Method for preparing transplantable cartilage tissue

Abstract

The present invention relates to a method for preparing transplantable cartilage tissue, a method for preparing cartilage tissue by selecting and picking from chondrocytes or spheroids, and the use of said cartilage tissue as a pharmaceutical composition, a medicament, a graft. In particular, the present invention relates to markers for identifying suitable chondrocytes for preparing cartilage tissue, which is particularly for transplantation purposes.

Description

[0001] This application is a divisional application of a Chinese application with application number 201880048374.7.

[0002] The present invention relates to a method for preparing cartilage tissue by selecting and picking from chondrocytes or spheroids, and to the use of said cartilage tissue as a pharmaceutical composition, a medicament or a graft. In particular, the present invention relates to markers for identifying suitable chondrocytes for preparing cartilage tissue, which is particularly for transplantation purposes.

[0003] Although articular cartilage shows notable resistance, this tissue has no or very low capacity for self - repair and regeneration, and untreated damage can lead to, for example, osteoarthritis. This low potential for spontaneous regeneration has led to the development of cell therapies, such as autologous chondrocyte transplantation (ACT), in an attempt to provide a functional and painless repair of articular cartilage defects. There is a high demand for methods for cartilage regeneration, for example, in young and active patients with traumatic damage or even with symptoms of cartilage degeneration, or for treating cartilage defects.

[0004] Biochemical and molecular studies on human chondrocytes are hindered by a series of factors, such as the insufficient availability of human tissue associated with a very small number of cells available in biopsies, the limited proliferative capacity of cultured chondrocytes and their high phenotypic instability.

[0005] Chondrocytes are cells that originate from chondroblasts and settle in cartilage tissue. Together with the cell interstitium (extracellular matrix (ECM)), chondrocytes constitute the main component of cartilage, particularly articular cartilage.

[0006] It has been known that by mimicking certain processes of embryonic development, such as culturing human chondrocytes three - dimensionally on an agarose substrate, cell aggregates are produced that are superior to monolayer cells in terms of their differentiation state and have, for example, chondroid characteristics. These characteristics, which reflect as accurately as possible the natural osteoarticular tissue, are characterized by the expression of collagen II (the main structural protein of the extracellular matrix of hyaline cartilage), proteoglycans (such as aggrecan) and the chondrocyte - specific protein S100 intracellularly.

[0007] In addition, it is desirable that the expression of collagen I, which is necessarily upregulated during the cell expansion phase of monolayer cultures, is again reduced in cell aggregates, since this protein is not present in native articular cartilage. Since 2002, the cell aggregates (spheroids) thus produced have been used by the applicant for the treatment and therapy of cartilage defects caused by injuries in young patients. For this purpose, the patient's own articular cartilage tissue is removed, more precisely, a biopsy of hyaline cartilage is taken from a healthy cartilage area. The chondrocytes located in the cartilage tissue are isolated by enzymatic digestion of the cartilage tissue with a collagenase solution and are expanded in a cell culture flask (monolayer culture) under standard cell culture conditions in the presence of autologous serum, and sufficient chondrocytes are provided for the preparation of transplantable cartilage tissue. The expanded cells form cell aggregates (so-called spheroids), which can be transplanted. However, the cell aggregates thus prepared are limited in their differentiation state and most show a relatively weak local expression of important collagen II and marginal synthesis of proteoglycans, while there is a relatively strong expression of unwanted collagen I. In order to further improve the differentiation of these cell aggregates, the culture medium can be enriched with certain bioactive substances. This primarily includes the growth factors TGF-β1-3, which have been described in the literature many times as cofactors for collagen synthesis, and L-ascorbic acid (vitamin C). However, on the one hand, this biochemical stimulation is often not sufficient to induce chondrocyte differentiation in 3D cell aggregates to the extent of producing cartilage-typical structures, and on the other hand, the use of growth factors such as TGF-β is particularly questionable for clinical applications in humans.

[0008] The cultivation of human cells in the form of three-dimensional cell aggregates (for example in the form of spheroids) for clinical applications in humans, such as for use as autologous cartilage grafts, has been described in DE 100 13 223, and a method for the in vitro preparation of three-dimensional cartilage tissue and bone tissue from bone marrow stem cells, chondral stem cells or mesenchymal stem cells is involved. In this case, the cells are first cultivated as monolayer cultures and subsequently as suspensions for such a long time until cell aggregates are produced, which contain at least 40% by volume of extracellular matrix in which differentiated cells are embedded.

[0009] In the said method, cell aggregates are prepared by cultivating the cells in a cell culture vessel coated with agarose for at least 1 - 2 weeks.

[0010] US 7,887,843 B2 discloses a method for the in vitro production of three-dimensional cartilage tissue and bone tissue, in which transplantable spheroids are prepared from bone marrow stem cells, chondral stem cells or mesenchymal stem cells by culturing 1×10 5 cells for at least two weeks.

