Advanced device and process for mimicking tissues in vitro

Abstract

A device (100, 200, 300, 400) for in-vitro modeling a tissue of an organ, capable of simulating mechanical force exposure, wherein the device (100, 200, 300, 400) comprises a cell culture chamber (104, 204) and a pressurized chamber (105, 205, 305), wherein the chambers (104, 105, 204, 205, 305) are physically separated by an elastic pressure-tight membrane (108, 208, 308) and wherein the pressurized chamber (105, 205, 305) comprises at least one partition wall (107, 207) for providing the pressurized chamber (105, 205, 305) with channels (109, 209, 409, 509) and / or subspaces (336, 337) for elastic deformations of different areas of the membrane (108, 208, 308) under different pressure conditions and a process for mimicking the behavior of a tissue of an organ with a device (100, 200, 300, 400, 500).

Description

[0001] DESCRI PTION

[0002] Title

[0003] ADVANCED DEVICE AND PROCESS FOR MIMICKING TISSUES IN VITRO

[0004] Technical Field

[0005] The present invention relates to a device for in-vitro modeling a tissue of an organ as well as a process for mimicking the behavior of a tissue of an organ with a device, which is especially an in vitro process.

[0006] Background Art

[0007] US2017299578 A1 discloses an array of culture membranes (porous) that can be deflected in 3D, on which lung alveolar epithelial cells or other cells are cultured. The size of the alveoli is given by openings in the “substrate”, meaning the middle solid part on which the flexible membrane is attached. This document further discloses the pumping of a fluid in a bottom chamber which is used to create a pressure difference across the membrane between the lower and the upper chamber leading to the deflection of the suspended membrane.

[0008] WO 2009039640 A1 discloses a device with an array of non-porous culture membranes, each deflected in 3D. They serve as the substrate for the cells cultured exclusively on the apical side of the membrane. The suspended membrane undergoes positive deflection from the basolateral side, propelled by a post that is vertically actuated through pneumatic activation.

[0009] US2023365908A1 discloses a device with an array of culturing membranes that can be deflected in 3D. Like the state of the art mentioned above the size of the culturing membrane (alveoli) is given by the opening in a middle plate.

[0010] US2023287324A1 discloses a device with a basolateral chamber having a spiral form on which a porous (and stretchable) membrane is located. A difference with this invention is that the fluidic channel has a spiral shape aimed at increasing the resident time of the fluid within the system.

[0011] WO2015032889A1 discloses a process with a membrane wherein the 3D deflection of the thin, porous membrane is induced by a second membrane, the microdiaphragm, located in the basolateral chamber. However, the size of the alveolus does not mimic the in-vivo situation and in addition, does not mimic an array of alveoli. Further to this the size of the deflected membrane is given by holes in the substrate as also described in the documents mentioned above.

[0012] W02018096054A1 discloses a device with a biodegradable membrane made of proteins found in the lung extracellular matrix. Cells can be cultured on both sides of the membrane to mimic for instance lung alveoli. The membrane is integrated on a thin metallic scaffold having an array of large hexagonal pores, similar to the in-vivo surface of the alveoli. Like in the state of the art mentioned above the size of the alveoli is given by the hexagonal pores in the thin scaffold (gold mesh).

[0013] Thus, in the predescribed cases the device incorporates openings of diverse sizes and shapes situated in the plate supporting the membrane. However, in none of these instances is the pressurized chamber partitioned to intentionally induce membrane deflection at varying amplitudes. The inventive device provides a more realistic modeling of a tissue and its behavior.

[0014] Disclosure of the invention

[0015] An inventive device is used for the modelling a tissue of an organ capable of simulating mechanical force exposure. This may include subjecting a tissue to a movement. Said tissue can have an artificial design which may correspond to the construction of a real tissue. Numerous different types of tissues can be designed.

[0016] Likewise different types of movements for the said tissue can be performed by said device, such as different types of contraction and compression exerted on various types of tissues.

[0017] Said device comprises a cell culture chamber for the arrangement of a cell culture. Some cell cultures require a surface or an artificial substrate such that the cells are able to adherent for the formation of a monolayer. For these cultures the bottom of said culture chamber might be a suitable surface. Cells can also be embedded within a scaffold, which refers to a 3D structured part commonly recognized in the tissue engineering field. The scaffold, such as a hydrogel matrix, enabling the cells to proliferate, migrate, differentiate, and perform other cellular activities unhindered either within the scaffold and / or at its surface. The scaffold may be enclosed within the cell culture chamber. A suspension culture can also be inserted in the cell culture chamber. It is also possible that a tissue, such as an artificial tissue, as a network of cells is inserted in said cell culture chamber. A combination thereof is also envisaged. A second part of said device is a pressurized chamber. This chamber is designed for the introduction of a pressurized gas and / or pressurized liquid. The gas could be air. It could also be a gas mixture, such as 02 and N2, with varying concentrations of 02, to create for instance a hypoxia gradient across the membrane and the cell culture. This can take place if a gas-permeable membrane made for instance of PDMS, is used. Said pressure can be an overpressure or a negative pressure compared to atmospheric pressure, especially a vacuum.

[0018] The fluid in the pressurized chamber could also be a liquid, such as water or mineral oil. This offers the advantage of reducing internal reflections and refractions at the interfaces of the membrane and of the bottom plate, resulting in improved microscopy images.

[0019] Both chambers are physically separated from each other by means of an elastic, pressure-tight membrane preferably for an overpressure up to at least 800 mbar, more preferably up to at least 1 ,5 bar.

