WO2013113883A1 - Procédé de réalisation de structures tridimensionnelles et structures ainsi réalisées - Google Patents

Procédé de réalisation de structures tridimensionnelles et structures ainsi réalisées Download PDF

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Publication number
WO2013113883A1
WO2013113883A1 PCT/EP2013/052046 EP2013052046W WO2013113883A1 WO 2013113883 A1 WO2013113883 A1 WO 2013113883A1 EP 2013052046 W EP2013052046 W EP 2013052046W WO 2013113883 A1 WO2013113883 A1 WO 2013113883A1
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Prior art keywords
tissue
liquid
dimensional structure
printing
living cells
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PCT/EP2013/052046
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German (de)
English (en)
Inventor
Wilhelm Jahnen-Dechent
Horst Fischer
Andreas BLAESER
Daniela Filipa DUARTE CAMPOS
Sabine Neuss-Stein
Michael Weber
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Rwth Aachen
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Publication of WO2013113883A1 publication Critical patent/WO2013113883A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2521/00—Culture process characterised by the use of hydrostatic pressure, flow or shear forces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/76—Agarose, agar-agar

Definitions

  • the present invention relates to a process for producing a three-dimensional structure from a structural material comprising the step of printing or extruding the structural material in a liquid to obtain a three-dimensional structure, said liquid being a high-density liquid. Furthermore, the present invention relates to such produced three-dimensional structures and corresponding printing devices for forming this three-dimensional structure, in particular for use according to the invention in the inventive method.
  • the inventive method is particularly suitable for the production of three-dimensional structures containing at least partially living cells.
  • tissue engineering In recent years, the biotechnological production of biomaterials has increased and, in particular in the field of tissue engineering, various fields of application have been discussed.
  • One of the goals of tissue engineering is to remedy the lack of donor organs by implanting autologous organs or tissues.
  • tissue substitutes There are various possibilities for generating corresponding tissue substitutes.
  • cells are taken from the patient and propagated in vitro. After receiving a sufficient number of cells, the cells are then cultured using various methods, for example in a bioreactor. Depending on the application, the cells are placed there in or on a shaping scaffold, the so-called scaffold.
  • This Scaffold or scaffold can be of biological origin or synthetic. Such Sca fo / c / materials can be present both as degradable materials and permanent materials.
  • tissue engineering derived tissue substitutes include skin as well as other soft or hard tissues. These include, but are not limited to, bone or cartilage tissue, as well as various forms of vessels including blood vessels, trachea, or esophagus.
  • Other tissue engineered structures include heart valves or venous valves.
  • the aim of tissue engineering is the provision of entire functional tissues or organs.
  • the tissue substitute to be produced is usually cultured in bioreactors until use. Depending on the application, the tissue substitute can be stored with appropriate means.
  • biodegradable structural materials which are present as polymeric gels, for example collagen, fibrin, alginate or agarose gels, are preferably used for this purpose.
  • polymeric gels for example collagen, fibrin, alginate or agarose gels.
  • the shaping of these three-dimensional structures, the scaffolds, which optionally contain living cells, can be carried out by various methods. Casting, extrusion or printing of the three-dimensional structures are possible here.
  • the low stability of these gels as structural material presents problems in producing corresponding three-dimensional structures. Previous results, however, only allowed the production of very small three-dimensional polymer constructs.
  • Fluorohydrocarbons are non-aqueous (hydrophobic), biocompatible, highly oxygenated fluids with high buoyant density. Perfluorohydrocarbons or perhalocarbons are often inert fluids. Fluorocarbons have been studied in experiments with liquid respiration since the 1950s. It has been shown that suspension cultures in perfluorocarbons can survive very well (eg, Maillard E. et al, Biomaterials, 201 1, 32, 9282-9). Brief description of the invention
  • the present invention provides a method for producing a three-dimensional structure from a structural material optionally containing living cells comprising the step of printing or extruding the structural material optionally in admixture with living cells immersed in a liquid to a three-dimensional structure optionally containing living cell obtained, characterized in that this liquid is a liquid of high density, ready.