[0011] Anderer et al. (Journal of Bone and Mineral Research (2002), New York, NY, US, Vol. 17, No. 8, pp. 1420-1429) also disclose a method for generating three-dimensional cartilage tissue in vitro. According to this method, 1×10 5 or 2×10 5 chondrocytes are cultured for 5 days, 2 weeks, 1 month, 2 months, and 3 months to prepare spheroids.

[0012] The cartilage tissue prepared in vitro as described in the prior art does not show an expression pattern of basic matrix proteins such as type II collagen similar to those of natural tissues. More precisely, the composition of the extracellular matrix (ECM) produced by chondrocytes in vitro is significantly different from that of natural cartilage tissue. However, the composition of the ECM is decisive for the biological functions of cartilage (e.g., mechanical load-bearing capacity). Therefore, there is a need for new methods for preparing cartilage tissue (also for quality control).

[0013] "Transplantable cartilage tissue" means an ex vivo or in vitro method of preparation, in which the prepared tissue largely corresponds to or is the same as natural tissue. Within the scope of the present invention, the transplantable cartilage tissue, for example, largely corresponds to natural tissue in terms of the expression or expression pattern of the extracellular matrix, such as in terms of the expression or expression pattern of structural proteins or proteins such as type II collagen and / or S100 protein and / or tissue-specific proteoglycans (e.g., cartilage-specific proteoglycans, such as aggrecan). The detection of the expression or expression pattern can be carried out, for example, in a histological or immunohistological manner.

[0014] The tissue from which chondrocytes are isolated is, for example, selected from musculoskeletal tissue, bone tissue, cartilage, bone, meniscus, intervertebral disc, bone marrow, joint, or tendon. In this case, the transplantable cartilage tissue prepared according to the method of the present invention is similar to natural tissue in terms of composition and protein expression (see above).

[0015] Natural articular cartilage tissue has the following proportional composition of extracellular matrix: approximately 60-80% water, relative to the wet weight of the cartilage tissue. The high water content is important for the mechanical load-bearing capacity of cartilage tissue and is important for the "sponge effect" together with proteoglycans. In addition to water, natural cartilage joints also contain structural proteins or matrix proteins, such as collagen, proteoglycans, and non-collagen proteins. In this case, the structural macromolecules total approximately 20-40% of the wet weight of the articular cartilage tissue.

[0016] In the case of natural articular cartilage tissue, the collagen fraction is 60%, the proteoglycan fraction is 25 - 35%, and the non - collagen protein and glycoprotein fraction is 15 - 20%, relative to the dry weight of the cartilage. Type II collagen amounts to approximately 90 - 95% of the total collagen content of natural articular cartilage tissue.

[0017] Human natural articular cartilage tissue contains approximately 1 - 5% chondrocytes, relative to the tissue volume. In this case, chondrocytes are the basic producers of the matrix molecules of the functional natural tissue.

[0018] There is a high demand for providing such chondrocytes that have sufficient quality and can produce transplantable cartilage tissue corresponding to natural tissue, so that a successful treatment can be expected. The success of this treatment depends decisively on the quality of the transplantable cartilage tissue.

[0019] Therefore, the object of the present invention is to provide improved transplantable cartilage tissue, which particularly has improved properties, especially sufficient quality, such as consistency with natural chondrocytes and improved - quality spheroids, so that high - quality grafts can be obtained, especially for the successful treatment and therapy of articular cartilage defects.

[0020] Surprisingly, the inventors were able to identify such markers in isolated chondrocytes that allow sufficient quality for the preparation of transplantable cartilage tissue. The markers according to the invention particularly allow the determination of the chondrocyte consistency (consistency marker) of isolated chondrocytes in culture with natural chondrocytes.

[0021] Suitable chondrocyte consistency in turn allows the preparation of suitable transplantable cartilage tissue, because on the one hand, sufficient extracellular matrix (ECM) of cartilage can be obtained, and on the other hand, improved treatment of articular cartilage defects can be achieved.

[0022] In a further embodiment, the markers according to the invention allow the determination of the purity or contamination of isolated chondrocytes or spheroids ("purity marker").

[0023] The above - mentioned markers ensure sufficient quality and are thus "quality markers".

[0024] Therefore, the present invention relates to a method for selecting chondrocytes or spheroids for the preparation of transplantable cartilage tissue, wherein at least one quality marker, that is, a consistency marker or a purity marker, is determined.

[0025] A method for selecting chondrocytes or spheroids for preparing transplantable cartilage tissue according to the present invention includes: expanding the chondrocytes in a monolayer culture in a first step (see above, the so-called two-dimensional expanded culture), and aggregating into spheroids in a second step (see above, the so-called 3D-culture), so that the chondrocytes aggregate into spheroids, and the spheroids can be selected.

[0026] In a preferred embodiment, the quality marker or the consistency marker or the purity marker is selected from the group consisting of: KAL1, CRTAC1, NRN1 or EBF3, which are measured in vitro.

