HK1133035A - Methods to maintain, improve and restore the cartilage phenotype of chondrocytes - Google Patents

Description

Methods of maintaining, improving and restoring cartilage phenotype of chondrocytes

Technical Field

The present invention relates to the field of chondrocyte expansion and cartilage repair. The invention also relates to a controlled population of cells of cartilage that can be used to maintain, improve or restore the cartilage phenotype of chondroblasts such as chondrocytes or chondroblasts.

Background

Both articular cartilage and meniscal cartilage play an important role in normal joint motion mechanisms. Articular cartilage covers the ends of the bones of the knee joint. It consists of sparsely packed cells (chondrocytes) surrounded in an extracellular matrix consisting of collagen, various proteins/glycoproteins, and proteoglycans of high water retention carrying negatively charged polysaccharides called mucopolysaccharides. Meniscal cartilage is fibrocartilage characterized by the presence of cells in lacunae arranged in rows and bundles of collagen fibers in the matrix. Damage to articular cartilage and/or meniscal cartilage is a common condition for imaging millions of people. The injury is complicated by the weak regeneration of adult articular cartilage and the dyskinesia and pain associated with the injury. Several modes of cartilage repair have been proposed in recent years. In one mode, the "autologous chondrocyte migration" technique (ACT) collects cells from healthy shopping cartilage, cultures and expands to give a sufficient number of cells, and replantates them into articular or meniscal cartilage defects [ Brittberg et al (1994) N Engl. JMed.331, 889-895 ]. Cell expansion must be obtained from small biopsied cartilage to repair the cartilage defect. ACT technology has limitations such as the propensity of chondrocyte expansion in monolayer culture to de-differentiate with increasing numbers of passages. This dedifferentiation will result in a composition of the matrix with different biochemical and biomechanical properties than the composition in the original tissue. There is a need to optimize cell culture conditions and limit the number of passages to maintain the phenotypic properties of chondrocytes, including the ability to form stable, glassy cartilage in vivo, to resist vascular erosion and endochondral osteogenesis. All currently employed ACT techniques use cell therapy consisting of an expanded population of chondrocytes taken from a primary biopsy by cell culture methods. Admittedly, these cell populations contain varying numbers of dedifferentiated cells that retain their phenotypic stability and are unable to differentiate stably towards the articular chondrocytes that are formed. This condition was demonstrated in clinical studies of the quality of tissue repair as a high degree of variability from hyaline cartilage on fibrocartilage to no repair was shown [ Peterson et al (2000) Clin ortho Res.374, 212-. This suggests that the number of chondrocytes expanded is not sufficient to achieve a successful transplantation.

Various efforts have been undertaken to improve the quality of chondrocytes or to use alternative sources of chondrocytes. (WO0124833) describes the use of molecular markers as quality control for chondrocyte expansion or passage. The use of these markers allows one to determine whether the number of passages of a population of chondrocytes will be such that cartilage-forming ability is not significantly lost after transplantation. Different approaches have been used to increase the outcome of ACT by adding different matrix components (e.g. perlecan) to chondrocytes [ French et al (1999) j.cell. biol.145, 1103-1115 ].

An image of the ability of the environmental matrix to regenerate chondrocytes has also been reported. Graff et al [2003, Biotechnol. Bioeing.82, 457-464] describe a population of cells from cartilage which are characterized by the presence of a native extracellular matrix ("chondroprotein") and describe that these cells have enhanced chondrogenic properties relative to chondrocytes whose extracellular membranes have been reconstituted in vitro.

Cartilage biopsies have been described to include: in addition to typical chondrocytes, there are small fractions of cells with different morphology and behavior. Kamil et al [2004, Tissue Eng.10, 139-144] describe the removal of a population of cells commonly referred to as "foam cells" from a cartilage biopsy in an expansion protocol for higher yields and how these cells can still be expanded. The report suggests that other parts of cartilage biopsy than the classical adherent chondrocyte part can be used as a source of chondroblasts.

Despite the various methods of expanding chondrocytes and verifying their quality, it is not possible to obtain sufficient numbers of phenotypically stable chondrocytes in a defined passage number to improve the properties of low quality chondrocytes or to restore cell properties after inappropriate treatment (e.g., over-hatching or expansion). The need for methods to produce stable numbers of high quality cells in ACT requires methods that ensure consistency and independence of chondrocyte quality and number in biopsy.

Summary of The Invention

The present invention relates to a population of cells obtainable from cartilage which have a modulating effect when combined with a population of cells having chondrogenic capacity, thereby increasing, inducing or restoring the phenotypic stability of chondrocytes. This population of regulatory cells naturally appears as part of a freshly isolated cartilage biopsy, but may be enriched and/or isolated based on its physical and/or physiological properties. It has been demonstrated that the cell population retains its regulatory capacity when frozen, making it possible to store the population of regulatory cells (in enriched or non-enriched form), for example during which mature chondrocytes have to be expanded to a sufficiently large number for transplantation. Once a sufficient number of chondrocytes are obtained, the chondrocyte population is combined with a regulatory cell population, and the combined cell population is ready for transplantation.

