Classifications

• • 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
  • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
• • 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
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
• • 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
  • A61L27/14—Macromolecular materials
  • A61L27/26—Mixtures of macromolecular compounds
• • 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
  • A61L27/3839—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 characterised by the site of application in the body
  • A61L27/3843—Connective tissue
  • A61L27/3852—Cartilage, e.g. meniscus
• • 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
  • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y70/00—Materials specially adapted for additive manufacturing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y80/00—Products made by additive manufacturing
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/04—Alginic acid; Derivatives thereof
• • 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
  • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus

Definitions

• the present invention generally relates to the field of tissue engineering, particularly tissue engineering in the context of cartilaginous tissues. More specifically, the invention relates to a three-dimensional structure comprising a sufficient number of chondrocytes and a cross-linked biopolymer formulation, wherein said three- dimensional composition has a mechanical stability suitable for implantation into a subject in need thereof. In further aspects, the invention relates to a method for the preparation of such three-dimensional compositions, e.g. via bio-printing, a composition obtainable by such method, and medical uses of the three-dimensional compositions. BACKGROUND OF THE INVENTION
• a rare congenital disease called anotia or microtia affects 1 in 3,800 newborn babies and is characterized by the complete absence (anotia) or a deformation (microtia) of the external ear.
• the current standard of care for young microtia patients is autologous costal cartilage grafting based on the harvesting of several ribs to build an ear template.
• Several surgeries are required to reconstruct the external ear and a steep learning curve for surgeons limits the number of surgical experts around the world that can perform these procedures. Patients often experience high donor site pain from the rib harvest and the patient-reported outcome is often poor due to the low aesthetic outcome of the reconstruction.
• a few synthetic implants are available, for example under the tradename Medpor ® , but since they are made from polyethylene, they are prone to foreign body reactions and high complication rates are seen. In addition, because of the rigidity of such implants, patients experience difficulty with sleeping. Prosthetic reconstruction is a possibility that can be used particularly for elderly patients, as the surgery is minimally invasive, but requires continuous management and is not ideal from a psychological perspective, as the prosthesis is not the patient’s own organ. Additive manufacturing or “three-dimensional printing " is a versatile technology that has greatly facilitated industrial production of complex shapes, also in very small production sizes at meanwhile reasonable prices. The technology is based on computer-controlled assembly of liquid material, usually in a layer-wise fashion, which is subsequently solidified to produce three-dimensional objects.
• biofabrication techniques based on additive manufacturing bear a huge potential, as they may enable to produce living, patient specific tissues and organs for use in regenerative medicine.
• biofabrication techniques including three- dimensional bioprinting are based on layer-by-layer assembly, in this case of living cells and biomaterials to manufacture three-dimensional (3D) biological structures.
• 3D three-dimensional
• These structures can be designed based on clinical 3D models of individual patients to produce personalized tissue grafts.
• External ear or nose reconstruction is one clinical application that could be significantly improved with bioprinted personalized grafts. Such grafts or implants might even be produced with autologous cells.
• Cohen et al. (2018; PLoS ONE 13(10): e0202356) describe a study investigating a full-scale, patient-based human ear generated by implantation of human auricular chondrocytes and human mesenchymal stem cells in a 1 :1 ratio.
• the implant is created by molding. Considering that a full-sized pediatric ear requires over 200 million cells and is about 10 mL in volume, the authors indicate that a non-deforming biopsy would provide insufficient cells to populate a pediatric-sized ear.
• auricular chondrocytes proliferate in vitro, but can dedifferentiate when cultured in monolayer; thus, combination of auricular chondrocytes with mesenchymal stem cells is proposed.
• a further problem observed in this study was significant shrinkage of ear constructs during subcutaneous implantation into lab animals.
• BMSCs bone marrow stromal cells
• the BMSCs used were of animal origin and polyglycolic acid (PGA) was used as scaffold. It was observed that pellets formed by passage 3 (P3) microtia chondrocytes showed looser tissue structures with weaker glycosaminoglycan (GAG) and collagen II expression, indicating an obvious decline in chondrogenic ability.
• PGA polyglycolic acid
• the method comprises providing particles and/or fibres, an aqueous solution of a gelling polysaccharide and mammalian cells; mixing particles/fibres polysaccharide and cells to obtain a printing mix; and depositing the printing mix in a three-dimensional form.
• Kesti et al. (Adv. Funct. Mater. 2015; DOI 10.1002/adfm.201503423) present a clinically compliant bioprinting method which yields patient-specific cartilage grafts with good mechanical and biological properties. They found that mechanical properties of the printed structures, which were cultured for a maximum of 8 weeks in vitro, were inferior compared to the native cartilage.
• tissue-engineered implants to have suitable mechanical properties to withstand the stress associated with surgery, apart from being compatible with the subject the implant is made for.
• the present inventors have surprisingly found that with a long in vitro maturation period of at least 8 weeks, in particular 16 weeks, a high mechanical strength in combination with a high cell viability of three-dimensional compositions comprising chondrocytes and biopolymer formulation, namely a viability of at least 70%, in particular at least 80%, at least 85%, at least 90% or at least 95% can be achieved.
• This unexpected advantageous combination of features makes the inventive compositions particularly suitable for implantation into a subject in need thereof, such as a human patient.
• a three-dimensional composition with a mechanical stability suitable for implantation into a subject in need thereof which comprises chondrocytes and a cross-linked biopolymer formulation.
• a method for the preparation of such a three-dimensional composition as well as medical uses of the composition and related implants comprising chondrocytes.
• the present invention is directed to a three-dimensional composition
• a three-dimensional composition comprising at least about 6 x 10 6 chondrocytes per mL of composition and a cross- linked biopolymer formulation, wherein said three-dimensional composition has a mechanical stability suitable for implantation into a subject in need thereof.
• the composition particularly has an elastic modulus (E) of at least 180 kPa, at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.
• the chondrocytes may be derived from a variety of source tissues, particularly from auricular chondrocytes, particularly human auricular chondrocytes.
• the composition may be substantially free of stem cells, such as bone marrow-derived stem cells, and/or substantially free of progenitor cells, such as chondrogenic progenitor cells.
• the composition is particularly free of at least one of added tissue particles, added fibers, microbeads, and nanoparticles, more particularly free of all of these components.
• the biopolymer formulation comprises gellan gum and alginate.
• the biopolymer formulation may be a homogeneous cross-linked gellan gum / alginate formulation, wherein the gellan gum content may be from about 2% (w/v) to about 5% (w/v), particularly from about 2.0% (w/v) to about 3.0% (w/v), more particularly about 2.5% (w/v), based on the total volume of biopolymer formulation, and/or wherein the alginate content may be from about 1 % (w/v) to about 3% (w/v), particularly from about 1 .0% (w/v) to about 2.0% (w/v), more particularly about 1 .5% (w/v), based on the total volume of biopolymer formulation.
• the biopolymer formulation is a CaCh-cross- linked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of biopolymer formulation.
• the form of the three-dimensional composition can be freely selected according to the specific needs and in accordance with the possibilities of manufacturing.
• the composition may be a wedge, a tissue-engineered human nose or human auricle or a part thereof.
• the invention is further directed to a method for the preparation of a three- dimensional composition according to any one of the preceding claims, comprising the steps of: a.