[0027] In a preferred embodiment, the quality marker or the consistency marker is selected from the group of KAL1, CRTAC1, and / or the purity marker is selected from the group of EBF3, NRN1. In particular, synovial cells are an adverse contamination, which can be measured in vitro by the purity marker EBF3. Such contamination reduces the quality of chondrocytes and spheroids.

[0028] In a further preferred embodiment, in addition to the markers mentioned above, according to the present invention, so-called potential markers can also be sought, which allow prediction of the activity and regeneration ability of the transplantable cartilage tissue to be obtained from the spheroids. Such potential markers according to the present invention are preferably proteoglycans, particularly preferably aggrecan (ACAN).

[0029] Table 1: Marker genes according to the present invention

[0030]

[0031]

[0032] The relevant DNA sequences can be clearly identified by accession numbers (e.g., NCBI).

[0033] The in vitro measurement of the markers according to the present invention is preferably carried out by measuring the gene expression of these markers and the gene expression levels in the isolated chondrocytes and the obtained spheroids, which can be determined, for example, routinely by polymerase chain reaction (PCR, particularly qPCR), see Examples.

[0034] According to the present invention, the markers show gene expression and increased or decreased gene expression in the method according to the present invention, thus having the ability as markers.

[0035] In a further embodiment, the determination of gene expression or gene expression levels can also be carried out in the presence of and in comparison with a reference gene and its gene expression or gene expression levels. This allows for the normalization and quantification of gene expression, also with the aid of software support, see Michael W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Research, 2001, Vol. 29, No. 9.

[0036] Table 2: Reference genes according to the invention

[0037]

[0038] The relevant DNA sequences can be unambiguously identified by accession numbers (e.g., NCBI).

[0039] Within the scope of the present invention, "selection" means, for example, discarding such chondrocytes or spheroids that do not have gene expression of the markers according to the present invention or have relatively lower gene expression or gene expression levels compared to other chondrocytes or spheroids. A person skilled in the art can distinguish stronger gene expression from weaker gene expression for one or more chondrocytes / spheroids according to the obtained gene expression patterns and accordingly select such chondrocytes or spheroids.

[0040] Thus, in a further preferred embodiment, selected and picked are such isolated chondrocytes and the resulting spheroids after amplification that have the following gene expression ratios with one or two reference genes (R) (more precisely, B2M and / or TOP1):

[0041]

[0042] Further preferred ratios are, for example

[0043]

[0044] The values given are understood as suitable thresholds.

[0045] Thus, the present invention relates to a method for selecting chondrocytes or spheroids for the preparation of transplantable cartilage tissue, wherein the markers according to the present invention are determined in comparison with reference genes.

[0046] In a further preferred embodiment, during the determination of the purity marker EBF3, the proportion of contaminating synovial cells in the chondrocytes isolated after amplification or in the spheroids obtained is less than or equal to 9%, particularly less than or equal to 5%, compared to the total number of cells.

[0047] The method according to the invention allows the selection of chondrocytes and spheroids specifically selected for the cartilage tissue to be transplanted. This is based on the special properties of the markers according to the invention (i.e., quality markers or conformity markers or purity markers). Thus, such chondrocytes and spheroids are selected which have a specific gene expression and the corresponding gene products can be detected, for example, by PCR.

[0048] Accordingly, the invention relates to isolated chondrocytes or isolated spheroids obtainable by the method according to the invention. It also includes transplantable cartilage tissue, particularly in the form of a medicament or drug or in the form of a pharmaceutical composition (comprising such chondrocytes or spheroids), which is particularly useful for use in the treatment of articular cartilage defects and / or autologous chondrocyte transplantation.

[0049] In a further preferred embodiment of the invention, the transplantable cartilage tissue according to the invention is administered to a patient by means of an applicator (see EP2345450B1 (co.fix 150 of the applicant) or DE102009025347A1). Preferably, the transplanted cartilage tissue in the form of a pharmaceutical composition can be kept in physiological saline together with other auxiliaries and additives.

[0050] The following examples serve to explain the invention, but do not limit the invention to the examples.

[0051] Example 1:

[0052] Isolation of chondrocytes from cartilage biopsies and cell culture

[0053] Cartilage-specific cells (chondrocytes) were isolated from cartilage biopsies by enzymatic digestion using collagenase (350 units / ml). The enzymatic digestion was carried out for 4 to 6 hours and at 37 °C. The cartilage biopsies were obtained from the femoral condyles of the knee joint. After enzymatic digestion, the chondrocytes were centrifuged, washed, counted and seeded in tissue culture flasks together with cell culture medium (αMEM / Ham’s F12 1:1, 1% glutamine) and autologous serum (10 - 15% human serum). For expansion, the cells were cultured in vitro at 37 °C in a two-dimensional expansion culture (monolayer) for 7 to 38 days. The cell culture medium was changed every 3 - 6 days. When the cell culture reached confluence, the cell culture was passaged by means of papain and distributed in multiple culture flasks.

