CA3185014A1 - Matured three-dimensional printed compositions and uses thereof - Google Patents

Abstract

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.

Description

Matured three-dimensional printed compositions and uses thereof Description FIELD OF THE INVENTION
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
Damage to or complete lack of the aesthetic features in the face, such as nose and ears, for whatever cause, can subject affected persons to psychosocial problems throughout their lives. For example, ears and nose are affected in the vast majority of burns involving the head and neck of injured persons. If affected, the external ear is often completely destroyed and total ear reconstruction by surgery is required, albeit this is a very challenging task.
Accidents and dog bite injuries are a further common cause for the need of reconstructive surgery, in particular if parts of the face, such as nose and/or ear, are affected. Facial penetrating injuries can lead to loss of function with soft-tissue deformity of form and contour that require multiple surgical interventions and concomitant psychological trauma (Alizadeh et al., Plast Reconstr Surg Glob Open.

2 2017 Oct; 5(10): e1431). Every year, about 30,000 patients require reconstructive surgery as a result of dog bites in the United States alone.
Another cause for damage or loss of aesthetic features in the face are diseases, for example cancer. 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. In the field of medicine, 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. Of particular interest is the combination of cells with supporting structures.

3 PCT/EP2021/066301 Like non-medical additive manufacturing, 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.
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.
However, large hydrogel-based structures often have limited mechanical strength, making them unsuitable for handling in the context of implantation, because they do not withstand the mechanical stress during surgery.
An overview of bioprinting technologies for auricular cartilage tissue engineering is given by Kesti (2018; DOI 10.3929/ethz-b-000280389).
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 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. In addition, 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.
Zhang et al. (Biomaterials (2014): vol. 35, pp. 4878-4887) describe regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with bone marrow stromal cells (BMSCs). 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.

4 WO 2016/092106 Al relates to a method of providing a graft scaffold for cartilage repair, particularly in a human patient. 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.
Kesti et al. (BioNanoMat 2016; DOI 10.1515/bnm-2016-0004) describe a parametric study with the Vivoflow bioink to investigate the most important material and bioprinting process parameters to obtain reproducible bioprinting with the scope to establish a basis for best practice guidelines.
In summary, to be useful in reconstructive surgery, it is crucial for 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.
BRIEF SUMMARY OF THE INVENTION
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.
Accordingly, provided herein is 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. Also provided is a method for the preparation of such a three-dimensional composition, as well as medical uses of the composition and related implants comprising chondrocytes.
Thus, the present invention is directed to a three-dimensional composition

5 comprising at least about 6 x 106 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.
Mechanical stability may be quantified by determination of the elastic modulus (E).
Accordingly, 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.
In particular, 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. Moreover, 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.
According to the invention, a great variety of biopolymers may be used. In some embodiments, the biopolymer formulation comprises gellan gum and alginate. In particular, 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`)/0 (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.

6 In specific exemplary embodiments, the biopolymer formulation is a CaCl2-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.
For example, 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. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 107 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. maturing the three-dimensional composition, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition with suitable mechanical stability for implantation, wherein the chondrocytes are particularly derived from auricular chondrocytes, more particularly human auricular chondrocytes, more particularly human autologous auricular chondrocytes.
According to specific embodiments of the method, 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

7 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 106 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 107 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. 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.
The invention further relates to medical uses of the three-dimensional compositions described. According to some embodiments, a cell composition is provided for use in medicine, which comprises at least about 6 x 106 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.
As already mentioned above, maturation is particularly performed in vitro.
Also provided is a cell composition for use in medicine, which comprises at least about 6 x 106 chondrocytes per mL of composition and which is provided within a biopolymer formulation and has an elastic modulus (E) of at least 180 kPa.

