Sterile isolator, cover and method for printing biological three-dimensional structure

Abstract

The invention relates to a sterile isolator (2) intended for printing a three-dimensional biological structure, said isolator comprising:-a support (4),-a cover (6) comprising a rigid part (22) and a flexible part (24) extending in an extension of said rigid part, said cover (6) being sealed to said support (4) so as to define an internal chamber (32), at least a portion of the rigid member is a septum (30) adapted to be pierced by a printing needle for printing a biological three-dimensional structure in the internal chamber. The invention also relates to a biological printing device and a printing method.

Description

Technical Field

[0001] The invention relates to a sterile isolator, a cover and a method for printing 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 house 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. Sterilizing the printer or the 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 during the cell culture process to place the container in another room. Summary of the invention

[0005] A first object of the present invention is to propose a sterile isolator that allows the print head to be easily displaced in at least two orthogonal directions, in particular in three orthogonal 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 sterile isolator 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.

[0006] A second 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 is significantly reduced.

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

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

[0009] A fifth 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.

[0010] The sixth object of the present invention is to provide a method for printing and culturing three-dimensional biological structures.

[0011] These objects have been achieved by the sterile isolator, the cover and the method as defined in the independent claims. Secondary features of the sterile isolator and the method 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 cover according to a first embodiment of the present invention;

[0013] Figure 2 yes Figure 1 A radial cross section of the cover shown;

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

[0015] Figure 4 is a schematic diagram of a cross section of a sterile isolator and a fluid source according to a first embodiment of the present invention;

[0016] Figure 5 is a schematic diagram of a cross section of a bioprinting apparatus according to the present invention, a fluid source, and a robotic arm during the steps of a printing method according to the present invention;

[0017] Figure 6 is a schematic diagram of a cross section of a bioprinting apparatus according to the present invention, a fluid source, and a robotic arm during another step of a printing method according to the present invention;

[0018] Figure 7 is a flow chart showing the steps of a method according to the present invention;

[0019] Figure 8 is a schematic diagram of a cross section of a sterile isolator and a fluid source according to a third embodiment of the present invention. DETAILED DESCRIPTION

[0020] The sterile isolator 2 according to the present invention is intended to be fixed to a printing cartridge or a printing syringe for printing biological three-dimensional structures in the sterile isolator.

[0021] The printing syringe or printing cartridge may include a bio-ink composition. A bio-ink composition is a biological material that allows a hydrogel including a cell population to be made in a controlled three-dimensional shape. An example of a bio-ink is described in WO 2017115056.

[0022] 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.

[0023] In the following description, the term "printing" refers to manual deposition or injection of bio-ink or 3D printing with a displaceable robotic arm. Similarly, the term "printing needle" is used to refer to a needle or deposition nozzle.

[0024] refer to Figure 4 , a sterile isolator 2 according to a first embodiment of the present invention comprises a support 4, a cover 6 and a fixing element 8, which is configured to fix the cover to the support 4 in a sealed and airtight manner.

[0025] In the embodiment shown, the fixing element 8 is a clamping ring. Alternatively, the fixing element 8 may be a hook, for example hinged to the support and adapted to wedge the edge of the cover into the edge of the upper surface of the support. Alternatively, the cover may be sealed in another way, by welding, gluing, etc.

[0026] The support 4 comprises a plate 10 having a substantially flat upper surface 12 suitable for receiving the bio-ink. The support has for example the shape of a disc.

[0027] The support 4 comprises at least one fluid inlet 14 for injecting fluid into the internal chamber and a fluid outlet 16 for discharging fluid from the internal chamber. In the embodiment shown, the fluid inlet 14 is connected to a fluid source. The fluid inlet 14 and the fluid outlet 16 are provided with controllable valves 17.

[0028] The support 4 also comprises at least one inlet port 18 and an outlet port 20 for circulating a cell culture medium. The cell culture medium is used to feed the printed biological three-dimensional structure.

