Print needle holder device, sterile isolator and method for printing and culturing biological cells

Abstract

The present invention relates to a sterile isolator (91) comprising a container (92) and a print needle holder device (58) secured to the container. The container comprises: a base wall (92); a side wall (94), the side wall and the base wall defining an interior chamber; an inlet port and an outlet port; and an orifice (24). The print needle holder device (58) includes: a needle hub; a print needle; and a flexible sheath having a first end edge fixed to the needle hub and a second end edge fixed to the aperture of the container, a three-dimensional biological structure being intended to be printed on the base wall (92), the internal chamber being intended to receive a cell culture medium.

Description

Technical Field

[0001] The invention relates to a printing needle holder device and a sterile isolator for printing and containing biological three-dimensional structures. The invention also relates to a method for printing and culturing biological cells. Background Art

[0002] It is known to print out biological three-dimensional structures 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 that is suitable for containing and controlling the culture medium and also includes 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, as it must house the printer or movable robotic arm. Therefore, when a biological three-dimensional structure has been formed, the printer or 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. Sterilizing the printer or movable robotic arm and keeping it sterile can be problematic due to its size and its lubricated mechanical parts. Since the container is large and cumbersome, it may be difficult to move the container to place it in another room during the cell / culture process. Summary of the invention

[0005] A first object of the invention is to propose a printing pin holder device which can be fixed to different containers which may have different sizes or different properties specific to the biological structure to be printed.

[0006] A second object of the present invention is to provide a printing pin holder device that can be fixed to a disposable sterile isolator, such as for industrial use, or to an isolator that can be sterilized and used multiple times, such as for laboratory experimental purposes.

[0007] A third object of the present invention is to propose a less bulky, less cumbersome and less cumbersome sterile isolator.Since the printer or a part thereof is not included in the sterile isolator, the size and weight of the sterile isolator are significantly reduced.

[0008] A fourth object of the present invention is to propose a sterile isolator that can be easily moved and stored after the printing process.

[0009] A fifth object of the invention is to propose a sterile isolator that allows the printer to be reused immediately after the biological three-dimensional structure has been formed.

[0010] The sixth object of the present invention is to propose a sterile isolator that allows the print head to be easily displaced in at least two vertical directions, in particular in three vertical directions, without stress on the needle and without the risk of needle bending or breaking. Therefore, 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 print needle holder device allows the print head to be easily moved in this direction. Therefore, a less complex printer can be used. Similarly, because the resistance applied to the robot arm is small, the printer can use a smaller motor.

[0011] A seventh object of the present invention is to provide a modular sterile isolator. Since different types of printing pin holders can be fixed to different types of containers, the sterile isolator can be adapted to different uses and requirements.

[0012] An eighth object of the present invention is to propose a sterile isolator that simplifies the printing of biological three-dimensional structures and their cultivation in a single container.

[0013] The ninth 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.

[0014] These objects have been achieved by a printing pin holder device, a sterile isolator and a method as defined in the independent claims.Secondary features of the printing pin holder device and the sterile isolator are set out in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a perspective view and a schematic diagram of a printing pin holder device according to a first embodiment of the present invention;

[0016] Figure 2 is a perspective view and a schematic diagram of a printing pin holder device according to a second embodiment of the present invention;

[0017] Figure 3 is a perspective view and a schematic diagram of a printing pin holder device according to a third embodiment of the present invention;

[0018] Figure 4 is a perspective view and a schematic diagram of a printing syringe and a printing needle holder device in a holder according to a fourth embodiment of the present invention;

[0019] Figure 5 is a perspective view and a schematic diagram of a printing pin holder device according to a fifth embodiment of the present invention;

[0020] Figure 6 is a perspective view and a schematic diagram of a printing pin holder device according to a seventh embodiment of the present invention;

[0021] Figure 7 is a perspective view and a schematic diagram of a container according to a first embodiment of the present invention and a schematic diagram of a printing pin holder of a sterile isolator;

[0022] Figure 8 is a perspective view and a schematic view of a container according to a second embodiment of the present invention and a schematic view of a printing pin holder of a sterile isolator;

[0023] Fig. 9 is a perspective view and a schematic diagram of a container according to a third embodiment of the present invention and a schematic diagram of a printing pin holder of a sterile isolator;

[0024] Fig.10 is a perspective view and a schematic diagram of a sterile isolator according to a fourth embodiment of the present invention;

[0025] Fig.11 is a perspective view and a schematic diagram of a sterile isolator according to a fifth embodiment of the present invention;

[0026] Fig.12 is a flow chart representing the steps of a method according to the present invention. DETAILED DESCRIPTION

[0027] In the following description, the term “printing” refers to manual deposition or injection of the bio-ink composition in a sterile isolator and 3D printing with a displaceable robotic arm.