8 Particular medical uses according to the invention are treatment of anotia or microtia or facial injuries with persistent damage to ears and/or nose.
Finally, the invention is directed to an implant for use in the improvement of hearing, comprising at least about 6 x 107 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.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is, in a first aspect, concerned with providing a three-dimensional composition, comprising at least about 6 x 106 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. In exemplary embodiments, 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. In other exemplary embodiments, 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. As an example, 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, in particular the human 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.
In exemplary embodiments, the three-dimensional composition may comprise a total of about 2 x 107 chondrocytes, 4 x 107 chondrocytes or 6 x 107 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. In further particular embodiments, 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 5 being mechanically stable if specific combinations of extracellular matrix proteins are observed in combination. By way of non-limiting example, 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 50X9, may be classified as being of sufficient mechanical stability.
According to the invention, chondrocytes derived from a variety of sources may be used. Generally, the chondrocytes are derived from cells of mammalian origin, particularly of human origin. For tissue engineering, usually 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. In particular embodiments, the chondrocytes of the three-dimensional composition are derived from auricular chondrocytes, particularly human nasal or auricular chondrocytes. According to certain preferred embodiments, the chondrocytes are derived from human autologous auricular chondrocytes.
One advantageous approach to obtain the chondrocytes is 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.
As already mentioned, it has surprisingly been found that according to the invention, a high mechanical strength of the three-dimensional composition can be combined with a high cell viability. In particular, 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. In particular embodiments of the present invention, 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). Likewise, 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.
Still further, it is unnecessary according to the present invention to add components such as tissue particles and/or fibers and/or microbeads and/or nanoparticles to the three-dimensional composition. In particular, the composition according to the invention is free of at least one of added tissue particles; added fibers;
microbeads;
and nanoparticles. In certain preferred embodiments, the composition according to the invention is free of added tissue particles, added fibers, microbeads, and nanoparticles.

"Tissue particles" as used herein refers to minced tissue, particularly cartilaginous tissue. Examples of such tissue 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.
"Microbeads" are generally synthetic polymer particles with a diameter of about 0.1 rn to about 5 mm.
"Nanoparticles" as used herein 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.
Likewise, it is unnecessary according to the present invention that poorly soluble calcium or strontium compounds (solubility in water at 20 C < 1 g/100 mL), such as calcium carbonate, calcium phosphate or hydroxyapatite, are added to the three-dimensional composition. Accordingly, in certain embodiments, no calcium carbonate (CaCO3), calcium phosphate (Ca2(PO4)3) and hydroxyapatite, respectively, is externally added to the composition.
To determine the pattern of genes expressed, at the level of transcription, under specific circumstances or in a specific cell to give a global picture of cellular function, gene expression profiling can be used. In the context of the present invention, according to certain embodiments, 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. For instance, the gene expression of the selected marker genes Collagen type I, Collagen type II, Aggrecan and Interleukin-113 (IL-113) can be determined. In particular, the gene expression is characterized by relative gene expression profiles, i.e. relative expression between a housekeeping gene and selected marker genes. According to the certain embodiments, 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).
When using chondrocytes of autologous origin, there will of course be donor-to-donor variability. In any case, the gene expression profile, particularly of the selected marker and housekeeping genes, will particularly be matching with that of the initial autologous biopsy.
As an example, 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 Interleukin-113 (IL-113) may then be de determined as 2-Act values. As an example, the 2-Act value for Collagen type II / Collagen I ratio may be equal to or greater than 1.10-4, the 2-Act value for Collagen type II may be equal to or greater than 1.10-2, the 2-Act value for Aggrecan may be equal to or greater than 3.5.10-2, and the 2-Act value for IL-113 may be lower than 5.10-6.
A variety of biopolymers for use in the present invention is available. As used herein, the term "biopolymer'' 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.
For 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.
In the context of the present invention, the biopolymer formulation comprises one or more of biopolymers suitable for use in tissue engineering. In particular, the biopolymer formulation comprises gellan gum and alginate. More particularly, 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-I33, 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.
In particular embodiments 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.
Accordingly, in these particular embodiments, the three-dimensional composition contains only a single, cell-laden biopolymer formulation (also referred to as (cellular) "bio-ink'), i.e. the composition is not reinforced by acellular additional parts, e.g.
layers, of biopolymers.
In particular, 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).
According to some embodiments, 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.
The term 5 "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

10 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. In particular, 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 15 chloride. In certain embodiments, calcium chloride is used, particularly in a concentration from about 40 mM to about 120 mM, such as about 50 mM.
Where gellan gum is used as a biopolymer according to the invention, its amount within the biopolymer formulation can vary significantly. In particular, 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. Likewise, where alginate is 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.
Accordingly, in some embodiments of the invention, 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.
According to some embodiments, 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.
In certain preferred embodiments of the invention, the biopolymer formulation may be a CaCl2-cross-linked 2.5% (w/v) gellan gum/1.5 A (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 106 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 A
(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.
As indicated above, 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. In particular, the three-dimensional composition of the invention does not display significant shrinkage following in vivo implantation. In other words, the composition is suitable for use in plastic or reconstructive surgery.
In a further aspect, 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.

