Cell culture apparatus for producing tissues

Abstract

A cell culture apparatus for producing tissues comprises a tissue cassette, and a cell media reservoir, wherein the cell media reservoir comprises an enclosure for enclosing cell culture media, wherein the cell media reservoir has an opening for inserting the tissue cassette into the cell media enclosed in the cell media reservoir, wherein the tissue cassette has a cassette housing comprising a first main side having a first aperture covered by a first membrane, and at least one second main side having a second aperture covered by a second membrane, such that a culture chamber is formed between the first membrane and the second membrane, wherein the first membrane and the second membrane are attached to the cassette housing, wherein the tissue cassette has an injection opening extending through the cassette housing into the culture chamber for feeding cells into the culture chamber, and wherein at least one of the first membrane and the second membrane is gas-permeable.

Description

[0001] Cell culture apparatus for producing tissues

[0002] Technical field

[0003] The invention relates to a cell culture apparatus for producing tissues, and a method for 3D culturing of cells for producing tissues.

[0004] Background of the invention

[0005] The field of tissue engineering and regenerative medicine has seen significant advancements in the development of methodologies for culturing cells and creating tissue scaffolds. Traditional methods for culturing cells include two-dimensional (2D) plate culture and suspension culture. However, these methods may fall short in replicating the complex three-dimensional (3D) structures and microenvironments found in vivo. As a result, there has been a growing interest in developing 3D cell culture systems that can better mimic the natural cellular environment and improve the functionality of engineered tissues.

[0006] Several approaches have been explored to achieve 3D cell cultures, including the use of bioreactors, microcarriers, and scaffolds. Bioreactors, such as those described in WO 2004 / 078954 Al, provide a controlled environment for cell growth and tissue development by continuously supplying nutrients and removing waste products. These systems often incorporate semi -permeable membranes to facilitate nutrient exchange while preventing contamination.

[0007] Microcarriers, as discussed in WO 2023 / 187018A1, are another method for 3D cell culture. These are microparticles that provide a surface for cell attachment and growth, allowing for high-density cell cultures. However, microcarrier-based systems require sufficient agitation to prevent aggregation and ensure uniform cell distribution, which can be challenging to achieve in large-scale cultures.

[0008] Scaffold-based approaches, such as those described in EP 2 653 531 Al and WO 2002 / 042421 A2, involve the use of porous materials that support cell attachment and proliferation. These scaffolds can be made from various materials and are designed to mimic the extracellular matrix (ECM) of native tissues. The scaffolds can be configured in different shapes and sizes to accommodate various tissue engineering applications.

[0009] Recent innovations have focused on improving the design and functionality of 3D cell culture systems. For instance, US 2018 / 0016547 Al discloses a method for mass culturing cells using porous polyimide films, which can be arranged in various configurations to optimize cell growth and nutrient exchange. This method highlights the importance of material selection and structural design in developing effective 3D cell culture systems.

[0010] US 2021 / 0230526 relates to a cell enclosure device, a tissue-type chip, an organtype chip, an organ-type chip system, a method for culturing cells using the cell enclosure device, and a cell transportation method using the cell enclosure device.

[0011] Advanced Functional Materials, vol. 32, no. 1, 26 August 2021, XP093264097 discloses a scalable method for the engineering of vascularized tissue constructs inspired by the vasculogenesisinduction method.

[0012] US 2009 / 0111180 relates to a method and apparatus for growing and conditioning tissue engineered medical products and in particular to a method and apparatus for a servocontrolled bioreactor with a dynamic pressurization system and a chamber with an integrated nutrient reservoir for conditioning tissue engineered medical products (TEMPs) in the orthopedic, secretory organ and vascular areas.

[0013] Summary of the invention

[0014] Despite the advancements discussed above, there remains a need for more efficient and versatile 3D cell culture systems that can be easily employed, sterilized, and used for various tissue engineering applications. The cell culture apparatus according to the present invention meets this need.

[0015] It is proposed a cell culture apparatus for producing tissues, comprising a tissue cassette, and a cell media reservoir, wherein the cell media reservoir comprises an enclosure for enclosing cell culture media, wherein the cell media reservoir has an opening for inserting the tissue cassette into the cell media enclosed in the cell media reservoir, wherein the tissue cassette has a cassette housing comprising a first main side having a first aperture covered by a first membrane, and at least one second main side having a second aperture covered by a second membrane, such that a culture chamber is formed between the first membrane and the second membrane, wherein the first membrane and the second membrane are attached to the cassette housing, wherein the tissue cassette has an injection opening extending through the cassette housing into the culture chamber for feeding cells into the culture chamber, and wherein at least one of the first membrane and the second membrane is gas-permeable.

[0016] The tissue cassette of the cell culture apparatus comprises a cassette housing, which serves as a structural support for the cells and the membranes, defining and maintaining a shape of the culture chamber. In the context of the cell culture apparatus, the term “cassette” may be understood as a container that may be inserted or removed from the cell media reservoir. It may be a replaceable item, preferably dimensioned to be received through a fitting opening in the cell media reservoir as the host. When inserted, it has the main sides with the membranes acting as functional interfaces to the cell media, allowing a simple adaption to user requirements and modularity. The cassette may have a substantially cuboid shape, in particular a flat cuboid shape. In some variants, the tissue cassette may have a more complex shape with kinks, bends, round or rounded parts or other geometrical characteristics that deviate from purely planar or cuboid parts. The tissue cassette may comprise one second main side, two second main sides, three second main sides, and so on. The shape of the cassette housing and the number of second main sides, i.e. the number of main sides in total, can be chosen according to the desired shape of the tissue to be produced.

