CN115916276A

CN115916276A - Cured three-dimensional printing composition and use thereof

Info

Publication number: CN115916276A
Application number: CN202180043391.3A
Authority: CN
Inventors: M·J·J·凯斯提; K·S·U·库伯
Original assignee: Reagan Biotherapy Corp
Current assignee: Reagan Biotherapy Corp
Priority date: 2020-06-17
Filing date: 2021-06-16
Publication date: 2023-04-04
Also published as: CA3185014A1; EP4168058A1; KR20230025788A; JP2023529561A; AU2021290995A1; IL299029A; MX2022015741A; BR112022021958A2; WO2021255123A1

Abstract

The present invention relates generally to the field of tissue engineering, and in particular to tissue engineering in the context of cartilage tissue. More specifically, the present invention relates to a three-dimensional structure comprising a sufficient number of chondrocytes and a cross-linked biopolymer preparation, wherein the three-dimensional composition has mechanical stability suitable for implantation into a subject in need thereof. In a further aspect, the invention relates to a method for preparing such a three-dimensional composition, for example by bioprinting, a composition obtainable by such a method, and medical uses of the three-dimensional composition.

Description

Cured three-dimensional printing composition and use thereof

Description of the invention

Technical Field

The present invention relates generally to the field of tissue engineering, and in particular to tissue engineering in the context of cartilage tissue. More specifically, the present invention relates to a three-dimensional structure comprising a sufficient number of chondrocytes and a cross-linked biopolymer preparation, wherein the three-dimensional composition has mechanical stability suitable for implantation into a subject in need thereof. In a further aspect, the invention relates to a process for preparing such a three-dimensional composition, for example by bioprinting, to a composition obtainable by such a process, and to the medical use of the three-dimensional composition.

Background

For whatever reason, impaired or complete loss of facial aesthetic features (e.g., nose and ears) can subject affected persons to psychosocial problems throughout their life. For example, in most burns involving the head and neck of an injured person, the ears and nose are affected. If affected, the outer ear is usually completely destroyed, requiring a total ear reconstruction by surgery, although this is a very challenging task.

Accidents and dog bites are another common cause of the need for reconstructive surgery, particularly when certain parts of the face (e.g., the nose and/or ears) are affected. Facial penetrating injuries can lead to loss of function with malformations of soft tissue shape and contours, requiring multiple surgical interventions and concomitant psychological trauma (Alizadeh et al, plant Reconsr Surg Glob open.2017, 10 months; 5 (10): e 1431). Each year, approximately 30,000 patients in the united states alone require reconstructive surgery due to bites by dogs.

Another cause of impaired or lost facial aesthetic features is disease, such as cancer. A rare congenital disease called anhedonia or microcephaly affects 1 of 3,800 newborns and is characterized by complete loss (anhedonia) or distortion of the outer ear (microcephaly). The current standard of care for young patients with small ear deformities is autologous costal cartilage grafts, which are based on the harvesting of several ribs to construct an ear template. Reconstructing the outer ear requires multiple procedures and the steep learning curve of the surgeon limits the number of surgical specialists that can perform these procedures around the world. Patients often experience pain in the donor area due to rib acquisition and often report poor results due to poor aesthetic results of reconstruction.

Some synthetic implants may be used, for example, under the trade name

The implants of (1), however, are prone to foreign body reactions and have a high incidence of complications because they are made of polyethylene. Furthermore, due to the rigidity of such implants, it is difficult for the patient to fall asleep.

Prosthetic reconstruction is a possibility particularly suitable for elderly patients, since the surgery is minimally invasive, but requires constant management and is not ideal from a psychological point of view, since the prosthesis is not the patient's own organ.

Additive manufacturing or "three-dimensional printing" is a multifunctional technology that greatly facilitates the industrial production of complex shapes while also achieving very small production sizes at a reasonable price. This technique is based on computer controlled assembly of liquid materials, usually in a layered fashion, followed by curing to produce a three-dimensional object. In the medical field, additive manufacturing based bio-fabrication techniques have great potential as they can produce living, patient-specific tissues and organs for regenerative medicine. Of particular interest is the combination of cells and support structures.

As with non-medical additive manufacturing, bio-fabrication techniques, including three-dimensional bio-printing, are based on layer-by-layer assembly, in which case living cells and biological material fabricate three-dimensional (3D) biological structures. These structures can be designed according to clinical 3D models of individual patients to produce personalized tissue grafts. External ear or nasal reconstruction is a clinical application that can be significantly improved by bioprinted personalized implants. Such grafts or implants may even be produced from autologous cells.

However, large structures based on hydrogels are often of limited mechanical strength, making them unsuitable for handling during implantation, as they cannot withstand the mechanical stresses during surgery.

Kesti (2018.

Cohen et al (2018, PLoS ONE 13 (10): e 0202356) describe a study investigating full-size, patient-based human ears produced by implantation of human auricular chondrocytes and human mesenchymal stem cells at a ratio of 1. The implant is made by molding. Considering that a full-size pediatric ear requires more than 2 million cells and is about 10 milliliters in volume, the authors indicate that non-deformed biopsies do not provide enough cells to fill a pediatric-sized ear. In addition, auricular chondrocytes proliferate in vitro, but can dedifferentiate in monolayer culture; therefore, the binding of auricular chondrocytes to mesenchymal stem cells is proposed. Another problem observed in this study was that during subcutaneous implantation in experimental animals, the ear structure significantly contracted.

Zhang et al (Biomaterials (2014): volume 35, pages 4878-4887) describe the regeneration of human auricular cartilage by coculturing human auricular 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 (pel lets) formed by passage 3 (P3) auricular malformed chondrocytes showed a looser tissue structure, and expression of glycosaminoglycans (GAG) and type II collagen was weak, indicating a significant decrease in chondrogenic capacity.

WO 2016/092106 A1 relates to a method for providing a graft scaffold for cartilage repair, in particular in human patients. The method comprises providing an aqueous solution of particles and/or fibres, a gel-forming polysaccharide and mammalian cells; mixing the particles/fibrous polysaccharide and the cells to obtain a printing mixture; and depositing the printing mixture in three dimensions.

Kesti et al (adv. Funct. Mater.2015; DOI 10.1002/adfm.201503423) propose a bioprinting method that meets clinical criteria and produces patient-specific cartilage grafts with good mechanical and biological properties. They found that printed constructs cultured in vitro for up to 8 weeks had inferior mechanical properties compared to native cartilage.

Kesti et al (BioNanoMat 2016.

In summary, to function in reconstructive surgery, a tissue engineering implant must have suitable mechanical properties to withstand the stresses associated with the surgery, in addition to being compatible with the object from which the implant is made.

Summary of The Invention

The inventors have surprisingly found that by a long in vitro maturation period of at least 8 weeks, in particular 16 weeks, the three-dimensional composition comprising chondrocytes and biopolymer preparation has a high mechanical strength and a high cell viability, i.e. the activity may reach at least 70%, in particular at least 80%, at least 85%, at least 90% or at least 95%. This unexpected combination of advantageous features makes the compositions of the present invention particularly suitable for implantation into a subject in need thereof, such as a human patient.

Thus, provided herein is a three-dimensional composition having mechanical stability suitable for implantation into a subject in need thereof, comprising chondrocytes and a cross-linked biopolymer formulation. Also provided is a method of making such a three-dimensional composition, as well as the medical use of the composition and associated implants comprising chondrocytes.

Accordingly, the present invention is directed to a three-dimensional composition comprising at least about 6 x 10 per milliliter of the composition ⁶ A chondrocyte and a cross-linked biopolymer formulation, wherein the three-dimensional composition has mechanical stability suitable for implantation into a subject in need thereof.

Mechanical stability can be quantified by measuring the modulus of elasticity (E). Thus, the composition especially has an elastic modulus (E) of at least 180kPa, at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa or at least 260kPa.

Chondrocytes may be derived from a variety of sources, particularly from auricular chondrocytes, especially 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. Furthermore, the composition is in particular free of at least one of added tissue particles, added fibers, microbeads and nanoparticles, more in particular free of all of these components.

According to the present invention, a wide 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 the biopolymer formulation, and/or wherein the alginate content may be from about 1% (w/v) to about 3% (w/v), particularly from about 1.0% (w/v) to about 2.0% (w/v), more particularly about 1.5% (w/v), based on the total volume of the biopolymer formulation.

In certain exemplary embodiments, the biopolymer formulation is CaCl ₂ Crosslinked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation based on the total volume of the biopolymer formulation.

The shape of the three-dimensional composition can be freely chosen according to the specific needs and the manufacturing possibilities. For example, the composition may be a wedge, a tissue engineered human nose or human pinna or a portion thereof.