In a first step (step a.), isolated chondrocytes are expanded in vitro. By this expansion, at least about 6 x 107 chondrocytes are obtained from harvested culture.
In some embodiments, the expansion may optionally be combined with a cryopreservation step.
According to some embodiments, 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).
According to yet further embodiments, step a. may further comprise the sub-step 4) cell expansion until the end of passage 3 (P3).
For instance, isolated chondrocytes (passage 0 cells) are seeded in a suitable amount, e.g. about 105 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.).
In step b., the expanded chondrocytes are mixed with a biopolymer formulation.
The biopolymers that can be used for this formulation are those described hereinabove.
In particular, the biopolymer formulation is a gellan gum / alginate formulation.
According to particular embodiments, 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. As a result of this mixing step, a bio-ink (i.e. cells +
biopolymer formulation) is obtained. This bio-ink is suitable for, e.g., bio-printing. In particular, 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. Accordingly, in these embodiments, a single biopolymer formulation is applied throughout the whole three-dimensional composition.

Following step b., the bio-ink is deposited in layers onto a surface (step c.). By way of 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.
According to particular embodiments, 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.
Thereby, a three-dimensional composition consisting only of cell-laden biopolymer formulation layers is obtained.
Subsequently, 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. According to particular embodiments, step d. is a chemical cross-linking step, particularly chemical cross-linking with polyvalent ions.
For providing the polyvalent ions, particularly alkaline earth metal salts may be used.
Accordingly, 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. In certain preferred embodiments, 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. In exemplary embodiments, cross-linking is carried out by immersing the printed composition into medium containing CaCl2 and having a temperature of e.g. 4 C, or adding CaCl2 to the printing vessel.
In the final mandatory step of the method (step e.), 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. Formation of sufficient ECM is key to achieve the suitable mechanical stability. Important ECM components are for example collagen I and II. In particular, 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. As indicated above, the elastic modulus (E) may serve as a parameter for suitable mechanical stability.
Accordingly, in some embodiments of the invention, the three-dimensional composition at the end of step e. of the method has an elastic modulus (E) of at least 180 kPa. In further particular embodiments, 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 In particular, 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 in 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 other embodiments, 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. In certain preferred embodiments, the three-dimensional composition is subjected to in vitro maturation for 16 weeks.
When the maturation is performed in vitro, a so-called 3D medium may be used.
In particular, such 3D medium may be based on standard Dulbecco's modified eagle medium (DMEM) with HAMs F12, to which a suitable concentration of TGF-I33, Insulin, transferrin, selenium and ascorbic acid are added. 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% 02) or hypoxic (e.g. about 5-15% 02).

In other embodiments of the invention, 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 5 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. For example, the chondrocytes may be derived from articular cartilage, nasal cartilage or auricular cartilage. In particular embodiments, the chondrocytes used are derived from nasal or auricular chondrocytes, particularly human auricular chondrocytes. According to certain preferred embodiments, the chondrocytes are derived from human autologous auricular chondrocytes.
According to some embodiments, 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 107 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. maturing the three-dimensional composition, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition with suitable mechanical stability for implantation;
as described above.

According to a yet further aspect, the invention relates to a three-dimensional composition, comprising at least about 6 x 106 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 107 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. maturing the composition for at least 8 weeks, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition.
By performing these steps, a three-dimensional composition having an advantageous mechanical stability is obtained. In particular, the three-dimensional composition thus obtained has an elastic modulus (E) of at least 180 kPa. In further particular embodiments, 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 between about 180 kPa and about 260 kPa. According to some embodiments, the method consists of steps a.
to e. described above.
In yet a further aspect, the invention relates to medical uses of chondrocytes within a biopolymer formulation, for instance in the field of reconstructive surgery.

