Sterile mask and method for printing and culturing three-dimensional structures of organisms

Abstract

The invention relates to a sterile mask (2) and to a method for printing and culturing three-dimensional biological structures in said sterile mask. The sterile cover comprises: a base wall (10), a flexible side wall (8); the invention relates to a printing device (1) comprising a base wall (10),-a flexible side wall (8) which together with the base wall (10) forms a flexible housing (12) defining an internal chamber (14),-a fluid inlet port (22) and a fluid outlet port (24) which allow circulation of a culture medium,-a needle adapter (4) secured to the flexible side wall (8), the needle adapter (4) holding a printing needle (6), the flexible housing protects the biological three-dimensional structure from contamination and simultaneously authorizes movement of the needle adapter (4) in three directions during printing of the biological three-dimensional structure in the internal chamber.

Description

Technical Field

[0001] The present invention relates to a sterile enclosure and a method for printing and culturing a biological three-dimensional structure. Background Art

[0002] It is known to print out a biological three-dimensional structure onto a printing platform of a three-dimensional printer. After the structure is formed, the printed component is immersed in a culture medium for a cell culture process. Therefore, once printed, the biological three-dimensional structure must be transferred to a separate bioreactor for the cell culture process. However, it is difficult to maintain sterile conditions during the transfer of the biological three-dimensional structure to the bioreactor.

[0003] In order to avoid this difficulty, it is also known to print out biological three-dimensional structures in a container suitable for containing and controlling the culture medium and also including a printer or a movable robotic arm equipped with a print head. After printing, it is no longer necessary to transfer the biological three-dimensional structure in a separate bioreactor for the cell culture process.

[0004] The disadvantage of this mode of operation is that the container is cumbersome and bulky, since it must contain the printer or the movable robotic arm. Therefore, when a biological three-dimensional structure has been formed, the printer or the movable robotic arm equipped with the print head cannot be used to print out another structure, because the container must remain hermetically closed during the cell culture process. Due to its size and its lubricated mechanical parts, sterilizing the printer or the movable robotic arm and keeping it sterile can be problematic. Since the container is large and cumbersome, it may be difficult to move the container during the cell culture process to place the container in another room. Summary of the invention

[0005] A first object of the invention is to propose a sterile cover that allows the print head to be freely displaced in at least two orthogonal directions, in particular in three orthogonal directions, without generating stress on the needle and without the risk of the needle bending or breaking. Thus, the shape of the biological three-dimensional structure can be better controlled. Another result is that the printing platform does not need to be movable in the vertical direction, because the sterile cover allows the print head to be freely moved in this direction. Thus, a less complex printer can be used. In the same way, the printer can use a smaller motor because the resistance applied to the robot arm is less.

[0006] A second object of the invention is to propose a less bulky, less cumbersome and less heavy sterile hood.Since the printer or a part thereof is not included in the sterile hood, the size and weight of the sterile hood are significantly reduced.

[0007] A third object of the present invention is to provide a sterile cover that can be freely moved and stored after the printing process.

[0008] A fourth object of the invention is to propose a sterile cover that allows the printer to be reused immediately after the biological three-dimensional structure has been formed.

[0009] A fifth object of the present invention is to propose a sterile cover that simplifies the printing of biological three-dimensional structures and their cultivation in a single container.

[0010] A sixth object of the present invention is to propose a method for printing and culturing a three-dimensional biological structure, which ensures that the biological ink remaining in the needle after printing does not contaminate the printed biological three-dimensional structure.

[0011] These objects have been achieved by the sterile hood and the method as defined in the independent claims. Secondary characteristics of the sterile hood are set out in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 are a perspective view and a schematic diagram of a sterile cover according to a first embodiment of the present invention;

[0013] Figure 2 are a perspective view and a schematic diagram of a sterile cover according to a second embodiment of the present invention;

[0014] Figure 3 is a perspective view and a schematic diagram of a sterile cover according to a third embodiment of the present invention;

[0015] Figure 4 are a perspective view and a schematic diagram of a sterile cover according to a fourth embodiment of the present invention;

[0016] Figure 5 is a perspective view and a schematic diagram of a sterile cover according to a fifth embodiment of the present invention;

[0017] Figure 6 is a perspective view and a schematic diagram of a sterile cover according to a sixth embodiment of the present invention;

[0018] Figure 7 is a flow chart representing the steps of a method according to the present invention. DETAILED DESCRIPTION

[0019] The sterile cover 2 according to the invention is intended to be fixed to the barrel of a printing cartridge or a printing syringe for printing biological three-dimensional structures in the sterile cover.

