JPWO2020084262A5 - - Google Patents

Description

The present invention relates to the field of regenerative medicine, called "tissue engineering", which makes it possible to artificially create organs and biological tissues. The need for tissue and organ transplants has increased dramatically throughout the world in the past decade. The main reasons are the need to reduce the increase in life expectancy, the occurrence of vital organ dysfunction and degenerative diseases, and the consequences of tumor removal. In the European Union alone, more than 63,000 patients are waiting for an organ transplant (kidney, liver, heart, cornea, etc.) due to this organ defect, and six new patients are added to the waiting list every hour. will be added to. In contrast, only about 33,000 donors were recognized in 2016. Although some countries have taken steps to increase the number of organ donations, many patients waiting for a transplant will not receive one at the right time. Therefore, clinicians require tissue and organ substitutes that are well-characterized, safe, patient-specific, and potentially "off-the-shelf."

In this regard, scientists and industrialists in the field of tissue engineering apply biological and engineering principles to develop functional replacements that restore, maintain, or improve tissue function.

From a regulatory perspective, tissue engineering products belong to the category of advanced medical products (ATMPs).

They are different from conventional pharmaceuticals and therefore present unique challenges. They require quality assurance and complex logistics, meeting important regulatory requirements such as Advanced Medical Pharmaceutical Products (ATMP), as well as Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP) regulations. , must be implanted into the patient by a qualified surgeon.

Tissue engineering approaches traditionally rely on the use of biocompatible materials shaped to form 3D scaffolds that are seeded with living cells prior to maturation in a bioreactor. Then, as the cells proliferate, they colonize the scaffold and synthesize extracellular matrix to create the 3D tissue.

Despite significant investments being made to meet clinical and commercial expectations and impressive scientific achievements during the preclinical research stage, these traditional tissue engineering approaches have been slow to deliver clinical results. and are struggling to improve both cost-effectiveness. In fact, only a small number of tissue engineering products have received marketing approval to date, and even in these cases, therapeutic efficacy has not met expectations and sales have not been profitable. This is demonstrated by the fact that despite adequate safety and efficacy, three of these ATMPs have been withdrawn from the market: "Provenge" (2015), "Chondroselect" (Registered Trademark) (2016), and MACI (Registered Trademark) (Suspended).

Therefore, in order to meet clinical and commercial expectations, the manufacture of tissue engineering products is subject to several challenges that must be solved. these are,
- Industrialization and automation of manufacturing methods to increase robustness, recognizing that ATMP manufacturing is still primarily manual, unsupervised and involves multiple open stages;
- Compliance with regulatory requirements;
- the specialized labor required to supply consumables, install and own infrastructure (e.g. clean rooms) and equipment, and carry out various preparation, manufacturing and quality control operations for small individual batches of small volumes; Improving profitability and cost-effectiveness by reducing the high costs of
- The ability to reproduce the complexity of natural human tissues (i.e., the biochemical, mechanical, and physical stimuli that control the spatiotemporal distribution of cells and the cell microenvironment and govern cell behavior and tissue function) ),
- the need to promote rapid vascularization after transplantation to maintain cell viability during tissue growth, and
- In some cases, relates to the ability to customize tissue engineering products to provide patient-specific treatments (eg, by integrating autologous cells and/or patient anatomical data).

Various bioprinting approaches have been proposed to address the limitations of traditional tissue engineering approaches. According to published works, these printing methods are called biological printing, microprinting of biological elements, or bioprinting. These use the principles of 3D printing and proceed by layer-by-layer assembly of biological tissue components (such as cells and extracellular matrix) according to a predefined organization by digital design. Note that despite similarities in principle, bioprinting differs from 3D printed artificial joint production in the nature of the deposited material (living rather than inert) and in the technology used. There is a need to.

The main applications of bioprinting relate to the preparation of synthetic biological tissues for experimental research by replacing tissues harvested from living organisms to avoid regulatory and ethical issues. In the long term, bioprinting can be used to manufacture hollow structures such as epidermis, bone tissue, parts of the kidney, liver and other vital organs, heart valves, blood vessels, etc. for transplantation without the risk of rejection. would make it possible.

The global 3D bioprinting market was estimated at €450 million in 2014 and will increase significantly during the next decade with an annual growth rate of approximately 16.7%, reaching €8.2 billion by 2025. It is estimated that According to analysts, the medical segment of the bioprinting market is expected to account for more than 30.0% in 2022 due to the introduction of bioprinters capable of producing ATMP.

PRIOR ART US Pat. No. 5,001,000 describes a portable device known in the state of the art and comprising a first element for dispensing a structural material and a second disposable element for dispensing a biological material. do.