• the chondrocytes are particularly derived from auricular chondrocytes, more particularly human auricular chondrocytes, more particularly human autologous auricular chondrocytes.
• the biopolymer formulation may be a gellan gum / alginate formulation, particularly a 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of biopolymer formulation.
• Step e. of the method according to the invention is particularly performed in vitro, particularly for at least 8 weeks, more particularly for 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly for 16 weeks. Also encompassed by the invention is that step e. may be performed in vivo.
• the invention is further directed to a three-dimensional composition obtainable by a specific method. Accordingly, the invention relates to a three-dimensional composition, comprising at least about 6 x 10 6 chondrocytes per ml_ of composition and a cross-linked biopolymer formulation, which is obtainable by a method comprising the steps of: a. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 10 7 chondrocytes from harvested culture; b. mixing the expanded chondrocytes with a biopolymer formulation, thereby obtaining a bio-ink; c.
• the bio-ink in layers onto a surface, thereby obtaining a three- dimensional composition; d. cross-linking the biopolymer formulation within the three-dimensional composition; e. maturing the three-dimensional composition for at least 8 weeks, thereby allowing the chondrocytes to produce extracellular matrix to form the three- dimensional composition with suitable mechanical stability for implantation.
• a cell composition for use in medicine, which comprises at least about 6 x 10 6 chondrocytes per ml_ of composition and which is provided within a biopolymer formulation and which has undergone a maturation period of at least 8 weeks, particularly 10 to 24 weeks, more particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks.
• maturation is particularly performed in vitro.
• a cell composition for use in medicine which comprises at least about 6 x 10 6 chondrocytes per ml_ of composition and which is provided within a biopolymer formulation and has an elastic modulus (E) of at least 180 kPa.
• E elastic modulus
• Particular medical uses according to the invention are treatment of anotia or microtia or facial injuries with persistent damage to ears and/or nose.
• the invention is directed to an implant for use in the improvement of hearing, comprising at least about 6 x 10 7 chondrocytes, which is provided within a biopolymer formulation and has undergone a maturation period of at least 8 weeks, particularly 10 to 24 weeks, more particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks.
• the present invention is, in a first aspect, concerned with providing a three- dimensional composition, comprising at least about 6 x 10 6 chondrocytes per mL of composition and a cross-linked biopolymer formulation, wherein said three- dimensional composition has a mechanical stability suitable for implantation into a subject in need thereof.
• the composition comprises 6-9 million cells per mL composition, such as 6, about 7, about 8 or about 9 million cells per mL composition.
• the composition comprises 12 million cells per mL composition or up to 15 million cells per mL composition.
• the three-dimensional composition may be prepared using layer-by-layer deposition methods such as bio-printing. Accordingly, the composition can have a great variety of shapes as required, among them coupons, wedges, whole auricles or noses, in particular human auricles or noses, or parts of auricles or noses.
• a “coupon” as used herein is a three-dimensional basic geometric form. For instance, coupons may have a spherical, a lenticular, a cylindrical, a disc-like, a cubic, a cuboid, or a conical shape.
• a “wedge” as used herein is a three-dimensional form bearing significant resemblance in form and size to an anatomical structure, such as a part of a human nose or human auricle or even a full human nose or a full human auricle.
• a wedge can have the form of the capital letter “D”, where, e.g., the left, straight part is thinner than the right, curved part.
• Parts of auricles or pinnae, in particular parts of a human auricle or pinna may be the helix, the anti-helix, the concha, the tragus, the anti-tragus, or a substantial fragment thereof.
• Parts of the nose may be the septum, the alae, or a substantial fragment thereof.
• a “substantial fragment’ of an auricle or nose part as used herein, in particular a human auricle part or human nose part, corresponds to at least about one third, i.e. about 33% by volume of the complete auricle part or nose part. It is encompassed by the invention to combine several auricle or nose parts or substantial fragments thereof. For example, the helix and anti-helix may be combined with a fragment corresponding to half of the concha, which may be suitable for use in reconstructive surgery.
• the three-dimensional composition may comprise a total of about 2 x 10 7 chondrocytes, 4 x 10 7 chondrocytes or 6 x 10 7 chondrocytes.
• a parameter which allows to determine whether the mechanical stability of a three- dimensional composition described herein is suitable for the uses according to the invention is the elastic modulus (E).
• the elastic modulus (E) measures the resistance of the material to elastic deformation. Accordingly, in some embodiments of the invention, the three-dimensional composition has an elastic modulus (E) of at least 180 kPa.
• the elastic modulus is at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa. Accordingly, the elastic modulus may, in some embodiments of the invention, range from between about 180 kPa to about 260 kPa, such as from 180 kPa to 260 kPa or from 200 kPa to 260 kPa.
• One established method to determine the elastic modulus is by unconfined indentation testing. There, a stress or strain is applied to the surface of the three- dimensional composition via a small indenter, and the resulting response is monitored over time. The stress-relaxation is observed. From the obtained data, the elastic modulus (E) can be determined (see Examples, section “Methods"). Accordingly, in some embodiments of the invention, the elastic modulus of the three- dimensional composition is determined by unconfined indentation testing.
• a further possibility to determine the mechanical stability of the three-dimensional composition according to the invention, which may be used to supplement elastic modulus determination or as an alternative thereto, is histological and/or immunohistochemical analysis of samples of the composition.
• Such analysis encompasses different stainings of ECM components well-known to the skilled person.
• the samples are qualitatively analyzed and are, for example, classified as being mechanically stable if specific combinations of extracellular matrix proteins are observed in combination.
• samples in which stainings for collagen type II, glycosaminoglycans (GAG) and Elastin in combination are positive, optionally further in combination with a positive staining for SOX9, may be classified as being of sufficient mechanical stability.
• chondrocytes derived from a variety of sources may be used.
• the chondrocytes are derived from cells of mammalian origin, particularly of human origin.
• allogeneic cells i.e. cells from another donor, or autologous cells, i.e. cells from the individual patient itself are advantageously used.
• Chondrocytes may be derived from a variety of source tissues, for example from articular cartilage, nasal cartilage or auricular cartilage.
• the chondrocytes of the three-dimensional composition are derived from auricular chondrocytes, particularly human nasal or auricular chondrocytes.
• the chondrocytes are derived from human autologous auricular chondrocytes.
• chondrocytes are by cell expansion and maturation from isolated primary chondrocytes, particularly human isolated primary chondrocytes.
• the chondrocytes are, according to the invention, distributed within the biopolymer formulation. This can for example be achieved by mixing a population of cells with biopolymer before assembling the three-dimensional composition, e.g. by bio printing as described herein.
• the cell viability of the chondrocytes of the three-dimensional composition according to the invention is at least 70%. More particularly, the cell viability of the chondrocytes is at least 80%, or at least 85%. In certain preferred embodiments, the cell viability of the chondrocytes is at least 90%, or even at least 95%.
• a variety of established methods for determining the viability of cells are known to the skilled person, for instance methods based on automated cell sorting (in particular flow cytometry) or hemocytometry.
• the cell viability of the chondrocytes is determined by hemocytometry, based on the European Pharmacopoeia monograph on Nucleated Cell Count and Viability (Ph. Eur. 2.7.29.). In accordance therewith, cell viability may be determined by trypan blue staining and microscopic examination using a hemocytometer with manual or automated counting.