[0020] The printing syringe or printing cartridge may include a bio-ink composition. The bio-ink composition is a biological material that can form a hydrogel and a cell population in a controlled three-dimensional shape. An example of a bio-ink is described in WO 2017115056.

[0021] Preferably, for the printing operation, the printing syringe or ink cartridge is mounted on a displaceable printer, for example on a displaceable robotic arm. Printing can also be done manually with a printing nozzle.

[0022] In the following description, the term “printing” refers to manual deposition or injection of bio-ink or 3D printing with a displaceable robotic arm.

[0023] In the following description, the term "flexible enclosure" will be used both for an enclosure that is completely flexible and for an enclosure that has a lower rigid portion and is an "at least partially flexible enclosure".

[0024] refer to Figure 1 The sterile cover 2 according to the first embodiment of the present invention comprises a needle adapter 4 , a printing needle 6 held by the needle adapter, a flexible side wall 8 tightly fixed to the needle adapter 4 , and a flexible base wall 10 .

[0025] The needle adapter 4 used here is a tip that is usually connected to the open end of the barrel of a medical syringe. The needle adapter includes a needle hub that holds the needle and a connector portion that is configured to connect to a corresponding connector portion of the barrel of the syringe.

[0026] The needle adapter comprises a connector part 5 which is adapted to be connected to a complementary connector part of a printing cartridge or a printing syringe. The connector part and the corresponding connector part are for example Luer lock connectors or another sterile connector, such as the connector commercialized by Sartorius

[0027] The side wall 8 forms together with the base wall 10 a gas-tightly closed flexible casing 12 . This flexible casing 12 delimits an inner chamber 14 .

[0028] In the embodiment shown, the needle adapter 4 is placed on top of the at least partially flexible housing 12 .

[0029] Alternatively, the needle adapter 4 is a needle adapter that is integral with the printing cartridge or printing syringe.

[0030] Preferably, the printing needle 6 is a biological printing needle. The needle adapter 4 and the printing needle 6 constitute a printing head.

[0031] In the illustrated embodiment, the flexible base wall and the flexible side walls may be made of one of a thermoplastic elastomer and a thermosetting material.

[0032] Specifically, the flexible base wall 10 and the flexible side wall 8 can be composed of a multilayer film, which includes a contact layer in contact with the medium filling the container, a barrier layer and an outer layer in contact with the external environment. The three layers are connected to each other by a connecting layer. If the container is to be filled with a biopharmaceutical product, the contact layer should be made of a material that can contact this biopharmaceutical product without causing degradation of the film and the biopharmaceutical product. In addition, the container itself must be sealable. For this purpose, the material is usually selected from polyolefins, such as polyethylene.

[0033] The barrier layer blocks the passage of gases such as oxygen, carbon dioxide and is typically made of ethylene vinyl alcohol (EVOH).

[0034] The outer layer contributes to the mechanical strength of the wall. For this purpose, it must be flexible enough to withstand high mechanical stresses, but not stretch too much to prevent the shell from deforming when filled with product. The outer layer may comprise a polyolefin alone or in admixture with a copolymer of ethylene and an alpha-olefin. For example, a multilayer film as described in EP 20945799 may be used.

[0035] For example, when the flexible shell has a cylindrical shape, the flexible side wall 8 can be made of a unique sheet. For example, when the flexible shell has a parallelepiped shape, the side wall 8 of the flexible shell 12 can also be made of several sheets. In this patent application, the term "side wall" will be used to represent these two possibilities in order to simplify the description.

[0036] The base wall 10 may also be made of one or several sheets.

[0037] Alternatively, the base wall and side walls may be made of silicone.