[0020] Said membrane can be the bottom of said cell culture chamber. It is at least part of said cell culture chamber. There could be further elements such a non-elastic frame around said membrane. The membrane could be further provided with one or more functional layers, such as a scaffold, for instance one or several layers of different hydrogels. Due to its elasticity, it is possible to provide a deformation either in the direction of the cell culture chamber under overpressure or away from the cell culture chamber under negative pressure. The pressure-tight characteristic of the membrane enables the pressure to remain stable in the pressurized chamber to create the desired mechanical stimuli to the tissue. The membrane can be gas permeable but pressure tight to pressures of at least 800 mbar. However, in such a scenario, the diffusion flux should be minimal to maintain a stable pressure so that it does not interfere with the experiment being performed.

[0021] Said pressure tight membrane can preferably be defined as a non-porous membrane with no pores larger than 1 pm, thus cells cannot migrate through the membrane. Therefore, a non-porous membrane might have pores of less than 1 pm, for example 0,05-0,9 pm. As the cells have a typical size between 10-20 pm they are unable to migrate through the membrane.

[0022] The pressurized chamber comprises at least one partition wall for providing the pressurized chamber with channels and / or sub-spaces for elastic deformations of different areas of the membrane under different pressure conditions. Thus, the direction of the simulated motion is at least partly designed by the structure of these channels and / or sub spaces.

[0023] Further advantageous embodiments are subject matter of the dependent claims.

[0024] The membrane can be provided as a pressure-tight membrane, preferably made of polydimethylsiloxane (PDMS), polyurethane, or other elastic materials or a combination thereof. PDMS has been proven to be suitable for this use.

[0025] The device is provided with an inlet, preferably an aseptic inlet connected with said cell culture chamber for the introduction of cells and a cell culture medium inside said cell culture chamber. An aseptic inlet is needed to fill said chamber with different media without contaminations and with reduced effort for cleaning.

[0026] The device may further be provided with a pressurized fluid inlet, preferably a nonaseptic inlet, connected with said pressurized chamber, for the introduction or removal of a pressurized fluid to or from the said pressurized chamber. This chamber does not need to be cleaned. Since it has no connection to the cell culture chamber, especially not through the membrane, there is no danger of contamination.

[0027] Said pressurized fluid inlet is provided with a fluid pressure regulation device, for the regulation of at least one pressure inside said pressurized chamber, preferably as a fluid valve. More preferably there are individual fluid pressures in the different channels and / or sub spaces that are regulated by said fluid pressure regulation device. An example would be a valve that can be switches to different channels and / or sub spaces so that these channels and / or sub spaces can be provided with a predetermined individual pressure.

[0028] To prevent a bypass between said channels or sub spaces, it is of advantage that said partition wall has a material connection with the elastic pressure-tight membrane over a part of its longitudinal extension, preferably at least 80% of its longitudinal extension. Said material connection could be an adhesive connection or a melted connection, such as a welded connection.

[0029] In a further preferred embodiment, the pressurized chamber and / or a plate as a part of said pressurized chamber, which comprises the partition walls of the pressurized chamber is demountable connected to cell culture chamber. In one first embodiment the interface between the pressurized chamber and / or a middle plate and the cell culture chamber can be between the membrane as a part of the bottom of the cell culture chamber and the rest parts of the cell culture chamber. In this first embodiment, the exchange is the pressurized chamber and / or said middle plate together with the membrane, wherein the bottom of the cell culture chamber is removed.

[0030] In one second embodiment the interface between the pressurized chamber and / or the cell culture chamber can be between the membrane as a part of the lid of the pressurized chamber and the rest of the middle plate and / or of said pressurized chamber, wherein the lid of the pressurized chamber is removed.

[0031] In one third embodiment the membrane comprises at least two layers which are parallel positioned to each other and preferably pressed together. This way each of said both chambers remain intact.

[0032] This way it is possible to exchange either the geometry of the channels and / or sub spaces within the device and / or the membrane, depending on the tissue and / or the form of movement that should be part of said modelling.

[0033] Said partition wall could have a corrugated shape, preferably a sinusoidal shape, and / or a meandering shape to simulate a specific geometry and movement of said tissue.

[0034] The pressurized chamber could be provided with multiple partition walls dividing said pressurized chamber in multiple channels and / or sub spaces. Thus, the movement of the surface may vary depending on the pressure in each of said channels and / or sub spaces.

[0035] To simulate a wavy movement a part of the said multiple partition walls may have different length but identical shape.

[0036] For a plane surface under normal pressure, it is of advantage if at least a part, preferably all, of the partition walls have identical height over their whole longitudinal course and have a corrugated shape. This way the zeroing and calibration is possible.

[0037] The channels have cross-sections of varying sizes over their longitudinal extension, such that the smallest cross-section has a diameter of less than 50%, preferably between 10-40%, compared to the diameter of the largest cross section. Some tissues have movements with different larger and smaller spaces in-between. This helps to control the amplitude of the movement.

[0038] The channels may have different cross-sections. Thus, the channels are not identical.

[0039] Alternatively, or additionally the pressurized chamber might be provided by at least two sub-spaces by said partition wall, wherein each sub-space is provided with a separate pressurized fluid inlet. Thus, one sub space may be provided with an overpressure and another sub space may be provided with a negative pressure.

[0040] It is of advantage if the device is provided with a separate pressurized fluid outlet which is connected to said pressurized chamber. To perform the movement the pressure inside the pressurized chamber can change. The pressure can be provided by a single in- and outlet by switching a regulation device such as a valve. In this embodiment the switching is not necessary thus movement-change can be faster if the pressurized fluid inlet or outlet are separate.