• the present inventors have also surprisingly found that the three-dimensional composition according to the present invention can be successfully used even if the composition is substantially free of stem cells, such as mesenchymal stem cells (MSC) and bone marrow-derived stem cells (BMSC).
• stem cells such as mesenchymal stem cells (MSC) and bone marrow-derived stem cells (BMSC).
• BMSC bone marrow-derived stem cells
• the composition according to the invention can be successfully used even if it is substantially free of progenitor cells, such as chondrogenic progenitor cells.
• Substantially free of as used herein means that less than about 2%, particularly less than 1%, less than 0.5% or even less than 0.1% of cells based on the total number of cells within the three-dimensional composition are stem cells or progenitor cells.
• tissue particles and/or fibers and/or microbeads and/or nanoparticles are added to the three-dimensional composition.
• the composition according to the invention is free of at least one of added tissue particles; added fibers; microbeads; and nanoparticles.
• the composition according to the invention is free of added tissue particles, added fibers, microbeads, and nanoparticles.
• tissue particles refers to minced tissue, particularly cartilaginous tissue.
• tissue examples include articular cartilage, nucleus pulposus, meniscus, trachea, nasal cartilage, rib cartilage, ear cartilage, synovial fluid, tracheal cartilage, vitreous humor, brain, liver, spinal cord, muscle, connective tissues and subcutaneous fat, intrapatellar fat pad and small intestinal submucosa.
• the tissue particles may have been subject to further treatment in addition to mincing.
• Fibers as used herein may be synthetic fibers such as polymethyl methacrylate (PMMA) or natural fibers, such as elastin, resilin, silk and their derivatives.
• PMMA polymethyl methacrylate
• natural fibers such as elastin, resilin, silk and their derivatives.
• Microbeads are generally synthetic polymer particles with a diameter of about 0.1 pm to about 5 mm.
• Nanoparticles refers to a wide class of materials that include particulate substances, which have one structural dimension of less than 100 nm.
• “Free of as used in the context of added tissue particles, added fibers, microbeads, and nanoparticles means that the composition contains a specific compound at a percentage of less than about 0.5%, in particular less than 0.25%, more particularly less than 0.1 % or even 0.0% by weight of the total composition.
• poorly soluble calcium or strontium compounds such as calcium carbonate, calcium phosphate or hydroxyapatite
• poorly soluble calcium or strontium compounds such as calcium carbonate, calcium phosphate or hydroxyapatite
• the cells of the claimed composition may be analyzed with regard to the expression of a selected housekeeping gene or several housekeeping genes, e.g. glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and selected marker genes associated with chondrocyte differentiation, ECM production and/or inflammation.
• GPDH glyceraldehyde 3-phosphate dehydrogenase
• selected marker genes associated with chondrocyte differentiation, ECM production and/or inflammation.
• the gene expression of the selected marker genes Collagen type I, Collagen type II, Aggrecan and lnterleukin-1 b (IL-1 b) can be determined.
• the gene expression is characterized by relative gene expression profiles, i.e. relative expression between a housekeeping gene and selected marker genes.
• the relative gene expression profile e.g. of a sample of suspended cells from the composition according to the invention, is determined by quantitative Polymerase Chain Reaction (qPCR).
• chondrocytes of autologous origin there will of course be donor-to- donor variability.
• the gene expression profile, particularly of the selected marker and housekeeping genes, will particularly be matching with that of the initial autologous biopsy.
• the so-called Ct value or “threshold cycle” i.e. the cycle number at which the amount of amplified product is sufficient to yield a detectable fluorescent signal for GAPDH as a reference gene is set to a range from about 12 - about 18, particularly from 12-18.
• An exemplary range is 12.54 - 17.57. Since the Ct value is measured in the exponential phase when reagents are not limited, real-time qPCR can be used to reliably and accurately calculate the initial amount of template present in the reaction.
• the exemplary selected marker genes Collagen type I, Collagen type II, Aggrecan and lnterleukin-1 b may then be de determined as 2 _ACt values.
• the 2 _ACt value for Collagen type II / Collagen I ratio may be equal to or greater than 1 -1 O 4
• the 2 _ACt value for Collagen type II may be equal to or greater than 1 -1 O 2
• the 2 _ACt value for Aggrecan may be equal to or greater than 3.5-1 O 2
• the 2 _ACt value for IL-1 b may be lower than 5-1 O 6 .
• biopolyme for use in the present invention is available.
• biopolyme is understood to mean a polymeric material derived from renewable resources such as plants, animals and microorganisms, which is biocompatible (compatibility between material and host, e.g. histocompatibility and blood compatibility), non-toxic to organisms, in particular to mammalians, degradable in vivo, in particular enzymatically degradable, and can provide a certain degree of mechanical stability to structures, e.g. in its cross-linked from.
• tissue engineering applications for instance agarose, alginate, cellulose, collagen, fibrin, gelatin, hyaluronan, dextran and gellan gum have been investigated. Also encompassed are sulfated versions of such biopolymers.
• the biopolymer formulation comprises one or more of biopolymers suitable for use in tissue engineering.
• the biopolymer formulation comprises gellan gum and alginate.
• the biopolymer formulation consists of gellan gum and alginate, i.e. high molecular weight compounds gellan gum and alginate are the only structural components of the biopolymer formulation, but small molecules (MW ⁇ 1000 Da), and compounds with a molecular weight above 1000 Da, which are not structural components, such as growth factors, may optionally be present as well.
• a growth factor that may advantageously be present is TGF- 3, e.g. in a concentration of 10 ng/ml.
• “Structural components” as used herein are chemical compounds that are, as such or after cross-linking, required for determining and maintaining the form of the three- dimensional composition of the invention.
• the complete amount of biopolymer formulation used in the three-dimensional composition is mixed with cells, i.e. the three-dimensional composition comprises a homogeneous biopolymer formulation.
• the three-dimensional composition contains only a single, cell-laden biopolymer formulation (also referred to as (cellular) “bio-ini c”), i.e. the composition is not reinforced by acellular additional parts, e.g. layers, of biopolymers.
• the biopolymer formulation used in the three-dimensional composition of the invention may be a cross-linked gellan gum / alginate formulation.
• Cross- linking may be performed with different means, which may be roughly classified into physical cross-linking (e.g. stereocomplex and thermal cross-linking) and chemical cross-linking (for example via radical initiators, cations, or enzymes).
• the biopolymer formulation is a chemically cross-linked formulation, particularly a chemically cross-linked gellan gum / alginate formulation, more particularly a ionically cross-linked gellan gum / alginate formulation.
• gellan gum / alginate formulation as used herein means that the formulation does not contain further compounds that serve as structural components of the composition, such as further biopolymers.
• An exemplary way to perform cross-linking according to the invention is by using polyvalent ions, particularly alkaline earth metal ions.
• the biopolymer formulation of the inventive three-dimensional composition is particularly ionically cross-linked with calcium ions or strontium ions.
• the polyvalent ions are provided from a well water-soluble cation source (solubility in water at 20°C > 25 g/100 ml_); for example, the polyvalent ions may be provided by strontium chloride or calcium chloride.
• strontium chloride or calcium chloride may be provided by strontium chloride or calcium chloride.