[0038] Preferably, the silicone may be one of the following: an unsaturated rubber that can be cured by sulfur vulcanization, natural polyisoprene: cis-1,4-polyisoprene natural rubber (NR) and trans-1,4-polyisoprene gutta-percha, synthetic polyisoprene (IR stands for isoprene rubber), polybutadiene (BR stands for butadiene rubber), chloroprene rubber (CR), polychloroprene, chloroprene rubber, Bayer, etc., butyl rubber (copolymer of isobutylene and isoprene, IIR), halogenated butyl rubber (chlorinated butyl rubber: CIIR; brominated butyl rubber: BIIR), styrene-butadiene rubber (copolymer of styrene and butadiene, SBR), also known as Buna N rubber: nitrile rubber (copolymer of butadiene and acrylonitrile, NBR), hydrogenated nitrile rubber (HNBR) Therban and Zetpol (unsaturated rubbers can also be cured by non-sulfur vulcanization if necessary), saturated rubbers that cannot be cured by sulfur vulcanization, EPM (ethylene propylene rubber, copolymer of ethylene and propylene) and EPDM rubber (ethylene propylene diene rubber, terpolymer of ethylene, propylene and diene components), epichlorohydrin rubber (ECO), polyacrylic rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FVMQ), fluoroelastomers (FKM and FEPM) Viton, Tecnoflon, Fluorel, Aflas and Dai-El, perfluoroelastomers (FFKM) Tecnoflon PFR, Kalrez, Chemraz, Perlast, polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), (Hypalon), ethylene-vinyl acetate (EVA) and thermoplastic elastomers (TPE).

[0039] The flexible housing 12 has a generally trumpet shape here. Specifically, in the embodiment shown, the needle adapter 4 is fixed between two joints 16 of the upper end edge 18 of the side wall. At each joint 16, a portion of the end edge 18 of the flexible side wall is fixed to another portion of the end edge 18 of the flexible wall to form a gusset 20.

[0040] Alternatively, the flexible shell 12 of the sterile cover may have a cup shape, a belt shape, a tube shape, or a folded tube shape.

[0041] The sterile hood 2 further comprises a fluid inlet port 22 and a fluid outlet port 24 which allow for the circulation of culture medium during the cell / culture process.

[0042] The fluid inlet port 22 and the fluid outlet port 24 have a tri-clamp system or hose connections.

[0043] Advantageously, the sterile cover 2 may comprise a replenishment port, not shown in the figures. The replenishment port is used to fasten sensors for controlling different parameters of the culture medium, such as its temperature or its pH, for example.

[0044] Preferably, the sterile cover 2 comprises a support armature 26 which holds the lower portion 28 of the flexible shell to keep the lower portion of the side wall 8 away from the inner face of the base wall 10. Thus, the lower portion 28 of the flexible shell cannot contact or fall into the printed three-dimensional biological structure laid on the base wall and damage the printed three-dimensional biological structure.

[0045] Advantageously, the support bracket 26 holds only the lower portion 28 of the flexible shell 12 .

[0046] Advantageously, the upper portion 30 of the flexible housing authorizes displacement of the printing needle 6 along the vertical direction Z while maintaining the sterility of the three-dimensional biological structure during the printing process. Thus, it is no longer necessary to use a 3D printer in the form of a vertically movable printing plate.

[0047] Advantageously, the upper portion 30 of the flexible housing authorizes the displacement of the printing needle 6 in two horizontal and orthogonal directions X, Y relative to a printing platform not depicted, while maintaining the sterility of the three-dimensional biological structure during the printing process.

[0048] The sterility of the internal chamber 14 is maintained during the movement of the printing syringe. Therefore, the printing process does not need to be performed under a laminar flow hood or a biological safety cabinet.

[0049] exist Figure 1 In the illustrated embodiment, the support bracket 26 is outside and spaced apart from the flexible shell 12. The sterile cover 2 includes an attachment link 32 fixed to the support bracket 26 and the side wall 8 at an attachment point 34 to keep the lower portion 28 of the flexible side wall 8 at a distance from the inner face of the base wall 10. Preferably, the attachment points 34 are regularly spaced around the flexible shell 12. The lower portion 28 of the flexible shell is positioned below the attachment points 34. The upper portion 30 of the flexible shell is positioned above the attachment points 34.

[0050] In the embodiment shown, the support bracket 26 includes a frame 36 and columns 38 supporting the frame. The frame 36 surrounds the flexible shell. In the non-limiting embodiment shown, the frame 36 has an annular shape. Alternatively, the frame can have a rectangular or square shape.

