Bioreactor system

Abstract

The present invention relates to a biofabrication system for bioprinting and / or patterning three dimensional scaffold and retention structures using biofilm producing bacteria as the means of synthesizing geometries. The machine comprises a negative mobile mold of the final three dimensional shape desired and one or more oxygen blocking floaters to allow biofilm growth only in the positive part of the aforementioned mold. The bioprinter movement is calibrated relative to the culture medium surface level to maintain continuous biofilm growth. The bioreactor may be used along with the ability to control temperature, humidity, air flow and culture media conditions and nutrients as the three dimensional synthesis requires.

Description

[0001] Bioreactor system

[0002] Cross-reference i to related

[0003] This patent application claims the benefit of priority of EC SENADI-2023-78895 filed 24 October 2023 and which is herein incorporated in its entirety.

[0004] The abstract filed herewith is also incorporated in its entirety.

[0005] Technical field

[0006] The present invention relates to a three-dimensional (3D) bioprinter.

[0007] More particularly, the invention proposes a 3D bioprinting system and method that uses bacterial cellulose (BC) as a structural polymer with increased strength and mechanical stability.

[0008] Background to the invention

[0009] 3D bioprinting is an additive manufacturing technique related to 3D printing, based on the deposition, usually on a micrometer scale, of biomaterials that either encapsulate cells or are loaded with cells subsequently to form comparable planar or three- dimensional structures, to living tissues.

[0010] Three-dimensional bioprinting aims to replicate and, if possible, enhance natural biological structures, taking advantage of the influence of the morphological configurations and spatial distribution in cell development.

[0011] Despite being complex and initially inaccessible, this transformative technology is revolutionizing in numerous fields. To speed up the innovation and promote discoveries, a versatile and globally accessible bioprinting system is needed. These advances have the potential to revolutionize multiple fields and facilitate breakthroughs inspired by nature's designs in animals, plants, fungi and bacteria.

[0012] In most cases, a three-axis (X, Y, Z) mechanical platform is used which controls the movements of the extruders that print the bioink in the required algorithm and shape.

[0013] A distinction can be made between two groups of mixtures used for bioprinting; bioinks, which consist of a combination (in the form of aqueous gel) of biomaterials such as growth factors, living cells, etc. and thermopolymer-based materials.

[0014] There are also various bioprinting methods such as:

[0015] • Extrusion, in which the bioink is extruded through a nozzle, with the necessary pressure is transferred to the bioink by means of a piston, compressed air or screw. • Ink jet where the bio-ink is deposited in microscopic droplets by means of a piezoelectric or thermal actuator similar to that of desktop printers.

[0016] • Stereolithography where the biomaterial used is cured by light or ultra violet.

[0017] • Laser-assisted methods, where the heat energy of a laser beam is concentrated on microscopic areas of a metallized foil whose underside is impregnated with a thin layer of bio-ink.

[0018] Most b / oprinting techniques are currently based on extrusion systems for extrusion of mainly gelling materials / bioinks which, although they are great culture media for animal, human, plant and bacterial cells, do not present ideal mechanical strength and stability for applications such as scaffolds for the creation of artificial organs, biocatalysts, biofilters, bioreactors, and other technical and scientific applications such as biomimicry and synthetic biology.

[0019] Synthetic materials have had limited success as implants due to lack of biocompatibility and biomechanical performance mismatch. Natural polymers are much more suitable as biomaterials due to their better biocompatibility and greater tissue integration. Cellulose is attractive as a biomaterial due to a its good mechanical properties.

[0020] Bioprinting and biofabrication have primarily been used in biomedical applications, but the technology's potential in various disciplines has been hindered by complexities, limited scale, and high costs. These advances as stands today represent a powerful tool that allows leaps on innovation and bio fabrication techniques. The main setback for a more mainstream use of bioprinting is the scale that still limits the kind and size of constructs made, since traditional methods of depositing bioinks allows only for small scale geometries, where these methods are intrinsically limited by the material used.

[0021] The development of affordable and scalable alternatives to traditional bioprinting is crucial for broader adoption in biological, agronomic, and ecological fields. An essential focus in this area is the selection of suitable bioinks for cell-laden scaffold deposition. These bioinks must meet biocompatibility, water retention, mechanical stability, and printing resolution requirements. However the development of suitable gel-like bioinks for printing is complex and they are expensive to produce.

[0022] WO2019197333 relates to a system comprising a robotic arm configured to position a pneumatically driven or inkjet-based microfluidic device during and / or after 3D bioprinting - which disposes of bioink cellular material, where the cellular material is dispensed, molded or extruded in a predefined pattern programmed by a computer.

[0023] US2016288414 describes a bioprinter having a processor, a support assembly and two printheads. Each printhead has an arm assembly having a proximal portion and a distal portion. It has a set of nozzles that can be configured to simultaneously receive and dispense multiple biomaterials. WO2017184839 relates to systems and methods which utilise a bioprinter with a single printhead which may include a plurality of outputs, each connected to a plurality of tanks for manufacturing multi-material biomedical constructions in vivo.

[0024] Bacterial cellulose (BC), which is a biofilm produced by several microorganisms, shows favourable characteristics such as printability, resolution, mechanical strength and stability, ideal for tissue engineering and other biofabrication applications. Its biodegradability enables gradual replacement by natural tissues, and its customizability supports tailored applications. Additionally, BC promotes vascularization, ensuring adequate nutrient supply in large-scale constructs. The sustainable and cost-effective nature of BC production makes it an attractive choice for a more diverse range of applications beyond biomedical purposes. BC grows in the air-water interface of a biological reaction medium for BC producing bacteria with an agitation free environment, forming a thick biofilm that takes the form of water level cross section of the receptacle with the enriched biological reaction medium it's in.

[0025] WO2011038373 relates to inkjet printing, which enables the production of three- dimensional biosynthetic cellulose structures with controlled shape and porosity. This system is based on a polymeric or metallic perforated material designated to be overgrown with cellulose membrane during the cultivation in a bioreactor and utilizes a microfluidic delivery system for a specified time and in specific conditions, in which the media are continuously aggregated in the air / bacteria interface. The materials lower edge is successively dropped into the medium. Such materials are reported to have applications as implants and scaffolds for tissue engineering and regenerative medicine.

[0026] DE102012201272A1 relates to a device for preparing hollow bodies made of microbial cellulose, which includes a template, a first reservoir, which is filled with a mixture comprising a liquid culture medium and a polymer-forming microorganism, a wetting device, and a housing. The template is a female form of a cavity of the hollow body to be produced. The device further comprises a moving mean by which the template is rotated around spatial axes.

[0027] Nevertheless it will be appreciated that improved devices capable of producing strong printed or moulded biomaterials without unnecessary waste and complexity would provide a useful contribution to the art.

[0028] Brief description of the invention

[0029] An object of the present invention is to overcome the drawbacks and / or disadvantages of the prior art with respect to bioprinting systems of complex structure and high cost.