[0054] After a sufficient number of cells from the monolayer culture was reached, 200,000 cells / well were seeded in microtiter plates coated with 2% agarose. The cells aggregated and formed three-dimensional round structures (spheroids). During the culture of the spheroids, the cell culture medium (αMEM / Ham’s F12 1:1, 1% glutamine) supplemented with 10% autologous serum was changed regularly (every 3 - 6 days). This 3D-culture lasted for 14 to 42 days.

[0055] After spheroid culture, chondrocyte grafts for transport and implantation were prepared by collecting the spheroids in a pharmaceutical carrier (0.9% NaCl) and subsequently transferring them to an applicator system co.fix 150 or a syringe.

[0056] In summary, the preparation of chondrocyte grafts consisted of: a monolayer culture period for the expansion of the isolated chondrocytes, and a 3D-culture period for the aggregation of chondrocytes into spheroids. The chondrocyte grafts were intended for autologous application, i.e., implantation into the knee of the same patient.

[0057] Example 2:

[0058] a.) RNA isolation

[0059] Chondrocytes were cultured as a two-dimensional expanded culture (monolayer) until spheroids were prepared, and then dissolved from the bottom of the cell culture flask and from the cell aggregates by means of an enzyme solution (papain). 1×10E6 of these chondrocytes were taken from the cell suspension for consistency testing and transferred to a reaction vessel, while the remaining cells were used for subsequent spheroid preparation. The monolayer cells relevant for testing were centrifuged down, and the cell pellet was lysed in the corresponding lysis buffer (PeqGOLD, Peqlab), snap-frozen and stored at -70 °C until RNA isolation. For marker testing on chondrocyte-spheroids, after a two-week culture duration, 8 spheroids were taken from the well plate, washed with PBS, and lysed with the corresponding lysis buffer (PeqGOLD, Peqlab). For optimal cell disruption, the spheroids received in the lysis buffer were homogenized mechanically by drawing up and pushing out the spheroids with a syringe and cannula. Subsequently, the lysate was snap-frozen in liquid nitrogen and stored at -70 °C until RNA isolation.

[0060] Total RNA was extracted from chondrocyte-spheroids and chondrocyte-monolayers using the peqGOLD Total RNA Kits from Peqlab or the RNeasy Plus Mini Kit from Qiagen according to the manufacturer's instructions. The isolated RNA was eluted in nuclease-free water and stored at -70 °C until cDNA synthesis. After RNA isolation, the quantity, quality, and integrity of the RNA were determined using a spectrophotometer (NanoDrop, Thermo Scientific) and by means of a bioanalyzer (Agilent) according to the manufacturer's instructions.

[0061] b.) cDNA synthesis

[0062] cDNA synthesis was performed with the aid of Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. For each sample, 80 ng of total RNA was transcribed by using random hexamer primers. A template-primer mixture was placed in a sterile reaction vessel for a reaction volume of 20 μl. The following additional components were pipetted into the template-primer mixture: Transcriptor reverse transcriptase reaction buffer, Protector RNase inhibitor, deoxynucleotide mixture, Transcriptor reverse transcriptase. The reaction batches were mixed, centrifuged, and then incubated in a thermal cycler. The reverse transcribed cDNA was then diluted with nuclease-free water to a final concentration of 1 ng / μl. The samples were stored at -20°C until further use in qPCR analysis.

[0063] c.)qPCR

[0064] For qPCR analysis, use a 384-well format 480Instrument II (Roche) qPCR technology. Primers were HPLC purified and dissolved in nuclease-free water to a concentration of 10 μM. Probes were used at a concentration of 10 μM. For markers to be measured, first establish determined qPCR reaction conditions for primer concentration and probe concentration and for the use of additives. For all samples, a master mix was prepared according to the reaction conditions of specific primers. Each 8 μl of a specific reaction mixture for each target gene was pipetted into the corresponding wells of a 384-well plate, and then 2 μl of cDNA sample (1 ng / μl) was added, resulting in a total volume of 10 μl for each qPCR reaction.

[0065] qPCR analysis is based on the exponential increase of fluorescently labeled PCR amplicons of the gene of interest. qPCR data are evaluated with the aid of 480 software was performed by using the Advanced Relative Quantification software module (Roche). The detection format used was the so-called single-color hydrolyzed sample / UPL sample program with the following parameters: excitation filter 483 nm, emission filter 533 nm, filter combination FAM. qPCR data were generated and analyzed using the software module for relative quantification. Absolute qPCR data (CP values) for each target gene were generated using the Abs Quant / 2nd Derivative Max method. The Cp value (crossing point or cycle threshold) is the number of PCR cycles necessary to exceed the determined fluorescence signal threshold. Fluorescence signal acquisition was performed at 72°C at the end of each cycle.

[0066] The normalization of the mRNA expression level of the target gene relative to a reference gene is carried out by means of the so-called E-(efficiency)-method using the Advanced Relative Quantification software module (Roche). Two reference genes (TOP1 and B2M) are each measured together as internal normalization controls for each sample. The expression of the target gene is normalized against these reference genes, and the normalized data are automatically provided by the Advanced Relative Quantification software module as target / reference values (T / R). The mathematical model underlying this software module is based on the relationship between the Cp values of the target gene compared to the reference gene and takes into account in particular the primer-specific amplification efficiency.

