were 1.7 ± 0.0 pM in the empty Costar Transwell®, 1 ± 0.0 pM in the hydrogel-loaded Costar Transwells®, 2.6 ± 0.1 pM in the empty HMPs (which equals a complete diffusion) and 0.7 ±0.1 pM in the hydrogel-loaded HMPs (Fig. 5i). The Papp values, which were calculated based on the CB concentrations, yielded a permeability of 281.0 ± 3.2 nm / s for empty Costar Transwells®, 168.0 ±6.9 nm / s for hydrogel-loaded Costar Transwells®, 2034.0 ±75.1 nm / s for empty HMPs and 568.0 ± 96.5 nm / s for hydrogel-loaded HMPs (Fig. 5j), showing that - based on Papp values as a measure of barrier integrity - acellular Costar Transwells® already constitute an increased barrier to small molecule diffusion compared to HMPs.

[0118]

[0119] 3.1 Aim of the Experiment

[0120] The aim of this experiment is to identify geometries and materials of the cell culture unit according to the invention that enable the formation of a free-standing coherent structure of the liquid matrix material spanning across the opening and subsequently the formation of a cured membrane, i.e. the success of hydrogel loading. Specifically, the diameter d defined by the opening and the diameter D defined by the confining outer edge are varied. Furthermore, different materials for the membrane support structure and different hydrogels are investigated. Additionally, viscosities of the hydrogels and water as well as surface contact angles of the materials used are determined, to investigate the influence of these parameters on the formation of hydrogel membranes.3.2 Experimental Protocol

[0121] The cell culture units tested corresponded to the embodiment of Fig. 1a-b. Thereby, an array of structures with varying diameters d (2, 3, 4, 5, 6 and 7 mm), defined by the opening interposed between the apical and basal chamber, and diameters D (6, 8 and 10 mm), defined by the confining outer edge, was tested.

[0122] Furthermore, a range of materials with different surface properties, i.e. cyclic olefin copolymer COC (mcs-foil 015, Microfluidic ChipShop), polymethyl methacrylate PMMA (106-630-62, goodfellow) with thicknesses of 50 pm and 250 pm and a hydrophilic film of PET (62580, Tesa), were used to produce the material layer corresponding to the support face. To produce the material layer corresponding to the confining outer edge, cyclic olefin copolymer COC was used throughout all test series. Sessile drop contact angle experiments were performed with a Theta Flow Auto 4 (Attension Theta Flow Optical Tensiometer, Biolin Scientific) using Young-Laplace fit to determine the surface contact angle in drop shape analysis. Surface contact angles were measured with distilled water (4 pL, n = 6 per material).

[0123] The success of hydrogel loading was investigated with photochemically crosslinkable 2 (w / v)% PEG hydrogels (in the following “PEG 2 %”) (produced and cured analogously to the description provided above in Experiment 1), temperaturesensitive 5.5 mg / mL COL hydrogels (in the following “COL 5.5 mg / mL”) and ionically crosslinked VitroGel® RGD gels (TWG003, TheWell Bioscience), which were fabricated according to the vendor’s protocols. The COL 5.5 mg / mL hydrogel was prepared from neutralized TeloCol-6 Type I bovine collagen solution (COL; 5.5 mg / mL after neutralization, 5225-50ML, Advanced BioMatrix). Briefly, the VitroGel® RGD solution was mixed with DMEM (4.5 g / L D-Glucose, 41965-039, Gibco) in a 2:1 ratio. After an initial crosslinking of 15 min at 37°C, more DMEM was added according to the vendor’s protocols for a complete crosslinking with the calcium ions present in the medium.

[0124] Furthermore, viscosity measurements of these hydrogels and of water as a control liquid were performed with a Rheosense m-VROC RP Sample Volume Viscometer (M-A100066, RheoSense). Since the investigated materials had different viscosities, the tested shear rates were chosen for each material independently. Except for COL 5.5 mg / mL, which was measured at 10°C corresponding to the processing conditions,all viscosities were measured at room temperature. Water was measured with a shear rate ranging from 995.3 to 3486.2 s’1. For both PEG 2 % and COL 5.5 mg / mL the tested shear rate ranged from 48.9 to 248.4 s’1and VitroGel® RGD was characterized at a constant shear rate of 5.4 s’1.

[0125] 3.3 Results

[0126] The results are shown in Fig. 6-9. As shown in Fig. 6a, the hydrogels that were tested for producing suspended membranes varied in their viscosity properties. For water, being the control liquid, the viscosity was measured to range from 1.6-2.3 mPa*s at a shear rate of 995.3-3486.2 s’1. PEG 2 % was shown to be a shear-thinning material with a viscosity of 16.7 mPa*s at a shear rate of 48.9 s’1that was reduced to 7.5 mPa*s at 248.4 s’1. Similarly, COL 5.5 mg / mL behaved as a shear-thinning material over the same shear rate range, with 207.7 mPa*s at 48.9 s’1and 28.6 mPa*s at 248.4 s’1. The viscosity of VitroGel® RGD was measured to be 197.4 mPa*s at a constant shear rate of 5.4 s’1.

