Method of assembling bioreactor having biomaterial deposition end that moves with top portion to allow bioprinting

Abstract

A bioreactor (4) allows bioprinting within an internal volume to form a side wall (SW) extending upwardly from a substrate provided with a target support (20) for the bioprinting. Once the adapter (5) has been coupled to the bioreactor top portion (4b) to plug the corresponding aperture (O4), the biological material deposition end (8) may be maintained in an inserted state so as to extend through and / or be adapted to be fed through the aperture (O4). The base and the top portion (4b) are interconnected by a sealing operation to form a sealed enclosure (E) comprising the sidewalls and ports. Bio-printing is carried out by driving externally from the deposition end (8) using a change in volume to deliver biological material onto the support (20), and then a culture and / or maturation stage of the printed tissue can be initiated inside the mask (E) without displacing the printed tissue.

Description

Technical Field

[0001] The present disclosure relates to systems and enclosures for bioprinting. More specifically, the present disclosure relates to a method of assembling a bioreactor and a bioreactor, typically a single-use bioreactor, suitable for bioprinting while also being used for subsequent steps without the need to move the printout. Background Art

[0002] Forming three-dimensional (3D) objects using biological materials or coating such 3D objects under sterile conditions requires a significant effort to maintain sterile conditions and assemble the necessary equipment for forming or coating under sterile conditions. Hereinafter, bioprinting is defined as the use of 3D printing techniques using materials containing living cells to, for example, produce tissue for reconstructive surgery.

[0003] Known 3D bioprinters include a drive system having a frame, a reservoir, and a nozzle / needle in the form of a dispenser that allows movement in the XYZ direction. The nozzle / needle has a biomaterial deposition end for discharging biomaterial. In order to maintain sterile conditions, the bioprinter must be contained in a large printing chamber. Document US2022 / 0105686A1 discloses a method for controlling a 3D bioprinter, wherein a printing chamber is used to accommodate the bioprinter. Printing living tissue in a sterile environment and ensuring continuous operation of CO2 incubation can be performed in the printing chamber without moving the printout after printing the living tissue. The transfer material is then used to mature while maintaining sterile conditions, usually after being introduced into a bioreactor.

[0004] WO 2022064125 A1 discloses a bioprinting process in which a sterile isolator is provided to define a bioprinting space. Printing components and associated mechanical components are housed within this bioprinting space. The sterile isolator has a window that allows a laser beam to pass through for scanning and activating the bioprinting source. The sterile isolator also includes a temperature-regulated space for maturation of tissue sheets and a sterile airlock for exchange between the interior and exterior of the isolator.

[0005] Document EP 3194150 B1 discloses a sterilizable container that defines an internal volume, wherein a printer assembly is housed within this internal volume. The container can serve as a bioreactor. To maintain sterile conditions, the printing assembly can be removed through a transfer hatch formed in the side wall. The assembly of the printing assembly and the subsequent removal of this assembly represent a complexity. Document US2020 / 0406542 discloses a bioprinting device that can be laterally interfaced with a bioreactor so that transfer can be performed without opening the door of the device housing that defines the internal volume. However, the printout requires displacement.

[0006] The current construction of the sterilizable container that forms the printing chamber is complicated. More generally, although the additive manufacturing of biological systems has the potential to revolutionize the engineering of soft structures, bioprostheses and scaffolds for tissue repair, some limitations need to be overcome. During the whole process (bioprinting, then maturation), for example, an aqueous environment with pH, ​​ionicity, temperature and sterility controlled within strict tolerances is used to keep cells alive. Expensive bioprinters that attempt to solve this challenge have been tested, but these expensive bioprinters mean the harsh steps and workload of maintaining sterile conditions after the bioprinting step. Therefore, there is still room for improvement in the specific areas of the present disclosure. Summary of the Invention

[0007] To improve the situation, embodiments of the present invention provide a method of assembling a bioreactor, which may be a single-use, sterilizable bioreactor, allowing bioprinting within the interior volume of the bioreactor using a biomaterial deposition tip to deliver biomaterial to a target support, the method comprising:

[0008] - providing a target support for bioprinting on or above a substrate portion (said substrate portion being adapted / intended to be a lower portion of a bioreactor);

[0009] - coupling the adapter to the bioreactor, for example to the bioreactor top part (which may be distinct from the base part while contributing to delimiting the interior volume directly by the inner face of the top part), at an aperture which is plugged airtightly by the adapter;

[0010] - forming a side wall to define an interior volume of the bioreactor, wherein the side wall is integral with the base portion, the side wall being adapted to taper and / or extend longitudinally upwards / away from the base portion in an upright configuration of the bioreactor;

[0011] - providing one or two connection ports for direct fluid communication with the internal volume, so as to form a fluid inlet port and a fluid outlet port, such that the bioreactor can be used for a cultivation and / or maturation phase of printed material (obtained by bioprinting) within the internal volume after bioprinting; and

[0012] - The base portion and the top portion are interconnected by at least one sealing operation to form a sealing cover comprising side walls, wherein the sealing cover is at least partially flexible and movable so as to allow the biomaterial deposition tip inserted through the aperture or fed / filled via the aperture to be movable in at least two lateral directions within the internal volume while being fixed to the adapter.

[0013] Printed materials do not need to be moved. The same container (here, the bioreactor) can be used for all process steps from bioprinting to final tissue recovery, including cell culture (and potentially storage and transport of printed tissue). This helps maintain sterile conditions, while the size and design of the bioreactor can be easily varied. The sealed enclosure is compatible with bioprinting constraints.

[0014] Optionally, in a step performed before all the wall parts of the enclosure are assembled, at least one functional component can be attached to the wall parts defining the interior volume. Such functional components can include stirring components, pH and / or oxygen sensors, temperature sensors, aerators.

[0015] In some options, prior to bioprinting and after fluid communication between the deposition tip and a reservoir portion of the printer unit (e.g., cartridge / syringe, etc.), a collector can be used to deliver a certain amount of product (biomaterial or the like) into the sealed enclosure without reaching the target support until a suitable flow rate of the flowable product for the bioprinting phase is achieved. This corresponds to a calibration step, for example, using the piston, screw system, and / or pneumatic tool of the printer unit.

[0016] The method provides a base portion that can be fixed as it extends upward from the bioreactor. This allows a first print to be made within the internal volume, targeting a given location, followed by culturing cells at the same location based on the tissue obtained from the first print, and then a second print to be initiated without any change in location. The base portion can optionally have a peripheral portion with an annular shape that is rigid or rigidly fixed to maintain its shape and circumference.

[0017] The adapter can further be moved along a third direction that is substantially perpendicular to the base portion (the Z direction, in which case the transverse direction is the XY direction). The adapter can be arranged in the top portion, away from the side walls, for example using a rigid spacing structure. In some options, the base portion can have a movable portion that is displaced to bring the target support closer to the adapter, possibly without longitudinally moving the top portion. The latter option can be performed without any displacement of the printed tissue relative to the target support, and thus without changing the fixation of the cells to the supports arranged in the base portion.

[0018] At least when the adapter is in the lowered position, an annular space can be obtained around the adapter thanks to a spacer member included in or arranged around the top portion. Due to the flexibility of the top portion, an injection needle or similar deposition tip can be moved in three orthogonal directions, enabling bioprinting by the needle / deposition tip on at least a substantially planar area that matches a target support (printing platform / support) that is integral with or included in the base portion.

[0019] The bioreactor can advantageously be used directly for maturation, cell culture, etc., without transferring the bioprinter, and possibly without any changes to the geometry / configuration at the level of the base portion. If not reused for further printing operations, the drive system can simply be disconnected. After printing the tissue or pattern, the inlet port can be used to introduce nutrients, fluids, and any desired products for growth / maturation, as programmed by a control unit associated with the drive system that moves the printing portion (including the adapter and deposition tip). At least one fluid outlet port is provided for the removal of waste materials, etc.

[0020] The inlet port and / or the outlet port can be a connection port forming a nozzle with a barb. They are suitable for sterile fluid connection with a flexible hose.

[0021] Near the base portion, the sidewall of the bioreactor can be relatively wider / relatively wider than the bioreactor top opening or adapter flange. The sidewall cross-section can vary in an annular shape about the central axis. Regardless of how the sidewall cross-section varies (gradually decreasing or increasing, with or without an undulating profile), the sidewall has an upward longitudinal extension in the extended configuration, such that the lower annular end of the sidewall extends opposite the upper end of the sidewall.

[0022] The bioreactor can be equipped with multiple connectors, including connection ports. The adapter allows for connection to the drive system outside the containment enclosure. Ports for one or more sensors can be sterile connections. In some options, a single interface connector can be provided, including several such sterile connections. At least one connection and disconnection can involve a clamp system, each associated with a port. The adapter includes means for rigidly securing the position of the needle forming the deposition tip, such that the relative position between the needle tip and the adapter remains unchanged.

[0023] Thanks to this assembly of the sealing cover, the target support and the printing pin, nozzle, or similar deposition tip (print head insertion part) can be provided in the internal volume without any additional bulky embedded part of the printing system (e.g., no reservoir, drive system, etc. are present in the bioreactor). Specifically, the adapter can move together with the flexible part of the sealing cover: this flexible part follows the movement of the adapter and thus moves together with the adapter, without any drive part (driving the movement of the adapter) being present in the internal volume. The top part can directly cover / delimit the internal volume, forming the top of the bioreactor opposite the base part.

[0024] When assembling the side walls, the following options are available:

[0025] - the side wall has at least one welding area for joining two edges of corresponding size provided in at least one flexible sheet, such that a weld is obtained in the side wall;

[0026] - The base material of the side wall is transparent and / or any part of the side wall is made of a transparent material.

[0027] - a side wall surrounding a target support extending inside the containment enclosure;

[0028] The side wall has a lower end of annular shape which is directly connected to the base part using an annular flange or frame which has been provided in the side wall before connection to the base part.

[0029] The side wall has a lower end comprised in a flexible sheet of plastic material, this lower end being attached at a sealing area of ​​annular shape to a peripheral connection part provided in the base portion.

[0030] After connecting the base portion to the side wall, a receiving portion is defined, wherein at least two connection ports are already provided in said receiving portion.

[0031] Each connection port may comprise a nozzle or similar fluid connector, which preferably projects outwards, for example relative to the circumference of the receiving portion.

[0032] - The method comprises forming at least one of a fluid inlet port and a fluid outlet port on the receiving portion.

[0033] - After connecting the base part to the side wall, the containing part is defined as having at least one fixing part / member protruding radially outwards from the peripheral edge of the base part, which fixing part allows stabilizing the bioreactor when it is fixed to a support or a holding and retaining structure / device.

[0034] The containing part is intended to be fastened airtightly to the covering part to form a sealed enclosure, said sealed enclosure being sterilizable; sterilization can be performed to sterilize the sealed enclosure; in a variant, sterilization of the containing part and the covering part is performed when these parts are not assembled together.

[0035] In particular, the sidewall and base portions define a single containment portion of the bioreactor for receiving a biopharmaceutical product (during tissue printing or cell culture). Optionally, the wall members of the bioreactor can each form / comprise a respective outer surface of the containment enclosure, which is the outer surface of the bioreactor in the absence of any additional bioreactor containment portion surrounding the enclosure.

[0036] The base part, the side walls and the top part may be assembled at respective joint edges to form an outer wall of the bioreactor, thereby obtaining an inner volume, preferably without any dividing walls within the inner volume.

[0037] Optionally, the top portion may be connected to or comprised in a piece involved in forming the annular sidewall.For example, the top portion may be a flap or extension of at least one flexible membrane that already forms the sidewall.

[0038] The base portion can be more rigid than the flexible portion of the bioreactor, which allows the adapter to be inserted into the lower compartment of the internal volume. In addition, the target support can be an integral part of the base portion, thereby forming a rigid portion. Alternatively, the base portion is formed as an at least partially flexible lower wall, which is optionally made of a flexible film. Stabilizing lugs or fixing members can be provided to retain the base portion (when the base portion is flexible or when the base portion includes a flexible zone).

[0039] According to one embodiment, the at least one sealing operation is performed before any connection of the adapter to the drive system (eg comprising the end arm), preferably before any connection between the adapter and a cartridge or reservoir containing biomaterial.

[0040] The bioreactor can be a single-use container, typically sterile, that is connected to the printing unit (using a connection piece that selectively engages an adapter, for example having a fastening area arranged outside the sealing cover). The adapter can protrude outwardly (for example axially, away from the base portion) relative to the outer surface of the sealing cover.

[0041] The cartridge or reservoir portion of the printing unit can be removably connected to the adapter to allow the biomaterial to flow in the needle containing the deposition end and over the central area of ​​the adapter. The needle is in fluid communication with the reservoir and extends below the reservoir. The adapter can extend completely below the reservoir portion (having a higher cross-section than the needle / deposition end) or, when the drive system is fixed to the cartridge / reservoir portion at an outer area of ​​the cartridge / reservoir portion beyond the adapter and the top portion, the adapter can surround the reservoir portion at an annular area.

[0042] In some options, the assembly is adapted to allow multiple cycles of bioprinting and cell culture. The bioreactor may comprise a transparent plastic material to facilitate inspection of the progress of the bioprocess. Optionally, the top portion is provided with an adapter forming a first adapter, and a second adapter is also coupled to a wall member, which may be the top portion. This allows the use of two needles or similar deposition tips to deposit different materials. In some options, collagen, another compound that is soluble upon hydrolysis, a water-soluble compound may be exemplary materials deposited via a needle or similar deposition tip associated with the second adapter. Combinations of such materials may be used.

[0043] In one embodiment, a common adapter can cover two apertures provided in the same sheet portion or similar flexible portion. With this configuration, the two deposition ends can be controlled by the same external drive system, which is selectively connected directly or indirectly to the outer connection portion of the adapter. In variations using one or two septum portions, the two apertures can correspond to the piercing areas. Such septum portions can optionally be provided in the same adapter with an annular periphery for connection to the sheet portion or similar flexible portion. Alternatively, the two septum portions can be contained in the same adapter, forming a single flange or similar annular fastening member.

[0044] Before the sealing operation, a storage part can be provided, allowing the depositing tip to be stored. When several materials must be deposited, there is a common needle suitable for depositing materials fed from different reservoir parts, or different printing needles (or similar depositing tips) are provided. When two separate ink cartridges are used to feed two separate needles, each connected to the top part, the two drive systems can optionally be connected to corresponding adapters after the sealing cover is achieved.

[0045] Prior to the sealing operation, the base portion may be formed into a rigid structure suitable for holding the printed tissue (obtained from bioprinting). Possibly, the target support is formed by this rigid structure extending parallel to and / or along the base portion, and this structure is rigidly fixed to the lowermost layer of the sealing cover using one or more fixing members.

[0046] For example, an additional layer of material that is different from the material of the base portion can be fixed to the inner surface of the base portion. This additional layer can be rigid to form a substrate, optionally in the form of a porous material (such as wool or a membrane), and welding can be used to fix this layer. The base portion can also be a support for a plastic wall or a similar additional layer printed (using 3D printing) or overmolded on the inner face of the base portion. This additional wall portion contained in the internal volume or forming the inner face of the sealing cover can be configured according to the tissue to be printed.