According to some embodiments, a cell composition comprising at least about 6 x 106 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 in 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.
In certain preferred embodiments of the invention, 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.
In some embodiments, 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.
Particular medical indications in which 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.
According to some embodiments, the cell composition for use in medicine comprises at least about 6 x 106 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. According to other embodiments, 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. Accordingly, the elastic modulus may, in some embodiments of the invention, range from between about 180 kPa to about 260 kPa.
In some embodiments, E is at least 250 kPa. Determination of elastic modulus may be performed as described hereinabove, e.g. by unconfined indentation testing.

Again, 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. According to some embodiments, the chondrocytes present in the cell composition for use in medicine are derived from human auricular chondrocytes, particularly human autologous auricular chondrocytes.
According to a yet further aspect, 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 107 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). Advantageously, 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. For example, 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.
In particular, 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. Depending on the required form of the implant, 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.
In order to be sufficiently stable for resisting mechanical stress during implantation, 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.

According to some embodiments, the implant comprises at least about 6 x 107 chondrocytes within a biopolymer formulation and has an elastic modulus (E) of at least 180 kPa. According to other embodiments, 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. In some embodiments, E is at least 250 kPa. Determination of elastic modulus may be performed as described hereinabove, e.g. by unconfined indentation testing.
In yet a further aspect, 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.
The invention is further illustrated by the following examples and figures.
LEGENDS TO THE FIGURES
Figure 1: Production Flow Chart 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 m.
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 5 collagen II stains.
EXAMPLES
Methods Unconfined indentation testing Mechanical evaluation of 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. During the test, 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.
Specifically, 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.
Cell culture The obtained cartilage biopsies were washed in phosphate-buffered saline and connective tissue was removed from the biopsies 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 was carried out with regular medium changes until approximately 80 % cell confluency was observed. 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. Sufficient number of cells were thawed and expanded in 2D
culture conditions (DMEM + 25 pg/mL ascorbic acid + 10 % FBS) until approximately 80 %

confluent was observed. Cells were then passaged to P2 and again cultured until approximately 80 % confluency was reached. At this point the cells were harvest from 30 cell culture flasks. After the completed harvest, the cells were pooled into a single cell suspension. This suspension was centrifuged and resuspended in appropriate volume of medium to be ready for mixing with biopolymer formulation.
Biopolymer formulation and bio-ink preparation 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.
Bio-printing Bioprinting may generally be carried out as described in co-owned application WO 2019/106606 Al, 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.

Crosslinkinq Immediately after the printing process the construct set is transferred to a shaker bed. Crosslinking solution containing 50 mM of CaCl2 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.
Maturation 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-B3 +
25 pg/mL ascorbic acid + 1`)/0 ITS is used and medium changes take place every to 4 days. Produced constructs were matured for 16 weeks 7 days.
Example 1: Cell isolation and expansion Cells from one donor are 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. 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.
After the harvest of each processing block, 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 GelIan 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.
For bio-printing, modified bioprinter CellInk Inkredible+ was used for printing. For both productions 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 CaCl2 before being transferred into maturation medium.
From each production, coupons were matured together in one T175 culture flask with removable lid. The following 3D medium was used: DMEM Hams F12 with glutaMAX + 10 ng/mL TGF-63 + 25 pg/mL ascorbic acid + 1% ITS. Per flask a minimum volume of 70 mL or 3.5 mL per test coupon was used. Medium was changed every 3 to 4 days. Coupons were matured for periods of 3 weeks, 8 weeks, 13 weeks and 17 weeks 3 days.

Example 3: Testing of drug substance, biopolymer formulation and drug product 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.
Table 1: selected gene expression markers Gene Description GAPDH Ubiquitous glycolytic enzyme (housekeeping gene). Used as a reference gene.
Collagen type I Fibrous protein in bone, synovium and the skin.
Collagen type II Structural protein in cartilage tissue.
Aggrecan Structural protein in cartilage with high negative charge to improve water retention in the tissue.
Interleukin-113 Inflammatory marker in cells.
Elastin Elastic type protein in tissues including auricular cartilage.
FGFR3 Protein growth factor associated with cell proliferation.
MMP-1 Matrix metalloprotease is an extracellular matrix remodelling enzyme.
ACE Angiotensin converting enzyme is a marker in fibroblasts.
ALK-1 TGF-beta receptor that mediates cartilage homeostasis.
Biogolymer formulation testing After biopolymer formulation was completed it was characterized for printing properties and safety, in particular sterility, rheology, pH and osmolarity were tested.