  • the present invention is directed to a method for producing a three-dimensional structure containing at least partially living cells and suitable as a tissue substitute, the preparation being effected by printing or extruding the structure immersed in a liquid, the liquid preferably being Fluorocarbon, in particular a perfluorocarbon is.
  • This liquid in particular the fluorohydrocarbon which can be used according to the invention, is a liquid of high density.
  • the fluorocarbons such as the perfluorocarbons
  • the fluorocarbons are, due to their inert properties, not further miscible and reactive with the printed structural material present in liquid form, if appropriate also containing solvent. Due to the high density of the liquid, which is higher than the density of the structural material when printed in liquid form, stabilization and support of the printed or extruded structural material is achieved.
  • the application is directed to three-dimensional structures, in particular those containing living cells, obtainable by means of the method according to the invention.
  • These three-dimensional structures are in particular artificial soft or hard tissue.
  • a further aspect is a printing device for, in particular, site-specific design of these three-dimensional structures.
  • This device can selectively or selectively extrude the structural material with the aid of structural geometric data on the three-dimensional structure.
  • the present invention is directed to the use of these three-dimensional structures optionally containing living cells as artificial tissue, in particular as soft tissue or hard tissue.
  • the present invention provides the use of a high density liquid, in particular fluorocarbons, such as perfluorocarbons, as a medium for printing or extruding a three-dimensional structure, optionally containing living cells.
  • a method for producing a three-dimensional structure from a structural material optionally containing living cells comprising the step of printing or extruding the structure optionally immersed in a liquid in a mixture with living cells in order to obtain a three-dimensional structure optionally containing a living cell, characterized in that this liquid is a liquid of high density.
  • this high-density liquid acts on the printed or extruded structural material in such a supportive manner that the three-dimensional structures can be easily produced by extrusion and printing to obtain this structure.
  • this liquid allows a location-selective structure of the structure.
  • the liquid be one that is immiscible with the structural material and / or non-reactive.
  • the liquid is thus preferably a high density inert liquid which supports and stabilizes the printed or extruded structural material.
  • the high density liquid is one that is immiscible with the structural material and non-reactive.
  • structural material herein is meant a compressible or extrudable material which can form a three-dimensional structure, in particular, this structural material is one which forms a stable three-dimensional structure, in particular, this stable three-dimensional structure can be obtained by cooling that for extruding or Printing heated structural material or other curing of this structural material, for example by crosslinking etc, as by induction of curing by energy supply.
  • thermosensitive synthetic polymers As structural materials both thermosensitive synthetic polymers and hydrogels can be used.
  • the hydrogels As structural materials both thermosensitive synthetic polymers and hydrogels can be used.
  • Hydrogels are those of a material selected from agarose, collagen, fibrin, alginate, chitosan, hyaluronan or synthetic hydrogels including polyethylene glycol, poly (N-isopropylacrylamide) and copolymers. re, polylactides, polyurethanes or polyvinyl alcohols or mixtures of both natural and synthetic polymers, in particular hydrogels, which are highly hydrated and thus are particularly suitable in interaction with living cells.
  • Silica-based gels in particular those which are at least partially prepared with a Si-N precursor, may also be used as polymers.
  • the person skilled in suitable systems are known.
  • Various structural materials can be used to produce the three-dimensional structure. Either such that different structural materials are used in different layers or that different structural materials are used in a printed or extruded layer to form regions of different properties. As a result, it is possible to produce structures which have different cell types in different subareas or which only have cells in sections, while other sections are cell-free.
  • high density liquid herein is meant a fluid, especially a liquid, which has a density greater than or equal to the density of the structural material which is printed or extruded into the high density liquid at the time of printing or extrusion 0.2 g / cm 3 , preferably greater than 0.3 g / cm 3 , compared to the density of the structural material
  • the high-density liquid is one whose density is higher than the density of the structural material printed in liquid form.
  • the density is a measure of the mass of the substance per unit volume and is completely independent of the viscosity of a fluid.