[0047] In some embodiments, the top portion is formed as an apex region of a dome-shaped flexible portion, wherein the dome-shaped portion (e.g., having a hemispherical shape) encompasses all or part of the sidewall. This shape is achieved in an expanded state of the flexible portion, wherein the lower annular end of the flexible portion is secured to the base portion. Thus, the sidewall can taper upward from the base portion, or at least from a sidewall region disposed adjacent to / above the base portion.

[0048] The top portion and / or the side walls may comprise a stretchable, flexible material, possibly a silicon material. Of course, the sheet material used to form the stretchable portion (including the side walls) may be flat in its initial configuration prior to the expansion step using the injection of an inert gas (typically from the bottom side of the containment enclosure).

[0049] In some options, a treatment (eg, laser treatment) may be provided to change the physical and / or chemical properties of the inner surface of the base portion. This treatment may be suitable for forming a rigid or flexible material of the base portion.

[0050] In a variant or in addition, the recess is formed as a printing area / target area as a cavity within the base part of the sealing cover; such a recess can be obtained by thermoforming of a thin plastic sheet or by manufacturing an injected or printed rigid plate (for example by 3D printing).

[0051] Depending on some options, integration of the adapter may involve one or more of the following details:

[0052] - The adapter is positioned away from the side wall.

[0053] - The method comprises: forming the top portion using at least one film (possibly plastic film) sheet or silicon material; and annularly sealing the annular outer edge of the top portion to the side wall region, preferably to the upper end of the side wall.

[0054] The annular outer edge may be cut and / or sized such that the deformable surface of the top portion is defined by the outer edge and the inner volume is variable in the upright configuration depending on the configuration of the top portion.

[0055] The adapter may be surrounded by a sheet material portion forming a deformed continuous annular surface.

[0056] - The surface bounded by the outer edge deforms without changing its area (deformation at constant surface area); this excess of surface allows the configuration of the top portion to be modified for following the movements of the adapter when driven from the outside by a drive system extending outside the internal volume.

[0057] The method comprises maintaining the upper end of the side wall in an annular configuration by means of a spacing member directly coupled to a flexible portion of the sealing cover surrounding the adapter, said flexible portion forming all or part of the top portion.

[0058] - the adapter is configured as a rigid connectable end portion or extension of a drive system arranged outside the bioreactor, whereby the flexible portion can be displaced and follow the movement of the adapter when bioprinting is performed and biomaterial is delivered onto a target support.

[0059] - The adapter is positioned to extend distally from a containing portion of the housing / bioreactor, said containing portion comprising a base portion and side walls.

[0060] The annular configuration of the upper end of the side wall is maintained by a rigid structure forming part of the side wall and / or by pulling two opposing parts of the side wall radially outwards (this arrangement allows a minimum gap to be maintained between the two opposing parts).

[0061] According to a particular feature, the membrane forming the sheet of cover, optionally adapted to surround the adapter in a continuous annular leak-proof contact, may have a thickness greater than or equal to 100 μm. For example, each membrane / sheet in the cover may have a thickness between 150 μm and 450 μm.

[0062] Alternatively, a thicker film or sheet can be used (at least in the unstretched state of the sheet). For example, a silicon sheet / film of about 1 or 2 mm can be used. When such a stretchable sheet is used to form the sidewalls and / or top portion, this thickness is defined before stretching. It may be possible to obtain a dome shape or a similar expanded sphere shape starting from a flat sheet. Alternatively, the dome shape can be obtained as a result of a preforming step, for example, using silicon material.

[0063] According to an option for forming a flexible sealing cover, four wall members can be sealed together, wherein two wall members are spaced apart gussets or bellows. Each of the membranes constituting the first wall member, the second wall member, the first gusset, and the second gusset, respectively, has a thickness locally along the weld seam that is not less than the average thickness of the membrane.

[0064] Corresponding films can each consist of at least three non-metallic plastic layers.In addition, the films can be transparent or translucent.

[0065] When the deformable surface is optionally formed away from the base portion, the deformable surface may be sized so that in the upright configuration the top portion is movable from an intermediate folded state, which is preferably a flat state, to:

[0066] an outwardly directed expanded state of increased internal volume, wherein the top portion is dome-shaped such that the adapter extends above the side walls in said upright configuration, and

[0067] - an inwardly curved state of reduced internal volume, wherein the top portion forms a bioreactor recess for the passage of a drive system intended to be connected to the adapter while the adapter is surrounded by side walls.

[0068] The deformability of the surface at the top portion may be due to a surface area that is higher than a corresponding planar surface area / segment defined by the upper annular end of the sidewall in the upright configuration.

[0069] The method may comprise changing the capacity of the internal volume due to movement of the top portion towards a position (possibly a lower position) in which the top portion telescopes and is adapted to be pressed downwardly by the adapter when the adapter is driven from the outside by the drive system. The reduction in the capacity of the cover may be actuated by moving the drive system after the connecting step so that the adapter is movable (with the same movement) as the end portion / extension of the drive system.

[0070] In some options, prior to and / or during bioprinting, the method includes exhausting gas contained in the interior volume, possibly via an inlet port and / or an outlet port associated with the hood.

[0071] In certain embodiments, with the top portion telescoping, gas venting and displacement of the adapter toward the lower position may occur simultaneously.

[0072] Regardless of the manner in which the internal volume is modified, the telescoping of the top portion can preferably be achieved while the bioreactor is still in an upright configuration. Due to the flexibility of the plastic material forming the top portion, this allows the adapter to be moved, preferably by driving a robotic arm arranged outside the bioreactor.

[0073] In some options, the method includes changing a level of pressurization in the interior volume to move the top portion between the telescoping state and the expanded state.

[0074] In some options, the outer edge may be polygonal. The method may include using a cover portion to seal the outer polygonal edge to the side wall, for example in an annular shape at an upper end, the upper end (of annular shape) preferably also being polygonal to match the shape and size of the polygonal outer edge.

[0075] The surface of the top portion defined by the polygonal outer edge can be sized in a sizing step (e.g., using a cutting step) so that this top portion surface is higher than the corresponding area defined by the upper end of the side wall (the cross-sectional area defined by the annular upper end).

[0076] According to one option, the forming of the top portion (using at least one plastic film sheet) comprises sizing and / or cutting a rounded peripheral outer edge that is preferably continuously rounded and / or substantially circular in shape. The upper end may also be rounded to match the shape and size of the outer edge.

[0077] In some options, the method includes attaching a first elastic return member to the sidewall and / or top portion, the first elastic return member:

[0078] - is involved in maintaining the annular configuration of the upper annular end, and

[0079] - fixed to an external fixing structure provided around the bioreactor.

[0080] The adapter can be displaced in one of the at least two transverse directions to move away from the first elastic return member until it reaches a deflected position in which the first elastic return member extends to modify the annular configuration and locally narrow the periphery of the upper end of the side wall. In some variants, no elastic return member is provided on the outside of the cover, and deformation is allowed in the intermediate side wall area between the fastening areas provided for connection to the external structure.

[0081] According to particular features, the method comprises providing at least one annular hinge in the top portion or along the annular upper end of the side wall for hingedly connecting the top portion.

[0082] According to one option, a rigid structure or a structure provided with a biasing member is provided around the sidewall to maintain the sidewall so that it extends distally around the target support. Accordingly, the bioreactor is maintained in an upright configuration, and the structure with the associated sidewall linkage prevents contact between the target support and the sidewall. The adapter can move within the region surrounded by the rigid structure (within the interior volume).

[0083] According to an embodiment, the method includes one or more of the following features:

[0084] - Performing sealing / fastening of several flexible panels to each other such that when the bioreactor is in an upright configuration they form at least a top portion and side walls.

[0085] - In the base portion, at least a portion thereof is made flexible and an outer marginal portion of the base portion is provided with radially protruding outer extensions adapted to hold the base portion substantially planar or flat along the base plane.

[0086] In a variation, the base portion may be a rigid plate.

[0087] Depending on the option of performing a sealing operation to define an interior volume, the method may include:

[0088] Prior to the sealing operation interconnecting the base part and the top part, a receiving surface is made and / or mounted on the inner face of the base part delimiting the internal volume so as to define all or part of the target support.

[0089] - Provide surface treatment at the target support.

[0090] - optionally using the peripheral edge of the base portion surrounding the receiving surface for fastening together with the complementary portion in order to obtain a sealed enclosure.

[0091] The complementary part comprises the top part and has a flexible material to delimit the inner volume from above.

[0092] - The complementary parts are pre-assembled before being connected to the base part.

[0093] When the complementary parts are preassembled (forming the cover part), the adapter can be coupled to the top part and arranged to protrude outwardly relative to the outer surface of the top part before being fastened to the base part. The protruding portion of the adapter can include an outer connecting portion, optionally forming a male portion, which can be connected to a female portion provided in the drive system, and vice versa (female portion at the outer connecting portion of the adapter).

[0094] According to an embodiment, the adapter, preferably the needle holder, is permanently connected to the base material of the flexible plastic sheet forming all or part of the top part of the bioreactor.

[0095] The coupling of the adapter may comprise: clamping the annular edge of the top portion between two rigid rings of the adapter; or welding the annular edge of the top portion to the rigid peripheral portion of the adapter.

[0096] The adapter and / or the printing platform forming the target support can be obtained from at least one printable material by means of three-dimensional (3D) printing technology, preferably the printable material and / or the base material being heated during printing so that the printed adapter (respectively, the printing platform) is connected to the base material.

[0097] When all or part of the side wall is flexible, the side wall can be obtained by welding at least two sheets, at least one of which is made of a plastic film, preferably a multilayer film. Possibly, this at least one sheet is provided with a fluid inlet port and / or a fluid outlet port.

[0098] In some options, one or more gusseted sections are provided to form all or part of the side wall, each of the one or more gusseted sections being welded or leak-tightly fastened to the base section, preferably welded to the base section in a folded state. The base section may also be fully or partially flexible.

[0099] In some embodiments, the method comprises a spacing step for spacing apart two opposing portions of the side wall having an annular shape in the upright configuration of the bioreactor by a spacing member, preferably in the form of a reinforcing member or a retaining member. The spacing member can prevent the two opposing portions of the side wall from moving inwardly toward each other.

[0100] In some options, fluid inlet ports and / or fluid outlet ports are provided in such opposing portions of the sidewalls.

[0101] Optionally, the method comprises reinforcing the annular joint between the side wall and the top portion, said side wall and top portion being made of a flexible material, preferably of the same multilayer film material.

[0102] Optionally, all or part of the top portion is made of a rigid material and is configured to be movable due to the flexibility of the side walls. When this arrangement is produced to form a sealed enclosure, the method can further include a spacing step for spacing the top portion from the base portion, for example using a return member (possibly with a tensioning effect), a biasing member, or a similar retainer that is made separate from the side walls. In some embodiments, the return member / retainer is coupled to the top portion and is configured to apply an upward biasing force, whereby the top portion is spaced a predetermined distance from the base portion by default. After bioprinting, such a return member can be used to ensure that the side walls of the bioreactor do not remain in a telescoping state.

[0103] When the top portion is made flexible (preferably made of one or more flexible films), the adapter is rendered displaceable relative to the base portion. The adapter can be displaced between the following positions:

[0104] a first position remote from the base portion, in which the adapter is surrounded by a flexible portion of the top portion which tapers upwards or is dome-shaped and protrudes upwards,

[0105] and a plurality of second positions in which the adapter is extended proximally relative to the base portion with the flexible portion folded downwardly (forming an upwardly open outer recess in the cover).

[0106] In a particular embodiment, the method comprises varying the level of pressurization in the internal volume so as to move the top portion between a telescoping state and an expanded state, the expanded state allowing movement of the adapter over the target support, preferably by actuating a robotic arm arranged outside the bioreactor, due to the flexibility of the plastic material forming the top portion.

[0107] Pressure can be applied from above on the top portion in the expanded state to bring the adapter and deposition tip into position for bioprinting.

[0108] After bioprinting, the deposition tip may be allowed to return to the telescoping state after being displaced away from the target support and / or removed. The internal volume may thus be reduced. The telescoping state may simply mean that the top portion is brought closer to the base portion, and does not necessarily mean complete telescoping of the flexible wall portion.

[0109] According to another particular embodiment, the method comprises a connecting step for fastening the side walls at respective opposite axial ends, each axial end comprising a rigid disk, at least one rigid ring or a similar rigid component. The fastening of the side walls may comprise:

[0110] - a first connecting step for connecting the side wall of the rigid part comprising or having a substantially annular shape to the base part; and

[0111] - A second connecting step for connecting the side walls to the top part made of at least one flexible plastic sheet.

[0112] Optionally, the second connection may be obtained before or after the first connection, or such connections may be obtained simultaneously.

[0113] When the base portion is rigid, at least a portion of the side wall may also be rigid so that the housing portion of the cover is substantially / completely rigid. In a variant, the base portion is at least partially flexible, for example made of plastic film, and is welded to the annular lower end of the rigid side wall.

[0114] The method optionally includes severing an uppermost portion of the top portion after bioprinting and after welding or clamping two opposing portions of the top portion at the welding area so as to reduce the internal volume, the severed uppermost portion extending completely above the welding area.

[0115] According to a particular embodiment using a rigid coupling at or adjacent the top portion, the method comprises:

[0116] - fastening in an airtight manner a rigid coupling of annular shape to a sheet of flexible material, at a position opposite the base portion, said sheet of flexible material being suitable for delimiting the internal volume from above and optionally for forming one or more flaps belonging to the side wall (the side wall being made of a plurality of flexible sheets);

[0117] - sealing a sheet assembly to the rigid coupler to cover the opening of the rigid coupler, the sheet assembly having an outer edge or flange fastened directly to the rigid coupler;

[0118] - The top covering surface of the sheet assembly extending above the rigid connector is sized so that it is suitable for folding inwardly and outwardly relative to the rigid connector, whereby the adapter can be moved in the at least two lateral directions under the rigid connector without the need for any driving part to be accommodated in the internal volume.

[0119] The top portion may be formed from a flexible sheet defining an opening that mates with the rigid coupler (and corresponding opening) and a sheet assembly that is made separate from the flexible sheet.

[0120] Optionally, the method comprises connecting a moving part of the stirring device / a stirring internal part to the containing portion such that the moving part extends inside the internal volume after forming the sealed enclosure.

[0121] It will be appreciated that the single internal volume may be defined by a sealed enclosure.When a stirring / moving member is provided, the movement of this member may directly participate in homogenising the fluid circulating at / along the target support (and associated printing structure).

[0122] When bioprinting is performed, in the upright configuration of the bioreactor, the top portion can conform to an upwardly projecting protrusion in its expanded state, potentially having a 3D shape that forms an increased volume of the internal volume. Optionally, the top portion can be domed due to the remaining area compared to the upper open area formed at the upper annular end of the sidewall. This upper annular end can remain substantially fixed during bioprinting.