• calcium chloride is used, particularly in a concentration from about 40 mM to about 120 mM, such as about 50 mM.
• gellan gum is used as a biopolymer according to the invention, its amount within the biopolymer formulation can vary significantly.
• the gellan gum content may range from about 2% (w/v) to about 5% (w/v), based on the total volume of biopolymer formulation, more particularly from about 2.0% (w/v) to about 3.0% (w/v), more particularly about 1.5% (w/v) based on the total volume of biopolymer formulation.
• Gellan gum may be prepared for use for instance by dissolving it in a suitable amount of aqueous glucose solution (e.g. about 300 mM), which may be buffered.
• aqueous glucose solution e.g. about 300 mM
• alginate when used as a biopolymer according to the invention, its amount within the biopolymer formulation may range from about 1% (w/v) to about 3% (w/v), particularly from about 1 .0% (w/v) to about 2.0% (w/v), more particularly about 1.5% (w/v), based on the total volume of biopolymer formulation.
• Alginate may be prepared for use for instance by dissolving it in a suitable amount of aqueous glucose solution (e.g. about 300 mM), which may be buffered.
• the biopolymer formulation is a cross-linked gellan gum / alginate formulation, wherein the gellan gum content is from about 2% (w/v) to about 5% (w/v), particularly from about 2.0% (w/v) to about 3.0% (w/v), more particularly about 2.5% (w/v), based on the total volume of biopolymer formulation.
• the biopolymer formulation is a cross-linked gellan gum / alginate formulation, wherein the alginate content is from about 1 % (w/v) to about 3% (w/v), particularly from about 1.0% (w/v) to about 2.0% (w/v), more particularly about 1.5% (w/v), based on the total volume of biopolymer formulation.
• the biopolymer formulation according to the present invention does not contain more than 3.5% (w/v) alginate.
• the biopolymer formulation may be a CaC -cross-linked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of biopolymer formulation.
• An exemplary specific embodiment of the three-dimensional composition comprises at least 6 x 10 6 chondrocytes derived from human auricular chondrocytes with a cell viability of at least 95%, and a cross-linked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of biopolymer formulation, wherein the composition is substantially free of stem cells and progenitor cells and free of added tissue particles, added fibers, microbeads and nanoparticles, and wherein the composition has an elastic modulus (E) of at least 250 kPa.
• E elastic modulus
• the composition according to the invention may be a wedge, a tissue-engineered human auricle or a part thereof.
• Such composition is suitable to be located on the skull of a patient outside the ear canal.
• the three- dimensional composition of the invention does not display significant shrinkage following in vivo implantation.
• the composition is suitable for use in plastic or reconstructive surgery.
• the present invention relates to a method for the preparation of a three-dimensional composition as described herein.
• the method comprises at least five mandatory steps as follows.
• a first step step a.
• isolated chondrocytes are expanded in vitro.
• the expansion may optionally be combined with a cryopreservation step.
• step a. may comprise three sub-steps, i.e. sub step 1) - cell expansion of isolated primary chondrocytes until the end of passage 1 (P1); sub-step 2) cryopreservation of the chondrocytes after P1; and sub-step 3) thawing and cell expansion until the end of passage 2 (P2).
• step a. may further comprise the sub-step 4) cell expansion until the end of passage 3 (P3).
• isolated chondrocytes (passage 0 cells) are seeded in a suitable amount, e.g. about 10 5 cells, in medium, such as supplemented DMEM and cultured until the end of passage 1. Then, the cells may be collected, evenly split into aliquots and cryopreserved. Subsequently, when needed, the required number of cell aliquots is thawed and cultured until the final harvest before proceeding to the next step (step b.).
• a suitable amount e.g. about 10 5 cells
• medium such as supplemented DMEM
• the expanded chondrocytes are mixed with a biopolymer formulation.
• the biopolymers that can be used for this formulation are those described hereinabove.
• the biopolymer formulation is a gellan gum / alginate formulation.
• the biopolymer formulation is a 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, wherein the percentages of gellan gum and alginate, respectively, are each based on the total volume of biopolymer formulation.
• a bio-ink i.e. cells + biopolymer formulation
• This bio-ink is suitable for, e.g., bio-printing.
• the expanded chondrocytes may be mixed with the complete amount of biopolymer formulation used in the three-dimensional composition, yielding a homogeneous biopolymer formulation with cells evenly distributed therein.
• a single biopolymer formulation is applied throughout the whole three- dimensional composition.
• the bio-ink is deposited in layers onto a surface (step c.).
• this deposition which may be performed via a layer-by-layer deposition method such as bio-printing, a three-dimensional composition is obtained.
• This composition may have any shape as described herein, for example the shape of a human nose, human auricle or part thereof.
• no chemically or physiologically different layers e.g. acellular layers vs. cellular layers or layers of different chemical composition (such as differing chemical constituents or differing concentrations of the same constituents), are used for the deposition step c., particularly bio-printing, i.e. deposition is carried out with a single homogeneous biopolymer formulation.
• a three-dimensional composition consisting only of cell-laden biopolymer formulation layers is obtained.
• the biopolymer formulation within the three-dimensional composition is cross-linked (step d.).
• Step d. may encompass any physical or chemical cross- linking as described above.
• step d. of the method particularly encompasses cross-linking with an alkaline earth metal salt, more particularly cross-linking with a well water-soluble calcium salt or a strontium salt, more particularly cross-linking with calcium chloride or strontium chloride.
• the cross-linking step d. is carried out using calcium chloride, for example at a concentration of from about 40 mM to about 120 mM, such as about 50 mM.
• cross- linking is carried out by immersing the printed composition into medium containing CaC and having a temperature of e.g. 4°C, or adding CaC to the printing vessel.
• the three-dimensional composition is subjected to maturation.
• Maturation allows the chondrocytes to produce extracellular matrix (ECM) to form a three-dimensional composition with suitable mechanical stability.
• ECM extracellular matrix
• the composition shall have a mechanical stability such that it can withstand the mechanical stress during an implantation, e.g. to a human patient.
• the biopolymer formulation is very stable over the maturation time so the concentration of the biopolymer will not change significantly.
• the elastic modulus (E) may serve as a parameter for suitable mechanical stability.
• the three-dimensional composition at the end of step e. of the method has an elastic modulus (E) of at least 180 kPa.
• the elastic modulus is at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.
• the elastic modulus may, in some embodiments of the invention, range from between about 180 kPa to about 260 kPa
• the maturation step e. is performed in vitro.
• In vitro maturation is for example carried out in culture flasks under suitable conditions (see below). The inventors have surprisingly found that a long in vitro maturation shows a positive effect both on cell viability and on development of the mechanical stability of the resulting three-dimensional composition. Accordingly, maturation, particularly//? vitro maturation, is carried out for at least 8 weeks. More particularly, in vitro maturation may be carried out for 10 to 24 weeks, i.e. for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks. More particularly, in vitro maturation may be carried out for at least 10, 12, 13, 14, 15, 16 or 17 weeks.
• in vitro maturation may be carried out for 18, 19, 20, 21, 22, 23 or 24 weeks. This longer maturation may be needed in some cases where patient surgery scheduling changes during maturation.
• the three-dimensional composition is subjected to in vitro maturation for 16 weeks.