[0029] Alternatively, the culture medium can be replaced with a physiological liquid solution (NaCl 0.15 M).

[0030] exist Figure 1 and 4 In the embodiment shown, the cover 6 presents the general shape of a flat pad, comprising a hollow cylinder 21 extending perpendicularly relative to the flat pad.

[0031] It comprises a more rigid part 22 and a more flexible part 24 surrounding the more rigid part.

[0032] To simplify the description, the most rigid component 22 will be referred to below as a “rigid component”, and the most flexible component will be referred to below as a “flexible component.” The term “flexible” here means that it bends easily without breaking.

[0033] The flexible part 24 extends in the extension of the rigid part. The rigid part 22 is preferably located in the center of the flexible part. In order to facilitate the understanding of the invention, the rigid part 22 has been shown in different ways with hatching.

[0034] The hollow cylinder 21 belongs to the rigid part 22 and is made of the same material as the rigid part. The hollow cylinder 21 is placed over a portion of the flat pad. The hollow cylinder is blocked at its lower end by a portion of the rigid part forming a spacer.

[0035] The hollow cylinder 21 is intended to receive a needle connector 26 of a printing syringe or ink cartridge 28, such as Figure 5 The part of the rigid component located in the hollow cylinder will be pierced by the printing needle 29 of the printing syringe or ink cartridge. The part of the rigid component located in the hollow cylinder 21 forms a septum 30.

[0036] The needle connector 26 is firmly held by the hollow cylinder 21. Advantageously, the hollow cylinder 21 increases the stability of the printing needle 29 when the printing needle moves in two horizontal directions to print a biological three-dimensional structure.

[0037] Preferably, the rigid part 22 has a size greater than the hollow cylinder 21. In particular, the rigid part 22 extends around the hollow cylinder 21 a radial distance D comprised between two and five times the inner diameter d of the hollow cylinder.

[0038] Advantageously, the fact that the rigid part 22 has dimensions greater than the diameter d of the hollow cylinder 21 helps to avoid tearing of the cap 6 under the pressure of the printing syringe and under the pressure of the fluid contained in the cap.

[0039] Preferably, the cover 6 is made of an elastic material that can be easily bent, extended and expanded. For example, the cover 6 can be made of an elastomer.

[0040] Specifically, the rigid component 22 can be made of silicone. More preferably, the rigid component 22 can be made of liquid silicone rubber (LSR), and more preferably made of liquid silicone rubber of series 4300. For example, LSR called LSR4370 can be used.

[0041] Alternatively, the rigid component 22 may be made from a thermoplastic elastomer (TPE).

[0042] The rigid component 22 has a Shore hardness comprised between A40 Shore and A100 Shore.

[0043] The rigid part 22 has an elongation comprised between 100% and 600%. The elongation is the ratio of the extension of a material to the length of the material before stretching.

[0044] The flexible member 24 is made of silicone and in particular of liquid silicone rubber (LSR), and more preferably of liquid silicone rubber of series 4300. For example, liquid silicone rubber called LSR4301 may be used.

[0045] The flexible part 24 of the cover is elastic and has an elongation comprised between 800% and 1100%.

[0046] The flexible part 24 of the cover has a Shore hardness comprised between A0 Shore and A30 Shore and preferably comprised between A0 Shore and A15 Shore.

[0047] The flexible part 24 of the cover has a viscosity comprised between 3 and 9 millijoules per square centimeter and preferably comprised between 6 and 9 millijoules per square centimeter. The viscosity is measured, for example, by using the viscosity probe method described on the Internet at the following address: https: / / adhesives.specialchem.com / selection-guide / test-methods-to-evaluate-tack.

[0048] Advantageously, this adhesiveness allows the cover 6 to adhere to the support 4 and to keep the internal chamber 32 airtightly closed.

[0049] The cover may be formed by bi-material molding.