[0067] The normalized expression values are used to examine the quality of the chondrocyte grafts. The key quality attributes are consistency, purity, and potential, where for each quality parameter at least one specific marker is identified, which can be measured to detect sufficient quality. In this case, the normalized expression values of the markers must meet the acceptance limits specified to detect sufficient quality. The values regarding the quality parameters are based on the mRNA expression levels in monolayer chondrocytes (KAL1) or spheroids (CRTAC1, NRN1, EBF3, and ACAN).

[0068] Table 3 shows the application of the described selection for chondral graft products. In summary, 48 chondral graft products are prepared as described and are subjected to tests for consistency, purity (contamination), and potential using the markers according to the invention. Forty-eight of the 48 chondral grafts meet the criteria for consistency in monolayer (KAL1) and in spheroids (CRTAC1). Four of the 48 chondral grafts do not meet the purity (contamination) criteria (EBF3, NRN1), while 19 of the 48 chondral grafts do not meet the potential criteria. In summary, 22 of the 48 chondrocyte grafts do not meet the chondrocyte quality (consistency, purity / contamination, or potential) criteria. Twenty-six of the 48 chondrocyte grafts meet the chondrocyte quality (consistency, purity / contamination, or potential) criteria and are suitable for the preparation of transplantable cartilage tissue and are selected.

[0069] Table 3: Selection according to the given thresholds

[0070]

[0071]

[0072] n.d. = not determined. ID = identifier. T / R = target - to - reference ratio.

[0073] Example 3:

[0074] Chondrocytes amplified in monolayer culture with defined quality (here: consistency) are used to prepare cartilage tissue for transplantation.

[0075] From a patient with a cartilage defect to be treated, a biopsy of hyaline cartilage is taken from a healthy cartilage area. Chondrocytes located in the cartilage tissue are isolated by enzymatic digestion of the cartilage tissue with a collagenase solution and are amplified in a cell culture flask (monolayer culture) in the presence of autologous serum under standard cell culture conditions, such that the maximum population doubling level (PDL) does not exceed 10, and sufficient chondrocytes are provided for the preparation of transplantable cartilage tissue. A part of the chondrocytes amplified in monolayer is used to determine chondrocyte consistency, while the rest is used to prepare transplantable cartilage tissue.

[0076] Markers for chondrocyte consistency in monolayer culture indicate the presence of chondrocytes in the chondrocyte culture. For chondrocyte consistency in monolayer culture, total RNA of chondrocytes cultured in monolayer is isolated using common molecular biology techniques and transcribed into cDNA using reverse transcriptase. The expression of at least one reference gene (see above, Table 2) and the consistency marker "KAL1" is determined using common polymerase chain reaction (PCR). The expression of "KAL1" is correlated with the expression of the reference gene, and thus a normalized gene expression value for the consistency marker "KAL1" is obtained. The expression of "KAL1" indicates the presence of chondrocytes with sufficient quality (consistency) for the preparation of transplantable cartilage tissue.

[0077] To prepare transplantable cartilage tissue, at least 100,000 chondrocytes amplified in monolayer with sufficient quality (consistency) are transferred into a cell culture container in the presence of standard cell culture medium and autologous serum. The cell culture container prevents the adhesion of chondrocytes, for example, by an agarose coating on the bottom of the cell culture. After a few days, the chondrocytes adhere to each other and form aggregates (so - called spheroids). The spheroids are cultured for at least a period of 1 - 2 weeks and form a cartilage - like extracellular matrix (spheroid culture). This cartilage tissue (chondrocytes with sufficient quality (consistency) and cartilage - like extracellular matrix) is suitable for establishing cartilage replacement tissue after being introduced into the cartilage defect of the patient to be treated, which fills the defect and assumes the function of natural cartilage tissue. Cartilage tissue with insufficient quality (consistency in monolayer culture) is not suitable for establishing cartilage replacement tissue and is discarded.

[0078] Example 4:

[0079] Prepare cartilage tissue for transplantation using chondrocytes located in spheroid cultures with a defined quality (here: consistency).

[0080] Use the chondrocytes isolated in Example 3 and expanded in monolayer for the preparation of transplantable cartilage tissue.

[0081] As described in Example 3, transfer the chondrocytes expanded in monolayer to a special cell culture vessel that prevents chondrocyte adhesion, so that aggregates (so-called spheroids) are formed after a few days. Culture the spheroids for at least a period of 1 - 2 weeks and form a cartilage-like extracellular matrix (spheroid culture).