  • the viscosity is a measure of the viscosity of a fluid. This viscosity is independent of the density of the substance Density can have a low viscosity and is nevertheless suitable according to the invention. Hydrocarbons such as perfluorocarbons on a high density at low viscosity.
  • the method according to the invention is particularly suitable for producing three-dimensional structures, wherein these structures may at least partially contain living cells. These living cells can then be used in the context of tissue engineering (also referred to in German as tissue engineering or tissue engineering). With the aid of the method according to the invention, the disadvantages of known methods can be overcome, namely the production of three-dimensional structures of good mechanical stability and in dimensions that have not yet been achieved due to the low stability of the gels, such as hydrogels used in the tissue engineering field could become.
  • the high-density liquid is preferably a liquid having a density of at least 1.5 g / cm 3 , particularly preferably a liquid having a density of at least 1.7 g / cm 3 .
  • These liquids are preferably hydrophobic liquids.
  • Particularly suitable high-density liquids according to the invention are fluorohydrocarbons, in particular perfluorocarbons.
  • the liquids which can be used according to the invention and into which the three-dimensional structures are extruded or printed, in particular in a location-selective manner are those which are preferably nonaqueous, biocompatible and readily oxygenatable.
  • the high density liquids are preferably such that they face the structural material inert, immiscible and / or non-reactive.
  • the perfluorocarbons allow one to produce three-dimensional structures that may at least partially contain living cells.
  • Hydrocarbons and especially perfluorocarbons are known as liquids in which cells can survive. They are z.
  • liquids which exhibit the ability to release respiratory gases such as oxygen, nitrogen and carbon dioxide.
  • it is in the Perfluorkohlenwasserstof- fen as high density liquid to perfluorocarbons selected from the group consisting of perfluorotributylamine (C 12 F 27 N), perfluorodecalin (CI OF-I S), Perfluorhexamethylprisman (Ci 2 Fi 8).
  • Perfluorohydrocarbons such as perfluorodecalin or perfluorohexamethylprisman are chemically inert so that reaction with the structural material or with cell components does not occur.
  • Perfluorohydrocarbons or perfluorocarbon emulsions which in the present case fall under the term perfluorocarbons, are known as oxygen carriers. They are z. B. as a blood substitute (EP 0307087 B1) or described in the context of liquid ventilation. Tan, Q., et al., Tissue Engineering Part A, 2009, 15 (9): 2471-2480 discloses the use of perfluorohydrocarbon emulsions to oxygenate cultured cells and tissues.
  • such high-density liquids such as fluorohydrocarbons and in particular perfluorocarbons
  • the means for extruding or printing are immersed in the high-density liquid, and then the structural material optionally containing living cells is extruded three-dimensionally or printed in layers.
  • Corresponding methods of extruding and printing structures are generally commonly known. For example, methods known from “rapid prototyping” can be used to extrude or print the structural material in a spatially selective manner, typically on the basis of structural geometric data provided. "This data gives the detailed geometry of this fabric to be fabricated, such as tissue. again.
  • the method is a method for producing a three-dimensional structure containing living cells.
  • This structure containing living cells is then cultured in further steps by means of known cell culture methods in order finally to provide artificial three-dimensional structures as a tissue substitute.
  • Structural material which is used according to the invention is at least partially a mixture of the structural material and living cells in order to produce three-dimensional structures which at least partially contain living cells.
  • the living cells are preferably eukaryotic cells, in particular cells of human origin. It is particularly preferred that the cells are primary cells, e.g. Cells of xenogeneic, allogenic or autologous origin. These cells are provided as isolates accordingly.
  • the cells may be correspondingly tissue-specific cells.
  • tissue-specific or organ-specific cells is meant, first of all, all cells that are in the organ or tissue (including cells not restricted to the particular tissue or organ).
  • These cells include various types of cells including muscle cells, epithelial cells, nerve cells, connective tissue cells, but also stem cells.
  • cells of the type muscle cells can be smooth muscle cells, striated muscle cells or cardiac muscle cells, epithelial cells close a intestinal cell.