[0127] Different materials, onto which the hydrogel solutions were dispensed, were characterized for their hydrophilicity / hydrophobicity by measuring the static water surface contact angle. In Fig. 6b, the measured surface contact angles are shown to be 104.3 ± 1.4° for COC, 84.7 ± 1.2° for PMMA (50 pm thickness), 79.0 ± 1.7° for PMMA (250 pm thickness), 74.8 ± 2.6° for PET and 33.5 ± 1.9° for the hydrophilic film, where surfaces with contact angles >90° are considered hydrophobic and surfaces with contact angles <90° hydrophilic.

[0128] Counting the success of hydrogel loading, VitroGel® RGD membranes could be produced with 100% success rate for all materials and for all combinations of diameter D and diameter d (Fig. 7). The ratio d / D, which resulted in a successful hydrogel loading, ranged from 0.25 to 0.70.

[0129] With COL 5.5 mg / mL, all membranes with diameters D of 6, 8 and 10 mm in combination with diameters d of 2, 3, 4 and 5 mm were successfully formed (Fig. 8). In this relation, successful ratios d / D ranged from 0,25 to 0,67. An opening with a diameter d = 6 mm could be covered with a success rate of 50% with membrane support structures comprising COC and the hydrophilic film.Using PEG 2 %, free-standing membranes could be produced with 100% success rate on all plate materials and with diameters D of 6, 8 and 10 mm in combination with diameters d of 2, 3 and 4 mm (Fig. 9). In this relation, successful ratios d / D ranged from 0.25 to 0.50. For D = 10 mm in combination with d = 5 mm, membranes could be formed with PMMA with thicknesses of 50 pm and 250 pm and with the hydrophilic film. Furthermore, membranes could be formed with membrane support structures comprising COC, PMMA with a thickness of 50 pm and with the hydrophilic film for D = 6 mm in combination with d = 4 mm.

[0130]

[0131] 4.1 Aim of the Experiment

[0132] Due to its hydrophilicity, synthetic PEG hydrogels have a tendency to swell upon crosslinking. Since this could negatively affect the shape and stability of the hydrogel membrane suspended inside the cell culture unit, the aim of this experiment is to identify a PEG hydrogel formulation that comprises low swelling upon being crosslinked as a suspended membrane.

[0133] 4.2 Experimental Protocol

[0134] A mass-based swelling test was performed, wherein PEG hydrogel formulations ranging from 2-5 (w / v)% were prepared from 8-arm PEG-SH and PEG-Norbornene with a molecular weight of 20 kDa. The hydrogels were photocrosslinked directly inside pre-weighed Eppendorf tubes for 6 min under UV-C light using 3 mM LAP as a photoinitiator. Directly after crosslinking, the hydrogels were weighed inside the Eppendorf tubes to determine their crosslinked weight mgc(Mettler Toledo AT261 DeltaRange Scales, Mettler Toledo). Then, hydrogels were swollen overnight in PBS at room temperature and upon careful PBS removal, the Eppendorf tubes were closed immediately and the swollen weight mgswas measured. Given that the hydrogels have a water content of >95%, their density p was approximated as 1 , resulting in mgc« Vgcand mgs« Vgs. To calculate the swelling ratio after crosslinking Q’, the following equation was used: Q' =Vgs / Vgc.

[0135] 4.3 Results

[0136] As shown in Fig. 10, for the tested 8-arm PEG hydrogels, swelling after crosslinking seemed uncoupled from the polymer concentration. Indicated by a Q’ of 1, thevolumes stayed the same in both crosslinked and swollen states for hydrogels ranging from 2-5 (w / v)%. Hence, the 8-arm PEG hydrogels within this concentration range could be considered non-swelling and thus particularly suitable for the formation of suspended hydrogel membranes.

[0137]

[0138] 5.1 Aim of the Experiment

[0139] The aim of this experiment is to investigate the suitability of the cell culture unit and the membrane formed therein for culturing human intestinal epithelium compared to the state of the art. A specific goal is to compare properties of membranes formed in cell culture units according to the invention and of Caco-2-derived intestinal monolayers cultured on these membranes with properties of Costar Transwells® and such monolayers cultured on these Costar Transwells®. The investigated properties comprise the apparent paracellular permeability (Papp). Furthermore, the morphology of the cell cultures is investigated.

[0140] 5.2 Experimental Protocol

[0141] Caco-2 immortalized cells from human colorectal adenocarcinoma (HTB-37, ATCC) were cultured according to the supplier’s instruction. Briefly, they were expanded in DMEM (4.5 g / L D-Glucose, 41965-039, gibco) + 10% fetal bovine serum (FBS, Qualified One Shot™, A31605-01, gibco) + 100 U / mL Penicillin-Streptomycin (P / S; 15140-122, gibco) in TC-treated cell culture flasks (353136, Coming) at 37°C, 5% CO2 and 95% humidity. At 80-100% confluence, the cells were split in a 1:10 ratio. Medium was exchanged three times per week.

