[go: up one dir, main page]

WO2025087952A1 - Système de bioréacteurs - Google Patents

Système de bioréacteurs Download PDF

Info

Publication number
WO2025087952A1
WO2025087952A1 PCT/EP2024/079921 EP2024079921W WO2025087952A1 WO 2025087952 A1 WO2025087952 A1 WO 2025087952A1 EP 2024079921 W EP2024079921 W EP 2024079921W WO 2025087952 A1 WO2025087952 A1 WO 2025087952A1
Authority
WO
WIPO (PCT)
Prior art keywords
mold
bioprinter
biofilm
reaction
bioreactor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/079921
Other languages
English (en)
Inventor
Esteban PONGUILLO BETANCOURT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad San Francisco De Quito Usfq
Original Assignee
Universidad San Francisco De Quito Usfq
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad San Francisco De Quito Usfq filed Critical Universidad San Francisco De Quito Usfq
Publication of WO2025087952A1 publication Critical patent/WO2025087952A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus

Definitions

  • the present invention relates to a three-dimensional (3D) bioprinter.
  • the invention proposes a 3D bioprinting system and method that uses bacterial cellulose (BC) as a structural polymer with increased strength and mechanical stability.
  • BC bacterial cellulose
  • 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.
  • 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.
  • 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.
  • 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.
  • bioprinting methods such as:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BC Bacterial cellulose
  • BC Bacterial cellulose
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the main reaction chamber is transparent e.g. made of tempered silicate boron glass, or plastic.
  • the chamber could also be made of stainless steel with corresponding viewing windows of transparent material.
  • 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.
  • 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).
  • 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.
  • Such 3D printer controllers are well known in the art and commercially available e.g. https://www.lasercontrolcard.com/3d-laser-printing-controller-product
  • 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.
  • 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.
  • 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)).
  • FDM fused deposition modeling
  • SLA stereolithography
  • SLS selective laser sintering
  • the mold may be preferable to prepare the mold from a material that dissolves in a solvent not present in the biological reaction.
  • a material that dissolves in a solvent not present in the biological reaction An example would be PolyVinyl Butyral (PVB) that dissolves in isopropanol.
  • 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.
  • organs or organoids e.g. luminal organs
  • biocatalysts e.g. biocatalysts
  • biofilters e.g. synthetic biology and industrial biotechnology
  • bioreactors e.g. a cell proliferation
  • Other utilities for biofilms may include nutrition.
  • 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.
  • 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.
  • 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.
  • manufacture e.g. Computer-aided design (CAD) and Computer-aided manufacturing (CAM)
  • CAD Computer-aided design
  • CAM Computer-aided manufacturing
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the floating devices are low-density solid forms that fit into and around the actual mold.
  • the floating devices are small low-density granules that are automatically disposed outside of the reaction zone in the mold.
  • the mold carriage upon completion of the reaction, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Troubleshooting and testing features may be included in the firmware such as automatic check of biofilm thickness variability viability and speed reaction.
  • 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.
  • bioreactor condition data temperature, pH, humidity, etc
  • FIG. 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.
  • Figure 2 illustrates a perspective isometric lower view of the complete embodiment of the three-dimensional BC bioprinter on its submerged position.
  • 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.
  • the electronic components such as the Z movement stepper motor, ventilations vents and various entry points for probes, sensors and liquid recirculation.
  • 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
  • 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).
  • 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.
  • 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).
  • Figure 9 illustrates a bottom isometric exploded view of the final three-dimensional shape formed in the mold system.
  • Figure 10 illustrates a cylindrical mold formed by simple extrusion.
  • Figure 11 illustrates a spiral form mold
  • Figure 12 illustrates molds having positive and negative tapers forming frustoconical shapes.
  • 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.
  • Figure 15 illustrates asymmetrical shapes having compound geometries achievable with the system of the invention.
  • 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).
  • Figure 17(a) and (b) shows a close up of the reaction chamber.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the bioprinter may include relative positioning-means based on mechanical or sensor end-stops.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • a Secondary controller Raspberry Pi in the embodiments described herein.
  • a Programable Logic Control may be used.
  • 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.
  • links PC - 3D Printer Direct Control (Open-source)
  • 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% .
  • 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
  • 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.
  • 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.
  • 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.
  • the main electronic component such as the Z movement stepper motor, ventilations vents and various entry points for probes, sensors and liquid recirculation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • the filter system in the drawing is merely illustrative since the filtration of a recirculation system depends heavily on the application of the biomaterial.
  • 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.
  • 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.
  • 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.
  • these considerations and techniques greatly improve the rate of growth and material properties.
  • 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.
  • 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.
  • FIGs. 10-15 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.
  • FIGs. 16-18 illustrate a device according to the invention, and an example 3D printed product.
  • 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;
  • 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.
  • 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.
  • Bioreactor system 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.
  • a floating device 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.
  • 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%.
  • Bioreactor system 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).
  • 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).
  • 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).
  • Bioreactor system 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.
  • 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).
  • reaction zone (46) 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).
  • 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.
  • reaction zone (46) is always at the water level (45) regardless of the position of the mold (47).
  • the support hooks allow it to continue the reaction without the material sliding into the liquid, stopping the reaction.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un système de biofabrication pour la bio-impression et/ou la formation de motifs en trois dimensions d'échafaudage et de structures de rétention à l'aide de bactéries productrices de biofilm en tant que moyen de synthèse de géométries. La machine comprend un moule mobile négatif de la forme tridimensionnelle finale souhaitée et un ou plusieurs flotteurs de blocage d'oxygène pour permettre la croissance de biofilm uniquement dans la partie positive du moule susmentionné. Le mouvement de bio-imprimante est étalonné par rapport au niveau de surface du milieu de culture pour maintenir une croissance continue de biofilm. Le bioréacteur peut être utilisé avec la possibilité de réguler la température, l'humidité, le débit d'air et les conditions des milieux de culture et des nutriments selon les besoins de la synthèse tridimensionnelle.
PCT/EP2024/079921 2023-10-24 2024-10-23 Système de bioréacteurs Pending WO2025087952A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ECDI202378895 2023-10-24
ECSENADI-2023-78895 2023-10-24