[0028] The printing pin holder device 2 according to the invention is intended to be fixed to a container 4 to form a sterile isolator 6. Different examples of containers will be described in the following description.

[0029] The printing pin holder device 2 according to the invention is also intended to be fixed to a printing cartridge or cartridge of a printing injector 8 for performing a printing operation.

[0030] The printing syringe or printing cartridge may include a bio-ink composition. A bio-ink composition is a biological material that can form hydrogels and cell groups in a controlled three-dimensional shape. An example of a bio-ink is described in WO 2017115056.

[0031] For printing operations, the printing syringe or ink cartridge may be mounted on a displaceable printer, for example on a displaceable robotic arm.

[0032] refer to Figure 1 The printing pin holder device 2 according to the first embodiment of the present invention comprises a pin connector 10, a printing pin 12 held by the pin connector, and a flexible sheath 14 having a first end edge 16 tightly fixed to the pin connector.

[0033] The needle connector 10 used here is a tip that is usually connected to the open end of the barrel of a medical syringe. The needle connector 10 includes a needle hub that holds the needle and a connector part that is configured to connect to a corresponding connector part of the barrel of the syringe. The connector part and the corresponding connector part are, for example, Luer lock connectors.

[0034] Alternatively, the needle connector 10 is a needle connector that is integral to the print cartridge.

[0035] Preferably, the printing pin 12 is a biological printing pin.

[0036] The flexible sheath defines an internal cavity open to the outside. The flexible sheath comprises a first end edge 16 tightly fixed to the needle hub and a second end edge 18 defining an opening open to the outside. The second end edge 18 is intended to be fixed to the container 4. Preferably, as shown in the figure, the second end edge is opposite to the first end edge.

[0037] The flexible sheath 14 may be made of one of thermoplastic elastomers, thermosetting materials, and silicone.

[0038] In particular, the silicone may be one of the following materials: 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), nitrile rubber (copolymer of butadiene and acrylonitrile, NBR), also known as Buna N rubber, 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, a copolymer of ethylene and propylene) and EPDM rubber (ethylene propylene diene rubber, a 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] As a non-limiting example Figure 1 In the first embodiment shown, the flexible sheath 14 is made of a thermoplastic elastomer that is welded to the needle hub.

[0040] The flexible sheath 14 forms a flexible sleeve.

[0041] In the embodiment shown, the flexible sheath 14 forms a skirt extending around the needle hub. The flexible sheath here has a generally trumpet shape. Alternatively, the flexible sheath 14 can have a cup shape, a band shape, a tube shape or a folded tube shape.

[0042] Advantageously, the sheath allows the needle to be displaced in the vertical direction. Thus, it is no longer necessary to use a printer in the form of a vertically movable printing plate.

[0043] Advantageously, during the printing process of the three-dimensional biological structure in the sterile isolator, the sheath allows the needle to be displaced in both horizontal and vertical directions relative to the container which is in a fixed position on the printing platform.

[0044] When the needle holder device is fixed to a rigid container, the size of the opening is at least as large as the size of the biological structure to be printed. When the needle holder device is fixed to a flexible container, the opening can have a smaller size of the biological structure to be printed because the flexibility of the container will be used to allow movement of the needle.

[0045] Preferably, the second end edge 18 defines an opening 20 which is at least two times larger than the opening defined by the first end edge fixed to the needle hub.

[0046] Preferably, the printing needle 12 is a biological printing needle. The needle connector 10 and the printing needle 12 form a printing head.

[0047] The printing pin holder device 2 according to the first embodiment further comprises a rigid flange 22 fixed to the second end edge 18. In the embodiment shown, the rigid flange 22 presents an annular shape. The rigid flange can be circular, rectangular or square. The rigid flange can be made of plastic or stainless steel, for example.

[0048] The rigid flange 22 is intended to be fixed to the orifice 24 of the container 4. To this end, the rigid flange 22 can advantageously have a first connector part 26, which is suitable for fixing to a complementary connector part of the rigid flange 28 of the container, as described below. The first connector part 26 can be a sterile connector or a non-sterile connector. It can involve a clamp system, in particular a three-clamp system.