[0030] Novel bioreactor or bioprinter

[0031] The present inventor has provided a novel bioreactor which allows layered, selective, bioprinting of moulded articles in a bioreactor design. The novel system is not based on extrusion, has only one axis, uses fixed molds and variables to model biofilm produced on the surface (air-liquid interface) of the medium into a three-dimensional shape. This is capable of creating scaffolds for a myriad of applications.

[0032] As described herein the bioprinter system is based on a bioreactor housed inside a reactor chamber. The chamber, typically transparent, comprises suitable liquid growth media, and the bioreactor is in direct contact with the liq uid / gas interface within the chamber. A controller maintains the appropriate living conditions of the biofilm producing bacteria and production of the articles, while the chamber supports suitable mechanical and sensor components to achieve this.

[0033] According to a embodiment preferred of the invention, the bioprinter comprises a bioreactor where a bacterial biofilm is formed at the air-water interface of the medium and is patterned in cross-sections of the final three-dimensional shape to be bioprinted using a 2-part system consisting of an open negative mold of the form gradually emerging from the biological reaction medium for the bacteria that continuously reveals the cross-section of the form and one or more floating devices that block biofilm growth where growth is not necessary in the reaction zone.

[0034] Also provided is a bioprinter containing a bioreactor that maintains appropriate conditions of temperature, humidity, airflow and culture media nutrients for precise production of a biofilm in three-dimensional form, with increased mechanical strength, stability and biodegradability according to claim 1.

[0035] Also provided is a new bioprinting method using a two-part system: a movable open negative mold for bacterial biological reaction media and one or more floating devices that block unnecessary biofilm growth.

[0036] In addition to the one or more floating devices, the system may also utilise a thin layer of a non-aqueous or oil based fluid in the zones that biofilm growth is not required.

[0037] Reaction chamber

[0038] According to one embodiment of the invention the main reaction chamber is transparent e.g. made of tempered silicate boron glass, or plastic. However in other embodiments the chamber could also be made of stainless steel with corresponding viewing windows of transparent material.

[0039] Movement and control of the mold height

[0040] According to one embodiment of the invention, the vertical movement of the mold upwards during creation of the article is realized by means of a stepper motor coupled to a mold carriage. The carriage and mold can be moved vertically by a motor engaging the carriage by appropriate means. For example the carriage and mold can be moved via a worm screw and support rods (e.g. two rods) that are connected to the mold. Spacers may be used to improve and adapt the depth of reach and align with the floating devices (discussed in more detail below).

[0041] The initial position of the mold is set with respect to the inner hooks of the mold which supports the first layers of cellulose growth bacterial cellulose. From this point, the motor first moves the hooks a couple of microns below the water surface lever to create solid layers of bacterial biofilm before encountering the support hooks that act as attachment points for the freshly biofilm layer layers to avoid sliding / slipping of the piece into or out the mold, stopping and / or hindering the reaction in a complete continuous manner.

[0042] Controller

[0043] Since the speed and consistency of synthesis of each new layer may change in thickness over time, the movement may be monitored by optical and water level sensors to confirm the position of the current bacterial cellulose layer in the reaction zone. This information is processed and transmitted to the 3D printer controller for adjustment of depth of the mold according to its needs. This is particularly important in achieving accurate geometry of cross-sectional change, where oxygen exchange and nutrient accessibility can be slowed or accelerated depending on the available area.

[0044] Such 3D printer controllers are well known in the art and commercially available e.g. https: / / www.lasercontrolcard.com / 3d-laser-printing-controller-product

[0045] Typically such systems include at least 2 sensors, the first one a laser displacement sensor and the second one a camera to assess the height and thickness of the current layer being synthesised. This information is received by the controller and then translated and then sent to the MCU of the 3D printer. Controllers can be based on single board computes (for example a Raspberry Pi) to process the relevant calculations and height compensations and way have a web or app interface (see e.g. https: / / octoprint.org / ). The MCU of the 3D printer can have a modified version of the firmware compatibility with the printer controller to accept live changes. In the present context the growth of the organism is relatively slow allowing pauses and height adjustment (microns at a time) resuming the print when the position is correct. This is done slowly to avoid disruption of the biofilm synthesis.

[0046] Molds and articles

[0047] According to another embodiment of the invention, the method and system presented in this bioprinter can have multiple types of molds for all types of geometries. These include simple extrusions, spirals, cylinders etc. The molds may have positive or negative tapers, allowing the formation of cones or frustoconical forms. Asymmetrical forms can be readily produced, since the mold itself is only moved in one dimension (upwards during growth). The molds can be made using any traditional 3D printing technology (Fused deposition modeling (FDM), stereolithography (SLA), and selective laser sintering (SLS)). Where the geometry is simple and the molds form permits it, the mold can be made in any material that is resistant to sterilisation techniques (e.g. from polycarbonate (PC) or polypropylene (PR)).

[0048] As explained below, for preparing biofilm articles which are complex and could be trapped in the mold, it may be preferable to prepare the mold from a material that dissolves in a solvent not present in the biological reaction. An example would be PolyVinyl Butyral (PVB) that dissolves in isopropanol.

[0049] Thus the system has utility in creating a wide variety of articles such as scaffolds for artificial organs or organoids (e.g. luminal organs), biocatalysts, biofilters, bioreactors, and other technical and scientific applications in biomimicry, synthetic biology and industrial biotechnology. After production, scaffolds can be seeded with appropriate cells, particularly stem cells, and cultured using methods known in the art (see e.g. US1021471B2; WO2017042232 etc). Other utilities for biofilms may include nutrition.

[0050] The method and system presented in this bioprinter can have multiple types of molds for all kind of geometries as long as it complies the needs of the system such as an entry and exit point for both gas and liquid circulation to maintain proper synthesis of the biofilm during the movement of the molds through a proper floating device that can block biofilm growth where it is not required.

[0051] The mold material must be able to exchange gases to prevent the formation of bubbles within the biofilm and to ensure adequate oxygen ingress to the bacteria.

[0052] In one embodiment of the mold, the material used is not breathable but during manufacture (e.g. Computer-aided design (CAD) and Computer-aided manufacturing (CAM)) microholes and ducts are incorporated into the design to improve gas circulation in and out of the reaction zone.

[0053] In another embodiment, the material of the mold itself is made of a breathable micro porous material that allows for continuous gas exchange between the air space inside the reactor and the reaction zone, in addition to avoid the formation of air bubbles from the biological reaction process.

[0054] Movement and positioning of the mold carriage

[0055] According to another embodiment of the invention, the mold itself is connected to a plurality of (e.g. 3) spacers which allow the levelling of the hook supported within the culture medium of the material and provide additional depth in the liquid medium.