  • epithelial cells skin epithelial cells, endothelial cells, pneumocytes, nerve cells include neurons including motor and sensory neurons as well as interneurons, glial cells, etc .; as well as connective tissue cells with fibroblasts, chondrocytes, osteoblasts, adipocytes, myofibroblasts, pericytes.
  • tissue-specific and organ-specific cells can also be introduced.
  • the structural material or the mixture with the living cells and optionally further additives may further contain excipients to assist the cultivation process.
  • excipients are in particular cell adhesion molecules, differentiation factors, immunologically active cytokines / chemokines and / or antibiotics. These may optionally be conjugated with the structural material.
  • Other additives include ions, nutrients including amino acids, peptides, proteins, lipids, carbohydrates, nucleic acids and other necessary nutrients that promote cell proliferation and differentiation.
  • the structural material can furthermore contain additives in liquid or solid form.
  • additives for example, for the construction of bone or cartilage substitutes corresponding calcium phosphate may be present.
  • corresponding calcium phosphate may be present.
  • components eg. In the form of encapsulated compounds allowing for a time-delayed release of compounds present in the capsule.
  • the structural materials are polymers that allow good cohesion between the individual printed or extruded layers.
  • the structural material may be a perma- material or a biodegradable material.
  • a biodegradable material may be advantageous. This biodegradable material temporarily takes over the function of the extracellular matrix, which is subsequently secreted by the cells themselves. Areas of application here are in particular vessels, etc.
  • the method according to the invention is particularly suitable for the formation of tissue substitutes, in particular of artificial soft tissue or hard tissue.
  • Soft tissue or hard tissue include
  • the method according to the invention for producing the three-dimensional structure may comprise further steps.
  • the high-density liquid may be removed to further cultivate the three-dimensional structure containing living cells, if any. Cultivation may be carried out according to conventional techniques known from tissue engineering to obtain the target tissue.
  • the method according to the invention is a printing method.
  • the structural material optionally containing living cells is sequentially printed site-selectively.
  • the printing takes place in several layers one above the other, in order to allow the formation of the three-dimensional structure, possibly with living cells, by this multiple printing.
  • the layers may be formed of the same or different structural materials.
  • This printing is especially complex three-dimensional Structures can be produced.
  • Such three-dimensional structures include heart valves as well as other types of hard and soft tissue as well as vessels, e.g. B. vessels with outgoing vessel parts (vascular branches), etc.
  • Especially in complex three-dimensional structures are free spaces, such as lumens and cavities, which can be prepared by the method according to the invention.
  • One known method comprises an inkjet based system as described in the literature.
  • Other processes are pneumatic or extruder based.
  • the present invention is directed to three-dimensional structures containing optionally living cells obtainable by the method according to the invention.
  • These three-dimensional structures are, in particular, tissue substitutes, such as artificial soft tissue or hard tissue, including bone tissue, cartilage tissue, blood vessels, trachea, esophagus, heart valve or organs.
  • tissue substitutes such as artificial soft tissue or hard tissue, including bone tissue, cartilage tissue, blood vessels, trachea, esophagus, heart valve or organs.
  • These three-dimensional structures, possibly containing living cells can be used as artificial tissue and patients implanted. They can be implanted as transient or permanent tissue.
  • the three-dimensional structures which can be produced with the aid of the method according to the invention can in particular represent complex structures which were not attainable with the hitherto known methods due to the instability of the structures produced, or which can only be achieved by use. additional auxiliary structures.
  • the structural geometric data that is to say the detailed geometry of the three-dimensional structure to be reproduced, in particular of the tissue or of the organ, allows the control of the extruding or printing device, in particular of the printing head or of the extrusion nozzle, in order to print the spatial form of the desired three-dimensional structure in a location-selective manner.
  • These structural geometric data are using known methods based on original structures, eg. B. vessels, organs, etc., and then implemented accordingly using the device according to the invention.
  • the present application is directed to a device, such as a printing device or extrusion device, for forming a three-dimensional structure optionally containing living cells.