[0049] Alternatively, the rigid flange 22 may be fixed to the orifice 24 of the container by any fixing means, such as attachment links, rubber, etc. The rigid flange 22 may also be welded to the flexible membrane of the container 4, as described below.

[0050] exist Figure 1 In the embodiment shown, the needle connector 10 comprises a second connector part 30, which is suitable for fixing to a complementary connector part of a syringe or a printing ink cartridge. The second connector part 30 can be a sterile connector or a non-sterile connector. It can involve a clamp system, in particular a three-clamp system.

[0051] Advantageously, Figure 1 The printing pin holder device 2 shown comprises a pin protection cap 32 adapted to snap onto an abutment ring of the pin hub 10. The pin protection cap avoids drilling of the flexible sheath.

[0052] Advantageously, the printing pin holder device 2 may include a receiving sleeve 34 suitable for receiving and holding the printing pin 12 before or after printing. Figure 1 In the embodiment shown, the receiving sleeve 34 is integral with the rigid flange 22 .

[0053] Alternatively, the receiving sleeve 34 is integral with the flexible sheath, such as Figure 6 In the latter embodiment, the receiving sleeve is preferably made of the same material as the flexible sheath.

[0054] refer to Figure 2 , the printing pin holder device 36 according to the second embodiment of the invention is similar to the printing pin holder device according to the first embodiment, except for the fact that the flexible sheath presents a different shape. 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. In this second embodiment, the pin connector 10 is fixed between two joints 38 of the first end edge 16 of the flexible sheath. At each joint, a part of the first edge 16 of the flexible sheath is fixed to another part of the first end edge to form a gusset 40.

[0055] refer to Figure 3 The printing pin holder device 42 according to the third embodiment of the invention is similar to the printing pin holder device according to the first embodiment, except for the fact that the flexible sheath 14 presents two sets of pin connectors 10 and needles. 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.

[0056] In this embodiment, the printing pin holder device 42 comprises two pin connectors 10, 44 holding two printing pins 12. To this end, the flexible sheath 10 presents two peaks 46, 48 or fingers on its upper side. One part of the first end edge 16 is assembled or molded to form a first peak 46 tightly fixed to the first pin connector 10. Similarly, another part of the first end edge 16 is assembled or molded to form a second peak 48 and tightly fixed to the second pin connector 44.

[0057] Preferably, this embodiment is made of molded silicone.

[0058] Alternatively, the printing pin holder device 42 may include three, four or five sets of pin headers and pins.

[0059] This embodiment can be used, for example, when performing multiple printing steps of the same bio-ink separated by interruptions. For example, cells can be cultured during these interruptions. Advantageously, a different needle and needle adapter is used for each printing step to avoid contamination of the already printed three-dimensional structure by residual material remaining in the needle.

[0060] When performing several printing steps with different bio-inks to print different types of cells or tissues (e.g., muscle and skin cells), this embodiment can also be used. In this case, different bio-inks are introduced in each printing needle 12 and needle connector 10, 44.

[0061] refer to Figure 4 , a printing pin holder device 52 according to a fourth embodiment of the present invention comprises a rigid frame 54, a diaphragm 56 supported by the rigid frame, and a flexible sheath 14 tightly fixed to the rigid frame.

[0062] The membrane 56 constitutes a septum 56 intended to be pierced by a printing needle of a syringe, such as Figure 4 For example, the syringe is housed in a housing of a movable robotic arm.

[0063] exist Figure 1 In the non-limiting embodiment shown, the rigid frame 54 has a generally oblong shape. The septum 56 is tightly sealed to the inner face of the rigid frame. The septum is configured so that it can be pierced. To this end, the septum is made of a material commonly used to make septa.

[0064] In the embodiment shown, the flexible sheath 14 presents the same shape as the pin holder device of the second embodiment. The flexible sheath may also have the same shape as the pin holder device of the first embodiment or the shape of the third embodiment with several rigid frames and membranes.

[0065] As for other characteristics, the flexible sheath 14 of the device according to the fourth embodiment is similar to the flexible sheath 14 of the device according to the first embodiment. It will not be described again.

[0066] Advantageously, the printing pin holder according to this embodiment also allows displacement of the rigid frame and the printing pins in two perpendicular horizontal directions as well as in the vertical direction during printing of a three-dimensional biological structure in a container.