[0142] In order to characterize the barrier integrity of Caco-2 -derived intestinal monolayers, Caco-2 cells were seeded on membranes formed of MG / COL (1:3) hydrogel (as described in Example 1) in HMPs with D = 6 mm and d = 3 mm (as described in Example 1) and Costar Transwell® Permeable Supports (24 well plate inserts, 0.4 pm pores, transparent PET membrane, 3470, Coming) at a density of 3’000 cells I mm2. The Caco-2 monolayers were cultured for 21 days in DMEM + 10% FBS + 100 U / mL P / S at 37°C, 5% CO2 and 95% humidity. Medium was exchanged three times per week (600 pL in the basal chamber, 200 pL in the apical chamber). To track epithelium maturation, the apparent paracellular permeability (Papp) was measuredevery 3-4 days. For details on the methods used for determining the Papp values, it is referred to “Method 1”. On day 21, Caco-2 monolayers were treated for 48h with pro-inflammatory cytokines: the medium in the basal chamber was exchanged for fresh medium containing recombinant human TNFa (20 ng / mL, 210-TA, R&D Systems) or a combination of recombinant human TNFa + recombinant human IFNy (20 ng / mL each, 285-IF, R&D Systems). After this treatment period, the Caco-2 monolayers in both HMPs and Costar Transwells® were fixed in 4 % paraformaldehyde solution (J19943.K2, Thermo Fisher Scientific) for 30 min at room temperature and kept in PBS until processing for immunohistochemical stainings. For details on the methods used for immunohistochemical stainings, it is referred to “Method 2 and “Method 3

[0143] Furthermore, bright field images of the cell cultures were taken over the course of 18 days.

[0144] 5.3 Results

[0145] The results are shown in Fig. 11. The bright field images taken over the course of 18 days (Fig. 11a), show comparable proliferation, confluency and morphology of Caco-2 cells cultured in HMPs and Costar Transwells®. In the acellular MG / COL (1:3) membrane of the HMP Papp values ranging from 670-1140 nm / s were measured, while the detected Papp values of the acellular Costar Transwells® were around 210-290 nm / s. This shows that the Costar Transwells® already have a certain baseline barrier function, compared to the suspended hydrogel membranes that allow for more CB diffusion across the acellular membrane. In confluent Caco-2 monolayers cultured in Costar Transwells®, Papp values decreased from 13.8 nm / s on day 2 to 0.0 nm / s on day 23, while the Caco-2 cells cultured on suspended MG / COL (1:3) membranes had a Papp value of 22.9 nm / s on day 2 that then decreased to 0.0 nm / s on day 23.

[0146]

[0147] 6.1 Aim of the Experiment

[0148] The aim of this experiment is to investigate the suitability of the cell culture unit and the membrane formed therein for culturing epithelial monolayers compared to the state of the art. A specific goal is to compare properties of membranes formed in cell culture units according to the invention and of primary intestinal cells derived frommouse small intestinal organoids cultured as epithelial monolayers on these membranes with properties of Costar Transwells® and such epithelial monolayers cultured on these Costar Transwells®. In this regard, the apparent paracellular permeability (Papp) is investigated. Furthermore, the morphology of the cell cultures is investigated.