[0017] The first main side and the at least one second main side may be arranged at a distance. If two main sides, i.e., a first main side and a single second main side, are used, they may be parallel to each other. In this case, their distance to each other defines the thickness of the tissue cassette. The main sides each indicate a spatial boundary plane of the housing.

[0018] In embodiments where the tissue cassette has a flat cuboid shape, the thickness of the tissue cassette may be smaller than the length and height of the tissue cassette. For example, the length and / or height of the tissue cassette may be at least 5 times, or preferably at least 10 times, larger than the thickness of the tissue cassette. However, other variants are not ruled out.

[0019] The cassette housing defines apertures in the main sides. The apertures may be understood as openings or cutouts, where the respective main side lacks material. The remaining parts of the cassette housing outside the apertures do not necessarily need to be completely fluid-tight, as long as a cell paste may be held inside the culture chamber.

[0020] The cassette housing may comprise a plurality of parts that are assembled to form the cassette housing. The parts may be attached to each other through friction, snap fit, fastening elements or through other fastening means. For example, the parts are placed onto each other to form a stack, wherein separate clamps are used for clamping the parts together to secure the stack. The parts may also be glued together, as long as the glue that is used is biocompatible and still allows for some kind of disassembly that does not disrupt the tissue. At least a part of the gaps between the individual parts may remain unsealed but may be small enough to prevent cell paste from leaking through the gaps.

[0021] The apertures arranged in the cassette housing serve for supplying nutrients and disposing of waste. The apertures may comprise a single opening on the respective main side. They may also comprise a plurality of openings on at least one of the main sides. The apertures may extend over a majority of the surface area of the respective main side. It is to be understood that the apertures are associated with the respective main sides, even if they are not necessarily completely flush with the spatial boundary plane of the tissue cassette at the respective main side. Instead, the apertures may be offset inward by a comparably small distance relative to the spatial boundary. Nevertheless, their location and orientation clearly indicate that they belong functionally and structurally to the corresponding main side. To provide structural support to the cell paste and the growing tissue, the apertures are covered by the membranes. It is to be understood that the respective membrane is preferably arranged on an inner side of the apertures.

[0022] The gas-permeable nature of the membranes allows for the exchange of gases between the culture chamber and the exterior of the cassette housing required for maintaining the appropriate conditions for cell growth. When attaching the membranes to the cassette housing, a leakage from the culture chamber can be avoided if remaining gaps between the membranes and the respective part of the tissue cassette are minimized or sealed. The attachment of the membranes may be accomplished at an inner side of the cassette housing or at an exterior side of the cassette housing. Further below, examples of suitable attachments are mentioned.

[0023] The injection opening is provided for introducing the cell paste into the culture chamber. The injection opening may allow a precise and sterile introduction of cells into the culture chamber. A tubular element, such as a cannula, a tip of a pipette, a tube or hose may be inserted into the injection opening. The tubular element may reach into the culture chamber when introducing the cell paste. Preferably, the injection opening allows the cells to be evenly distributed. Larger culture chambers may comprise more than one injection opening. For example, two injection openings may be arranged on two opposite locations on the tissue cassette.

[0024] The injection opening may be closable through a lid, a cap or a cover. The injection opening may preferably be configured that the culture environment inside the culture chamber is not contaminated. The injection opening may be placed in an edge region or a rim of the cassette housing and not on one of the main sides. Using a dedicated injection opening facilitates an easy and controlled introduction of cells and allows achieving consistent and reproducible results in cell culture processes.

[0025] The culture chamber may be filled with the desired volume using either a pipette, syringe or a pump. Preferably, a syringe with a connected cannula is used, even more preferably a syringe using a 20G PTFE cannula.

[0026] The tissue cassette may comprise a leakage port, which relieves the pressure inside the culture chamber during filling and prevents overfilling of the culture chamber. This may be similar to air vents in plastic injection molding. The leakage port may be closable or coverable after filling the culture chamber. The cell culture apparatus includes a cell media reservoir configured to receive the tissue cassette. The enclosure of the cell media reservoir may be liquid-tight in some of its sides to be able to enclose cell culture media. The cell media reservoir is provided for holding cell or culture media used to provide nutrients to the cells inside the culture chamber through one or both of the membranes. This allows the cells present in the culture chamber to grow. During the growth of the cells, metabolic wastes generated by the cells are secreted to the outside of the culture chamber through at least one membrane. At the same time, nutrients and oxygen, present outside of the culture chamber, are provided into the culture chamber through at least one membrane. Nutrients and oxygen that have penetrated the respective membrane are supplied to the cells, which may then uniformly grow throughout the culture chamber into a desired shape. The cell type and objectives shall be considered when selecting the culture media to ensure optimal growth, viability and reproducibility.

[0027] The cell media reservoir may be designed to allow the tissue cassette to be placed in, for example, an upright or horizontal position. For introducing the tissue cassette, the cell media reservoir has an insertion opening. For example, the insertion opening may be arranged on a top side of the cell media reservoir. The insertion opening may have an opening cross-section that is larger than at least one cross-section of the tissue cassette. The cell media reservoir does not need to have a design that strictly corresponds to the tissue cassette. Instead, the use of already commercially available receptacles that allow to hold cell media and to submerge the tissue cassette may be usable. The cell media reservoir may be made from a biocompatible material, which may include a plastic material, glass, a metal material or other suitable materials. The cell media reservoir may thus be customized or an off-the-shelf jar or any other off-the-shelf laboratory container.

