EP3645067A1

EP3645067A1 - Method for producing transplantable cartilaginous tissue

Info

Publication number: EP3645067A1
Application number: EP18740519.6A
Authority: EP
Inventors: Giulietta ROÉL; Christian Kaps; Claudia ESCHEN
Original assignee: Co Don AG
Current assignee: CO.DON GMBH
Priority date: 2017-06-25
Filing date: 2018-06-22
Publication date: 2020-05-06
Also published as: CN110944684A; CA3067770A1; EA202090133A1; JP2020525107A; CN114540285A; EP3417888A1; WO2019002148A1; BR112019027474B1; JP2023001205A; US20200147269A1; BR112019027474A2; CN110944684B; BR112019027474B8

Abstract

The present invention relates to a method for producing cartilaginous tissue by means of selection of and sampling from chondrocytes or spheroids and the use thereof as pharmaceutical composition, drug or transplant. The invention specifically relates to markers for identifying suitable chondrocytes for producing cartilaginous tissue, in particular for the purpose of transplantation.

Description

Method for producing transplantable cartilage tissue

description

The present invention relates to a method for

Production of cartilage tissue by selection and selection from chondrocytes or spheroids and its use as

pharmaceutical composition, pharmaceutical or

Graft. In particular, the invention relates to markers for identifying suitable chondrocytes for producing cartilage tissue, in particular for transplantation purposes. Although articular cartilage shows remarkable resilience, this tissue has no or very little ability to self-repair and regenerate, and untreated lesions, for example, can cause osteoarthritis. This low potential for spontaneous regeneration led to the development of cell therapies, such as the autologous

Chondrocyte transplantation (ACT), in an effort to

functional and painless repair of

To provide articular cartilage defects. There is a great need for methods of cartilage regeneration, for example, in young active patients with traumatic lesions or even with symptoms of cartilage degeneration or

Treatment of cartilage defects.

Biochemical and molecular studies with human chondrocytes have been hampered by a number of factors, including the

lack of availability of human tissue in conjunction with the very low number of cells involved in a biopsy

the limited proliferation capacity and high phenotypic instability of cultured chondrocytes.

A chondrocyte (cartilage cell) is a cell derived from chondroblasts and resident in cartilage tissue. Together with the intercellular substances (extracellular matrix (ECM)), the chondrocytes are the main constituents of the cartilage, in particular articular cartilage.

It is already known, by mimicking certain processes of embryonic development, e.g. human cartilage cells

cultivate three-dimensionally on an agarose base, so that cell aggregates are formed, which in their

Differentiation status superior to monolayer cells and e.g. have cartilage-like properties. These

Properties that are as close as possible to the native

Reflecting articular cartilage tissue is characterized by the expression of collagen II (main structural protein of the extracellular matrix of hyaline cartilage) of

Proteoglycans, e.g. Aggrecan and the intracellular

chondrocyte-specific protein S100.

Moreover, it is desirable that the expression of collagen I, which is inevitably upregulated in the monolayer culture during the cell proliferation phase, be included in the

Cell aggregates is reduced again, since this protein does not occur in the native articular cartilage. The so generated

Cell aggregates (spheroids) have been used since 2002 by the Applicant for the treatment and treatment of injuries

Cartilage defects used in younger patients. For this purpose, the patient's body's own articular cartilage tissue, namely a biopsy of hyaline cartilage taken from a healthy cartilage area. By enzymatic digestion of the cartilage tissue with a collagenase solution, those in the cartilage tissue

located cartilage cells (chondrocytes) isolated and using standard cell culture conditions in cell culture bottles

(Monolayer culture) in the presence of autologous serum

increased, and sufficient chondrocytes for the production of transplantable cartilage tissue. The increased cells form cell aggregates, so-called

Spheroids that can be transplanted. However, the cell aggregates thus produced are in their

Differentiation status limits and usually shows a relatively weak local expression of the important collagen II and a marginal synthesis of proteoglycans with relatively strong expression of the unwanted collagen I. To the

To further improve differentiation of these cell aggregates, it is possible to enrich the culture medium with certain bioactive substances. These include, above all, the growth factors TGF-β1-3 described extensively in the literature and L-ascorbic acid (vitamin C) as cofactor for collagen synthesis. However, this biochemical ranges

On the one hand, stimulation is often not enough for the

To induce differentiation of cartilage cells in the 3D cell aggregate to the extent that cartilage-typical constructs arise and on the other hand, the use of growth factors such as TGF-ß in particular for a clinical application in humans

questionable.

The cultivation of human cells in the form of

three-dimensional cell aggregates, e.g. in the form of spheroids, is already in clinical use in humans, e.g. described as autologous cartilage graft in DE 100 13 223 and relates to a process for the in vitro production of

three-dimensional cartilage and bone tissue from bone, cartilage or mesenchymal stem cells. The cells are first cultivated in a Monolayerkultur and then in suspension until a cell aggregate is formed, which contains at least 40% by volume extracellular matrix, in which differentiated cells are embedded. In the process, cell aggregates are prepared by cultivating cells for at least 1-2 weeks in agarose-coated cell culture vessels.