[0051] Advantageously, the posts 38 are regularly spaced around the flexible housing 12 .

[0052] The inner face of the edge of the flexible side wall 8 is fixed to the inner face of the peripheral edge of the base wall 10 to form an extension 40 .

[0053] The extension 40 protrudes from the base wall 10 in a horizontal plane.

[0054] Alternatively, the extension 40 is made of a complementary circular flexible sheet or membrane welded to the outside of the base wall 10. In this case, the edge of the flexible membrane forms the extension.

[0055] In the illustrated embodiment, the extension 40 has perforated tabs 42 regularly spaced around the circular sheet.

[0056] Advantageously, the lower ends of the posts 38 of the support bracket are positioned in the holes 44 of the tabs 42. Advantageously, therefore, the extension and the base wall 10 are kept extended and flat by the support bracket.

[0057] Advantageously, the extension 40 also stabilizes the positioning of the flexible housing on the support.

[0058] The extension 40 can also be used to reference the position of the flexible housing relative to a support, such as a printed plate form. To this end, the tab 42 can, for example, include two holes: one hole is mounted in a boss formed on the printed plate form for referencing its position, and the other hole is received by the lower end of a post 38.

[0059] Advantageously, the sterile cover 2 may include a receiving sleeve 46 suitable for receiving and retaining the printing needle 6 before or after printing. Figure 1 In the embodiment shown, the receiving sleeve 46 is integral with the side wall. The receiving sleeve 46 is preferably made of the same material as the flexible side wall.

[0060] This receiving sleeve may have a sufficient volume to receive and isolate the bio-ink during the priming phase of the needle. For example, a volume comprised between 0.5 ml and 3 ml may be used.

[0061] Advantageously, Figure 1 The illustrated sterile cover 2 may include a needle protection cover 48 adapted to snap onto the abutment ring of the needle adapter. The needle protection cover 48 prevents the needle from drilling a hole into the flexible housing.

[0062] According to an embodiment not shown, the sterile cover comprises several needle adapters and printing needles. According to this embodiment, the flexible housing may for example have the shape of a glove. The needle adapters and printing needles may be fixed at the end of each finger. According to this embodiment, at least the side wall is made of silicone.

[0063] Alternatively, the base wall 10 is rigid. For example, the base wall is made of biocompatible plastic. The side walls are fixed to the edges of the rigid base wall in a sealed manner.

[0064] refer to Figure 2, the sterile cover 50 according to the second embodiment of the invention is similar to the sterile cover 2 according to the first embodiment, except for the fact that the flexible shell presents a different shape and the support bracket is different. The technical elements of the second embodiment that are identical to the technical elements of the first embodiment have been referenced by the same reference numerals and will not be described again. Only the differences between the second embodiment and the first embodiment will be described in detail.

[0065] In this second embodiment, the sterile cover 50 comprises a flexible shell 52 having a cylindrical shape in its lower part and a generally trumpet shape in its upper part. The upper part 30 has, for example, the shape of a truncated cone or a truncated pyramid.

[0066] The support bracket 54 comprises several support beams 56 extending in the vertical direction. The lower end of the support beams has feet 58 to keep the beams vertical. The support beams 56 are regularly spaced around the flexible shell and are attached directly against the side wall 8.

[0067] Advantageously, the support beams 56 maintain the lower portion 28 of the side wall in a vertical position. Thus, even when the printing syringe causes the upper portion 30 of the side wall to drop, the side wall will not contact the printed three-dimensional biological structure laid on the inner face of the base wall.

[0068] In the embodiment shown, the inner face of the edge of the flexible side wall is fixed to the inner face of the peripheral edge of the base wall 10 to form an extension 40 extending in a horizontal plane.

[0069] The extension surrounds the lower cylindrical portion of the flexible housing 52. In the embodiment shown, the extension 40 does not include a perforated tab. Alternatively, the extension includes a perforated tab that is used to reference the position of the flexible housing relative to the printing platform.

[0070] Alternatively, according to an embodiment not shown, the sterile cover 50 comprises a rigid base hard frame. The flexible membrane of the base wall is fixed to the rigid base hard frame so that the membrane is stretched to form a flat surface.