[0123] Of course, due to the flexibility, the top portion can have many different configurations, possibly with an assembly between heterogeneous parts.

[0124] During assembly, the top portion can be provided as a folded or gusseted portion, or similar structure, wherein excess membrane material is used to achieve the folding in an upright configuration of the bioreactor (e.g., with the side walls generally expanded) at least when the top portion extends generally parallel to the base portion (flat portion).

[0125] Indeed, in this configuration the expanded length of the top portion measured between its two opposite ends (with the adapter provided between these two ends) may be significantly higher than the largest transverse characteristic dimension (possibly a diameter) of the annular upper end of the sidewall.

[0126] The method includes securing the adapter to the connection of the printer unit without inserting any articulated, movable components of the drive system of the printer unit into the housing. The method may also include locking the connection, possibly allowing an unlocking action to disengage the drive system without displacing the biomaterial deposition end relative to the adapter.

[0127] This connection may involve an outer connection portion of the adapter that extends outside the housing.

[0128] In some embodiments, the sidewalls and top portion can be formed as a single piece of a transparent (possibly silicone) flexible plastic material. This piece can be provided with a dome shape. In a variation, the sidewalls and top portion are made from flexible assembled sheet material portions to form a dome-shaped assembly. The flexible portion of the cover can consist of or include the dome-shaped assembly.

[0129] In some options, the cover comprises a body portion and a sheet assembly provided with a deposition end, the sheet assembly being connected to the side wall via a shoulder portion of the cover.

[0130] According to another aspect, an embodiment provides a bioreactor for continuously bioprinting a biomaterial and allowing a maturation and / or cell culture phase based on the printed biomaterial (tissue), the bioreactor comprising:

[0131] a sealing cap comprising a receiving portion and a covering portion, said receiving portion being provided with a base portion intended to form all or part of a lower end of the sealing cap, said covering portion preferably comprising a top portion to which a biomaterial deposition end is fixed via an interface so as to protrude inwardly in an internal volume of the bioreactor, said internal volume being delimited by said receiving portion and said covering portion, said base portion being provided with a target support;

[0132] a flexible portion made of one or more plastic sheets adapted to follow the movement of said interface relative to said base portion in order to locally reduce the internal volume of the enclosure;

[0133] - an upper end of the cover, which is provided with an aperture and comprises a plug member adapted to plug the aperture and move relative to the base portion, the plug member being comprised in the cover portion to form all or part of the interface; and

[0134] A plug component adapted to carry a needle or nozzle forming a deposition end is integrated in the sealing cover to (functionally) interact with the flexible portion such that during the bioprinting phase, the plug component can move together with the deposition end in at least two lateral directions within the internal volume to deposit the biological material on the target support.

[0135] In some options, a silicone material or an elastomeric material may be used, preferably transparent. More generally, the flexible material has a Shore hardness of between 0.05 or 0.1 and 5, preferably transparent.

[0136] In some options, the sealing cover is provided with another (second) flexible portion that can serve as a pressure compensating member. This pressure compensating member is configured to deform and expand along with the sealing cover, for example, in response to a simultaneous downward displacement of the plug member and the (first) flexible portion relative to the target support from a distal position to a proximal position.

[0137] The bioreactor may comprise one or two connection ports to form a fluid inlet port and / or a fluid outlet port adapted to be opened after bioprinting for direct fluid communication between the external circuit and the internal volume, such that the bioreactor may be used for a culture and / or maturation phase of the printed material / tissue within the internal volume after bioprinting.

[0138] After bioprinting, the bioreactor with the sealed cover is adapted to remain in a closed state, possibly without any uncontrolled ingress of fluids. Of course, at least one connection port is used, for example, in the phase of introducing culture medium (to be circulated and / or mixed) into the bioreactor, this phase being controlled by an operator, possibly using a control unit associated with sensors (temperature sensors, gas sensors, pH sensors are exemplary sensors that can be embedded in the bioreactor).

[0139] The adapter can define an annular sealing contact area having a circular shape. In some options, the adapter extends distally from a receiving portion of the bioreactor comprising the base portion and the sidewalls due to a stable configuration of the sidewalls and / or at least one spacer contained in or connected to the sealing cover, whereby a biomaterial deposition tip inserted through the aperture when secured to the adapter can move while being prevented from laterally contacting the receiving portion of the sealing cover.

[0140] The stable configuration may be due to a rigid material used to form and / or define the side wall.The adapter may be centrally arranged in a cover portion connected to the side wall.

[0141] In some embodiments, the spacing member coupled to the annular peripheral area of ​​the top portion is configured to prevent the peripheral area of ​​the top portion (4b) from moving toward the base portion. Possibly, the spacing member is movable relative to two directions parallel to the bottom formed by the base portion, the spacing member being part of an external structure for positioning, holding and / or retaining the top portion.

[0142] In some embodiments, due to the flexibility of the top portion, the adapter can move in three orthogonal directions, possibly without modifying the shape and / or perimeter of the side walls (which are reinforced, at least partially made of a rigid material, or held in place by an external retention system).

[0143] In some embodiments, the bioreactor includes a gas injection unit configured to inject gas (e.g., an inert gas) into the interior volume to achieve expansion of at least one flexible sheet at a position relative to the base portion and to maintain the annular peripheral region of the top portion above the annular side wall while allowing the needle / deposition tip or similar bioprint head to be displaced in three directions.

[0144] The bioreactor is preferably an assembly obtained by the above method, which assembly comprises:

[0145] a receiving portion (housing portion) defining a longitudinal axis and having a longitudinal opening therethrough, said longitudinal opening forming an aperture, said receiving portion comprising a base portion and side walls;

[0146] an adapter coupled to the cover distally from the receiving portion, said adapter being adapted to fit the deposition tip, said adapter extending distally from the receiving portion and allowing the deposition tip to be inserted through the longitudinal opening; and

[0147] - a sealing member mounted within the longitudinal opening of the receiving portion, the sealing member being configured to receive the deposition tip therethrough in a generally sealed relationship therewith, the adapter being adapted to move together with the flexible top portion of the hood in at least a radial direction relative to the longitudinal axis, the sealing member maintaining a generally sealed relationship between the receiving portion and the movable portion including the flexible top portion.

[0148] The top portion may be composed of a flexible plastic material, have a given inner surface for defining the inner volume, and deform while the inner surface remains constant.

[0149] A bioreactor is a bioprocessing bag provided with several flexible panels / wall parts which are sealed to each other such that when the bioreactor is in an upright configuration they form at least the top and the side surfaces of the bag (side surfaces of the side walls).

[0150] At least one of the flexible panels / wall members may form a top portion and when the bioreactor is deformed, in the upright configuration, the top portion will define a downwardly tapering projection of the bioreactor carrying the deposition end with an upper surface of the bag having a depression due to the adapter moving and pressing the top portion downwardly.

[0151] Optionally, the recess is open only upwards.

[0152] In a variant, the top portion is deformable so that the recess opens both upwards and towards the side wall.

[0153] In some embodiments, the top portion or another portion of the sealing enclosure is provided with a vent allowing compensation for volume changes within the sealing enclosure.

[0154] The vent may lead to / communicate with another deformable container (usually also sealed), the volume of which may increase when the internal volume of the bioreactor decreases, and vice versa.

[0155] The bag / housing of the bioreactor may be obtained by sealing together flexible panels of polygonal shape, such as rectangular or hexagonal shape.

[0156] According to an embodiment, the base portion extends in a planar manner and may be provided with / attached to retaining protrusion members which protrude radially outwards and are configured to retain a given planar position of the base portion, the given planar position being a fixed position.

[0157] According to another aspect, a bioprinting system is provided, comprising a bioreactor and a printer unit, wherein the printer unit comprises:

[0158] - a reservoir portion, which is filled with the original contents of the biological material,

[0159] - a feed assembly, and

[0160] a connection portion for coupling the printer unit to the bioreactor via an adapter, said adapter carrying a needle provided with a biomaterial deposition end, such that said needle is a dispensing end portion in the circuit of the printer unit,

[0161] Wherein separating the connecting portion from the bioreactor separates the needle and / or adapter from the printer unit and enables the internal volume (V) to remain gas-tightly closed.

[0162] In one embodiment, the bioprinting system comprises a drive system for providing relative displacement between the bioprint head and the target support, wherein the drive system and the reservoir of the feed assembly / cartridge extend completely outside the bioreactor.

[0163] The drive system may comprise or consist of a robotic arm or an electromechanical actuator.

[0164] Optionally, the system includes a support structure for holding the side wall and / or the upper annular end of the side wall fixed or maintaining a substantially constant annular shape.

[0165] Furthermore, the system may comprise a print head mount or deposition head coupled to an XYZ positioning arrangement (here forming part of the drive system) such that the print head mount directly drives displacement of the deposition head in three orthogonal directions. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] The figures of the drawings will now be briefly described.

[0167] Figure 1 is a schematic diagram of the steps involved in producing a sealed container according to an embodiment of the present invention.

[0168] Figure 2 is used, for example, via Figure 1 A perspective view of the steps to obtain an exemplary printing system for a bioreactor.

[0169] Figure 3 is a diagram illustrating the operation of forming and then using a bioreactor that is particularly suitable for transferring living biological material within a sealed enclosure without inserting a drive system into its interior volume.

[0170] Figure 4A and Figure 4B is a schematic longitudinal sectional view showing the needle assembly fastened to the receiving portion of the bioreactor in an expanded configuration and a retracted configuration, respectively, the expanded configuration being adapted for the adapter and accessible thanks to e.g. Figure 2 The connection between the cartridges is shown being moved by a drive system, the retracted configuration being suitable for bioprinting once the outer upper end of the adapter has been secured to the drive system.

[0171] Figure 4C A sealing cover made of a flexible sheet material is shown, wherein retaining and holding structures allow the flexible containing portion of the cover to be kept in an expanded state, wherein a wide access opening communicates with an upper portion adapted to mount a needle adapter.

[0172] Figure 5A and 5B Two further embodiments of a bioreactor are shown having an adapter configured as a needle holder or a septum, using different means to allow relative movement of the adapter with respect to a base part, here a fixed base part.

[0173] Figure 6A non-limiting way of obtaining a full or partial sealing enclosure using four complementary membrane portions adapted for welding is depicted.

[0174] Figure 7A Schematically illustrated are assembly steps with sealing operations to obtain a sealing enclosure with another movable top part, here with flap portions or curved sides extending downwards to form sub-portions of the side walls.

[0175] Figure 7B A cut sheet of plastic film is shown which is suitable for forming a top portion before coupling with an adapter, said top portion here having a polygonal shape in a flat configuration, this top portion sheet portion being suitable for use in Figure 7A Type of assembly shown.

[0176] Figure 8A Show for example Figure 1 A needle assembly is shown in which the deposition end has been secured to an adapter surrounded by a flexible material tapered or dome shaped sheet portion, the deposition end being protected by a sheath and a sheet portion provided with an end flange.

[0177] Figure 8B shows a 3D bag structure, which can be used, for example, Figure 6 An assembly of or similar is obtained from several flexible sheets, wherein a top coupler of annular shape allows fastening a movable needle assembly at a position opposite to the base portion.

[0178] Figure 8C A needle assembly according to another embodiment is shown, which is obtained from several flexible sheets with the formation of the gussets.

[0179] Figure 8D Another needle assembly is shown which, when integrated into a sealing cover, forms an annular weld separating the side wall from the top portion of the bioreactor.

[0180] Figure 9 The connection means are shown disposed outside the internal volume and comprise an external connection portion of the adapter and a corresponding connection portion formed on the printer cartridge.

[0181] Figure 10 is a perspective view showing the printing system before the adapter and the top portion of the sealing cover are lowered to a lower level, wherein a deposition tip held via the adapter can deposit biological material on a target support.

[0182] Figure 11A and 11B A bioreactor and a displaceable part of a bioreactor according to a particular embodiment are schematically shown, respectively, the bioreactor being provided with two deposition ends which can be associated to respective materials.

[0183] Figure 12 A bioreactor according to another particular embodiment is schematically shown, said bioreactor being provided with two deposition ends which can be associated with respective materials.

[0184] Figure 13 Exemplary steps are shown for making the top part of the mask movable using a sheet of flexible material conforming to a dome, wherein an adapter (here not yet associated with a needle) is arranged at the displaceable apex of the dome. DETAILED DESCRIPTION

[0185] The following provides detailed descriptions of several embodiments of the present invention with reference to examples and accompanying drawings.

[0186] In the various figures, the same reference numerals are used to denote the same or similar elements. For the sake of clarity, some dimensions may be exaggerated. For example, the width or height of the containment portion RC of some of the illustrated bioreactors may be exaggerated to improve the illustration.

[0187] See Figure 1 , shows exemplary assembly steps for forming a single-use sealing cover suitable for sterilization. A flexible portion made of one or more plastic film sheets 2 can be used to make this cover to allow the internal volume V to vary. The internal volume V can optionally correspond to the following juxtapositions:

[0188] a first compartment C1 defining a lower volume intended to be filled with fluid during the biological treatment phase,

[0189] - a second compartment C2 forming an upper volume having an annular space surrounding the printed portion extending in the internal volume V, said second compartment C2 being in direct communication with said first compartment C1 .

[0190] In a variant, the internal volume V may not be divided. For example, the internal volume V may be as follows Figure 5B As shown, the side wall fixed to the base portion 4a is part of the dome-shaped wall DW. In other words, the receiving portion RC can be laterally delimited by upwardly tapering side walls, which may have a gradually curved / rounded profile. Assembly can be facilitated by using a simple sheet assembly 2 to form both the side walls and the upper end of the sealing cover E.

[0191] The containment enclosure E can have a relatively small lower volume, for example, less than 300 or 500 mL, and may have an average or maximum height of less than or equal to 100 mm. This containment enclosure E is intended to be used during the bioprinting phase and then during the maturation phase. The containment portion RC can be sized to accommodate less than 200 mL of culture medium, for example, about 100 mL or less. The containment portion RC can have a capacity of, for example, as low as 10 or 25 mL.

[0192] like Figure 2 As shown, the sealing cover E can be connected at its top to the drive system DS of the printer unit 3, which has a feed part filled with biological material. The sealing cover E is provided with an adapter 5, which is a connection part preassembled with the wall part of the bioreactor 4 and is used to attach to the cartridge 6, 106 containing the biological material BM (e.g., living cells) (see also Figure 9-12 ) or a similar reservoir portion. Such materials may be viscous, at least in the reservoir. Collagen or hydrogel may also be contained in this reservoir together with or separately from the cells to be introduced. The deposition end 8, 108 with an open free end is used to release a filamentous stream or similar stream (e.g., a continuous stream or droplets) of biomaterial.