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

Other et al. (Journal of Bone and Mineral Research (2002), New York, NY, US, Vol. 17, No. 8, pp. 1420-1429) also discloses a method for the in vitro production of

three-dimensional cartilage tissue. Following this procedure, 1 × 10 ⁵ or 2 × 10 ⁵ chondrocytes are cultured for 5 days, 2 weeks, 1, 2, and 3 months to produce sphaerides.

The in vitro produced cartilage tissues described in the prior art do not show expression patterns of essential matrix proteins like collagen type II, which are similar to those of the native tissues. Rather, the composition of the extracellular matrix (ECM), which differs in vitro from the

Chondrocytes is produced, clearly from the native

Cartilage tissue off. However, the composition of the ECM is crucial for the biological functionality of the cartilage, for example its mechanical strength. There is therefore a need for new methods for the production of cartilage tissue, also for quality control.

"Transplantable cartilage tissue" means production ex vivo or by means of an in vitro method, whereby the tissue produced corresponds to a large extent to the native tissue or is identical to the native tissue For the purposes of this invention, the transplantable cartilage tissue corresponds for example, in terms of expression or

Expression pattern of the extracellular matrix largely that of the native tissue, for example in relation to the

Expression or expression patterns of structural proteins or proteids such as collagen type II and / or S100 protein and / or the tissue-specific proteoglycans, e.g. Cartilage-specific proteoglycans, such as aggrecan. The detection of the expression or expression pattern can e.g. histologically or

immunohistologically. The tissue from which the chondrocytes are isolated is, for example, selected from musculoskeletal tissue,

Skeletal tissue, cartilage, bone, meniscus, disc,

Bone marrow, joint or tendon. The transplantable cartilage tissue produced according to the method of the invention resembles the native tissue in terms of composition and protein expression (supra).

Native articular cartilage tissue has a proportional

Composition of the extracellular matrix of about 60 to 80% water in relation to the wet weight of the cartilage tissue. The high water content is important for the mechanical

Resilience of cartilage tissue and together with the

Proteoglycans for the "sponge effect." In addition to water, the native cartilage joint contains the structural proteins or

Proteins of the matrix, such as collagens, proteoglycans and non-collagen proteins. The structural ones do

Macromolecules about 20 to 40% of the wet weight of the

Articular cartilage tissue.

In native articular cartilage, the collagen content is 60%, the proteoglycan content is 25 to 35%, and the non-collagen protein and glycoprotein content is 15 to 20% on the cartilage dry weight. Type II collagen accounts for approximately 90 to 95% of the total collagen content of the native

Articular cartilage tissue.

Native articular cartilage tissue in humans contains about 1 to 5% of cartilage cells (chondrocytes) in terms of tissue volume. The cartilage cells are thereby the essential producers of the matrix molecules of the functional native tissue.

There is a great need to provide chondrocytes of sufficient quality and quality

can produce suitable transplantable cartilage tissue, which corresponds to the native tissue, so that a therapeutic success can be expected. This therapeutic success largely depends on the quality of the transplantable cartilage tissue.

Therefore, the object of providing a

improved transplantable cartilage tissue, in particular with improved properties, in particular of sufficient quality, such as identity with native chondrocytes and

qualitatively improved spheroids, allowing high quality

Transplants can be obtained, in particular for

successful treatment and therapy of

Articular cartilage defects.

Surprisingly, the inventors were able to identify markers in isolated chondrocytes, which allow a sufficient quality for the production of transplantable cartilage tissue. The markers according to the invention allow in particular the

Determination of cartilage cell identity ("identity marker") from the isolated chondrocytes in one culture to the native chondrocytes.

An appropriate cartilage cell identity in turn allows the Production of suitable transplantable cartilage tissue, since on the one hand a sufficient cartilaginous extracellular matrix (ECM) can be achieved, on the other hand a

improved treatment of articular cartilage defects is achieved.

In a further embodiment, the allow

Marker according to the invention, the determination of the purity or contamination of the isolated chondrocytes or spheroids ("purity marker") .The aforementioned markers ensure a sufficient

Quality and therefore represent "quality markers".

Therefore, the invention relates to a method for the selection of chondrocytes or spheroids for the production of

transplantable cartilage tissue, wherein at least one

Quality marker or identity marker or purity marker is determined.

The inventive method for the selection of chondrocytes or spheroids for the production of transplantable

Cartilage tissue includes in a first step that the

Chondrocytes can be propagated in a monolayer culture (supra, so-called two-dimensional expansion culture), and in a second step can aggregate to spheroids (supra, so-called 3D cultivation), allowing the chondrocytes to

Spheroids aggregate, and spheroids can be selected.

In a preferred embodiment, the quality marker or purity marker is selected from the group KALI, CRTAC1, NRN1 or EBF3, which is (are) determined in vitro. In a preferred embodiment, the quality markers or identity markers are selected from the group KALI, CRTAC1, and / or the purity markers are EBF3, NR 1. In particular, synoviocytes are detrimental contaminants that can be determined by the purity marker EBF3 in vitro. Such impurities reduce the quality of chondrocytes as well as spheroids.