The invention also relates to a process for preparing a three-dimensional composition according to any one of the preceding claims, comprising the following steps:

a. expanding the isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, to obtain at least about 6 x 10 from the harvested culture ⁷ Individual chondrocytes;

b. mixing the expanded chondrocytes with a biopolymer preparation, thereby obtaining a bio-ink;

c. depositing the bio-ink layer-wise onto a surface, thereby obtaining a three-dimensional composition;

d. crosslinking the biopolymer formulation within the three-dimensional composition;

e. maturing the three-dimensional composition, thereby allowing the chondrocytes to produce extracellular matrix to form a three-dimensional composition having mechanical stability suitable for implantation,

wherein the chondrocytes are derived in particular from auricular chondrocytes, more particularly from human autologous auricular chondrocytes.

According to a particular embodiment of the method, the biopolymer preparation may be a gellan gum/alginate preparation, in particular a gellan gum/1.5% (w/v) alginate preparation based on the total volume of the biopolymer preparation.

Step e. Of the method according to the invention is in particular performed in vitro, in particular for at least 8 weeks, more in particular for 10, 12, 13, 14, 15, 16 or 17 weeks, more in particular for 16 weeks. The invention also includes that step e can be performed in vivo.

The invention further relates to a three-dimensional composition obtainable by a specific process. Accordingly, the present invention relates to three-dimensional compositions comprising at least about 6 x 10 per milliliter of the composition ⁶ A chondrocyte and cross-linked biopolymer preparation obtainable by a process comprising the steps of:

e. the three-dimensional composition is matured for at least 8 weeks, thereby allowing the chondrocytes to produce extracellular matrix to form a three-dimensional composition having mechanical stability suitable for implantation.

The invention also relates to the medical use of the three-dimensional composition described. According to some embodiments, there is provided a cell composition for use in medicine, comprising at least about 6 x 10 cells per milliliter of the composition ⁶ A chondrocyte, and which is provided within a biopolymer preparation 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 mentioned above, the maturation is in particular carried out in vitro.

Also provided is a cell composition for use in medicine comprising at least about 6 x 10 cells per milliliter of the composition ⁶ And which is provided in a biopolymer formulation and has an elastic modulus (E) of at least 180kPa.

A particular medical use according to the invention is the treatment of otoceless or microcephaly or facial lesions with persistent damage to the ears and/or nose.

Finally, the invention relates to an implant for improving hearing, comprising at least about 6 x 10 ⁷ Chondrocytes provided in a biopolymer formulation and subjected to 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

In a first aspect, the present invention is directed to providing a three-dimensional composition comprising at least about 6 x 10 per milliliter of the composition ⁶ A chondrocyte and a cross-linked biopolymer formulation, wherein the three-dimensional composition has mechanical stability suitable for implantation into a subject in need thereof. In exemplary embodiments, the composition comprises 6 to 9 million cells per milliliter of composition, for example 6, about 7, about 8, or about 9 million cells per milliliter of composition. In other exemplary embodiments, the composition comprises 1200 ten thousand cells per milliliter of the composition or up to 1500 ten thousand cells per milliliter of the composition.

Three-dimensional compositions can be prepared using layer-by-layer deposition methods such as bioprinting. Thus, the composition may have a variety of shapes as desired, particularly a coupon, a wedge, an entire pinna or nose, particularly a pinna or nose, or a portion thereof, of a human. As used herein, a "specimen" is a three-dimensional basic geometry. For example, the sample may have a spherical shape, a lenticular shape, a cylindrical shape, a disc shape, a cube, a rectangular parallelepiped, or a conical shape. As used herein, a "wedge" is a three-dimensional form that is substantially similar in form and size to an anatomical structure, such as a human nose or a portion of a human pinna or even an entire human nose or an entire human pinna. For example, the wedge may have the form of a capital letter "D", wherein, for example, the straight portion on the left is thinner than the curved portion on the right.

Parts of an auricle or pinnae, in particular of a human, may be the helix (helix), the antihelix (anti-helix), the concha (concha), the tragus (tragus), the antitragus or essential fragments thereof. A part of the nose, in particular a human nose, may be the nasal septum, the alar nasal septum or substantial fragments thereof. As used herein, an "essential segment" of a pinna or nose portion, particularly a human pinna portion or a human nose portion, corresponds to at least about one third of a complete pinna portion or nose portion, i.e., about 33% by volume. The present invention contemplates combining several pinna or nose portions or substantial segments thereof. For example, the helix or antihelix may be combined with fragments corresponding to half of the concha, which may be suitable for reconstructive surgery.

In exemplary embodiments, the three-dimensional composition may comprise a total of about 2 x 10 ⁷ Cartilage cell, 4X 10 ⁷ Cartilage cell or 6X 10 ⁷ And (4) chondrocytes.

The parameter that allows determining whether the mechanical stability of the three-dimensional compositions described herein is suitable for use according to the invention is the elastic modulus (E). The modulus of elasticity (E) measures the resistance of a material to elastic deformation. Thus, in some embodiments of the invention, the three-dimensional composition has an elastic modulus (E) of at least 180kPa. In further particular embodiments, the elastic modulus is at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa, or at least 260kPa. Thus, in some embodiments of the invention, the elastic modulus may be in a range of between about 180kPa to about 260kPa, such as 180kPa to 260kPa or 200kPa to 260kPa.

One established method of determining the modulus of elasticity is by the unconfined indentation test. There, stress or strain is applied to the surface of the three-dimensional composition by a small indenter and the resulting response is monitored over time. Stress relaxation was observed. From the data obtained, the modulus of elasticity (E) can be determined (see the examples, "methods" section). Thus, in some embodiments of the invention, the elastic modulus of the three-dimensional composition is determined by the unconfined indentation test. Another possibility to determine the mechanical stability of the three-dimensional composition according to the invention is the histological and/or immunohistochemical analysis of the composition samples, which can be used in addition to or as an alternative to the elastic modulus determination. Such assays include differential staining of ECM components well known to those skilled in the art. The sample is qualitatively analyzed, for example, if a particular combination of extracellular matrix proteins is observed, it is classified as mechanically stable. As a non-limiting example, a sample in which type II collagen, glycosaminoglycan (GAG), and elastin stain positive in combination, optionally further combined with a SOX9 positive stain, may be classified as having sufficient mechanical stability.

According to the present invention, chondrocytes from a variety of sources may be used. Generally, chondrocytes are derived from cells of mammalian origin, in particular of human origin. For tissue engineering, it is often advantageous to use allogeneic cells, i.e. cells from another donor, or autologous cells, i.e. cells from the individual patient himself. Chondrocytes can be derived from a variety of source tissues, such as articular cartilage, nasal cartilage, or ear cartilage. In particular embodiments, the chondrocytes of the three-dimensional composition are derived from auricular chondrocytes, in particular human nasal or auricular chondrocytes. According to certain preferred embodiments, the chondrocytes are derived from human autologous auricular chondrocytes.

One advantageous method of obtaining chondrocytes is by cell expansion and maturation from isolated primary chondrocytes, particularly isolated human primary chondrocytes.

According to the invention, the chondrocytes are distributed in a biopolymer preparation. This can be accomplished, for example, by mixing the cell population with a biopolymer prior to assembly of the three-dimensional composition, such as by bioprinting as described herein.

As already mentioned, it has surprisingly been found that the high mechanical strength of the three-dimensional composition can be combined with a high cell viability according to the invention. In particular, the chondrocytes of the three-dimensional composition according to the invention have a cell viability of at least 70%. More specifically, the chondrocytes have a cell viability of at least 80%, or at least 85%. In certain preferred embodiments, the chondrocytes have a cell viability of at least 90%, or even at least 95%.

A variety of established methods for determining cell viability are known to the person skilled in the art, for example methods based on automated cell sorting (in particular flow cytometry) or blood cell counting. In a particular embodiment of the invention, the cell viability of the chondrocytes is determined by cytometry, based on the monograph of the european pharmacopoeia on the count and viability of nucleated cells (ph. Eur.2.7.29.). Accordingly, cell viability can be determined by trypan blue (trypan blue) staining and microscopy using a hemocytometer with manual or automated counting.

The inventors have also surprisingly found that the three-dimensional composition according to the invention can be successfully used even if it is substantially free of stem cells, such as Mesenchymal Stem Cells (MSC) and bone marrow derived stem cells (BMSC). Also, even compositions according to the invention that are substantially free of progenitor cells, such as chondrogenic progenitor cells, may be used successfully.

As used herein, "substantially free" means that less than about 2%, particularly less than 1%, less than 0.5%, or even less than 0.1% of the cells are stem or progenitor cells, based on the total number of cells within the three-dimensional composition.

Still further, it is not necessary 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 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.

As used herein, "tissue particles" refers to minced tissue, particularly cartilage tissue. Examples of such tissues 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 tissue and subcutaneous fat, intra-patellar fat pad and small intestine submucosa. In addition to the morcellation, the tissue particles may be further processed.

As used herein, "fiber" may be a synthetic fiber such as Polymethylmethacrylate (PMMA) or a natural fiber such as elastin, resilin, silk and derivatives thereof.

"Microbeads" are generally synthetic polymer particles having a diameter of about 0.1 μm to about 5 mm.