The delivery system also includes a sterilizable enclosure, at least one robotic arm assembly, and a delivery device within the sterilizable chamber. The robotic arm is configured to move a delivery device that includes a first delivery member and a second delivery member, the first delivery member being removably connectable to the second delivery member.

Figures 10 and 11 of this patent, unlike the present invention, primarily relate to the handling of well plates, where both the print head and activation module are housed in a sterile enclosure.

This solution does not allow additive printing production of biological tissues under optimal sterile conditions.

WO 2006/000002 is also known and relates to an apparatus for depositing layers, the apparatus comprising:
- a frame to which an enclosure is attached, further comprising:
- a table intended to support the object to be manufactured;
- a material dispensing device intended to place material onto a table in order to form said object;
- a frame supporting the extrusion means;
It includes a control member intended to control the deposition of material on the table.

Inside the enclosure at least the plate and the end of the nozzle are arranged, and outside the enclosure at least the means for moving the table and the dispenser and the control means are arranged.

This document does not pertain to systems for bioprinting biological tissues.

US Pat. No. 6,001,200 describes a prior art method comprising steps for creating pharmaceutical products by 3D printing. The product is printed according to the instructions. At least one attribute of the product is measured and compared to a desired attribute of a desired final product. If there is a difference between the product attributes and the desired attributes, changes are made to the set of instructions and a new product is printed. Safe and accurate medical products are created when there is a match between product attributes and desired attributes.

It is not a solution for biological tissue manufacturing.

US Pat. No. 5,020,000 describes a method of printing at least one biological ink, the method comprising: depositing at least one droplet of the at least one biological ink on a deposition surface of a receiving substrate; using at least one laser-type printhead, the printing method includes depositing at least one droplet of the at least one biological ink on the same deposition surface of the receiving substrate as by the laser-type printhead; Characterized by the use of at least one nozzle printhead. This is definitely the closest state of the art, but it requires a very restricted clean room type environment for the operator.

US Pat. No. 5,021,002 relates to a sterilizable container intended to receive one or more bioactive fluids and/or one or more preparation fluids, the container comprising:
- an area within the container configured to receive at least one sterilizable 3D printer assembly;
- a sterilizable 3D printer assembly, comprising:
- at least one printing platform;
- at least one printhead for dispensing structural material onto the printing platform to form a three-dimensional structure thereon; and
- a 3D printer assembly comprising a movement mechanism that allows relative displacement between the print head and the printing platform, the area being configured such that fluid can reach the printer assembly; including.

US Patent Application Publication No. 2017/0335271 US Patent Application Publication No. 2010/0206224 US Patent Application Publication No. 2018/0290386 US Patent Application Publication No. 2017/0320263 US Patent Application Publication No. 2016/0297152

The solution presented by US Pat. No. 5,001,201 is used, which involves the treatment of large volumes of air, implying a large air flow rate to generate the overpressure necessary to accommodate the bioprinting device in sterile conditions. Whatever the bioprinting technology, it has the disadvantage of involving a high risk of turbulence within the bioprinting area, which is particularly prohibitive for print reproducibility.

Also, such flow rates require powerful fans and filters, which are developed to allow the passage of a large amount of air flow while retaining all the particles that cause vibration and noise pollution. do.

Additionally, the presence of mechanical devices within the sterilization chamber results in the production of particulates that are carried by the air flow into the bioprinting space, which is contrary to the intended purpose. It may therefore be emphasized that in the two examples cited in the prior art, the printing means are directly integrated into the sterilizable area. Currently, these elements are one of the main sources of particle contamination within confined spaces.

Finally, when changing bioprinting sequences, there is a high risk of contamination or cross-contamination between samples, as different samples accompany each other in the same chamber and are also exchanged outside the sterilization step.

To ameliorate these drawbacks, the present invention relates to a method and a unit for producing biological tissue by bioprinting using three separate modules, the three separate modules comprising:
- a printing module that is unavoidably located in a sterilizable enclosure and that integrates a print head;
- an activation module (laser, transducer, etc.), necessarily located outside the sterilizable enclosure, for transferring the biological object to the target via the printhead;
- A module placed inside or outside the sterilizable enclosure for moving the print head relative to the target.

The enclosure includes an interaction zone with the activation module.

The invention particularly relates to the addition of biological tissue by integral deposition onto a target of elements containing living cells as well as other components that make it possible to reconstitute a living biological tissue. The present invention relates to a method and unit for digital printing.

In its most general sense, the technical features of the dependent claims are taken in accordance with the recitation of claim 1 and optionally, alone or in technically feasible combinations of other features. A bioprinting system including is described below.

The object of the present invention is to treat objects of biological significance, sometimes comprising living cells of different types (e.g. pluripotent stem cells or any other differentiated cells), as well as collagen and, more generally, It is the transport of extracellular matrix material from a source to a target.