In a further preferred embodiment, in addition to the aforementioned markers, a so-called potential marker can be applied according to the invention, which allows a prediction of the activity and regeneration capacity of the transplantable cartilaginous tissue to be obtained from the spheroids. Such potential markers according to the invention are preferably proteoglycans, more preferably aggrecan (ACAN). Table 1: Marker genes according to the invention

Functions in the

Accession

Marker designation Quality test

No.

as well as

selection

homo sapiens

cartilage acidic

Chondrogenic protein 1

CRTAC1 NM_018058 Identity marker

(CRTAC1).

for spheroids transcript

variant 1. mRNA

homo sapiens

neuritin 1 chondrogener

NRN1 NM_001278710 (NRN1). Purity marker transcript for spheroids variant 2. mRNA

homo sapiens

KALI Chondrogener

NM_000216 anosmin 1

(ANOS1) Identity marker

(ANOS1). mRNA

For Monolayer culture

homo sapiens

Chondrogenic early B-cell

EBF3 NM_001005463 Purity marker factor 3 (EBF3).

for spheroids mRNA

homo sapiens

aggrecan (ACAN). Potentiality markers

ACAN NM_001135

transcript for spheroids variant 1. mRNA

The associated DNA sequences can be uniquely identified by accession number (e.g., NCBI).

The in vitro determination of the markers according to the invention is preferably carried out by determining the gene expression of these markers as well as the level of gene expression in the isolated chondrocytes and also preserving spheroids, which

For example, routinely with the polymerase chain reaction (PCR, in particular qPCR) can be found, see examples. According to the invention, the markers show gene expression as well as increased or decreased gene expression in the method according to the invention, so that they are suitable as markers.

In a further embodiment, the determination of the gene expression or gene expression level can also be carried out in the presence and in the comparison of reference genes and their gene expression or gene expression level. This allows one

Normalization of gene expression as well as their

Quantification, also by means of software support, see Michael W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Research, 2001, Vol. 9th

Table 2: Reference genes according to the invention Functions in the

Accession

Reference gene Designation Quality test

No.

as well as

selection

homo sapiens

beta-2-

B2M NM_004048 reference gene

microglobulin

(B2M). mRNA

homo sapiens

topoisomerase

TOP1 NM_003286 reference gene

(DNA) I (TOP1).

mRNA

The associated DNA sequences are accessible via accession no.

to uniquely identify (e.g., NCBI).

In the context of this invention, "selection" means that

For example, such chondrocytes or spheroids are discarded which have no gene expression of the marker according to the invention or in relation to other chondrocytes or spheroids have a comparatively low gene expression or gene expression level. The person skilled in the art is able, on the basis of the gene expression patterns obtained, to differentiate a stronger gene expression from a weaker gene expression to one or more chondrocytes / spheroids and accordingly to select such chondrocytes or spheroids.

In a further preferred embodiment, therefore, such isolated chondrocytes after multiplication as well as spheroids obtained therefrom are to be selected as well as selected, the following ratios of gene expression to one or both reference genes (R) namely B2M and / or TOP1

respectively :

CRTAC1 / R> 0.003 NRN1 / R> 0, 1

KALI / R> 1.0

EBF3 / R <0.4

ACAN / R> 0, 29 Further preferred ratios are such as

CRTAC1 / R> 0.006, in particular> 0.009 and> 0.012

NRN1 / R> 0.2, in particular> 0.3 and> 0.4

KALI / R> 2.0, in particular> 3.0 and> 4.0

EBF3 / R ^ 0.3, in particular <0.2 and <0.1 ACAN / R> 0.39, in particular> 0.49 and> 0.59

The stated values are suitable

Thresholds.

Transplant ierbarem cartilage tissue, wherein in comparison to a reference gene, the markers of the invention are determined.

In a further preferred embodiment, the proportion of synoviocytes as an impurity in the isolated chondrocytes after multiplication or in the spheroids obtained in relation to the total number of cells is less than or equal to 9%, in particular less than or equal to 5%, in the course of the determinations carried out by the purity smarker EBF3. ,

The inventive method allows the selection of

Chondrocytes as well as spheroids specific for one too transplantable cartilage tissue. This is due to the particular properties of the markers according to the invention, namely quality markers or identity markers or

Purity marker. Consequently, such chondrocytes as well as spheroids are selected which have specific gene expression and corresponding gene products can be detected, for example by means of PCR.

Therefore, the invention relates to isolated chondrocytes or isolated resulting spheroids obtainable by

inventive method. Furthermore includes is a

transplantable cartilage tissue, in particular in the form of an agent or pharmaceutical composition or in the form of a pharmaceutical composition containing such chondrocytes or

Spheroids, in particular for use in the treatment of articular cartilage defects and / or autologous

Chondrocyte transplantation.

In a further preferred embodiment of the invention, the transplantable cartilage tissue according to the invention is made into a patient with the aid of an applicator (see EP 2345450B1 (co.fix 150 of the applicant) or DE102009025347A1)

administered. Preferably, the transplanted

Cartilage tissue in the form of a pharmaceutical composition in a physiological saline solution, together with other auxiliaries, additives are kept. The following figures and examples serve to illustrate the invention without restricting the invention to the figures and examples.

