ES2928610B2 - BIOPRINTER WITH PORTABLE STERILE CHAMBER - Google Patents

Description

DESCRIPTION

BIOPRINTER WITH PORTABLE STERILE CHAMBER

OBJECT OF THE INVENTION

The object of the invention falls within the sector of mechanical engineering applied to tissue engineering and regenerative medicine.

Specifically, the object of the invention is a bioprinter that deposits the materials inside a small, portable, sterilizable chamber and isolated from the outside in a hermetic and reversible manner by means of an elastic membrane, allowing the installation and operation of said bioprinter and chamber in non-sterile environments.

BACKGROUND OF THE INVENTION

3D bioprinting is an additive manufacturing technique related to 3D printing, based on the deposition, generally at a micrometric scale, of biomaterials that either encapsulate cells or are subsequently loaded with them to form flat or three-dimensional structures comparable to living tissues. .

In most cases, a three-axis mechanical platform controls the movements of the extruders that print the bioink in the required algorithm and shape.

Starting from digital three-dimensional files of the object to be produced (obtained by CAD design, photogrammetry, 3D scanning, etc.), computer programs called “slicers” are used to digitally divide said files into thin layers and generate the series of commands that control the deposition of material and guide the movement of the extruder head in the three axes.

Biomaterials used in bioprinting must be printable, non-cytotoxic, and biodegradable within an organism.

Ideally, they should be able to generate an environment that stimulates tissue formation until its complete resorption and replacement by host tissue, so that secondary surgery for implant removal is avoided. The mechanical resistance of the materials must provide sufficient support to the seeded cells, as well as allow their manipulation and implantation.

Printed biomaterials used in tissue engineering must also promote cell adhesion and maturation, as well as their migration, proliferation and differentiation.

We can distinguish between two groups of mixtures used in bioprinting:

- Bioinks, which consist of a combination, in the form of an aqueous gel, of biomaterials, growth factors, living cells, etc.

- Thermopolymer-based materials such as poly-lactic acid (PLA), poly-glycolic acid (PLG), poly (lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL), among others.

All of them have excellent biocompatibility and mechanical resistance properties. They are often combined with ceramic materials such as hydroxyapatite (HA), calcium phosphate (CaP), tricalcium phosphate (TCP) and dicalcium silicate (C ₂ S). With this class of materials, relatively rigid porous scaffolds are printed that are implanted as is or previously seeded with cells.

Additionally, there are various bioprinting methods already known:

- EXTRUSION: The bioink is extruded through a nozzle. The necessary effort is transferred to the bioink using a piston, compressed air (pneumatic extrusion) or screw.

- INK JET: The bioink is deposited in microscopic droplets using a piezoelectric or thermal actuator similar to that of desktop printers.

- STEREOLITHOGRAPHY: The biomaterial used is cured using UV light. - LASER ASSISTED: The heat energy of a laser beam is concentrated in microscopic areas of a metallized sheet whose underside is impregnated by a thin layer of bioink. The instantaneous expansion and subsequent collapse by cavitation of the vaporized bioink volume located just below the metallized sheet area hit by the laser generates jets of bioink microdroplets that can reach cellular resolution.

The use of living cells requires that the 3D bioprinting process be carried out under conditions of sterility and isolation from the work environment, to guarantee the absence of non-viable particles in the environment.

Obtaining and maintaining these working conditions are complex and expensive processes that require the sterilization of all elements in contact with the raw materials used and with the bioprinted elements, which, because bioprinting, according to the state of the art, is performed in open mode, requires these machines to be operated in a sterile environment, generally inside a laminar flow cabinet or isolator.

On the other hand, regulatory requirements such as the Good Manufacturing Practices for cellular medicines (GMP) issued by the EMA ( European Medicines Agency) and which include tissue-engineered medicines, and the GTP ( Good Tissue Practices) ) dictated by the FDA ( Food and Drug Administration) can limit the translation to the clinical phase of tissues and organs produced by bioprinting, mainly due to the restrictions that these regulations establish for their manipulation after manufacturing.

However, bioprinting equipment has certain limitations, such as complete sterilization with ultraviolet light may be difficult due to the shielding produced by the elements that make up the bioprinters.

Another limitation to highlight is that bioprinters need adaptation to the shape and size of the laminar flow cabinets, limiting design and scaling possibilities and geometric variety.

Currently, bioprinting machines do not optimally resolve the requirement of minimal manipulation of bioprinted objects, such as loading raw materials, removal of bioprinted products or transfer to bioreactors.

Consequently, the design constraints related to sterility and adaptation to regulatory requirements cause high production costs and, consequently, high acquisition, operation and maintenance costs of bioprinters.