Objects of biological significance can be placed together in a fluid to form a "bioink" containing biological particles, such as, for example, living cells. These bioinks are then prepared and packaged in sterile form so that they can be used to print biological tissue when the time comes.

Within the meaning of this patent, "bioprinting" refers to a process for developing biological tissues and organs for tissue engineering, regenerative medicine, pharmacokinetics, and more generally biology research. Spatial structure of stacks of layers formed by discrete deposition of living cells and other biological products, especially objects of biological importance, by methods of performing computer-aided geometric structuring Refers to becoming. Bioprinting is used to create biological tissues such as skin, heart valves, cartilage, heart tissue, kidneys, liver, and other vital organs or hollow structures such as the bladder and vascular structures. Involves layer-by-layer co-deposition of living cells and biomaterials.

The invention also relates to a cassette for a bioprinting system as described above, characterized in that the cassette consists of a sterilizable and sealed enclosure, which constitutes the jacket of the printing module.

The present invention also relates to a bioprinting method for producing structured biological materials from materials consisting, at least in part, of biological particles (cells and cell derivatives), the method comprising: controlling, via the robot, the movement of at least one target in three dimensions facing at least one print head arranged in the enclosure, said print head being arranged in the enclosure in accordance with regulatory requirements in the clinical field. , supplied and controlled from outside the enclosure to ensure the safety of the tissue produced.

The invention will be better understood from the following detailed description of non-limiting examples of the invention, taken in conjunction with the accompanying drawings, in which: FIG.

1 is a schematic diagram of a first variant of a bioprinter according to the invention; FIG. 3 is a schematic diagram of a second variant of the bioprinter according to the invention; FIG. 3 is a schematic diagram of a third variant of the bioprinter according to the invention; FIG. FIG. 4 is a schematic diagram of a fourth variant of the bioprinter according to the invention. FIG. 6 is a schematic diagram of a fifth variant of the bioprinter according to the invention. FIG. 1 is a perspective front view of an embodiment of the present invention. FIG. 1 is a perspective side view of an embodiment of the invention. Figure 3 is a schematic diagram of an alternative embodiment in the form of a rigid cassette; Figure 3 is a schematic diagram of an alternative embodiment in the form of a flexible cassette; 1 is a diagram of a unit according to the invention with a sterile glove box; FIG. 1 is a diagram of a unit according to the invention with a sterile glove box; FIG. 1 is a diagram of a unit according to the invention with an alpha transfer system with an interlock fixed to a beta type container; FIG.

General Description of a Bioprinter Figures 1 to 5 show diagrams of different embodiments of the hardware architecture of a bioprinter according to the invention. It consists of several important parts, the important parts are:
- an additive printhead (1), the configuration of which depends on the technology used and which includes a bioink transfer region composed of biologically significant objects; and target (6),
- one or more sources (2) for supplying the additive printhead (1) with a bioink composed of biologically significant objects;
- a mechatronic assembly (3) ensuring a relative displacement of the transfer area with respect to the target (6);
・Optional maturity area (4);
- Printing activation means (5), the configuration of which varies depending on the technology used.

The term "bioprinting head" refers to a bioprint or, more generally, a support formed by a support that receives an object of biological significance that is transferred to a target, Refers to the part of a bioprinting system that presents an exit area for objects of interest, but does not include activation means, e.g.
- Regarding the activation means for laser bioprinting, the bioprinting head does not include a laser and optical components for pulsed laser bioprinting;
- Regarding the activation means for inkjet or microvalve bioprinting, the bioprinting head does not contain electricity generation means that allow the actuator (valve/piezo) to be activated;
- Regarding the activation means for microextrusion bioprinting, the bioprinting head does not include a syringe pump.

Generally, in biological bioprinting devices, two parts can be distinguished, the two parts are:
- within the meaning of this patent, the terminal part of the device, designated by "printhead (1)", which receives the bioink to be transferred and/or the object of biological significance; and a portion comprising an interface for an activation member;
- a part denoted by "activation means (5)" within the meaning of this patent, which is physically separable from the print head (1) and is in mechanical or electrical contact with the bioprinting head (1); , acoustically or optically coupled and containing all the means that make it possible to control the physical transfer of the biologically relevant objects present in the bioprinting head to the target.

The invention also aims at optimizing the sterile area and for this purpose:
a) the print head (1) must be placed within a sterilizable enclosure (10);
b) the printing activation means (5) must be located outside the sterilizable enclosure (10);
c) The leak-tight interaction zone (20) allows activation of the print head (1) by the printing activation means (5).

Other components of the bioprinter may be placed within the sterilizable enclosure (10) or outside of this enclosure (10).

In particular, the source (2) may be integrated with the printhead (1) within a sterilizable enclosure (10), which may then be introduced into the bioprinter and subsequently undergo maturation of the printed tissue. form a cassette that can be pulled out for use.