[0067] refer to Figure 5 The pin holder device 58 according to the fifth embodiment of the invention is similar to the pin holder device 2 according to the first embodiment, except for the fact that the pin holder device does not comprise a rigid flange.

[0068] Other technical elements of the fifth embodiment which are identical to those of the first embodiment have been referenced by the same reference numerals and will not be described again.The pin holder device 58 according to this embodiment is particularly suitable for fixing to a container comprising a rigid flange, as described below.

[0069] refer to Figure 6 The printing pin holder device 60 according to the sixth embodiment of the present invention is similar to the printing pin holder device according to the first embodiment, except that:

[0070] - the needle connector 10 has been replaced by a rigid frame 54 supporting a septum 56, and

[0071] The printing pin holder device 60 does not comprise a rigid flange fixed to the second end edge of the flexible sheath.

[0072] Other 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.

[0073] The printing pin holder device according to the present invention can be fixed to any existing container to form a sterile and airtight isolator. The present invention also relates to a sterile isolator. This sterile isolator comprises different containers that are particularly suitable for fixing to the printing pin holder device according to the present invention.

[0074] The sterile isolator 6 according to the first embodiment of the present invention comprises Figure 7 A container 4 and a schematically indicated printing pin holder device 2 are shown.

[0075] The container 4 can be fixed to Figures 1 to 4 Any of the printed needle holder devices shown is used to form a sterile and airtight isolator. After fixing, a sterilization process is performed, for example by gamma irradiation.

[0076] The container 4 includes a base wall 62, side walls 64, a top wall 66 and an orifice 24 adapted to be secured to the rigid frame 22 of any printing pin holder device, such as Figures 1 to 4 shown.

[0077] The side wall of the container may be a single side wall, for example when the container has a cylindrical shape, or may be a plurality of side walls, for example when the container has a parallelepiped shape. In this patent application, the term "side wall" will be used to represent both possibilities in order to simplify the understanding of the description. The base wall and the side walls may be made of one or several plastic sheets.

[0078] The side walls 64, base wall 62, top wall 66 and the printing pin holder device define an internal chamber 68. The fixing between the container 4 and the rigid frame 22 can be done by any fixing means (for example, by welding or by attaching connecting rods). The fixing must hermetically close the internal chamber.

[0079] The three-dimensional biological structure is intended to be printed on the base wall.The sterile isolator obtained by fixing the container 4 to the needle holding device is intended to receive the cell culture medium in the inner chamber.

[0080] In the embodiment shown, the top wall 66 comprises an aperture 24. This aperture 24 is closed by a printing pin holder arrangement.

[0081] Alternatively, the side wall 64 may include an aperture rather than a top wall.

[0082] In the illustrated embodiment, the base wall 62, the side walls 64 and the top wall 66 are made of a flexible film. The flexible film may be made of a thermoplastic elastomer or a thermosetting material.

[0083] The flexible film is usually 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 an adhesive 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.

[0084] The barrier layer blocks the passage of gases such as oxygen, carbon dioxide and is typically made of ethylene vinyl alcohol (EVOH).

[0085] 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 separator from deforming when filled with product.

[0086] The outer layer may comprise the polyolefin alone or in admixture with a copolymer of ethylene and an alpha-olefin.

[0087] The side wall 64 and the base wall 62 are realized from the same sheet material. Alternatively, the side wall 64 and the base wall 62 are realized from two different sheets material welded to each other.

[0088] Advantageously, the container 4 is flexible so that the side wall 64 can move during the printing process to follow the movement of the syringe if necessary.

[0089] The container 4 comprises an inlet port 70 and an outlet port 72 for circulating culture medium to culture the biological three-dimensional structure after the printing process.

[0090] Preferably, the container 4 comprises a replenishment port, not shown. The replenishment port is used to fix sensors for controlling different parameters of the culture medium, for example its temperature.

[0091] Preferably, the container 4 comprises an extension 74 protruding from the container and extending in a horizontal plane. The extension comprises a relief 76 to reference the position of the container relative to the support and in particular to the printing platform.

[0092] In the non-limiting 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 74 .

[0093] The extension 74 protrudes from the base wall 10 in a horizontal plane.

[0094] In the embodiment shown, the extension 74 has four perforated tabs 78 regularly spaced around the base wall. The holes of these tabs are suitable for placement in four reliefs made on the printing platform to reference the position of the sterile isolator 6.