Publications (1)

Publication Number Publication Date
WO2025087952A1 true WO2025087952A1 (fr) 2025-05-01

Family

ID=93258844

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/079921 Pending WO2025087952A1 (fr) 2023-10-24 2024-10-23 Système de bioréacteurs

Country Status (1)

Country Link
WO (1) WO2025087952A1 (fr)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1021471A (en) 1911-01-24 1912-03-26 Robert W Pain Adjustable music-box.
WO2011038373A2 (fr) 2009-09-28 2011-03-31 Virginia Tech Intellectual Properties, Inc. Bioimpressions tridimensionnelles d'implants de cellulose biosynthétique et d'échafaudages pour ingénierie tissulaire
DE102012201272A1 (de) 2012-01-30 2013-08-01 Kkf Ug Vorrichtung zur Herstellung von Hohlkörpern aus mikrobiellem Polymer
US20160288414A1 (en) 2013-11-04 2016-10-06 University Of Iowa Research Foundation Bioprinter and methods of using same
WO2017042232A1 (fr) 2015-09-07 2017-03-16 Ucl Business Plc Ingénierie tissulaire
WO2017184839A1 (fr) 2016-04-20 2017-10-26 The Brigham And Women's Hospital, Inc. Systèmes et procédés pour bio-impression à matériaux multiples in vivo
US20180221129A1 (en) * 2015-08-05 2018-08-09 Universitätsklinikum Jena Medical implant based on nanocellulose
WO2019197333A1 (fr) 2018-04-11 2019-10-17 Cellink Ab Dispositif microfluidique pour la formation de motifs sur un matériau cellulaire dans un environnement extracellulaire 3d
WO2021025823A2 (fr) * 2019-07-12 2021-02-11 University Of Rochester Impression 3d de biofilms