[0071] Alternatively, the base wall 10 is rigid. For example, the base wall is made of biocompatible plastic. According to this alternative, the side walls are fixed to the edges of the rigid base wall in a sealing manner.

[0072] refer to Figure 3 , the sterile cover 60 according to the third embodiment of the invention is similar to the sterile cover 2 according to the first embodiment, except for the fact that the flexible housing comprises a rigid frame 64 with a septum 62 instead of the needle adapter 4. The technical elements of the third embodiment that are identical to the technical elements of the first embodiment have been referenced by the same reference numerals and will not be described again. Only the differences between the first and third embodiments will be described in detail.

[0073] exist Figure 3 In the non-limiting embodiment shown, the rigid frame 64 has a generally oval shape. The rigid frame comprises a central orifice. The diaphragm 62 covers the central orifice in a sealed manner. The diaphragm is fixed to the inner face of the rigid frame. The diaphragm constitutes a septum intended to be pierced by a printing needle of a syringe or a printing cartridge. The upper edge of the flexible side wall 8 is fixed to the outer periphery of the rigid frame.

[0074] refer to Figure 4 , the sterile cover 66 according to the fourth embodiment of the invention is similar to the sterile cover 50 according to the second embodiment, except for the fact that the sterile cover comprises a rigid frame 64 with a septum 62 instead of the needle adapter 4. The technical elements of the fourth embodiment that are identical to the technical elements of the second embodiment have been referenced by the same reference numerals and will not be described again. Only the differences between the second and fourth embodiments will be described in detail.

[0075] exist Figure 4 In the non-limiting embodiment shown, the rigid frame 64 has a generally oval shape. The rigid frame comprises a central orifice. The diaphragm 62 covers the central orifice in a sealed manner. The diaphragm is fixed to the inner face of the rigid frame. The diaphragm constitutes a septum intended to be pierced by a printing needle of a syringe or a printing cartridge. The upper edge of the flexible side wall 8 is fixed to the outer periphery of the rigid frame.

[0076] The sterile cover 68 according to the fifth embodiment of the present invention comprises Figure 5 The flexible and airtight housing 69 is depicted and Figure 5 Support bracket not depicted.

[0077] Technical elements of the fifth embodiment that are identical to those of the first embodiment have been referenced by the same reference numerals and will not be described again.

[0078] The flexible shell 69 is made of a first sheet 70, the edges of which are welded to the edges of a second sheet 72. The sheets are made of a thermoplastic elastomer or a thermosetting material.

[0079] The first sheet 70 forms the base wall 10 and the lower portion 28 of the side wall. The second sheet 72 constitutes the upper portion 30 of the side wall and the top of the flexible shell.

[0080] The sterile cover 68 according to the fifth embodiment of the invention comprises a needle adapter 4 fixed in an opening of a second sheet 72. The second sheet 72 is fixed in a sealing manner around the needle adapter 4. The lower edge 74 of the second sheet is fixed to the first sheet 10 in a sealing manner, preferably by welding.

[0081] The printing needle 6 is fixed to the needle adapter 4 .

[0082] The supporting bracket, not depicted, is adapted to hold the lower portion 28 of the flexible shell 69 such that the lower portion 28 of the side wall is not in contact with the inner face of the base wall 10 or with the printed three-dimensional biological structure deposited thereon.

[0083] Alternatively, the sterile cover 68 does not include a support bracket. In this case, the lower part 28 of the flexible shell 69 can be placed in a rigid container. The latter does not need to be sterilized.

[0084] In the illustrated embodiment, the first sheet 70 and the second sheet 72 are circular. Alternatively, the first sheet and the second sheet have a square or rectangular shape.

[0085] Advantageously, this flexible housing 69 is easy to manufacture.

[0086] refer to Figure 6 , a sterile cover 76 according to a sixth embodiment of the present invention comprises a partially flexible shell 77. The shell 77 comprises a rigid base wall 78, four flexible side walls 80 and a flexible top wall 82. Other technical elements of the sixth embodiment that are identical to those of the first embodiment have been referenced by the same reference numerals and will not be described again.

[0087] The rigid base wall 78 is made of plastic, for example.