[0095] Advantageously, the sterile isolator 6 can be removed from the printing platform after the first printing cycle to be placed in another position for the cell / culture process. Afterwards, the sterile isolator can be placed again on the printing platform for the second printing cycle at the same position as for the first printing cycle. Correct positioning is important for printing biological three-dimensional structures, because the biological three-dimensional structures must be precisely shaped.

[0096] Alternatively, the extension portion 74 includes only two embossments 76 having a circular arc shape. The embossments 76 are through holes.

[0097] Advantageously, the extensions 74 also allow the container to be stabilized on the support since they extend in a horizontal plane.

[0098] Advantageously, the sterile isolator 6 according to this first embodiment can be placed in a rigid container. The latter does not need to be sterilized.

[0099] In the embodiment shown, the container 4 has the general shape of an inverted truncated pyramid. The container may have any other shape, for example Fig. 9 The parallelepiped shape or the inverted frustum shape shown.

[0100] Similarly, the side wall 64 may have any cross section among a circular cross section, a parallelepiped cross section, a square cross section, and a rectangular cross section.

[0101] The side wall 64 may be formed from a single sheet, or from several sheets welded to one another.

[0102] The container 4 may also comprise a base rigid frame fixed to the base wall made of the flexible film to form a flat surface. The base rigid frame is not represented on the illustrated embodiment.

[0103] refer to Figure 8 , a sterile isolator 81 according to a second embodiment of the present invention comprises a container 80 and a schematically indicated printing pin holder device 58 .

[0104] Alternatively, the sterile isolator 81 may be secured by any fixing element. Figure 6 The printing pin holder device 60 is shown fixed to Figure 8 The container 80 shown is made.

[0105] Container 80 is similar to Figure 7 The container is shown except that it also includes an upper rigid flange 28 which is tightly secured to the top wall 66 of the container around the orifice 24 .

[0106] Technical elements of the sterile isolator of the second embodiment that are identical to technical elements of the first embodiment have been referenced by the same reference numerals and will not be described again.

[0107] Alternatively, Figures 1 to 4 The print pin holder device shown can also be fixed to Figure 8 The container 80 is shown. In this case, the rigid flange 28 preferably comprises a connector part 82 adapted to be fixed to the first connector part 26 of the printing pin holder device.

[0108] Advantageously, both the first connector part 26 of the rigid flange 22 of the print holder device and the connector part 82 of the container comprise a clamp system, in particular a three-clamp system. Thus, the print holder device and the container can be sterilized separately and fixed together if necessary.

[0109] The connector component 82 may also be a non-sterile connector.

[0110] The rigid flange 28 is made of plastic, for example.

[0111] The rigid flange 28 may have a septum which hermetically closes the orifice 24 of the container before the container is secured to the needle holder device.

[0112] refer to Fig. 9 , a sterile isolator 85 according to a third embodiment of the present invention comprises a container 84 and a schematically indicated printing pin holder device 58 .

[0113] Container 84 is similar to Figure 8 The container is shown, except that it has a parallelepiped shape.

[0114] Technical elements of the sterile isolator of the third embodiment that are identical to technical elements of the first embodiment have been referenced by the same reference numerals and will not be described again.

[0115] Figure 6 The printing pin holder device 60 shown can also be fixed to Fig. 9 Container 84 is shown.

[0116] refer to Fig.10 A sterile isolator 91 according to a fourth embodiment of the present invention comprises a container 90 having a rigid base wall 92, rigid side walls 94 and a printing pin holder device 58 suitable for the fifth embodiment.

[0117] In this embodiment, the container 90 does not include a top wall. The second end edge 18 of the needle holder device is tightly fastened to the top portion of the side wall. The printing needle device 58 forms the entire top wall of the sterile isolator. In the illustrated embodiment, the printing needle holder device 58 is tightly fixed to the container 90 by an attachment rod.

[0118] In the illustrated embodiment, the side wall 94 has a cylindrical shape. The extension 74 includes four opposed perforated tabs 78 that protrude horizontally from the base wall to reference the position of the sterile isolator.

[0119] Advantageously, the inner face of the side wall has a receiving sleeve 96 suitable for receiving the printing pin 12 before or after a printing operation. The receiving sleeve 96 is preferably arranged against the side wall.

[0120] Preferably, the inner face of the side wall includes a vessel 98. The vessel is used to collect residual bio-ink during the priming phase to adjust the flow rate of the printing needle. The vessel is also used to clean the residual bio-ink in the needle. After printing, the residual bio-ink remaining in the needle can be advantageously cleaned in the vessel. To simplify Fig.10 and 11 , the vessel has been represented by the same element as the receiving sleeve 96.