• 3D medium When the maturation is performed in vitro, a so-called 3D medium may be used.
• 3D medium may be based on standard Dulbecco’s modified eagle medium (DMEM) with HAMs F12, to which a suitable concentration of TGF- 3, Insulin, transferrin, selenium and ascorbic acid are added.
• DMEM Dulbecco modified eagle medium
• the cell compositions plus biopolymer as described herein are particularly matured at a temperature between about 36°C to about 38°C, particularly about 37°C.
• Atmospheric conditions are particularly normoxic (e.g. 21% O2) or hypoxic (e.g. about 5-15% O2).
• the maturation step e. is performed in vivo.
• In vivo maturation may for example be carried out if surgeons want to perform biobanking in some cases before implantation to the reconstruction site. Like in vitro maturation, in vivo maturation is carried out for at least 8 weeks, particularly for 10 to 24 weeks, i.e. for 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 weeks. More particularly, in vivo maturation may be carried out for at least 10, 12, 13, 14, 15, 16 or 17 weeks. In other embodiments, in vivo maturation may be carried out for 18, 19, 20, 21 , 22, 23 or 24 weeks. In certain preferred embodiments, the three- dimensional composition is subjected to in vivo maturation for 16 weeks.
• the chondrocytes used in the method for preparation may be derived from a variety of sources as described hereinabove.
• the chondrocytes may be derived from articular cartilage, nasal cartilage or auricular cartilage.
• the chondrocytes used are derived from nasal or auricular chondrocytes, particularly human auricular chondrocytes.
• the chondrocytes are derived from human autologous auricular chondrocytes.
• the method does not comprise any other steps than those mentioned above, i.e. the method consists of the steps a. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 10 7 chondrocytes from harvested culture; b. mixing the expanded chondrocytes with a biopolymer formulation, thereby obtaining a bio-ink; c. depositing the bio-ink in layers onto a surface, thereby obtaining a three- dimensional composition; d. cross-linking the biopolymer formulation within the three-dimensional composition; e.
• the invention relates to a three-dimensional composition, comprising at least about 6 x 10 6 chondrocytes per ml_ of composition and a cross-linked biopolymer formulation, which is obtainable by a method comprising the steps of: a. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 10 7 chondrocytes from harvested culture; b.
• the three-dimensional composition thus obtained has an elastic modulus (E) of at least 180 kPa.
• the elastic modulus is at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.
• the elastic modulus may, in some embodiments of the invention, range between about 180 kPa and about 260 kPa.
• the method consists of steps a. to e. described above.
• the invention relates to medical uses of chondrocytes within a biopolymer formulation, for instance in the field of reconstructive surgery.
• a cell composition comprising at least about 6 x 10 6 chondrocytes per ml_ of composition is provided for use in medicine, wherein the cell composition is within a biopolymer formulation, particularly a homogeneous biopolymer formulation.
• This composition has been subjected to a maturation period of at least 8 weeks, which has particularly been performed in vitro. More particularly, the maturation period has lasted 10 to 24 weeks, such as 10, 11 , 12, 13, 14, 15, 16 17, 18, 19, 20, 21 , 22, 23 or 24 weeks. More particularly, the maturation period, particularly //? vitro maturation period, has lasted 10, 12, 13, 14, 15, 16 or 17 weeks. In other embodiments, the maturation period, particularly in vitro maturation period, has lasted 18, 19, 20, 21 , 22, 23 or 24 weeks.
• the cell composition within the biopolymer formulation as described has undergone maturation, particularly in vitro maturation, for 16 weeks.
• Maturation conditions, in particular for in vitro maturation are particularly those described above, i.e. 3D medium, about 36°C to about 38°C and normoxic or hypoxic atmosphere.
• the biopolymer formulation and chondrocytes are arranged in a three-dimensional structure, particularly in the form of a wedge, a human auricle or part thereof.
• the cell composition within the biopolymer formulation after maturation may be used are the treatment of anotia or microtia or facial injuries with persistent damage to ears and/or nose, for example caused by burns or dog-bites. Accordingly, some embodiments of the present invention relate to a three-dimensional composition as described hereinabove for the treatment of anotia or microtia or facial injuries with persistent damage to ears and/or nose.
• the cell composition for use in medicine comprises at least about 6 x 10 6 chondrocytes per ml_ of composition and is provided within a biopolymer formulation, particularly homogeneous biopolymer formulation and has an elastic modulus (E) of at least 180 kPa.
• the elastic modulus (E) is at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.
• the elastic modulus may, in some embodiments of the invention, range from between about 180 kPa to about 260 kPa.
• E is at least 250 kPa.
• Determination of elastic modulus may be performed as described hereinabove, e.g. by unconfined indentation testing.
• an exemplary medical use is treatment of anotia or microtia or facial injuries with persistent damage to ears and/or nose.
• the chondrocytes in the cell composition for use in medicine may be derived from a variety of sources as described hereinabove.
• the chondrocytes present in the cell composition for use in medicine are derived from human auricular chondrocytes, particularly human autologous auricular chondrocytes.
• the invention relates to an implant for use in the improvement of hearing. It is well-known that a central function of the auricle is to collect, amplify and direct sound waves into the external auditory canal. Accordingly, reconstructive surgery to restore the damaged, deformed or absent auricle can improve the auditory sense of patients significantly and reproducibly.
• Such an implant according to the invention comprises at least about 6 x 10 7 chondrocytes provided within a biopolymer formulation.
• Exemplary implants may have a volume of about 8 ml to about 10 ml and a size of about 5-5.5 cm x about 3- 3.5 cm x about 0.8-1.3 cm (full-sized adult ear).
• the implant has a suitable three-dimensional structure, in particular the form of a mammalian, particularly human nose or human auricle or a part thereof.
• such implant may be one or several parts of the nose, in particular the human nose, such as the septum, the alae, or a substantial fragment thereof as defined herein.
• such implant may be one or several parts of auricles or pinnae, in particular parts of a human auricle or pinna, such as the helix, the anti-helix, the concha, the tragus, the anti-tragus, or a substantial fragment thereof as defined herein.
• a human auricle or pinna such as the helix, the anti-helix, the concha, the tragus, the anti-tragus, or a substantial fragment thereof as defined herein.
• several parts of an auricle or nose or substantial fragments of such parts may be combined either in one implant or provided as separate implants.
• the implant has undergone a maturation period of at least 8 weeks, particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks.
• the maturation is particularly carried out in vitro.
• the implant comprises at least about 6 x 10 7 chondrocytes within a biopolymer formulation and has an elastic modulus (E) of at least 180 kPa.
• the elastic modulus (E) is at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.
• E is at least 250 kPa. Determination of elastic modulus may be performed as described hereinabove, e.g. by unconfined indentation testing.
• the invention is directed to a method of treating anotia or microtia or facial injuries with persistent damage to ears and/or nose, comprising the step of implanting a three-dimensional composition as described hereinabove into a subject in need thereof.
• Figure 2 Summary of cell viability as determined via hemocytometer at different time points for two production batches. Viability testing after bioprinting Day 1 - week 17 was performed from test coupons. Error bars indicate standard deviation. Where no deviation is given, sample size was one.
• Figure 3 Results of indentation of coupon slices at different maturation durations, across two production batches, both containing cellular (solid line) and acellular (dashed line) coupons.