[0193] Regardless of the design of the cartridge 6, 106 and the manner in which the adapter 5 is formed on the sealing cover E, the deposition end 8 can have a section that is calibrated to be able to deliver a continuous flow of bio-ink. A tapered profile can be provided. The dimensional parameters at the free end of the tapered deposition end 8 can be in the following ranges, for example: 25-999 μm diameter (sub-millimeter size) or having a diameter of 1 mm, possibly less than 3 mm in diameter. Other parameters can vary. The biomaterial can exhibit a low viscosity (<10 mPa s). The cell density in the material BM can also be low (e.g., <106 cells ml-1). In some variations, biomaterial deposition can also be performed point by point, and / or a microvalve can be used just above the nozzle / deposition end.

[0194] The drive system DS can be an articulated arm or any suitable electromechanical assembly that ensures relative displacement of the print transfer area with respect to the target. In some options, when constructing a structure for cell growth (e.g., a cube-shaped structure) or perhaps when bioprinting tissue T, two drive systems DS can be used to guide the movement of the two material deposition ends 8, 108. Of course, as Figure 3 The step 44 shown is not necessarily tissue production and may depend on the material deposited on the target support 20. Referring to Figures 11-12, two deposition tips 8, 108 are shown, which can be received in the first compartment C1 at the same time or at different times. Figure 11 shows a configuration with two adapters to allow the deposition tips 8, 108 to be driven by corresponding drive systems DS. In this latter case, only one deposition tip 8 or 108 can be displaced close to the target area 20.

[0195] Each adapter 5 , 105 may be surrounded by an annular edge E1 of the sheet portion, thereby defining an aperture O4 when the adapter is coupled to the membrane sheet away from the outside edge of the sheet.

[0196] The bioreactor 4 has a base portion 4a on which the target support 20 can be formed. Positioning reliefs 4h can be provided at the base portion 4a, for example to interact with complementary reliefs of the support portion that contacts the hood from below. The base portion 4a can be polygonal in shape, preferably rectangular. Alternatively, the base portion can be continuously rounded, for example having a circular shape. In the upright configuration of the bioreactor 4, the internal volume V is laterally delimited by the side walls SW. In the upright configuration of the bioreactor 4, this internal volume V extends / delimited between the base portion 4a and the top portion 4b. The aperture O4 of the top portion 4b is plugged by a sheet or assembly belonging to the adapter 5. When (at a specific step 40) the adapter 5 is connected to the sheet portion 2 or to a rigid upper plate / component arranged opposite the target support 20, an annular seal is obtained to prevent gas from passing around the adapter 5. Step 40 can be performed before another attachment step to define the internal volume V.

[0197] In some options, several adapters 5, 105 are typically arranged at the top portion 4b, each in a movable section. FIG11 shows a top portion 4b comprising two flexible sections that can move in opposite directions. Reinforcements may be provided around these flexible sections, or the top portion 4b may be provided with a flange 24 or frame, thereby defining a top access path for communicating with compartment C1.

[0198] In addition, the adapter 5 may include at least one internal channel that is intended to be filled with, for example, a material that flows downward. A needle or similar deposition tip 8 may have the adapter 5 extending or inserted therethrough, with a rigid fastener preventing relative movement of the adapter relative to the deposition tip 8. The adapter 5, 105 may be a rigid plug component that seals the corresponding aperture O4 after a heat welding operation or similar welding. The adapter 5, 105 may be a tri-clamp adapter that is optionally adapted to clamp a needle or similar printhead, thereby forming the deposition tip 8, 108. The adapter 5, 105 may be a Luer lock adapter that allows the deposition tip 8 to be inserted into the internal volume V.

[0199] The adapter 5, 105 may be provided with any suitable external connection portion to allow feeding of the biomaterial(s) to the deposition end 8. In some options, the adapter 5 may be a septum member having a channel formed upon piercing the septum S50 (see Figures 5A-5B ). In addition, the adapter 5 may be provided with a sealing membrane SM (see Figure 1 or Figure 9 ) of the sterile connector, the sealing membrane is, for example, a breathable membrane or an airtight membrane. This membrane SM serves as a sterile barrier, which covers the open end of the outer connection part FC1 of the adapter 5. Figure 8A and 9As shown in the exemplary adapter 5 of , the sealing membrane SM may be provided with a disc-shaped covering portion 5a (covering the upper end of the adapter 5) and a flexible pulling portion or similar tab 5b having a base connected to the covering portion 5a.

[0200] The side walls SW may be able to form a self-supporting structure or may be coupled to the holding structure HR. Figure 1 A rigid (or at least partially rigid) containing portion RC is shown comprising a side wall SW of generally annular shape, but the side wall may alternatively be made of a flexible plastic material having an inner face adapted for contact with biopharmaceutical fluids. Figure 4A An example is shown where the receiving portion RC is flexible (possibly with the exception of the base portion 4a). In some options, a semi-rigid or rigid component, possibly a clamping component 54 or an outer fastening ring or flange, may be provided at a fastening region RF between the flexible sheet portion 2 forming the top portion 4b and the annular upper end of the receiving portion RC, which is intended to be filled with a biopharmaceutical fluid after the bioprinting step 44. Welding may be performed to ensure airtightness at this fastening region RF.

[0201] A rigid or semi-rigid collar can be provided as a seal, for example, by welding, between the two corresponding annular ends of the flexible sheet material of the side wall SW and the flexible sheet material of the top portion 4b. This collar can be rigid or semi-rigid, forming a coupling already mounted on one of the top portion 4b and the side wall SW. Before being connected to form the annular fastening region RF, this coupling is provided with an associated annular seal on the flexible sheet material at its peripheral region. Furthermore, the adapter 5 is already coupled to the top portion 4b.

[0202] More generally, the bioreactor 4 comprises a top portion 4b opposite a base portion 4a, said top portion being provided with an adapter 5 which carries or is traversed by a tool for forming a printing transfer zone. Such a tool may be a needle, a nozzle or a similar deposition tip for depositing biomaterial in a bioprinting stage 44. Figure 3 As shown, this bioprinting stage 44 can be performed after a preparation stage (40, 41, 42) in which the bioreactor 4 is assembled (usually pre-assembled and usually after a connection step 43) so that the adapter 5 is integral with / rigidly fixed to the moving part of the printer unit 3. This moving part may contain a reservoir part or an ink cartridge 6. The bioreactor 4 can also be sterilized in a sterilization step 42a.

[0203] A sterilization step 42a may occur before the bioprinting stage 44. In some embodiments, this sterilization step 42a may be performed after the connection step 43. Sterilization is important to allow bioprinting and subsequent culturing in the same chamber (in the interior volume V) in an aseptic manner. Possibly, the sterilization step 42a may be performed when the parts are assembled at the connection step 43, or just before this connection is performed.

[0204] One or more sealing operations to achieve a sealed enclosure E can be performed prior to any connection between the adapter 5 and the cartridge 6 or reservoir containing the biomaterial BM. In this sealed state, the initially created bioreactor 4 can initially conform to a telescoping or substantially flattened state of at least the top portion 4b, and, when the sidewalls SW are flexible, folded to limit the height. In some variations, the enclosure E is already created in an expanded state, possibly with rigid components or entirely rigid sidewalls maintaining the axial spacing between the base portion 4a and the top portion 4b.

[0205] Regardless of how the housing E is produced / assembled, the bioreactor 4 is partially flexible to define the interior volume V. Specifically, the top portion 4b is flexible and / or laterally displaceable. As the sidewalls SW extend about the longitudinal axis A (where the bioreactor 4 extends upward in a stable configuration), the top portion 4b is adapted to move laterally and radially displace the aperture O4 in various directions. While some options provide, for example, for the aperture O4 to be spanned by the longitudinal axis A in the upwardly expanded state of the top portion 4b, this aperture O4 may also be slightly displaced to initially be adjacent to the outer edge E2 of the top portion 4b.

[0206] Considering the longitudinal axis A' of the side wall SW, the top portion 4b can be provided with an adapter 5 at the aperture O4, coinciding or not coinciding with this axis A'. When displaced in the transverse direction X or Y, the initially centered adapter 5 can be displaced transversely in any radial direction. Figure 4B The position of the adapter 5 when depositing the bio-ink / material is reflected, wherein the adapter longitudinal axis A is significantly displaced compared to the longitudinal axis A' (here the central axis) of the side wall SW.

[0207] The target support 20 constituting the printing platform (e.g., substantially planar) may extend transversely relative to a longitudinal axis A, which is typically a vertical axis. After bioprinting, an adapter storage position may be reached, which is:

[0208] an uppermost position of the adapter 5 or a similar elevated position such that the deposition end 8 situated in the volume V remains away from the target support 20 ,

[0209] - or laterally displaced position.

[0210] The connection to the external holder may be used to prevent the adapter 5 from moving downwards towards the target support 20 .

[0211] When the position of the adapter 5, 105 is laterally displaced, a needle storage compartment C3 can be provided in the internal volume V. When the needle is stored in this compartment C3, which can be provided with a bottom, purging of the needle can be performed. A sheath 28, a housing or an annular retainer can be provided on the inner side of the sealing cover E having a retaining portion 9. The retaining portion 9 can be an integral part of the sealing cover, made of the same material as used in the wall part of the containing portion RC. In an exemplary embodiment, the retaining portion 9 includes an upper abutment edge for contacting the bottom surface of the adapter 5. A suitable passage section is defined by the upper retaining portion 9, allowing a needle or similar deposition end 8 to pass from above until inserted and retained to prevent accidental displacement of the adapter, wherein this end 8 is in a central area of ​​the internal volume, just above the target support 20 for bioprinting.

[0212] In some options, the deposition tip 8, 108 can be coupled to a hose, thereby allowing for purging of biomaterial or the like (which may include cells, extracellular matrix (ECM) components, growth factors, and other biomaterials) prior to the bioprinting stage 44. A calibration step 43b can be performed after forming the sealing cap E (and after associating the adapter and deposition tip with the printer unit 3), wherein a quantity of biomaterial not used for bioprinting on the target support 20 is retrieved. A collector can be embedded within the bioreactor 4. For example, the sheath 28 can include / form such a collector for collecting a quantity of material delivered from the needle, hose, or similar deposition tip 8 during the calibration step 43b.

[0213] In addition, in some embodiments, the flow rate of the flowable biomaterial / bio-ink can be adjusted. A section of the nozzle, end hose, or similar deposition end 8, 108 can be adapted to a given flow rate range or a given flow rate value. Optionally, the deposition end 8, 108 can be adapted to continuously deposit material according to a grid design, for example, having several loops, without interrupting the flow. Thus, a continuous filament can be deposited. In some options, an overlay grid of such material can be printed, for example, to promote cell colonization of the printed matrix using a bottom-up approach. The flow rate is calibrated at calibration step 43b so that the deposition end 8 can deliver a filament with a constant / substantially constant cross-section.

[0214] For example, the inner diameter of the deposition tip 8, 108 is less than or equal to 3 mm, such as less than or equal to 0.8 mm, and greater than or equal to 25 μm. The cross-section at this deposition tip 8, 108 for the flow of the flowable material to be dispensed during the bioprinting phase 44 may be between 200 and 800 μm. In addition or independently thereof, the cross-section of the needle or similar hollow deposition tip 8, 108 measured inside the bioreactor 4 may be much smaller (at least 10 smaller) than the cross-section of any port P4, P4' of the bioreactor 4.

[0215] When the adapter is a rigid portion surrounded by a flexible sheet material, the adapter may include an annular ring or flange to which the edge E1 is secured. This flange may have a diameter of less than 30 or 40 mm, such that the flexible material of the sheet portion 2 or 2' is spaced less than 20 mm from the longitudinal axis A of the adapter 5, 105. The sidewall SW may be maintained at a distance of at least 20 or 30 mm; perhaps at least 50 mm from the longitudinal axis A, due to the portion of the sidewall SW being maintained or made rigid. Thus, at a specific step 44 prior to bioprinting, possibly after the sealing operation at step 41, the spacing is created when the sidewall SW is substantially flexible.

[0216] The adapter 5, 105 can be designed with a leak-proof interaction component that is arranged to define at least one continuous annular sealing contact. Such a leak-proof interaction component can be provided to accommodate / adapt the deposition end 8, 108. Typically, in some options, the leak-proof interaction component can be a perforable cap or a leak-proof seal (septum S50, Luer lock, etc.).

[0217] In some options (including options compatible with the absence of the retaining portion 9), the bioreactor 4 can have a flexible top portion 4b that is long enough to allow welding at two complementary inner portions of the top portion before cutting to remove the adapter 5 having the deposition end 8 after the bioprinting stage 44 (an intermediate step of welding and cutting to reduce the volume V before bioprinting). More generally, the top portion 4b can optionally be configured to allow at least a portion thereof to be severed or removed. For example, after bioprinting and welding the two opposing portions of the top portion 4b at the welding region, the uppermost portion of the top portion 4b is severed, with the severed uppermost portion extending completely above the welding region.

[0218] Furthermore, the bioreactor 4 can be configured to release access to the base portion 4a by means of an unlocking device, for example by allowing the side wall SV to be displaced / separated over the base portion 4a, to facilitate retrieval of the tissue after the maturation step 45. Alternatively, a circular cutting step can be performed to sever at least one of the side wall SW and the top portion 4b.

[0219] After fixing the containment part RC to the flexible / movable upper part, the sealing enclosure E is obtained. Figure 4B It can be seen that the connector or similar part for obtaining the fastening zone RF defines a passage for the passage of a needle or similar deposition tip 8 between the second / upper compartment C2 and the first / lower compartment C1, thereby allowing the deposition tip 8 to be inserted in this lower compartment C1 delimited by the side wall SW. Figure 4B A downward folding of the top portion 4b is shown, which may extend at least partially below the annular upper end 4c of the side wall SW.

[0220] Therefore, the bioreactor 4 is provided with a highly flexible portion, here at the sheet portion 2 forming the top portion 4b. When viewed from the outside of the containment enclosure E, the outer surface of this top portion 4b can have a convex surface (convex top portion) which, due to the displacement of the adapter 5 when driven by the drive system DS, changes to a concave configuration after applying and maintaining downward pressure or a similar downwardly directed force in the region adjacent to the aperture O4 of the top portion 4b. When bioprinting tissue on a target support 20 that remains fixed to the base portion 4a via the deposition tip 8, movement along the XYZ directions can be achieved. This base portion 4a can be prevented from moving, for example, by using a holding and retaining structure HR that blocks the base portion 4a at a lug, similar to a fixing extension MF, integrally formed with the base portion 4a and / or the side wall SW.

[0221] This fixed state can be partially or completely maintained during the preparation phase, during the bioprinting step 44, and then optionally during the maturation phase, using one or more fluids (i.e., the biopharmaceutical fluid received in the containment portion RC) in order to obtain a sealed enclosure. The fluid movement or agitation can be performed while still maintaining the base portion 4a fixed, for example using the holding and retaining structure HR and without disturbing the bioprocess inside the bioreactor 4 in the upper position of the adapter 5. Figure 8B A non-limiting example of a stirring member 29 is shown, which is arranged here in the base portion 4a and is laterally displaced relative to the target support 20. Any suitable stirring member 29 can be mounted on the base portion 4a before forming the fastening region RF with the flexible portion of the sealing cover E, i.e. before mounting the cover part (2, 2').