[0056] Since the process only requires the mold to be submerged and the water level is generally flat except in the areas touching the inner edges and walls of the mold due to surface tension, the levelling process allows the liquid to flow properly in and out of the mold during the process. In one embodiment the spacers connect the mold to a mold carriage. The mold carriage is capable of linear movement (up down). The movement may be achieved via a combination of a worm (infinite) screw linked to the carriage e.g. via a nut, and actuated by a motor (e.g. a stepping motor). The carriage movement may be stabilised with one or more support rods connected to the carriage via bearings. For example the carriage may be connected with two bearings on two support rods providing the linear movement and a nut for the infinite screw that connects its movement to the carriage from the stepper motor.

[0057] All of these components inside the bioreactor are moisture and rust resistant, eliminating the possibility of rust and friction, thus maintaining the precise movement of the mold inside the carriage. In the case of electronics, all the components are located outside the bioreactor to avoid damage from moisture and biological agents.

[0058] Floating devices

[0059] According to another embodiment of the invention, the function of the floating devices is to block the growth of bacterial cellulose that occurs outside of the reaction zone in the molds, eliminating the access to oxygen in the air-water interface of the culture medium.

[0060] In one embodiment there are two or more floating devices to "block" the liquid surface within the perimeter of the mold and outside the perimeter of the mold respectively.

[0061] For example, where the mold has a cylindrical cross section, and the first floating device would be essentially circular, and float within the inner wall of the mold, dimensioned to closely match its internal diameter. The second device would be essentially annular, and float outside the outer wall of the mold, dimensioned to closely match the mold's external diameter. The external diameter of the second device would closely match the internal diameter of the reaction chamber.

[0062] Where the mold has a different cross section (whether symmetrical or asymmetrical) the same principles apply: an inner floating device matching the mold's inner profile, and an outer floating device matching the mold's outer profile and fitting within the reaction chamber.

[0063] For complex and variable cross-section geometries, in addition to a floating device, blockage can be achieved by depositing a thin layer of a non-aqueous or oil based fluid in the zones where biofilm growth is not required. This may be advantageous for replacing the 'outer floating device' above to adapt to the relevant cross section.

[0064] In one embodiment, the floating devices are low-density solid forms that fit into and around the actual mold. However, in another embodiment, the floating devices are small low-density granules that are automatically disposed outside of the reaction zone in the mold.

[0065] Removing the formed biofilm from the system and mold

[0066] According to another embodiment of the invention, upon completion of the reaction, the mold carriage emerges completely from the culture medium and the mold is removed from the machine. The support hooks are designed to separate easily from the mold and the three-dimensional form of bacterial cellulose is removed from the mold. In the case of shapes based on extrusions or geometries more complex, the invention provides two options to release the form. The first embodiment is a multi-part mold to release the biofilm form and the second embodiment utilises molds that can be dissolved in the presence of specialised solvents which do not react with bacterial cellulose.

[0067] Sensors and environmental control

[0068] All mechanical components may be connected to the reactor lid where the stepper motor and linear rods can be attached. This lid also contains the inlet and outlet fans to maintain an adequate oxygen level in the reactor air space.

[0069] The intake fan must purify and sterilise the air incoming into the reactor via filters to reduce the possibility of contamination. The air introduced in the reactor and the gases produced by the biological reaction process leave across the outtake fan passing through a one direction valve to reduce the possibility of contamination entering through it.

[0070] The reaction chamber or the mechanical lid itself contains entry points for sensor probes. For example there may be three probes: temperature for both the bioreactor atmosphere and liquid medium as well as a third probe for humidity. The other enter points serve as conduits to renew the liquid culture medium, one is for intake and other for outtake; the last one is to place humidity in the form of water vapour to avoid dryness of material printed once outside the liquid medium and to aid the synthesis process.

[0071] Biomaterials

[0072] The three dimensional printer in the Examples below utilises BC by way of exemplification, but those skilled in the art will recognise that other biofilms which are formed at a liquid -air interface could be produced using the same system. Table 1

[0073] ("SCOBY" is a Symbiotic Consortium Of Bacteria and Yeast)

[0074] The BC producing bacteria (Acetobacter xylinum was used in the Examples herein) need a constant temperature of 28° C to 31° C and a relative humidity in the air space of 70 to 80% to improve the mechanical and biological properties of the material. However, as explained above, the conditions used will depend on the strain, which may optionally be genetically enhanced strains which would greatly improve the reaction time and properties of bacterial cellulose. The culture media can range from sugar enriched laboratory grade mediums, to industry waste from food and agronomic industries. Generally the strains can undergo the BC synthesis in pure medium where only one species is included. However, the use of a consortium can also be beneficial , since the conditions of speed, temperature and humidity are automatically corrected according to the growth of each layer in the mold where the machine system doesn't depend on a fixed rate of BC growth. Also the medium is constantly renewed to avoid equilibrium of the reaction and accumulation of metabolites that can hinder or stop the reaction, allowing a continuous, stable and homogeneous formation of BC biofilm coupled with the automated electronic system of printing control and bioreactor conditions, that allows to have a more permissive environment to form three dimensional shapes form BC, that doesn't require expensive culture media or specialised strains to work properly.

[0075] The automatic system of the three-dimensional BC bioprinter is based on growth curves of the strains, sensor data of the current reaction undergoing in the bioreactor system as well as configurations based on the type of strain, medium and BC pellicle formed.

[0076] Troubleshooting and testing features may be included in the firmware such as automatic check of biofilm thickness variability viability and speed reaction.

[0077] Such checks may utilise the laser and camera sensors of the printer controller, coupled with the bioreactor condition data (temperature, pH, humidity, etc) to gather data about growth of the strain to inform the user of the machine if parameters go outside or predetermined limits.

[0078] It will be apparent that the system itself can work in many scales and is not limited in any way or form in respect of the final volume of the mold since bacterial growth and biofilm growth can grow in many different conditions.

[0079] In order to overcome the disadvantages of the current systems and methods available , as described above, the aforementioned improvements have for the purpose of demonstrating the advantages and new features of the invention set forth, which will become more apparent from the detailed description which follows, when taken in conjunction with the accompanying drawings.

[0080] Brief description of the drawings

[0081] Both the summary, the appended drawings and the following description are meant to be read together to fully understand the complete function and capabilities of the system proposed in this invention

[0082] The drawings are mostly illustrative to describe the three-dimensional printer capabilities. The system is designed to be scalable and adapted to multiple needs ranging from printing volume, type of bacteria to consortium used and culture media available Figure 1 illustrates a perspective isometric upper view of the complete embodiment of the three-dimensional BC bioprinter inside the reactor chamber of the bioreactor system in accordance with the present invention.

[0083] Figure 2 illustrates a perspective isometric lower view of the complete embodiment of the three-dimensional BC bioprinter on its submerged position.

[0084] Figure 3 illustrates a top view of the main lid containing the electronic components such as the Z movement stepper motor, ventilations vents and various entry points for probes, sensors and liquid recirculation.

[0085] Figure 4 illustrates an isometric upper view of the main lid containing a more detailed view of the different entry and coupling points for the bioprinting and reaction function as well as the support points for the linear rods

[0086] Figure 5 illustrates an isometric bottom view of the detailed function of the mold system and floating devices (inside and outside), as well as a detailed view (A) of the mold system and floating devices (inside and outside).