A mechatronic assembly (3) for moving the target (6) relative to the source also includes a print head in a sterilizable enclosure (10), with or without the source (2). (1) can be integrated.

The maturation chamber (4) is also integrated with the print head (1) in the enclosure (10), with or without the supply source (2) and/or the mechatronic movement assembly (3). can be done.

ALTERNATIVE EMBODIMENTS The invention may be implemented in different ways.

FIG. 1 shows a general diagram of a sterilizable enclosure (10), which includes:
- as a requirement, a maturation enclosure (4) in which the printhead (1) and, optionally, the target for receiving the biologically significant object to be transferred are located;
- optionally a supply source (2), for example a bioink reservoir and a supply system, which according to the invention can also be placed in a non-sterile area and into a sterilizable enclosure (10); , a source (2) comprising a connector for sterile transfer of objects of biological significance, and
- optionally a moving means (3) for moving the print head (1), which according to the invention can also be placed in a non-sterile area and is provided with a sterile mechanical connector; Includes transportation means (3).

The activation means (5), for example a laser and associated optics, are arranged below this sterilizable enclosure (10), and a transparent window (20) forms an interface allowing the passage of the light beam. . The activation means (5) are not placed in a sterile area.

Figure 2 shows a variant in which all components except the activation means (5) are arranged in a sterilizable enclosure (10).

Figure 3 shows a variant in which all components except the activation means (5) and the movement means (3) for moving the print head (1) are arranged in a sterilizable enclosure (10). Give an example. The coupling between the print head (1) and the movement means (3) is achieved, for example, by magnetic transmission or by a sterile joint passing through the wall of the sterilizable enclosure (10).

Figure 4 shows a variant in which only the print head (1) and the maturation area (4) are arranged in a sterilizable enclosure (10). All other components, in particular the activation means (5) and the movement means (3), as well as the source (2), are outside the sterilizable enclosure.

Figure 4 shows a variant in which only the print head (1) and the maturation area (4), together with the movement means (3), are arranged in a sterilizable enclosure (10). All other components, in particular the activation means (5) and the source (2), are outside the sterilizable enclosure.

Figure 5 shows that the printhead (1), the maturation region (4) and the robotic arm (3) ensuring relative displacement of the printhead (1) with respect to the target are arranged in a sterilizable enclosure (10). A modification example is shown below. Only the activation means (5) and the source (2) are present outside the sterilizable enclosure.

DESCRIPTION OF THE EMBODIMENTS The bioprinter consists of a frame whose (non-sterilizable) lower part (11) contains activation means (5), such as an optical head for a laser printer, a laser, and an imaging system.

This frame carries a sterilizable enclosure (10) consisting of a positive pressure chamber supplied by a blower (15) via a filter cartridge (16). A robotic arm (3) placed within this sterilizable enclosure (10) ensures the movement of the target (6) relative to the print head (1). A leaktight window (20) allows the transmission of the laser beam and the imaging beam between the sterilizable enclosure (10) and the printing activation means (5) located in the non-sterilizable area.

The enclosure (10) is sealed by a glazed wall (22) through which an interface (21) for sterile handling gloves passes.

It has a transfer system (23) sealed by an alpha part (24), which transfers materials, e.g. bioink cartridges, to non-sterile areas by means of a leak-free and risk-free connection. through which it is possible to move from one sterile area to another.

Such a system makes it possible to meet the requirements related to Good Manufacturing Practice (GMP), established by the European Commission within the framework of the development of "Quality Procedures", limiting two categories of risks: The two categories are:
- the risk of cross-contamination of products (from another product or from internal and external contaminants);
- Risk of confusion, especially regarding labeling and identification of components.

They emphasize hygiene and organizational practices that must be implemented at all levels.

Variant implementing a “glove box” or leak-tight hatch According to a variant not shown, the sterilizable enclosure (10) has a control mechanism for a leak-tight device, the control mechanism comprising: It is possible to carry out these tasks for manual handling of objects or products within this enclosure in a sterile or dust-free atmosphere. For example, the wall of the enclosure (10) has a flange, the outer periphery of which is attached to an ear that is intended to cooperate with a recess in the flange of the glove box.

The flange of the enclosure (10) is inserted into the flange of the glove box.

The sterilizable enclosure (10) may also be provided with a hatch attached to the flange for communicating with another sterilizable enclosure, for example for post-printing maturation.

Variant using a cassette According to a variant of the invention, the encapsulated bioprinting system is based on a sterilizable cassette containing the target (6) and the print head. The system consists of sterile/leak-tight interfaces (double leak-tight transfer doors, beta bags, optical windows, injectors, etc.) and allows the cassette to contain biologically important objects that can be transferred for printing. one or more activation means, mechanical coupling means for displacement of the target (6) relative to the head, and elements necessary for maturation (medium, CO ₂ , O ₂ etc.). Cassettes can be used for maturation. It can be transported between the printing system and the incubator.