[0028] A proper positioning of the cassette housing within the cell media reservoir may ensure that the culture chamber is fully immersed in the culture media, allowing for efficient and continuous supply of nutrients essential for the growth and development of the tissue. The shape and volume of the cell media reservoir and of the tissue cassette may be adapted to each other in such a way that the culture chamber may reliably be fully immersed in the culture media. However, it is not ruled out that in some variants a full immersion is not needed. This may be the case if the cassette housing includes a section into which the culture chamber does not extend.

[0029] To prevent the tissue cassette from floating due to buoyant forces exerted by the culture media, holding elements may be used. For example, this may be a lid of the cell media reservoir, which may be attachable to the cell media reservoir. In another embodiment, holding elements may be formed by individually, strategically positioned elements, such as downward-facing clamps, resilient tension arms, or other mechanical locking mechanisms, configured to engage an upper or side edges of the tissue cassette. In a simple variant, the insertion opening and the tissue cassette may be configured to provide a snap-fit connection. The holding elements may apply a constant downward force, counteracting buoyancy while maintaining the tissue cassette being in a stable orientation during submersion. In another variant, the tissue cassette may comprise a weight that allows to compensate the buoyant forces.

[0030] However, in some applications a full submersion is not required. For example, the tissue cassette is only partially submerged in the cell media so that only one of the membranes is in contact with the cell media to form an air-liquid-interface. In other words, one of the membranes is in contact with the cell media and other ones are in contact with the air. This may be done by controlling the media volume, or by letting the tissue cassette float.

[0031] The cell media reservoir may have a cylindrical shape having a circular or rectangular base surface and a cylinder height, wherein the size of the base surface may exceed a corresponding cross-sectional surface of the tissue cassette. For example, the base surface may be at least two or three times the size of the corresponding cross-sectional surface of the tissue cassette. This allows for providing a sufficient amount of culture media and its agitation.

[0032] The tissue cassette and the inside of the culture media reservoir should be kept sterile during the culture process.

[0033] As explained above, at least one of the first membrane and the second membrane may be permeable to nutrients and metabolic waste, wherein the cassette housing may provide structural support and define the culture chamber geometry, and wherein the tissue cassette may be removably insertable into the cell media reservoir, which has an insertion opening dimensioned to receive the tissue cassette.

[0034] As will be explained further below, the first aperture and / or the second aperture may have at least one divider for dividing the respective aperture into smaller openings and for an additional support of the respective membrane on the respective aperture.

[0035] At least one of the membranes may be configured for cell attachment or adhesion. In order to keep a defined tissue geometry, the respective membrane allows for cell attachment or cell adhesion. The respective membrane may have intrinsic properties that allow for cells to attach, such as being hydrophilic or having a specific surface structure or charge. A membrane’s ability to allow for cell attachment may be improved by, e.g., adding a surface coating, such as laminin or poly-L-lysine (PLL), providing a surface charge, e.g. by a plasma treatment, or by providing a surface structure, e.g. by nanopatterning. Depending on the application, the tissue should be able to be completely removed from the tissue cassette, or in some cases, the tissue should be removed together with the membrane. If the tissue need to be removed completely from the tissue cassette, the membrane should have enough cell attachment to keep the tissue geometry, but low enough attachment that it can still be removed from the tissue without destroying (ripping, tearing) the tissue.

[0036] The cassette housing may be made from a biocompatible material. For example, the cassette housing may be made from a biocompatible resin. It may be a biocompatible thermoplastic material, or a biocompatible metallic material, or a biocompatible elastomer material. The biocompatible material may be used in a casting, cutting, and / or additive manufacturing process. For example, a resin or a thermoplastic material may directly be used in an additive manufacturing process to manufacture a single one or a small batch of cassette housings with a desired shape and size. Using a biocompatible material may be particularly important for applications where the cells or tissues grown in the cell culture apparatus are intended for therapeutic use. The biocompatible material thus ensures safety and compatibility of the cell culture environment with the biological materials being used. For example, if the chosen material is a resin, it may be a commercially available photopolymer resin composed of methacrylic acid esters and photoinitiators. This type of resin may be transparent. The resin may be compliant with ISO 13485 and / or USP Class VI certified. The biocompatible resin allows to create the cassette housing by using a stereolithography process, or the like.

[0037] The membranes of the tissue cassette may be biocompatible, and be permeable to culture media and cell waste products, but not to cells. The membranes may be compatible with at least one sterilization method, such as gamma radiation, autoclave, ethylene oxide gas, and other.

[0038] The membranes may also be made out of a degradable or biodegradable material, which would naturally be absorbed or removed over time.

[0039] The membranes may be optically transparent. The optical transparency of the membranes enables real-time observation and monitoring of the cell culture during filling the cell paste into the culture chamber as well as during the growth process without disturbing any condition in the culture chamber or the cell environment.

[0040] The membranes may be made from polyethylene terephthalate (PET) with a pore size of 3 to 4 pm and a thickness of 12 to 23 pm. Thus, the membranes are porous to allow nutrients and waste products to pass the membranes. The pore size allows for the exchange of nutrients and waste products while preventing the passage of cells. The thickness of the membranes ensures that the membranes are durable enough to withstand the culture conditions. PET membranes with the above- mentioned specifications may provide an optimal balance between permeability and structural integrity. The membranes provide a reliable and effective barrier that supports cell growth while maintaining the integrity of the culture environment. The pores may be evenly distributed over substantially the whole surface area of the membranes. Furthermore, using PET allows to sterilize the tissue cassette, for example by an autoclaving process.