[0222] In general, in the present disclosure, "biopharmaceutical fluid" should be understood to mean a product produced by biotechnology (culture medium, cell culture, buffer solution, artificial nutrient liquid, blood product and derivatives of blood product), or a pharmaceutical product or the like intended for use in the medical field (e.g., regenerative medicine). This product can be in liquid / fluid form. Gas can also be produced during the bioprocess, and the sealing cover E can be airtight. In some options, a sterile exhaust port can be added, for example, containing a filter. This exhaust port can be provided according to the cell culture to be prepared in the bioreactor 4. When performing the bioprocess (culturing / maturation), at least when the culture medium (with nutrients) is initially introduced, the materials BM, M can be a minor portion of the cover contents.

[0223] In some embodiments, the deposition end 8 may extend within a sub-volume defined by the flexible portion in its expanded state. The containment portion RC may define a nominal volume, which is the first volume of the containment enclosure. The sub-volume corresponds to a second volume that may vary. In the expanded state of the flexible portion, the following relationship may be satisfied, for example:

[0224] 0.1 <V2 / V1<1

[0225] Therein V1 denotes the first volume (corresponding to the first compartment C1 ), and V2 denotes the volume of the upper / second compartment C2 obtained if the flexible portion is expanded so as to extend the adapter completely above the first compartment C1 .

[0226] More generally, the internal volume V may vary. The deposition tip 8 is provided in a needle or nozzle protruding in the internal volume V so as to maintain a determined length L (which may be greater than or equal to 9 mm). This may be suitable for depositing bio-inks, typically droplets of viscous printing fluid containing living cells.

[0227] The side wall SW may be greater than the length L, and the movable flexible assembly 2, 2' (forming all or part of the cover portion) containing the adapter 5 and connected to the top of the receiving portion RC may also have a height greater than the length L, for example the maximum height of the movable assembly may be greater than 20 or 30 mm. This height corresponds to the height of the upwardly tapering sheet portion, without taking into account the adapter 5, 105, which is generally rigid and protrudes axially outward.

[0228] Assembly options with external retainer

[0229] The side walls SW may be flexible, rigid or partially flexible. Figure 5AA non-limiting embodiment is shown in which the top portion 4b is a rigid component 50, here a rigid plate articulated and / or supported by a partially flexible retainer 51 (here with the ability to bend according to the bend, possibly without a twisting effect), which exerts a return force toward the raised / upwardly extended configuration of the bioreactor. Here, the flexibility of the sidewall SW compensates for the rigidity of the top portion 4b, and the flexible retainer can be an arch with a varying curvature. The sidewall can be a bag-like sidewall SW fixed to the annular peripheral region RF' of the base portion 4a by welding or similar suitable sealing. Thus, the sidewall SW is maintained at its opposite ends by axially opposing portions 4a, 4b (with corresponding fixing regions RF, RF'), knowing that the top portion 4b is suspended by the retainer 51. When pressure is applied to the adapter 5 to reach the lower position for bioprinting with the deposition end 8, a reduction in the radius of curvature of at least some of the retainers 51 (here, elongated retainers of elastic material (e.g., silicone or the like)) occurs. Hinge links 52, 53 are provided at two opposite ends of each arm / holder 51 for respectively:

[0230] - at the lower end of the holder 51 , connected to the base portion 4 a via a lower pivot 52 , thus ensuring a hinge link; and

[0231] - At the upper end of the holder, it is connected to the top part 4b via an upper pivot 53, ensuring a hinge link.

[0232] Any type of assembly with elastic retainers (e.g., with at least three or four retainers) can be suitable for guiding the movement of the rigid part 50. With this configuration, the adapter 5 can be contained in the plane of the rigid part 50, with the septum S50 arranged in its central region. Thanks to the supports / retainers, the needle / deposition tip 8 can be inserted through the plasma septum or similar septum without causing the side walls SW to telescope.

[0233] In the embodiment with flexible side walls SW, the housing / hood E of the bioreactor 4 defines an internal volume of varying delimitation, wherein the base portion 4a forms a fixed portion, possibly the only fixed portion.

[0234] When this side wall SW is made of a flexible plastic film / material (e.g., a silicon material), the side wall SW can expand from a relatively flat configuration to an expanded configuration. In both configurations, the side wall SW is part of a cover E connected in an annular shape to the base portion 4a, and the base portion remains flat or planar along the base plane P. The upright configuration of the bioreactor 4 can correspond to this expanded configuration. For example, polyethylene can be included as an inner layer of a single or multilayer film included in the side wall SW and / or the top portion 4b. Alternatively or in addition, the side wall SW can be at least partially made of a silicon material, possibly for a wall portion intended to be in contact with the culture medium after bioprinting.

[0235] When flexible, the side wall SW and / or base portion 4a can be maintained (having a retaining effect) by the retaining and holding structure HR to define a substantially constant volume for the compartment C1. This retaining effect can be achieved by applying a radially outward pulling force using lugs or similar extension / fixing members MF provided laterally on the side wall SW. Additionally or alternatively, an upwardly directed force can be applied to the upper end of the side wall SW.

[0236] See Figure 10 , shows a holding and retaining structure HR comprising an L-shaped section for guiding the position of the base portion 4a and at least one side of the side wall SW. The bottom of the structure HR may be a base plate that extends at least partially below the base portion 4a to maintain the base portion 4a substantially flat along the base plane. The peripheral portion of the base portion 4a may also be configured to remain fixed, for example, due to rigidity considerations.

[0237] Here, a substantially vertical rigid wall is shown transverse to the bottom, allowing, for example, a tab or similar fixing member MF' to be clamped or otherwise secured to the cover E provided on the side wall SW, away from the inlet and outlet at the top portion for the deposition end 8. The fixing member MF arranged at or near the side of the base portion can extend relatively closer to the target support 20 (collection plate, flat surface, etc.). In addition, regardless of the method used to provide flexibility in the sealing cover E to allow displacement of the adapter 5, the retaining and holding structure HR can have one or more guiding undulations (protrusions or recesses) optionally arranged in the central area of ​​the base plate BP. By utilizing complementarity in shape or a docking or friction effect, possibly using a rotational lock, the corresponding surface S20 of the base portion 4a can be fixed to the base plate BP of the structure HR. More generally, when the base portion 4a is not inherently rigid enough to remain flat, the sealing cover E can be located on the lower portion of the retaining and holding structure, possibly with a stretching effect. The inner surface forming surface S20 can be planar and configured to be free of wrinkles or undulations.

[0238] The structure HR may optionally surround the side wall SW. Figure 4C , the holding and retaining structure HR can be provided with a ring or similar surrounding portion that is connected to several attachment fixing members MF' of the cover E. This surrounding portion can be connected to a lifting rod, a side column or a similar support member 16 distributed around the cover E. When the gusset G2 is provided in the cover E, the structure HR allows the gusset G2 to remain substantially unfolded, so that there is a spacing between the two opposing main flexible sheets 7. Prior to the sealing step 41 of forming the cover E, the fixing members MF' can be formed on the corresponding sheets (7 and / or G2), and then these sheets are assembled at step 41. The fixing members MF' can be added away from the welding area or similar sheet fastening area.

[0239] Possibly, one or more elastic return members may be incorporated for connecting the side wall SW of the cover E to the fixed part of the structure HR. This may allow slight deformations, for example for compensating for small increases or decreases in pressure in the lower zone of the internal volume V.

[0240] At least two, three, or four fixing means MF' may be distributed in the same annular area of ​​the hood E, possibly at the same height level as the upper end of a similarly wide hood portion delimiting the containment portion RC or the ripening stage and delimiting the compartment C1. Due to the retaining effect of the lower fixing means MF, the lower portion of the hood E is less susceptible to deformation than the upper portion. More generally, any suitable structure may be provided to make the hood more susceptible to deformation in its top portion 4b than in its lower portion, including all or part of the side wall SW.

[0241] See Figures 4A to 4C , except at the holding area RH, in particular at the fixing means MF' or a similar fixing area RF, the cover E may:

[0242] - a slight movement in the side wall SW between the base portion 4a and the region RH or RF, with limited local deformation; and

[0243] - can be largely moved beyond the zone RH or RF (at the top portion 4b and / or in the flexible sheet portions 2, 2'), possibly with the ability to fold the plastic material of the cover E so that its rigid upper end can be displaced in the compartment C1.

[0244] In some options, the rigid end may comprise a transverse weld joint SL' for joining the membrane sheet and / or a top clamping member held outside the internal volume V of the bioreactor 4. In some embodiments, a tightening system such as a cable tie may be involved. The rigid end may also be provided with an adapter 5 and an associated deposition end 8 ( Figure 4C The rigid end may form an aperture O4 through which the longitudinal axis A of the adapter spans. In the uppermost extended configuration of the top portion 4b, this axis A may be the axis of symmetry or the central axis of the side wall SW, possibly the central axis of the sealing cover E.

[0245] In some options, the inner face of the respective wall members delimiting the internal volume V of the sealing enclosure E can be made of a given plastic material. More generally, such wall members allow a change in the configuration of the enclosure E, typically with a reduced volume when the deposition tip 8, 108 is in position to transfer biomaterial onto the target support 20, while said volume can be increased after the bioprinting step 44 in order to maintain the deposition tip 8 away from the target support 20. This change in configuration can be achieved by using folds and / or undulations to create an excess of surface in the top portion 4b, possibly forming one or more annular hinges around the aperture O4 of the top portion 4b.

[0246] See Figure 4A , due to the flexibility of the film forming at least the side walls SW and the top flexible sheet assembly 2, the flexible portion contained in the top portion can be folded inwardly. The lower compartment C1 can be similar to Figure 1 , but with flexibility in the side walls SW and / or using the same material to delimit the respective compartments C1 , C2.

[0247] In some modifications, at least one hinge can be provided, preferably each hinge has an annular shape. Even when the top portion is dome-shaped as shown in the figure due to the flexibility of the membrane material, one or more external folds can be provided in the top portion 4b. Possibly, a reinforcement can be provided in the annular region to have a predefined hinge effect and better control the change of the configuration of the top portion 4b. In any case, the top portion 4b can be well adapted to be inserted in the compartment C1 with a high degree of freedom about the movement (here along the XYZ direction) of the adapter 5. Due to the change of configuration, the flexible sheet portion 2 provided at the top of the bioreactor 4 can be deformed to obtain a constant surface area. Optionally, the wavy profile of the sheet material of the top portion 4b can be provided so that there are several grooves (not shown), such as continuous annular grooves or discontinuous grooves with annularly distributed groove portions.

[0248] Figure 4A The design is suitable for obtaining the top portion 4b and the side walls from one or more flexible sheets.

[0249] The sheet portion 2 may have an outer edge E2 that delimits the periphery p of the top portion 4b, which remains at substantially the same height level of the upright bioreactor 4, and the configuration of the top portion 4b obtained in the assembled state of the sheet portion 2 may be such that it protrudes upward or outward relative to the height level of the periphery p (e.g., without reducing or significantly reducing the compartment C1). The different parts of the sealing cover E may be varied to achieve the ability of the region of the top portion 4b provided with the aperture O4 to shift within the volume V as the configuration of the upper compartment C2 changes. Some examples of options for producing the bioreactor 4 and for assembling the different parts will be described below.

[0250] Sealing cover structure

[0251] Reference Figure 3 , it can be seen that the enclosure E can be obtained from different pieces so as to define the internal volume V. The methods for assembly include, for example:

[0252] - two preliminary steps consisting in defining a portion 2 and / or 2 ′ (covering portion) intended to cover the containing portion RC and providing apertures O and / or O4 in this portion;

[0253] a first assembly comprising a coupling step 40 for mounting the adapter 5 as a fitting inside the aperture O4; this step 40 can be performed after the two preliminary steps or can be obtained when the part is envisaged, for example in the case where the adapter can be obtained directly when forming / molding the part 2, with the adapter 5 then being included / overmolded on the part 2;

[0254] - providing side walls SW and a base portion 4a, and optionally fixing a printing platform on the inner face of the base portion 4a; the base portion 4a may optionally consist of at least one planar portion separate from a top portion.

[0255] - securing one or two connection ports P4, P4' to the base portion 4a and / or the side wall SW (eg at a location adjacent to the base portion 4a), optionally with barbed male connectors adapted for connection to flexible hoses.

[0256] - Providing the peripheral area with outwardly extending lugs or similar fixing means MF, or incorporating rigid material and / or retaining portions in the side walls SW.

[0257] - forming 41 a sealing enclosure E by a welding operation or similar gastight connection, possibly at one or several annular joints / overlapping segments (which may be distributed in the edges of the polygonal periphery of the base part 4a and / or the top part 4b).

[0258] A sterilization step can also be performed, for example, just after the sealed cover is formed, to sterilize the cover E. Figure 3 The sterilization step 42a is shown as being performed after the adapter 5 is attached, but the precise order of the steps may vary: of course, steps 43 and 42a may be performed before step 42 for preparing the structure for holding the bioreactor 4. Once the enclosure E is obtained at step 41, the sterilization step 42a may be performed, with or without integrating the adapter 5 before this step 42a. In some variants, the sterilization is performed just before the bioprinting step 44.

[0259] More generally, the method may mean forming / obtaining a flexible portion distributed in at least one of the cover portion (which may be the sheet portion 2) and the side wall SW of the containment portion RC, and then assembling the flexible portion and the other wall component by a sealing operation at each joint so as to obtain a sealed enclosure E. Figure 3 As shown, before the formation 41 of the hood E, the adapter 5 can already be mounted on the covering part.

[0260] The side wall SW can be sized so that its upper annular end 4c is larger than the outer diameter of the adapter 5, 105, and may be at least the same size as the annular lower end of the base portion 4a. In a variant, the side wall SW can taper upwards, provided that the top portion 4b is also adapted to taper in the same direction (upwards) and / or the side wall SW is directly connected to the adapter 5, 105.

[0261] Regarding the base portion 4a, preliminary steps for adapting the inner surface and / or for securing additional layers or components to the base portion 4a may be involved in order to improve the adhesion of the printed tissue. In fact, depending on the application (or the type of plastic used for the base portion), the adhesion of the printed tissue to the target support can be increased to prevent any portion of the printed tissue from moving within the bioreactor 4.

[0262] The method may include the step of adding or modifying an inner layer at the base portion. Laser treatment or other physical and chemical treatments may be performed before sealing to obtain the cover E. More generally, a special portion at the base portion may be provided to ensure the rigidity and / or stability of the target support 20, which special portion may be directly located on the base portion or included as an inner layer of the base portion 20.

[0263] A recess may be provided which is open inwardly (inside the cover E) so that the target support 20 is accessible by a projection or annular wall P20 (see Figure 3 ) is defined and surrounded by a protrusion or annular wall extending away from the side wall SW (displaced radially inwardly compared to the side wall SW). The recess can be obtained by reducing the thickness of the base portion or a similar inner layer. Alternatively, the recess can be defined by an annular protrusion, which may be formed as a ring, which is printed by an additive method (3D printing) or appropriately / rigidly fastened to the inner layer of the base portion 4a. In other options, the base portion 4a can be thermoformed to have undulations and / or similar recesses defined at the target support 20, with or without an inner protrusion.