[0087] Figure 6 illustrates a bottom view of the mold and floating devices(inner and outer), as well as a full view of the air outlets to promote air flow to the reaction zone.

[0088] Figure 7 illustrates an exploded view of the filtering system and air inlet, as well as a fan adapter and fan outlet grilles.

[0089] Figure 8 illustrates front views of the mold system undergoing layered BC synthesis during depth control emerging from the water level, as well as the BC three-dimensional formed within the reaction zone of the mold as a cross-section (A-A and B-A).

[0090] Figure 9 illustrates a bottom isometric exploded view of the final three-dimensional shape formed in the mold system.

[0091] Figure 10 illustrates a cylindrical mold formed by simple extrusion.

[0092] Figure 11 illustrates a spiral form mold.

[0093] Figure 12 illustrates molds having positive and negative tapers forming frustoconical shapes.

[0094] Figure 13 illustrates interior geometries achievable with the system of the invention. The bioprinter can be used to prepare both solid and hollow forms. Where the geometry creates the risk of airlocks which could prevent access for media, it may be preferred to include venting holes (at appropriate angles) to allow gasses to escape, and ensuring optimal orientation of the mold itself. Figure 14 illustrates other spiralular forms achievable with the system of the invention.

[0095] Figure 15 illustrates asymmetrical shapes having compound geometries achievable with the system of the invention.

[0096] Figure 16 shows an embodiment of the invention. The numbered parts are as follows: 1 3D Printer Body (From a Creality ENDER 3); 2 Z Axis Stepper Motor; 3 Mold Carriage; 4 Mold; 5 MCU Housing; 6 Breather Fan; 7 Fan Wiring; 8 Stepper Motor Wiring; 9 LCD Display (Printing and Reaction Conditions).

[0097] Figure 17(a) and (b) shows a close up of the reaction chamber. In this embodiment 2 Systems used to block cellulose synthesis, a floaher in the interior part of the mold and a oil layer in the exterior to accommodate the irregular form of the reaction chamber (in this case a mason jar). The hooks are located in the reaction zone where the cellulose form layer by layer.

[0098] Figure 18 shows an example bioprinted bacterial cellulose 3D-article. The image is taken after the removal of the hooks and postprocessing steps to remove the bacteria and bleach the cellulose to achieve transparency. The final form has an imprint from the 3D printed mold and maintains dimensional accuracy (if maintained in a saline solution).

[0099] ***

[0100] Detailed description of the invention

[0101] From this description a more comprehensive and complete view of the whole system, using the drawing and previous information should take place. However, it does not reflect the whole capabilities and potential of the machine since the design itself allows multiple changes in bacteria, medium, printing and reaction conditions. In addition, unless otherwise stated, any description herein is mostly illustrative of the operation of the system. Hence, any design considerations should be taken as exemplary only. The terminology also implies various degrees of importance of a mechanism or system component and will be addressed in due time since several components are designed in a specific way to work and others are mostly for illustrative purposes. The embodiments of certain parts mentioned as well, are to be taken as modular parts of the three- dimensional biofilm bioprinter to be interchangeable to form different iterations of the same base system related in this invention.

[0102] The present invention is related to bioprinters, biomaterials and bioreactors capable of realizing two and three-dimensional constructions, as well as the constructions themselves obtained or obtainable from the systems.

[0103] The biopolymer (e.g. bacterial cellulose, BC) bioprinted material itself can be subjected to various post-processing procedures in order to adapt to different applications, such as cell, tissue and organ engineering, catalysts and biological filters, food engineering, biomimetics of biological morphologies and their various bioengineering applications and synthetic biology.

[0104] The methods involved use design and fabrication computer-aided mold design requires specific conditions to function properly in the system described in the present invention. In each embodiment, the final synthesized three-dimensional shape is inherently contaminated, as the system itself requires a (BC) / biofilm producing bacterium or consortium to function. Therefore, always a sterilization process is necessary for any biological application.

[0105] This bioprinter replaces gel-type bioinks and other related systems, improving the cost, volume and versatility of the possible constructions to be made, since the bacteria and / or consortium used are economical to maintain, as are the culture media used. A preferred example polymer used is bacterial cellulose (BC), which meets the requirements of a material to be used in bioengineering, such as biocompatibility, excellent mechanical resistance, biodegradability, customization, among others.

[0106] In some aspects the bioprinter acts as a three-dimensional replicator, reading from a digital file into a physical object, which can also be used as a fabrication system for multiple identical shapes both 2D or 3D.

[0107] As discussed above, the mold itself can be prepared using traditional 3D printing techniques, incorporating hooks in the first layers to improve adhesion to the mold during the first stages of synthesis and also to avoid slippage of the formed parts. The molds are designed so both oxygen and culture media can access the mold at all times with no blockages, using venting holes as required. In the system of the invention there is a clear separation between the reaction zone and the blocked zone which is achieved by a fixed geometry floater that can move independently from the mold or by multiple smaller floaters. The reactor itself works as continuous fermentation (i.e chemostat) where media is constantly and simultaneously replaced by fresh media. Changes of water level are controlled by a controller as discussed previously and below.

[0108] The system is preferably automated since there are multiple factors that can modify or disrupt the biopolymer pellicle so a constant correction and adjustment is preferably incorporated. The bioprinter can work with different kinds of biofilm as long as it grows in a liquid medium and the pellicle is formed in the liquid-gas interface, and / or with BC using various conditions of growing that change the properties of the material.

[0109] The bioprinter is designed as a system, meaning that more than one mold, motor systems with linear carriages and automated regulation can also be incorporated at the same time independently inside the same bioreactor; since the nutrient conditions are maintained at a stable, enriched and homogeneous levels across the medium at all times. The reaction occurs at the same rate proportional to the area and printing volume, where multiple forms can be synthesised at once, with same shape or different geometries working as a large-scale production of multi-customizable machine. The molds that the machine depends on for BC biofabrication shapes are fabricated with traditional 3D technology, where the geometry and material used can greatly improve and change the final characteristics of the outcome. However, basic 2D patterns that could be extruded and don't require specialised technology skills or fabrication methods (as long as the molds comply with the specific requirements described herein) are preferred. The precise identity of the fabrication material is not critical, as long as they are non reactive or disruptive to the used bacteria. Likewise in relation to the bioreactor, since most biofilm producing bacteria require non-complex mediums and conditions, the maintenance and precision of the temperature, nutrients, oxygen and air flow is forgiving and similar to a food grade fermentation that can be deployed under industry standard conditions.

[0110] Those skilled in the art will appreciate that the specific application will affect the requirements of sterility and precision, for example medical and bioengineering applications, where there are multiple machine embodiments and modules that could be added and removed depending on the shape and object. The system described in the invention is based mainly in bioprinting and the bioreactor aims to control a proper production of a biofilm pellicle and avoid contamination that can impede the biological process.