  • the device is designed such that it is suitable for carrying out the method according to the invention.
  • This device comprises means for printing or extruding, which means are designed such that it allows location-selective printing or extruding of the structural material optionally containing living cells and allows printing or extrusion in a high-density liquid.
  • the device according to the invention has a device for receiving the fluid. high density.
  • the means for printing or extruding is characterized in that the means for extruding or printing are formed so as to enable a submerged in a liquid extruding or printing.
  • the device for printing or extrusion is designed such that it allows with the aid of an optionally motorized X, Y, Z control positioning of these means for printing or extrusion in a predetermined position in all three dimensions. Furthermore, this means for printing or extrusion is preferably provided with a unit which allows heating of the structural material to be printed or extruded. Corresponding control elements, including valves, permit a precise quantity delivery of the structural material to be extruded or printed.
  • the device for receiving the liquid of high density can also be designed to be movable, i. H. also be designed to be movable in the X, Y and Z directions. If the receiving device is designed to be movable for receiving the device for the high-density liquid, the means for printing and extruding may also be designed to be stationary.
  • the device for receiving the high-density liquid is suitable for incubating the three-dimensional structure optionally containing living cells at temperatures of at least 30 ° C.
  • this device is one that represents a bioreactor itself or that can be transferred to a bioreactor, or that the structure printed or extruded in the device can be transferred to a nutrient medium.
  • cultivate the three-dimensional structure prepared by extrusion or printing with contained living cells to provide a corresponding three-dimensional structure suitable as a tissue substitute.
  • the printing device may be provided under appropriate Culture conditions are maintained.
  • FIG. 1 shows the printing process as a first step, the incubation at 37 ° C., ie. H. the cultivation of the printed three-dimensional structure containing living cells as a second step and the three-dimensional structure obtained in the third step.
  • the plate may be present in a receiving device which allows movement in the X, Y and Z directions (not shown).
  • the extrusion head or print head is designed so that the smallest amounts of structural material can be printed or extruded. As a result, a plurality of layers can be printed or extruded one above the other.
  • the means for printing or extruding is heatable 4 to heat the structural material and thus to keep it in a corresponding liquid state for printing or extrusion.
  • the control means for controlling the means for printing or extruding or for moving the receiving means for the device for receiving the high-density liquid are not shown.
  • Structure 5 extruded or printed. Possibly. It is also possible to use a plurality of extrusion or pressure agents, ie multiple heads or nozzles For example, areas with or without cells to print or extrude or to print or extrude different mixtures of structural material and cells.
  • a plurality of extrusion or pressure agents ie multiple heads or nozzles
  • areas with or without cells to print or extrude or to print or extrude different mixtures of structural material and cells.
  • the extrusion of the respective flowable structural material takes place such that the printhead or the extrusion die is immersed in the high-density liquid and a predetermined amount of the structural material is extruded at the predetermined position.
  • the liquid By printing in the liquid, also referred to as “submerged printing” or “submerged extrusion”, it is possible to provide three-dimensional structures of a desired size. In particular, it is possible to provide these structures such that no deformation of the three-dimensional structure takes place during printing or extrusion or later cultivation. As a result, the size of the produced three-dimensional structure is no longer limited as it is in the prior art. Furthermore, complex structures including cavities and lumens, or complex structures such as vascular branches, etc. are also possible.
  • the high-density liquid-containing receptacle and the fabricated three-dimensional structure are taken out of the printing device and placed in a culture device placed. Previously, the liquid of high density is at least partially removed and replaced by appropriate culture medium 6.
  • the three-dimensional structure is then cultured according to known methods, for. B. a tissue substitute 7, such as artificial soft tissue or To provide artificial hard tissue.
  • a tissue substitute 7 such as artificial soft tissue or To provide artificial hard tissue.
  • an agarose gel after autoclaving was brought to about 37 ° C. and mixed with cells of a cell line, here MG-63 human osteosarcoma cell line, or isolated and cultured human mesenchymal stem cells to obtain a concentration of 200,000 cells / ml.