[0082] Markers for chondrocyte consistency in the spheroid culture indicate the presence of chondrocytes in the spheroid culture. To determine chondrocyte consistency in the spheroid culture, isolate the total RNA of a part of the chondrocytes cultured as spheroids using common molecular biology techniques and transcribe it into cDNA using reverse transcriptase. Use common polymerase chain reaction (PCR) to determine the expression of at least one reference gene (see above, Table 2) and the consistency marker "CRTAC1". Correlate the expression of "CRTAC1" with the expression of the reference gene, and thus obtain the normalized gene expression value for the consistency marker "CRTAC1". The expression of "CRTAC1" indicates the presence of chondrocytes with sufficient quality (consistency) in the spheroids for transplant cartilage tissue.

[0083] This cartilage tissue (chondrocytes with sufficient quality (consistency) and cartilage-like extracellular matrix) is suitable for establishing cartilage replacement tissue after being introduced into the cartilage defect of the patient to be treated, which fills the defect and undertakes the functions of natural cartilage tissue. Cartilage tissue with insufficient quality (consistency in the spheroid culture) is not suitable for establishing cartilage replacement tissue and is discarded.

[0084] Example 5:

[0085] Prepare cartilage tissue for transplantation using chondrocytes located in spheroid cultures with a defined quality (here: purity).

[0086] Use the chondrocytes isolated in Example 3 and expanded in monolayer for the preparation of transplantable cartilage tissue.

[0087] As described in Example 3, chondrocytes amplified in a single layer were transferred to a special cell culture vessel that prevents chondrocyte adhesion, and aggregates (so-called spheroids) were formed after several days. The spheroids were cultured for at least a period of 1-2 weeks to form a cartilage-like extracellular matrix (spheroid culture).

[0088] Markers for chondrocyte purity indicate the content of chondrocytes in the spheroid culture. To determine the chondrocyte purity in the spheroid culture, total RNA of a portion of the chondrocytes cultured as spheroids was isolated using common molecular biology techniques and transcribed into cDNA using reverse transcriptase. Common polymerase chain reaction (PCR) was used to determine the expression of at least one reference gene (see above, Table 2) and the purity marker "NRN1". The expression of "NRN1" was correlated with the expression of the reference gene, and thus the normalized gene expression value for the purity marker "NRN1" was obtained. If the expression level of "NRN1" exceeds a previously defined purity threshold, such as 1.0, then this indicates that chondrocytes of sufficient quality (purity) are present in the spheroids for transplanting cartilage tissue. If the purity threshold is not exceeded, then chondrocytes of insufficient quality (purity) are present in the spheroids for transplanting cartilage tissue.

[0089] This cartilage tissue (chondrocytes of sufficient quality (purity) and cartilage-like extracellular matrix) is suitable for establishing cartilage replacement tissue after being introduced into the cartilage defect of a patient to be treated, which fills the defect and assumes the function of natural cartilage tissue. Cartilage tissue of insufficient quality (reduced purity) is not suitable for establishing cartilage replacement tissue and is discarded.

[0090] Example 6:

[0091] Prepare the cartilage tissue to be transplanted using chondrocytes in the spheroid culture with a defined cell contamination

[0092] The chondrocytes isolated in Example 3 and amplified in a single layer were used to prepare transplantable cartilage tissue.

[0093] As described in Example 3, spheroids were formed and cultured for at least a period of 1-2 weeks to form a cartilage-like extracellular matrix (spheroid culture).

[0094] Markers for chondrocyte contamination indicate the content of cell contamination with synoviocytes, osteocytes, and / or adipocytes in spheroid cultures. To determine chondrocyte cell contamination in spheroid cultures, total RNA of a portion of chondrocytes cultured as spheroids is isolated using common molecular biology techniques and transcribed into cDNA using reverse transcriptase. Common polymerase chain reaction (PCR) is used to determine the expression of at least one reference gene (see above, Table 2) and the contamination marker "EBF3". The expression of "EBF3" is correlated with the expression of the reference gene, and thus a normalized gene expression value for the contamination marker "EBF3" is obtained. If the expression level of "EBF3" does not exceed a previously defined threshold for contamination, e.g., 0.8, then this indicates that there is no excessive cell contamination by synoviocytes in the spheroids for transplantable cartilage tissue. If the threshold for contamination is exceeded, then there are chondrocytes of insufficient quality (contamination) in the spheroids for transplantable cartilage tissue.

[0095] This cartilage tissue (chondrocytes and chondroid extracellular matrix with sufficient quality (contamination)) is suitable for establishing cartilage replacement tissue after being introduced into the cartilage defect of a patient to be treated, which fills the defect and assumes the function of natural cartilage tissue. Cartilage tissue with insufficient quality (elevated content of cell contamination) is not suitable for establishing cartilage replacement tissue and is discarded.

[0096] Example 7:

[0097] Prepare transplantable cartilage tissue using chondrocytes with defined potential in spheroid cultures

[0098] The chondrocytes isolated in Example 3 and amplified as a monolayer are used to prepare transplantable cartilage tissue.

[0099] As described in Example 3, spheroids are formed and cultured for at least a period of 1 - 2 weeks to form a chondroid extracellular matrix (spheroid culture).