[0111] In some aspects also the bioprinter may include relative positioning-means based on mechanical or sensor end-stops. In these aspects, to maintain proper calibration and levelling of the printing zone in the mold and to avoid damages to the carriage, the machine homes the Z axis by an assisted manual process where it is lowered until a sensor located at the same level of the support hooks senses conductivity from the liquid medium. From this set point, a couple micrometres are lowered to ensure a proper full layer of BC so the carriage can rise until the cross section shape grabs around the hooks with a newly synthetized BC layer. The control of synthesis is required to maintain a proper layer density and thickness, whereas little time in a layer could cause layer separation and a print failure due to incorrect or weak layer production; too much time in a layer leads to accumulation of material and inefficient use of culture medium. As mentioned before, there are several requirements for the mold and one is the minimum thickness of the wall / perimeter used, which is heavily dependent on the strain and surface tension of the liquid. The layer synthesis control is maintained by optical sensors, proofing the reaction zone, this verification is coupled with simple magnetic induction sensors embedded inside the floating devices and the mold to ensure correct water and mold alignment into the reaction zone. The calibration of the machine requires growth curve, reaction speed and chemical equilibrium data to function properly and adjust parameters correctly. However, redundant systems such as the analogs and manual ones mentioned before ensure proper pellicle formation. Since this is a mechanical and biological process undergoing at the same time, precision is very important in this system. Living organisms do not always grow or react the same, regardless of genetic and strain stability, any calibration step ensures proper layer synthesis. FIG. 1 illustrates an upper isometric perspective view of the complete embodiment of the three-dimensional BC bioprinter inside the reactor chamber of the bioreactor system. The bioprinter system comprises a reaction chamber 1 that encapsulates all of the mechanical and bioprinting components that are in direct contact with the liquid and / or the gas interface inside the bioreactor, two linear rods 16 are driven by a infinite (worm) screw 17 on the Z axis connected by a screw nut 14 and linear bearing 8 to the mold Z carriage 7 into three distinct spacers 10 that function to provide extra depth intro the liquid medium and to adjust the levelling of the mold 11 to ensure proper printing conditions. First layers grip into internal supports and proper gas evacuation for biological processes. An outer floating device 18 blocks the BC to form in undesired zones by blocking the interchange of gasses and dissolution of oxygen in said zones, while allowing correct flow of the linear movement system, this flotation device also functions as a water level sensor 18 containing magnetic conducting material embedded while a sensor in the periphery of the mold 13 and reaction chamber senses its changes.

[0112] This linear movement system is driven by a stepper motor 3 that is supported by the main lid 6 that likewise supports the air flow system composed of an air intake fan 5 and the air outtake fan 15 that connects to a one side valve 4 to reduce the possibility of contaminating agents into the bioreactor. The main lid 6 also contains multiple entry points 2 for the electronic water-proof wiring as well as liquid and gas piping to maintain proper reaction conditions. The electronic components are the standard used for 3D printing

[0113] The 3D printing controller or MCU is a standard-use mainboard for 3D printing however the data collected such as water-level, bioreactor conditions and height corrections may be processed by a secondary controller (Raspberry Pi in the embodiments described herein). Those skilled in the art will recognise that based on the disclosure herein a Programable Logic Control may be used. Preferably the control system is housed in a separate unit comprising a main board, drives, relays and analog and digital components for the bioreactor sensor and probes. In the non-limiting example herein the following components were utilised: Links: PC - 3D Printer Direct Control (Open-source)

[0114] (for calibration and test) s

[0115] Motors, MCU, and wiring used from this printer.

[0116] The bioreactor and printing conditions are adjustable according to the strain and medium conditions. Temperature and humidity parameters in the case of the air space or atmosphere in the reactor and the liquid phase or medium, have a medium recirculation and temperature control. Other parameters such as pH can be controlled inside the reactor as well with the use of additional automated electronics. The biological conditions where bacterial growth can be directly controlled in the medium reservoir in which the fresh culture media is deposited. The system mainly works in moderate temperatures, being able to set from 25 to 35° C and humidities from 0 to 100% . However, in other embodiments a cooling down and drying feature serves to stop the reaction once the form is completed. The speed of reaction and rise speed of the mold carriage depends greatly on factors mentioned before, where low stepping speeds and ranges are used with high precision encoded stepper motors if high resolution is required

[0117] FIG. 2 illustrates a lower isometric perspective view of the complete embodiment of the three-dimensional BC bioprinter on its submerged position. This embodiment shows the filtration system 20 for the intake fan 5 and the outtake valve 19 that restricts the flow of air only from the inside to the outside of the bioreactor.

[0118] All these components are coupled by an adapter for the intake fan 5 and the outlet valve 19 a screen for the outtake fan 21. In this perspective, a worm screw rigid coupling to drive the rotational movement of the motor across the infinite screw into the linear rods and into the carriage 9, this system is supported in the bottom base of the main lid by friction screw fasteners 24. The mold 11 itself has a hollow open cavity for the liquid medium to enter, limited by an exterior wall 22 and an interior wall 27 that act as the negative limits of the geometry to be formed. All this is contained and separated by a gasket 23 in the periphery of the internal part of the main lid into the reaction chamber border 1.

[0119] FIG. 3. illustrates a top view of the main lid containing the main electronic component such as the Z movement stepper motor, ventilations vents and various entry points for probes, sensors and liquid recirculation.

[0120] The bioprinter contains all components that have direct contact with the gas or liquid interface of the bioreactor on the main lid, it contains multiple entry points 2 for probes, cable sensors and hosing for liquid and gaseous entering and leaving the reaction chamber 1, as well as the previously mentioned movement system driven by the stepper motor 3 and the air recirculation system composing of the intake fan 5 and the filtration system 20, the outtake fan 15, and its valve 4. The BC producing bacteria are intrinsically aerobic meaning that they needs oxygen to survive (and hence form a floating cellulose biofilm in the air-water interface of a liquid medium, so as to utilise the oxygen available). For this reason a constant oxygen renewal inside the air space is important to continue a proper reaction, hence also the continual renewal of the medium. As with any living organism, once the population reaches an equilibrium with the current resources available they start to decay, and consequently in this case stop producing BC. A continuous flow of oxygen and nutrients is used for a constant, homogenous and undisrupted BC pellicle formation. Suitable chemostats are well known in the art - see e.g. Continuous Cultures (Chemostats), Editor(s): Thomas M. Schmidt, Encyclopedia of Microbiology (Fourth Edition), Academic Press, 2019, Pages 743-761, ISBN 780128117378, https: / / doi.org / 10.1016 / B978-0-12-801238-3.02490-9.