[0149] 6.2 Experimental Protocol

[0150] Mouse intestinal crypts were isolated from the small intestine of euthanized C57BL / 6J naive mice. The obtained intestinal tissue was extensively washed in cold PBS + Primocin (0.1 mg / mL, ant-pm-1, InvivoGen). The small intestine was located between the stomach and the cecum and opened lengthwise using autoclaved surgical scissors. The luminal side was gently scraped with a frozen glass slide to remove luminal content and villous structures. Following further washing steps in PBS + Primocin, the tissue segment was cut into small pieces with a scalpel and transferred to 20 mL of 8 mM EDTA (R1021, Thermo Scientific) in PBS. After 30 min incubation at 4°C, with intermittent snap-shaking every 10 min, the supernatant was collected and run through a 70 pm filter (MACS® SmartStrainers (70 pm), 130-110-916, Miltenyi Biotec). The remaining tissue fragments were washed in PBS + 0.1% BSA (MACS® BSA Stock Solution (10%), 130-091-376, Miltenyi Biotec) and the supernatant was again collected and filtered. The filtered supernatants were centrifuged at 4°C and 400g for 5 min and the resulting pellets were resuspended in Advanced DMEM / F12 medium (12634-010, gibco) + IXGlutamax (35050-061, gibco) + 10 mM HEPES (15630-056, gibco) + 100 U / mL P / S + 0.1% BSA. After another centrifugation step at 4°C and 400g for 5 min, the pelleted crypts were resuspended in 50% MG (diluted with Advanced DMEM / F12 medium) and pipetted as 25 pL domes in pre-warmed 24-well plates (CytoOne). After 15-30 min of crosslinking at 37°C, ENR medium consisting of Advanced DMEM / F12 medium + 1X Glutamax + 10 mM HEPES + 100 U / mL P / S + 2% B27 supplement without Vitamin A (12587-010, gibco) + 0.5% Rspondin3 from conditioned medium (R001, ImmunoPrecise Antibodies) + 0.5% Noggin from conditioned medium (N002, ImmunoPrecise Antibodies) + 1 mM N-acetylcysteine (A9165, Sigma) + 50 pg / mL Primocin + 50 ng / mL recombinant human EGF (AF-100-15, Peprotech) was added to the MG-embedded intestinal crypts. For the first 24-48h after crypt isolation, 10 pM Y-27632 (72305, STEMCELL Technologies) was added to prevent apoptosis. Intestinal organoids were incubated at 37°C, 5% CO2 and 95% humidity and ENR medium was exchanged three timesper week. Every 4-7 days the growing mouse intestinal organoids were mechanically dissociated and passaged in a 1:4 - 1:6 ratio. After passaging, 10 pM Y-27632 was added to the medium for the first 24-48h.

[0151] To generate mouse small intestinal monolayers, organoids were dissociated into a single cell suspension in TrypLE™ Express (12604-013, gibco) + 10 pM Y-27632 + 500 U / mL DNAse I recombinant (04536282001, Roche) for 10 min at 37°C and strained over a 40 pm strainer. Mouse small intestinal single cells were seeded on membranes formed of MG / COL (1:3) hydrogel (as described in Example 1) in HMPs with D = 6 mm and d = 3 mm (as described in Example 1) and Costar Transwell® Permeable Supports at a density of 5’000-7’000 cells I mm2. Prior to cell seeding, the Costar Transwell® Permeable Supports were coated with 0.5 mg / mL Cultrex 3-D Culture Matrix Laminin I (3446-005-01 , R&D Systems) + 0.5 mg / mL Type IV Collagen from human placenta (C5533, Sigma-Aldrich). The coating was done overnight at 4°C, followed by a 1h incubation at 37°C directly before cell seeding. The mouse intestinal monolayers were cultured for 7 days in ENRW-Th medium (ENR + 0.15 nM Wnt surrogate (N001, ImmunoPrecise Antibodies) + 2.5 pM Thiazovivin (04-0017, Stemgent)) at 37°C, 5% CO2 and 95% humidity. Medium was exchanged daily (600 pL in the basal chamber, 200 pL in the apical chamber). Papp measurements were performed every day. For details on the method used for determining the Papp values, it is referred to “Method 1”. At the end of the experiment, the mouse intestinal monolayers in both HMPs and Costar Transwells® were fixed in 4% paraformaldehyde solution for 30 min at room temperature and kept in PBS until processing for immunohistochemical stainings. For details on the methods used for immunohistochemical stainings, it is referred to “Method 2” and “Method 3”.

[0152] Furthermore, bright field images of the cell cultures were taken over the course of 7 days.

[0153] 6.3 Results

[0154] As shown in the bright field images in Fig. 12a, confluent mouse intestinal epithelium could be grown in both setups for up to 7 days of culture with comparable morphologies. The IF-stained image in Fig. 12b reveals that the mouse intestinal cells cultured on suspended MG / COL (1:3) membranes express the epithelial cell marker E-Cadherin. The data shown in Fig. 12c shows similar Papp results for the acellularcontrols as previously displayed in Fig. 11b: with 160.6 nm / s, permeability of acellular Costar Transwells® was clearly lower already at baseline than the 679.9 nm / s measured in acellular MG / COL (1:3) membranes inside HMPs. The Papp values measured for mouse intestinal epithelial confirmed the visual assessment of the existence of a confluent barrier with Papp values of 43.9 ± 6.8 nm / s in suspended MG / COL (1 :3) membranes and 29.6 nm / s in Costar Transwells® measured on day 7 of culture. The H&E-stained sections depicted in Fig. 12d confirm that in both cell culture setups, the intestinal epithelial cells grow as monolayer.

[0155]

[0156] 7.1 Aim of the Experiment

[0157] The aim of this experiment is to investigate the suitability of the cell culture unit and the membrane formed therein for coculturing. A specific goal is to compare properties of cocultures of mouse intestinal epithelium with fibroblasts to properties of monocultures of mouse embryonic fibroblasts and of mouse intestinal epithelium, each prepared in cell culture units according to the invention. The investigated properties are the trans-epithelial electrical resistance (TEER) and the apparent paracellular permeability (Papp). Furthermore, the morphology of the cell cultures is investigated.