As used herein, "nanoparticle" refers to a broad class of materials, including particulate matter, having a structural dimension of less than 100 nm.

"free" as used in the context of added tissue particles, added fibers, microbeads, and nanoparticles means that the composition contains less than about 0.5%, specifically less than 0.25%, more specifically less than 0.1% or even 0.0% of a particular compound as a percentage of the total weight of the composition.

Also, according to the present invention, it is not necessary to use a poorly soluble calcium or strontium compound (solubility in water at 20 ℃ C.)<1g/100 mL) such as calcium carbonate, calcium phosphate or hydroxyapatite is added to the three-dimensional work. Thus, in certain embodiments, there is no calcium carbonate (CaCO) ₃ ) Calcium phosphate (Ca) ₂ (PO ₄ ) ₃ ) And hydroxyapatite were added externally to the composition, respectively.

In order to determine the pattern of genes expressed at the transcriptional level, under specific circumstances or in specific cells, to give a global picture of the cell function, gene expression profiling can be used. In the context of the present invention, according to certain embodiments, the cells of the claimed compositions can be analyzed with respect to the expression of a selected housekeeping gene or genes, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and selected marker genes associated with chondrocyte differentiation, ECM production and/or inflammation. For example, the gene expression of the selected marker genes type I collagen, type II collagen, aggrecan, and interleukin-1 β (IL-1 β) can be determined. In particular, gene expression is characterized by a relative gene expression profile, i.e., the relative expression between the housekeeping gene and the selected marker gene. According to certain embodiments, the relative gene expression profile, e.g. I/O of a suspension cell sample from a composition according to the invention, is determined by quantitative polymerase chain reaction (qPCR).

When autologous chondrocytes are used, there will of course be donor to donor differences. In any case, the gene expression profile, in particular the expression profile of the selected marker and housekeeping gene, will specifically match the expression profile of the initial autologous biopsy.

For example, the so-called Ct value or "threshold cycle", i.e., the number of cycles at which the amount of amplification product is sufficient to produce a detectable fluorescent signal for GAPDH as the reference gene, is set to about 12 to about 18, especially 12 to 18. An exemplary range is 12.54-17.57. Since the Ct value is measured in the exponential phase without reagent restriction, real-time quantitative PCR can be used to reliably and accurately calculate the initial amount of template present in the reaction.

Exemplary selected marker genes type I collagen, type II collagen, aggrecan, and interleukin-1 beta (IL-1 beta) can then be identified as 2 ^-ΔCt The value is obtained. For example, type II collagen/type I collagen ratio of 2 ^-ΔCt The value may be equal to or greater than 1 · 10 ^-4 Type II collagen 2 ^-ΔCt The value may be equal to or greater than 1 · 10 ^-2 Aggrecan 2 ^-ΔCt The value may be equal to or greater than 3.5 · 10 ^-2 2 of IL-1. Beta ^-ΔCt The value may be less than 5.10 ^-6 。

A variety of biopolymers for use in the present invention are 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 the material and the host, e.g. histocompatibility and hemocompatibility), non-toxic to organisms, in particular to mammals, degradable in vivo, in particular enzymatically degradable, and can provide a degree of mechanical stability to the structure, e.g. in its cross-linked form. For tissue engineering applications, for example, agarose, alginate, cellulose, collagen, fibrin, gelatin, hyaluronan, dextran, and gellan gum have been investigated. Also included are sulfated forms of such biopolymers.

In the context of the present invention, the biopolymer preparation comprises one or more biopolymers suitable for tissue engineering. In particular, the biopolymer formulation comprises gellan gum and alginate. More specifically, the biopolymer preparation consists of gellan gum and alginate, i.e. the high molecular weight compounds gellan gum and alginate are the only structural components of the biopolymer preparation, but small molecules (MW ≦ 1000 Da) and compounds with molecular weights above 1000Da are not structural components, e.g. growth factors, and may optionally be present. A growth factor which may advantageously be present is TGF-. Beta.3, for example at a concentration of 10ng/ml.

As used herein, a "structural component" is a compound that is required, either by itself or after crosslinking, to determine and maintain the form of the three-dimensional composition of the invention.

In a particular embodiment of the invention, the entire amount of biopolymer preparation used in the three-dimensional composition is mixed with the cells, i.e. the three-dimensional composition comprises a homogeneous biopolymer preparation. Thus, in these particular embodiments, the three-dimensional composition comprises only a single, cell-laden biopolymer preparation (also referred to as a (cellular) "bio-ink"), i.e., the composition is not enhanced by cell-free extra portions (e.g., layers) of biopolymer.

In particular, the biopolymer formulation used in the three-dimensional composition of the invention may be a cross-linked gellan gum/alginate formulation. Crosslinking can be carried out in different ways, broadly divided into physical crosslinking (e.g. stereocomplex and thermal crosslinking) and chemical crosslinking (e.g. by free radical initiators, cations or enzymes). According to some embodiments, the biopolymer formulation is a chemically cross-linked formulation, in particular a chemically cross-linked gellan gum/alginate formulation, more particularly an ionically cross-linked gellan gum/alginate formulation. The term "gellan/alginate formulation" as used herein means that the formulation does not comprise other compounds, such as other biopolymers, that serve as structural components of the composition.

An exemplary method of crosslinking according to the present invention is to use multivalent ions, in particular alkaline earth metal ions. The biopolymer preparation of the three-dimensional composition of the invention is specifically crosslinked with calcium ions or strontium ions. Specifically, the multivalent ion is provided by a cation source with good water solubility (solubility in water at 20 ℃ is more than or equal to 25g/100 mL); for example, the multivalent ion may be provided by strontium chloride or calcium chloride. In certain embodiments, calcium chloride is used, particularly at a concentration of about 40mM to about 120mM, e.g., about 50mM.

When gellan gum is used as a biopolymer according to the present invention, its amount in the biopolymer formulation may vary significantly. Specifically, the gellan gum content can range from about 2% (w/v) to about 5% (w/v), more specifically from about 2.0% (w/v) to about 3.0% (w/v), more specifically about 1.5% (w/v), based on the total volume of the biopolymer formulation. Gellan gum may be prepared for use, for example, by dissolving it in an appropriate amount of aqueous glucose solution (e.g., about 300 mM), which may be buffered. Likewise, when alginate is used as the biopolymer according to the present invention, its amount in the biopolymer formulation may range from about 1% (w/v) to about 3% (w/v), particularly about 1.0% (w/v)) to about 2.0% (w/v), more particularly about 1.5% (w/v), based on the total volume of the biopolymer formulation. Alginates may be prepared for use, for example, by dissolving them in a suitable amount of aqueous dextrose solution (e.g., about 300 mM), which may be buffered.

Thus, 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), specifically from about 2.0% (w/v) to about 3.0% (w/v), more specifically about 2.5% (w/v), based on the total volume of the 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), specifically from about 1.0% (w/v) to about 2.0% (w/v), more specifically about 1.5% (w/v), based on the total volume of the biopolymer formulation. The biopolymer formulation according to the invention does not contain more than 3.5% (w/v) alginate.

In certain preferred embodiments of the invention, the biopolymer preparation may be CaCl ₂ Crosslinked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation based on the total volume of the biopolymer formulation.

Exemplary embodiments of the three-dimensional compositions comprise at least 6 x 10 ⁶ Chondrocytes derived from human auricular chondrocytes and having a cell viability of at least 95%, and a cross-linked 2.5% (w/v) gellan gum/1.5% (w/v) alginate preparation, based on the total volume of the biopolymer preparation, wherein the composition is substantially free of stem 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 250kPa.

As mentioned above, the composition according to the invention may be a wedge, a tissue engineered human pinna or a part thereof. Such a composition is suitable for positioning on the skull outside the ear canal of a patient. In particular, the three-dimensional compositions of the present invention do not exhibit significant shrinkage after 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 preparing a three-dimensional composition as described herein. The method comprises at least the following five mandatory steps.

In a first step (step a.), the isolated chondrocytes are expanded in vitro. By this amplification, at least about 6X 10 is obtained from the harvested culture ⁷ And (4) chondrocytes. In some embodiments, amplification may optionally be combined with a cryopreservation step.

According to some embodiments, step a. May comprise three substeps, namely substep 1) -cell expansion of the isolated primary chondrocytes until the end of passage 1 (P1); substep 2) cryopreservation of chondrocytes after P1; and substep 3) thawing and cell expansion until the end of passage 2 (P2).

According to still further embodiments, step a. May further comprise the sub-step 4) of cell expansion until the end of passage 3 (P3).

For example, isolated chondrocytes (passage 0 cells) are seeded in a suitable amount, e.g. about 10 ⁵ The individual cells are cultured in medium, such as supplemented DMEM, to the end of passage 1. The cells can then be collected, evenly divided into aliquots and cryopreserved. Subsequently, when necessary, aliquots of the desired number of cells are thawed and cultured until final harvest, before proceeding to the next step (step b.).