And it has been found that the population of regulatory cells can be isolated from chondrocytes from a cartilage biopsy by harvesting over a period of time, such as about 2 to 5 days after seeding and/or when adherent cells reach at least 30% confluence of the cell non-adherent fraction of the culture vessel.

In one aspect, the invention provides methods for enriching a population of regulatory cells of the invention. More specifically, the present invention provides a method of enriching a population of regulatory cells from an isolated cartilage sample, the method comprising the steps of: (a) mechanically and/or enzymatically treating the isolated cartilage sample to obtain individual cells; (b) transferring the obtained independent cells to a cell culture container to expand the chondrocytes in a monolayer; (c) subjecting the cell culture vessel to suitable cell culture conditions, thereby obtaining a population of adherent cells and a supernatant comprising a population of non-adherent cells; (d) collecting a supernatant comprising a population of non-adherent cells from the cell culture vessel after at least 2 days and/or when adherent cells reach at least about 30% confluence; (e) the non-adherent cell population corresponding to the regulatory cell population of the invention is collected from the supernatant.

According to one embodiment, the cartilage sample used in the enrichment method of the invention is a sample of articular cartilage or meniscal cartilage.

The enrichment method of the present invention generally begins with a cartilage sample treated with collagenase a enzyme.

In another aspect, the invention provides an enriched population of regulatory cells that are non-adherent, non-passaged populations of regulatory cells obtained using the enrichment methods described herein.

In yet another aspect, the invention provides different uses of the enriched regulatory cell populations of the invention.

According to a first embodiment, the enriched population of regulatory cells obtained with the method of the invention is used as a medicament.

More specifically, the present invention provides a method of treating a cartilage disorder comprising: patients with cartilage disorders are infused with the enriched population of regulatory cells of the invention. Accordingly, the present invention provides pharmaceutical compositions comprising the enriched regulatory cell populations of the present invention.

More specifically, the present invention provides a therapeutic method that involves the formation of a micro-vent at a defect in articular cartilage. The microfractures formed in the affected area allow the underlying bone stem cells to access the articular cartilage defect. In vivo cartilage reconstruction is then formed by applying a cellular therapy comprising a population of cells with regulatory functions according to the invention. During this reconstitution process the regulatory cell populations of the present invention direct mesenchymal stem cells into the chondroblast line.

According to yet another embodiment, the enriched population of regulatory cells is used to improve, maintain or restore chondrocyte phenotypic stability of an expanded or passaged chondrocyte population in vitro.

According to yet another embodiment of the invention, mesenchymal stem cells are differentiated in vitro into chondroblast lineage using an enriched population of regulatory cells.

In another aspect, the invention provides compositions comprising one or more populations of different cells of a population of regulatory cells (in enriched form or freshly isolated chondrocytes as described herein).

More specifically, the invention provides compositions comprising a combination of two or more different cell populations comprising:

-a cell population comprising regulatory cells, which is (i) a freshly isolated chondrocyte population obtained from a cartilage biopsy, or (ii) an enriched regulatory cell population of non-adherent cells by collecting a culture supernatant of chondrocytes P0 taken from a fresh isolation of a cartilage biopsy; and

-one or more cell populations selected from the group consisting of: a passaged chondrocyte cell population, a mesenchymal stem cell population, and a chondroblast cell population.

More specifically, the present invention provides compositions comprising a combination of two or more different cell populations comprising: an enriched population of regulatory cells, the relative amount of enriched regulatory cells typically ranging from 1 to 75% of the total number of cells of the combined composition.

According to a particular embodiment, the population of cells comprising regulatory cells present in the combined composition of the invention is taken from articular cartilage or meniscal cartilage.

The invention also provides different applications of the combination of a regulatory cell population (enriched form or freshly isolated chondrocytes as described herein) with different cell populations.

More specifically, a combined cell population comprising a cell population of the regulatory cells of the invention is used as a medicament. The present invention thus provides pharmaceutical compositions comprising a combined cell population as described herein. The combined cell population can also be used to support a subject for therapeutic use.

The invention provides the use of a combination of a regulatory (optionally enriched) cell population of the invention and a second cell population for the preparation of a cellular therapeutic for the treatment of a cartilage disorder. Thus, there is provided a method of treating a cartilage disorder comprising administering to a cartilage disorder a composition comprising a population of regulatory cells of the invention and one or more additional cell populations

The regulatory cell population in combination with other cell populations may be used, for example, to provide higher passage numbers of chondrocytes for transplantation. Furthermore, the method of the present invention makes it possible to generate a large amount of chondrocytes through repeated passaging. The eventual reduction of chondrocyte phenotype after over-passaging can be restored by the addition of the regulatory cell populations of the present invention.

The use of the cell populations of the invention thus enables cellular therapy of larger defects, since a large number of chondrocytes with a stable phenotype are available. Alternatively, the quality of the population of regulatory cells of the invention can be improved using it, independent of the number of chondrocytes available.

The use of the regulatory cell populations of the present invention improves the ACT method that is commonly used.