[0050] Alternatively, the rigid component 22 is made by 3D printing and the flexible component 24 is formed by molding.

[0051] Before printing, the fluid source 15 is configured to introduce fluid into the fluid inlet 14. Under the action of the fluid pressure, Figure 1 The flat pad of the cover shown expands to form a Figure 4 The hemisphere shown. The cover 6 and the support 4 form a hermetically sealed housing. This housing defines an internal chamber 32, in which biological three-dimensional structures can be printed.

[0052] The fluid introduced into the interior chamber by source 15 may be a gas, such as sterile air, carbon dioxide, oxygen, ammonia, or an inert gas, such as nitrogen.

[0053] The gas is suitable for expanding the volume of the internal chamber. The gas exerts pressure on the inner surface of the cover 6 to expand it.

[0054] Alternatively, the fluid introduced into the internal chamber can be a gel. In this case, the cover is filled with gel so that the suspension printing of the biological three-dimensional structure can be performed inside.

[0055] In this case, the gel is commercialized, for example, by Merck (registered trademark) at a concentration of 15% to 30% in water or HBSS medium. Acid F-127 gel, carbomer gel and gelatin particle suspension.

[0056] according to Figure 3 In the second embodiment shown, the cover 34 does not include the hollow cylinder 21. The cover is in the shape of a flat pad. According to this embodiment, the entire surface of the rigid part 22 can be pierced by the printing needle. The rigid part 22 forms the spacer 30.

[0057] Alternatively, the cover 6 has a hemispherical shape like a bell or a cup.

[0058] The support is preferably made of medical grade material.

[0059] The support 4 may be a disposable support, for example made of a rigid polymer material, such as a thermoplastic or a thermosetting plastic.

[0060] Advantageously, this disposable support can be set up very quickly at any time and anywhere.

[0061] Advantageously, the support is disposable. Significant time gain can be achieved between prints because it does not require washing and sterilization. No special detergents need to be purchased and stored.

[0062] Alternatively, the support may be made of an inorganic material such as glass or ceramic, or of a metal such as stainless steel.

[0063] Advantageously, the metal support can be washed and sterilized several times.

[0064] The sterile isolator may include a cover, not shown, adapted to be received in the hollow cylinder 21 to close the sterile isolator after printing.

[0065] Alternatively, the sterile isolator includes a clamp adapted to grip the cover after printing to separate the septum from the interior chamber.

[0066] Alternatively, the cover 6, 34, 58 may be made of the following materials:

[0067] Unsaturated rubbers that can be cured by sulfur vulcanization:

[0068] Natural polyisoprene: cis-1,4-polyisoprene natural rubber (NR) and trans-1,4-polyisoprene gutta-percha

[0069] Synthetic polyisoprene (IR stands for isoprene rubber)

[0070] Polybutadiene (BR stands for butadiene rubber)

[0071] Chloroprene rubber (CR), polychloroprene, chloroprene rubber, Bayer, etc.

[0072] Butyl rubber (copolymer of isobutylene and isoprene, IIR)

[0073] ○Halogenated butyl rubber (chlorinated butyl rubber: CIIR; brominated butyl rubber: BIIR)

[0074] Styrene-butadiene rubber (copolymer of styrene and butadiene, SBR)

[0075] Nitrile rubber (copolymer of butadiene and acrylonitrile, NBR), also known as Buna N rubber

[0076] ○Hydrogenated nitrile rubber (HNBR) Therban and Zetpol

[0077] (Unsaturated rubbers can also be cured by non-sulfur vulcanization if desired.)