[0121] FIG. 4 illustrates an isometric upper view of the main lid containing a more detailed view of the different entry and coupling points for the bioprinting and reaction function as well as the support points for the linear rods. The main centre of the bioprinter for the mechanical parts is the main lid 6 that seals and isolates liquid medium from the exterior using a set of two gaskets in the interior perimeter of the main lid coupling 6, 23 meaning all components and supports are held inside by multiple connecting points for stainless steel screws 28, 30, and where the stepper motor fits its shaft 27 and other pieces with the help of an adapter space 29. and multiple entry points mentioned in previous descriptions. The movement along the Z axis requires linear rods to drive the movement precisely and without interruption , this is accomplished by fixing the rods into coupling 32 that fits along the rod and secure using screw 31 and nut 24 friction fasteners.

[0122] FIG. 5 illustrates an isometric lower view of the mold and floating devices system (inner and outer molds) detailing their function, as well as a detailed view (A) of the support hood system inside the reaction zone. The main system of the bioprinter is the mold 11 and floating devices 18 inside and outside the mold itself to block BC synthesis in undesired zones.

[0123] The starting reaction zone A-36 is microns higher that the supporting hooks A-35 to ensure grip of the consequent lawyers being formed into the mold, the liquid medium enters and leaves from the open space in the base of the mold 23 and in one embodiment interexchange gases across the material itself 34. This system is connected by the spacers into the carriages by three different stainless steel screws in coupling zones 33 to provide proper depth of the mold while allowing extra printing volume. During this process the emergence of the mold has to be slow and gentle to avoid layer separations, since the synthesis has to occur uninterruptedly, if any disturbance occurs the bacteria in the bioreactor starts to synthesise a new pellicle that is not crosslinked with the previous one can damage the printing process. The support hooks are a precaution measure since the BC pellicle forms with the exact shape of the mold, but once a layer is formed and emerges, the conditions of humidity and water absorption change, causing slight deformation that can make the shape slip into the medium, stopping the continuous synthesis of BC.

[0124] In one embodiment a continuous spray of enriched medium and bacteria is poured into the emerged BC pellicle to maintain humidity and continue secondary formation of BC in the form.

[0125] In some aspects the machine has the capability of recovering from a synthesis failure by dipping inside the culture medium previously synthesised layers by bio-welding the next layers

[0126] FIG. 6 illustrates a bottom view of the mold and floating devices (inner and outer) as well as a comprehensive view of the air vents to promote air flow into the reaction zone. The bioprinter mold also contains ventilation vents 37 to promote oxygen entry into de reaction zone 36 and the aforementioned flotation 18 devices in the rest of the air-water interface, the support hooks 35 and coupling points 33 can be seen more clearly in this view. The support hooks form a radial pattern that completely grips the BC form across the reaction; furthermore these hooks are designed to be broken to release the three dimensional BC form.

[0127] FIG 7. Illustrates an exploded view of the air intake and filter system as well as an adapter for the fan and a screen for the outtake fan. The air intake for the bioprinter system comprises an intake fan 2 that pulls in air filtered by means of a microporous materials 39 and is trapped by two screen plates 38, 40. The filtered air exits across the outlet flange 43 of the fan and enters the bioreactor with the help of an adapter gasket to avoid leaking. 41 This adapter also contains an outlet screen 42 for the outtake air exit to increase turbulence and better distribute the air across the bioreactor gas atmosphere. It is very important that the air contains adequate amounts of oxygen and is free of biological and environmental contamination. The BC pellicle itself is a potential substrate for other microorganisms to grow (indeed this is why this material is a good scaffold for bioengineering and cell tissue applications). Moreover, if the BC producing bacteria are contained in a consortium of other microorganisms (such as a Kombucha SCOBY) the growth conditions can be less restrictive in terms of sterility, medium composition, pH levels and variability during synthesis, as well as more resistant to general changes which might otherwise affect the production of the biofilm.

[0128] The filter system in the drawing is merely illustrative since the filtration of a recirculation system depends heavily on the application of the biomaterial.

[0129] FIG 8. A-B illustrates front views of the mold system undergoing layered BC synthesis as during depth control emerging from water level, as well as the three-dimensional BC formed inside the reaction zone of the mold as a cross section (A-A and B-A). The bioprinter mold starts the process in a lowered position inside the culture media 44 where the first layer forms microns higher form the support hooks position below the water lever 45 in which a continuous formation of BC layers are synthesised according to the mold form 23, the formation of the last BC layer always occurs inside the medium in the air-water interface which has also been referred as the reaction zone 46. Once the printer has synthesised a layer, a continuous process of elevation occurs at the rate of synthesis of the BC producing bacteria in the bioreactor. The reaction time depends primarily on temperature and nutrient condition, but once the layer has been formed the machine stops for X set time to let the pellicle to be completely formed into the desired geometry before elevating into the next layer / cross section of the mold. The reaction zone 46 is always at water level 45 regardless of the mold position 47. This is the basic concept that permits bioprinting to work and allows various kinds of geometries to be synthesised. The culture media inside 48 and outside 49 must be blocked for optimal growth and air flow into the reaction zone otherwise BC will grow more prominently in this zone since the access to oxygen is more direct compared with the reaction zone. Finally, once the bioprinter has correctly synthesised the first layers, the support hooks allow it to continue the reaction without slipping of the material into the liquid, stopping the reaction. The mold itself in some embodiments allows the medium to enter in the lowered position and interchange of gases once the mold reaches the top position either by incorporating microholes into the mold, or by using permeable materials to fabricate them. There are several considerations to promote crosslinking of BC, these considerations and techniques greatly improve the rate of growth and material properties.

[0130] FIG. 9 illustrates an isometric exploded lower view of the final three-dimensional shape formed in the mold system form BC. The final form printed by the system 51 comprises the positive form of the mold inserted in the liquid medium with no extra geometries formed in the interior cavity of the mold 48. Once the reaction has completed, the support hooks are manually removed from the three-dimensional BC form. The internal 23 and external 34 walls of the mold that forms the three-dimensional negative of the final form can be shaped into all kinds of geometries as long as each meets the requisites for the synthesis to take place. The first one is that the mold has to have two open ends, the lower one allows the liquid culture medium to enter the mold and the second upper allows gases from biological reactions to escape and oxygen to diffuse into the reaction zone. The mold also allows water to flow properly into internal cavities and oxygenation in general.

[0131] Some mold geometries are illustrated in FIGs. 10-15. Other geometries will be apparent to those skilled in the art in the light of these.

[0132] FIGs. 16-18 illustrate a device according to the invention, and an example 3D printed product.

[0133] ***

[0134] The following numbered embodiments represent aspects of the invention:

[0135] 1. Three-dimensional biofilm synthesizing bioreactor system based on bacterial cellulose that comprises: - A reaction chamber (1) that encapsulates all mechanical and bioprinting components that are in direct contact with the liquid and / or gas interface within the bioreactor and maintains the appropriate living conditions of the cellulose-producing bacteria;

[0136] - A main cover (6) containing multiple entry points (2), for waterproof electronic wiring, as well as liquid and gas pipes to maintain proper reaction conditions;

[0137] - A stepper motor (3) that drives the linear movement system and is supported on the main cover (6); Likewise, the air flow system composed of an air inlet fan (5) and the air outlet fan (15) that connects to a valve on one side (4) to reduce the possibility of agents entering, contaminants to the bioreactor; characterized in that the bioreactor has the function of modeling the bacterial cellulose film formed at the air-water interface in cross sections of the final three-dimensional shape that is to be bioimprinted by means of a mobile open negative mold (11) for bacterial biological reaction media 5 and a floating device (18) that blocks unnecessary biofilm growth.