More specifically, the present invention provides a method of preparing a cell population for ACT transplantation, the method comprising the steps of:

a) mechanically and/or enzymatically treating the isolated cartilage sample to obtain individual cells,

b) transferring the obtained individual cells into a cell culture container to expand the chondrocyte monolayer,

c) maintaining the culture vessel under suitable cell culture conditions to obtain a population of adherent cells and a supernatant comprising a population of non-adherent cells,

d) collecting supernatant comprising a population of non-adherent cells from the culture vessel after at least 2 days or upon at least about 30% confluence of adherent cells in the culture vessel,

e) collecting the non-adherent cells from the supernatant,

f) the obtained non-adherent cell population is combined with a chondroblast population (which is not an enriched regulatory cell population).

According to an embodiment of the method of preparing a cell population for ACT transplantation, the chondrogenic cell population is a mature chondrocyte population taken from the same or a different isolated cartilage sample. More specifically, the method further comprises the steps of:

g) expanding or passaging the adherent cell population obtained in step (c),

h) collecting the expanded and passaged adherent cell population,

i) in step (f), the non-adherent cell population from step (e) is combined with the expanded and passaged adherent cell population from step (h).

Typically, in the process of the invention, collagenase a is used for the enzymatic treatment in step (a).

In a particular embodiment of the method of the invention the cartilage sample isolated is a semilunar cartilage sample. Optionally, the population of chondrogenic source cells is from a meniscal cartilage biopsy. Alternatively, the chondrogenic cell population is a mesenchymal stem cell population.

Other embodiments of the methods of the invention for preparing a population of ACT transplant cells further comprise the step of seeding the combined cell population obtained in step (f) or (i) onto a support.

The present invention thus relates to methods of improving cartilage reconstruction in vivo by administering a cell therapy comprising a mixture or composition of the present autologous or allogeneic conditioned chondrocyte cell population and expanded autologous or even allogeneic chondrocyte cells having a fibroblast morphology.

The invention also relates to methods of improving cartilage remodeling in vivo by administering a cell therapy agent comprising a mixture of a population of regulatory cells of the invention and mesenchymal stem cells.

Compositions comprising a combination of a population of cells of the invention and chondrocytes are capable of accelerating cartilage formation in vitro. Thus, it is contemplated that the combined implantation of chondrocytes and regulatory cell populations of the present invention results in enhanced cartilage reconstruction, which when injected into the body, directs a faster regeneration process and high quality cartilage formation.

And it was observed that cartilage produced by the cell population of the present invention in particle culture showed rapid formation of mechanically stable particles even when combined with higher passage dedifferentiated chondrocytes. This not only makes the involvement of the population of chondrocytes available for transplantation more flexible, but also predicts an effect on local chondroblasts. The ability to induce a chondrocyte phenotype stabilized by partially dedifferentiated cells indicates that the regulatory cells of the invention may be used to induce cartilage formation, for example in the case of osteoarthritis, when local chondrocytes within the joint have been affected by the breakdown process.

Detailed Description

Defining:

the term "chondrogenic" when used in reference to a cell or population of cells refers to the inherent ability of the cell or population of cells to produce cartilage or stimulate cartilage growth, as appropriate. The chondroblasts include chondrocytes and cells that differentiate into chondrocytes themselves, such as precursor cells belonging to the osteochondral line.

As used herein, "chondrogenic factor" refers to a compound that promotes cartilage differentiation, for example, certain growth factors such as TGF- β.

The term "chondrocyte phenotypic stability," when used in reference to a cell population, refers to the ability of the cell population to produce cartilage in vivo. This ability can be achieved by injecting a partial population of cells (at least about 1-20X 10) into a mammal (in vivo), such as an immunodeficient mouse⁶Individual cells) were tested to determine (a time span of about 3 weeks) the progression of the cartilage implant without vascular erosion or signs of endochondral osteogenesis. A population of phenotypically stable chondrocytes, characterised by the presence of markers of phenotypic stability, as described in WO 124833. The chondrocyte phenotypic stability is gradually lost in mature chondrocytes after passage.

The term "freshly isolated cells" (FI) refers to a population of cells taken from a biopsy after digestion of tissue. The term "freshly isolated chondrocytes" as used herein refers to a population of cells obtained directly from chondrocyte-containing tissue, such as cartilage, that has not been passaged through digestion. It is within the scope of the invention to isolate a population of cells freshly either for direct use (i.e., within 48-120 hours after isolation) or frozen for later use. Alternatively, the characteristics of a freshly isolated cell population may be maintained under certain culture conditions for extended periods of time, e.g., at high densities without subculturing in ultra-low contact plates of an incubator for extended periods of time.

The term "modulate" when used with respect to a cell or cell population refers to the ability of the cell or cell population to affect chondrogenesis of the cell population, and/or to initiate chondrocyte expansion, and/or to affect chondrocyte phenotypic stability of other cell populations when contacted with one another.

The term "enrichment" as used in the present invention for the regulatory cell population relates to the following: from the freshly isolated cells obtained from the cartilage biopsy, cells that developed mature chondrocytes are removed, resulting in a higher percentage of regulatory cells relative to the total number of regulatory cells in the freshly isolated cell population. One particular method of enriching the regulatory cell population according to the invention is based on its non-adherent nature.