The source of biologically significant movable objects for the printing method can be connected to a cell culture automaton configured by a dedicated reservoir.

This solution allows biofabrication according to Good Manufacturing Practice (GMP) compliant methods in a sealed enclosure to avoid the risk of biological contamination.

The cassette is coupled to a means for characterizing the tissue or organ (on-line characterization of the tissue during or after manufacture) (imaging, Raman spectroscopy, OCT, physicochemical analysis).

The cassette may provide a single window, multiple windows, double-sided (front and back) windows, etc.

The system optionally includes means for controlling and regulating elements for the maturation stage (bioreactor), the elements comprising:
-pH (pH detection range 0-14, accuracy 0.01 by NaOH/ _CO2 adjustment),
- temperature (controlled by Peltier, double jacket, cryostat);
- dissolved oxygen (e.g. by gas flow measurement),
-Hygrometer,
- means for removing or replacing the medium;
- stirring and injection means,
-Other.

The cassette optionally includes an identifier of the tissue to be printed and associated printing parameters (sequence, source, CAD model, etc.) linked to a database (connected to the cassette). The identifier can be of a graphic type (for example a barcode or a QR code matrix code) or of a digital type, for example in the form of a numerical sequence recorded in the memory of a radio frequency tag of the RFT type. obtain. Such a solution makes it possible to ensure the printing process. The cassette can optionally be sent to the user in real time by SMS, email, etc., to control manufacturing and data collection methods, to manage alarms, to save experiments on a local hard drive. Equipped with an Ethernet port type network interface card to perform notification.

Unit with Glove Box Figures 10 and 11 show an example of a unit with a glove box (25).

The implementation of the printing method in the context of a glove box with a leak-tight hatch (21) can take the following form.
- Laser printing: The laser printing module (1) consists of at least one laser head and a target (6). The laser head consists of a transparent flat surface (also called a donor) on which an ink film is deposited. The head's ink supply (2) can be provided by the use of a SAS, by a reservoir placed in a sterile enclosure, or by manual application of ink to the donor surface (with a leak-proof glove or by a robot integrated with a pipettor). (spreading) or by a reservoir and a pump (operating under pressure or flow) located outside the enclosure (automatic spreading of the ink by fluid means). The activation means (5) for activating the transfer of ink to the target (6) consist of a laser located outside the enclosure. The leaktight interaction means (20) is in this case a leaktight optical window transparent to the wavelength of the laser. The means (3) for relative displacement of the print head with respect to the target (6) consist of a six-axis robot arm.
- Microvalve printing: The microvalve printing module (1) consists of one or more valves with a target and a needle, which can have different shapes, lengths and opening diameters. The ink supply source (2) for the valve consists of a reservoir and a pressure pump located outside the enclosure. The activation means (5) for activating the transfer of ink to the target (6) consist of a piezoelectric or solenoid shutter. The sealed interaction means (20) can in this case be a connector, a pierceable cap (septum) or a leak-tight seal. The means (3) for relative displacement of the print head with respect to the target (6) consist of a six-axis robot arm.
- Extrusion printing: The extrusion printing module (1) consists of a target and a final reservoir with a needle that can have different shapes, lengths and opening diameters. The ink supply (2) of the extruder is constituted by an initial reservoir and a pressure pump, located outside the enclosure. The activation means (5) for activating the transfer of said biological matter to the target (6) consist of a pressurization system. The sealed interaction means (20) can in this case be a connector, a pierceable cap (septum) or a leak-tight seal. The means (3) for relative displacement of the print head with respect to the target (6) consist of a six-axis robot arm.

With such a configuration, it is possible to manufacture tissue that satisfies GMP manufacturing standards. Thus, clinical batches can be safely manufactured for patients. By way of illustration, mention may be made of the example of the production of autologous or allogeneic skin substitutes for regenerative medicine applications. Thus, the various modalities mentioned here in turn make it possible to produce the following products:
- Dermis composed of alternating layers of biomaterial (collagen type) printed either by microvalves or extrusion and cell layers (fibroblast type) printed by laser. The precision of the cell patterns makes it possible to guarantee the structure and function of the dermis after maturation, close to those of the native skin.
- Epidermis composed of a dense layer of laser-printed cells (keratinocyte type).

This dermal maturation is performed within the incubator either inside or outside a sterile enclosure, depending on the incubator configuration. At the end of this step, the skin has reached maturity and can be transplanted into the patient.

Application for the manufacture of biological tissues The cassette configuration is particularly suitable for the manufacture of one or more tissues or biological organs, intended for example for the manufacture of autologous tissues.