In conclusion, the demonstrated usefulness and great potential of bioprinting, together with the high cost, the difficulty of use or the deficiencies identified in the equipment that is part of the state of the art, justify an invention such as the one described in this report, which It simplifies and cheaper its implementation and expands its possibilities of use.

DESCRIPTION OF THE INVENTION

The product object of the present invention aims to solve the problems mentioned above, maintaining in conditions of isolation from the work environment and sterility the stages involved during bioprinting, such as: the preparation and transfer of the bioinks to the printer, the bioprinting process and the handling and transfer of the products obtained, so that the bioprinter does not need to be installed in a sterile environment and in a classified air work environment.

The invention consists of a bioprinter that comprises a portable sterile chamber, in which the sterile environment in which the bioprinting is produced is restricted to the interior of the portable sterile chamber, allowing the coupling and detachment of said chamber with respect to the bioprinter without losing sterility.

The portable sterile chamber is a container open at the top and hermetically and reversibly closed by a lid.

The lid comprises a rigid perimeter frame that is intended to be attached to the edge of the container and a membrane that is sealed along its perimeter to said frame and that is intended, in turn, to isolate the container from the outside.

The aforementioned membrane is impermeable to liquids, gases and, mainly, elements that can cause biological contamination such as cells, viruses and spores.

Said membrane comprises an adapter for a bioprinter head in its central part intended to be linked with said head in a reversible manner.

The adapter is made up of a series of closed access openings through which bioprinting needles penetrate into the portable sterile chamber. Likewise, the access openings comprise elastomer membranes with cross cuts, intended to receive the bioprinting nozzles.

The sterile chamber includes openings for the exchange of fluids with its interior. Additionally, during the bioprinting process, the membrane adapter is linked to the head of the bioprinter, moving in solidarity with said head, the membrane being deformed by stretching and/or bending, preserving the tightness of the interior of the portable sterile chamber.

The head consists of a fixed part linked to the moving elements of the machine and a magazine that adapts reversibly to the fixed element, said magazine housing the biomaterials with which cartridges are loaded, which can be syringes, also included in the magazine. . Each cartridge consists of a plunger.

Additionally, the charger includes, in its lower part, a mobile element which includes the nozzles that penetrate into the interior of the sterile chamber through the access openings of the membrane adapter.

The fixed part of the head includes a piston for each cartridge, as well as automatic mechanisms that are intended for piston-piston detection and coupling.

The automatic mechanisms make it possible, on the one hand, for each cartridge to contain a different volume of material and, on the other hand, to act on the pistons in both directions of the Z axis of geometric coordinates, both extruding and retracting material, allowing the obtaining of constructs. higher quality bioprints.

The machine can be connected to a filtered gas pumping installation with particle, temperature and humidity control; and has a series of injectors located on the lower face of the fixed part of the head, intended to fulfill the function of serving guide for the assembly of the charger and to transmit a stream of filtered gases into the sterile chamber passing through the interior of the charger. A positive pressure is generated inside the sterile chamber during the bioprinting process, making it difficult for microorganisms and non-viable particles to enter.

The bioprinting process is produced by extruding mixtures of gel-like consistency biomaterials, generally called bioinks, loaded into cartridges mounted on the magazine and equipped with needles that do not enter any part of the device at any time, neither during bioprinting nor during the previous or subsequent operations.

To guarantee this action, the charger consists of a mobile element that carries on its underside the nozzles that penetrate the sterile chamber.

Upon coupling between the moving element of the charger and the membrane adapter, the moving element passively slides upward, releasing the needles inside the portable sterile chamber. The forces generated by the movement of the head are communicated to the membrane through the membrane adapter, keeping the cartridges isolated, free of tensions that could cause errors during the bioprinting process.

The head includes a digital camera to monitor manufacturing processes.

Likewise, the portable sterile chamber can be used as a bioreactor, both when coupled to the rest of the bioprinter and separated from it, allowing liquid and gas exchange with the interior using the aforementioned filtered air injection system and accessing with needles. sterile through the access openings provided with the membrane adapter.

The invention described is compatible with bioprinting with multiple nozzles, with bioprinting processes other than extrusion and with mixed heads for bioprinting with bioinks and with thermopolymers.

Among the advantages of the device, the possibility of installing said device and carrying out the bioprinting process in non-working environments stands out. necessarily sterile, for example, a conventional laboratory in a hospital, in a research center or in an industry R&D department.

Another advantage it presents is the possibility of scaling bioprinting processes for large elements, as well as simplifying the design of the machine with a consequent reduction in manufacturing, operation and maintenance costs, making it possible, for example, to develop the process bioprinting with structure and elements typical of FDM 3D printing.