[0078] Saturated rubbers that cannot be cured by sulfur vulcanization:

[0079] EPM (ethylene propylene rubber, a copolymer of ethylene and propylene) and EPDM rubber (ethylene propylene diene monomer rubber, a terpolymer of ethylene, propylene and a diene component)

[0080] ·Epichlorohydrin rubber (ECO)

[0081] ·Acrylic rubber (ACM, ABR)

[0082] Silicone rubber (SI, Q, VMQ)

[0083] Fluorosilicone rubber (FVMQ)

[0084] Fluoroelastomers (FKM and FEPM) Viton, Tecnoflon, Fluorel, Aflas and Dai-El

[0085] Perfluoroelastomer (FFKM) Tecnoflon PFR, Kalrez, Chemraz, Perlast

[0086] Polyether block amide (PEBA)

[0087] Chlorosulfonated polyethylene (CSM), (Hypalon)

[0088] Ethylene vinyl acetate (EVA)

[0089] Various other types of 4S elastomers:

[0090] Thermoplastic elastomer (TPE)

[0091] Arthropod protein and elastin

[0092] Polysulfide rubber

[0093] Elastic polyolefins, elastic fibers for fabric production

[0094] Poly(dichlorophosphazene), an "inorganic rubber" made by polymerizing hexachlorophosphazene.

[0095] refer to Figure 8 The sterile isolator 54 according to the third embodiment of the present invention is similar to the sterile isolator 2 according to the first embodiment, except that the cover is different from Figure 1 and 3 Specifically, Figure 1 The hollow cylinder 21 is not blocked by a part of the rigid part forming the septum. In this third embodiment, the cover 58 comprises a rigid part 22 and a flexible part 24 extending in the extension of the rigid part. The rigid part 22 comprises a through hole in the continuation of the hollow cylinder 21. The needle connector 26 passes through the through hole of the cover. The needle connector 26 is sealed to the inner surface of the hollow cylinder and the inner surface of the through hole.

[0096] The needle connector 26 is a tip that is typically connected to the open end of the barrel of a medical syringe. The needle connector 26 includes a needle hub that holds the needle and a connector component 56 that is configured to connect to a corresponding connector component of the barrel of a syringe or cartridge. The connector component and the corresponding connector component are, for example, Luer lock connectors.

[0097] Advantageously, Figure 8 The illustrated sterile isolator 54 may include a needle protection cover adapted to snap onto an abutment ring of the needle hub.

[0098] Other technical elements of the second embodiment which are identical to those of the first embodiment have been referenced by the same reference numerals and will not be described again. In particular, the cover, the needle connector 26 and the support define a hermetically closed housing.

[0099] Figure 5 The bioprinting device 35 according to the present invention is schematically shown. The bioprinting device 35 includes Figures 1 to 4The sterile isolator 2, the printing cartridge 28 or the printing syringe described. The printing cartridge or the printing syringe 28 has a needle connector 26 holding a printing needle 29. The printing needle 29 passes through the septum 30 of the sterile isolator. The printing cartridge or the printing syringe 28 is fixed to a robot arm 36, which is suitable for moving along the three directions X, Y, Z of an orthogonal coordinate system.

[0100] refer to Figure 7 , the method of printing a biological three-dimensional structure begins by injecting 40 a fluid into the internal chamber 32 for the first time to inflate the cover 6 .

[0101] Preferably, the fluid introduced into the interior chamber by source 15 is a gas, such as sterile air, carbon dioxide, oxygen, ammonia, or an inert gas.

[0102] When the printing is suspension printing, the fluid introduced into the internal chamber is a gel.

[0103] When using Figure 5 When the bioprinting device is shown, during step 42, the printing needle or deposition nozzle penetrates the septum 30 and is introduced into the internal chamber 32. The needle hub 26 is inserted into the hollow cylinder 21. The needle hub 26 is held in the hollow cylinder 21.

[0104] When using Figure 8 When the sterile isolator is shown, during step 42, the printing syringe or ink cartridge is fixed to the connector 56. The printing syringe or printing ink cartridge is fixed to the robot arm, such as Figure 5 Visible above.

[0105] During step 44 , a deposition material, in particular a bio-ink, is deposited into the inner chamber through the inserted needle or nozzle.