[0264] Furthermore, in some options, the shape of the top portion 4b may be dome-shaped in the default configuration due to thermoforming or similar forming operations, so that the adapter 5 is arranged at the vertex of this top portion 4b. This facilitates the sealing step 41 of fastening the annular outer edge E2 of the top portion 4b to the other wall parts to obtain the sealed enclosure E, while the deposition end 8 may already protrude from the inner face of the top portion.

[0265] While at least one barbed nozzle or similar connection port for forming an inlet and / or outlet for nutrients (culture medium) is present in at least one of the wall parts delimiting the internal volume V, other connectors may be provided, for example for the circulation of gases such as CO 2 and O 2 (or filtered air flowing through a HEPA filter). Furthermore, a temperature sensor and / or a pH sensor may be provided in the internal volume, such sensors being embedded due to the connection (to the wall parts of the enclosure E) performed before closing the seal 41.

[0266] Furthermore, the connection ports P4, P4' can be placed as desired, for example on a flexible element or on a rigid wall member, possibly at the periphery of the base portion 4a, e.g. Figures 5A-5B As shown. Figure 1-2 In the embodiment, the connection ports P4, P4' are placed on the rigid side walls, but at least one of such connection ports may also be provided in a flexible, panel-like wall, such as Figures 7A-7B Reflected.

[0267] Possibly, the needle / deposition tip 8 for transferring the biomaterial may already be present before the cover is formed. To ensure sealing without disturbing the deposition tip 8:

[0268] - this deposition end 8 is pre-positioned in a configuration that does not protrude beyond the lower end of the adapter 5 or protrudes less than in the operating configuration,

[0269] - or the needle / deposition end 8 is already protruding and the flexible sheet portion 2 may conform to a dome or similar convex configuration so that the needle is displaced away from the compartment C1 and does not protrude beyond the fixation region RF.

[0270] When a rigid base is provided to form the base portion 4a and / or the rigid side wall SW, a ring-shaped silicone seal may optionally be sandwiched between two rigid parts of the ring-shaped clamping frame. Figure 5B This clamping frame CF is shown here when a deformable sheet 2 is used to form the top portion 4b and has one or more connection ports forming the inlet P4 and the outlet P4', which are fixed to a rigid disk D forming all or part of the base portion 4a. Of course, this clamping fixture can be used in many other situations where the bioreactor 4 does not necessarily include the ports P4, P4' in this rigid part.

[0271] The sealing cover E can be Figure 5B In the expanded state shown, the base portion 4a is bounded by the sheet or sheet assembly 2 configured in a dome shape. The sealing step 41 may involve welding, clamping or any suitable means to obtain an annular sealing contact between the peripheral annular area of ​​the assembly 2 and the base portion 4a. Pre-positioning step 40a ( Figure 3 ) can be performed before welding or similar sealing to obtain the cover E.

[0272] The sheet assembly 2 may be provided with an apex region E14 or a similar movable top portion, wherein the adapter 5 is coupled to the sheet or sheet assembly 2. Figure 3 All or some of the steps shown can be applied to this configuration, in particular:

[0273] - a coupling step 40 of coupling the adapter 5 directly to the flexible sheet or assembly of sheets 2 , ensuring that the central opening of the assembly of sheets 2 (similar to the opening O4 ) is sealed;

[0274] a sealing step 41 to ensure that the sheet assembly comprising the apex area E14 or a similar top portion provided with the adapter 5 is connected as a whole to the base portion 4 a ;

[0275] - A sterilization step 42a, wherein the sealing cover obtained at step 41 is sterilized.

[0276] Here, the flexible sheet material assembly 2 is provided with an annular border forming an annular end. Figure 13 , shows an embodiment in which the sheet 2 is made of a silicone material or a similar flexible plastic (preferably transparent). Prior to the cutting operation, the original sheet material may have a rectangular shape. A circular cut may be performed to define the outer edge / border (here having a circular shape) of the sheet 2, possibly after the multilayer region is formed by coupling the sheet 2 to the diaphragm layer, so that the diaphragm layer already covers the central opening O4. The adapter 5 may include this diaphragm layer, having a diaphragm S50 and a rigid upper guide member provided with a flange 5c protruding upward from the multilayer region. The sheet member including the flange 5c and the tubular guide member may be made of silicone that is harder (having a greater Shore hardness) and / or more rigid than the silicone material of the sheet 2.

[0277] At least one welding operation can be performed at the flange 5c of the adapter 5 to secure the adapter 5 (with the membrane S50) to the area of ​​the sheet 2 surrounding the opening O4. After the adapter 5 is coupled at step 40, the adapter 5 can be fixed using a Figure 5BThe sealing is achieved between the rigid plate or disk D provided with the base portion 4a and the sheet 2 using a clamping frame in the enclosure or using any suitable fixing means to maintain the annular boundary in a fixed configuration. After the sealing step 41, the sheet 2 can be inflated by the sterile gas present in the enclosure E. All or part of this sterile gas can be introduced into the enclosure E via a gas inlet or port. In this way, a dome shape, i.e. a shape close to a spherical or hemispherical shape, can be obtained. Then, at step 42a, the sheet 2 can be inflated by the sterile gas present in the enclosure E. Figure 13 The tray D or similar substrate may comprise / be made of metal or plastic material. The tray or substrate may be recycled or reused as needed, provided that appropriate cleaning / sterilization has been provided. In some variations, the sheet 2 may be preformed to already have a dome shape prior to the sealing step 41 and / or prior to any pre-positioning step 40a.

[0278] This assembly 2 is adapted to taper in an expanded state towards the central portion of the assembly 2. This expanded state may have been achieved when the connection step 43 is performed so that the outer end of the adapter 5 (extending outside the cover E) is fixed to the movable part (drive system DS) of the external printer unit 3. Of course, the structure of the adapter 5 may vary greatly and is not limited to Figure 5B The example shown. The adapter 5 only needs to be sealed to the sheet assembly 2, which can form a passage for depositing biomaterial BM via the deposition tip 8 extending inside the interior volume V. The adapter 5, having an annular flange 5c and an upper tube or similar guiding upper end, facilitates the introduction of a needle or similar deposition tip 8. By piercing the septum S50, this deposition tip can be easily inserted through the opening O4 into the interior volume V. After steps 44 and 45, the needle can be removed, and the septum S50 can optionally automatically close the opening O to maintain the sealed state.

[0279] When a needle or similar deposition tip is mounted on a flexible sheet assembly, which may be dome-shaped, via an adapter 5, movement along a transverse direction parallel to the base portion 4a is facilitated, as the curvature can vary on different sides of this dome-shaped sheet assembly 2 or 2'. For example, when the adapter 5 is moved toward a given side, there may be no (or no significant) downward movement, and the assembly 2 or 2' may locally increase its curvature on this given side, while obtaining a loose profile (e.g., with a slope of approximately 45°) on the opposite side. On the one hand, due to the flexibility of the material, and due to the absence of any significant / undesirable folding that could impair the displacement of the adapter 5, the dome-shaped (wall DW) or spherical (i.e., with a curved profile) silicone material wall can easily follow the movement of the needle during the bioprinting phase 44. Due to the connection of the adapter 5 to the printer unit 3, this sheet assembly 2 is prevented from telescoping. In addition, due to the presence of gas / air in the internal volume V, this lightweight sheet assembly cannot telescope, so that no significant fold is formed at or around the top portion 4b. Of course, silicon material is merely one exemplary material, and any other translucent or transparent elastic plastic having suitable flexibility may be used.

[0280] Optionally, one or more cutting steps may be provided, for example after the welding operation to obtain the weld. Figure 6 , the sealing cover E can be obtained by welding the plastic material sheets and then cutting the outer part. Similarly, it is also possible to perform the cutting of the peripheral edge of the bottom / base part after joining with the side wall SW. Although Figure 3 The rectangular shape of the sheet 2 is shown coupled to the adapter 5 in the central region, with the cover portion 5a of the adapter 5 overlapping the aperture O4, but other external shapes can be provided to form the outer boundary of the top portion 4b. A flange can form the cover portion 5a, with the axial central passageway contained within this flange. The respective sides 2a, 2b, 2c, 2d of the sheet 2, when extended in the upright / expanded configuration (e.g., when forming a flap), can be positioned on different sides of the cover E. Alternatively, these sides 2a, 2b, 2c, 2d can mate with respective sides of the panel or the upper side of the sidewall SW.

[0281] exist Figure 6 、 7A In the non-limiting examples of Figures 7 and 7B, it can be seen that at least the side wall SW is made of several plastic sheets that can be assembled at their side edges. Figure 6 In the case of a weld zone for assembling the sheets, the weld zone is more rigid than the non-welded portion. Each protruding strip defined by the weld seam can have a maximum thickness that is approximately twice the thickness of the flexible side wall SW in the flexible zone, making it possible to vary the internal volume of the bioreactor 1. In some variations, only a portion of the side wall SW has this flexibility, possibly using a Figure 6 or Figures 7A-7B Same type of assembly.

[0282] In some embodiments, as Figure 8A The assembly 2' shown can be used with Figures 7A-7B The top portion 4b is associated with the .

[0283] In some variants, the bioreactor 4 defines a container that makes stirring possible, as described in document EP 2 326412 (see in particular the present document). Figure 1 E to 1H and Figure 2 ).

[0284] Furthermore, when welding flexible sheets or panels, the joint can be produced by local heating during a sufficiently long period (e.g., which may be of the order of a few seconds or perhaps 10 seconds) of exposure to heat or heating by low-voltage electric pulses (e.g., up to 9 pulses) generated by the welding head. The technique of heating by low-voltage electric pulses can be used so that the appearance of the visible surface remains unchanged while ensuring good weld quality: in fact, no high pressure is required during welding.

[0285] Pulse welding, heat welding or laser welding techniques can make it possible to obtain durable welds SL. Such welds can include longitudinal seams extending longitudinally at least in the side wall SW. FIG4 shows an example of using four membranes to obtain a 3D cover whose volume can be varied due to the flexibility of the membranes.

[0286] After the four films 102, 103, 111, and 112 are supplied and ready for use, they can be unwound, for example, using rollers (not shown) that unwind them in the same general direction (referred to as the longitudinal rolling direction). Assembly can be performed. This rolling direction is, of course, simply used as a reference point for illustrating the drawings, and it is of course possible to lay out the films in one or more different directions (it is not necessary for the conveying direction to correspond to a straight layout).

[0287] refer to Figure 6 、 7B 8B, an example of an embodiment for obtaining a side weld seam SL with or without forming a gusset in an initially flat state is given. Such a seam ML can be formed during the production of the housing E of the bioreactor 4 (here a flexible bioreactor) in the longitudinal direction (which can correspond to the longitudinal direction of the rolling direction of the membrane strip).

[0288] On the production line, the insertion of the gussets 11 and 12 can be carried out in a manner known per se: see Figure 6, a step 41a of supplying and making available the films 102, 103, 111 and 112, and a step 41b of folding the films 111, 112 towards the inside, are intended to form the gussets 11, 12. In order to weld each gusset 11, 12, the respective film 111, 112 can be kept supported against the guides already used for folding or against equivalent abutment elements.

[0289] See Figure 8C As can be seen, gussets G2, for example a pair of opposing gussets, can also be provided in a sheet assembly 2' prepared separately from the receiving portion RC of the bioreactor 4, prior to the final tightening step using the annular flange 24. This final tightening step can be performed using a variety of flexible assemblies 2, 2' belonging to the cover portion of the bioreactor 4. This sheet portion 2 or 2' can form a 3D structure with a wide annular part that defines the passage O2 for the deposition end 8, 108 and possibly also for the adapter 5, 105. This sheet portion 2, 2' is adapted to expand with an upwardly tapering geometry (tapering from the annular part / flange 24) and, when driven downwardly by the drive system DS, expand with a downwardly tapering geometry to have a folded portion passing through the passage O2. The movable flexible portion of the top portion 4b can be included in the assembly 2 or (additionally or complementary) in the assembly 2'.

[0290] Furthermore, the adapter 5 can also be fixed to the sheet assembly 2' when the sheets are welded. Figure 8C As shown, aperture O4 is defined between two sheets (here, sheet edges). The two sheet edges are distributed between two corresponding sheets of plastic film material, which can sandwich gusset G2 in a flat state. The two sheet edges are welded at welded joint SL'. Here, widening the sheet assembly 2' is significant for improving the freedom of movement of the adapter 5. Similarly, passageway O2 (matching the access opening O or open end provided at the top of side wall SW) can open into a wider space, which is compartment C1 used for bioprocessing (cell maturation, etc.).

[0291] Therefore, for at least one of the receiving portion RC and the covering portion 2, 2', film cutting and assembly may be involved.

[0292] At this stage, a number of connection ports P4, P4' and / or top apertures O4 or other openings O suitable for receiving rigid interfaces may already be provided in the main part of the bioreactor 4: in the containment section RC, and in the complementary parts 2, 2' to cover the containment section RC. Supplying the membrane in an initially flat state can facilitate the formation of specific openings and / or the installation of the ports P4, P4'. In a variant, the base part 4a and / or other parts of the containment section RC are made of rigid molded plastic, thereby facilitating the integration of nozzles for the ports P4 and / or P4', for example.

[0293] like Figure 6 As shown in the non-limiting example of FIG, four side welds SL can be formed simultaneously during the welding step 41c, which makes it possible to assemble four membranes 102 and 103 (a first pair of membranes facing each other), 111 and 112 (a second pair of membranes facing each other and arranged transversely relative to the first pair of membranes). The fold lines FL1 and FL2 can remain unfolded during this welding step 41c (defining a minimum transverse space between the first gusset plate 11 and the second gusset plate 12 in a flat configuration).

[0294] The weld seam length can be adapted. For example, it can be longer than the final length of the side weld seam SL, particularly in the case of an option that allows for an oblique cut. This pair of membranes 102 and 103 makes it possible, after the cutting step 41d, to form a rectangular sheet from which the corresponding wall elements 2 and 32 can be obtained (when the element 32 here forms the base portion 4a). In this non-limiting example, a gusset is obtained that is also not cut obliquely and can also have the same length (in the rolling direction) as the rectangular sheet. Figure 8B An exemplary expanded configuration of the cover E is shown, which here has a generally parallelepiped configuration when the pressure is low, since the gussets 11, 12 are not folded. When the pressure increases, for example if the flexible portion 2' to which the adapter 5 is connected is displaced downwards by a compression effect, the membranes 102, 103, 111 and 112 can bend slightly.

[0295] The cutting step 41d may be optional. The material of the four membranes 102, 103, 111, 112 may be the same. More generally, it will be understood that the first wall member 2, the second wall member 32, the first gusset 11, and the second gusset 12 are defined by polygonal sheets, such as rectangular sheets, which optionally have a common multilayer structure, wherein the layers define an inner surface suitable for contact with biopharmaceutical fluids.