In step b, the expanded chondrocytes are mixed with a biopolymer preparation. Biopolymers which can be used in the formulation are those described above. In particular, the biopolymer formulation is a gellan gum/alginate formulation. According to a specific embodiment, the biopolymer formulation is 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, wherein the percentages of gellan gum and alginate, respectively, are based on the total volume of the biopolymer formulation. As a result of this mixing step, a bio-ink (i.e. cell + biopolymer formulation) is obtained. The bio-ink is suitable for bio-printing and the like. In particular, the expanded chondrocytes may be mixed with the entire amount of biopolymer preparation used in the three-dimensional composition, resulting in a homogeneous biopolymer preparation in which the cells are evenly distributed. Thus, in these examples, a single biopolymer formulation is applied throughout the three-dimensional composition.

After step b, the bio-ink is layered onto the surface (step c). By such deposition, which may be performed by a layer-by-layer deposition method such as bioprinting, a three-dimensional composition is obtained. The composition may have any shape described herein, for example the shape of a human nose, a human pinna, or a portion thereof.

According to a particular embodiment, no chemically or physiologically different layer, such as a cell-free layer versus a cell layer or a layer of different chemical composition (e.g. different chemical composition or different concentration of the same composition), is used for the deposition step c. Thereby, a three-dimensional composition consisting only of a layer of the cell-loaded biopolymer preparation is obtained.

Subsequently, the biopolymer preparation in the three-dimensional composition is crosslinked (step d.). Step d. May include any physical or chemical crosslinking as described above. According to a particular embodiment, step d. In order to provide multivalent ions, alkaline earth metal salts may be used in particular. Thus, step d. Of the process comprises in particular cross-linking with an alkaline earth metal salt, more in particular with a calcium or strontium salt which is well water-soluble, more in particular with calcium chloride or strontium chloride. In certain preferred embodiments, the crosslinking step d.is performed using calcium chloride, e.g., at a concentration of about 40mM to about 120mM, e.g., about 50mM. In an exemplary embodiment, crosslinking is performed by dipping the printed composition into a solution containing CaCl ₂ And at a temperature of, for example, 4 deg.C, or CaCl is added to the printing container ₂ 。

In the final mandatory step of the process (step e.), the three-dimensional composition is subjected to curing. Maturation allows chondrocytes to produce extracellular matrix (ECM) to form a three-dimensional composition with suitable mechanical stability. The formation of sufficient ECM is critical to achieving suitable mechanical stability. Important ECM components are, for example, type I and II collagen. In particular, the composition should have mechanical stability such that it can withstand mechanical stress during implantation, for example, in a human patient. The biopolymer formulation is very stable over the maturation time, so that the concentration of the biopolymer does not change significantly. As mentioned above, the modulus of elasticity (E) can be used as a parameter for suitable mechanical stability. Thus, in some embodiments of the invention, at the end of step E. In further particular embodiments, the elastic modulus is at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa, or at least 260kPa. Thus, in some embodiments of the present invention, the elastic modulus may be in a range between about 180kPa to about 260kPa.

In particular, the maturation step e. In vitro maturation is for example performed in culture flasks under suitable conditions (see below). The inventors have surprisingly found that prolonged in vitro maturation shows a positive influence on both the cell viability and the development of the mechanical stability of the resulting three-dimensional composition. Thus, maturation, in particular in vitro maturation, is performed for at least 8 weeks. More specifically, in vitro maturation may be performed for 10 to 24 weeks, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks. More specifically, in vitro maturation may be performed for at least 10, 12, 13, 14, 15, 16, or 17 weeks. In other embodiments, in vitro maturation may be performed for 18, 19, 20, 21, 22, 23, or 24 weeks. This longer maturation time may be required in some patients where the surgical schedule changes during maturation. In certain preferred embodiments, the three-dimensional composition undergoes in vitro maturation for 16 weeks.

When maturation is performed in vitro, so-called 3D media may be used. In particular, such 3D medium may be based on standard Dulbecco's Modified Eagle Medium (DMEM) and HAM F12 to which appropriate concentrations of TGF- β 3, insulin, transferrin, selenium and ascorbic acid are added. The cell composition plus biopolymer as described herein is specifically matured at a temperature between about 36 ℃ and about 38 ℃, specifically about 37 ℃. Atmospheric conditions are in particular normoxic (e.g. 21% ₂ ) Or low oxygen content (e.g. about 5-15% ₂ )。

In other embodiments of the invention, the curing step e. For example, in vivo maturation may be performed if the surgeon wants to perform a library of biological samples in some cases prior to implantation at the reconstruction site. As with in vitro maturation, in vivo maturation is carried out for at least 8 weeks, in particular 10 to 24 weeks, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks. More specifically, in vivo maturation may be performed for at least 10, 12, 13, 14, 15, 16, or 17 weeks. In other embodiments, in vivo maturation may be performed for 18, 19, 20, 21, 22, 23, or 24 weeks. In certain preferred embodiments, the three-dimensional composition undergoes in vivo maturation for 16 weeks.

The chondrocytes used in the preparation method may be derived from a variety of sources as described above. For example, the chondrocytes may be derived from articular cartilage, nasal cartilage or otic cartilage. In a particular embodiment, the chondrocytes used are derived from nasal or auricular chondrocytes, in particular 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 further steps than the above-described steps, i.e. the method consists of the steps as described above:

a. expanding the isolated chondrocytes in vitro, optionally in combination with a cryopreservation step, to obtain at least about 6 x 10 from the harvested culture ⁷ A plurality of chondrocytes;

c. depositing the bio-ink layer by layer onto a surface to obtain a three-dimensional composition;

e. the three-dimensional composition is matured, thereby causing the chondrocytes to produce extracellular matrix to form a three-dimensional composition having mechanical stability suitable for implantation.

According to another aspect, the present invention relates to a three-dimensional composition comprising at least about 6 x 10 per milliliter of the composition ⁶ A chondrocyte and cross-linked biopolymer preparation obtainable by a process comprising the steps of:

e. the composition is allowed to mature for at least 8 weeks, thereby allowing the chondrocytes to produce extracellular matrix to form a three-dimensional composition.

By performing these steps, a three-dimensional composition with advantageous mechanical stability is obtained. In particular, the three-dimensional composition thus obtained has an elastic modulus (E) of at least 180kPa. In further particular embodiments, the elastic modulus is at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa, or at least 260kPa. Thus, in some embodiments of the invention, the modulus of elasticity may be in a range between about 180kPa and about 260kPa. According to some embodiments, the method consists of steps a.

In another aspect, the invention relates to the medical use of chondrocytes in biopolymer formulations, for example in the field of reconstructive surgery. According to some embodiments, there is provided a composition comprising at least about 6 x 10 per milliliter of the composition ⁶ A cellular composition of individual chondrocytes for use in medicine, wherein the cellular composition is within a biopolymer formulation, particularly a homogeneous biopolymer formulation. The composition has been aged for at least 8 weeks, which is particularly done in vitro. More specifically, the maturation period lasts 10 to 24 weeks, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks. More specifically, the maturation period, in particular the in vitro maturation period, has lasted 10, 12, 13, 14, 15, 16 or 17 weeks. In other embodiments, the maturation period, particularly the 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 in the biopolymer preparation has been matured, in particular in vitro, for 16 weeks. Maturation conditions, in particular in vitro maturation, in particular the above conditions, i.e. 3D medium, about 36 ℃ to about 38 ℃ and normoxic or hypoxic atmosphere.

In some embodiments, the biopolymer preparation and the chondrocytes are arranged into a three-dimensional structure, particularly in the form of a wedge, a human pinna, or a portion thereof.

A particular medical indication for which the cell composition in the cured biopolymer formulation may be used is the treatment of aneural or small ear deformities or facial damage accompanied by a lasting damage to the ears and/or nose, such as caused by burns or dog bites. Accordingly, some embodiments of the present invention relate to a three-dimensional composition as described above for use in the treatment of an aneural or lesser ear deformity or a facial injury with persistent damage to the ears and/or nose.

According to some embodiments, the cell composition for use in medicine comprises at least about 6 x 10 cells per milliliter of the composition ⁶ Chondrocytes and is provided within a biopolymer formulation, particularly a homogeneous biopolymer formulation, and has an elastic modulus (E) of at least 180kPa. According to other embodiments, the elastic modulus (E) is at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa, or at least 260kPa. Thus, in some embodiments of the present invention, the elastic modulus may be in a range between about 180kPa to about 260kPa. In some embodiments, E is at least 250kPa. The determination of the elastic modulus can be performed as described above, for example by an unlimited indentation test. Also, an exemplary medical use is the treatment of an ear-free or small ear deformity or facial damage accompanied by a lasting damage to the ear and/or nose.

Chondrocytes in a cell composition for use in medicine may be derived from a variety of sources as described above. According to some embodiments, the chondrocytes present in the cell composition for use in medicine are derived from human auricular chondrocytes, in particular human autologous auricular chondrocytes.