The terms "non-adherent", "floating", or "non-contact", when used to refer to a cartilage-derived cell or cell population, refer to the following: when cells or cell populations are introduced into a culture container (e.g., a culture flask), the suspension remains unattached to the container surface. More specifically, the non-adherent cell population (NAC) of the invention is present in the supernatant of a chondrocyte monolayer of freshly isolated chondrocytes (i.e. during the first expansion under standard culture conditions) for at least two days or when adherent cells have obtained at least 30% confluence.

The "mature chondrocytes" used in the present invention refers to mature cells of cartilage tissue, which are derived from chondroblasts and are capable of producing cartilage. Mature chondrocytes are obtained from a fresh isolated cell population of cartilage by culture. When the matrix or support is accessible (e.g., in monolayer culture), freshly isolated cells flatten and acquire a polygonal or fibroblast morphology, losing their extracellular matrix to mature chondrocytes. Thus, as used herein, a "mature chondrocyte population" refers to a cell population obtained by culturing a chondrocyte monolayer, for example in a culture flask, with mature chondrocytes adhered to the surface of the culture flask and passaged one or more times.

The term "expansion", when used with respect to a cell population derived from, for example, cartilage tissue, indicates that the cell population has been subjected to conditions such that the number of cells has been increased by proliferation. Briefly, expansion is achieved by subjecting a population of cells to a culture vessel with an appropriate medium, optionally until confluence.

"first expansion" of a cell population isolated from a tissue refers to the culturing of the cell population prior to the first passage. In this first expansion, a portion of the freshly isolated cells will adhere to the surface of the vial and become mature chondrocytes (P0 mature chondrocytes), while a portion of the freshly isolated cells remain as non-adherent cells (non-adherent cell population) in the supernatant. The first expansion is usually about 15 days, depending on the density of the seeded cells. Once passaged, the supernatant of the cultured cells was removed and adherent cells were detached from the culture vessel, aliquoted and transferred again to a culture vessel containing fresh medium (at this stage relative to P1). The P1 cells were re-attached to flasks for further passage. P1 cells or higher passage cells are generally referred to herein as "passaged cells".

The term "cartilage defect" refers herein to any condition resulting from cartilage loss or damage. The most common occurs in joints such as, but not limited to, knee, elbow, ankle, and is therefore commonly referred to as articular cartilage defects. Cartilage defects are also often referred to as nearby bones, such as femoral condyle defects, humeral defects, and the like. Fibrocartilage loss/tear of the meniscus is also known as a meniscus defect.

The present invention is based on the following observations: freshly isolated cells taken from a cartilage biopsy, except for a population of cells that will differentiate into mature chondrocytes, a population of cells that adhere to the surface of the flask and can expand when introduced into the culture flask; and non-adherent cell populations that are not only phenotypically stable, but also serve as regulatory cell populations. The regulatory activity of this cell population is shown by the following: which is capable of maintaining chondrocyte phenotypic stability of the chondrocyte population and restoring chondrocyte phenotypic stability of the dedifferentiated cell population. Thus, this cell population, present in freshly isolated cells taken from cartilage, is of particular interest for cartilage production and repair. Thus, the present application provides a novel application of freshly isolated chondrocytes, exploiting the characteristics of the subpopulation of cells regulated therein. Also, the non-adherent nature of the regulatory cell population provides a means for enriching the regulatory cell population of the present invention. Thus, although the first expanded non-adherent cells traditionally taken from chondrocyte culture were discarded, the present invention provides for the use of such non-adherent cells not only as chondroblast cell populations but also as regulatory cell populations capable of significantly affecting chondrocyte phenotypic stability of other cell populations.

It has been observed that the population of regulatory cells of the invention comprises cells having an extracellular matrix. Chondrocytes have a round or semi-round appearance when present in cartilage tissue. Digestion of cartilage tissue destroys the natural extracellular matrix that is present around the cells. In the first 48 hours of culture, the cells again (albeit different) get extracellular matrix. When contacted with a matrix or support, such as a monolayer culture, most freshly isolated cells flatten and form a polygonal or fibroblast morphology, again losing their extracellular matrix. The regulatory cell populations of the present invention are characterized by having a round cell shape, with such recovered extracellular matrix retained therearound.

According to the invention, the regulatory cell populations of the invention are present in freshly isolated cells obtained from various cartilage biopsies. The invention thus provides methods involving the use of such regulatory cell populations, either in freshly isolated cells or enriched cell populations, in chondrogenesis in vitro or in vivo. The invention also provides methods for enriching a population of cells for regulatory function.

The regulatory cell populations of the invention are either a portion of a fresh isolated cell population or an enriched regulatory cell population, typically obtained from an isolated cartilage sample. According to the invention, the isolated cartilage is fibrocartilage and/or articular cartilage obtained from any part of the human or animal body. And more particularly to selecting sites that are readily accessible in vivo for taking a cartilage biopsy. If the separation of the regulatory cell population is the cell used to achieve ACT as described herein, the ideal cartilage biopsy is from a non-weight bearing site with little contact with other bone, such as the intercondylar notch of the femur. Alternatively, the cartilage sample used in the method of the invention is herniated disc cartilage or meniscal cartilage (fibrocartilage).