Example 1 : Isolation of cartilage cells (chondrocytes) from cartilage biopsy and cell culture

Cartilage-specific cells (chondrocytes) were isolated from cartilage biopsy by enzymatic digestion using collagenase (350 units / ml). The enzymatic digestion took between 4 and 6 hours and took place at 37 ° C. Cartilage biopsy was taken from the femoral condyle of the knee joint. After enzymatic digestion, the chondrocytes were centrifuged, washed, counted and placed in tissue culture flasks together with cell culture medium (Alpha MEM / Ham's F12 1: 1, 1% glutamine) and autologous serum (10-15%).

Human serum). For expansion, the cells were cultured in vitro for 7 to 38 days at 37 ° C in the two-dimensional expansion culture (monolayer). The exchange of cell culture medium took place every 3-6 days. If the

Cell cultures were confluent, the cell culture was passaged using papain and several culture bottles

divided up .

After reaching a sufficient number of cells from the monolayer culture each 200,000 cells per well in

Dried microtiter plates coated with 2% agarose. The cells aggregate and form one

three-dimensional, round structure (spheroid). During the

Cultivation of the spheroids was periodically changed (every 3-6 days) to cell culture medium (Alpha MEM / Ham's F12 1: 1; 1% glutamine) supplemented with 10% autologous serum. The 3D cultivation took between 14 and 42 days.

After spheroid cultivation, the

Cartilage cell graft prepared for transport and implantation by placing the spheroids in the drug carrier (0.9% NaCl) and then into the applicator system co. fixed 150 or a syringe convicted.

In summary, there is the production of

Cartilage cell transplants from a monolayer culture phase to the isolated chondrocytes

and to aggregate a 3D culture phase around the chondrocytes into spheroids. The cartilage cell graft is intended for autologous use, ie implantation in the knee of the same patient. Example 2: a.) RNA isolation

The cartilage cells were transformed into a two-dimensional expansion culture (monolayer) until the spheroids were produced

cultivated and then using an enzyme solution

(Papain) from the bottom of the cell culture bottle and from the

Cell structure solved. lxlOE6 of these cartilage cells were removed from the cell suspension for identity verification and transferred to a reaction vessel, while the remaining cells were used for the subsequent production of the spheroids. The test-relevant monolayer cells were centrifuged off and the cell pellet was lysed in the appropriate lysis buffer (PeqGOLD, Peqlab), snap-frozen and stored at -70 ° C. until RNA isolation. For marker testing on cartilage cell spheroids, 8 spheroids were removed from the well plate after two weeks of culture, washed with PBS and lysed with the appropriate lysis buffer (PeqGOLD, Peqlab). The spheroids incorporated in the lysis buffer were mechanically prepared for optimal cell disruption by means of a syringe and cannula

Homogenized by the spheroids were added and removed. The lysate then became liquid nitrogen

Shock frozen and stored at -70 ° C until RNA isolation.

Extraction of total RNA from the cartilage cell spheroids and monolayer cells was carried out using the peqGOLD Total RNA Kit from Peqlab or with 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 measured using a spectrophotometer (NanoDrop, Thermo Scientific) and a Bioanalyzer (Agilent) according to the instructions of the

Manufacturer determined. b.) cDNA synthesis

The cDNA synthesis was carried out using the Transcriptor First

Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. For each sample, 80 ng of total RNA was transcribed using random hexamer primers. In a sterile reaction vessel, the template-primer mix for a reaction volume of 20 μΐ was set. The following additional components were added to the template-primer mix:

Transcriptor Reverse Transcriptase Reaction Buffer, Protector RNase Inhibitor, Deoxynucleotide Mix, Transcriptor Reverse Transcriptase. The reaction mixtures were mixed,

centrifuged down and then in a thermocycler

incubated. The reverse-transcribed cDNA was then diluted with nuclease-free water to a final concentration of 1 ng / μΐ. The samples were stored at -20 ° C in the qPCR analyzes until further use. c.) qPCR For the qPCR analyzes, the qPCR technique with the LightCycler® 480 Instrument II (Roche) in 384-well format was used. The primers were HPLC purified and were in

nuclease-free water to a concentration of 10 μΜ dissolved. The probes were used at a concentration of 10 μΜ. For the markers to be measured previously defined qPCR reaction conditions with respect to the primer and

Probe concentration and established for the use of additives. For all samples, a master mix was prepared according to the reaction condition of the specific primers.

In each case 8 μΐ of the reaction mixture specific for each target gene was pipetted into the corresponding well of the 384-well plate and then 2 μΐ cDNA sample (1 ng / μΐ)

added, which resulted in a total volume of 10 μΐ per qPCR reaction.