[0041] Said device can be provided with an outlet, preferably an aseptic outlet, connected with said cell culture chamber for the circulation of a cell culture medium inside said cell culture chamber. The sterilization of said device such as sterilization in place (SIP) can be easier provided with a separate in- and outlet.

[0042] The device can be provided in a very compact embodiment with a chip comprising said the cell culture chamber, the membrane and the pressurized chamber. The device can be the chip only or a superstructure comprising the chip and further elements.

[0043] The device could be provided with a medium reservoir which is connected to said cell culture chamber and preferably to said chip.

[0044] As a pressurized fluid, a fluid with a defined oxygen concentration can be preferably be applied in the pressurized chamber in a preferred embodiment of the invention. Thus, a hypoxic gradient across the membrane is achieved

[0045] The device could be further provided with a pressure generating device, such as a pressurized fluid storage device and / or a pressurized fluid generating device. The cell culture chamber can be provided with a cell culture restrain system positioned at the bottom of the cell cultivation chamber which restrain system is permeable for cell culture medium. Thus, during the movement of the cell culture, the cell culture medium can evade by passing said restrain system while the further ingredients of the cell culture, such as cells, remain in an area of the cultivation chamber defined by said restrain system.

[0046] The pressurized chamber and / or at least a middle plate of the device comprising the partition wall or partition walls of the pressurized chamber can be demountable connected to said elastic, pressure-tight membrane and / or to said cell culture chamber. Thus, several different geometries of channel arrangements can be used to imitate different movements of a tissue.

[0047] Further part of the current invention is a process for mimicking the behavior of a tissue of an organ with a device, preferably the inventive device mentioned above.

[0048] Said device comprise a cell culture chamber for the arrangement of a cell culture separated by a membrane and a pressurized chamber comprising channels and / or sub-spaces, wherein at least one fluid pressure is applied to said membrane in such a way that the membrane is provided with different deflections in response to the applied pressure. Thus, the pressure may vary over a short sequence of time.

[0049] In a preferred embodiment of the inventive process at least a part of said channels and / or sub-spaces have different diameters and / or at least a part, such as two or more, of said channels have cross-sections of varying sizes over its longitudinal extension. The process is not limited to channels. Also, sub-spaces of varying sizes can be provided. By different geometries in the channels and / or sub-spaces the deflection of the membrane is different. Since there are differences in the amplitude of the membrane due to the geometry of the pressurized chamber, the pressure in said channels and / or sub spaces can be uniform.

[0050] A similar effect of different amplitudes can be achieved if the channels and / or sub spaces are individually filled with different fluid pressure. This way a different amplitude can be achieved even if the geometries of the channels and / or sub spaces is equal.

[0051] For mimicking different tissues, the cell culture chamber, preferably with the membrane as a part of the bottom of said cell culture chamber, can be covered with hydrogel containing ingredients, such as cells, forming a layer to create an epithelium and / or multiple hollow bodies formed by cell layers, preferably resembling alveoli, and / or a microvasculature structure.

[0052] The invention and some further preferred embodiments are further described in the following paragraphs.

[0053] The inventive device can preferably be provided for in-vitro modelling in-vivo tissues of organs. It may comprise a first body portion with at least one culturing chamber, a second body portion with at least one pressurized chamber and a culturing membrane dividing the culturing chamber with the pressurized chamber and a third body portion that closes the pressurized chamber so it can be pressurized.

[0054] The term “in-vivo modeling a tissue” in the context of the invention can relate to modelling in-vivo conditions of tissues of the organs, such as, e.g., the lung, and more particularly to the lung alveolar epithelium. The first, second and third body portions can be formed as distinct physical units, which can be mounted together, or can be combined, such as the first body portion can integrate the culturing membrane, and the second and third body portion can form a single physical unit. Alternatively, the second and third body portions can be combined in a culturing membrane and a pressurized chamber.

[0055] In some embodiments, the culturing membrane can be flexible, and can be coatable with cells on its upper side to create an epithelium, similar to the alveolar epithelium. It is however not limited to the lung alveolar epithelium but can be for instance be used to mimic the gut epithelium, The term “flexible” in this context can relate to the deflection of the membrane when a pressure, negative or positive, is applied in the pressurized chamber. The deflection of the culturing membrane induces a three-dimensional deformation of the membrane and of the cells cultured on this membrane. This mechanical stress can be similar to the physiological cyclic mechanical stress induced by the breathing motions, typically comprised between 5 and 12% linear strain under standard conditions. It can also mimic the much larger mechanical stress, typically larger than 20% linear strain, that can take place when patients are intubated and the lungs are over-inflated, which can cause ventilated induced lung injury (VI L I), which may lead to respiratory failure.

[0056] In a further preferred embodiment of the present invention is in the pressurized chamber that is preferably divided in parallel channels with sinusoidal walls forming a series of circular chambers that are connected to each other. In some embodiments, these circular chambers have a surface that is comparable to the in-vivo surface of one alveolus. These chambers are covered with the culturing membrane and can form an array of alveoli with surfaces that are comparable to the in-vivo dimensions. When a pressure is applied in the pressurized chamber, the culturing membrane deflects, either positively or negatively, depending on the pressure applied, and the cells cultured on the membrane are exposed to a gradient of mechanical stress. This results in areas where the mechanical stress is not existent and others where it is maximal.