[0088] Advantageously, in the embodiment shown, the base wall 78 comprises a plate 79 surrounded by a vertically extending rim 81. The rim 81 forms the rigid lower portion 28 of the flexible shell. The flexible side wall 8 forms the upper portion 30 of the flexible shell. In the embodiment shown, the height of the rim 81 corresponds to less than 20% of the total height of the shell. Alternatively, the rim may have a height representing 50% of the total height of the shell.

[0089] The flexible side walls 80 and top wall 82 are made of a thermoplastic elastomer sheet or a thermosetting material sheet. The side walls and top wall together with the rigid base wall form a hermetically closed housing 77. This housing 77 is partially flexible because the side walls 80 and top wall 82 are flexible.

[0090] In the embodiment shown, the partially flexible housing 77 has the general shape of a parallelepiped. Other shapes may also be used.

[0091] The top wall 82 is formed by welding triangular portions of each side wall. Other shapes may also be used.

[0092] The sterile cover 76 comprises a rigid frame 64 fitted with a membrane 62. The rigid frame 64 is fixed to the top wall in a sealed manner.

[0093] Alternatively, the rigid frame 64 and septum 62 are replaced by the needle adapter 4 and the printing needle held by the needle adapter.

[0094] Alternatively, the base wall 10 comprises only a flat rigid plate and a support bracket 36 similar to that described with respect to the first embodiment. The support bracket is placed around the flexible shell. The attachment links 32 are fixed to the side walls 80 to prevent them from falling on the base wall 10.

[0095] Alternatively, the base wall 10 comprises a rigid plate and a support beam 56 fixed to the rigid plate. In this embodiment, the flexible side wall 80 is integral with the support beam to keep the lower portion 28 of the flexible shell at a distance from the base wall 10.

[0096] All alternative embodiments described in conjunction with the first and second embodiments also apply to the other embodiments. In order to simplify the description, these embodiments are not described again.

[0097] The present invention also relates to a method for printing and culturing three-dimensional biological structures in a sterile hood.

[0098] When using a sterile cover as described in the first, second and fifth embodiments, the printing needle is fixed to the needle adapter 4, and the method starts with the step of fixing the printing syringe or printing cartridge to the connector 5 of the needle adapter 4. When using a sterile cover as described in the third, fourth and sixth embodiments, the method starts with the step of piercing the septum 62 forming the septum by the printing syringe.

[0099] Alternatively, the printing needle may be integral to the cartridge and the method begins by filling the cartridge with bio-ink.

[0100] During the startup phase, the flow rate to the printing needle is regulated.

[0101] During step 84, a three-dimensional biological structure is printed or deposited on the base wall 10 of the flexible housing. The upper portion 30 of the flexible housing 12 authorizes the printing needle 6 to move in the vertical direction Z and in two orthogonal horizontal directions X, Y during the bioprinting step while maintaining the sterility of the internal chamber.

[0102] Step 84 is followed by step 86 during which two opposing portions of the side wall 8 are clamped and fixed together along a horizontal plane to separate the upper portion of the interior chamber including the printing needles 6 from the lower portion of the interior chamber to avoid contamination of the printed three-dimensional structure by residual bio-ink remaining in the needles. The fixing can be done with a clamp or by welding the two portions of the side wall together.

[0103] During step 88 , culture medium is introduced into the inner chamber for feeding the three-dimensional biological structure.

[0104] Alternatively, step 86 may be performed after step 88 .

Claims

1. A sterile cover (2, 50, 68), comprising: - a base wall (10), - at least one flexible side wall (8); said at least one side wall (8) together with said base wall (10) forms an at least partially flexible shell (12, 52, 69) delimiting an internal chamber (14), at least one fluid inlet port (22) and at least one fluid outlet port (24) allowing the circulation of a culture medium, characterized in that the sterile cover comprises a needle adapter (4) tightly fixed to the at least one flexible side wall (8), the needle adapter (4) being intended to hold a printing needle (6), The at least partially flexible housing (12, 52, 69) is intended to protect the biological three-dimensional structure from contamination and simultaneously authorize the needle adapter (4) to move in at least two directions (X, Y) during aseptic printing of the biological three-dimensional structure in the internal chamber.