[0100] Markers regarding the potential of chondrocytes indicate that chondrocytes in spheroid cultures have the ability to form a cartilage-like extracellular matrix. To determine the potential of chondrocytes in spheroid cultures, total RNA of a portion of chondrocytes cultured as spheroids was isolated using common molecular biology techniques and transcribed into cDNA using reverse transcriptase. The expression of at least one reference gene and the potential marker "aggrecan" was determined using common polymerase chain reaction (PCR). The expression of "aggrecan" was correlated with the expression of the reference gene, and thus a normalized gene expression value for the potential marker "aggrecan" was obtained. If the expression level of "aggrecan" exceeds a previously defined threshold for potential, such as 0.1, then this indicates that chondrocytes with sufficient quality (potential) are present in the spheroids for transplantation of cartilage tissue. If the threshold for potential is not exceeded, then chondrocytes with insufficient quality (insufficient potential to form a cartilage-like extracellular matrix) are present in the spheroids for transplantation of cartilage tissue.

[0101] This cartilage tissue (chondrocytes with sufficient quality (potential) and a cartilage-like extracellular matrix) is suitable for establishing cartilage replacement tissue after being introduced into a cartilage defect of a patient to be treated, which fills the defect and assumes the functions of natural cartilage tissue. Cartilage tissue with insufficient quality (potential) is not suitable for establishing cartilage replacement tissue and is discarded.

[0102] Example 8:

[0103] Prepare cartilage tissue for transplantation using chondrocytes of determined quality

[0104] The chondrocytes isolated in Example 3 and amplified as a monolayer were used to prepare transplantable cartilage tissue.

[0105] As described in Example 3, spheroids were formed and cultured for at least a period of 1 - 2 weeks to form a cartilage-like extracellular matrix (spheroid culture).

[0106] To qualitatively detect high-quality spheroid cultures for transplantation and for regenerating articular cartilage defects, concordance was determined using the concordance marker "KAL1" as described in Example 3 and using the concordance marker "CRTAC1" as described in Example 4. In addition, as described in Example 5, the purity marker "NRN1" was determined, and / or as described in Example 6, the contamination marker "EBF3" was determined. Further, as described in Example 7, the potential marker "aggrecan" was determined.

[0107] Cartilage tissue (chondrocytes and chondroid extracellular matrix with sufficient quality (homogeneity in monolayer culture and / or in spheroid culture, sufficient purity and / or acceptable degree of cell contamination, and sufficient potential to form a cartilage-like extracellular matrix)) is suitable for establishing cartilage replacement tissue after being introduced into a cartilage defect of a patient to be treated, which fills the defect and assumes the function of natural cartilage tissue. Cartilage tissue with insufficient quality (absence of homogeneity in monolayer culture and / or in spheroid culture, insufficient purity and / or insufficient degree of cell contamination, and insufficient potential to form a cartilage-like extracellular matrix) is not suitable for establishing cartilage replacement tissue and is discarded.

[0108] Example 9:

[0109] The procedure described in Example 8 will be retrospectively applied to the cartilage tissue to be transplanted in 20 patients, in whom cartilage tissue for regenerating knee cartilage injuries is used. The ability of the transplanted cartilage tissue to establish cartilage replacement tissue, i.e., the clinical success of the treatment, is clinically evaluated according to the established cartilage repair score (KOOS – Knee injury and Osteoarthritis Outcome Score: Roos EM & Lohmander LS, Health and Quality of Life Outcomes 2003, I:64). For this purpose, the KOOS (baseline value) of each patient is determined by means of a patient questionnaire before treatment with the cartilage tissue to be transplanted. One year after treatment with the cartilage tissue to be transplanted, the KOOS (1-year follow-up value) of each patient is again determined by means of a patient questionnaire. When the difference (KOOSΔ) between the 1-year follow-up value and the baseline value is at least 8 points, a clinical or therapeutic success of the treatment is obtained (Roos EM & Lohmander LS, Health and Quality of Life Outcomes 2003, I:64). If the difference (KOOSΔ) between the 1-year follow-up value and the baseline value is less than 8 points, then there is no clinical improvement caused by the treatment, i.e., the therapy is not successful.

[0110] It is demonstrated that in the transplantable cartilage tissues of 4 out of 20 patients (5697 - 1607, 5862 - 1311, 6070 - 2413, 6988 - 2423), the potential marker "aggrecan" is below the determined limit value, while the consistency of chondrocytes can be determined by means of the consistency markers "KAL1" or "CRTAC1". The expression value is above the determined limit value. The purity of the cartilage tissue to be transplanted can also be measured and given by means of the contamination marker "EBF3"; here, the expression value is below the determined limit value. Therefore, these 4 cartilage tissues to be transplanted show insufficient quality for establishing cartilage replacement tissue and are discarded. This corresponds to a rejection rate of 20% (4 out of 20 cartilage tissues to be transplanted).