The cartilage biopsy used to generate cells in the present invention may be obtained from young or elderly individuals, healthy or diseased cartilage (e.g., osteoarthritic cartilage).

The first aspect of the present invention relates to a method for enriching a population of regulatory cells of the present invention. In its most general form, the method comprises the steps of:

mechanical and/or enzymatic treatment of the isolated cartilage sample to obtain a single cell population,

transferring the obtained single cell population to a cell culture vessel to expand the chondrocytes in a monolayer,

-maintaining the culture vessel under suitable cell culture conditions, and

-collecting the supernatant comprising the non-adherent cell population from the cell culture vessel, the non-adherent cell population corresponding to the regulatory cell population of the invention.

According to the present invention, culture of freshly isolated cells from cartilage tissue provides a means for physically isolating the cells into "adherent" cell populations, including the requisite chondrocytes, as well as non-adherent cell populations with regulatory characteristics.

In the first step of the enrichment process according to the invention, the isolated cartilage is treated to obtain a population of individual somatic cells ("freshly isolated chondrocytes"). According to a specific embodiment, the treatment comprises an enzymatic treatment. Enzymes suitable for digestive cartilage biopsy include, but are not limited to: collagenase NB4, collagenase a or dispase/collagenase II. In a particular embodiment of the invention the cartilage biopsy is treated with collagenase a. Other proteases suitable for cartilage breakdown include enzymes selected from the group consisting of (alone or in combination with insulin): aspartase enzymes such as cathepsin D, cysteine enzymes such as cathepsin B, L, S, K and calcium-activated neutral proteases I and II, serine proteases such as neutrophil elastase, cathepsin G and protease 3 and metalloproteinases such as MMP 1-20. "treating" according to the present invention includes contacting the isolated tissue with an amount of enzyme for a sufficient period of time to digest the tissue into individual cells. Exemplary enzyme concentrations and digestion periods are described in the examples section herein, and other treatments may be readily determined by one skilled in the art. In a particular embodiment of the invention collagenase a is used in an amount of 0.1 to 0.3% for 8 to 16 hours, more particularly 12 hours.

Alternatively, cartilage biopsies can be processed using mechanical methods to obtain individual cell populations, for example using low speed mechanical homogenization as described by Poole et al (1998) J.Orthop.Res 6, 408-419.

In the enrichment process of the present invention, individual cells obtained from a cartilage biopsy ("freshly isolated chondrocytes") are introduced into a "culture vessel" to allow separation of "adherent" and "non-adherent" cell populations therein. Suitable culture vessels for use in the present invention are standard culture flasks for matrix-dependent cell culture (such as, but not limited to, Falcon from BD corporation (Becton Dickinson)^TMBottles, TPP bottles from Tekeno plastics Products (Techno Plastic Products), Greiner tissue culture treated bottles, and the like). Typically, these flasks contain an inner surface or bottom to which a surface treatment has been applied. Flasks of different shapes or sizes may be used and these aspects are not strictly required in the context of the present invention. The only limitation is that the bottle is closed, i.e., the culture medium above the adherent cell population is allowed to retain the mature chondrocyte population for the period of time required to adhere to the bottom surface, thereby allowing physical separation of the adherent and non-adherent cells and recovery of the non-adherent cells from the culture medium. According to a specific embodiment, the culture medium and conditions used for this step of the enrichment method of the invention are standard chondrocyte culture media. The media used in the chondrocyte expansion process typically comprises DMEM, HAMS/F12 or other optional cell culture media supplemented with 5 to 15% serum. Alternatively, serum-free media suitable for chondrocyte culture may be used. These media may include mixed vitamins, growth factors, hormones, sugars, and the like. According to a specific embodiment, DMEM medium containing serum and antibiotics is used. The cell culture was standardized at 37 deg.C with 5% CO₂It is desirable that slight variations in these conditions do not significantly affect the adhesion of the cell population to the surface of the culture vessel of the present invention. According to a specific embodiment of the invention, the supernatant of the culture flask containing the population of non-adherent cells will remain in the culture flask for a period of time, such as 2 to 5 days after seeding or until the adherent cells have reached at least about 20%, more specifically about 30% confluence before collection. This will depend on the size of the vial and the number of cells initially introduced therein. In the first expansion, confluency is achieved within about two weeks, usually 175cm, for the adherent cells in the culture flask²5,000 and 20,000 freshly isolated chondrocytes were introduced into the flask of (1). The techniques for culturing chondrocytes are well known to the skilled artisan.