[0041] In an alternative, at least one of the membranes may be provided in the form of a flexible electrospun poly -caprolactone (PCL) membrane. It may have a thickness of about 20 pm and be formed as a mesh with an average pore size of about 5 pm. It may be plasma-treated to promote cell attachment. It may be sterilizable by ethylene oxide. Such a PCL membrane is biodegradable and may be gradually broken down and eventually removed in vivo.

[0042] In a still further alternative, the membranes may be made from a metallic material. A metallic membrane may be made out of a thin metal foil or grid with a defined pore structure tailored to the desired permeability as discussed above.

[0043] As mentioned above, the first aperture and / or the second aperture may comprise at least one divider. A divider may be understood as a partition arranged inside the respective aperture. For example, the respective aperture may be divided into several openings, which are arranged in a row structure or a matrix structure and mutually form the respective aperture. The respective aperture may thus comprise a grid structure with a more or less strong subdivision into partial openings. The at least one divider allows an additional support of the respective membrane on the aperture, thereby preventing a bulging or buckling of the membrane especially when large apertures are required. The at least one divider may be formed by a frame or a grid component attached to the respective main side of the tissue cassette. It may comprise a thickness that is sufficient to provide the structural support to the tissue, depending on the material selection and the size of the apertures.

[0044] The cassette housing may be made from flexible silicone. Flexible silicone provides a durable and at the same time mechanically adaptable material for the tissue cassette, allowing it to accommodate various shapes and sizes of culture chambers. This flexibility may be particularly useful for applications that require the culture of tissues with more complex geometries. Due to the flexible material, the cassette housing allows to be flexed or deformed when removing the tissue from the culture chamber. The material thickness of the cassette housing may be chosen to form a sufficient structural rigidity to hold a cell culture and the membranes.

[0045] The membranes may be thermally bonded to the cassette housing. Thermal bonding ensures a secure and leak-proof seal between the membranes and the cassette housing, preventing a leakage of the cells inside the culture chamber or entry of culture media into the culture chamber. This method of attachment may also provide a strong and durable bond that can withstand the conditions of the cell culture. The membranes may, however, also be clamped or attached into the cassette housing. It is important to prevent leakage of the cell paste at an interface between the membranes and the cassette housing. Besides thermal bonding, the membranes may be attached by ultrasonic welding, gluing or other material bonding methods.

[0046] The cell media reservoir may be configured to allow a full submersion of the cassette housing in the cell culture media. The cell media reservoir may comprise a base surface and at least one side wall, each of which may include rounded, curved, or polygonal surfaces. The height of the cell media reservoir, i.e. its extension away from the base surface, may be selected to be at least equal to the height, i.e. the associated dimension, of the cassette housing to ensure full submersion. The surface area of the cell media reservoir at its widest point may be designed to exceed that of the cassette housing, thereby allowing sufficient clearance around all sides.

[0047] The cell culture apparatus may be designed to allow for mechanical stimulation of the tissue through vibration, media agitation, or cyclic pressure gradients. Cells and tissues may develop differently depending on external signals they receive. One of the inputs that influence cell behavior is that of mechanotransduction or mechanical stimulation. Several types of mechanical stimulation can be applied, such as shear stress or push / pull forces. Mechanical stimulation may be applied to the tissue in the tissue cassette by vibration, media agitation or through cyclic pressure gradients across or around the tissue cassette. A media agitation may be induced through a pump, a stir bar or by external movement, such as an orbital shaker. An agitation of the cell media also ensures that nutrients are evenly distributed and that waste products are removed efficiently. The agitation may also help to simulate the dynamic conditions of a natural tissue environment, promoting an improved cell growth and development. A cyclic pressure gradient over the tissue cassette may be induced through a peristaltic pump. Shear stress may be induced by having a parallel flow over the cassette housing surfaces.

[0048] Additionally, the cell media inside of the cell media reservoir or the whole cell media reservoir may be exchanged during the culture process, either manually or automatically.

[0049] The cell culture apparatus may be configured to prevent leakage of the cell paste between the first membrane, the at least one second membrane and the cassette housing. As stated above, this may be accomplished by dimensioning remaining gaps between the parts of the cassette housing to be small enough to avoid leakage of the cells. It may also be feasible to seal the remaining gaps, e.g. through sealing elements arranged between the individual parts of the cassette housing. The parts of the cassette housing may be glued together to prevent the formation of gaps when it can be guaranteed to at least partly disassemble the cassette housing to remove the tissue from the culture chamber. Different types and shapes of tissues can be cultured in tissue cassettes in several configurations. Several tissue cassettes may be placed in the same culture media reservoir. During the culture process, it is possible to move the tissue cassette into other cell media reservoirs for treatments or processing. It is also possible to keep the tissue cassette in the same cell media reservoir and exchange different cell media for treatments or processing.

[0050] The cell culture apparatus may comprise a liquid exchange system coupled with the enclosure of the cell media reservoir to selectively and / or automatically exchange cell culture media in the enclosure. For example, the enclosure of the cell media reservoir may comprise a tubing or openings for facilitating the exchange and refreshing of the cell culture media. When paired with a liquid exchange system, such as a peristaltic pump or pressure-driven pumping system, the cell culture apparatus can be automated. Preferably, the liquid exchange system is an automated liquid exchange system. Automation may be particularly advantageous for scaling up, as the modular design of the tissue cassette, combined with automated media exchange, allows for efficient production of a large number of tissues. Additionally, a precise control over media exchange may enable a better control of the overall process. This may be especially useful for applying different culture conditions or post-processing steps to the tissues at specific time points. For example, growth factors may be introduced during specific stages of culture, or chemicals may be added to remove biomaterial introduced in the initial tissue or cell paste. Another application may be the introduction of chemicals for tissue decellularization.