[0296] The adapter 5 coupled to the flexible membrane 2' can be welded or sealed in an annular area surrounding the opening O of the top sheet portion 2. Using this concept, it is possible for the side wall SW to comprise two opposing flaps 14, 15 of the respective main sheet 2, 4a and gussets 11, 12. In a variant, the gussets 11, 12 can be incorporated to form the base portion 4a and the top portion 4b. More generally, the orientation of the bioreactor can vary.

[0297] Now refer to Figures 7A-7B , the welding can be similar to form different seams, except that no gussets are formed. This reflects another way of performing step 41 of sealing the wall parts. Several flexible panels W1, W2, 2 are welded to each other so that when the bioreactor 4 is in an upright configuration, they form at least the top part 4b and the side walls SW. For example, two panels of generally hexagonal shape can be assembled to form a seam, which may be similar to the weld seam SL. When all or part of the base part 4a is flexible, radially protruding external extensions or similar fixed extensions may have been provided around / along the outer circumference of the base part before sealing. In a variant, the base part is rigid at its outer peripheral part and is reinforced or stabilized externally by pulling at its opposite ends.

[0298] This assembly is suitable for combining several flexible parts in a cover E without integrating rigid parts, with the possible exception of the base part 4a. Figures 7A-7B The adapter 5 designed as a needle holder is shown, the plug of which is used to form the aperture O4 of the top sheet 2 of the top part 4b, but another way of forming the flexible part moving with the adapter 5 may be provided.

[0299] For example, Figure 8B As shown, a relatively wide opening O can be provided in the sheet portion 2, allowing a rigid coupling 56 of tubular shape to be installed, and then a movable deposition end assembly 2' comprising a flexible membrane having an aperture O4 plugged by the adapter 5 and sealed and rigidly fastened to the outer edge of the coupling 56 can be installed to be movable through the access opening O. Here, the assembly 2' is configured as an additional wall member of the cover portion 2 (so that the cover portion 2 extends to plug / seal the opening O defined by the coupling 56).

[0300] refer to Figure 8A Or 8C, the assembly 2 'is a needle single-use system, which can be sterile and closed. The material of the sheet involved in this assembly 2 ' (stretchable material or non-stretchable material) can be transparent, so as to form a film, for example. Figure 8AIn the example of , this sheet can be attached to the adapter 5 at the annular inner edge delimiting the aperture O4 and optionally annularly attached to an annular flange 24 fixed to the outer edge of the sheet (also annular in shape). The periphery of the assembly 2 'can have a shape that remains unchanged, for example due to the rigidity of the flange 24 delimiting the outer periphery of the assembly 2 ' (at least in the substantially flat configuration of the assembly 2 ').

[0301] See Figures 8A to 8C , the coupling 56 can be coupled to the flange 24 at the annular outer edge or directly to the sheet. Before this coupling, the passage O2 can be hermetically closed, for example, by a suitable membrane 26 in annular sealing contact with the surface of the annular flange 24. Thus, a sterile and closed needle single-use system (here, assembly 2 ') is provided. Of course, as needed (see, for example, Figure 5B and Figure 9-12 ), any other sheet assembly (including or not including the side walls SW) can have this film 26 to protect the corresponding compartments C1 and / or C2.

[0302] The flange 24 may be involved in any of the following exemplary connections: a Luer lock fitted from or assembled on a needle; a triclamp port, a screw port, or a sterile connection at the annular end of the side wall SW.

[0303] When there is a wide opening O allowing access to the lower / main compartment C1 of the sealing enclosure E, any suitable deposition tip assembly 2' can be pre-assembled with the flexibility of its sheet material, which surrounds the adapter 5 carrying the needle / nozzle forming the deposition tip 8. This pre-assembled assembly 2', here forming the upper compartment C2 when extending / projecting upwards, can be airtightly fastened to a frame or similar annular-shaped coupling provided at the top of the 3D bag containing the base portion 4a and obtained by welding one or more sheets of film material forming at least the side walls SW. After sealing the assembly 2, 2' onto the opening O and / or over the side walls SW, the sealing enclosure E is obtained, wherein the assembly 2, 2' has suitable mobility to be partially inserted in the lower compartment C1, as Figure 8B shown (the same logic applies to Figure 10 or 12, but without limiting the possibilities).

[0304] Since the target support 20 is already present at the base portion 4a , with suitable connection ports P4 and / or P4 ′, the sealed enclosure E obtained at step 41 is directly the bioreactor 4 provided with the movable assembly 2 ′ to allow bioprinting at step 44 .

[0305] Example external connections provided by the adapter

[0306] So far, the adapter 5, 105 has been described as a coupling (carrying a needle or similar deposition tip 8) arranged at a position opposite the base portion, so as to plug the aperture O4 centrally located in the top portion 4b of the bioreactor 4, possibly forming a septum S50. More generally, the adapter 5 can be provided at any suitable position on the cover E, remote from the base portion 4a and compatible with the movement of the drive system DS above the target support 20 provided on or above the base portion 4a.

[0307] The adapter 5, 105 may be provided with an outer connection portion FC1, which is preferably rigid. Figure 2 、 4A Or 12, the sealing cover E can be obtained before the ink cartridge 6, 106 is preferably fixed to the adapter 5, 105 using the external connection part FC1. The ink cartridge 6, 106 is designed to contain all or part of the material to be printed, and optionally uses any suitable form of fastening member 6f to serve as a mechanical interface with the drive system DS. For example, this member can be a mechanical and / or magnetic fastening member.

[0308] This cartridge 6, 106 can remain generally upright when connected to the adapter 5, 105 and in fluid communication with the needle or similar deposition tip 8. In some variations, this cartridge 6 may already be connected to the adapter 5, which may be part of the top portion 4b, when the top portion is secured to the wall member of the containment portion RC. When the adapter 5 is already present in the top portion 4b without the cartridge 6, this cartridge 6, 106 can be supplied and sterilized separately and connected to the printing needle / deposition tip 8, 108 via the adapter 5, 105. Here, the adapter can be arranged at the apex of the dome-shaped top portion, which can be a storage location during bioprocessing, where culture medium enters compartment C1 of the interior volume V to interact with the cells of the printed tissue T.

[0309] The adapter 5, 105 can be pre-assembled with a needle or similar deposition end 8, 108 protruding from the lower surface of the adapter before being connected to the sheet portion 2, 2' and / or before being included in a similar upper portion covering the compartment C1 from above. In some options, a molding step or overmolding is performed to include the needle in the sheet forming the outer body of the adapter. Thus, a central / longitudinal channel can be provided in this sheet, at least at the deposition end 8, for the flow of biomaterial BM, M. At a position opposite the deposition end 8, the adapter 5, 105 can be provided with an inlet portion, thereby forming the first longitudinal end of the adapter 5. In some options, a check valve or similar automatic closing component can be provided at the first longitudinal end of the adapter. The second end can be nozzle-shaped to reduce the diameter, thereby allowing the delivery of a flow with a reduced cross-section. The closing component can be actuated when pressed (e.g., pressed downward) by a push component belonging to a cartridge unit provided with at least one cartridge 6, 106. The cartridge can be a syringe with a piston. A return component (elastic return member, such as a coil spring, etc.) may be provided to allow automatic closing when the cartridge is disconnected. In some options, two or more cartridges may converge towards the same feed channel in cooperation with the same inlet portion of the adapter outer connection part FC1.

[0310] refer to Figure 9 A needle or similar biomaterial deposition tip 8 can be secured to the insertion portion of the adapter 5, here configured as a connector that allows fluid communication with the cartridge 6 when the two connection parts FC1, F2C are assembled and locked together. The cartridge 6 is provided with a connection part FC2 that extends around the fluid passage opening at the axial aperture O6. In the adapter 5, the external connection part FC1 can comprise a single piece made of molded plastic material, provided with a tubular portion defining the fluid passage. For example, the material of the connection parts FC1, FC2 is incompressible and therefore cannot deform.

[0311] Here a connection arrangement is obtained when the connection parts FC1 , FC2 provided in the attachment assembly 25 , 25 ′ are coupled using mechanical fastening. Figure 9 In the embodiment of the present invention, the connection parts FC1 and FC2 are provided completely outside the internal volume V and each comprises a corresponding annular portion, optionally of complementary shape. The annular portion of the first connection part FC1 provided in the adapter 5 can be fixedly fastened radially to a central tube segment of the facing annular portion provided in the second connection part FC2. The free end of this central tube segment defines an aperture O6 (a second opening facing the inlet and outlet opening of the adapter 5). An end flange or annular rim is provided around this aperture O6 to form a receiving surface to which the membrane SM' covering the aperture O6 is removably bonded.

[0312] Similarly, the membrane SM covers the access opening of the adapter 5. When the two connection parts FC1, FC2 are attached via their attachment components 25, 25', each membrane SM, SM' can be optionally folded and unfolded. The connection parts FC1, FC2 can be a female component (such as Figure 9 The type of attachment assembly 25 shown, wherein the female assembly uses a locking slide and clamp or similar locking member to the male assembly ( Figure 9 The attachment assembly 25') shown cooperates.

[0313] An exemplary similar sliding connection is provided in the patent application from the applicant. Within the scope of the SFT sterile connector. The connecting parts FC1, FC2 allow for rapid and reliable sterile connection and sterile fluid transfer between two separate pre-sterilized process components in a biopharmaceutical manufacturing operation. Each membrane SM, SM' can be a sterilization grade polyethersulfone (PES) membrane or the like. The coupling or attachment assembly 25, 25' involved in the locking connection can be provided with an anti-mistake member, which, for example, needs to be slid or similarly guided in a direction perpendicular to the longitudinal direction of the adapter 5 (formed at the coupling area, or needs to be rotated along the longitudinal axis of the adapter). Here, the deposition end 8 is shown as an elongated needle or the like, preferably straight and extending along the longitudinal axis A1 of the adapter 5. However, other configurations for this deposition end 8, for example with an offset angle and / or bend, can be adopted.

[0314] Regarding the membranes SM, SM', they can be configured to be coupled, for example, at a pulling end (which in some options becomes a common pulling part, the combined pulling member 5b), thereby allowing the simultaneous removal of both membranes SM, SM'. This coupling step for associating the membranes can be performed before the connection between the locking components 25, 25'. Of course, the structure of the external connection part FC1 can be varied (with or without the use of such membranes SM, SM'), and the locking can possibly be performed using a separate piece. Typically, the cartridge connection allowed by the adapter 5, 105 can be suitable for single-use situations, and the deposition end 8, 108 is not intended to be used with other cartridges after bioprinting.

[0315] The printing unit 3 is obtained as the cartridge 6, 106 can be driven by the drive system DS according to the program to move the Cartesian 3D printer. The adapter 5 is arranged as the end of the cartridge part, which protrudes into the internal volume V. The adapter 5 is a print head for biological deposition, without any pumping part and optionally without any rotating part or joint J3, which are contained in the external drive system DS (which extends completely outside the cover E).

[0316] Bioreactors as single-use vessels

[0317] As disclosed above, the top portion 4b can be arranged directly on the side wall SW as a flexible (usually transparent) plastic part formed into at least one funnel, or mounted on an annular frame / interface arranged on a sheet portion covering the side wall SW. The bioreactor 4 can be produced with one or more functional components 30 already extending inside the internal volume V. Connectors 30c associated with these components / sensors are arranged at the external interface and can be connected to the control unit 35 of the printer unit 3.

[0318] Whatever flexibility is achieved in the cover E, it is particularly suitable for being sterilized.

[0319] The sterilizable sealing cover E is provided with a first port P4 and / or a second port P4' which are different from the aperture O4 plugged by the fitting / adapter 5, 105. Such ports P4, P4' allow for the formation of a fluid inlet and a fluid outlet. The sterilizable sealing cover E has an inner face suitable for receiving a biopharmaceutical product and is suitable for carrying out the following two steps:

[0320] - Automatic (preferably layer-by-layer) printing of bio-ink material (step 44- Figure 3 );

[0321] - Perform maturation or cell culture of the printed tissue / portion T (step 45 ).

[0322] For example, a sterilization step can be performed before step 42 to ensure that the bioreactor 4 is ready for the bioprinting step 44. More generally, this sterilization step 42a can be performed before the bioprinting stage 44 and possibly before some of the preparatory steps 42, 43 associated with the bioreactor 4. In some options, the sterilization step 42a is performed in the telescoping state of the bioreactor 4. Alternatively, the sterilization operation can be performed in the expanded state of the bioreactor 4.

[0323] Of course, step 43 of coupling the adapter may be performed before step 42 .

[0324] refer to Figure 1-3 The enclosure E can first be installed in an operational configuration, wherein the base portion 4a is stabilized with a fixed target support 20 and / or connected to a holding and retaining structure HR to limit / prevent any telescoping effect at the side wall SW of the bioreactor 4. This stage is suitable when a deformable side wall SW is used. At this stage, a spacing step 42 is performed to separate the two opposing portions of the flexible side wall SW, possibly using several fixing members MF'. In any case, the side wall SW is constructed or assembled with this structure HR so as to maintain a generally annular shape in the upright configuration of the bioreactor 4.

[0325] Figure 8DAn option is shown in which step 41 provides a welded joint / seam SL that improves the stability of the upper annular end 4c of the side wall SW, possibly without any retaining contact (via fixing member MF) at the base portion 4a. The sealing cover E can be formed as a D2 bag (here, only two flexible plastic material sheets are provided, without gussets) with a soft or rigid support member on the bottom surface. Pre-molding can be optionally performed to strengthen the bottom of the base portion 4a. Of course, in the upward upright configuration of the bioreactor 4, the outer periphery can have any shape (circular, square, other polygonal shapes) at the bottom and top of the annular side wall SW. Port P4 can be welded on any suitable cover surface, for example, on the bottom or in the seam SL. Plastic film can be selected so that if the adapter 5 is disconnected from the drive system DS, the seam SL prevents the sheet portion 2 from telescoping in the default configuration. After bioprinting, the adapter 5 and the deposition end 8 can be raised as shown so that they do not interfere with the bioprocess performed in the compartment C1 of the cover E.

[0326] Although Figure 8D A specific embodiment is shown that is compatible with a reduction in the number of flexible parts to be assembled, but another cover can be obtained, at least in the upright configuration of the bioreactor, using side walls SW and top part 4b that taper in opposite directions (towards the base part 4a and the adapter 5, respectively) with the upper sheet assembly (defining compartment C2) expanding upwards.

[0327] Furthermore, a grooved profile may be applied to the top portion 4b, which has a groove (not shown), possibly with a concentric arrangement of grooves. The grooves may optionally be obtained by thermoforming at least one sheet of the top portion 4b, possibly with a generally flat or dome-shaped default geometry. Expansion at the grooves may facilitate displacement of the adapter 5, 105 within the interior volume V.

[0328] The choice of spacing member can be varied, for example to provide a stretching effect. In some options, the spacing member takes the form of a reinforcement or retaining element of the structure HR. This spacing member prevents the two opposing portions of the side wall SW from moving inward. Thus, when the adapter 5 is moved over the target support 20 within the interior volume, it prevents contact between the target support or adapter 5 and the side wall SW. When spacing is performed, the periphery of the top portion 4b may be prevented from moving, or may only move to a small extent (e.g., less than 5 mm).