  • the agarose-hydrogel suspension used had a final concentration of 3%.
  • the printhead was immersed in perfluorotributylamine (C 12 F 27 N) and the hollow body sequentially printed layer by layer to print a more than 3 cm long hollow body. After making this over 3 cm long hollow body, the perfluorocarbon was removed and replaced by cell medium (37 ° C). After culturing for 24 hours, it was possible to demonstrate by means of live / dead staining of the cells that the majority of the printed cells were living in the three-dimensional structure and correspondingly distributed in the hollow body. When checking the three-dimensional structures after 2 or 3 weeks, the good results were confirmed. Staining for a proliferation marker (Ki-67) confirmed proliferation of the cells present in the structure 2 and 3 weeks after culturing, respectively.
  • a proliferation marker Ki-67

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  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention concerne un procédé de réalisation d'une structure tridimensionnelle à partir d'un matériau structural, comprenant l'étape d'impression ou extrusion du matériau structural dans un liquide afin d'obtenir une structure tridimensionnelle, ledit liquide étant un liquide de densité élevée. La présente invention concerne en outre les structures tridimensionnelles ainsi réalisées, ainsi que des systèmes d'impression correspondants pour former cette structure tridimensionnelle, destinés en particulier à être utilisés dans le procédé de l'invention. Le procédé de l'invention est particulièrement adéquat pour réaliser des structures tridimensionnelles, qui peuvent contenir des cellules au moins en partie vivantes.
PCT/EP2013/052046 2012-02-02 2013-02-01 Procédé de réalisation de structures tridimensionnelles et structures ainsi réalisées WO2013113883A1 (fr)

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EP3018531A1 (fr) 2014-11-10 2016-05-11 Technische Universität Berlin Procédé et dispositif de production d'un objet multi-cellulaires en trois dimensions
ES2600324A1 (es) * 2016-07-26 2017-02-08 Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (Ciemat) Procedimiento de consolidación de estructuras ligeras obtenidas por fabricación aditiva
EP3427949A1 (fr) 2017-07-12 2019-01-16 Albert-Ludwigs-Universität Freiburg Bio-encres mécaniquement syntonisables pour bio-impression
EP3766957A1 (fr) * 2019-07-18 2021-01-20 Axenoll Life Sciences AG Procédé de génération et / ou d'agencement de cultures cellulaires
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GB201902862D0 (en) 2019-03-04 2019-04-17 Univ Dublin A print head and insert for use with a three-dimensional bioprinter

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WO2016001442A1 (fr) * 2014-07-04 2016-01-07 Rwth Aachen Modèle tissulaire, procédé de fabrication et utilisation dudit modèle tissulaire
EP3018531A1 (fr) 2014-11-10 2016-05-11 Technische Universität Berlin Procédé et dispositif de production d'un objet multi-cellulaires en trois dimensions
WO2016075103A1 (fr) 2014-11-10 2016-05-19 Technische Universität Berlin Procédé et dispositif de fabrication d'un objet tridimensionnel à plusieurs cellules
US10299940B2 (en) 2014-11-10 2019-05-28 Technische Universität Berlin Method and device for producing a three-dimensional, multi-cell object
US11549097B2 (en) * 2016-03-01 2023-01-10 Oxford University Innovation Limited Phase transfer of a cargo laden scaffold
ES2600324A1 (es) * 2016-07-26 2017-02-08 Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (Ciemat) Procedimiento de consolidación de estructuras ligeras obtenidas por fabricación aditiva
EP3427949A1 (fr) 2017-07-12 2019-01-16 Albert-Ludwigs-Universität Freiburg Bio-encres mécaniquement syntonisables pour bio-impression
EP3766957A1 (fr) * 2019-07-18 2021-01-20 Axenoll Life Sciences AG Procédé de génération et / ou d'agencement de cultures cellulaires
WO2021009320A1 (fr) * 2019-07-18 2021-01-21 Axenoll Life Sciences Ag Procédé pour générer et/ou agencer des cultures cellulaires

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