Additionally, another notable advantage is that the portable sterile chamber can act as a bioreactor.

Likewise, compliance with GMP/GTP requirements is simplified since by allowing the bioprinting process and the handling of the objects produced without losing their sterility, the needs for handling and classifying the ambient air in the production environment are reduced. job.

The maintenance of the sterility of the objects and the bioprinting process, the bioreactor function of the sterile chamber and the precise placement and repositioning on the platform makes it possible to address advanced operations such as bioprinting on scaffolds previously made with other bioprinting or printing techniques. 3D, as well as bioprinting in successive stages separated in time or space, using several identical devices installed in distant laboratories.

Finally, the bioprinting device is compatible with a variety of bioprinting processes.

DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, in accordance with a preferred example of its practical implementation, a set of drawings is attached as an integral part of said description. where, for illustrative and non-limiting purposes, the following has been represented:

FIG. 1 - Shows a perspective view of the complete invention.

FIG. 2 - Shows a plan view of the sterile chamber.

FIG. 3 - Shows a perspective view of the head.

FIG. 4 - Shows a lower perspective view of the fixed part of the head.

FIG. 5 - Shows a side view of the head and a detail of the piston-piston coupling mechanism.

FIG. 6 - Shows a perspective and sectional view of the magazine with cartridges inside.

Fig. 7 - Shows a perspective and sectional view of the container.

PREFERRED EMBODIMENT OF THE INVENTION

A preferred embodiment of a bioprinter with a portable sterile chamber is described below, with the help of Figures 1 to 8.

FIG. 1 shows a perspective view of the bioprinter with a portable sterile chamber that comprises a head (2) and a sterile chamber (1), as well as elements typical of a delta-type 3D printer that are part of the technique, such as a structure (3), arms (4), a platform (5) and a chassis (6).

The sterile chamber (1) is coupled and detached from the platform (5), said sterile chamber (2) comprising a rigid container (7) inside which the bioprinted object is produced, as well as a lid (9) that allows keep said container (7) of the sterile chamber hermetically and reversibly closed.

FIG. 2 shows a plan view of the sterile chamber (1) in which a latex membrane (10) attached perimeter and hermetically to a frame (11) is observed. Also hermetically attached to the membrane (10), there is a membrane-head adapter (12) through which the interior of the sterile chamber (1) is accessed to produce the bioprinted object.

Likewise, the membrane-head adapter (12) comprises first access openings (13A) with thin elastomer discs (14) provided with cross cuts, one of said first access openings (13A) being arranged in a central area. and hermetically closed with a transparent lens (15).

Articulated fixing fins (16) are also observed, intended to keep the membrane-head adapter (12) in a central position during periods of inactivity of the invention.

The lid (9) additionally comprises second access openings (13B), sealed with an elastomer disc (17), said second access openings (13B) being intended for the exchange of fluids with the interior of the sterile chamber (1), as well as a particle filter (18) located in the upper part of said cover (9) is also shown.

In FIG 3, a perspective view of the head (2) is seen, which comprises a fixed part (22) connected to the chassis (6) and a removable part called charger (23), reversibly assembled to the lower face of the part. fixed (22) and intended to house biomaterials (19) as a rack.

In turn, the charger (23) includes cartridges (20) that transfer the biomaterial, a mobile element (24) that carries nozzles (25) that penetrate into the interior of the sterile chamber (1). Additionally, the head (2) comprises an injection system (39) towards the interior of the sterile chamber.

FIG 4 shows a lower perspective of the fixed part (22) of the head (2), in which the presence of a digital camera (36) intended for controlling the bioprinting process is observed.

Likewise, the connection between the fixed part (22) to the head chassis (2) is observed, the location of some pistons (28) and some injectors (26) intended to serve as a guide for the assembly of the charger (23) and transmit a stream of filtered gas into the interior of the sterile chamber (1) circulating inside said charger (23).

FIG 5 shows a side view of the head (2) in which the position of the stepper motors (29) and nut-spindle mechanisms (30) that cause the movement of the pistons (28) and that These, in turn, drive pistons (27) located in the cartridges (20) linked to the lower part of said pistons (28).

At the same time, at least one piston-piston coupling mechanism (31) can be seen that comprises an optical sensor (32), a servo (33), an axis (34) and a shoe (35) intended to detect the position of the plunger (27) of each cartridge (20) and, therefore, the filling level of each biomaterial (19) that will be transferred to the sterile chamber (1) through respective needles (21) of the cartridges (20).

To do this, the servo (33), linked to the piston (28), moves upwards an axis (34) that deforms the shoe (35), remaining blocked against the internal face of the piston (27) of the cartridge (20).