Tissues or organs may be based on the use of several cassettes due to differentiated maturation, compatibility of the printing module with a single cell type, etc.

Another advantage of the cassette lies in the fact that it can be introduced into the operating room after the final sterilization step and opened as close as possible to the patient.

The substrate may be housed in a module that is removable from the cassette for placement in a dedicated bioreactor.

The cassette may be wholly or partially disposable.

The cassette can be reused after internal and external sterilization.

The jacket of the cassette can be rigid or flexible (ethylene vinyl acetate, PVC, PU type thermoplastic, infusion material type low density polyethylene, silicone, metal (stainless steel 304L, 316L), or plastic (polycarbonate, etc.) It can be a type.

Rigid Cassette Figure 8 shows an embodiment of the invention in the form of a rigid cassette. It consists of major equipment items consisting of complex and expensive parts: activation means with one or more printheads, such as lasers, means of relative displacement of the printheads with respect to the target (3), and all Allows to use IT and optical means.

The cassette consists of a sterilizable sealed enclosure (10) configured to allow it to be placed in this item of equipment for a specific operation.

The cassette contains within a sealed and sterilizable enclosure (10) certain means, the certain means being:
- a printing module (100) consisting of a target (6) (110), on which objects of biological importance constituting the tissue to be produced are deposited;
- connecting rods (131, 132, 133) that ensure the connection with the relative displacement means and pass through the enclosure (10) with leak-tight seals (134, 135, 136); 133) is intended to transmit the movement of the relative displacement member of the target (6) with respect to the source along three axes X, Y, Z),
- a fluid carrying a movable object of biological significance, with a supply duct (141) connected to a reservoir (143, 144) outside the enclosure (10) and an outlet duct (142); A printing module (1) constituted by a flow support.

The activation means (5) are arranged outside the sterilizable sealed enclosure (10) and interact with the printing module (1) via the window (140).

The connection between the cassette and the fixed equipment item is ensured by a magnet surrounding the window (140).

This cassette allows tissue to be prepared by placing the cassette in the main equipment item and then ensuring maturation of the bioprinted tissue in another location, e.g. in a maturation enclosure. do.

Flexible Cassette FIG. 9 shows an embodiment of the invention in the form of a flexible cassette. It is a rigid cassette in that the sterilizable enclosure has a flexible wall (200) connecting a rigid top surface (210) and a rigid bottom surface (220) presenting a window (140). It is different from.

The top surface of the sterilizable enclosure is for receiving a target (6) on which a biological element is deposited , which is transferred from a bioink consisting of a movable object (148) of biological significance. It has two prongs (211, 212) located inside.

Other Embodiments Regardless of the bioprinting technology used, bioprinters traditionally consist of several key components, the key components being:
- one or more printheads comprising a bioink transfer region, composed of biologically significant objects;
- one or more sources (2) for supplying the print head (1) with a bioink consisting of biologically significant substances;
-Target (6),
- a mechatronic assembly (3) ensuring a relative displacement of the transfer area with respect to the target (6);
- activation means (5);
- optionally a maturation region (4).

According to a first variant, the biological ink is stored in a reservoir and passes through a nozzle or capillary to form droplets that are transferred to the target (6).

Variants of this first so-called nozzle printing include bioextrusion, inkjet printing or microvalve printing. In particular, methods have been developed to print biological elements without a nozzle in order to make it possible to achieve higher levels of resolution and increase the viability of printed cells. This printing method, called laser bioprinting, is also known under the name "laser-assisted bioprinting" (LAB).

Regarding bioextrusion, various means of activating printing have been implemented, the means being a worm, a piston, or a pneumatic system. It allows working with cell densities as high as 100 million cells per milliliter and resolutions as low as 1 millimeter. Bioextrusion consists of mechanically pushing a biological element placed in a microsyringe through a nozzle or needle several hundred micrometers in diameter. The advantage of this technology is its low cost and simplicity of implementation. However, it suffers from limitations associated with coarse resolution and non-negligible cell mortality associated with the shear imposed on the cells while passing through the nozzle.

Regarding inkjet bioprinting, it consists of ejecting microdroplets of liquid containing cells or biomaterial onto a substrate. Ejection is caused by thermal or piezoelectric methods. Thermal inkjet printing works by temporary activation of electrical resistance (with strong thermal effects), which generates a vapor bubble that propels the droplet through an orifice with a diameter of 30-200 μm. Piezoelectric inkjet printers use electrical pulses that create a change in the shape of a piezoelectric crystal that causes the ink reservoir to contract. Relaxation of the crystal results in droplet ejection. This technique has the advantage of being fast, both in terms of preparation time and printing speed. However, the main drawbacks of this technique are the cell concentration (less than 5 million cells/mL), which cannot be exceeded during printing to prevent clogging of the printhead, and the shear on the cells during the time of passage through the orifice. Associated with stress-induced cell death. Inkjet printing, whether piezoelectric or thermal, offers a better resolution of about 10 μm.