[0057] In some embodiments, the pressurized chamber can be divided in several compartments herein also referred to as sub-sections, which are more preferably pneumatically separated from each other, so that the culturing membrane can be positively deflected in some circular chambers and negatively deflected or not deflected in others. This is for instance interesting when the peristalsis movement of the gut epithelium is to be reproduced.

[0058] The device allows more complex cellular systems to be mimicked. In some embodiments, the elastic, pressure-tight membrane also referred to as culturing membrane can be coated with one of several layers of proteins or hydrogels containing or not cells. A feeder layer embedded or not in a hydrogel layer can be created that enhances the culture of cells cultured in or on top of the hydrogel layer.

[0059] If this hydrogel layer contains endothelial cells, they can self-assemble in a vasculo- genesis process to form a microvasculature network. Patterned vessels can be integrated in this hydrogel layer in which endothelial cells can be loaded and cultured. Patterned vessels can be created by 3D bioprinting of a sacrificial layer, such as Plu- ronic F127, or using a viscous fingering approach or a molding and removal of a solid filament, such as a needle, or a combination thereof. The vasculogenesis process can also be combined with the patterning process to create more complex bioartificial vessels of different sizes.

[0060] In one embodiment of the process a microvasculature network mimicking the alveolar capillaries is of high interest and can be placed and / or formed inside said cell culture chamber. It could be exposed to a physiological or non-physiological mechanical stress, induced by the breathing and / or the pressure applied to the lungs during mechanical ventilation. This microvasculature can be perfused as it is known in the literature, allowing to recreate a functional microvasculature.

[0061] In a further embodiment of the process a lung alveolar epithelium can be coated or formed on top of the hydrogel layer containing the lung capillaries. This is of high interest as the whole air-blood barrier could be reproduced by this mean. In sharp contrast to the conventional methods, which typically involve a 2D layer of epithelial cells and a 2D layer of endothelial cells separated by a porous membrane, this approach offers a much more physiological gas exchange and cell-cell interactions.

[0062] In a further embodiment, immune cells may be introduced and / or cultured in the cell culture chamber to mimic for instance a lung inflammation, a lung infection (bacterial, viral, pathogen- or fungal- induced). This can also be of high interest in the context of immunotherapies, where immune cells (such as T-cells) are used to treat the tumor.

[0063] Further according to an embodiment of the invention, various types of cells can be grown on the culturing membrane. Such cells can include any procariotic and eucari- otic cell type from a multicellular structure, including nematodes, amoebas, and bacteria, up to mammals such as humans. Cell types implanted or grown on culturing membrane of the device depend on the type of organ or organ function one wishes to mimic and the tissues that comprise those organs. Also, various stem cells, such as bone marrow cells, induced adult stem cells, embryonic stem cells or stem cells isolated from adult tissues can be co-cultured on the culturing membrane. Using different culture media in the culturing chamber feeding each layer of cells, one can allow different differentiation cues to reach the stem cell layers thereby differentiating the cells to different cell types. One can also mix cell types on the same side of the culturing membrane to create co-cultures of different cells without membrane separation.

[0064] Brief Description of the Drawings

[0065] The device according to the invention is described in more detail herein below by way of exemplary embodiments and with reference to the attached drawings, in which:

[0066] Fig. 1 A shows a top-view of the in-vitro mimicking device;

[0067] Fig.1 B shows a perspective view of the in-vitro mimicking device;

[0068] Fig. 1 C shows a cross-section view of the in-vitro mimicking device;

[0069] Fig. 2A shows the bottom view of the middle plate of the in-vitro mimicking device; Fig. 2B shows a detail of the bottom view of middle plate of the in-vitro mimicking device;

[0070] Fig. 2C shows the cross-section of the culturing membrane with the pressurized chamber and the cell culture chamber of the in-vitro mimicking device;

[0071] Fig. 2D shows a positively deflected culturing membrane of the in-vitro mimicking device;

[0072] Fig. 2E shows a negatively deflected culturing membrane of the in-vitro mimicking device;

[0073] Fig. 3A show the bottom view of the middle plate of a second embodiment of the in- vitro mimicking device;

[0074] Fig. 3B shows a detail of the bottom view of the middle plate of a second embodiment of the in-vitro mimicking device;

[0075] Fig. 3C shows a detail of a cross-sectional view of a second embodiment of the in- vitro mimicking device;

[0076] Fig. 4A shows the bottom view of the middle plate of a third embodiment of the in- vitro mimicking device;

[0077] Fig. 4B shows a detail of the bottom view of the middle plate of a third embodiment of the in-vitro mimicking device;

[0078] Fig. 4C shows a detail of the bottom view of the middle plate of a fourth embodiment of the in-vitro mimicking device;

[0079] Fig. 5A shows a detail cross-sectional view of the culturing membrane and a hydrogel layer containing a microvasculature and a layer of epithelial cells on top of the hydrogel of a fifth embodiment of the in-vitro mimicking device;

[0080] Fig. 5B shows a top view of a fifth embodiment of the in-vitro mimicking device;

[0081] Fig. 5C shows a detail of a vasculature formed in a hydrogel layer located on top of the culturing membrane of a fifth embodiment of the in-vitro mimicking device; Fig. 5D shows a smaller detail of a vasculature formed in a hydrogel layer located on top of the culturing membrane of a fifth embodiment of the in-vitro mimicking device;

[0082] Fig. 5E shows a top view of the in-vitro mimicking device;

[0083] Fig. 5F shows a bottom view of the top plate of the in-vitro mimicking device;

[0084] Fig. 6 shows a cross-sectional view of a sixth embodiment of the in-vitro mimicking device with a hydrogel layer containing a microvasculature and several lung organoids;

[0085] Fig. 7 A shows a cross-sectional view of a seventh embodiment on the in-vitro mimicking device with lung organoids half-embedded in the hydrogel layer;

[0086] Fig. 7B shows a detailed view of a cross-sectional view of a seventh embodiment on the in-vitro mimicking device with lung organoids half-embedded in a hydrogel layer; and

[0087] Fig. 7C shows the same detailed view as in Fig. 7B, but at a later time point with open-up lung organoids.