The qPCR analysis is based on the exponential increase of the fluorescence-labeled PCR amplicons of the gene of interest. The evaluation of the qPCR data was carried out with the aid of

LightCycler® 480 software using the software module Advanced Relative Quantification (Roche). The used

Detection format was the so-called mono-color hydrolysis

Sample / UPL sample program with the following parameters:

Excitation filter 483 nm, emission filter 533 nm,

Filter combination FAM. qPCR data was used with the

Software modules for relative quantification generated and analyzed. Absolute qPCR data (CP values) of the respective target genes were generated using the method Abs Quant / 2nd Derivative Max. The Cp value (Crossing point) is the number of PCR cycles required to reach a defined fluorescence signal threshold exceed. Fluorescence signal acquisition was performed at 72 ° C at the end of each cycle.

The normalization of the mRNA expression levels of the target genes compared to reference genes was using the so-called E (efficiency) method using the software module

Advanced Relative Quantification (Roche) performed. Two reference genes, TOP1 and B2M, were each considered internal

Normalization control measured for each sample. The

Expression of the target genes was normalized to these reference genes, and normalized data was automatically provided as Target / Reference Values (T / R) by the Advanced Relative Quantification software module. The mathematical model underlying this software module is based on the relationship between the Cp values of the target gene compared to the reference genes and takes into account i.a. the primer-specific

Amplification efficiency.

The normalized expression values are used for testing the quality of cartilage cell transplants. critical

Quality attributes represent identity, purity and potential, with at least one for each quality parameter

specific marker that can be measured to demonstrate sufficient quality. The

normalized expression values of a marker must be defined for the detection of sufficient quality

Fulfill acceptance limits. The value for the quality parameters is based on mRNA expression levels in monolayer cartilage cells (KALI) or spheroids (CRTAC1, NRN1, EBF3 and ACAN).

Table 3 shows the application of the described selection to cartilage graft products. Altogether 48 Cartilage graft products prepared as described and subjected to a test for identity, purity (contamination) as well as potential with the markers according to the invention. Forty - eight of the 48 cartilage grafts met the

Identity criteria in monolayer (KALI) and spheroid

(CRTAC1). Four of the 48 cartilage grafts did not meet the criteria of purity (contamination) (EBF3, NRN1) and 19 of the 48 cartilage grafts did not fulfill the criterion of

Potential. Altogether 22 of the 48 completed

Cartilage cell transplants are not the criterion of

Cartilage cell quality (identity, purity / contamination or potential). Twenty-six of the 48 cartilage cell transplants met the criterion of cartilage cell quality (identity, purity / contamination or potential) and are known as

Production of transplantable cartilage tissue and were selected.

Table 3: Selection according to the specified thresholds

Example 3:

Production of cartilage tissue to be transplanted using quality defined in monolayer culture of cartilage cells, here: identity

From the patient with the cartilage defect to be treated, a healthy cartilage area becomes a biopsy of hyaline cartilage

taken. By enzymatic digestion of the cartilage tissue with a collagenase solution, those in the cartilage tissue

(Monolayer culture) in the presence of autologous serum

multiplied so that a maximum population doubling level (PDL) of 10 is not exceeded and sufficient

Chondrocytes for the production of transplantable

Cartilage tissues are available. Part of the cartilage cells propagated in the monolayer is used to determine the

Cartilage cell identity and the remainder of the preparation of transplantable cartilage tissue.

The marker for cartilage cell identity in monolayer culture indicates that cartilage cells are involved in cartilage cell culture

available. For cartilage cell identity in the Monolayerkultur is using conventional molecular biology techniques the

Total RNA of the monolayer cultured chondrocytes isolated and transcribed by means of reverse transcriptase into cDNA.

The expression of at least one reference gene (supra, Table 2) and the identity marker "KALI" is determined by means of conventional polymerase chain reaction (PCR) .The expression of "KALI" is set in relation to the expression of the reference gene and thus yields a normalized gene expression value for the identity marker "KALI." The expression of "KALI" indicates that chondrocytes of sufficient quality (identity) for the production of transplantable cartilage tissue

present.

To produce the transplantable cartilage tissue, at least 100,000 of the monolayer-enhanced chondrocytes of sufficient quality (identity) are transferred to a cell culture vessel in the presence of standard cell culture media and autologous serum. The cell culture vessel prevents attachment of the chondrocytes by means of, for example, an agarose coating of the cell culture soil. After a few days, the chondrocytes adhere to each other and form aggregates, so-called spheroids. The spheroids are cultured for a period of at least 1-2 weeks and form a cartilaginous extracellular matrix (spheroid culture). This cartilage tissue (chondrocytes of sufficient quality (identity) and cartilaginous

extracellular matrix) is suitable for insertion into the cartilage defect of the patient to be treated

Build cartilage replacement tissue that fills the defect and takes over the function of the natural cartilage tissue. Cartilage tissue of insufficient quality (identity in the

Monolayer culture) is not suitable for building cartilage replacement tissue and is discarded.

Example 4: Production of Cartilaginous Tissue to be Transplanted by Spheroid Culture Cartilage Cells

Quality, here: identity

The isolated in Example 3 and multiplied in the monolayer

Cartilage cells are used to make transplantable cartilage tissue.

As described in Example 3, the chondrocytes propagated in the monolayer are transferred to a special cell culture vessel, which prevents attachment of the chondrocytes, so that after a few days aggregates, so-called

Spheroids, training. The spheroids are cultured for a period of at least 1-2 weeks and form a

cartilaginous extracellular matrix from (spheroid culture).