[0111] In the transplantable cartilage tissues of 16 out of 20 patients (6266 - 2601, 6658 - 2420, 7494 - 2284, 8528 - 2286, 9070 - 2709, 9110 - 2294, 5669 - 2263, 9110 - 2294, 6094 - 1312, 6340 - 2277, 6611 - 2418, 8315 - 2434, 8916 - 2440, 8931 - 1126, 8948 - 2706, 8966 - 2441), the potential marker "aggrecan" is above the determined limit value, and the consistency of chondrocytes can be determined by means of the consistency markers "KAL1" or "CRTAC1". The expression value is also above the determined threshold value. The purity of the cartilage tissue to be transplanted can also be measured and given by means of the contamination marker "EBF3"; here, the expression value is below the determined threshold value. Therefore, these 16 cartilage tissues to be transplanted show sufficient quality for establishing cartilage replacement tissue and are used for transplantation. This corresponds to a success rate of 80% (16 out of 20 cartilage tissues to be transplanted).

[0112] Thus, the transplantable cartilage tissue of 4 out of 20 patients (5697 - 1607, 5862 - 1311, 6070 - 2413, 6988 - 2423) showed cartilage tissue with insufficient quality (absence of consistency in monolayer culture and / or in spheroid culture, insufficient purity and / or insufficient degree of cell contamination, and insufficient potential to form a cartilage-like extracellular matrix), while the transplantable cartilage tissue of 16 out of 20 patients (6266 - 2601, 6658 - 2420, 7494 - 2284, 8528 - 2286, 9070 - 2709, 9110 - 2294, 5669 - 2263, 9110 - 2294, 6094 - 1312, 6340 - 2277, 6611 - 2418, 8315 - 2434, 8916 - 2440, 8931 - 1126, 8948 - 2706, 8966 - 2441) had cartilage tissue with sufficient quality (presence of consistency in monolayer culture and / or in spheroid culture, sufficient purity and / or sufficient degree of cell contamination, and sufficient potential to form a cartilage-like extracellular matrix).

[0113] Regarding the clinical or therapeutical success of the treatment, it was demonstrated (see Table 4) that in the 4 patients out of 20 (5697 - 1607, 5862 - 1311, 6070 - 2413, 6988 - 2423) (in whom transplantable cartilage tissue with insufficient quality was retrospectively determined), there was no clinical improvement caused by the treatment. The difference between the KOOS 1-year follow-up value and the KOOS baseline value was less than 8 points, so the therapy was not successful. In contrast, it was demonstrated that in the 16 patients out of 20 (6266 - 2601, 6658 - 2420, 7494 - 2284, 8528 - 2286, 9070 - 2709, 9110 - 2294, 5669 - 2263, 9110 - 2294, 6094 - 1312, 6340 - 2277, 6611 - 2418, 8315 - 2434, 8916 - 2440, 8931 - 1126, 8948 - 2706, 8966 - 2441) (in whom transplantable cartilage tissue with sufficient quality was retrospectively determined), there was clinical improvement caused by the treatment. The difference between the KOOS 1-year follow-up value and the KOOS baseline value was at least 8 points, so the therapy was successful.

[0114] Table 4: Quality markers for consistency, purity / contamination, and potential of 20 patients with known clinical therapy outcomes (KOOSΔ)

[0115]

[0116]

[0117] KOOS: Knee injury and Osteoarthritis Outcome Score. n.d. = not determined.

Claims

1. Use of a marker in the preparation of a reagent for selecting chondrocytes for the preparation of transplantable cartilage tissue, wherein the transplantable cartilage tissue is prepared by a method comprising: Determining a concordance marker and a purity marker in vitro, wherein the marker is determined in comparison with a reference gene B2M or TOP1, and determining the ratio of the following gene expressions: CRTAC1 / R > 0.003 NRN1 / R ≥ 0.1 KAL1 / R > 1.0 EBF3 / R ≤ 0.4 and ACAN / R ≥ 0.29; Amplifying the chondrocytes in a monolayer culture, and Aggregating the amplified chondrocytes into spheroids.

2. The use according to claim 1, characterized in that, Selecting the spheroids.

3. The use according to claim 1, characterized in that, i.) Determining chondrocytes in a monolayer culture with a concordance marker KAL1 in vitro, and / or ii.) Determining chondrocytes in spheroids with at least one concordance marker CRTAC1 in vitro.

4. The use according to claim 1, characterized in that, The proportion of synovial cells in the isolated chondrocytes after amplification or in the obtained spheroids is less than or equal to 9% compared to the total cell number.

5. The use according to claim 1, characterized in that, The proportion of synovial cells in the isolated chondrocytes after amplification or in the obtained spheroids is less than or equal to 5% compared to the total cell number.

6. Isolated spheroids obtainable by the method defined in one of claims 1 to 5.

7. An isolated spheroid obtainable by a method as defined in any one of claims 1 to 5, characterized in that, The proportion of synovial cells in the spheroids is less than or equal to 9% compared to the total cell number.

8. Transplantable cartilage tissue comprising the spheroids according to claim 7.

9. Use of the transplantable cartilage tissue according to claim 8 in the preparation of a reagent for treating articular cartilage defects or for autologous chondrocyte transplantation.

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