[0158] 7.2 Experimental Protocol

[0159] Mouse embryonic fibroblasts (MEF; ATCC, SCRC-1040) were obtained from ATCC and expanded according to the supplier’s instructions. MEFs were expanded in ATCC-formulated Dulbecco's Modified Eagle's Medium (30-2002, ATCC) + 15 % FBS in TC-treated cell culture flasks at 37°C, 5% CO2 and 95% humidity. At 80% confluence, the cells were split in a 1 :8 ratio and medium was exchanged three times per week.

[0160] For the coculture of mouse intestinal epithelium with stromal cells, MEFs were embedded in membranes formed of MG / COL (1:3) hydrogel (as described in Example 1) in HMPs with D = 6 mm and d = 3 mm (as described in Example 1) at a density of 5’000 cells I pL, followed by a top-seeding of 5’000-7’000 mouse intestinal organoid-derived single cells I mm2. The cells were kept in coculture for 7 days in ENRW-Th medium at 37°C, 5% CO2 and 95% humidity. Control samples containedeither only embedded MEFs or only top-seeded organoid-derived single cells. Medium was exchanged daily (600 pL in the basal chamber, 200 pL in the apical chamber). To track epithelium maturation in mono- and fibroblast coculture, TEER and Papp measurements were performed every day. For details on the methods used for determining the TEER and Papp values, it is referred to “Method 1”. At the end of the experiment, the samples were fixed in 4% paraformaldehyde solution for 30 min at room temperature and kept in PBS until processing for immunohistochemical stainings. For details on the methods used for immunohistochemical stainings, it is referred to “Method 2 and “Method 3

[0161] Furthermore, bright field images of the cell cultures were taken over the course of 7 days.

[0162] 7.3 Results

[0163] The reults are shown in Fig. 13. An important advantage of the HMP compared to the Costar Transwells® is the possibility of incorporating cells inside the hydrogel membrane separating the apical from the basal chamber, thereby allowing the culture of stromal cells in a 3D environment, similarly to the ECM in vivo. Fig. 13a shows bright field images of embedded mouse embryonic fibroblasts (MEFs) inside suspended MG / COL (1:3) membranes, top-seeded organoid-derived mouse intestinal epithelium (mlE) and the coculture of mlE + MEF in HMPs. As visualized in Fig. 13b and the zoom-in in Fig. 13c, the addition of MEFs to the mlE resulted in an increased barrier integrity, indicated by the reduced Papp values of 7.7 ± 4.5 nm / s in the coculture compared to the 43.9 ± 6.80 nm / s of the mlE monoculture after 7 days of culture. The Papp values of the MEFs without the addition of epithelial cells were 927.8 ± 26.4 nm / s and thus comparable to the Papp measurements of acellular MG / COL (1:3) membranes shown in Experiments 5 and 6. The TEER measurements shown in Fig. 13d further support the Papp measurements in showing a beneficial effect of the coculture of mlE + MEFs in achieving an epithelium with improved barrier function. After 7 days of culture, the mlE measured 58.6 ± 0.3 Q*cm2, while the coculture of mlE + MEFs achieved TEER values of 74.7 ± 2.5 Q*cm2. The culture of MEFs again resulted in similar results as the acellular control with 35.0 ± 0.0 Q*cm2compared to 31.83 ± 2.3 Q*cm2. The H&E-stained sections shown in Fig.

[0164] 13e confirm the presence of a cell monolayer in both conditions and show the close proximity of fibroblasts and epithelial cells in the coculture.

[0165]

[0166] 8.1 Aim of the Experiment

[0167] The aim of this experiment is to investigate the suitability of the cell culture unit and the membrane formed therein for culturing human intestinal organoid-derived monolayers compared to the state of the art. A specific goal is to compare properties of membranes formed in cell culture units formed in cell culture units according to the invention and of human intestinal organoid-derived monolayers cultured on these membranes with properties of Costar Transwells® and such monolayers cultured on these Costar Transwells®. The investigated properties comprise the apparent paracellular permeability (Papp). Furthermore, the morphology of the cell cultures is investigated.

[0168] 8.2 Experimental Protocol

[0169] Human small intestinal crypts were isolated from biopsies upon getting the patient’s consent, and they were expanded in 100% MG dome cultures. The human small intestinal organoids were cultured in the previously described FUJI medium (Fujii et al. 2018), which consists of Advanced DMEM / F12 medium + 1X Glutamax + 10 mM HEPES + 100 U / mL P / S + 2% B27 supplement without Vitamin A + 2% Rspondin3 from conditioned medium + 2% Noggin from conditioned medium + 1 mM N-acetylcysteine + 50 pg / mL Primocin + 0.1 pg / mL human IGF-1 (590908, BioLegend) + 50 ng / mL human FGF2 / FGF-basic (3718-FB, R&D Systems) + 50 ng / mL human EGF + 0.5 pM A83-01 (ALK4 / 5 / 7 inhibitor, 2939, R&D Systems) + 0.15 nM Wnt surrogate + 0.01 pM human Gastrin I (HY-P1097 / CS-0027717, Medchem Express). The organoids were expanded at 37°C, 5% CO2 and 95% humidity with three medium changes per week. Once a week, the organoids were mechanically dissociated and passaged in fresh 100% MG domes. In order to facilitate the mechanical fragmentation, the domes were incubated with Gentle Cell Dissociation Reagent (100-0485, STEMCELL Technologies) as suggested by the manufacturer. After passaging, 10 pM Y-27632 was added to the medium for the first 24-48h.