[0051] It is further proposed a method for mass culturing of cells for producing tissues, comprising the steps of injecting a cell paste through an injection opening into a culture chamber formed between a first membrane and at least one second membrane of a cassette housing of a tissue cassette, to fill the culture chamber; submersing the tissue cassette in a cell culture media bath within a cell media reservoir, wherein the cell culture media bath is agitated to provide nutrients to the cells through the membranes; incubating the submersed tissue cassette under suitable conditions for cell growth; and disassembling the tissue cassette to retrieve the cultured tissue.

[0052] As discussed above, the cassette housing provides structural support and defines the culture chamber geometry. The tissue cassette may be removably insertable into the cell media reservoir, which may have an insertion opening dimensioned to receive the tissue cassette.

[0053] The method for mass culturing of cells may involve providing a cell culture apparatus as described further above.

[0054] The cell culture media bath may be agitated using a magnetic stir-bar. This may be done by simply arranging a suitable stir-bar in the cell media reservoir and place the cell media reservoir on top of a stirrer device, in which another rotating magnet or an assembly of electromagnets is operated. The magnetic stir bar may be coated with a biocompatible material to avoid contamination of the cell media.

[0055] The cell culture apparatus may be incubated at 37°C, 5% CO2 in a humidified incubator. This step ensures that the cells are maintained in an environment that supports their growth and development.

[0056] Tissues may be cultured in the tissue cassette in several configurations. Since the modular nature of the tissue cassette, parts can be added or removed during the culture process. Several tissue cassettes may be placed in the same culture media reservoir. The cell media inside of the cell media reservoir or the whole cell media reservoir may be exchanged during the culture process, either manually or automatically. During the culture process, it may be possible to move the tissue cassette into other cell media reservoirs for treatments or processing. It may also be possible to keep the tissue cassette in the same cell media reservoir and exchange different cell media for treatments or processing.

[0057] One of the first membrane and the at least one second membrane may be removed after a predetermined time in culture. This may improve media access and / or induce shear stress to the surface of the tissue.

[0058] The tissue cassette may be placed horizontally or vertically during the culture process.

[0059] The cell culture media may be agitated using a pump, a stir bar, or an orbital shaker.

[0060] Short description of the drawings

[0061] In the following description this invention will be further explained by way of exemplary embodiments shown in the drawings:

[0062] Fig. 1 shows a tissue cassette in an isometric view.

[0063] Fig. 2 shows a cell media reservoir in an isometric view.

[0064] Fig. 3 shows the tissue cassette and the cell media reservoir in an upright position in a spatial view.

[0065] Figs. 4 and 5 show another example of a tissue cassette with a non-cuboid shape.

[0066] Figs. 6 and 7 show a still further example of a tissue cassette with a non-cuboid shape. Detailed description of the invention

[0067] Fig. 1 shows a first exemplary embodiment of a tissue cassette 2 in a three- dimensional view. The tissue cassette 2 has a cassette housing 4 that is defined by a first main side 6 and a parallel second main side 8, which each indicate a spatial boundary plane of the cassette housing 4. In this exemplary embodiment, the main sides 6 and 8 are planar and are arranged at a constant distance to each other.

[0068] The tissue cassette 2 has height h, a width w and a thickness t. The height h and the width w correspond to main extension dimensions of the cassette housing 4, while the thickness t corresponds to the extension of the cassette housing 4 orthogonal to the main sides 6 and 8. Both the height h and the width w equal a plurality of the thickness t. Thus, the cassette housing 4 is a flat, compact structure with two parallel main sides 6 and 8, at which the cassette housing 4 has a substantially rectangular shape.

[0069] A circumferentially closed, strip-like frame 10 runs along a closed and substantially rectangular path. At each corner of this path, the frame 10 transitions smoothly into a rounded curvature, eliminating sharp angles and creating a gentle, curved contour. Exemplarily, this rounding is consistent across all four corners. The frame 10 holds a first grid part 12 and a second grid part 14 at a distance and parallel to each other. The frame 10 and the grid parts 12 and 14 form the cassette housing 4 and define the volume of the tissue cassette 2. The first grid part 12 is arranged at the first main side 6 and the second grid part 14 is arranged at the second main side 14.

[0070] The first grid part 12 and the second grid part 14 are positioned directly opposite each other and are held in place by the frame 10, that runs along their outer contour. The width of the frame 10 in a direction orthogonal to the main sides 6 and 8 defines the thickness t of the tissue cassette 2. Exemplarily, the frame 10 is designed to be perpendicular to the grid parts 12 and 14 when assembled.