The cartilage cell identity marker in the spheroid culture indicates that cartilage cells are present in the spheroid culture. To determine the cartilage cell identity in the spheroid culture, the total RNA of a part of those cultivated in spheroids is by means of common molecular biological techniques

Chondrocytes isolated and transcribed by means of reverse transcriptase into cDNA. By means of conventional polymerase chain reaction (PCR), the expression of at least one reference gene

(supra, Table 2) and the identity marker "CRTAC1"

certainly. The expression of "CRTAC1" is set in relation to the expression of the reference gene and thus gives a normalized gene expression value for the identity marker "CRTAC1". The expression of "CRTAC1" indicates that

Chondrocytes of sufficient quality (identity) in the spheroid present for the transplantation of cartilage tissue.

This cartilage tissue (chondrocytes of sufficient quality (identity) and cartilaginous extracellular matrix) is

suitable to build up after insertion into the cartilage defect of the patient to be treated a cartilage replacement tissue, which fills the defect and takes over the function of the natural cartilage tissue. Cartilage tissue inadequate

Quality (identity in the spheroid culture) is not for

Construction of cartilage replacement tissue suitable and is discarded. Example 5:

Production of Cartilage Graft to be Transplanted by Spheroid Culture Cartilage Cells

Quality, here: purity

The isolated in Example 3 and multiplied in the monolayer

Cartilage cells are used to make transplantable cartilage tissue.

cartilaginous extracellular matrix from (spheroid culture).

The marker for cartilage cell purity indicates the content

Cartilage cells in the spheroid culture. To determine the cartilage cell purity in the spheroid culture, the total RNA of a part of the chondrocytes cultivated in spheroids is isolated by means of conventional molecular biological techniques and transcribed into cDNA by means of reverse transcriptase.

By means of conventional polymerase chain reaction (PCR) is the

Expression of at least one reference gene (supra, Table 2) The expression of "NRN1" is set in relation to the expression of the reference gene and thus gives a normalized gene expression value for the purity marker "NRN1." If the expression level of "NRN1" exceeds the previously defined threshold value for the NRN1

Purity, e.g. 1.0, this indicates that chondrocytes

sufficient quality (purity) in the spheroid

Transplantation of cartilage tissue. Is the

Threshold for purity falls below

Chondrocytes of insufficient quality (purity) in the spheroid for the transplantation of cartilage tissue before.

This cartilage tissue (chondrocytes of sufficient quality (purity) and cartilaginous extracellular matrix) is suitable for building up a cartilage replacement tissue after introduction into the cartilage defect of the patient to be treated

Fills defect and takes over the function of natural cartilage tissue. Cartilage tissue of inferior quality (reduced degree of purity) is not suitable for building up cartilage replacement tissue and is discarded. Example 6:

Production of Cartilage Tissue to be Transplanted by Spheroid Culture Cartilage Cells and Defined Cellular Impurities

The isolated in Example 3 and multiplied in the monolayer

Cartilage cells are used to make transplantable cartilage tissue.

As described in Example 3, spheroids are formed and cultured for a period of at least 1-2 weeks to form a cartilaginous extracellular matrix

(Spheroid culture).

The marker for cartilage cell contamination indicates the content cellular impurities with synovial cells, bone and / or fat cells in the spheroid culture. To determine cellular impurities of cartilage cells in

Sphäroidkultur is isolated by means of conventional molecular biology techniques, the total RNA of a part of the chondrocytes cultivated in spheroids and by means of reverse

Transcribed into cDNA transcriptase. By means of common

Polymerase chain reaction (PCR) is the expression of at least one reference gene (supra, Table 2) and the

The expression of "EBF3" is set in relation to the expression of the reference gene and thus results in a normalized gene expression value for the contaminant marker "EBF3"

Expression level of "EBF3" the previously defined threshold for contamination, eg 0.8, indicates that there is no excessive cellular contamination by synoviocytes in the spheroid for transplanting the cartilaginous tissue. When the threshold for contamination is exceeded, chondrocytes are of insufficient quality (contamination ) in the spheroid for the transplantation of cartilage tissue.

This cartilage tissue (chondrocytes sufficient quality

(Contaminant) and cartilaginous extracellular matrix) is suitable, after introduction into the cartilage defect of the patient to be treated, to build up a cartilage replacement tissue which fills the defect and takes over the function of the natural cartilaginous tissue. Cartilage tissue inadequate

Quality (increased content of cellular contaminants) is not suitable for building up cartilage replacement tissue and is discarded. Example 7: Production of Cartilaginous Tissue to be Transplanted by Sphiloid Culture Cartilage Cells

potential

The isolated in Example 3 and multiplied in the monolayer

Cartilage cells are used to make transplantable cartilage tissue.

(Spharoid culture).

The marker for cartilage cell potential indicates that

Cartilage cells in the sphoid culture have the ability to form cartilaginous extracellular matrix. To determine the potential of cartilage cells in spheroid culture, the total RNA of a part of the chondrocytes cultivated in spheroids is isolated by means of conventional molecular biological techniques and transcribed into cDNA by means of reverse transcriptase.