[0170] To generate human small intestinal monolayers, organoids were dissociated into a single cell suspension in TrypLE™ Express + 10 pM Y-27632 + 500 U / mL DNAse for 10 min at 37°C and strained over a 40 pm strainer. Human small intestinal single cells were seeded on membranes formed of MG / COL (1:3) hydrogel (as described inExample 1) in HMPs with D = 6 mm and d = 3 mm (as described in Example 1) and Costar Transwell® Permeable Supports at a density of 3’500 cells I mm2and cultured at 37°C, 5% CO2 and 95% humidity. Prior to cell seeding, the Costar Transwell® permeable supports were coated with 1 (v / v)% collagen I solution in PBS (TeloCol-6 Type I bovine collagen solution, 6.0 mg / mL, 5225-50ML, Advanced BioMatrix). The coating was done overnight at 4°C, followed by a 1 h incubation at 37°C directly before cell seeding. From d0-d2, the human intestinal monolayers were cultured in FUJI medium without A83-01 + 10 pM Y-27632, from d2-d4 in full FUJI medium and from d4 onwards in FUJI medium without EGF to induce epithelium differentiation. Medium was exchanged every two days (600 pL in the basal chamber, 200 pL in the apical chamber) and epithelium maturation was tracked with Papp measurements that were performed every other day. For details on the methods used for determining the Papp values, it is referred to “Method 1”. On day 7, intestinal monolayers were treated for 48h with pro-inflammatory cytokines: the medium in the basal chamber was exchanged for fresh medium containing recombinant human TNFa or a combination of 20 ng / mL recombinant human TNFa + 20 ng / mL recombinant human IFNy. At the end of the experiment, the human intestinal monolayers in both HMPs and Costar Transwells® were fixed in 4% paraformaldehyde solution for 30 min at room temperature and kept in PBS until processing for immunohistochemical stainings. For details on the methods used for immunohistochemical stainings, it is referred to “Method 2 and “Method 3

[0171] 8.3 Results

[0172] H&E- and IF-stained sections of human intestinal epithelium, cultured in HMPs and treated with pro-inflammatory cytokines from day 7 to day 9, are shown in Fig. 14a-c. The H&E staining revealed a reduced epithelium thickness after combinatory treatment of TNFa + IFNy (Fig. 14c), while the TNFa treatment alone (Fig. 14b) did not result in a morphology similar the untreated control (Fig. 14a). The IF staining shows that all conditions resulted in expression of intestinal markers E-Cadherin, Ki67, Fabpl, Muc2 and ZO1 after 9 days of culture and an increased expression of Caspase 3 in the TNFa + IFNy condition. Characterization of human epithelial barrier integrity resulted in Papp measurements of 0.0 ± 0.0 nm / s in Costar Transwells® and 7.7 ± 6.5 nm / s on suspended MG / COL (1 :3) membranes at day 9 (Fig. 14d).

[0173]

[0174] 9.1 Aim of the Experiment

[0175] The aim of this experiment is to further investigate the suitability of the cell culture unit and the membrane formed therein for coculturing. A specific goal is to compare properties of cocultures of human intestinal epithelial cells (hiE) with LAD2 human mast cells (MC) to properties of monocultures of human intestinal epithelial cells (hiE) and of LAD2 human mast cells (MC), each prepared in cell culture units according to the invention. The investigated properties are the trans-epithelial electrical resistance (TEER) and the apparent paracellular permeability (Papp). Furthermore, the morphology of the cell cultures is investigated.

[0176] 9.2 Experimental Protocol

[0177] LAD2 human mast cells were cultured as described in Radinger et al. 2010. LAD2 cells were expanded in StemPro-34 SFM medium + StemPro-34 nutrient supplement (10639011, gibco) + 2 mM L-glutamine (25030-024, gibco) + 100 ll / rnl penicillin / 100 pg / ml streptomycin (15140-122, gibco) + 100 ng / ml recombinant human stem cell factor (rhSCF; 300-07, PeproTech) in TC-treated cell culture flasks at 37°C, 5% CO2 and 95% humidity. The semi-adherent LAD2 cells were hemi-depleted once per week, i.e. half of the medium was replaced by addition of an equal volume of fresh LAD2 medium containing 2X rhSCF. The cells were always kept at a concentration of 0.5 -1.0 x 106 / mL for optimal expansion.