FIG 6 shows a perspective and sectional view of the magazine (23) with cartridges inside (20) in which neodymium magnets (37) intended to fix the mobile element (24) of the magazine (23) can be seen. of the head (22) with the membrane-head adapter (12), as well as fixing the charger (23) to the fixed part (22) of the head.

Finally, FIG 7 shows a perspective and elevation view of a container (38) intended to house the charger (23) of the head (2) and to guarantee the sterility of said charger (23).

Claims (23)

1. - Bioprinter with portable sterile chamber, comprising: - a head (2) intended for transferring the bioprinting material, and - a sterile chamber (1) attachable and detachable to the head that additionally comprises a membrane (10) intended to keep the interior of said sterile chamber (1) isolated from the outside, acting as a barrier for biological elements and adapting to the relative movement between the head (2) and the sterile chamber (1) in the axes of the x-y-z space, said membrane (10) additionally comprising a head-membrane adapter (12), which receives the head (2) of the bioprinter, and through which the bioprinting manufacturing process occurs inside the sterile chamber (1), The bioprinter being characterized in that it comprises a cover (9) which, in turn, comprises fixing fins (16) of the head-membrane adapter (12). 2. - Bioprinter with a portable sterile chamber according to claim 1 in which the sterile chamber (1) comprises a container (7) and the lid (9) comprises a frame (11) to which the membrane (10) is fixed. 3. - Bioprinter with a portable sterile chamber according to claim 1 in which the head-membrane adapter (12) is formed by at least a first access opening (13A) into the interior of the sterile chamber (1) to carry out the printing process. bioprinting. 4 - Bioprinter with a portable sterile chamber according to the previous claims, characterized in that the membrane (10) adapts by stretching and/or bending to the relative movement between the head (2) and the sterile chamber (1) during the bioprinting process. 5. - Bioprinter with a portable sterile chamber according to claim 1 wherein the head (2) consists of a fixed part (22) and a charger (23) that is coupled to said fixed part (22) and is intended to house biomaterials. (19). 6. - Bioprinter with a portable sterile chamber according to claims 1 and 5, the charger (23) additionally comprising cartridges (20) with plungers (27) intended for the transfer of biomaterials (19) during the bioprinting process. 7.- Bioprinter with a portable sterile chamber according to claim 6 characterized in that the cartridge (20) is a syringe. 8. - Bioprinter with a portable sterile chamber according to claims 1, 5 and 6 that comprises piston-piston coupling mechanisms (31) intended for the piston (27) of each cartridge (20) to move in the z coordinate axis. both to extrude and retract material. 9. - Bioprinter with portable sterile chamber according to claim 1 characterized in that the charger (23) is reversibly coupled to the membrane-head adapter. ⁽ 12 ^). 10. - Bioprinter with a portable sterile chamber according to claims 1 and 5, characterized in that it comprises a transport container (38) intended to house the charger (23). 11. - Bioprinter with a portable sterile chamber according to claim 1 characterized in that the membrane (10) is an elastomer. 12. - Bioprinter with portable sterile chamber according to claims 1 and 11 characterized in that the elastomer is latex. 13. - Bioprinter with a portable sterile chamber according to claim 1 characterized in that the sterile chamber (1) comprises second access openings (13B) intended for the exchange of fluids with the interior. 14.- Bioprinter with a portable sterile chamber according to claims 1, 2 and 13, characterized in that it comprises at least one gas injection system (39) into the sterile chamber (1). 15. - Bioprinter with a portable sterile chamber according to claims 1 and 14, characterized in that the gas injection system (39) into the sterile chamber (1) acts through the head (2). 16. - Bioprinter with a portable sterile chamber according to claims 1,2, 13 and 14 characterized in that the gas injection system (39) injects the gases into the sterile chamber (1) through the sterile chamber itself ( 1). 17. - Bioprinter with a portable sterile chamber according to claims 1 and 14, characterized in that a positive pressure is generated inside the sterile chamber. ( OR 18. - Bioprinter with a portable sterile chamber according to claim 1 characterized in that the head (2) is multi-material. 19. - Bioprinter with a portable sterile chamber according to claim 1 characterized in that the bioprinting material includes cells in its composition. 20. - Bioprinter with a portable sterile chamber according to claim 1 characterized in that the bioprinting material does not include cells in its composition. 21. - Bioprinter with a portable sterile chamber according to claims 1 and 17 characterized in that the bioprinting material is a thermopolymer. 22. - Bioprinter with portable sterile chamber according to claims 1, 17 and 18 characterized in that the thermopolymer is polycaprolactone. 23.- Bioprinter with a portable sterile chamber according to claims 1, 2, 13 and 14 characterized in that it can function as a bioreactor.