[0088] Description of Embodiments

[0089] In the following description certain terms are used for reasons of convenience and are not to be interpreted as limiting. The terms “right”, “left”, “up”, “down”, “under" and “above" refer to directions in the figures. The terminology comprises the explicitly mentioned terms as well as their derivations and terms with a similar meaning.

[0090] Fig. 1A-C shows a device 100, herein also referred to as a system, for in-vitro modelling in-vivo tissues of organs as a first embodiment of a device according to the invention. The system is made of a top plate 101 , a bottom plate 102 and a middle plate 103. The top plate 101 defines and / or is part of a cell culture chamber 104 that is accessible from the top to add cells or cell culture medium or a hydrogel to form a hydrogel layer or a combination thereof. The top plate has a number of access holes, a pneumatic connector hole 110 that is itself connected to a pneumatic channel 109 that is aimed at pressurizing a pressurized chamber 105. Then, two additional holes, a hydrogel inlet 113 and a hydrogel outlet 115 are connected to the cell culture chamber 104 via a hydrogel channel inlet 112 and / or a hydrogel channel outlet 114, respectively. These holes and fluidic channels are aimed at adding for instance a hydrogel layer 529, 629, 729 with or without cells on top of a culturing membrane 108. Four additional access holes are located on each side of the cell culture chamber 104. They include a medium channel bottom inlet 117 and a medium channel bottom outlet 118 that are connected with each other via a first medium channel 116. The first medium channel 116 is preferably arch-shaped and extend over a plane perpendicular to said bottom plate 102. The first medium channel 116 also connected to the cell culture chamber 104 via an array of micropillars at the bottom 122, 522 of said cell culture chamber 104. The situation is similar on the other side, with a medium channel top inlet 120 and a medium channel top outlet 121 that are connected with each other via a second medium channel 119. Said second medium channel 119 extending preferably over the same plane as the first medium channel 116, is preferably also arch shaped and is also preferably connected to the cell culture chamber 104 via an array of micropillars top 534. The pressurized chamber 105 is divided by thin sinusoidal walls 107 that create a series of circular and / or oval-shaped chambers 106. The first and second medium channel are positioned opposite to each other next to the cell culture chamber 104.

[0091] Fig. 2 shows a detailed view at the device 200 of the same embodiment, made of said top plate 201 , bottom plate 202 and middle plate 203. Said cell culture chamber 204 is located in the top plate 201 that is open and allows cells 211 to be pipetted and cultured on the culturing membrane 208. The cells 211 are nurtured with physiological medium 231 . The pressurized chamber 205 is structured with sinusoidal walls 207 that lead to the formation of interconnected circular and / or oval chambers 206. As shown in Fig. 2D, the pressurized chamber 205, also referred to as pressurized chamber, can be pressurized via the pneumatic channel 209 with a positive pressure, which leads to the positive deflection of the culturing membrane 208. It can also be pressurized with a negative pressure that leads to a negative deflection (Fig. 2E).

[0092] Fig. 3 shows a further embodiment as a device 300, where a pressurized chamber 305 is divided in at least two parts, a sub-space 326 on one side of the pressurized chamber and a pneumatic sub-space 327 on the other side of the pressurized chamber. Each sub-space 326, 327 is provided with a at least one pneumatic connector hole 310, 324 that is itself connected to a pneumatic channel 309, 323 that is aimed at pressurizing at least one part of the pressurized chamber 305. This defines a first longitudinal chamber 336 to be pressurized with a different pressure than a second longitudinal chamber 337. The chambers are separated by a rectangular wall 325 that reaches to the culturing membrane 308. As shown in Fig. 3C only the middle plate 303 and the bottom plate 302 of the device 300 are shown. As shown this configuration enables for instance the culturing membrane 308 to be deflected both positively and negatively, or through a combination of varying pressures, to mimic movements such as contraction and compression in peristalsis, which act on the gut epithelium. In such a scenario, the first longitudinal chamber 336 and the second longitudinal chamber 337 have a width of at least 50 pm, preferably 100 pm + / - 30 pm, but could also be larger 200 to 500 pm, to either mimic the distance between two crypts or between several crypts. The contraction rate of the peristalsis movements typically ranges from 3 to 15 contractions per minute. Higher contraction rates and amplitudes are also envisaged to mimic situations, such as in diarrhea-predominant irritable bowel syndrome. Reverse peristalsis, also called retroperistalsis, may also be mimicked, wherein muscle contractions move backward, typical in the case of reflux. The dimensions of the chambers can also mimic disease gut conditions, such as inflammatory bowel diseases (IBD). For instance, like Crohn's disease or ulcerative colitis, which are types of IBD, there may be changes in the crypt architecture including crypt distortion, branching, or irregular spacing.