Drug Product in-process and release testing From mixing the drug substance with the biopolymer until the final drug product is obtained, the samples were repeatedly tested for their physical, chemical and pharmacological properties. For example, after cell expansion process the released 5 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 10 modulus of the material.
Histological evaluation of the coupons was performed after fixation for the stains and immunohistochemical markers listed in Table 2.
15 Table 2: Brief description of selected histological markers Staining Description Staining observations H&E Standard pathology Cell nucleus are stained deep staining purple and other tissue in light violet Safranin 0 Cartilage marker staining Glycosaminoglycan for glycosaminoglycans structures are stained red and the cell nucleus in green Weigert's Elastin Elastin and elastic fibers Elastin fibers are stained staining black and cell nucleus in violet Alcian Blue Cartilage marker staining Glycosaminoglycan for glycosaminoglycans structures are stained blue and the cell nucleus in violet Collagen I ECM marker in repair Brown staining for collagen I
cartilage and connective and deep purple for cell tissue nucleus Collagen II Major Collagen type in Brown staining for collagen II
developed cartilage tissue and deep purple for cell nucleus Example 4: Study long term in vitro maturation In this preliminary study, two expansions from the same donor were used to produced cellular coupons which were matured up to 17 weeks. As the construct .. maturation is the process step in which the foundation for extracellular matrix production is achieved, a good understanding of this development over time is crucial. To deepen this understanding, formation of ECM such as Collagen I and Collagen II, the relationship to the mechanical properties of the construct, cell viability, functionality and number have been investigated over the timeframe of 17 weeks.
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 weeks. Acellular coupons were produced alongside the cellular coupons and cultured under the same conditions, following the same testing regime where applicable.
In both production batches, cell viability increased over time of maturation to 92.4 %
.. at 17 weeks for AUR-V047 and 81.2 % at 13 weeks in AUR-011 (see details below in Example 4.3). Both batches exhibit similar slope in viability increase.
Batch AUR-011 showed greater impact on cell viability when cells were mixed with the biopolymer, compared to AUR-V047. This greater loss in viable cells was visible throughout the maturation of 13 weeks, as the cell viability of AUR-011 remained below that of AUR-047.
Cell activity via MTS was recorded throughout the maturation process and is hypothesized to be correlating with cell viability and cell number. Overall an increase in cell activity was measurable between 3 and 9 to 13 weeks of maturation. In the last timepoint of each batch a decrease was recorded. As cell viability was increasing, limited diffusion through the coupon, caused by increasing amounts of ECM is likely the reason for lower MTS values.

Gene expression profiles of cells extracted from test coupons at different maturation timepoints show no change over time (see details below in Example 4.6). This indicates that evaluated genes were regulated early on in the maturation process and this level is maintained throughout maturation, resulting in deposition of ECM as observed histologically.
Visual appearance of coupons shows clear differences between cellular and acellular constructs with regards to color and transparency (see details below in Example 4.7). The color is dictated by the color of the medium which changes from red to yellow due to the presence of phenol red, which is a pH indicator. As cells are metabolizing and using the medium, more acidic conditions are created, causing the color change from red to yellow. No pH changes occur in acellular cultures, resulting in a red color of culture medium and hence a red coloration of the acellular coupons.
The deposition of extracellular matrix is causing the cellular coupons to become less transparent and more opaque in their appearance.
Overall shape of the coupons does not change throughout the maturation process.
No biopolymer degradation or degradation product were observed during the maturation period of up to 17 weeks supporting the theoretical stability of the biopolymer matrix. Acellular coupons exhibit white areas within the coupons, which increase in size over the duration of maturation. Currently the hypothesis is that the white areas are accumulation of ions (mainly calcium ions) precipitating.
Mechanical properties of coupons increase significantly between 3 weeks and 17 weeks of maturation (see details below in Example 4.4). After reaching the cation concentration equilibrium, the mechanical properties of acellular coupons are stagnating after 3 weeks of maturation. This proves that any increase in mechanical properties is caused by the production of ECM that is synthesized by the resident cells.
Histological analyses of elastin, glycosaminoglycans and collagen types I and II
support this correlation (see details below in Example 4.5). Figure 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 4.1: Printing outcome The batches obtained according to Example 2 were analyzed with regard to the cell and printing yield.
Table 3: Summary of cell yield and viability, used biopolymer amounts and number of printed constructs Cellular Acellular Cellular Acellular Production Production Production Production DS total cell yield 124.031 million - 58.734 million -cells cells DS viable cell 119.779 million - 54.99 million cells -yield cells DS viability 97 % 94 %
Added DS 1.066 mL 1.28 mL 0.9 mL 1.004 mL
volume (DM EM (DMEM only) only) BPM weight 10.66g 12.79 9.14g 10.04g Number of 25 22 19 18 printed constructs Even though the same donor was used in both DS expansions, the cell yield from 30 flask harvest was very different. This can be attributed to the difference in cell confluency at the point of harvest, which was estimated to be 75 % for AUR-while being estimated to around 85 % for AUR-011.