According to another aspect, the present invention relates to an implant for improving hearing. It is well known that the main function of the pinna is to collect, amplify and direct sound waves to the external auditory canal. Therefore, reconstructive surgery that repairs damaged, deformed, or missing pinnas can significantly and reproducibly improve the hearing of the patient.

Such implants according to the invention comprise at least about 6 x 10 ⁷ A chondrocyte, provided in a biopolymer formulation. Exemplary implants may have a volume of about 8ml to about 10ml and a size of about 5-5.5cm by about 3-3.5cm by about 0.8-1.3cm (full size adult ear). Advantageously, the implant has a suitable three-dimensional structure, in particular in the form of a mammal, in particular a human nose or a human pinna or a part thereof. For example, such an implant may be one of the noseOr several parts, in particular a human nose, such as the nasal septum, the alar part of the nose or a substantial segment thereof as defined herein. In particular, such an implant may be a pinna or one or more parts of a pinna, in particular a human pinna or a part of a pinna, such as the helix, the antihelix, the concha, the tragus, the antitragus or substantially a fragment thereof as defined herein. Depending on the desired form of the implant, several parts of the pinna or nose or most fragments of these parts may be combined in one implant or provided as separate implants.

In order to be sufficiently stable against mechanical stress during implantation, the implant undergoes a maturation period of at least 8 weeks, in particular 10, 12, 13, 14, 15, 16 or 17 weeks, more in particular 16 weeks. Maturation is especially carried out in vitro.

According to some embodiments, the implant comprises at least about 6 x 10 in the biopolymer formulation ⁷ Individual chondrocytes and having an elastic modulus (E) of at least 180kPa. According to other embodiments, the elastic modulus (E) is at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa, or at least 260kPa. In some embodiments, E is at least 250kPa. The determination of the elastic modulus can be performed as described above, for example by the unlimited indentation test.

In another aspect, the present invention relates to a method of treating an auricless or a microclaris or a facial injury with sustained damage to the ear and/or nose comprising the step of implanting into a subject in need thereof a three-dimensional composition as described above.

The invention is further illustrated by the following examples and figures.

Drawings

FIG. 1: production flow chart

FIG. 2 is a schematic diagram: cell viability determined by hemocytometer at different time points for the two production batches was summarized. Viability testing was performed from day 1 to week 17 after bioprinting from test specimens. Error bars indicate standard deviation. The sample size is one without a bias being given.

FIG. 3: the indentation results for sections of samples of different maturation durations in both production batches contained both cell (solid line) and cell-free (dashed line) samples.

FIG. 4 is a schematic view of: two cell production batches were subjected to histological analysis at 4 different time points. Natural ear cartilage was stained as a control.

FIG. 5 is a schematic view of: high magnification (10-fold) of cell samples at all maturation time points and native ear cartilage controls. All selections showed the highest staining intensity of the cross-section. Scale bar: 600 μm.

FIG. 6: histology of 2 production batches of cell-free specimens from 4 different time points.

FIG. 7 is a schematic view of: gene expression of 9 different genes in two production batches at different time points.

FIG. 8: representative images of cells and cell-free samples and average weights of cell samples at different maturation stages.

FIG. 9: mechanical properties of AUR-V047 during prolonged maturation and respective histological results of collagen type I and collagen type II staining.

Detailed Description

Method

Unlimited indentation test

Mechanical evaluation of indentation was performed using a universal testing machine (Zwick Z0.5; zwick/Roell) using the software testXpertIII (also known as Zwick/Roell) according to the operating instructions. The sample was uniaxially compressed at a constant speed until maximum strain was reached. During the test, the force and length changes on the sample were measured continuously, the associated compressive strain/compression curve was recorded, and the modulus of elasticity (E-modulus) was determined. In particular, the indentation is performed in the center of the sample, e.g. specimen, wedge, pinna, up to 14% strain, at a compression speed of 0.01 mm/s, using a flat cylindrical indenter (diameter of 2 mm). The indentation elastic modulus was analyzed at indentation depths equal to 1-5% strain.

Cell culture

The obtained cartilage biopsy was washed in phosphate buffered saline and connective tissue was removed from the biopsy until only cartilage remained. The cartilage is then minced and the cartilage material is digested with a collagenase solution to separate the cells from the tissue. Cell expansion was performed with regular medium changes until approximately 80% cell confluency was observed. Cells were then passaged into P1 and cultured again until approximately 80% confluency was reached. At this point the cells were harvested and prepared for cryopreservation. A sufficient number of cells were thawed and expanded in 2D culture conditions (DMEM + 25. Mu.g/mL ascorbic acid +10% FBS) until a degree of confluency of approximately 80% was observed. Cells were then passaged to P2 and re-cultured until approximately 80% confluency was reached. At this time, cells were harvested from 30 cell culture flasks. After harvesting was complete, the cells were pooled into a single cell suspension. The suspension was centrifuged and resuspended in the appropriate volume of medium to prepare for mixing with the biopolymer preparation.

Preparation of biopolymer formulations and bio-inks

The biopolymer formulation was prepared by mixing gellan gum and alginate in sterile, pyrogen-free glass vials. Sterile gellan gum is first weighed into a mixing bottle under aseptic conditions. The buffered glucose solution was transferred to a mixing flask containing gellan gum alone. Add to a magnetic mixer and place the bottle on a heated magnetic stirring apparatus at 90 ℃. After the gellan gum was completely dissolved, a pre-weighed amount of alginate was added to the mixing flask. Mixing was continued at 90 ℃ for 45 minutes until a homogeneous biopolymer mixture was obtained. After mixing, the bottles were transferred to sterile conditions and mixing was continued until a high viscosity paste formulation was obtained. The biopolymer formulation was collected and filled into a syringe, which was closed with a combination stopper and stored at 2-8 ℃ until further use.

The preparation of the bio-ink combines the biopolymer formulation and the cell suspension. The stored biopolymer formulation syringe was opened under sterile conditions and the appropriate amount for printing of the construct was weighed in a sterile container. The cell suspension is mixed directly with the biopolymer preparation after P3 cell harvest to achieve as high a cell viability as possible in the bio-ink. The bio-ink is loaded into the bio-printing syringe and may then be transferred to the bio-printing process.

Bio-printing

Bioprinting can generally be performed as described in commonly owned application WO 2019/106606 A1, which is incorporated herein by reference. In short, a printing syringe filled with bio-ink may be brought to the printer through a transfer box. The printing syringe may be attached to or inserted into a syringe holder of the printer. The printing injector nozzle is ready and any entrapped air can be removed from the printing system. In an additive manner, the biopolymer can be extruded from a printing syringe to form a cell construct.

Cross-linking

Immediately after the printing process, the set of constructs was transferred to a shaker. Will contain 50mM CaCl ₂ Is added to the printing container to crosslink the printing device. The crosslinking duration is 60. + -.5 minutes, before the printing device can be operated or transferred to the curing process.

Aging period

The maturation process is performed in an incubator that simulates physiological conditions. The production groups are matured together in a maturation vessel. The medium was changed every 3 to 4 days using a maturation medium containing DMEM Hams F12+10ng/mL TGF-. Beta.3 + 25. Mu.g/mL ascorbic acid +1% ITS. The constructs produced were matured for 16 weeks ± 7 days.

Example 1: cell isolation and expansion

This example describes cells from one donor. The donor was 31 years old and the cartilage biopsy resulted in non-particulate (non-mictic). The biopsy sample was 48.8mg of auricular cartilage with 21393 viable cells per mg of tissue. Tissue samples from which the final cell suspension was generated were obtained from a separate Program named Tissue Donation Program (Tissue Donation Program). Transportation is performed by certified suppliers using transportation materials that are certified and conform to international transportation qualifications of human organizations. The temperature of the procedure is limited to 2-8 degrees celsius.

The obtained biopsy was washed in phosphate buffered saline and connective tissue was removed with a scalpel and forceps until only cartilage remained. The cartilage is then minced and the cartilage material is digested with a collagenase solution to separate the cells from the tissue. Cell expansion was performed according to standard protocols until P1 was complete. Briefly, cells were cultured in 2D medium (DMEM + 25. Mu.g/mL ascorbic acid +10% FBS), with regular medium changes every three to four days until approximately 80% confluency. Cells were then passaged into P1 and cultured again until approximately 80% confluency was reached. At this point the cells were harvested and prepared for cryopreservation. Each containing 194.3 million cells, for a total of 12 vials, was stored 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 medium (DMEM + 25. Mu.g/mL ascorbic acid +10% FBS), with regular medium changes every three to four days until approximately 80% confluency. Cells were then passaged to P2 and re-cultured until approximately 80% confluency was reached. At this time, the drug substance harvest of 30 cell culture flasks (T175) in total was handled by two operators (two operators) in a harvest block of 10 flasks.

After harvesting each processing block, the cells were pooled into a single cell suspension. Before release for printing, the suspension was centrifuged and resuspended in a medium appropriate for the cell number volume.