For printing with microvalves, the means of activating printing is pressurization of the liquid with compressed air. The liquid to be printed is released by actuating a solenoid valve. Microvalve bioprinting is similar to inkjet technology, but differs in that microvalves are formed by pressurizing ink and then quickly opening a solenoid valve. This technology, which has the same limitations as inkjet bioprinting, has the advantage of being able to print more viscous solutions than inkjet systems. Typically the pressure is from a few hundred mbar to a few bars, making it possible to obtain lower cell densities on the order of millions of cells per milliliter and resolutions on the order of tens of μm.

Laser bioprinting can print inks with high cell densities on the order of 100 million cells per milliliter at 10 μm resolution. A device for printing biological elements by laser, based on the technique called "laser-induced forward transport " (LIFT), comprises a pulsed laser source for emitting a laser beam, a system for focusing and directing the laser beam, at least one a donor medium containing a biological ink, and a recipient substrate positioned to receive droplets ejected from the donor medium. According to this printing technique, the laser beam is pulsed and with each pulse a droplet is generated. Biological inks include a matrix, eg, an aqueous medium, in which are present the elements, eg, cells, to be deposited onto the recipient substrate. The donor medium is transparent to the wavelength of the laser beam and can be coated with an absorbing layer (metal, polymer, etc.) or not, depending on the configuration in which the biological ink is applied in the form of a film. (“Sacrificial Layer Free LIFT”), including slides.

Other optical/laser methods (donor surface imaging/point-and-shoot systems/photopolymerization, texturing, cavitation, cutting, etc.) can be combined with laser scanning using galvanometric mirrors, or instead of laser bioprinting. , which can be additionally implemented to generate a pattern on the recipient substrate.

Regardless of which technique is used, the production of biological tissue by bioprinting can be divided into five stages, which are:
- computer-aided design (CAD) preprocessing of the digital model, which will define the architecture of the biological tissue, i.e. the location in space (x, y, z) of all the components of the tissue;
- programming of printing parameters of bioinks (including cells and/or biomaterials), leading to defining the physical parameters of the printer and the trajectory of the printhead;
- Preparation and formulation of bioinks, consisting of transportable objects of biological significance;
- automated layer-by-layer printing of bioinks using bioprinters, using various techniques;
- A maturation run of the printed tissue, which is carried out in a bioreactor and allows the cells to self-organize until the specific biological function desired is manifested.

Upstream of this sequence, tomographic reconstruction may be performed using, for example, microscopic, histological, or medical images, and downstream, the bioprinted biological tissue is processed before being sent to the user. is adjusted to

The last three steps of the above manufacturing sequence, as well as the packaging step, must be performed in a completely sterile environment, free of any particles that could contaminate the bioprinted tissue; Contamination can alter the growth and functionality of the maturing tissue and/or contaminate the patient after tissue transplantation.

For this reason, these steps are performed within an enclosure that provides a controlled sterile atmosphere and an environment that promotes tissue development.

Laser Printing The printing assembly (1) consists of one or more laser heads and a target (6).

The laser head consists of a transparent flat surface onto which an ink film is deposited, either by manual glove box means, by a robot, or by fluid means coupled to a supply source (2).

The head supply (2) consists of a reservoir and a pressurization or flow pump.

The activation means (5) for activating the transfer of biological matter to the target (6) consist of a laser placed outside the enclosure.

The leaktight interaction means (20) is in this case a leaktight optical window transparent to the wavelength of the laser.

The means (3) for relative displacement of the print head with respect to the target (6) consist of a robotic arm or a mechatronic actuator.

The sterilizable hermetic enclosure corresponds to any one of the configurations described above.

Valve Printing The printing assembly (1) consists of one or more valves and a target (6).

The supply source (2) for the valve consists of a reservoir and a pressure pump.

The activation means (5) for activating the transfer of biological matter to the target (6) consist of a piezoelectric or solenoid shutter.

The leaktight interaction means (20) is in this case a pierceable cap or a leaktight seal.

The means (3) for relative displacement of the print head with respect to the target (6) consist of a robotic arm or a mechatronic actuator.

The sterilizable hermetic enclosure corresponds to any one of the configurations described above.

Extrusion Printing The printing assembly (1) consists of one or more extrusion nozzles and a target (6).

The supply source (2) for the nozzle consists of a reservoir and a pressure pump.

The activation means (5) for activating the transfer of biological matter to the target (6) can be a control for feeding the nozzle, for example a mechanical control by a worm or a piston, or a direct pneumatic control. It is.

The leaktight interaction means (20) is in this case a pierceable cap or a leaktight seal.