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1021471A (en) 1911-01-24 1912-03-26 Robert W Pain Adjustable music-box.
WO2011038373A2 (fr) 2009-09-28 2011-03-31 Virginia Tech Intellectual Properties, Inc. Bioimpressions tridimensionnelles d'implants de cellulose biosynthétique et d'échafaudages pour ingénierie tissulaire
DE102012201272A1 (de) 2012-01-30 2013-08-01 Kkf Ug Vorrichtung zur Herstellung von Hohlkörpern aus mikrobiellem Polymer
US20160288414A1 (en) 2013-11-04 2016-10-06 University Of Iowa Research Foundation Bioprinter and methods of using same
US20180221129A1 (en) * 2015-08-05 2018-08-09 Universitätsklinikum Jena Medical implant based on nanocellulose
WO2017042232A1 (fr) 2015-09-07 2017-03-16 Ucl Business Plc Ingénierie tissulaire
WO2017184839A1 (fr) 2016-04-20 2017-10-26 The Brigham And Women's Hospital, Inc. Systèmes et procédés pour bio-impression à matériaux multiples in vivo
WO2019197333A1 (fr) 2018-04-11 2019-10-17 Cellink Ab Dispositif microfluidique pour la formation de motifs sur un matériau cellulaire dans un environnement extracellulaire 3d
WO2021025823A2 (fr) * 2019-07-12 2021-02-11 University Of Rochester Impression 3d de biofilms

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Encyclopedia of Microbiology", 2019, ACADEMIC PRESS, article "Continuous Cultures (Chemostats", pages: 743 - 761
MANUEL SCHAFFNER ET AL: "3D printing of bacteria into functional complex materials", SCIENCE ADVANCES, vol. 3, no. 12, 1 December 2017 (2017-12-01), pages eaao6804, XP055491174, DOI: 10.1126/sciadv.aao6804 *
NING EVITA ET AL: "3D bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing", BIOFABRICATION, vol. 11, no. 4, 13 September 2019 (2019-09-13), pages 045018, XP055783656, Retrieved from the Internet <URL:https://iopscience.iop.org/article/10.1088/1758-5090/ab37a0> DOI: 10.1088/1758-5090/ab37a0 *

Similar Documents

Publication Publication Date Title
EP3430119B1 (fr) Système de bioréacteur et son procédé
Krujatz et al. Additive Biotech—Chances, challenges, and recent applications of additive manufacturing technologies in biotechnology
EP0353893B1 (fr) Réacteur de culture de cellules
EP3194150B1 (fr) Contenant destiné à recevoir un ou plusieurs fluide(s) bioactif(s) et/ou un ou plusieurs fluide(s) préparatoire(s), et un procédé associé
US5589112A (en) Constructing a high density cell culture system
US20240409866A1 (en) Lid configuration for a bioreactor
US20100151571A1 (en) Centimeter-scale, integrated diagnostics incubator for biological culturing
CN101120083A (zh) 利用透气性材料进行细胞培养的方法及装置
US9284521B2 (en) Pivoting pressurized single-use bioreactor
JP5922672B2 (ja) インキュベーションキャビティーへの簡単なアクセスのための蓋を備えるバイオリアクター
WO2008005998A2 (fr) Incubateur de diagnostic intégré à l&#39;échelle du centimètre pour cultures biologiques
CN102057033A (zh) 可缩放的细胞培养生物反应器和细胞培养方法
KR102575112B1 (ko) 바이오리액터
US20110319607A1 (en) Method For The Microbial Production Of A Body Containing Cellulose
Baba et al. Development of biomimetic system for scale up of cell spheroids-building blocks for cell transplantation
CN108121161A (zh) 一种高通量形成拟胚体的微阵列芯片的制备方法与应用
WO2025087952A1 (fr) Système de bioréacteurs
CN101402917B (zh) 一种生产组织工程产品的生物反应器
JP4584571B2 (ja) 細胞培養のための熱成形容器
CN117660183B (zh) 胚胎微流控动态培养皿及其培养方法
CN206188785U (zh) 一种细菌纤维素发酵生产设备
CN106047667A (zh) 一种细菌纤维素发酵生产设备
CN107446796A (zh) 一种细菌纤维素发酵生产设备
WO2025106382A1 (fr) Système et procédés pour la culture de tissus en 3d
de Haan et al. Organs-on-a-Chip

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24795137

Country of ref document: EP

Kind code of ref document: A1