Example 2: biopolymer preparations and mixing of cells with biopolymers

The study used gellan gum 2.5% and alginate 1.5% biopolymer formulations and was produced according to the process described above in the methods section.

For bioprinting, printing was performed using a modified bioprinter CellInk Inkredible +. For both products, a volume of 420 μ L of test sample was produced. Cell and cell-free samples were produced in both production batches. The printed samples were in 50mM CaCl prior to transfer to maturation media ₂ Medium crosslinking for 60 + -5 minutes.

From each product, the samples were matured together in a T175 flask with a removable lid. The following 3D media were used: DMEM Hams F12, containing glutaMAX +10ng/mL TGF-beta 3+25 μ g/mL ascorbic acid +1% ITS. The minimum volume used was 70mL per flask or 3.5mL per test sample. The medium was changed every 3 to 4 days. The maturation period of the samples was 3 weeks, 8 weeks, 13 weeks and 17 weeks ± 3 days.

Example 3: testing of drug substances, biopolymer formulations and drug products

Drug substance release test

Drug substance properties and safety were tested at different time points. Briefly, prior to harvesting the cells in P3, visual inspection was performed to confirm cell confluency and morphology. After confirming 75-85% confluency, samples of used media were collected for mycoplasma and sterility testing. After harvesting the last processing block, cells were pooled into a single batch of cell suspension. The cell suspension was centrifuged, and the supernatant was collected for endotoxin detection. In addition to these safety tests, cell viability and quantity were evaluated and cells were characterized by Polymerase Chain Reaction (PCR) gene expression analysis of the genes listed in the table below.

Table 1: selected gene expression markers

Biopolymer formulation testing

After the biopolymer formulation is completed, it is characterized for printing performance and safety, particularly by testing sterility, rheology, pH, and osmolarity.

Drug in-process and release detection

The physical, chemical and pharmacological properties of the samples were repeatedly tested from the mixing of the drug substance with the biopolymer until the final drug product was obtained. For example, after the cell expansion process, the released drug substance is mixed with the biopolymer preparation in a ratio of 1. The number of cells and the activity in the printed constructs were analyzed by hemocytometer. Gene expression in cells is performed with the sample after digestion. The genes analyzed are listed in table 1. Mechanical testing was performed using indentation to determine the elastic modulus of the material.

After staining and immunohistochemical marker fixation as listed in table 2, the specimens were evaluated histologically.

Table 2: brief description of selected histological markers

Example 4: study of Long-term in vitro maturation

In this pilot study, two amplifications from the same donor were used to produce cell samples matured for up to 17 weeks. Since construct maturation is a process step in which the basis for extracellular matrix production is achieved, it is crucial to have a good understanding of this development over time. To enhance this understanding, the formation of ECM (e.g. type I collagen and type II collagen), relationship to mechanical properties of the constructs, cell viability, function and number were studied over a 17 week time frame.

One batch of samples (AUR-V047) lasted up to 17 weeks in culture, while the second batch (AUR-011) matured up to 12 weeks. In both batches, the samples were tested after 3 weeks of maturation and additional process tests were performed at weeks 9, 12 and 17. Cell-free samples are produced together with the cellular sample and cultured under the same conditions, where applicable, following the same test protocol.

In both production batches, the cell viability of AUR-V047 increased to 92.4% at 17 weeks and to 81.2% at 13 weeks with the passage of maturation time in AUR-011 (see example 4.3 below for details). Both batches showed similar activity increase slopes. The AUR-011 batch had a greater effect on cell viability when the cells were mixed with the biopolymer than AUR-V047. This greater loss of viable cells is visible throughout 13 weeks of maturation, since the viability of AUR-011 cells is still lower than AUR-047.

Cell activity through MTS was recorded throughout maturation and was hypothesized to correlate with cell viability and cell number. Overall, the increase in cell activity was measurable between 3 and 9 to 13 weeks of maturation. At the last time point of each batch, a decrease was recorded. With increasing cell viability, limited diffusion through the sample (caused by increased number of ECMs) may be responsible for lower MTS values.

Gene expression profiles of cells extracted from test samples at different maturation time points showed no change over time (see details in example 4.6 below). This indicates that the genes evaluated are regulated early in the maturation process and that the levels remain unchanged throughout maturation, leading to the histologically observed ECM deposition.

The visual appearance of the specimens showed significant differences in color and clarity between the cells and the cell-free constructs (see example 4.7 below for details). The color is determined by the color of the medium, which changes from red to yellow due to the presence of phenol red, a pH indicator. As cells metabolize and use the medium, more acidic conditions are created, resulting in a color change from red to yellow. No pH change occurred in the cell-free culture, resulting in a red color of the medium and thus the cell-free sample. The deposition of extracellular matrix causes the appearance of the cell sample to become less transparent and more turbid.

The overall shape of the sample did not change throughout the curing process. No biopolymer degradation or degradation products were observed during the maturation period up to 17 weeks, supporting the theoretical stability of the biopolymer matrix. The cell-free sample showed a white area within the sample, which increased in size with the duration of maturation. The current hypothesis is that the white areas are the accumulation of ionic (mainly calcium) precipitates.

The mechanical properties of the samples increased significantly during the maturation period from 3 weeks to 17 weeks (see example 4.4 below for details). After reaching the cation concentration equilibrium, the mechanical properties of the cell-free samples remained unchanged after 3 weeks of maturation. This demonstrates that any increase in mechanical properties is caused by the production of ECM synthesized by resident cells.

Histological analysis of elastin, glycosaminoglycans, and type I and type II collagen supports this correlation (see example 4.5, infra, for details). Figure 9 shows the mechanical properties and histological analysis of type I and type II collagen over the entire maturation period of 17 weeks. It can be seen that the mechanical properties are continuously improved during this period. The same trend was seen for deposited type II collagen, whereas the presence of type I collagen reached a maximum at week 9 and decreased from then on. This indicates that the increase in mechanical properties is supported to a greater extent by the deposition of type II collagen rather than type I collagen. Type II collagen can be found in the natural otic cartilage matrix and is associated with a major component of tissue mechanical integrity. Both the improvement in mechanical properties and histological analysis showed that the 17-week matured samples did form cartilage-like ECM and over time functional auricular cartilage was expected to form.

Example 4.1: printed results

The batches obtained according to example 2 were analyzed with respect to cell and print yield.

Table 3: summary of cell yield and activity, amount of biopolymer used and number of printed constructs

Even though the same donor was used in both DS expansions, the yield of cells harvested from 30 flasks was very different. This is attributable to differences in cell confluency at harvest, with AUR-V047 estimated at 75% and AUR-011 estimated at about 85%.

Example 4.2: DS and DP safety-sterility, endotoxin and mycoplasma assay results all safety-related release criteria for the DS stage and all DP maturation stage samples were met, with no bacterial or mycoplasma contamination, and no increase in endotoxin levels found in spent media (table 4).

Table 4: summary of DS and DP approved sterility, endotoxin and Mycoplasma detection results

Example 4.3: cell viability and Activity assessment during maturation

The biopolymers obtained according to examples 1 and 2 and the printed samples were analyzed for cell viability and metabolic activity throughout the process. Figure 2 summarizes the results of the control during the pre-printing process until the maturation of the cell viability as determined by the hemocytometer is over.

Cell viability was high in both DS harvests (94-97%). After contact with the biopolymer, cell viability decreased to 63% and 76%, respectively. This phenomenon is a well-documented part of the production process according to the invention and is caused by the shear forces to which the cells are subjected when mixed with the biopolymer. In both production batches, the activity was lowest after 1-3 days of maturation. From there, a steady increase in viability was measured until the end of the corresponding maturation period. The viability of the two production batches developed over time was comparable.

Batch AUR-011 shows consistently lower activity than AUR-V047 from the point of view of mixing the cells with the biopolymer formulation. Since both batches used the same donor and biopolymer formulations, it can be assumed that this difference was caused by the mixing process. Overall, it can be concluded that the final viability, although increasing over time, depends on the viability that can be maintained during the mixing of the cells and the biopolymer.

Example 4.4: mechanical Properties of the test specimens

The mechanical properties of the two batches of cells and cell-free samples were analyzed throughout the maturation process (fig. 3). The initial modulus of elasticity (1-3 days after printing) was high and then dropped dramatically during the next 3 weeks of maturation. In the early stages of maturation (days 1-3), the mechanical properties of all constructs (cellular and acellular) are contributed only by the ionic crosslinking of the biopolymer matrix. During initial maturation, calcium ions are washed out of the construct to reach an equilibrium concentration in the medium while being replaced by other ions in the cell culture medium. This results in a decrease in hydrogel strength. Over time, a balance consistent with maturation medium conditions was reached, resulting in an elastic modulus of about 115kPa after 21 days of maturation culture. As shown in fig. 3, the mechanical properties of the cell-free constructs (dashed lines) remained consistent or slightly further decreased during the remaining maturation period of the two production batches. At the same time, a sharp increase in the mechanical properties of the cell construct (fig. 3, solid line) was observed from day 21 onwards. For both production batches, the peak in mechanical properties reached 256kPa and 296kPa at the end of week 13 and week 17, respectively. Compared with the cell-free samples, the mechanical properties of AUR-011 are increased by 227% and AUR-V047 by 384% at the respective end points. It is expected that the mechanical properties will further improve with further aging.