[0178] For the coculture of human intestinal epithelium with immune cells, LAD2 cells were embedded in membranes formed of MG / COL (1:3) hydrogel (as described in Example 1) in HMPs with D = 6 mm and d = 3 mm (as described in Example 1) at a density of 850 cells I pL, followed by a top-seeding of 3’500 human intestinal organoid-derived single cells I mm2. The cells were kept in coculture for 7 days in the same medium conditions as described above for human intestinal monolayers at 37°C, 5% CO2 and 95% humidity. Control samples contained either only embedded LAD2 cells or only top-seeded organoid-derived single cells. Medium was exchanged every other day (600 pL in the basal chamber, 200 pL in the apical chamber). To track epithelium maturation in mono- and immune cell coculture, TEER and Papp measurements were performed every two days. For details on the methods used for determining the TEER and Papp values, it is referred to “Method 1”. At the end of the experiment, thesamples were fixed in 4% paraformaldehyde solution for 30 min at room temperature and kept in PBS until processing for immunohistochemical stainings. For details on the methods used for immunohistochemical stainings, it is referred to “Method 2” and “Method 3”.

[0179] Furthermore, bright field images of the cells were taken over the course of 7 days.

[0180] 9.3 Results

[0181] Fig. 15a shows bright field images of embedded LAD2 cells inside suspended MG / COL (1:3) membranes, top-seeded organoid-derived human intestinal epithelium (hlE) and the coculture of hlE + MC in HMPs. As visualized in Fig. 15b, the addition of MCs to the hlE resulted in a faster establishment of a functional barrier, indicated by the lower Papp value on day 2 of the coculture (299.4 ± 197.2 nm / s) compared to the Papp value of the hlE in monoculture (816.2 ± 153.6). However, on the last day of the experiment, Papp values were comparable between the two conditions with 39.5 ± 34.0 nm / s for the hlE and 27.0 ± 24.1 nm / s for hlE + MC. The MCs alone showed similar permeability values as the acellular controls (1084.8 ± 185.3 nm / s vs.

[0182] 1201.3 ± 38.1 nm / s) over the course of 9 days of culture. The TEER measurements shown in Fig. 15c further support the Papp measurements in showing a beneficial effect of the coculture of hlE + MCs in achieving an epithelium with improved barrier function. After 9 days of culture, the hlE measured 26.9 ± 8.9 Q*cm2, while the coculture of hlE + MCs achieved slightly increased TEER values of 36.9 ± 6.7 Q*cm2. The culture of MCs alone resulted in clearly reduced values of 6.2 ± 0.7 Q*cm2.

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Claims

-46-C l a i m s1. Cell culture unit (100) for enabling a preparation of a membrane (5), comprising a well arrangement (1) with a membrane support structure (2),wherein said membrane support structure (2) comprises an opening (3), at least one support face (4) laterally extending from said opening (3) and at least one confining outer edge (6) for confining a curable liquid matrix material (7), which can be provided on said at least one support face (4) spanning across said opening (3), wherein said at least one confining outer edge (6) extends circumferentially around said opening (3) and said at least one support face (4).

2. Cell culture unit (100) according to claim 1, characterized in that said at least one confining outer edge (6) is stepped back from an inner edge face (10) of said opening (3) and / or that said at least one confining outer edge (6) is offset inwardly from an inner side wall face (1c) of said well arrangement (1)and / orwherein said opening (3) and said at least one confining outer edge (6) both define a same type of shape, preferably a circular shape, an elliptical shape, a square shape, preferably with rounded corners, or a rectangular shapes, preferably with rounded comers.

3. Cell culture unit (100) according to claim 1 or 2, characterized in that said opening (3) comprises a width d, in particular a diameter d, wherein said width d ranges from 1 to 15 mm, preferably from 2 to 7 mm,and / orwherein said at least one confining outer edge (6) comprises a width D, in particular a diameter D, wherein said width D ranges from 2 to 20 mm, preferably from 6 to 10 mm,and / orwherein said opening (3) comprises a width d, in particular a diameter d, and that said at least one confining outer edge (6) comprises a width D, in particular a diameter D, wherein a ratio of said widths d / D is less than or equal to 1 , preferably less than or equal to 0.7, more preferably less than or equal to 0.5.-47 -4. Cell culture unit (100) according to any one of claims 1 to 3, characterized in that said at least one confining outer edge (6) comprises a first material (11 a), wherein said first material (11a) comprises a liquid-repellent material, in particular a hydrophobic material, and / or a polymer, for example polyethylene terephthalate and / or cyclic olefin copolymer and / or polymethyl methacrylate, preferably polyethylene terephthalate and / or cyclic olefin copolymer,and / orwherein said at least one support face (4) and / or an inner edge face (10) of said opening (3) comprises a second material (11b), wherein said second material (11b) comprises a liquid-attracting material, in particular a hydrophilic material, and / or a polymer, for example polyethylene terephthalate and / or cyclic olefin copolymer and / or polymethyl methacrylate, preferably polymethyl methacrylate and / or polyethylene terephthalate.