Example 4.2: DS and DP safety - sterility, endotoxin and mycoplasma test results All safety related release criteria at the stages of DS and for all DP
maturation period samples were according to requirements, with no bacterial or mycoplasma contaminations or increased levels of endotoxins found in the spent culture medium (Table 4).
Table 4: Summary of sterility, endotoxin and mycoplasma test results for DS
and DP
release Para- Test Method Sample Specifi AUR-011 AUR-V047 meter -cation DS DP DS DP
Safety Sterility Membrane Culture No No No No No DS/DP filtration, medium growth growth growth growth growth USP<71>, ¨Last Ph.Eur. change 2.6.1 Endo- LAL, Culture <0,25 <0,25 <0,25 <0,25 <0,25 toxin USP<85>, medium EU/mL EU/mL EU/mL EU/mL EU/mL
DS/DP Ph.Eur. ¨ Last 2.6.14 change <2 EU/mL
Myco- PCR, Culture Nega- Nega- Nega- Nega- Nega-plasma Ph.Eur. medium tive tive tive tive tive DS/DP 2.6.7 ¨Last change Example 4.3: Cell Viability and Activity evaluation over the maturation period 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.
Cell viability was high in both DS harvests (94 ¨ 97 %). On contact with the biopolymer the cell viability dropped to 63 % and 76 %, respectively. This phenomenon is a well-documented part of the production method according to the present invention and caused by shear forces cells are exposed to when being mixed with the biopolymer. In both production batches, viability was lowest after day 1 ¨ 3 in maturation. From there a steady increase of viability was measured until the end of the respective maturation period. The development of viability over time is comparable in both production batches.

Batch AUR-011 displays consistently lower viabilities than AUR-V047 from the point the cells were mixed with the biopolymer formulation. As both batches used the same donor and biopolymer formulation, it can be assumed that this difference was caused by the mixing process. Overall it can be concluded that the final viability, albeit 10 increasing overtime, is dependent on the viability that can be maintained during the mixing of cells and biopolymer.
Example 4.4: Mechanical properties of coupons 15 The mechanical properties of cellular and acellular coupons of both batches were analyzed throughout the maturation process (Figure 3). Initial elastic moduli (1 ¨ 3 days after printing) are high, followed by a drastic drop over the following 3 weeks maturation period. At the early stage of maturation (Day 1 ¨ 3), the mechanical properties of all constructs, cellular and acellular, are contributed by the ionic cross-20 linking of the biopolymer matrix only. Over the period of initial maturation, calcium ions are washed out of the construct to reach an equilibrium concentration in the medium while being replaced by other ions from the cell culture medium. This leads to a decrease in strength of the hydrogels. Over time, an equilibrium is reached, consistent to the maturation medium conditions, resulting in an elastic modulus 25 around 115 kPa after 21 days of maturation culture. As can be seen in Figure 3, the mechanical properties of acellular constructs (dashed lines) stay either consistent or show a slight further decrease over the rest of the maturation period for both production batches. At the same time, a steep increase in mechanical properties can be observed for the cellular constructs (Figure 3, solid lines) from Day 21 onwards.
30 For both production batches the peak of mechanical properties is reached at the respective endpoint at 13 and 17 weeks with 256 kPa and 296 kPa. Compared to acellular coupons the mechanical properties have increased by 227 % in AUR-011 and 384 % in AUR-V047 at their respective endpoints. A further increase of mechanical properties with further maturation can be expected.