[0121] Advantageously, the side walls 94 are rigid so that they do not fall into and damage the printed three-dimensional structure.

[0122] When a rigid container is used, the opening 20 defined by the second end edge 18 of the printing needle holder has at least the size of the opening of the container. Similarly, the opening 20 defined by the second end edge 18 of the printing needle holder has at least the size of the biological structure to be printed.

[0123] refer to Fig.11 , a sterile isolator 88 according to a fifth embodiment of the invention is similar to a sterile isolator 91 according to the fourth embodiment of the invention, except that a container 92 has a flexible base wall 62 and a rigid side wall 94. In this embodiment, the flexible membrane of the base wall is secured to a cylindrical side wall to form the container.

[0124] Advantageously, the flexible membrane of the base wall is attached to the lower edge of the rigid side wall.Thus, advantageously, the flexible membrane remains flat during the printing process.

[0125] Other technical elements of the fifth embodiment that are identical to those of the fourth embodiment have been referenced by the same reference numerals and will not be described again.

[0126] According to another embodiment, not shown, the base wall is rigid and the side walls are fixed to the base wall to form the inner chamber.

[0127] The present invention also relates to a method for printing and culturing a three-dimensional biological structure in a sterile isolator according to any of the above embodiments. The method comprises starting with an initial step, during which

[0128] -according to Figures 1 to 3The needle connector 10 of the printing needle holder device of the embodiment shown in FIG. 5 is fixed to a printing ink cartridge or a syringe, or

[0129] -according to Figure 5 and 6 The septum 56 of the printing needle holder device of the illustrated embodiment is pierced by the printing syringe 8 .

[0130] At step 102, a three-dimensional biological structure is printed, and the flexible sheath allows the needle hub 10 and the needle to move in three perpendicular directions while maintaining the sterility of the interior chamber.

[0131] At step 104, a cell culture medium is supplied to the interior chamber,

[0132] At step 106 , a flexible sheath is assembled and secured together to separate the bioprinting needle from the internal chamber of the bioreactor to prevent residual bio-ink remaining in the needle from contaminating the printed three-dimensional structure.

[0133] Preferably, the fixing is performed by welding the two opposing parts of the flexible isolator.The clamp can also be used to separate the two parts of the flexible isolator.

Claims

1. A printing pin holder device (2, 36, 42, 58) adapted to be fixed to a container (4) having an orifice (24), the printing pin holder device comprising: - at least one needle connector (10), at least one printing pin (12) extending in a defined direction (Z), said at least one pin connector holding said printing pin (12), a flexible sheath (14) having a first end edge (16) tightly secured to said at least one needle connector and a second end edge (18) delimiting an opening (20) intended to be secured to said orifice (24) of said container, When the second end edge (18) is fixed to the container (4) to print a three-dimensional biological structure in the container, the printing pin holder (2) allows the printing pin (12) to be displaced in at least two perpendicular directions (X, Y), the two perpendicular directions (X, Y) being perpendicular to the defined direction (Z).

2. A printing pin holder device (52, 60) adapted to be fixed to a container (4) having an orifice (24), the printing pin holder device comprising: - at least one rigid frame (54) and a septum (56) supported by said rigid frame, said septum being designed to be pierced by a printing pin (12), a flexible sheath (14) having a first end edge (16) tightly fixed to said at least one rigid frame (54) and a second end edge (18) delimiting an opening (20) intended to be fixed to said orifice (24) of said container, When the second end edge (18) is fixed to the container to print a three-dimensional biological structure in the container, the printing pin holder (52, 60) allows the printing pin (12) to be displaced in at least two directions (X, Y), the two perpendicular directions (X, Y) being in a plane parallel to the spacer.

3. The pin holder device (2, 36, 42, 52, 58, 60) according to claims 1 and 2, comprising a rigid flange (22) fixed to the second edge (18), the rigid flange being intended to be fixed to the orifice (24) of the container.

4. The printing pin holder device (2, 36, 42, 52, 58, 60) according to claim 3, characterized in that: The rigid frame (22) has a first connector part (26) intended to be fixed to the container.

5. The printing pin holder device (2, 36, 42, 52, 58, 60) according to any one of claims 1 to 4, characterized in that The at least one flexible sheath (14) is made of a material selected from the group consisting of thermoplastic elastomer, thermosetting material and silicone.