2. A sterile cover (60, 66, 76), comprising: - a base wall (10), - at least one flexible side wall (8); said at least one side wall (8) together with said base wall (10) forms an at least partially flexible shell (12, 52, 77) delimiting an internal chamber (14), - at least one fluid inlet port (22) and at least one fluid outlet port (24) which allow the circulation of a culture medium, characterised in that the sterile cover comprises a fluid inlet port (22) tightly fixed to the at least one flexible side wall (8) a rigid frame (64) and a membrane (62) supported by the rigid frame, the membrane (62) forming a septum suitable for being pierced by a printing needle (6), The at least partially flexible housing (12, 52, 77) is intended to protect the biological three-dimensional structure from contamination and simultaneously authorize movement of the needle in at least two directions (X, Y, Z) during aseptic printing of the biological three-dimensional structure in the internal chamber.

3. A sterile cover (2, 50, 68; 60, 66, 76) according to any one of claims 1 and 2, comprising a support bracket (26) which holds a lower portion (28) of the at least partially flexible shell (12, 52, 69, 77) to keep the lower portion of the at least flexible side wall (8) away from the base wall (10).

4. The sterile cover (2, 50, 68; 60, 66, 76) according to any one of claims 1 to 3, characterized in that The inner face of the edge of the flexible side wall is sealed to the inner face of the peripheral edge of the base wall (10) to form an extension (40) extending in a horizontal plane.

5. The sterile cover (2, 68; 60, 76) according to any one of claims 3 and 4, characterized in that The support bracket (26) is outside the at least partially flexible shell (12, 69, 77) and is spaced apart from the at least partially flexible shell, and the sterile cover (2, 68; 60, 76) includes an attachment rod (32) fixed to the at least one side wall (8) and the support bracket (26) to keep the lower portion (28) of the at least flexible side wall at a certain distance from the base wall (10).

6. The sterile cover (2, 68; 60, 76) according to any one of claims 3 to 5, characterized in that The support bracket (26) includes a frame (36) and a post (38) secured to the frame, the frame (36) surrounding the at least partially flexible shell (12, 69, 77).

7. The sterile cover (2, 68; 60, 76) according to the combination of claims 4 and 6, characterized in that The at least one extension (40) includes a through hole (44), and wherein the post (38) of the support bracket (26) is positioned in the through hole (44).

8. The sterile cover (50, 66, 76) according to claim 3, characterized in that: The support bracket (26) comprises at least one support beam (56) integral with the at least flexible side wall (8), the at least one support beam (56) maintaining the lower portion (28) of the at least partially flexible shell (52, 77) at a distance from the base wall (10).

9. The sterile cover (2, 50, 68; 60, 66) according to any one of claims 1 to 8, characterized in that The base wall (10) is flexible.

10. The sterile cover (68) according to any one of claims 8 and 9, characterized in that The flexible shell (69) is made of a first sheet (70) welded to a second sheet (72); the first sheet (70) forms the upper portion (30) of the flexible shell; the second sheet (72) forms the base wall (10) and the lower portion of the flexible shell.

11. The sterile cover (76) according to any one of claims 1 to 8 and 10, characterized in that The base wall (10) is rigid.

12. The sterile cover (2, 50, 68) according to any one of claims 1, 3 to 11, characterized in that: The at least one needle adapter (4) comprises at least one sterile connector (5) adapted to be connected to a print cartridge in a sterile manner.

13. The sterile cover (2, 50, 68) according to any one of claims 1, 3 to 11, comprising a printing cartridge integral with the at least one needle adapter (4).

14. The sterile cover (2, 50, 68; 60, 66, 76) according to any one of claims 1 to 13, characterized in that The at least one side wall (8) is made of a material selected from the group consisting of a thermoplastic elastomer, a thermosetting material and silicone.

15. A method for printing and culturing a three-dimensional biological structure in a sterile cover according to any one of claims 1 to 14, wherein a needle (6) is fixed to the needle adapter (4); the method comprising the following steps in succession: - printing (84) a biological three-dimensional structure, the at least partially flexible housing (12, 52, 69, 77) authorizing the needle (6) to move in at least two directions (X, Y, Z) during the bioprinting step while maintaining the sterility of the internal chamber (14), - Supplying (88) culture medium to said inner chamber (14).

16. The method according to claim 15, further comprising the step of clamping and fixing two opposite parts of the side wall (8) to isolate the needle (6) from the biological three-dimensional structure.

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