Example 4.5: Histological evaluation of coupons At each maturation timepoint, sample coupons of both batches were analyzed using different histological and immunohistochemical (INC) stains. Figure 4 shows all images of coupons for different stains and at different timepoints. For reference, a native auricular cartilage sample was also included. These low magnification images are shown to establish overall impression and potential changes of the coupons at the different stages of maturation.
Since H&E is an overview stain to visualize cells, staining intensity is not particularly relevant. At this magnification number of cells and their morphology cannot be analyzed. Both Weigert's Elastin and Alcian Blue stains seem to increase in intensity with duration of maturation, indicating the increase of elastin, glycosaminoglycans and cartilage-like tissue. However, this trend is not visible in Safranin-O
stain, which, like Alcian Blue, also binds to glycosaminoglycans. Alcian Blue and Safranin-O
stains exhibit some level of unspecific background staining in acellular constructs (Figure 6), which shows no significant change over time of maturation. This suggest that an increase in stain intensity in Alcian Blue is not an artefact but indicates increased presence of glycosaminoglycans.
Weigert's Elastin intensity is much higher in native cartilage than in 17-week matured coupon, while the intensity of Alcian Blue and Safranin-O is mostly comparable between 17-week matured coupons and native cartilage.
At lower magnification the greatest differences can be observed in Collagen I
and II
stains over the period of maturation. 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. When comparing the 17-week matured construct to native cartilage the overall intensity appears to be indifferent at low magnification.
Figure 5 shows higher magnification (10x) of histology images of batch AUR-shown in Figure 4. In all stains cells show a healthy morphology with an increase in lacunae size and number over time. Visually, the cell number does not seem to increase over time of maturation, however full cell number analysis of H&E
stains is necessary to confirm this observation. Mostly 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. Compared to native cartilage, 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.
The greatest difference between 17-week matured samples and native cartilage is observed in samples stained with Weigert's Elastin and Collagen I. Using Weigert's Elastin, native auricular cartilage stains with much higher intensity and on close observation elastin fibers can be identified (dark strings between cells).
Very early formation of such fibers can be observed in close proximity to cells in 17-week matured samples (orange arrows). Histologically, 17-week matured samples are the furthest advanced and seem to be developing towards native auricular cartilage.
Figure 6 shows histological evaluation of acellular coupons cultured for the same time period as cellular coupons. With exception of Collagen I and Collagen II, which are specific stains, all stains exhibit some level of background. The biopolymer matrix has negatively charged structure, which attracts to positively charged staining molecules and entraps them within the matrix. The intensity of background staining appears to be stable throughout the maturation process, indicating that no significant loss of biopolymer occurs.
Example 4.6: Gene expression of cells during long-term maturation Gene expression profiles (Figure 7) of cells extracted from the test coupons after different times of maturation appear indifferent throughout the entire process. This is particularly interesting as the protein expression, as observed via histological analysis clearly changes over time for Collagen I, which increases early in the process before it decreases towards the end of 17-week maturation. This indicates that PCR analysis of cells can only be an indication of protein production within the 3D matured constructs. The small differences that can be observed in some genes between the donors are not significant and part of batch to batch variation.
Example 4.7: Visual appearance and weight of coupons 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.
Throughout the maturation process 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 106 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.
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.
5 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 10 the composition is free of at least one of a. added tissue particles;
b. added fibers;
c. microbeads; and d. nanoparticles, 15 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 20 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):
- GAPDH (reference gene): Ct = about 12¨ about 18 - Collagen type II / Collagen I ratio: 2-Act '1.10-4 - Collagen type II: 2-Act 1.10-2 - Aggrecan: 2-Act >3.5.10-2 - IL-113: 2-Act <5.10-6.

Item 12.
The composition according to any one of the preceding items, wherein the biopolymer formulation comprises gellan gum and alginate.
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.
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.
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 CaCl2-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 107 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. maturing the three-dimensional composition, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition with suitable mechanical stability for implantation.