By means of conventional polymerase chain reaction (PCR), the expression of at least one reference gene and the

Potential marker "Aggrecan" determines the expression of

"Aggrecan" is set in relation to the expression of the reference gene and thus gives a normalized gene expression value for the potential marker "aggrecan". Exceeds the

Expression level of "Aggrecan" the previously defined

Threshold for the potential, e.g. 0.1, this indicates that chondrocytes of sufficient quality (potential) in the

Spheroid for the transplantation of cartilage tissue present. If the threshold for the potential has been undershot, chondrocytes of insufficient quality (insufficient potential to form cartilaginous extracellular matrix) are present in the spheroid for transplanting the cartilaginous tissue.

This cartilage tissue (chondrocytes sufficient quality (Potential) and cartilaginous extracellular matrix) is suitable, after introduction into the cartilage defect of the patient to be treated, to build up a cartilage replacement tissue which fills the defect and takes over the function of the natural cartilaginous tissue. Cartilage tissue inadequate

Quality (potential) is not to build up

Cartilage replacement tissue suitable and is discarded.

Example 8:

Production of cartilage tissue to be transplanted by means of cartilage cells of defined quality

The isolated in Example 3 and multiplied in the monolayer

Cartilage cells are used to make transplantable cartilage tissue.

(Spheroid culture).

To detect high quality spheroid cultures for transplantation and regeneration of articular

Cartilage defects, as described in Example 3, the

Identity by means of the identity marker "KALI" and by means of the identity marker "CRTAC1", as described in Example 4, determined. In addition, as described in Example 5, the

Purity marker "NRN1" and / or as in Example 6

described, the impurity marker "EBF3" determined.

Furthermore, as described in Example 7, the

Potential marker "Aggrecan" determined.

Cartilage tissue (chondrocytes of sufficient quality (identity in the monolayer culture and / or in the spheroid culture, sufficient purity and / or acceptable level of cellular impurities and sufficient cartilaginous potential extracellular matrix) and cartilaginous extracellular matrix) is suitable for introduction into the cartilage defect of the patient to be treated of a cartilage replacement tissue

which fills the defect and takes over the function of the natural cartilage tissue. cartilage

insufficient quality (identity in the Monolayerkultur and / or in the spheroid culture not given, insufficient purity and / or insufficient degree of cellular

Impurities and insufficient potential to form cartilaginous extracellular matrix) is not suitable for building up cartilage replacement tissue and is discarded.

Example 9:

Retrospectively, this was described in Example 8

Procedure for cartilage tissue to be transplanted from 20

Patients on whom cartilage was used to regenerate knee cartilage damage. The ability of the transplanted cartilage tissue to build up cartilage replacement tissue was clinically established

Cartilage Repair Scores (KOOS - Knee Injury and Osteoarthritis Outcome Score: Roos EM & Lohmander LS, Health and Quality of Life Outcomes 2003, 1:64), ie assessed the clinical success of the treatment. For this purpose, the patient's KOOS was determined by patient survey before treatment with the cartilage tissue to be transplanted (baseline value). One year after treatment with the transplant

Cartilage tissue was again determined by patient survey of the KOOS of the respective patient (1 year

Follow-up value). The clinical or therapeutic

Success of the treatment is given if the difference (KOOS delta) between 1 year follow-up value and base value is at least 8 points (Roos EM & Lohmander LS, Health and Quality of Life Outcomes 2003, 1:64). Is the difference (KOOS delta) between 1 year follow-up value and

Underlying less than 8 points is not clinical

Improvement by the treatment before, i. the therapy has not been successful.

In 4 of the 20 transplantable cartilage tissues (5697 - 1607, 5862 - 1311, 6070 - 2413, 6988 - 2423), the potential marker "aggrecan" was found to be below the specified limit, while the identity of the

Cartilage cells using the identity marker "KALI" or "CRTAC1" could be detected. The expression values are above the defined limits. The purity of the cartilage tissue to be transplanted can also be determined by means of the

Pollution marker "EBF3" can be determined and is given, here are expression levels below the specified

Limits. These 4 to transplantable cartilage tissue therefore show insufficient quality to build up

Cartilage tissue and should be discarded. This corresponds to a failure rate of 20% (4 out of 20 to graftable tissue).

In the transplantable cartilage tissue of 16 of the 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 defined limit value, and the identity of the cartilage cells can be determined using the identity markers "KALI" and "CRTAC1 The expression values are also above the defined threshold values and the purity of the cartilage tissue to be transplanted could also be determined of the contaminant marker "EBF3" and is given, where the expression levels are below the established thresholds, indicating that these 16 graftable cartilage tissues are of sufficient quality to construct cartilaginous tissue and to be used for transplantation, corresponding to a success rate of 80% (16 from 20 to transplantable cartilage tissue).