The regulatory cell population of the present invention is characterized in that it is non-adherent, i.e., it does not adhere to the surface of the culture flask after 2 to 5 days of seeding the flask with cells from a cartilage biopsy or when adherent cells from chondrocytes reach about 30% confluence. According to the invention, the population of regulatory cells can be obtained by isolating adherent and non-adherent cells in a first expansion of a freshly isolated chondrocyte population. Mature chondrocytes, which typically appear in cell populations from cartilage biopsies without a native outer cell membrane, adhere to the surface of the (pre-treated) culture container over a 1 week period. This cell-contacting process is the basis for a method for enriching a population of regulatory cells, which is characterized by a resistance to the dedifferentiation and contacting processes over a prolonged period of time. It will be appreciated that the collection time of the regulatory cell population in the enrichment process of the invention may be less than 2 days or more than 5 days after seeding, but this will affect the efficiency of the enrichment process as the supernatant will contain a higher proportion of mature chondrocytes which either remain unadhered or no longer adhere to the surface of the culture vessel.

In the enrichment process of the present invention, the non-adherent cell population is physically removed from the adherent cell population. According to an embodiment of the invention, the population of non-adherent cells is collected with the supernatant from the culture vessel. More specifically, collection from the culture vessel of the non-adherent cell population comprises the step of applying gentle mechanical force (e.g., gentle tapping of the bottle) to detach the weakly adherent cells and collecting the culture supernatant. Recovery of cells from the supernatant uses standard techniques, such as, but not limited to, centrifugation (e.g., 1500rpm for 10 minutes). Other methods include other separation methods such as filtration.

Typically, the enrichment process of the invention results in an increase in the percentage of regulator cells present in the cell population by at least 10%, more particularly at least 20%, and most particularly at least 30% relative to the percentage of regulator cells present in a fresh isolation biopsy. Chondrocyte biopsies are generally found to contain 5-20% of regulatory cells. Since the properties of the cells are difficult to determine just after recovery from the biopsy, they can also be determined after the mature chondrocytes have begun to adhere to the surface of the flask. According to a specific embodiment, the enriched population of regulatory cells of the invention comprises at least 30%, more particularly at least 40%, most particularly between 50% and 95% regulatory non-adherent cells.

According to another embodiment, adherent and non-adherent cell populations are separated based on morphological and/or physiological characteristics. Thus, the regulatory cell populations of the present invention are characterized by a round cell morphology and extracellular matrix, and unlike the "cartilaginous" extracellular matrix described by Graff et al (2003, supra), are not native extracellular matrices, but rather are recovered from the cells in culture. The regulatory cell populations of the invention are also characterized by the expression of type II collagen and aggrecan. Thus other methods of isolating regulatory cells of the invention include the following: treatment of the cartilage biopsy to obtain individual cells as described above, followed by isolation of the common chondrocyte and the regulatory cell population using isolation techniques, such as FACS analysis or other cell isolation techniques.

Since the present invention provides methods for obtaining enriched populations of regulatory cells from the above described biopsies, it should also be noted that freshly isolated cells obtained from these biopsies comprise the regulatory cell populations of the present invention and may be used as such. Thus, the present invention contemplates the use of freshly isolated cells either as a source of regulatory cells for enrichment or directly for different applications of the regulatory cells of the invention as described below.

Thus, according to one embodiment of the invention, a population of regulatory cells in a freshly isolated population of cells is used for the various applications described herein. The fresh isolated cell population can be used directly, i.e., within 24 hours of isolation from cartilage tissue under standard culture conditions. It has further been established that the regulatory properties of a population of regulatory cells present in a freshly isolated population of cells can be maintained in vitro for a longer period of time under certain culture conditions, for example by storing the cells in a high density in an ultra-low contact plate of an incubator (available from e.g. Corning). Or a fresh isolated population of cells frozen within 24 hours of isolation for later use. Methods for freezing and thawing cell populations are well known in the art.

According to one embodiment, freshly isolated cells from a cartilage biopsy are divided into two portions, one of which is further expanded to increase cell number (using classical chondrocyte expansion techniques) and the other portion is frozen until expansion of the first portion is complete, after which the thawed portion of freshly isolated cells is combined with the expanded and passaged portion of chondrocytes to create a combined cell population that can be used as a cell therapeutic with the inventive increased phenotypic stability effect.

According to another embodiment of the invention, the population of regulatory cells is in the form of an enriched population of regulatory cells obtained by the method described herein for use in the different applications described herein. Again, the enriched population of regulatory cells can be used either directly or frozen for later use.

Other aspects of the invention provide different uses of the regulatory cell populations of the invention.

One aspect of the invention provides a combination of a) a population of regulatory cells of the invention (either "enriched" or not, i.e. in the form of a freshly isolated population of chondrocytes) and b) a mature, progenitor and/or dedifferentiated population of chondrocytes for use in a cellular therapeutic strategy.