[0093] In the embodiment of a device 400 shown in Fig. 4, it is envisaged that the circular chambers 106, 206 can have a different geometry, such as drawn in Fig. 4. Round chambers 427 as for the circular and / or oval chambers have for instance a surface that is similar to those of an alveolus in-vivo. Said chambers are aligned in rows divided by partition walls 428. It is also envisaged that the chambers can also be elongated and rectangular such as illustrated in figure 4C. This serves the purpose to mimic the gut epithelium more accurately. Longitudinal chambers have a length that corresponds the length of a crypt of the gut epithelium. We can also envisage to apply different pressures to adjacent chambers as discussed in embodiment of Fig. 3.

[0094] Fig. 5 A-F shows a further embodiment, wherein at least a hydrogel layer can be added on top of the cell culturing membrane of the device. Different types of cells can be integrated in the hydrogel, such as endothelial cells, fibroblasts, pericytes or other cell types that can be found in the lung parenchyma. As shown in the literature, endothelial cells and pericytes (or fibroblasts) can self-assembled in a functional microvasculature. The capillaries located around the alveoli can for instance be created and cyclically stretched. By adding lung alveolar epithelial cells (type I lung alveolar epithelial cells or type II lung alveolar epithelial cells or a mix of both), the air-blood barrier can be recreated and cyclically stretched.

[0095] Fig. 5E-F show the whole device, with the channels 512 aimed at adding the hydrogel to the top of the membrane. Physiological medium is added to the cells in the hydrogel layer by the channels and their respective inlets and outlets 520-519-521 and / or 517-516-518.

[0096] Fig. 6 shows another embodiment of the device and the process, with organoids 651 (lung or from other organs tissues, such as the gut or the brain, etc.) embedded in the microvasculature 650 to form a barrier, such as the air-blood barrier in the lung.

[0097] Fig. 7 A-C shows another embodiment of the inventive process to open up the organoids so that their epithelium can be exposed to air for example for the purpose of inhalation studies or the like. It is envisaged that by leaving part of the lung organoids exposed to air, wherein their size is larger than the thickness of the hydrogel, the organoids will open up, due to their exposition to air. Other embodiment of the process based on biochemical signaling can be used to do the same.

[0098] The inventive device in all aforementioned embodiments can have the form of a microfluidic chip. It can have one or more of the following preferred dimensions.

[0099] The thickness of the elastic pressure-tight membrane, herein also referred to as culturing membrane can be between 3 pm and 400 pm, more preferably 150 pm + / - 50 pm.

[0100] Said membrane material can preferably be a siloxane, more preferably PDMS polydimethylsiloxane. Said membrane may have a Youngs Modulus YM of 300kPa - 1 MPa under standard conditions.

[0101] Said membrane is preferably transparent. It can be easy to manufacture by spin coating, molding and / or casting.

[0102] It can preferably be biocompatible.

[0103] It can be coated with proteins for better cell adhesion, preferably with fibronectin, laminin, collagen, or a combination thereof. The membrane can also be made of other polymers, such as thiolene based material, preferably a photocurable off-stoi- chiometry thiolene, or polyurethane or other polymers.

[0104] Said pressurized chambers in the pressurized chamber can be circular of the same diameter. A preferred diameter can range from 100 pm to 1 mm, more preferably about 500 pm + / - 100 pm. The preferred thickness of the bottom, such as a bottom plate, of the pressurized chamber can be chosen as thin as possible for imaging purpose, but as thick as needed to ensure robustness without deformation under the typical fluid pressure applied. It could be 80 pm to 1 mm, more preferably 100 pm - 500 pm, more preferred 100-200 pm. It can be provided as a glass or an injected molded polymer preferably a PS (polystyrene), a PP (polypropylene), a COC (cyclo-olefine copolymers) and / or COP (cyclic olefin polymer) or similar thermoplastic materials or a combination thereof.

[0105] The pressurized chamber, as well as the channels and / or sub-spaces can be provided with a preferred height of 50 pm to 1 mm, more preferably 150 pm + / - 100 pm.

[0106] The one or more partition walls can be provided by a middle plate attached to the bottom plate. The cell culture chamber can be defined by a top plate which is attached to the middle plate. The middle plate can comprise the aforementioned membrane herein also referred to as culturing membrane. The material of the partition walls dividing the channels and / or sub spaces are either the same material as the culturing membrane, preferably PDMS, which presents a manufacturing advantage because there is no delamination of the membrane when a positive pressure is applied and the fabrication process of the membrane and the partition walls can be done in one step for example as a middle plate of the device. In this embodiment there is no alignment problem because the different plates of the device can be easily aligned by having the same diameter.

[0107] The said cell culture chamber should preferably be provided with dimensions that allow for a physiological medium volume sufficient for at least 24h. More preferred the volume of said cell culture chamber can be 50-500 pL more preferably 100 pL + / - 50 pl. If the volume is too small evaporation is an issue, if it is too large, then cytokines and other cellular products are diluted to such an extent that they cannot be analyzed (limit of detection of the analytical equipment) and in addition do not mimic the in-vivo situation (ratio blood volume to tissue).

[0108] The fluid pressure used for the device can typically be 0 to -800 mbar to 1 ,5 bar, more preferably -800 mbar to 1 bar, more preferably 0 + / - 800 mbar. -800 mbar defines a negative pressure.

[0109] The preferred overall size of the chip can be defined by an overall diameter, which is the largest extension of the device in a plane perpendicular to its longitudinal axis. This overall diameter is of about 15mm + / - 10%. Thus, the device fits in a 24-well plate format. It is however envisaged to miniaturize the device so that it can fit a higher throughput plate, such as a 48 or a 96 well format. In which case the overall diameter of the device is envisaged to be around 11 mm + / - 10% or 6.4mm + / - 10%, respectively.