5. Cell culture unit (100) according to any one of claims 1 to 4, characterized in that said at least one confining outer edge (6) forms part of a first material layer (9a) and / or that said at least one support face (4) and / or an inner edge face (10) of said opening (3) forms part of a second material layer (9b),preferably wherein said first and second material layers (9a, b) are connected to each other by means of adhesive bonding, preferably with a pressure-sensitive adhesive (9c).

6. Cell culture unit (100) according to any one of claims 1 to 5, characterized in that said membrane support structure (2) comprises at least one lateral face (13) extending from said at least one confining outer edge (6), preferably wherein said at least one lateral face (13) extends substantially vertically, and / or that said at least one support face (4) extends substantially horizontally,preferably wherein said at least one lateral face (13) comprises a height of at least 10 pm and a maximum of 1000 pm, preferably of at least 25 pm and a maximum of 500 pm, more preferably of at least 50 pm and a maximum of 250 pm, most preferably of 150 pm .

7. Cell culture unit (100) according to claim 6, characterized in that said at least one lateral face (13) extends from said at least one support face (4) forming at least one inner edge (14), preferably wherein said at least one inner edge (14) comprises-48-an angle (15) of less than or equal to 135°, more preferably of less than or equal to 90°.

8. Cell culture unit (100) according to any one of claims 1 to 7, characterized in that said at least one confining outer edge (6) comprises an angle (8) of less than or equal to 135°, more preferably of less than or equal to 90°,and / orwherein said well arrangement (1 ) comprises a main well (1a) with a first and a second chamber (18a, b), wherein said opening (3) is interposed between said first and second chambers (18a, b), preferably wherein said second chamber (18b) is fluidly connected to at least one auxiliary well (1 b) of said well arrangement (1 ), preferably wherein said at least one auxiliary well (1b) is at least partially arranged circumferentially around said main well (1).

9. System comprising a cell culture unit (100) according to any one of claims 1 to 8 and a curable liquid matrix material (7).

10. System according to claim 9, characterized in that said curable liquid matrix material (7) comprises a hydrogel,preferably wherein said hydrogel comprises a natural hydrogel (7), for example comprising a solubilized extracellular matrix (ECM), such as Matrigel® and / or VitroGel® RGD, and / or collagen, and / or comprises a synthetic hydrogel, for example comprising polyethylene glycol.

11. System according to claim 9 or 10, characterized in that preferably said opening (3) comprises a width d, in particular a diameter d, and said at least one confining outer edge (6) comprises a width D, in particular a diameter D, and that said curable liquid matrix material (7) comprises a viscosity of: at least 5 mPa*s, preferably wherein a ratio of said widths d / D is less than or equal to 0.5; preferably of at least 25 mPa*s, preferably wherein a ratio of said widths d / D is less than or equal to 0.67; more preferably of at least 150 mPa*s, preferably wherein a ratio of said widths d / D is less than or equal to 0.7.

12. Method for preparing a membrane in a cell culture unit (100) according to any one of claims 1 to 8, in particular with a system according to any one of claims 9 to 11 , comprising the steps of:- Applying (S1 ) said curable liquid matrix material (7) on said at least one support face (4) in a way that said curable liquid matrix material (7) spans across said opening (3); and- Curing (S3) said curable liquid matrix material (7) to form said membrane (5).

13. Method according to claim 12, characterized in that, prior to said curing (S3), a surplus amount of said curable liquid matrix material (7) is removed (S2), preferably wherein said surplus amount depends on a desired membrane thickness, and / orwherein said applying (S1) and / or removing (S2) of curable liquid matrix material (7) is performed by means of a pipetter, preferably with a circular motion of a pipette tip (20) of said pipetter,and / orwherein said formed membrane (5) comprises a thickness of a maximum of 500 pm, in particular of at least 150 pm and a maximum of 350 pm.

14. Cell culturing arrangement comprising at least one cell culture unit (100) according to any one of claims 1 to 8, preferably a plurality of such cell culture units (100),preferably wherein said cell culturing arrangement comprises at least one of the following: a single well plate; a multi well plate; a cell culture insert.

15. Method for manufacturing a cell culture unit (100) according to any one of claims 1 to 8 and / or a cell culturing arrangement according to claim 14, comprising the steps of:- Providing (S101) multiple material layers (9, 9a, 9b), preferably comprising a first material layer (9a) forming said at least one confining outer edge (6) and / or a second material layer (9b) forming said at least one support face (4) and / or an inner edge face (10) of said opening (3); and- Connecting (S104, S105, S106, S107) said multiple material layers (9, 9a, 9b) to each other, preferably by means of adhesive bonding, in particular with a pressure-sensitive adhesive (9c).

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