[0138] 2. Bioreactor system, according to embodiment 1, characterized in that the reaction chamber (1) comprises two linear towing rods (16) that are driven by a worm screw (17) in the Z axis, connected by a screw nut ( 14) and a linear bearing (8) to the mold carriage (7) of the Z axis in three different separators (10) whose function is to provide additional depth in the introduction of the liquid medium and adjust the leveling of the mold (11) to guarantee the adequate printing conditions of the first adjustment layers to the internal supports and adequate gas evacuation for biological processes.

[0139] 3. Bioreactor system, according to embodiment 1 and 2, characterized in that the reaction chamber (1) also comprises a floating device (18) that functions as a water level sensor (12) that contains magnetic conductors and embedded material while a sensor in the periphery of the mold (13) and the reaction chamber detects its changes.

[0140] 4. Bioreactor system, according to embodiments 1,2 and 3, characterized in that the system works at moderate temperatures, and can be configured from 25 to 35° C and humidity from O to 100%.

[0141] 5. Bioreactor system, according to embodiment 1, characterized in that the filtration system (20) for the inlet fan (5) and the outlet valve (19) restricts the air flow only from the inside to the outside of the bioreactor, which are coupled by an adapter for the inlet fan (5) and a screen for the outlet fan (21). A rigid motor worm coupling (25) serves to drive the rotational movement of the motor through the worm towards the linear rods and into the carriage (9), this system is supported on the lower base of the cover main using friction screw fasteners (24).

[0142] 6. Bioreactor system, according to embodiments 1 and 5, characterized in that the mold (11) has an open hollow cavity so that the liquid medium enters, limited by an outer wall (22) and an inner wall (27), which act as negative limits of the geometry to be formed. All of this is contained and separated by a gasket (23) on the periphery of the internal part of the main lid towards the edge of the reaction chamber (1). 7. Bioreactor system, according to embodiments 1 and 6, characterized in that the mold (11) contains ventilation vents (37) to promote the entry of oxygen to the reaction zone (36) and the flotation devices (18) at the air interface, -water, the support hooks (35) and the coupling points (33).

[0143] 8. Bioreactor system, according to embodiment 1, characterized in that the air inlet for the bioprinter system comprises an inlet fan (2) that sucks in the air filtered by microporous materials (39) that is trapped by two screen plates (38)(40). The filtered air leaves through the outlet flange (43) of the fan and enters the bioreactor with the help of an adapter gasket to prevent leaks (41). This adapter contains a screen for the air outlet (42) to increase turbulence and better distribute the air.

[0144] 9. A method to produce three-dimensional structures based on bacterial cellulose (CB), characterized in that:

[0145] - The bioprinter mold begins the process in a lowered position within the culture medium (44) where the first layer forms 20 microns higher than the position of the support hooks under the water lever (45) in which A continuous formation of layers (CB) is synthesized according to the shape of the mold (23).

[0146] - The formation of the last layer of (CB) always occurs within the medium at the air-water interface, which has also been called the reaction zone (46).

[0147] - Once the printer has synthesized a layer, there is a continuous process of raising the synthesis rate of the (CB) producing bacteria in the bioreactor.

[0148] - Reaction time depends mainly on temperature and nutrient condition, but once the layer has formed, the machine stops for X set time to allow the film to fully form into the desired geometry before rising to the next layer / cross section of the mold.

[0149] - The reaction zone (46) is always at the water level (45) regardless of the position of the mold (47).

[0150] - The culture media inside (48) and outside (49) must be blocked for optimal growth and air flow to the reaction zone, otherwise the (CB) will grow more prominently in this zone since that access to oxygen is more direct compared to the reaction zone.

[0151] - Finally, once the bioprinter has successfully synthesized the first layers, the support hooks allow it to continue the reaction without the material sliding into the liquid, stopping the reaction.

[0152] 10. A method according to embodiment 9, characterized in that the final shape printed by the system (51) comprises the positive shape of the mold inserted in the liquid medium without additional geometries formed in the interior cavity of the mold (48). Once the reaction has been completed, the support hooks are manually removed from the three dimensional shapes of (CB). The internal wall (23) and external wall (34) of the mold that forms the three-dimensional negative of the final shape can be molded in all types of geometries as long as it meets the requirements for the synthesis to be carried out.

[0153] *** A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0154] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0155] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0156] Ranges are often expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0157] Any subtitles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

[0158] The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by crossreference.

Claims

CLAIMS1 A bioprinter for producing a three-dimensional article(51) from bacterial biofilm, the bioprinter comprising:(i) a reaction chamber (1) for containing liquid culture medium (44) and maintaining suitable growth conditions for biofilm-producing bacteria;(ii) a bioreactor where growth of the biofilm-producing bacteria can produce a biofilm in a reaction zone (36) which is at the air-liquid interface (45) of the medium, which biofilm is laid-down in layers to produce the three-dimensional article to be bioprinted, characterised in that the bioreactor is a 2-part system having:(a) an open negative mold (11) of the form of the article to be bioprinted wherein in-use the mold is gradually raised above the culture medium surface thereby continuously revealing the cross-section of the article in the reaction zone in the mold, and(b) one or more floating devices (18) for floating on the culture medium to block biofilm growth outside of the reaction zone.***2 A bioprinter according to claim 1, wherein the mold (11) has two open ends, where the lower one allows the liquid culture medium to enter the mold and the second upper allows gasses from biological reaction of the biofilm-producing bacteria to escape and oxygen to diffuse into the reaction zone.3 A bioprinter according to claim 1 or claim 2 wherein the mold (11) has an open hollow cavity so that the liquid medium enters, limited by an outer wall (22) and an inner wall (27), which act as negative limits of the geometry of the article to be formed.4 A bioprinter according to any one of claims 1 to 3 wherein:(i) the mold (11) comprises holes or ducts, and / or(ii) the mold is made of a porous material, in order to permit gas circulation in and out of the reaction zone.5 A bioprinter according to any one of claims 1 to 4 wherein the mold (11) contains ventilation vents (37) to promote the entry of oxygen to the reaction zone (36) at the airliquid interface of the medium, the support hooks (35) and the coupling points (33).***6 A bioprinter according to any one of claims 1 to 5 either:(1) comprising two floating devices (18) for blocking biofilm growth within the internal periphery of the mould and outside the external periphery of the mold respectively, or(2) comprising one floating device (18) for blocking biofilm growth within the internal periphery of the mould and utilising a floating immiscible liquid layer for blocking biofilm growth outside the external periphery of the mold.***7 A bioprinter according to any one of claims 1 to 8 wherein the mold (11) is supported by a mold carriage (7) capable of linear movement within the chamber so as to change the relative position of the mold (11) with respect to the culture medium surface.8 A bioprinter according to claim 7 wherein the linear movement of the mold carriage is achieved by rotation of a vertical worm screw (17) linked to the carriage and actuated by a motor (3), wherein the mold carriage movement is stabilised with one or more vertical support rods (16).9 A bioprinter according to claim 8 wherein a rigid motor worm coupling (25) serves to drive the rotational movement of the motor through the worm towards the linear rods (16) and into the carriage (9), wherein optionally the rigid motor worm coupling (25) is supported on the lower base of the chamber lid, optionally by means of friction screw fasteners (24).10 A bioprinter according to claim 8 wherein the reaction chamber (1) comprises two linear tow rods (16) that are driven by a worm screw (17) in the Z axis, connected by a screw nut ( 14) and a linear bearing (8) to the mold carriage (7) of the Z axis and three separate spacers separators (10) whose function is to provide additional depth in the introduction of the liquid medium and adjust the level of the mold (11).***11 A bioprinter according to any one of claims 1 to 10 comprising means (4, 5, 15, 19, 20, 21) to adjust airflow into the reaction chamber.12 A bioprinter according to claim 11 wherein the means to adjust airflow into the reaction chamber is an air flow system composed of an air inlet fan (5) and an air outlet fan (15) and a valve (4) to reduce the possibility of agents entering which could contaminate the bioreactor.13 A bioprinter according to claim 12 wherein a filtration system (20) for the inlet fan (5) and the outlet valve (19) restricts the air flow only to the direction from the inside towards the outside of the bioreactor, which are coupled by an adapter for the inlet fan (5) and a screen for the outlet fan (21).