[0071] For holding the grid parts 12 and 14, the frame 10 may exemplarily comprise two circumferential grooves 13 or recesses at an inner side, i.e. facing into the interior of the frame 10, into which grooves or recesses the grid parts 12 and 14 are placed. The grooves 13 or recesses may be concave and a profile of the outer edge of the grid parts 12 and 14 may be rounded, such that the grid parts 12 and 14 may snap into the grooves 13. Other variants are not ruled out and the connection between the grid parts 12 and 14 and the frame 10 may be realized differently. For example, the grid parts 12 and 14 may simply be held in the frame 10 through friction. For this, the frame 10 has an inner radial edge, which forms a friction fit with each of the outer radial edges of the grid parts 12 and 14. Using a snap fit or friction fit connection may improve the ability to disassemble the cassette housing 4. At the first main side 6 and the second main side 8, the frame 10 defines a first aperture 16 and a second aperture 18, respectively. The apertures 16 and 18 exemplarily extend over the majority of the main sides 6 and 8. In the first aperture 16, the first grid part 12 is arranged. In the second aperture 18, the second grid part 14 is arranged.

[0072] At an inner side of the first grid part 12, a first membrane 24 is attached. At an inner side of the second grid part 14, a second membrane 26 is attached. For example, the membranes 24 and 26 are thermally bonded to the respective grid part 12 or 14. Hence, the membranes 24 and 26 are supported by the grid parts 12 and 14 and cover the apertures 16 and 18.

[0073] Exemplarily, both grid parts 12 and 14 have a plurality of partitions 20, which are arranged in a grid structure inside the respective grid part 12 and 14. The partitions 20 divide the first aperture 16 and the second aperture 18 into a plurality of smaller openings 22 that are arranged in a matrix shape. Thus, the grid parts 12 and 14 act as dividers and form “grids”, in which the respective aperture 16 or 18 is divided into smaller sections in the form of the openings 22.

[0074] The grid parts 12 and 14 thus do not only support the membranes 24 and 26 in an edge region, since the partitions 20 provide structural support for the membranes 24 and 26 throughout the entire area of the associated grid part 12 or 14. Thereby, excessive bulging or bucking is avoided to maintain their mechanical integrity.

[0075] The frame 10 and the grid parts 12 and 14 may be sealed to each other or attached to each other in a way that at least a leakage of cell paste from inside the cassette housing 4 to its exterior can be avoided, as further explained below.

[0076] A space between the first membrane 24 and the second membrane 26 as delimited by the frame 10 will be referred to as culture chamber 28, in which cells can be cultured to create a tissue. In this exemplary embodiment, the culture chamber 28 is configured to produce a flat and substantially rectangular piece of tissue with a certain thickness defined by the distance between the two membranes 24 and 26.

[0077] For feeding a cell paste into the culture chamber 28, an injection port 30 is arranged on an exterior side of the frame 10 and reaches from outside the culture chamber 28 into the culture chamber 28. In this example, the injection port 30 is designed as a pipe stub. A cannula, the tip of a pipette, or another tubular cell paste supply device can be inserted or clamped into the injection port 30 to inject cell paste. This may be done manually, e.g. through a syringe or a pipette, or automatically, e.g. through a pump.

[0078] The injection port 30 is arranged at a side 32 of the frame 10, which may be referred to as a top region 32. At the top region 32, the frame 10 has a greater extension in the thickness direction than in the remaining parts of the frame 10. The respective dimension is indicated with the letter “s” in Fig. 1, which exceeds the thickness t that corresponds to the extension of the frame 10 in all other regions. By letting the top region 32 have a larger extension than the rest of the frame 10, an upper rim with shoulders 34 is created that allows to insert the tissue cassette 2 into an insertion opening of a receptacle and let the shoulders 34 rest on an associated edge region of the insertion opening as end stops. This will be shown in Fig. 3 below.

[0079] Exemplarily, the frame 10 and the grid parts 12 and 14 are biocompatible. In addition, they are compatible with at least one sterilization technique, such as Gamma radiation, autoclave, Ethylene oxide gas, and other. As an example, but not ruling out other manufacturing technologies, the frame 10 and the grid parts 12 and 14 are made from a rigid 3D printed, biocompatible resin. However, other plastic materials and metal materials could also be used. For example, this could include polypropylene (PP), polyethylene (PE), polyethylene terephtalate (PET), polysulfone (PSU), silicone, stainless steel, some ceramic materials or even glass.

[0080] Fig. 2 shows a cell media reservoir 36 in a three-dimensional view. The cell media reservoir 36 has a fluid-tight enclosure 38 for enclosing a suitable cell culture media. It has an insertion opening 40, which is dimensioned to receive the tissue cassette 2. By inserting the tissue cassette 2 into the insertion opening 40, the tissue cassette 2 can be submerged in the cell media that is contained in an interior space 42 of the cell media reservoir 36. The cells inside the culture chamber 28 can be supplied with nutrients by the cell media and waste products can be removed into the cell media through the membranes 24 and 26.

[0081] Exemplarily, a feed pipe 44 and a return pipe 46 are attached to the cell media reservoir 36, wherein the pipes 44 and 46 are in fluid contact with the interior space 42. By using a pump arranged external to the cell media reservoir 36, the cell media in the interior space 42 can be agitated. The pump would then supply cell media through the supply pipe 44 into the interior space 42 and receive it from there through the return pipe 46. The agitation may be conducted alternatingly or continuously.

[0082] To further simplify this, instead of the pipes 44 and 46, a magnetic stir bar could be placed at a bottom side of the cell media reservoir 36, driven by a stirrer device beneath the cell media reservoir 36. By driving the magnetic stir bar, the cell media is agitated. Other variants are also possible, such as placing the cell media reservoir 36 onto an orbital shaker.

[0083] Fig. 3 shows a cell culture apparatus 48, which substantially consists of the cell media reservoir 36 and the tissue cassette 2. The width w of the tissue cassette 2 and the associated dimension of the insertion opening 14 are adapted to each other, such that the tissue cassette 2 can be easily inserted into the insertion opening 40. In doing so, the shoulders 34 rest on edge regions 50 of the cell media reservoir 38 and the tissue cassette 2 is held in the cell media reservoir 36.