Example 4.5: histological evaluation of test specimens

At each maturation time point, the two batches of sample specimens were analyzed using different histological and Immunohistochemical (IHC) stains. Fig. 4 shows all images of the sample at different stains and different time points. For reference, a sample of native ear cartilage is also included. These low-power images were displayed to determine the overall impression and potential changes of the specimens at different stages of maturation.

Since H & E is an overview stain that visualizes cells, the intensity of staining is not particularly relevant. At this magnification, cell numbers and morphology cannot be analyzed. The intensity of both the Weigert elastin and Alcian Blue stains appears to increase with aging time, indicating an increase in elastin, glycosaminoglycans and cartilage-like tissue. However, this trend is not seen in Safranin-O stain, which, like Alcian Blue, also binds to glycosaminoglycans. The Alcian Blue and Safranin-O stains showed some degree of non-specific background staining in the cell-free constructs (fig. 6), indicating that there was no significant change over maturation time. This indicates that the increase in staining intensity in Alcian Blue is not an artifact, but an increase in the presence of glycosaminoglycans.

Weigart's elastin strength was much higher in native cartilage than 17-week-old cartilage, while the strength of Alcian Blue and Safranin-O was essentially comparable between 17-week-old cartilage and native cartilage.

At lower magnification, the greatest difference in collagen type I and type II staining can be observed during maturation. The type I collagen strength was already high at 21 days maturation. The signal intensity further increased until 9 weeks, and then began to drop again. In addition, a different structural organization of collagen is observed during maturation. Type I collagen appears to be more fragile and disorganized at 21 days as time homogeneity and tissue levels increase. Even though a decrease in type I collagen was observed (from 13 weeks on), homogeneity (smooth transition, no holes) and tissue levels appeared to be still high. The strength of type I collagen was still high at 17 weeks compared to native cartilage. Since type I collagen is considered a repair cartilage, the decrease in type I collagen is expected to continue over time, eventually leading to the formation of type I collagen-free otic cartilage.

The staining intensity of type II collagen was weaker at 21 days of maturation, but increased dramatically at week 9, with further darkening observed in the samples at weeks 13 and 17, respectively. When comparing the 17-week matured construct to native cartilage, the overall strength does not appear to be different at low rates.

Fig. 5 shows a higher magnification (10 ×) of the histological images of the batch AUR-V047 shown in fig. 4. In all staining, cells showed a healthy morphology, increasing the size and number of lacunae (lacunae) with time. Visually, the cell number does not appear to increase with maturation time, but an analysis of the number of intact cells stained with H & E is necessary to confirm this observation. Usually single cells appear after 3 weeks of maturation, and a small population of cells called cartilage can be found at a later time point. After 17 weeks of maturation, most cells became part of the minicell population, and only a few single cells were found. Almost all cells show clearly visible lacunae, which is an ideal feature of healthy chondrocytes. The cell density was lower in 17-week-matured samples compared to native cartilage. The native cartilage samples showed a greater degree of tissue with smaller cell size and uniformly rounded lacunae compared to the 17-week-matured samples.

The greatest difference between the 17-week-matured sample and the native cartilage was observed in the samples stained with Weigert's elastin and type I collagen. With weibert's elastin, the staining intensity of the native ear cartilage is much higher and careful observation allows identification of elastin fibers (dark lines between cells). In the 17-week aged sample (orange arrow), early formation of such fibers can be observed in the vicinity of the cells. Histologically, the 17 week matured sample was the closest and appeared to be progressing to the native ear cartilage.

Fig. 6 shows histological evaluation of cell-free samples cultured over the same time period as the cell samples. All stains show a certain degree of background, except for type I and type II collagen as specific stains. The biopolymer matrix has a negatively charged structure, attracting positively charged dye molecules and trapping them within the matrix. The intensity of the background staining appeared to be stable throughout the maturation process, indicating that no significant loss of biopolymer occurred.

Example 4.6: gene expression in cells during long-term maturation

The gene expression profiles of cells extracted from the test samples after different maturation times (FIG. 7) did not show differences throughout the process. This is particularly interesting because, for type I collagen, the protein expression observed by histological analysis changes significantly over time, increasing early in the process and then decreasing until the end of 17 weeks maturation. This indicates that PCR analysis of the cells can only indicate protein production in the 3D maturation construct. The minor differences that can be observed in certain genes between donors are not significant and are part of the lot-to-lot differences.

Example 4.7: appearance and weight of the test specimens

Fig. 8 summarizes the visual appearance, size and weight of the samples for different maturation durations. The overall appearance of the cell construct remains unchanged throughout maturation. The yellow coloration is caused by the yellow color of the aged medium containing phenol red. Yellow indicates a decrease in pH caused by cell activity. The cell sample appeared uniform and opaque.

The red color of the cell-free sample is caused by the phenol red-containing medium, and the phenol red does not lower the pH and maintains the red color due to the lack of cells. Throughout maturation, white regions are increasingly present in the cell-free construct. The size of these regions may increase over time. Preliminary analysis supports the hypothesis that these white areas are caused by ion precipitation. There was no indication that this accumulation was in the cell construct. As can be seen from the values given in fig. 8, the average diameters of the cells and the cell-free samples did not change with the lapse of maturation time. The weight of the cell-free sample was not determined as part of the process and release control. The weight of the cell sample appeared to increase with increasing maturation time; however, it must be noted that the sample size is small and the increase may be a result of sample volume fluctuations. The potential increase in weight can be explained by an increase in extracellular matrix production within the sample as well as a potential increase in cell number. Further investigation is required to determine the validity of the observations and hypotheses.

The present invention is also characterized by the following items.

A three-dimensional composition according to item 1 comprising at least about 6 x 10 ⁶ Individual chondrocytes per milliliter of the composition and a cross-linked biopolymer preparation, wherein the three-dimensional composition has mechanical stability suitable for implantation into a subject in need thereof, wherein the biopolymer preparation is in particular a homogeneous biopolymer preparation.

The composition of item 1, wherein the composition has an elastic modulus (E) of at least 180kPa, at least 200kPa, at least 220kPa, at least 240kPa, at least 250kPa, or at least 260kPa.

The composition according to

item

1 or 2, wherein the chondrocytes are derived from auricular chondrocytes, in particular human auricular chondrocytes.

Composition according to any of the preceding items, wherein the chondrocytes are obtained by cell expansion and maturation from isolated primary chondrocytes, in particular isolated primary chondrocytes from humans.

The composition of any of the preceding items, wherein the chondrocytes have a cell viability of at least 70%, at least 80%, at least 85%, at least 90% or at least 95%.

The composition of any one of the preceding items, wherein the cell viability is determined by cytometry based on ph.

The composition of any of the preceding items, wherein the composition is substantially free of stem cells, such as bone marrow-derived stem cells.

The composition of any of the preceding items, wherein the composition is substantially free of progenitor cells, e.g., chondrogenic progenitor cells.

The composition of any of the preceding items, wherein the composition is free of at least one of:

a. added tissue particles;

b. added fibers;

c. microbeads; and

d. the number of the nano-particles,

in particular free of a, b, c, and d.

The composition of any of the preceding items, wherein

a. Is free of calcium carbonate, and/or

b. Without calcium phosphate, and/or

c. Without hydroxyapatite

Is additionally added to the composition, particularly wherein there is no calcium carbonate, no calcium phosphate and no hydroxyapatite, more particularly wherein there is no poorly water soluble calcium or strontium compound additionally added to the composition.

The composition of any of the preceding items, wherein the cells exhibit the following relative gene expression profiles as determined by quantitative polymerase chain reaction (qPCR):

GAPDH (reference gene): ct = about 12 to about 18

Type II collagen/type I collagen ratio: 2-delta Ct is more than or equal to 1 and 10 ^-4

-type II collagen: 2 ^-ΔCt ≥1·10 ^-2

-aggrecan: 2 ^-ΔCt ≥3.5·10 ^-2

-IL-1β：2 ^-ΔCt <5·10 ^-6 。

The composition of any of the preceding items, wherein the biopolymer formulation comprises gellan gum and alginate.

The composition of any of the preceding items, wherein the biopolymer preparation consists of gellan gum and alginate as the only structural components.

The composition according to any of the preceding items, wherein the biopolymer preparation is a cross-linked gellan gum/alginate preparation, in particular a chemically cross-linked gellan gum/alginate preparation.

The composition according to item 15, item 13 or 14, wherein the biopolymer preparation is crosslinked with multivalent ions, in particular with alkaline earth ions, more in particular with calcium or strontium ions.