[0110] The process and the device can be provided in at least two modes. Both modes of operation have their specific advantages. In general, the distance between the inferior part of the bottom plate to the cells may be kept minimal, which enables the imaging of the cells with a minimal focal distance. The small size of the circular chambers enables a smaller deflection amplitude to get a specific strain percentage. For example, for a 500 pm in diameter membrane, the deflection amplitude is about 100 pm that results in a 10% linear mechanical strain. The smaller size of the alveoli allows to reduce the focal distance and thus have better microscopy conditions, thus better microscopy images. A negative pressure is preferred to mimic the physiological condition. If one wants to mimic a pathophysiological stress, the positive pressure would be preferred as the membrane can deflect with a larger amplitude. One considers the physiological linear strain to be in the 5-12% range, whereas a pathophysiological strain is when it is larger than 20%. This scenario may arise during mechanical ventilation, particularly when portions of the lung are obstructed, potentially leading to ventilator-induced lung injury (VILI).

[0111] Although the present system cannot recreate the air-blood barrier made of a porous membrane sandwiched between a layer of epithelial cells cultured at the air-liquid interface and an endothelial layer, it still can recreate an air-blood barrier that is more physiological as envisaged in Fig. 5A, Fig. 6, Fig. 7. It is more physiological as it does not need any artificial or biological membrane, thus it is envisageable that the ultra-thin in-vivo thickness of the basal membrane (typically around 100nm thin) can be reproduced in-vitro. The device thereby allows simulation of the structure and function of a functional alveolar-capillary unit that can be exposed to physiological mechanical strain to simulate breathing or to both air-borne and blood-borne chemical, molecular, particulate and cellular stimuli to investigate the exchange of chemicals, molecules, and cells across this tissue-tissue interface through the pores of the membrane. The device may impact the development of in-vitro lung models that mimic organ-level responses, which are able to be analyzed under physiological and pathological conditions

[0112] In a further embodiment (no illustration), larger circular chambers can be envisaged to replicate the increased alveolar surface area observed in emphysema, resulting from the combination of alveoli. The inner walls between alveoli become weaker and eventually rupture, leading to the formation of larger air spaces instead of numerous smaller ones. Consequently, this diminishes the lung's surface area and reduces the gas exchange that takes place at the air-blood barrier.

[0113] In a further embodiment (no illustration), a combination of small and larger circular chambers to mimic small and larger alveoli can be envisaged. This can be of interest to study for instance the onset of emphysema, when some alveoli are still intact, and others are already damaged.

[0114] In a further embodiment (no illustration), the culturing membrane can only be deformed in part, while the other part is static to mimic either a stiffer environment, such as in fibrotic environment.

[0115] In a further embodiment (no illustration), the membrane may have different thicknesses for the creation of different elasticities in different areas.

[0116] The hydrogel layer on top of the culturing membrane can consist of several layers of different hydrogels, that include cells or not, that can be embedded in hydrogel or not. One can for instance envisage a first layer that consists of a feeder layer of cells (fibroblasts, pericytes,... ) mimicking the interstitial space, on which a hydrogel layer containing a vasculature component (endothelial cells, self-assembled endothelial lumens, patterned vessels, ... ) or a combination of vascular components, on which an epithelium is created either using epithelial cells, organoids, embedded or not in a hydrogel layer. The hydrogel layers can be directly pipetted from the top of the cell culture chamber,

[0117] The device can also be used for studying biotransformation, absorption, clearance, metabolism, and activation of xenobiotics, as well as drug delivery. The bioavailability and transport of chemical and biological agents across epithelial layers as in the lung, the intestine, the skin, the endothelial layers as in blood vessels, and across the blood-brain barrier can also be studied. The acute basal toxicity, acute local toxicity or acute organ-specific toxicity, teratogenicity, genotoxicity, carcinogenicity, and mutagenicity, of chemical agents can also be studied. Effects of infectious biological agents, biological weapons, harmful chemical agents and chemical weapons can also be detected and studied. Infectious diseases and the efficacy of chemical and biological agents to treat these diseases, as well as optimal dosage ranges for these agents can be studied. The response of organs in-vivo to chemical and biological agents, and the pharmacokinetics and pharmacodynamics of these agents can be detected and studied. The impact of genetic content on response to the agents can be studied. The amount of protein and gene expression in response to chemical or biological agents can be determined. Changes in metabolism in response to chemical or biological agents can be studied as well using the present device.