Item 25.
The method of item 24, wherein the chondrocytes are derived from auricular chondrocytes, particularly human auricular chondrocytes, more particularly human autologous auricular chondrocytes.
Item 26. The method of item 24 or 25, wherein step a. comprises the sub-steps 1) cell expansion of isolated primary chondrocytes until the end of passage 1 (P1);
2) cryopreservation of the chondrocytes after P1;
3) thawing and cell expansion until the end of passage 2 (P2).
Item 27.
The method of item 26, wherein step a. further comprises the sub-step 4) cell expansion until the end of passage 3 (P3).
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.
Item 31. The method of any one of items 24 to 30, wherein 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.
Item 37.
A three-dimensional composition, comprising at least about 6 x 106 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 107 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.
Item 38.
A cell composition comprising at least about 6 x 106 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.
5 Item 39. A cell composition comprising at least about 6>< 106 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, 10 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, 15 18, 19, 20, 21, 22, 23 or 24 weeks.
Item 41.
A cell composition comprising at least about 6 x 106 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 20 least 180 kPa, for use in medicine.
Item 42.
A cell composition comprising at least about 6 x 106 chondrocytes per mL of composition, which is provided within a biopolymer formulation, particularly 25 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.
30 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 107 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 107 chondrocytes, which is provided within a biopolymer formulation and has an elastic modulus (E) of at least 180 kPa.
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.

Claims (16)

Claims

1. A three-dimensional composition, comprising at least about 6 x 106 chondrocytes per mL of composition and a cross-linked homogeneous biopolymer formulation, wherein the biopolymer formulation comprises gellan gum and alginate, wherein said three-dimensional composition has a mechanical stability suitable for implantation into a subject in need thereof and wherein the composition has an elastic modulus (E) of at least 180 kPa.

2. The composition according to claim 1, wherein the composition has an elastic modulus (E) of at least 200 kPa, at least 220 kPa, at least 240 kPa, at least 250 kPa or at least 260 kPa.

3. The composition according to claim 1 or 2, wherein the chondrocytes are derived from auricular chondrocytes, particularly human auricular chondrocytes.

4. The composition according to any one of the preceding claims, wherein the composition is substantially free of stem cells, such as bone marrow-derived stem cells, and/or wherein the composition is substantially free of progenitor cells, such as chondrogenic progenitor cells, and/or 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..

5. The composition according to any one of the preceding claims, 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, and/or 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.

6. The composition according to any one of the preceding claims, wherein the biopolymer formulation is a CaCl2-cross-linked 2.5% (w/v) gellan gum/1.5%
(w/v) alginate formulation, based on the total volume of biopolymer formulation.

7. The composition according to any one of the preceding claims, which is a wedge, a tissue-engineered human nose or human auricle or a part thereof.

8. A method for the preparation of a three-dimensional composition according to any one of the preceding claims, comprising the steps of:
a. expanding isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, thereby obtaining at least about 6 x 107 chondrocytes from harvested culture;
b. mixing the expanded chondrocytes with a homogeneous biopolymer formulation comprising gellan gum and alginate, 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, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition with suitable mechanical stability for implantation, wherein the chondrocytes are particularly derived from auricular chondrocytes, more particularly human auricular chondrocytes, more particularly human autologous auricular chondrocytes.

9. The method of claim 8, 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.

10. The method of claim 8 or 9, wherein step e. is performed in vitro, particularly for at least 8 weeks, more particularly for 10 to 24 weeks, more particularly for 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly for 16 weeks.

11.A three-dimensional composition, comprising at least about 6 x 106 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 107 chondrocytes from harvested culture;
b. mixing the expanded chondrocytes with a homogeneous biopolymer formulation comprising gellan gum and alginate, 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 with suitable mechanical stability for implantation.

12.A cell composition comprising at least about 6 x 106 chondrocytes per mL of composition, which is provided within a homogeneous biopolymer formulation and has undergone a maturation period of 10 to 24 weeks, particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks, for use in medicine, particularly for use in a method of treating anotia or microtia or facial injuries with persistent damage to ears and/or nose.

13.A cell composition comprising at least about 6 x 106 chondrocytes per mL of composition, which is provided within a homogeneous biopolymer formulation and has an elastic modulus (E) of at least 180 kPa, for use in medicine, particularly for use in a method of treating anotia or microtia or facial injuries with persistent damage to ears and/or nose.

14. The composition for use according to any one of claims 11 to 13, wherein the maturation period is an in vitro maturation period.

15. An implant for use in the improvement of hearing, comprising at least about 6 x 107 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.