6. A printing pin holder device (2, 36, 42, 52, 58, 60) according to claims 1 to 5, comprising a receiving sleeve (34) adapted to receive the pin.

7. The printing pin holder device (52, 60) according to any one of claims 1, 3 to 6, comprising at least one needle protection cover (32) adapted to be snapped onto the needle connector (10).

8. The printing pin holder device (52, 60) according to any one of claims 1, 3 to 7, characterized in that: The at least one needle connector (10) comprises at least one second connector part (30) adapted to be connected to a complementary connector part of a print cartridge.

9. The printing pin holder device (52, 60) according to any one of claims 1, 3 to 7, comprising a printing ink cartridge integral with the at least one needle connector (10).

10. A sterile isolator (6, 91, 88), comprising a container (4, 90, 92), wherein the container comprises: - a base wall (62, 92), - at least one side wall (64, 94), said at least one side wall and said base wall defining an internal chamber (68), - at least one inlet opening (70) and at least one outlet opening (72), - an orifice (24), Characterized in that the sterile isolator comprises a printing pin holder device (2, 36, 42, 52, 58, 60) according to any one of claims 3 to 9, wherein the rigid flange (22) of the printing pin holder device is fixed to the container to close the orifice; the three-dimensional biological structure is intended to be printed on the base wall (62), and the internal chamber (68) is intended to receive a cell culture medium.

11. A sterile isolator (81, 85), comprising a container (80, 84), the container comprising: - a base wall (62, 92), - at least one side wall (64, 94), said at least one side wall and said base wall defining an internal chamber (68), - at least one inlet opening (70) and at least one outlet opening (72), - an upper rigid flange (28) which delimits the orifice (24), Characterized in that the sterile isolator comprises a printing pin holder device (2, 36, 42, 58) according to any one of claims 1, 4 to 9, the upper rigid flange (28) being fixed to the opening (20) of the printing pin holder device, the three-dimensional biological structure being intended to be printed on the base wall (62, 92), and the internal chamber (68) being intended to receive a cell culture medium.

12. The sterile isolator (6, 81, 85, 91, 88) according to any one of claims 10 and 11, characterized in that: The base wall (62) and the at least one side wall (64) are made of a flexible film.

13. The sterile isolator according to any one of claims 10 and 11, characterized in that: The base wall (92) is rigid and the at least one side wall (94) is made of a flexible film.

14. The sterile isolator (91) according to any one of claims 10 and 11, characterized in that: The base wall (62) is made of a flexible film and the at least one side wall (64) is rigid.

15. The sterile isolator (88) according to any one of claims 10 and 11, characterized in that: The base wall (92) and the at least one side wall (94) are rigid.

16. The sterile isolator (6, 81, 85, 91, 88) according to any one of claims 10 to 15, comprising a receiving sleeve (96) for receiving the printing pin.

17. A sterile isolator (6, 81, 85, 91, 88) according to any one of claims 10 to 15, comprising a vessel (98) for initiating the flow of biological ink in the printing needle and / or for cleaning the printing needle.

18. The sterile isolator (6, 81, 85, 91, 88) according to any one of claims 10 to 16, characterized in that: The base wall (62) includes at least one extension (74) protruding from the base wall in a horizontal plane.

19. The sterile isolator (6, 81, 85, 91, 88) according to claim 18, characterized in that: The extension (74) includes a relief to reference the position of the isolator relative to a support such as a print platform.

20. The sterile isolator (6, 81, 85, 91, 88) according to the combination of claims 1, 3 and 10, characterized in that: The rigid flange (22) of the pin holder device is fixed to an upper rigid flange (28) of the container.

21. The sterile isolator (6, 81, 85, 91, 88) of claim 19, comprising a septum secured to the upper rigid flange.

22. A method for printing and culturing a three-dimensional biological structure in a sterile isolator (6, 81, 85, 91, 88) according to any one of claims 9 to 21, the method comprising the following steps in succession: - printing (102) the three-dimensional biological structure, the flexible sheath allowing the needle hub and the printing needle to move in at least two perpendicular directions during the bioprinting step while maintaining the sterility of the internal chamber, - Supplying (104) cell culture medium to said inner chamber (68).

23. The printing method according to claim 22, further comprising fixing the flexible sheath (106) to separate the printing pin (12) from the internal chamber, such as by welding.

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