• Figure 4 Histological analysis of two cellular production batches at 4 different time points. Native auricular cartilage was stained as a control.
• Figure 5 High magnification (10x) of cellular coupons at all maturation timepoints and native auricular cartilage control. All selections show highest staining intensity of cross section. Scale bar: 600 pm.
• Figure 6 Histology of acellular coupons from 2 production batches at 4 different time points.
• Figure 7 Gene expression of 9 different genes across two production batches at different time points.
• Figure 8 Representative images of cellular and acellular coupons and average weight of cellular coupons at different maturation stages.
• Figure 9 Mechanical properties of AUR-V047 during the extended maturation process and the respective histological outcomes of collagen I and collagen II stains.
• indentation was performed with a universal testing machine (Zwick Z0.5; Zwick/Roell), using Software testXpert III (also Zwick/Roell) according to operating instructions.
• the samples are subjected to uniaxial compression with constant speed until maximum strain.
• the force at the sample and the change in length are continuously measured, the related compressive strain/compression curve is recorded, and the E-modulus is determined.
• indentation was performed in the center of the samples, e.g. coupons, wedges, auricles, until 14% strain with a compression speed of 0.01 mm/s using a flat cylindrical indenter (with 2 mm diameter).
• Indentation E-modulus was analyzed at indentation depth equal to 1-5% strain.
• the biopolymer formulation was prepared by mixing gellan gum and alginate in a sterile, pyrogen-free glass bottle. First the sterile gellan gum was weighed into the mixing bottle in aseptic conditions. Buffered glucose solution was pipetted into the mixing bottle containing only the gellan gum. A magnetic mixer was added and the bottle was placed on a heated magnetic stirring device at 90 °C. After gellan gum has been fully solubilized pre-weighed alginate was added to the mixing bottle. Mixing was continued at 90 °C for 45 minutes until a homogenous biopolymer mix is achieved. After mixing the bottle is transferred to aseptic conditions and continuously mixed until a high viscosity paste-like formulation is achieved. The biopolymer formulation is collected and filled into syringes which are closed with a combi-stopper and stored at 2-8°C until further use.
• Bio-ink preparation combines the biopolymer formulation and cells suspension.
• Stored biopolymer formulation syringe is opened in aseptic conditions and an appropriate amount for the construct printing is weighed in sterile container.
• the cell suspension is mixed directly after the P3 cell harvest with the biopolymer formulation to achieve highest possible cell viability in the bio-ink.
• Bio-ink is loaded into a bioprinting syringe that can then be transferred to the bioprinting process.
• Bioprinting may generally be carried out as described in co-owned application WO 2019/106606 A1, which is incorporated by reference herein. Briefly, a printing syringe filled with the bio-ink can be brought to the printer via pass box. The printing syringe can be attached to or inserted to a syringe holder of the printer. The printing syringe nozzle is primed to remove any entrapped air from the printing system. In an additive fashion, the biopolymer can be extruded from the printing syringe to form the cellular construct.
• Crosslinking crosslinking
• the construct set is transferred to a shaker bed.
• Crosslinking solution containing 50 mM of CaC is added into the printing container to crosslinking the printing set.
• Crosslinking duration is 60 ⁇ 5 minutes before the printing set can be manipulated or transferred to the maturation process.
• the maturation process was performed in an incubator which simulated physiological conditions.
• the production set was matured together in one maturation container.
• Maturation medium containing DMEM Hams F12 + 10 ng/mL TGF-P3 + 25 pg/mL ascorbic acid + 1 % ITS is used and medium changes take place every 3 to 4 days.
• Produced constructs were matured for 16 weeks ⁇ 7 days.
• Tissue samples from one donor were described in this example.
• the donor was 31 years of age and cartilage biopsy was non-microtic.
• the biopsy sample was 48.8 mg of auricular cartilage, with 21393 living cells per mg tissue.
• Tissue samples that created the final cell suspension were obtained from a separate program entitled the Tissue Donation Program. Shipping was performed by a certified vendor using shipping materials validated and qualified for international shipment of human tissue. A temperature limitation of 2-8 degrees Celsius is in place for this program.
• the obtained biopsies were washed in phosphate-buffered saline, connective tissue was removed by scalpel and tweezer, until only cartilage remained. The cartilage was then minced, and collagenase solution was used to digest the cartilage material allowing for isolation of the cells from the tissue.
• Cell expansion until the end of P1 was performed according to standard operation procedures. Briefly, cells were cultured 2D culture medium (DMEM + 25 pg/mL ascorbic acid + 10 % FBS) until approximately 80 % confluency with regular medium changes every three to four days. Cells were then passaged into P1 and again cultured until approximately 80 % confluency was reached. At this point cells were harvested and prepared for cryopreservation.
• DMEM + 25 pg/mL ascorbic acid + 10 % FBS 2D culture medium
• a total of 12 vials, each containing 1.943 million cells were preserved in liquid nitrogen.
• Two manufacturing batches were produced. For both manufacturing batches 2 vials of cryopreserved cells were thawed and expanded. Cells were cultured in 2D culture medium (DMEM + 25 pg/mL ascorbic acid + 10 % FBS) until approximately 80% confluent with regular medium changes every three to four days. Cells were then passaged to P2 and again cultured until approximately 80 % confluency was reached. At this point the drug substance harvest with a total of 30 cell culture flasks (T175) were processed in harvest blocks of 10 flasks with two operators.
• DMEM + 25 pg/mL ascorbic acid + 10 % FBS 2D culture medium
• the cells were pooled into a single cell suspension. This suspension was centrifuged and resuspended in the for the cell number appropriate volume of medium before being released for printing.
• Example 2 Biopolymer formulation and mixing of cells with biopolymer Gellan gum 2.5 % and alginate 1.5% biopolymer formulation was used in the study and produced according to the process described above in the method section.
• modified bioprinter Celllnk lnkredible+ was used for printing.
• 420 pL volume test coupons were produced.
• Cellular and acellular coupons were produced in both production batches.
• Printed coupons were cross- linked for 60 ⁇ 5 min in 50 mM CaC before being transferred into maturation medium.
• Drug substance release testing Drug substance characteristics and safety were tested at different time points. Briefly, before harvesting the cells in P3, a visual inspection was performed to confirm the cell confluency and morphology. After the confirmation of 75 - 85 % confluency a sample of the spent culture medium was collected for mycoplasma and sterility testing. After the harvest of the last processing block the cells were pooled to a single batch of cell suspension. This cell suspension was centrifuged, and supernatant was collected for endotoxin testing. In addition to these safety tests, the cell viability and number was evaluated, and the cells were characterized via polymerase chain reaction (PCR) gene expression analysis for the genes listed in the following table.
• PCR polymerase chain reaction
• the samples were repeatedly tested for their physical, chemical and pharmacological properties. For example, after cell expansion process the released drug substance was mixed in a ratio of 1:10 with the biopolymer formulation, the cell number and viability were confirmed via hemocytometer. Cell number and viability within the printed constructs were analyzed via hemocytometer. Gene expression of cells was performed coupons after digestion. Genes analyzed are listed in Table 1. Mechanical testing was performed using indentation to determine the elastic modulus of the material.