The means (3) for relative displacement of the print head with respect to the target (6) consist of a robotic arm or a mechatronic actuator.

The sterilizable hermetic enclosure corresponds to any one of the configurations described above.

Inkjet Printing The printing assembly (1) consists of one or more inkjet printheads and a target (6).

The supply source (2) of the inkjet printhead consists of a reservoir and a supply pump.

The activation means (5) for activating the transfer of biological matter to the target (6) are piezoelectric, acoustic, thermal, laser, etc. controls.

The leaktight interaction means (20) is in this case a pierceable cap or a leaktight seal.

The means (3) for relative displacement of the print head with respect to the target (6) consist of a robotic arm or a mechatronic actuator.

The sterilizable hermetic enclosure corresponds to any one of the configurations described above.

Acoustic Printing The printing assembly (1) consists of one or more acoustic printheads and a target (6).

The supply source (2) of the acoustic printhead consists of a reservoir and a supply pump.

The activation means (5) for activating the transfer of biological matter to the target (6) are transducers, lasers, etc.

The means (3) for relative displacement of the print head with respect to the target (6) consist of a robotic arm or a mechatronic actuator.

The sterilizable hermetic enclosure corresponds to any one of the configurations described above.

Of course, these variant embodiments are not limiting, and the present invention provides any suitable method for depositing biological elements towards a target (6) in a controlled manner for the production of biological tissues. technology.

Claims (19)

A bioprinting system for producing biological tissues, the system comprising:
a) a printing module comprising at least one printhead and at least one target (6) for printing transportable biological objects in a controlled manner for producing biological tissues; 1) and
b) a source (2) supplying the biological object to the print head for printing;
c) activation means (5) for transferring said biological matter to said target (6);
d) means (3) for relative displacement of the print head with respect to the target (6);
e) a sterilizable sealed enclosure (10);
the printing module (1) is located within the sterilizable sealed enclosure (10);
the activation means (5) being located outside the sterilizable sealed enclosure (10) and spaced apart from the printhead ;
Bioprinting system, characterized in that said sterilizable closed enclosure (10) has an interaction zone (20) with said activation means (5). 2. The bioprinting system of claim 1, wherein the sterilizable sealed enclosure constitutes a jacket for the printing module. 3. The bioprinting system of claim 2, wherein the sterilizable, sealed enclosure further includes at least one source for the biological matter. Bioprinting system according to claim 2 or 3, characterized in that the sterilizable sealed enclosure further comprises movement means for moving the target (6) . 5. The bioprinting system according to claim 4, wherein the moving means is constituted by a robot arm. 5. The bioprinting system of claim 4, wherein the moving means is constituted by a magnetic target support that interacts with a stator coil outside the sealed enclosure. 5. The bioprinting system according to claim 4, wherein the moving means comprises an electric moving platen. 5. The bioprinting system according to claim 4, wherein the moving means is constituted by a worm gear or a belt system. 2. The bioprinting system of claim 1, wherein said activation means (5) comprises a laser source associated with beam shaping optics, beam deflection means and focusing optics, and said interaction zone (20) comprises: A bioprinting system characterized by being composed of a transparent window. Bioprinting system according to claim 1, characterized in that the activation means (5) consist of a light source capable of interacting with the print head, and the interaction zone (20) is constituted by a transparent window. bioprinting system. Bioprinting system according to claim 1, characterized in that the target (6) consists of a volume containing a polymer resin and the printhead consists of an injection needle for transportable biological matter. bioprinting system. 2. The bioprinting system of claim 1, further comprising a bioreactor for maturing biological matter in the fully or partially printed target (6), said bioreactor comprising said sterile 1. A bioprinting system characterized in that it is placed in a sealed enclosure. 2. The bioprinting system of claim 1, further comprising a bioreactor for maturing the biological matter in the fully or partially printed target (6), said bioreactor being leak-tight . A bioprinting system characterized in that the bioprinting system is connected to said sterilizable hermetic enclosure by sexual means. Bioprinting system according to claim 12 or 13 , characterized in that the bioreactor comprises the supply of the fully or partially printed target (6) in a controlled atmosphere. system. 14. A bioprinting system according to claim 12 or 13 , characterized in that the bioreactor includes means for exchanging the culture medium. 14. Bioprinting system according to claim 12 or 13 , characterized in that the bioreactor comprises means for regulating physicochemical conditions. Bioprinting system according to claim 1, characterized in that it further comprises means for the physicochemical characterization of the maturation of the printed target (6) during printing. 2. A cassette for a bioprinting system according to claim 1, characterized in that the cassette consists of a sterilizable sealed enclosure constituting the jacket of the printing module. Cassette according to claim 18 , characterized in that it comprises at least one communication identification means capable of transmitting information relating to the printhead and/or the target (6) to the activation means (5). Characteristic cassette.