[0329] When starting step 44 or before this step, the cartridge 6, 106 is optionally connected to the bioreactor 4 using the adapter 5, which can be needleless or provided with a needle. This is the cartridge-adapter connection step 43. At this step 43, each deposition tip 8, 108 is connected to a corresponding microfluidic pump and / or the pump allows the biomaterial BM, M to be pushed downward to reach the deposition tip 8, 108.

[0330] The robotic arm may be equipped with one or more cartridges 6, 106. In some embodiments, when more than one cartridge is involved in the bioprinting step 44, two / several robotic arms are provided. In some options, the bioprinting step may involve printing multiple cell types. For example Figure 2 The robotic arm shown in FIG. 1 or 10 is given here as a non-limiting example for the drive system DS. Any mechanical device having a movable part assembled for the relative displacement of the print head / deposition end 8, 108 rigidly fixed to the adapter 5, 105 with respect to the target support 20 may be provided.

[0331] According to the instructions of the control unit 35, the robot arm or similar system DS can move in the X, Y and Z directions to print the tissue according to the 3D design. Depending on the programming of the printing parameters of the bio-ink (containing cells and / or biological materials), it is possible to define certain physical parameters of the printer unit 3 and the trajectory of the deposition tip 89, 108 forming the embedded print head.

[0332] In embodiments using the flexibility of the material at the top of the cover E, the portion 4b can wrap around an arm or similar of the drive system DS, at least during bioprinting. In other embodiments compatible with a rigid plate or similar rigid top portion 4b covering the containment portion RC, the flexibility of the side walls SW is such that they compensate for lateral movements of this top portion 4b, e.g. Figure 5A shown. Figure 5A The dashed lines in reflect the change in configuration and displacement of the outer holder 51, thereby allowing the lowering of the position of the deposition end 8. The top portion 4b can be tilted / slanted significantly without changing the position of the base portion 4a or the area where the target support 20 is located.

[0333] exist Figure 5B In the embodiment of the present invention, pressure management in the housing E can optionally be performed using at least one port 55a, 55b separate from the ports P4, P4'. The flexible wall of the housing E, at least in the expanded state, can be a dome-shaped wall DW adapted to taper from the lower periphery of the base portion 4a or from the sidewall portion. This wall DW can be elastic, deformable at a constant surface, or capable of increasing its surface. This wall can be controlled to telescope sufficiently to allow displacement of the adapter 5, which contains a septum S50 or (alternatively) a channel for accommodating the needle / deposition end 8 or communicating with the needle / deposition end.

[0334] exist Figure 5A and 5B In these embodiments, although only one deposition end 8 is shown, it should be understood that at least two deposition ends can extend in the same internal volume. A connection step 43 can be performed to associate corresponding ink cartridges or ink cartridge reservoir portions to corresponding deposition ends using the same adapter 5.

[0335] refer to Figure 3 , the connecting step 43 may occur before the optional spacing step 42 or after the spacing step 42. In some options, the cartridge 6, 106 may be filled at a later stage, i.e., after the connecting step 43 and before the bioprinting step 44. Before or at the connecting step 43, a deposition tip 8 is provided, which may be inserted through the aperture (O4) when fixed to the adapter 5. When the deposition tip 8 extends from the adapter 5 inside the cover E, the biomaterial BM or M contained in the reservoir of the cartridge 6, 106 may be deposited on the target support 20 once the adapter 5 is displaced to a precise position such that the deposition tip 8, 108 faces / reaches the target support 20.

[0336] During bioprinting to produce tissue T at step 44, the adapter 5, 105 and the deposition tip 8, 108 move together in at least two lateral directions within the internal volume V relative to the target support 20. Such lateral directions can be perpendicular to the Z direction and perpendicular to the direction of the longitudinal axis A of the adapter 5, 105. With regard to movement along the Z direction, the flexibility of the upper part of the cover and / or the flexibility provided at the side walls SW can be sufficient to move and lower the deposition tip 8, 108 at different height levels, and also to raise the deposition tip 8, 108. A storage step for storing the deposition tip may be involved after step 44 and before step 45. Alternatively, welding and possibly cutting can be performed to separate and extract the deposition tip 8, 108 from the receiving portion RC. In this case, welding allows at least the lower compartment C1 to be sealed airtight. Clamping can also be used instead of welding.

[0337] Since the internal volume V is already closed, the tissue T can be subjected to a bioprocess, such as a maturation step 45 or a similar stage with cell culture. Ports P4 and / or P4' provide for the appropriate introduction and circulation of the culture medium flowing in the containment portion RC. Functional components 30 (e.g. sensors) already present in the internal volume V can be used to deliver appropriate signals / information reflecting the key parameters of the bioprocess while the tissue is maintained in an appropriate position on the support 20. In addition, the stirring member 29 can be activated because the base portion 4a can have a suitable interface that may use magnetic interaction. The stirring member 29 can be associated with an interface provided in the base portion 4a or below the base portion, thereby allowing the introduction of a magnetic source adapter for magnetic coupling with the stirring member.

[0338] Regarding the printer unit 3, which is an external assembly and is arranged outside the bioreactor 4, it can contain any suitable feed source for feeding the biomaterial BM, M to the nozzle / needle or similar deposition tip 8, 108. The feed source can consist of or include a reservoir and a pump (feed pump, possibly with a pressurizing effect). Preferably, the activation / actuation means for activating the transfer of the biological object to the target support is completely external to the bioreactor 4. Such activation / actuation means can be a control part for feeding the deposition tip 8, 108, for example, a mechanical control part by means of a worm drive or a piston, or a direct pneumatic control part.

[0339] It should be understood that different steps 44, 45 (with the possibility of several bioprinting cycles) can occur in the same bioreactor 4, for example, to allow the cells to self-organize, possibly until a specific (sought-after) biological function is achieved / appears. After the last cell culture (end of the maturation step 44), the printed substance / tissue T can be retrieved. After this step 44, the bioreactor 4, including the adapter 5, 105, is optionally removed from the support and / or disconnected from the holding and retaining structure HR, and the bioreactor 4 is discarded. Disconnection can also be performed to remove the cartridge 6, 106, for example, before discarding a single-use bioreactor 4.

[0340] Of course, the internal volume V can be varied depending on the application, and the hood shape can be appropriately adjusted. For example, the height and / or width can be increased to provide greater freedom of movement. Those skilled in the art will appreciate that other variations and modifications can readily be made within the scope of the invention as defined by the appended claims, and it is intended that the invention be limited solely by the appended claims.

[0341] Any reference signs in the appended claims should not be construed as limiting the claims. It will be apparent that the use of the verb "to comprise" and its conjugated forms does not exclude the presence of any other elements than those specified in any claim. The word "a / an" preceding an element does not exclude the presence of a plurality of such elements.

Claims

1. A method of assembling a bioreactor (4) allowing bioprinting within an internal volume (V) of the bioreactor using a biomaterial deposition tip (8, 108) for delivering biomaterial (BM, M) to a target support (20), the method comprising: - providing the target support (20) on or above a base portion (4a) of the bioreactor (4) for the bioprinting; - connecting (40) the adapter (5, 105) to the top part (4b;) of the bioreactor having the aperture (O4); 2'), the adapter (5, 105) plugs the hole (O4) in an airtight manner; - forming a side wall (SW) integral with the base portion (4a) to define the internal volume of the bioreactor (4), the side wall (SW) being adapted to taper and / or extend longitudinally upwards away from the base portion (4a) in an upright configuration of the bioreactor; - providing at least one connection port (P4, P4') for direct fluid communication with the internal volume (V) so as to form a fluid inlet and a fluid outlet, so that after the bioprinting (44), the bioreactor (4) can be used for a cultivation and / or maturation phase of the printed material within the internal volume (V); as well as - The base part (4a) and the top part (4b; 2') are interconnected by at least one sealing operation to form (41) a sealing cover (E) including the side wall (SW), wherein the sealing cover (E) is at least partially flexible and movable so as to allow relative movement between the adapter (5, 105) and the target support (20), so that when fixed to the adapter (5, 105), the biomaterial deposition end (8, 108) inserted through the pore (O4) and / or suitable for feeding via the pore can move in at least two lateral directions within the internal volume (V) relative to the target support (20).

2. The method according to claim 1, characterized in that The at least one sealing operation is performed before any connection of the adapter to a drive system (DS), preferably before any connection between the adapter (5, 105) and a cartridge (6, 106) or reservoir containing the biological material (BM, M).

3. The method according to claim 1 or 2, characterized in that: The adapter (5, 105) is positioned away from the side wall (SW), the method comprising: - forming the top portion (4b; 2') using at least one flexible membrane sheet, such that the internal volume (V) is variable in the upright configuration at least according to the configuration of the top portion (4b; 2').

4. The method according to claim 3, characterized in that The top portion (4b; 2') is provided with a deformable surface, the deformable surface being dimensioned such that in the upright configuration the top portion (4b) can move from an intermediate folded state, preferably a flat state, to: - an outwardly directed expanded state increasing the internal volume (V), wherein the top portion (4b; 2') is dome-shaped so that the adapter (5, 105) extends above the side wall (SW) in the upright configuration, and - an inward curvature that reduces the internal volume (V), wherein the top portion (4b; 2') forms a bioreactor recess for the passage of a drive system intended to be connected to the adapter (5, 105) while the adapter (5, 105) is surrounded by the side wall (SW).

5. A method according to any one of the preceding claims, comprising: - the upper end (4c) of the side wall (SW) is maintained in an annular configuration by a spacing member directly connected to a flexible portion of a sealing cover (E) surrounding the adapter (5, 105), the flexible portion forming all or part of the top portion (4b, 2'); - The adapter (5, 105) is configured as a rigidly connectable end portion or extension of a drive system (DS) arranged outside the bioreactor (4), whereby the flexible portion is able to shift and follow the movement of the adapter (5, 105) when the bioprinting is performed while delivering the biomaterial (BM, M) on the target support (20).

6. A method according to any one of the preceding claims, comprising: - several flexible panels (W1, W2; 7, G2; 11, 12, 2, 32) are sealed to each other so that when the bioreactor (4) is in the upright configuration, they form at least the top portion (4b) and the side walls (SW); as well as - Providing in the at least partially flexible base portion (4a) a radially protruding outer extension (MF), said radially protruding outer extension being adapted to hold said base portion (4a) substantially planar or flat along a base plane (P).

7. A method according to any one of the preceding claims, comprising: - during the sealing operation, the base portion (4a) and the top portion (4b; 2') before interconnection, making and / or installing a receiving surface on the inner face of the base portion (4a) defining the internal volume (V) so as to define all or part of the target support (20); - and optionally using the peripheral edge of the base portion (4a) around the receiving surface for fastening together with a complementary portion, which comprises the top portion (4b) and has a flexible material to delimit the internal volume (V) from above, so as to obtain the sealing enclosure (E).

8. A method according to any one of the preceding claims, comprising changing the capacity of the internal volume (V) due to the movement of the top part (4b; 2') towards a certain position, in which position, when the adapter (5, 105) is driven from the outside by a drive system (DS), the top part (4b; 2') telescopes and is suitable for being pressed downwards by the adapter.

9. The method according to claim 1 or 2, characterized in that: The adapter (5, 105) is preferably a needle holder which is permanently connected with or to a base material of a flexible plastic sheet forming all or part of the bioreactor top part (4b; 2').

10. The method according to any one of the preceding claims, characterized in that The side wall (SW) is obtained by welding at least two sheets, at least one of which: - made of a plastic film (7, G; 102, 103, 111, 112; W1, W2), preferably a multilayer film, and - provided with the fluid inlet port (P4) and / or the fluid outlet port (P4').

11. A method according to any one of the preceding claims, comprising a spacing step (42) for spacing two relative parts of a side wall (SW) having an annular shape in an upright configuration of the bioreactor by means of a spacing member, the spacing member preferably being in the form of a reinforcing member or a retaining member, whereby the spacing member prevents the two relative parts of the side wall (SW) from moving inwardly toward each other.

12. The method according to claim 1 or 2, characterized in that: The top portion (4b) is made entirely or partially of a rigid material and is configured to be movable due to the flexibility of the side wall (SW), The method further includes spacing the top portion (4b) from the base portion (4a) using a return member separated from the side wall (SW), the return member being coupled to the top portion and applying an upward biasing force, whereby the top portion is spaced apart from the base portion (4a) by default by a predetermined distance.

13. The method according to any one of claims 1 to 3, characterized in that: The top part (4b; 2') is made flexible, preferably made of a flexible film, so that the adapter (5) can be displaced relative to the base part between the following positions: - a first position remote from the base portion (4a), in which the adapter (5) is surrounded by a flexible portion of the top portion (4b; 2') which tapers upwards or is dome-shaped and protrudes upwards, - and a plurality of second positions in which the adapter (5) is extended proximally relative to the base portion (4a) and in which the flexible portion is folded downwardly.

14. A method according to claim 13, comprising changing the level of pressurization in the internal volume (V) so as to move the top part (4b) between a collapsed state and an expanded state, wherein, due to the flexibility of the plastic material forming the top part (4b; 2'), the expanded state allows the adapter (5, 105) to be moved over the target support (20), preferably by driving a robotic arm arranged outside the bioreactor (4).

15. The method according to any one of the preceding claims 1 to 4, comprising: - a first connection step for connecting a side wall (SW) comprising or being a rigid part of substantially annular shape to said base portion (4a); as well as - A second connection step for connecting said side wall (SW) to a top portion (4b) made of at least one flexible plastic sheet (2).

16. A method according to any one of the preceding claims, comprising cutting off the uppermost portion of the top portion (4b; 2') after the bioprinting and after welding the two relative portions of the top portion (4b; 2') at the welding area so as to reduce the internal volume (V), the cut-off uppermost portion extending completely above the welding area.

17. The method according to claim 1, comprising: - fastening in a hermetically sealed manner, at a position opposite to said base portion (4a), a rigid coupling (56) of annular shape to a sheet of flexible material (2) suitable for delimiting said internal volume (V) from above and for forming one or more flaps (14, 15) belonging to said side wall (SW), said side wall (SW) being made of a plurality of flexible sheets; - sealing a sheet assembly (2') to the rigid coupler to cover the opening (O) of the rigid coupler, the sheet assembly (2') having an outer edge or flange (24) fastened directly to the rigid coupler (56); - The top covering surface of the sheet assembly extending above the rigid connector (56) is sized so that the top covering surface is suitable for folding inwardly and outwardly relative to the rigid connector (56), whereby the adapter (5, 150) is able to move along at least two lateral directions below the rigid connector (56) without the need for any drive part to be accommodated in the internal volume (V).

18. A method according to any one of the preceding claims, comprising connecting a mobile part (29) of a stirring device to a containing portion (RC) such that the mobile part (29) extends inside the internal volume (V) after forming (41) the sealing enclosure (E).

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