[0084] In Fig. 3, the pipes 44 and 46 are connected to a schematically illustrated liquid exchange system 51. The liquid exchange system 51 is configured to selectively and / or automatically exchange cell culture media in the enclosure 38. While a selective operation may require a manual trigger, the automatic operation may be controlled through a control unit, which is not shown herein.

[0085] Different shapes of tissue cassettes can be used for creating different shapes of tissues. For example, fig. 4 and 5 show a tissue cassette 52 having a cassette housing 53 with a first part 54 on a first main side 55, a second part 56 on an opposite second main side 57, and a frame 58. The frame 58 has a first aperture 60 covered by a first membrane 62 and a second aperture 64 covered by a second membrane 66. The first membrane 62 is held by the first part 54 and the second membrane 64 is held by the second part 56. The first part 54 and the second part 56 are held by the frame 58, e.g. through friction fit. The membranes 62 and 66 and the frame 58 form a culture chamber 68 in the form of a heart valve. This could be referred to as a “valve mold”. In the exemplary embodiment shown in Figs. 4 and 5, the valve mold has an oval shape with a bend along the short diameter.

[0086] As an example, the first part 54 comprises an injection port 70 for feeding the cell culture into the culture chamber 68. The injection port 70 may have substantially the same shape and function as the injection port 30 of the exemplary embodiment shown in Figs. 1 to 3. A suitable cell media reservoir would comprise an enclosure filled with cell media, into which enclosure the tissue cassette 52 can be inserted. For example, the cell media reservoir may be a simple off-the-shelf jar.

[0087] Figs. 6 and 7 show a further example of a tissue cassette 72 that has a hollow- cylindrical culture chamber 74. The tissue cassette 72 has a cassette housing 76 with a first grid part 78 in the form of a cylindrical component. The first grid part 78, and two second grid parts 80 and 82 can be assembled to form the cassette housing 76. The second grid parts 80 and 82 exemplarily have a semi-cylindrical shell with two opposite half circular edges and two parallel axial edges. Both second grid parts 80 and 82 have connecting flanges 84 arranged at their axial edges. The connecting flanges 84 of each of the second grid parts 80 and 82 are exemplarily on the same extension plane. When contacting the second grid parts 80 and 82 through their connecting flanges 84, their semi-cylindrical shells form a cylindrical grid.

[0088] When the second grid parts 80 and 82 are connected, they enclose the first grid part 78. For maintaining correct relative positions of the grid parts 78, 80 and 82 to form the hollow-cylindrical culture chamber 74, the first grid part 78 has two opposite axial end sections 79, in which an outer diameter is increased. In the remaining section arranged between the axial end sections 79, the outer diameter is reduced to define a distance to the second grid parts 80 and 82. The axial end sections 79 may be brought into a flush contact with corresponding sections 83 of the second grid parts 80 and 82.

[0089] In analogy to Fig. 1, the first grid part 78 and the second grid parts 80 and 82 have partitions 86 that divide a first aperture 88, belonging to first grid part 78, and second apertures 90, belonging to the second grid parts 80 and 82, into several openings 92. All openings 92 lay on a cylindrical surface.

[0090] At a radial outer side of the first grid part 78, a first membrane 94 is arranged. At radial inner sides of each of the grid parts 80 and 82, a second membrane 96 is provided. When assembling the cassette housing 76, the first grid part 78 holds the grid parts 80 and 82 at their half-circular edges in the sections 81 and a distance is created between the membranes 94 and 96. A radial inner surface of the first grid part 78 thus defines the first main side, while the radial outer surfaces of the second grid parts 80 and 82 define the second main side.

[0091] The tissue cassette 72 has an injection port 98 that reaches into the culture chamber 74 from a top side 100 radially between the first main side and the second main side. The grid parts 78, 80 and 82 may be held together through clamping the connecting flanges 84 together through clamps 102.