According to a particular embodiment of the invention, the population of regulatory cells and the population of chondroblasts used in the combined cell population of the invention are obtained from the same cartilage sample. According to a first embodiment, a portion of the freshly isolated cells obtained from the biopsy sample is frozen, or directly or within 24 hours of the first expansion, and a second portion is expanded to obtain an expanded population of chondrocytes (mature chondrocyte population). Alternatively, both the population of regulatory cells and the population of chondrocytes are derived from the same first expansion of a freshly isolated chondrocyte sample, wherein the regulatory cells correspond to the population of cells taken from the non-adherent cell fraction during the first expansion of chondrocytes and the mature chondrocytes correspond to one or more chondrocyte passages of the adherent cell population of the same first expansion of chondrocytes. Alternatively, it is contemplated that the combination of the population of regulatory cells and the population of chondrocytes or chondroblasts are derived from different cartilage biopsies or from a cartilage sample that has been divided into a first and a second fraction, then the population of regulatory cells is preferably isolated from the first fraction and the population of chondrocytes is preferably isolated from the second fraction.

According to the invention, the non-adherent cell population present in the freshly isolated cell population or obtained during the first phase of expansion of freshly isolated chondrocytes has regulatory properties which have a specific effect on chondrogenesis both in vitro and in vivo. Thus, other aspects of the invention relate to the use of the identified population of regulatory cells in various aspects of chondrogenesis and cartilage repair.

The different uses of the regulatory cell populations of the present invention are based on their ability to modulate the stability of the chondrocyte phenotype of a second cell population when mixed with the second cell population. Chondrocyte phenotypic stability of a cell population can be determined by implanting the cell population into a nude mouse model as described in WO 0124833. Usually about 5X 10⁶The population of cells passaged early to chondrocytes is capable of producing mature cartilage strains within 2-3 weeks. Molecular markers can be used to predict the outcome of this transplantation as described in WO0124833, and thus these molecular markers can be indicative of chondrocyte phenotypic stability of a cell population. In general, the presence of one or more positive markers (e.g., BMP-2 and/or FGFR-3) may allow the absence of one or more negative markers (e.g., ALK-1) to be used to identify populations of phenotypically stable chondrocytes.

According to one aspect, the present invention provides methods for maintaining, improving and/or restoring the phenotypic stability of chondrocytes into a chondrocyte population. In the methods of the invention, the regulatory cell population is combined or mixed with the chondroblast population. The chondrogenic cell population contemplated in the context of the present invention may be one or a mixture of cell populations selected from the group consisting of: chondrocytes, expanded passaged chondrocytes, Mesenchymal Stem Cells (MSCs), or precursor cells belonging to the osteochondral line are freshly isolated. In the method of the invention, the cells in the population of regulatory cells are combined with the population of chondroblasts, wherein the relative amount of regulatory cells is typically between 1 and 75%, more particularly between 10 and 50% of the total number of cells in the mixture. According to the invention, the population of regulatory cells can be combined with the population of chondrogenic cells, either in the form of a mixture (gentle mixing of the cell suspension to ensure complete mixing of the two cell populations) or in the form of so-called chondrocytes, which represent clusters of chondrogenic cells.

Accordingly, the present invention provides methods for improving, restoring and maintaining the phenotypic stability of chondrocytes from freshly isolated chondrocytes, expanded passaged chondrocytes, mesenchymal stem cells or precursor cells belonging to the osteochondral line. More specifically, the present invention provides methods for improving, restoring and maintaining chondrocyte phenotypic stability of cultured chondrocytes, wherein the culturing may include 6 to 10 or more subsequent passages.

Thus the methods of the invention allow the optional combination of a regulatory cell population (either a freshly isolated cell population or an enriched cell population) and a chondroblast cell population in one composition. Typically, when the resulting combined cell population is to be used for transplantation, both cell populations are autologous. Alternatively, it is also contemplated to use, for example, xenogenic chondrocytes or xenogenic MSCs in combination with autologous or even allogeneic regulatory cell populations.

A particular aspect of the invention relates to a method for obtaining a population of cells with improved chondrocyte phenotypic stability from a cartilage biopsy. The improvements envisaged by the methods of the invention may be different routes but are effective in the reconstruction of chondrogenesis and/or cartilage defects in vitro or in vivo. It has been demonstrated that contacting a population of chondrocytes obtained from a standard monolayer culture with a population of regulatory cells of the invention results in a significant improvement in the stability of the chondrocyte phenotype of the resulting cell population, over the additive effect of the two cell populations. This positive effect on chondrocyte phenotypic stability has been observed not only in the chondrocyte population at the first or second passage, but also on cells showing signs of dedifferentiation that have undergone high-order passage. Thus, according to this aspect of the invention, a method is provided for obtaining an improved chondrogenic cell population, for example for ACT. These methods are based on the combination of two cell populations, the first cell population being a mature chondrocyte population obtained from a cartilage biopsy using standard culture methods, and the second cell population being a regulatory cell population, obtainable as described above. The two cell populations can be treated independently and combined at any stage. The regulatory effect of the regulatory cell population on the chondrocyte population enables significant proliferation of the mature chondrocyte population without affecting the chondrocyte phenotypic stability of the final cell therapeutic.