Thus, the transplantable cartilage tissues of 4 of the 20 patients (5697-1607, 5862-1311, 6070-2413, 6988-

2423) a cartilage tissue of insufficient quality (identity in the monolayer culture and / or in the spheroid culture not given, insufficient purity and / or insufficient degree of cellular impurities and insufficient potential to form cartilaginous extracellular matrix), while the transplantable cartilage tissue of 16 of the 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) have a cartilage tissue of sufficient quality (given identity in the monolayer culture and / or in the spheroid culture, sufficient purity and / or

sufficient level of cellular contaminants and sufficient potential to form cartilaginous extracellular matrix).

With regard to the clinical or therapeutic success of the treatment (see Table 4) it can be seen that in those 4 of the 20 patients (5697 - 1607, 5862 - 1311, 6070 - 2413, 6988 - 2423) in whom a retrospectively transplantable

Cartilage tissue of insufficient quality was found to be no clinical improvement due to the treatment. The difference between the KOOS 1 year follow-up value and the KOOS base value was less than 8 points, the therapy was therefore unsuccessful. In contrast, 16 of the 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) in which a transplantable cartilage tissue of sufficient quality has been found retrospectively. there is a clinical improvement from the treatment. The difference between the KOOS 1 year follow-up value and the KOOS base value is at least 8 points, the therapy has been successful. Table 4: quality markers for identity,

Purity / Impurity and Potential of 20 Patients with Known Clinical Outcome (KOOS delta)

9110 - 2294 19.17 152.6 0.501 0.0706 2.221 Yes

5669 - 2263 20.51 12.05 2.576 0.0669 1.12 Yes

9110 - 2294 19.17 152.6 0.501 0.0706 2.221 Yes

6094 - 1312 14.00 25.97 2.537 0.0761 0.439 Yes

6340 - 2277 15.11 16.17 0.439 0.0459 0.2942 Yes

6611 - 2418 17.96 40.96 0.541 0.1905 0.9808 Yes

8315 - 2434 41.83 30.29 0.174 0.2405 2.628 Yes

8916 - 2440 25.47 19.51 1.513 0.1495 1.613 Yes

8931 - 1126 42.23 2.02 1.637 0.123 0.4226 Yes

8948 - 2706 42.15 39.52 3.028 0.1408 0.9095 Yes

8966 - 2441 9.48 30.05 0.2841 0.0482 0.5464 Yes

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

Claims

claims

Method for the selection of chondrocytes for the production of transplantable cartilage tissue, characterized in that at least one identity marker or purity marker, selected from the group KALI, CRTAC1, NRN1 or EBF3, is determined in vitro.

Method for the selection of chondrocytes according to claim 1, characterized in that, for the production of transplantable cartilage tissue, the chondrocytes are multiplied in a monolayer culture in a first step, and in a second step aggregate into spheroids.

Method for the selection of chondrocytes according to one of the preceding claims 1 or 2, characterized in that the chondrocytes are aggregated into spheroids, and spheroids are selected.

Method for the selection of chondrocytes according to one of the preceding claims, characterized in that the identity markers are selected from the group KALI, CRTAC1 and / or that the purity markers are selected from the group NRN1, EBF3.

Method for the selection of chondrocytes according to one of the preceding claims, characterized in that i.) Chondrocytes in a monolayer culture with the identity marker KALI is determined in vitro and / or ii.) Chondrocytes in spheroids with at least one identity marker CRTAC1 is determined in vitro. Method for the selection of chondrocytes according to one of the preceding claims, characterized in that in addition proteoglycans, in particular aggrecan are determined in vitro.

Method for the selection of chondrocytes according to one of the preceding claims, characterized in that the markers according to the invention are determined in comparison to a reference gene.

Method for the selection of chondrocytes according to claim 7, characterized in that at least one

Ratio of gene expression selected from the group CRTAC1 / R> 0.003

NRN1 / R> 0, 1

KALI / R> 1.0

EBF3 / R <0.4

ACAN / R> 0.29

is determined.

Ratio of gene expression selected from the group CRTAC1 / R> 0.006, in particular> 0.009 and> 0.012

NRN1 / R> 0, 2, in particular> 0.3 and> 0.4

KALI / R> 2, 0, in particular> 3.0 and> 4.0

EBF3 / R ^ 0.3, in particular <0.2 and <0.1

ACAN / R> 0.39, in particular> 0.49 and> 0.59 is determined. Method for the selection of chondrocytes according to one of the preceding claims, characterized in that the proportion of synoviocytes in the isolated chondrocytes after multiplication or the obtained spheroids in proportion to the total cell number is less than or equal to 9%, in particular less than or equal to 5%.

Isolated chondrocytes obtainable by a method according to one of claims 1 to 10.

Isolated spheroids obtainable by a process according to one of claims 1 to 10.

Isolated spheroids obtainable by a process according to any one of claims 1 to 10, characterized in that the proportion of synoviocytes in the spheroids in relation to the total cell number is less than or equal to 9%.

Transplantable cartilage tissue containing chondrocytes and / or spheroids according to any one of claims 10 to 13 or obtainable by a method according to any one of claims 1 to 9.

Transplantable cartilage tissue according to claim 14 for use in the treatment of articular cartilage defects, autologous chondrocyte transplantation.