• AUR-V047 One batch of coupons (AUR-V047) was kept in culture up to 17 weeks, while the second batch (AUR-011) was matured up to 12 weeks. In both batches, samples were tested after 3-week maturation with additional process tests at 9, 12 and 17 weeks. Acellular coupons were produced alongside the cellular coupons and cultured under the same conditions, following the same testing regime where applicable.
• FIG. 9 shows mechanical properties and histological analysis of collagen I and collagen II throughout the maturation time of 17 weeks. As can be seen, mechanical properties continuously increase throughout this period. The same trend can be seen for deposited collagen II, while collagen I presence reaches its maximum at 9 weeks and decreases from there onwards. This suggests that the increase in mechanical properties is supported to greater extent by the deposition of collagen II rather than collagen I. Collagen II can be found in the native auricular cartilage matrix and is associated to major component in mechanical integrity of the tissue. Both the increase in mechanical properties and histological analyses suggest that 17-week matured samples are indeed laying down a cartilage-like ECM and over time are expected to form functional auricular cartilage.
• Example 2 The batches obtained according to Example 2 were analyzed with regard to the cell and printing yield.
• Table 4 Summary of sterility, endotoxin and mycoplasma test results for DS and DP release
• FIG. 1 Cell viability and metabolic activity within the biopolymer and printed coupons obtained according to Examples 1 and 2 was analyzed throughout the entire process.
• Figure 2 summarizes the results from the in-process control before printing until the end of maturation for cell viability determined via hemocytometer.
• Collagen I intensity is already high at 21 Days of maturation. The intensity of the signal increases further until 9 weeks, before it starts to decrease again. Furthermore, the different structural organization of Collagen is observed over the maturation period. Collagen I appears to be more fragile and disorganized at 21 Days with homogeneity and level of organization increasing over time. Even where decrease of Collagen I is observed (from 13 weeks) homogeneity (smooth transitions, no holes) and level of organization appear to remain high. Compared to native cartilage Collagen I intensity is still high at 17 weeks. Since Collagen I is considered a repair cartilage the decrease of Collagen I is expected to continue with time to finally form Collagen I free auricular cartilage.
• Stain intensity of Collagen II is weak at 21 Days of maturation but increases drastically by 9 weeks, with further darkening observed in 13- and 17-weeks samples, respectively.
• the overall intensity appears to be indifferent at low magnification.
• Figure 5 shows higher magnification (10x) of histology images of batch AUR-V047 shown in Figure 4.
• cells show a healthy morphology with an increase in lacunae size and number over time.
• the cell number does not seem to increase over time of maturation, however full cell number analysis of FI&E stains is necessary to confirm this observation.
• Usually single cells are present after 3 weeks of maturation, small groups of cells, called chondrons, can be found at later timepoints. After 17 weeks of maturation most cells are part of small groups and only few single cells can be found. Almost all cells exhibit clearly visible lacunae, which is a desired characteristic of healthy chondrocytes.
• the cell density in 17-week matured samples is lower. Native cartilage samples show a greater degree of organization with cell size being smaller and lacunae being homogenously rounded compared to 17-week matured sample.
• Figure 8 summarizes the visual appearance, size and weight of coupons at different durations of maturation. Overall appearance of cellular constructs is constant throughout the maturation process. The yellow coloration is caused by the yellow color of phenol red containing maturation medium. The yellow color indicates a pH reduction, caused by cell activity. Cellular coupons appear homogenous and non- translucent.
• Coloration of acellular coupons is red, caused by the medium containing phenol red, which due to lack of cells does not decrease in pH and remains red in color.
• white areas increasingly appear within the acellular constructs. These areas increase in size over time. Initial analysis supports the assumption that these white areas are caused by ionic precipitation. There is no indication of such accumulations within the cellular constructs. Average diameters of cellular and acellular coupons do not change over the time of maturation as can be seen from the values given in Figure 8. Weight of acellular coupons was not determined as part of process and release controls. Weight of cellular coupons appears to increase with duration of maturation; however, it must be noted that sample sizes are small, and increase could be a result of fluctuations in coupon volume. The potential increase in weight could be explained by the increased production of extracellular matrix within the coupons as well as potentially increasing cell numbers. Further investigation is necessary to determine the validity of this observation and hypothesis.
• the invention is further characterized by the following items.
• Item 1 A three-dimensional composition, comprising at least about 6 x 10 6 chondrocytes per ml_ of composition and a cross-linked biopolymer formulation, wherein said three-dimensional composition has a mechanical stability suitable for implantation into a subject in need thereof, wherein the biopolymer formulation is particularly a homogeneous biopolymer formulation.
• Item 2 The composition according to item 1 , wherein the composition has an elastic modulus (E) of at least 180 kPa, at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.
• E elastic modulus
• Item 3 The composition according to item 1 or 2, wherein the chondrocytes are derived from auricular chondrocytes, particularly human auricular chondrocytes.
• Item 4 The composition according to any one of the preceding items, wherein the chondrocytes are obtained by cell expansion and maturation from isolated primary chondrocytes, particularly human isolated primary chondrocytes.
• Item 5 The composition according to any one of the preceding items, wherein the cell viability of said chondrocytes is at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%.
• Item 6 The composition according to any one of the preceding items, wherein the cell viability is determined by hemocytometry based on Ph.Eur. 2.7.29.
• Item 7 The composition according to any one of the preceding items, wherein the composition is substantially free of stem cells, such as bone marrow-derived stem cells.
• Item 8 The composition according to any one of the preceding items, wherein the composition is substantially free of progenitor cells, such as chondrogenic progenitor cells.
• Item 9 The composition according to any one of the preceding items, wherein the composition is free of at least one of a. added tissue particles; b. added fibers; c. microbeads; and d. nanoparticles, particularly free of a., b., c. and d..
• Item 10 The composition according to any one of the preceding items, wherein a. no calcium carbonate, and /or b. no calcium phosphate, and/or c. no hydroxyapatite are externally added to the composition, particularly wherein no calcium carbonate, no calcium phosphate and no hydroxyapatite, more particularly wherein no poorly water-soluble calcium or strontium compounds, are externally added to the composition.
• Item 11 The composition according to any one of the preceding items, wherein the cells show the following relative gene expression profile, as determined by quantitative Polymerase Chain Reaction (qPCR):
• Item 13 The composition according to any one of the preceding items, wherein the biopolymer formulation consists of gellan gum and alginate as the only structural components.
• Item 14 The composition according to any one of the preceding items, wherein the biopolymer formulation is a cross-linked gellan gum / alginate formulation, particularly a chemically cross-linked gellan gum / alginate formulation.
• Item 15 The composition according to item 13 or 14, wherein the biopolymer formulation is cross-linked with polyvalent ions, particularly with an alkaline earth metal ions, more particularly with calcium ions or strontium ions.
• Item 16 The composition according to any one of the preceding items, wherein the biopolymer formulation is a cross-linked gellan gum / alginate formulation, wherein the gellan gum content is from about 2% (w/v) to about 5% (w/v), particularly from about 2.0% (w/v) to about 3.0% (w/v), more particularly about 2.5% (w/v), based on the total volume of biopolymer formulation.
• the biopolymer formulation is a cross-linked gellan gum / alginate formulation, wherein the gellan gum content is from about 2% (w/v) to about 5% (w/v), particularly from about 2.0% (w/v) to about 3.0% (w/v), more particularly about 2.5% (w/v), based on the total volume of biopolymer formulation.