[0106] During step 46, the robot 36 moves the print cartridge or syringe 28 relative to the support 4 while the bio-ink is deposited into the interior chamber 32. During this step, the biological three-dimensional structure 36 is printed on the support, such as Figure 6 As shown, or in the case of suspension printing, printing is performed in a gel contained in an internal chamber 32.

[0107] Advantageously, the fluid pressure on the inner surface of the cover prevents the walls of the cover from coming into contact with the upper surface 12 of the support 4 or with the biological three-dimensional structure during the printing operation.

[0108] Advantageously, the pressure of the fluid in the inner chamber is chosen so that the tension on the cover is insignificant, resulting in little pressure on the printing pin 29. Thus, the movement of the printing pin 29 is not hindered by the cover, which protects the biological three-dimensional structure from non-sterile areas.

[0109] Advantageously, the printing process does not need to be performed under a laminar flow hood or a biological safety cabinet.

[0110] Advantageously, the volume of fluid introduced into the internal chamber 32 is adapted to the volume of the biological three-dimensional structure to be printed.

[0111] During step 48, if Figure 6 As shown, the fluid is injected into the internal chamber 32 for the second time to expand the volume of the cover under the pressure of the fluid. Therefore, during the printing process, the volume of the internal chamber gradually adapts to the size of the biological three-dimensional structure 38 that has been printed. Since the volume of the internal chamber may increase during the printing process, the tension on the printing pin 29 is less important. During the printing process, the pressure in the internal chamber 42 is adapted so that there is a short distance between the upper part 39 of the cover and the spacer 30. This short distance ensures that the tension caused by the presence of the cover on the printing pin is not important.

[0112] During step 50 , deposition material, in particular bio-ink, is deposited into the inner chamber through the inserted needle or nozzle. This deposition constitutes a second printing of a biological three-dimensional structure on the already printed three-dimensional biological structure 38 .

[0113] During step 52, the fluid is evacuated and culture medium is introduced into the interior chamber for feeding the biological three-dimensional structure.

[0114] Alternatively, the method comprises only steps 40 to 46 .

Claims

1. A sterile isolator (2) intended for printing biological three-dimensional structures, the isolator comprising: - a support (4), - a cover (6, 34) comprising a rigid part (22) and a flexible part (24) extending in the extension of the rigid part, the cover (6, 34) being sealed to the support (4) to define an internal chamber (32), at least a portion of the rigid part being a septum (30) suitable for being pierced by a printing needle (29) for printing a biological three-dimensional structure in the internal chamber.

2. The sterile isolator (2) according to claim 1, characterized in that: The cover (6, 34) comprises at least one hollow cylinder (21) protruding from the rigid part (22), the hollow cylinder being suitable for accommodating a needle connector (26) holding the printing needle (29), the part of the rigid part being located inside the hollow cylinder forming the septum (30).

3. A sterile isolator (54) intended for printing biological three-dimensional structures, the isolator comprising: - a support (4), a cover (58) sealed to the support (4) to delimit an internal chamber (32), the cover (58) comprising a rigid part (22) and a flexible part (24) extending in the extension of the rigid part, the rigid part (22) being provided with at least one hollow cylinder (21), and - A needle connector (26) which is sealed inside the hollow cylinder (21).

4. The sterile isolator (2, 54) according to any one of claims 1 to 3, characterized in that: The cover (6, 34, 58) is made of elastic material, preferably elastomer.

5. The sterile isolator (2, 54) according to any one of claims 1 to 4, characterized in that: The support (4) comprises at least one fluid inlet (14) for injecting fluid into the internal chamber (32), and the flexible part (24) of the cover (6, 34, 58) is suitable for expanding under the pressure of the fluid contained in the internal chamber (32).

6. The sterile isolator (2, 54) according to any one of claims 1 to 5, characterized in that: The rigid part (22) of the cover is made of thermoplastic elastomer (TPE), or of silicone and in particular of liquid silicone rubber (LSR), and more preferably of series 4300 liquid silicone rubber.