[0092] REFERENCE NUMERALS

[0093] 2 tissue cassette

[0094] 4 cassette housing

[0095] 6 first main side

[0096] 8 second main side

[0097] 10 frame

[0098] 12 first grid part

[0099] 13 groove

[0100] 14 second grid part

[0101] 16 first aperture

[0102] 18 second aperture

[0103] 20 partition

[0104] 22 openings

[0105] 24 first membrane

[0106] 26 second membrane

[0107] 28 culture chamber

[0108] 30 injection port

[0109] 32 top region

[0110] 34 shoulders

[0111] 36 cell media reservoir

[0112] 38 fluid-tight enclosure

[0113] 40 insertion opening

[0114] 42 interior space

[0115] 44 feed pipe

[0116] 46 return pipe

[0117] 48 cell culture apparatus

[0118] 50 edge region

[0119] 51 liquid exchange system

[0120] 52 tissue cassette

[0121] 53 cassette housing

[0122] 54 first part

[0123] 55 first main side 56 second part

[0124] 57 second main side

[0125] 58 frame

[0126] 60 first aperture

[0127] 62 first membrane

[0128] 64 second aperture

[0129] 66 second membrane

[0130] 68 culture chamber

[0131] 70 injection port

[0132] 72 tissue cassette

[0133] 74 culture chamber

[0134] 76 cassette housing

[0135] 78 first grid part

[0136] 79 end section

[0137] 80 second grid part

[0138] 81 section

[0139] 82 second grid part

[0140] 84 connecting flange

[0141] 86 partitions

[0142] 88 first aperture

[0143] 90 second aperture

[0144] 92 openings

[0145] 94 first membrane

[0146] 96 second membrane

[0147] 98 injection port

[0148] 100 top side

[0149] 102 clamp

Claims

CLAIMS1. A cell culture apparatus (48) for producing tissues, comprising: a tissue cassette (2, 52, 72), and a cell media reservoir (36), wherein the cell media reservoir (36) comprises an enclosure (38) for enclosing cell culture media, wherein the cell media reservoir (36) has an insertion opening (40) for inserting the tissue cassette (2, 52, 72) into the cell media enclosed in the cell media reservoir (36), wherein the tissue cassette (2, 52, 72) has a cassette housing (4, 53, 76) comprising a first main side (6, 55) having a first aperture (16, 60, 88) covered by a first membrane (24, 62, 94), and at least one second main side (8, 57) having a second aperture (18, 64, 90) covered by a second membrane (26, 66, 96), such that a culture chamber (28, 68, 74) is formed between the first membrane (24, 62, 94) and the second membrane (26, 66, 96), wherein the first membrane (24, 62, 94) and the second membrane (26, 66, 96) are attached to the cassette housing (4, 53, 76), wherein the tissue cassette (2, 52, 72) has an injection opening (30, 70, 98) extending through the cassette housing (4, 53, 76) into the culture chamber (28, 68, 74) for feeding cells into the culture chamber (28, 68, 74), and wherein at least one of the first membrane (24, 62, 94) and the second membrane (26, 66, 96) is gas-permeable.

2. The cell culture apparatus (48) of claim 1, wherein at least one of the first membrane (24, 62, 94) and the second membrane (26, 66, 96) is permeable to nutrients and metabolic waste, wherein the cassette housing (4, 53, 76) provides structural support and defines the culture chamber geometry, and wherein the tissue cassette (2, 52, 72) is removably insertable into the cell media reservoir (36), which has an insertion opening dimensioned to receive the tissue cassette (2, 52, 72).

3. The cell culture apparatus (48) of claim 1 or 2, wherein the first aperture (16, 60, 88) and / or the second aperture (18, 64, 90) has at least one divider for dividing the respective aperture (16, 18, 60, 64, 88, 90) into a plurality of smaller openings (22) and for an additional support of the respective membrane (24, 26, 62, 66, 94, 96) on the respective aperture (16, 18, 60, 64, 88, 90).

4. The cell culture apparatus (48) of any of the preceding claims, wherein at least one of the membranes (24, 26, 62, 66, 94, 96) is configured for cell attachment or adhesion.

5. The cell culture apparatus (48) of any of the preceding claims, wherein the cassette housing (4, 53, 76) is made from a biocompatible material.

6. The cell culture apparatus (48) of any of the preceding claims, wherein the membranes (24, 26, 62, 66, 94, 96) are made from polyethylene terephthalate (PET) with a pore size of 3 to 4 pm and a thickness of 12 to 23 pm.

7. The cell culture apparatus (48) of any of the preceding claims, wherein the membranes (24, 26, 62, 66, 94, 96) are thermally bonded to the cassette housing (4, 53, 76).

8. The cell culture apparatus (48) of any of the preceding claims, wherein the cell media reservoir (36) is configured to allow a full submersion of the cassette housing (4, 53, 76) in the cell culture media.

9. The cell culture apparatus (48) of any of the preceding claims, wherein the cell culture apparatus (48) is designed to allow for mechanical stimulation of the tissue through vibration, media agitation, or cyclic pressure gradients.

10. The cell culture apparatus (48) of any of the preceding claims, wherein the cell culture apparatus (48) is configured to prevent leakage of the cell paste between the first membrane, the second membrane and the cassette housing (4, 53, 76).

11. The cell culture apparatus (48) of any of the preceding claims, comprising a liquid exchange system (51) coupled with the enclosure (38) of the cell media reservoir (36) to selectively and / or automatically exchange cell culture media in the enclosure (38).

12. A method for mass culturing of cells for producing tissues, comprising: injecting a cell paste through an injection opening (30, 70, 98) into a culture chamber (28, 68, 74) formed between a first membrane (24, 62, 94) and at least one second membrane (26, 66, 96) of a cassette housing (4, 53, 76) of a tissue cassette (2, 52, 72), to fill the culture chamber (28, 68, 74), submersing the tissue cassette (2, 52, 72) in a cell culture media bath within a cell media reservoir (36), wherein the cell culture media bath is agitated to provide nutrients to the cells through the membranes (24, 26, 62, 66, 94, 96),incubating the submersed tissue cassette (2, 52, 72) under suitable conditions for cell growth, and disassembling the tissue cassette (2, 52, 72) to retrieve the cultured tissue.

13. The method of claim 12, comprising selectively and / or automatically exchanging cell culture media in the enclosure (38) of the cell media reservoir (36)..

14. The method of claim 12 or 13, wherein the cell culture apparatus (48) is incubated at 37°C, 5% CO2 in a humidified incubator.

15. The method of any of the claims 12 to 14, wherein one of the first membrane (24, 62, 94) and the at least one second membrane (26, 66, 96) is removed after a predetermined time in culture.

16. The method of any of the claims 12 to 15, wherein the tissue cassette (2, 52, 72) is placed horizontally or vertically during the culture process.

17. The method of any of the claims 12 to 16, wherein the cell culture media is agitated using a pump, a stir bar, or an orbital shaker.

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