14. A bioprinter according to claim 13 wherein the air inlet for the bioprinter system comprises an inlet fan (2) that sucks in the air filtered by microporous materials (39) that is trapped by two screen plates (38)(40), such that filtered air leaves through the outlet flange (43) of the fan and enters the bioreactor with the help of an adapter gasket to prevent leaks (41), wherein optionally this adapter contains a screen for the air outlet (42) to increase turbulence and better distribute the air.***15 A bioprinter according to any one of claims 1 to 14 wherein the bioreactor comprises a conductivity sensor such as to sense the conductivity of the medium thereby detecting positioning of the support hooks relative to the medium and thereby maintaining the reaction zone.16 A bioprinter according to claim 15 wherein a floating device (18) can function as a water level sensor (12) and contains magnetic conductors and embedded material while a sensor in the periphery of the mold (13) and the reaction chamber detects its changes.***17 A bioprinter according to any one of claims 1 to 16 which comprises temperature and / or humidity sensors and means to modify temperature and / or humidity in response to said sensors.18 A bioprinter according to claim 17 wherein the reaction chamber has a lid which includes one or more of:(i) entry points (2) for sensors;(ii) entry and exit conduits to renew the liquid culture medium;(iii) entry and exit conduits for water vapour to modify the humidity within the chamber.19 A bioprinter according to claim 18 comprising a gasket (23) on the periphery of the internal part of the lid towards the edge of the reaction chamber (1).20 A bioprinter according to any one of claims 1 to 16 which can be configured from 25 to 35° C and humidity from 0 to 100%.***21 A bioprinter according to any one of claims 1 to 20 wherein(i) the reaction chamber (1) encapsulates all mechanical and bioprinting components that are in direct contact with the liquid-gas interface within the bioreactor and maintains the appropriate living conditions of the biofilm-producing bacteria;(ii) a main cover (6) containing multiple entry points (2), for waterproof electronic wiring, as well as liquid and gas pipes to maintain proper reaction conditions; and supporting:(iii)a stepper motor (3) that drives a linear movement system and is supported on the main cover (6); and(iv) an air flow system composed of an air inlet fan (5) and the air outlet fan (15) that connects to a valve on one side (4) to reduce the possibility of agents entering which could contaminate the bioreactor.***22 A bioprinter according to any one of claims 1 to 21 comprising a plurality of bioreactors adapted to be suspended in the same culture medium and independently raisable from the culture medium.***23 A bioprinter according to any one of claims 1 to 22 wherein the biofilm-producing bacteria are present in a consortium of bacteria.24 A bioprinter according to any one of claims 1 to 23 wherein the biofilm-producing bacteria are selected from those shown in Table 1.25 A bioprinter according to any one of claims 1 to 24 wherein the biofilm is selected from those shown in Table 1.26 A bioprinter according to claim 25 wherein the biofilm is bacterial cellulose.***27 A method for producing a three-dimensional article from bacterial biofilm, the method comprising:(i) providing a bioprinter according to any one of claims 1 to 26;(ii) lowering the mold (11) into culture medium comprising biofilm-producing bacteria so that medium enters the mold (11) creating a reaction zone at the surface of the medium therein, but wherein the one or more floating devices inhibit growth of the bacteria elsewhere on the surface of the medium;(iii) raising the mold (ll)from the culture medium thereby revealing a cross-section of the article in a reaction zone and then stopping raising the mold;(iv) once a biofilm layer has fully formed into the desired geometry, repeating step (iii).28 A method according to claim 27 for producing three-dimensional structures based on bacterial cellulose (CB) wherein the bioprinter mold (11) begins the process in a lowered position within the culture medium (44) where the first layer forms about 20 microns higher than the position of the support hooks under the liquid lever (45) in which a continuous formation of layers (CB) is synthesised according to the shape of the mold (23), and once the bioprinter has successfully synthesised the first layers, the support hooks allow it to continue the reaction without the material sliding into the liquid, stopping the reaction.29 A method according to claim 1 or claim 28 wherein:(i) upon completion of the reaction the mold is raised above the culture medium and the mold is removed from the reaction chamber;RECTIFIED SHEET (RULE 91) ISA / EP(ii) the three-dimensional article is removed from the mold.30 A method according to claim 29 wherein the three-dimensional article is removed from the mold either by disassembling the mold or dissolving the mold.31 A method according to claim 30, characterised in that the final shape printed by the system (52) comprises the positive shape of the mold inserted in the liquid medium without additional geometries formed in the interior cavity of the mold (48) and once the reaction has been completed, the support hooks are manually removed from the three dimensional shapes of (CB).32 A method according to any one of claims 1 to 31 where the article is a scaffold, and the method further comprises ce 11 u la rization the article such as to produce a tissue, organ or organoid.***33 A three-dimensional article made from bacterial biofilm obtained or obtainable by the method of any one of claims 27 to 32***34 Use of the article according to claim 33 to prepare a scaffold for a tissue, organ or organoid, a catalyst, a biological filter, or a foodstuff.RECTIFIED SHEET (RULE 91) ISA / EP

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