7. The sterile isolator (2, 54) according to any one of claims 1 to 6, characterized in that: The rigid part (22) of the cover has a Shore hardness comprised between Shore A40 and Shore A100.

8. The sterile isolator (2, 54) according to any one of claims 1 to 7, characterized in that: The rigid part (22) of the cover (6, 34, 58) has an elongation comprised between 100% and 600%.

9. The sterile isolator (2, 54) according to any one of claims 2 to 8, characterized in that: The rigid component (22) extends around the hollow cylinder (21) over a radial distance (D) comprised between two and five times the inner diameter (d) of the hollow cylinder (21).

10. The sterile isolator (2, 54) according to any one of claims 1 to 9, characterized in that: The flexible component (24) is made of silicone and in particular of liquid silicone rubber (LSR), and more preferably of series 4300 liquid silicone rubber.

11. The sterile isolator (2, 54) according to any one of claims 1 to 10, characterized in that: The flexible part (24) of the cover (6, 34, 58) is elastic and has an elongation comprised between 800% and 1100%.

12. The sterile isolator (2, 54) according to any one of claims 1 to 11, characterized in that: The flexible part (24) of the cover (6, 34, 58) has a Shore hardness comprised between A0 Shore and A30 Shore, and preferably between A0 Shore and A15 Shore.

13. The sterile isolator (2, 54) according to any one of claims 1 to 12, characterized in that: The flexible part (24) of the cover (6, 34, 58) has a viscosity comprised between 3 millijoules per square centimeter and 9 millijoules per square centimeter, and preferably between 6 and 9 millijoules per square centimeter.

14. The sterile isolator (2, 54) according to any one of claims 1 to 13, comprising a clamping ring (8) configured to fix the cover (6, 34, 58) to the support (4) in an airtight and sealed manner.

15. The sterile isolator (2, 54) according to any one of claims 1 to 14, characterized in that: The support member (4) is made of a polymer rigid material, a metal or an inorganic material.

16. The sterile isolator (2, 54) according to any one of claims 1 to 15, characterized in that: The cover (6, 34, 58) is formed by bi-material molding.

17. The sterile isolator (2, 54) according to any one of claims 1 to 16, characterized in that: The rigid component (22) is made by 3D printing and the flexible component (24) is formed by molding.

18. The sterile isolator (2, 54) according to any one of claims 1 to 17, characterized in that: The support (4) comprises at least one inlet port (18) and an outlet port (20) for circulating a cell culture medium or a physiological fluid solution.

19. A method for printing a biological three-dimensional structure using a sterile isolator (2, 54) according to any one of claims 1 to 18 and a printing cartridge (28), wherein the printing cartridge is adapted to be fixed to or has a needle connector (26), wherein the needle connector (26) holds a printing needle (29), the method comprising: - injecting (40) a first fluid into the internal chamber (32) to inflate the cover (6, 34), and - Printing (44) a biological three-dimensional structure (38) for the first time in said inner chamber (32).

20. The printing method according to claim 19, further comprising the following steps: - injecting (48) a second time a fluid into the internal chamber so as to expand the cover (6, 34, 58) under the pressure of the fluid, - Printing (50) a biological three-dimensional structure a second time on the printed three-dimensional biological structure (38).

21. The method according to any one of claims 19 and 20, characterized in that The fluid injected into the inner chamber is a gel or a gas among inert gas, sterile air, carbon dioxide, oxygen and ammonia.

22. A cover (6, 34) for a sterile isolator, the sterile isolator being intended for printing biological three-dimensional structures, the cover comprising a rigid component (22) and a flexible component (24) extending in an extension of the rigid component, at least a portion of the rigid component forming a septum (30) suitable for being pierced by a printing needle for printing biological three-dimensional structures.

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