• Item 17 The composition according to any one of the preceding items, wherein the biopolymer formulation is a cross-linked gellan gum / alginate formulation, wherein the alginate content is from about 1 % (w/v) to about 3% (w/v), particularly from about 1.0% (w/v) to about 2.0% (w/v), more particularly about 1.5% (w/v), based on the total volume of biopolymer formulation.
• the biopolymer formulation is a cross-linked gellan gum / alginate formulation, wherein the alginate content is from about 1 % (w/v) to about 3% (w/v), particularly from about 1.0% (w/v) to about 2.0% (w/v), more particularly about 1.5% (w/v), based on the total volume of biopolymer formulation.
• Item 18 The composition according to any one of items 14 to 17, wherein the polyvalent ions are provided by calcium chloride or strontium chloride, particularly calcium chloride.
• Item 19 The composition according to any one of the preceding items, wherein the biopolymer formulation is a CaCh-cross-linked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of biopolymer formulation.
• Item 20 The composition according to any one of the preceding items, wherein the elastic modulus (E) is determined by unconfined indentation testing.
• Item 21 The composition according to any one of the preceding items, wherein the mechanical stability of the composition is determined histologically and/or immunohistochemically.
• Item 22 The composition according to any one of the preceding items, which is a wedge, a tissue-engineered human nose or human auricle or a part thereof.
• Item 23 The composition according to item 22, which is suitable to be located on the skull of a patient outside the ear canal.
• Item 24 A method for the preparation of a three-dimensional composition according to any one of the preceding items, comprising the steps of: a. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 10 7 chondrocytes from harvested culture; b. mixing the expanded chondrocytes with a biopolymer formulation, thereby obtaining a bio-ink; c. depositing the bio-ink in layers onto a surface, thereby obtaining a three- dimensional composition; d. cross-linking the biopolymer formulation within the three-dimensional composition; e.
• chondrocytes are derived from auricular chondrocytes, particularly human auricular chondrocytes, more particularly human autologous auricular chondrocytes.
• step a. comprises the sub-steps
• step a. further comprises the sub-step
• Item 28 The method of any one of items 24 to 27, wherein the biopolymer formulation is a gellan gum / alginate formulation, particularly a 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of biopolymer formulation.
• Item 29 The method of any one of items 24 to 28, wherein step c. is performed via layer-by-layer deposition method such as bio-printing.
• Item 30 The method of any one of items 24 to 29, wherein step c. is carried out with a single homogeneous biopolymer formulation.
• step d. is a chemical cross-linking step, particularly chemical cross-linking with polyvalent ions, more particularly cross-linking with an alkaline earth metal salt, more particularly cross- linking with a calcium salt or a strontium salt, more particularly cross-linking with calcium chloride or strontium chloride, more particularly cross-linking with calcium chloride.
• Item 32 The method of any one of items 24 to 31 , wherein step e. is performed in vitro.
• Item 33 The method of any one of items 24 to 31 , wherein step e. is performed in vivo.
• Item 34 The method of any one of items 24 to 33, wherein step e. is performed for at least 8 weeks, particularly for 10, 12, 13, 14, 15, 16 or 17 weeks.
• Item 35 The method of any one of items 24 to 34, wherein step e. is performed for 10 to 24 weeks, particularly for 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 weeks.
• Item 36 The method of any one of items 24 to 35, wherein step e. is performed in vitro for 16 weeks.
• a three-dimensional composition comprising at least about 6 x 10 6 chondrocytes per ml_ of composition and a cross-linked biopolymer formulation, which is obtainable by a method comprising the steps of: a. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 10 7 chondrocytes from harvested culture; b. mixing the expanded chondrocytes with a homogeneous biopolymer formulation, thereby obtaining a bio-ink; c. depositing the bio-ink in layers onto a surface, thereby obtaining a three- dimensional composition consisting of cell-laden biopolymer formulation layers; d. cross-linking the biopolymer formulation within the three-dimensional composition; e. maturing the three-dimensional composition for at least 8 weeks, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition.
• a cell composition comprising at least about 6 x 10 6 chondrocytes per ml_ of composition, which is provided within a biopolymer formulation, particularly a homogeneous biopolymer formulation, and has undergone a maturation period of at least 8 weeks, particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks, for use in medicine.
• a cell composition comprising at least about 6 x 10 6 chondrocytes per ml_ of composition, which is provided within a biopolymer formulation, particularly a homogeneous biopolymer formulation, and has undergone a maturation period of at least 8 weeks, particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks, for use in a method of treating anotia or microtia or facial injuries with persistent damage to ears and/or nose.
• Item 40 The cell composition according to item 38 or 39, which has undergone a maturation period of 10 to 24 weeks, particularly 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 weeks.
• a cell composition comprising at least about 6 x 10 6 chondrocytes per ml_ of composition, which is provided within a biopolymer formulation, particularly a homogeneous biopolymer formulation, and has an elastic modulus (E) of at least 180 kPa, for use in medicine.
• E elastic modulus
• a cell composition comprising at least about 6 x 10 6 chondrocytes per ml_ of composition, which is provided within a biopolymer formulation, particularly a homogeneous biopolymer formulation, and has an elastic modulus (E) of at least 180 kPa, for use in a method of treating anotia or microtia or facial injuries with persistent damage to ears and/or nose.
• E elastic modulus
• Item 43 The cell composition for use according to any one of items 37 to 42, wherein the chondrocytes are derived from human auricular chondrocytes, particularly human autologous auricular chondrocytes.
• Item 44 The cell composition for use according to any one of items 38 to 42, wherein the biopolymer formulation and chondrocytes are arranged in a three- dimensional structure, particularly in the form of a wedge, a human auricle or part thereof.
• Item 45 The cell composition for use according to any one of items 37 to 44, wherein the maturation period is an in vitro maturation period.
• Item 46 The method of any one of items 24 to 36 or the composition according to any one of items 37 to 45, wherein the maturation is carried out in 3D medium at about 36°C to about 38°C under normoxic or hypoxic conditions.
• Item 47 A three-dimensional composition according to any one of items 1 to 23 for the treatment of anotia or microtia or facial injuries with persistent damage to ears and/or nose.
• Item 48 An implant for use in the improvement of hearing, comprising at least about 6 x 10 7 chondrocytes, which is provided within a biopolymer formulation and has undergone a maturation period of at least 8 weeks, particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks.
• Item 49 The implant for use according to item 48, which has undergone a maturation period of 10 to 24 weeks, particularly 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 weeks.
• Item 50 The implant for use according to item 48 or 49, wherein the maturation period is an in vitro maturation period.
• Item 51 An implant for use in the improvement of hearing, comprising at least about 6 x 10 7 chondrocytes, which is provided within a biopolymer formulation and has an elastic modulus (E) of at least 180 kPa.
• E elastic modulus
• Item 52 The implant for use according to item 51 , wherein E is at least 250 kPa.
• Item 53. A method of treating anotia or microtia or facial injuries with persistent damage to ears and/or nose, comprising the step of implanting a three- dimensional cartilaginous composition according to any one of items 1 to 23 into a subject in need thereof.

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