[0118] Reference signs

[0119] 100 device

[0120] 101 top plate

[0121] 102 bottom plate

[0122] 103 middle plate

[0123] 104 cell culture chamber

[0124] 105 pressurized chamber

[0125] 106 chamber

[0126] 107 wall

[0127] 108 membrane

[0128] 109 channel

[0129] 110 pneumatic connector hole

[0130] 112 hydrogel channel inlet

[0131] 113 hydrogel inlet

[0132] 114 hydrogel channel outlet

[0133] 115 hydrogel outlet

[0134] 116 medium channel bottom

[0135] 117 medium channel bottom inlet

[0136] 118 medium channel bottom outlet

[0137] 119 medium channel top

[0138] 120 medium channel top inlet

[0139] 121 medium channel top outlet

[0140] 122 bottom

[0141] 200 device

[0142] 201 top plate

[0143] 202 bottom plate

[0144] 203 middle plate

[0145] 204 cell culture chamber

[0146] 205 pressurized chamber

[0147] 206 interconnected circular chambers

[0148] 207 walls

[0149] 208 membrane

[0150] 209 channel

[0151] 211 cells

[0152] 231 physiological medium 300 device

[0153] 302 bottom plate

[0154] 303 middle plate

[0155] 305 pressurized chamber

[0156] 308 membrane

[0157] 309 channel

[0158] 310 pneumatic connector hole

[0159] 312 hydrogel channel inlet

[0160] 313 hydrogel inlet

[0161] 314 hydrogel channel outlet

[0162] 315 hydrogel outlet

[0163] 316 medium channel bottom

[0164] 317 medium channel bottom inlet

[0165] 318 medium channel bottom outlet

[0166] 319 medium channel top

[0167] 320 medium channel top inlet

[0168] 321 medium channel top outlet

[0169] 323 pneumatic channel

[0170] 324 pneumatic connector hole

[0171] 325 rectangle wall

[0172] 326 top

[0173] 335 bottom

[0174] 336 longitudinal chamber

[0175] 337 longitudinal chamber

[0176] 400 device

[0177] 427 chambers

[0178] 512 channels

[0179] 509 channel

[0180] 510 pneumatic connector hole

[0181] 512 hydrogel channel inlet

[0182] 515 hydrogel outlet

[0183] 517 medium channel bottom inlet

[0184] 518 medium channel bottom outlet

[0185] 519 medium channel top

[0186] 520 medium channel top inlet

[0187] 521 medium channel top outlet

[0188] 522 bottom 529 hydrogel layer

[0189] 534 array of micropillars top

[0190] 550 Microvasculature

[0191] 629 hydrogel layer 651 organoid

[0192] 729 hydrogel layer

Claims

Claims1 . Device (100, 200, 300, 400) for in-vitro modeling a tissue of an organ, capable of simulating mechanical force exposure, wherein the device (100, 200, 300, 400) comprises a cell culture chamber (104, 204) and a pressurized chamber (105, 205, 305), wherein the chambers (104, 105, 204, 205, 305) are physically separated by an elastic pressure-tight membrane (108, 208, 308) and wherein the pressurized chamber (105, 205, 305) comprises at least one partition wall (107, 207) for providing the pressurized chamber (105, 205, 305) with channels (109, 209, 409, 509) and / or sub-spaces (336, 337) for elastic deformations of different areas of the membrane (108, 208, 308) under different pressure conditions.

2. Device according to claim 1 , characterized in that the pressure-tight membrane (108, 208, 308) is provided with a thickness of 3 pm -400 pm.

3. Device according to one of the preceding claims, characterized in that a pressurized fluid inlet in the pressurized chamber (105, 205, 305) is provided with a fluid pressure regulation device, for the regulation of at least one pressure inside said pressurized chamber (105, 205, 305), preferably as a pressurized fluid valve, wherein preferably the pressure regulating device is provided for the regulation of different fluid pressures in different channels (109, 209, 409, 509) and / or sub-spaces (336, 337) inside said pressurized chamber (105, 205, 305).

4. Device according to one of the preceding claims, characterized in that said partition wall (107, 207) has a material connection with the elastic pressure- tight membrane (108, 208, 308) over a part of its longitudinal extension, preferably at least 80% of its longitudinal extension.

5. Device according to one of the preceding claims, characterized in that said partition wall (107, 207) has a corrugated shape, preferably a sinusoidal shape, and / or a meandering shape.

6. Device according to one of the preceding claims, characterized in that the pressurized chamber (108, 208, 308) is provided with multiple partition walls (107, 207) dividing said pressurized chamber (108, 208, 308) in multiple channels (109, 209, 509).

7. Device according to one of the preceding claims, characterized in that a part of the said multiple partition walls (107, 207) have different length but identical shape.

8. Device according to one of the preceding claims, characterized in that at least a part, preferably all, of the partition walls (107, 207) have identical height over their whole longitudinal course and have a corrugated shape.

9. Device according to one of the preceding claims, characterized in that the channels (109, 209, 509) have cross-sections of varying sizes over their longitudinal extension, such that the smallest cross-section has a diameter of less than 50%, preferably between 10-40%, compared to the diameter of the largest cross section.

10. Device according to one of the preceding claims, characterized in that the channels (109, 209, 509) have different cross-sections.11 . Device according to one of the preceding claims, characterized in that pressurized chamber (308) is provided by at least two sub-spaces (336, 337) by said partition wall, wherein each sub-space (336, 337) is provided with a separate pressurized fluid inlet.

12. Device according to one of the preceding claims, characterized in that the device (300) is provided with a separate pressurized fluid outlet which is connected to said pressurized chamber (305).

13. Device according to one of the preceding claims, characterized in that the device (100, 200, 300, 400, 500) is provided as a chip, comprising said the cell culture chamber (104, 204), the membrane (108, 208, 308) and the pressurized chamber (105, 205, 305).

14. Device according to one of the preceding claims, characterized in that the device (100, 200, 300) is provided with a pressure generating device, such as a pressurized fluid storage device and / or a pressurized fluid generating device.

15. Process for mimicking the behavior of a tissue of an organ with a device, preferably a device (100, 200, 300, 400, 500) according to one of the preceding claims, wherein the device (100, 200, 300, 400, 500) comprises a cell culture chamber (104, 204), a membrane (108, 208, 308) and a pressurized chamber(105, 205, 305) comprising channels (109, 209, 409, 509) and / or sub-spaces (336, 337), wherein at least one fluid pressure is applied to said membrane (108, 208, 308) such that the membrane (108, 208, 308) is provided with different deflections in response to the applied pressure.

Images