The composition according to any 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 the biopolymer formulation.

The composition according to any of the preceding items, wherein the biopolymer preparation is a cross-linked gellan gum/alginate preparation with an alginate content of about 1% (w/v) to about 3% (w/v), in particular about 1.0% (w/v) to about 2.0% (w/v), more in particular about 1.5% (w/v), based on the total volume of the biopolymer preparation.

The composition according to any one of claims 14 to 17, wherein the multivalent ion is provided by calcium chloride or strontium chloride, in particular calcium chloride.

The composition of any of the preceding items, wherein the biopolymer formulation is CaCl ₂ Crosslinked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation based on the total volume of biopolymer formulation.

The composition of any of the preceding items, wherein the elastic modulus (E) is determined by the unconfined indentation test.

The composition according to any of the preceding items, wherein the mechanical stability of the composition is determined by histology and/or immunohistochemistry.

The composition of any of the preceding items, which is a wedge, a tissue engineered human nose or a human pinna, or a portion thereof.

The composition of item 23, item 22, adapted to be located on the skull of a patient outside the ear canal.

A method of making the three-dimensional composition of any of the preceding claims, comprising the steps of:

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.

The method of items 26, 24 or 25, wherein step a

1) Cell expansion of the isolated primary chondrocytes until the end of passage 1 (P1);

2) Cryopreservation of chondrocytes after P1 generation;

3) Thawing and cell expansion until the end of passage 2 (P2).

The method of item 27 item 26, wherein step a

4) Cells were expanded until the end of passage 3 (P3).

The method of any one of claims 24 to 27, wherein the biopolymer formulation is a gellan gum/alginate formulation, in particular 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation, based on the total volume of the biopolymer formulation.

Item 29. The method of any one of claims 24 to 28, wherein step c.

The method of any one of claims 24 to 29, wherein step c.

Item 31. The method according to any one of items 24 to 30, wherein step d.

The method of any one of claims 24 to 31, wherein step e.

Item 34 the method of any one of claims 24 to 33, wherein step e.is performed for at least 8 weeks, in particular 10, 12, 13, 14, 15, 16 or 17 weeks.

The method of any one of claims 24 to 34, wherein step e.is performed for 10 to 24 weeks, in particular 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks.

The method of any one of claims 24 to 35, wherein step e.

A three-dimensional composition according to item 37 comprising at least about 6 x 10 per milliliter of the composition ⁶ A chondrocyte and cross-linked biopolymer preparation obtainable by a process comprising the steps of:

b. mixing the expanded chondrocytes with a homogeneous biopolymer preparation, thereby obtaining a bio-ink;

c. depositing the bio-ink layer-wise onto the surface, thereby obtaining a three-dimensional composition consisting of a layer of the cell-laden biopolymer preparation;

e. the three-dimensional composition is allowed to mature for at least 8 weeks, thereby allowing the chondrocytes to produce extracellular matrix to form the three-dimensional composition.

Item 38A composition comprising at least about 6X 10 per milliliter ⁶ A cell composition of chondrocytes, which is provided in a biopolymer formulation, in particular a homogeneous biopolymer formulation, and which has undergone a maturation period of at least 8 weeks, in particular 10, 12, 13, 14, 15, 16 or 17 weeks, more in particular 16 weeks, for use in medicine.

Item 39A composition comprising at least about 6X 10 per mL ⁶ A cell composition of chondrocytes, which is provided in a biopolymer preparation, in particular a homogeneous biopolymer preparation, and which has undergone a maturation period of at least 8 weeks, in particular 10, 12, 13, 14, 15, 16 or 17 weeks, more in particular 16 weeks,

methods for treating aneural or lesser ear deformities or facial damage with persistent damage to the 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, in particular 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks.

Item 41A composition comprising at least about 6X 10 per milliliter ⁶ A cell composition of chondrocytes, provided in a biopolymer formulation, in particular a homogeneous biopolymer formulation, and having an elastic modulus (E) of at least 180kPa,

it is used in medicine.

Item 42. A composition comprising at least about 6X 10 per mL ⁶ A cell composition of chondrocytes provided in a biopolymer formulation, in particular a homogeneous biopolymer formulation, and having an elastic modulus (E) of at least 180kPa,

methods for treating aphakia or microcephaly or facial damage accompanied by persistent damage to the ears and/or nose.

The cell composition for use according to any one of claims 37 to 42, wherein the chondrocytes are derived from human auricular chondrocytes, in particular human autologous auricular chondrocytes.

The cellular composition for use according to any of claims 38 to 42, wherein the biopolymer preparation and the chondrocytes are arranged in a three-dimensional structure, in particular in the form of a wedge, a human pinna or a part thereof.

The cell composition for use according to any one of claims 37 to 44, wherein the maturation period is an in vitro maturation period.

The method of any one of claims 24 to 36 or the composition of any one of claims 37 to 45, wherein the maturation is performed in a 3D medium at about 36 ℃ to about 38 ℃ under normoxic or hypoxic conditions.

The three-dimensional composition according to any one of claims 1 to 23 for use in the treatment of an ear-free or small ear deformity or a facial injury with persistent damage to the ears and/or nose.

Item 48 an implant for improving hearing comprising at least about 6 x 10 ⁷ Chondrocytes provided in a biopolymer formulation and subjected to a maturation period of at least 8 weeks, in particular 10, 12, 13, 14, 15, 16 or 17 weeks, more in particular 16 weeks.

Item 49 the implant for use according to item 48, which has undergone a maturation period of 10 to 24 weeks, in particular 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks.

The implant of claim 50, wherein the maturation period is an in vitro maturation period.

Item 51. An implant for improving hearing comprising at least about 6 x 10 provided in a biopolymer formulation ⁷ And has an elastic modulus (E) of at least 180kPa.

The implant of item 52 used according to item 51, wherein E is at least 250kPa.

A method of treating an auricless or a small ear deformity or a facial injury accompanied by sustained damage to the ear and/or nose, comprising the step of implanting the three-dimensional cartilage composition according to any one of items 1 to 23 into a subject in need thereof.

Claims

1. A three-dimensional composition comprising at least about 6 x 10 per milliliter of the composition ⁶ A plurality of chondrocytes and a cross-linked homogeneous biopolymer preparation, wherein the biopolymer preparation comprises gellan gum and alginate, wherein the three-dimensional composition has mechanical stability suitable for implantation into a subject in need thereof, and wherein the composition has an elastic modulus (E) of at least 180kPa.

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

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

4. The composition of 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 the following

a. Added tissue particles;

b. added fibers;

c. microbeads; and

d. the number of the nano-particles,

in particular not containing a.

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 the 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 the biopolymer formulation.

6. The composition of any one of the preceding claims, wherein the biopolymer preparation is CaCl ₂ Crosslinked 2.5% (w/v) gellan gum/1.5% (w/v) alginate formulation based on the total volume of biopolymer formulation.

7. The composition of any one of the preceding claims, which is a wedge, a tissue engineered human nose or a human pinna or a portion thereof.

8. A method of making the three-dimensional composition of any of the preceding claims, comprising the steps of:

a. expansion of isolated chondrocytes in vitroOptionally in combination with a cryopreservation step, thereby obtaining at least about 6X 10 from the harvested culture ⁷ A plurality of chondrocytes;

b. mixing the expanded chondrocytes with a homogeneous biopolymer preparation comprising gellan gum and alginate, thereby obtaining a bio-ink;

d. crosslinking a biopolymer formulation within the three-dimensional composition;

e. maturing the three-dimensional composition, thereby allowing chondrocytes to produce extracellular matrix to form a three-dimensional composition having mechanical stability suitable for implantation,

9. The method of claim 8, wherein the biopolymer preparation is a gellan gum/alginate preparation, in particular a gellan gum/1.5% (w/v) alginate preparation based on the total volume of the biopolymer preparation.

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 10 ⁶ Individual chondrocytes/mL composition and cross-linked biopolymer preparation obtainable by a process comprising the steps of:

12. A composition comprising at least about 6 x 10 per milliliter ⁶ A cell composition of chondrocytes, provided in a homogeneous biopolymer preparation and subjected to a maturation period of 10 to 24 weeks, in particular 10, 12, 13, 14, 15, 16 or 17 weeks, more in particular 16 weeks,

methods for use in medicine, particularly for the treatment of otoleral or small ear deformities or facial injuries accompanied by persistent damage to the ears and/or nose.

13. A composition comprising at least about 6 x 10 per milliliter ⁶ A cellular composition of chondrocytes provided in a homogeneous biopolymer formulation and having an elastic modulus (E) of at least 180kPa,

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

15. An implant for improving hearing comprising at least about 6 x 10 ⁷ Chondrocytes provided in a biopolymer formulation and subjected to maturation for at least 8 weeks, particularly 10 to 24 weeks, more particularly 10, 12, 13, 14, 15, 16 or 17 weeks, more particularly 16 weeks.