Another embodiment of the invention relates to the use of a population of regulatory cells (e.g., a freshly isolated population of chondrocytes of the invention or an enriched population of regulatory cells) to restore chondrocyte phenotypic stability to a population of cells having reduced or decreased chondrocyte phenotypic stability. While the initial cells derived directly from explanted tissues (e.g., cartilage biopsies) remain producing and secreting extracellular components characteristic of native cartilage, specifically type II collagen and proteoglycans sulfate, it is known that during monolayer expansion in vitro, mature chondrocytes dedifferentiate and lose their ability to form hyaline cartilage in vivo. The regulatory cell populations of the present invention are capable of improving or restoring chondrocyte phenotypic stability to those cells that have lost their stable phenotype for further passage forward. In addition or alternatively, the regulatory cell populations of the invention are capable of improving or restoring chondrocyte phenotypic stability of chondrocytes that are absent or have reduced chondrocyte phenotypic stability due to a disease (e.g., osteoarthritic cartilage) or other condition. Once such a population of cells is combined with a population of regulatory cells of the invention, the chondrocyte phenotypic stability of the resulting population of cells is restored. For example, for transplantation purposes, this is beneficial because less restrictions are placed on tissues that can be used as a source of cells for autologous repair strategies, such as ACT, or allogeneic repair strategies.

Yet another embodiment of the invention relates to the use of a population of regulatory cells to direct the development of chondrocyte precursor cells towards cartilage forming chondrocytes and more particularly, in the context of the present invention, chondrocyte precursor cells are considered, such as Mesenchymal Stem Cells (MSC) or CDMP-1 positive precursor cells, such as described in WO 0125402. In fact, the present invention demonstrates that stem cells, and more specifically MSCs or osteochondral precursor cells, in combination with a population of regulatory cells of the invention are directed towards a cartilage-producing phenotype. Mesenchymal Stem Cells (MSCs) are capable of differentiating into different mesenchymal lineages, including adipose and connective tissue, bone, and cartilage. MSCs have been detected in human postnatal Bone Marrow (BM), peripheral blood, periosteum, muscle, adipose tissue and adult connective tissue. It has also been demonstrated that many fetal tissues contain MSCs, such as human BM (bone marrow) in the first trimester of pregnancy, liver, blood, and BM, liver, lung, spleen, pancreas, and kidney in the second trimester. Adult BM is currently the most commonly used source of MSCs for clinical purposes. The ability of the cell populations to modulate differentiation of pluripotent MSCs into chondrocyte lineages, and more particularly the ability to induce cartilage-like expression patterns, allows MSCs to be used for efficient production and/or regeneration of cartilage. Also, mesenchymal stem cells have the important advantage that they are self-renewing cell populations that modulate the immune system and reduce the extent of human xenorejection. Thus, mesenchymal stem cells have significant potential in the regenerative medicine field as they make it possible to repair or replace damaged tissues with HLA-mismatched (allogeneic) cells. The present invention therefore provides a method of repairing a cell population, the method comprising combining a population of regulatory cells of the invention with autologous or allogeneic MSCs.

It is to be understood that the above methods of improving, inducing or restoring the chondrocyte phenotypic stability of a chondrocyte cell population or a chondrocyte precursor cell population may be accomplished in different ways. In particular embodiments, the regulatory cell population of the invention is contacted with the other cell population prior to administration to the patient. In one embodiment, multiple cell populations are contacted within 1-30 minutes prior to administration, although contact times up to 1 hour or more are also contemplated. Optionally, the cell population may be contacted in the form of a cell suspension. Or placed together on a substrate for implantation.

Another aspect of the invention relates to the in vitro production of cartilage using the regulatory cell populations of the invention, optionally in combination with one or more chondrogenic cell populations.

Indeed, it may be beneficial in cartilage repair to form cartilage material in vitro, followed by implantation of a cartilage defect. The advantage of such synthetic cartilage material is that cartilage production can be monitored by morphological, biochemical and molecular characteristics prior to implantation. The regulatory cell populations of the present invention can be used either alone or in combination with a population of chondroblasts (selected from the group consisting of freshly isolated chondrocytes, expanded and optionally passaged chondrocytes, MSCs) for the expansion of chondroblasts in an anchor-dependent or non-anchor-dependent culture system. For the latter, cells of the regulatory cell population, optionally in combination with, for example, chondrocytes, can be clonally cultured in an agar gel. Alternatively, in another anchor-independent method, the population of regulatory cells, optionally in combination with, for example, chondrocytes, can be clonally cultured in suspension medium. In the anchor-dependent method, the regulatory cell populations of the invention are combined with, for example, chondrogenic cell populations isolated from primary tissue and grown in monolayers in a two-or three-dimensional matrix to produce cartilage-forming nodules.

In other aspects, the invention provides a population of regulatory cells for use as a medicament, and methods of treatment comprising administering the population of regulatory cells to a patient in need thereof. More specifically, it is believed that the population of regulatory cells is of therapeutic value for the treatment of osteochondral defects by administering the regulatory cells directly to the defect where chondrogenesis is desired. Typically, the regulatory cells of the invention will be administered to the area containing the chondrocytes, most commonly chondrocytes that are no longer capable of producing sufficient amounts of hyaline cartilage as a result of disease or mechanical defect. According to this aspect of the invention, the improvement or restoration of chondrocyte phenotypic stability of the cell population by the regulatory cell population of the invention occurs in situ following implantation into the cartilage defect. Again, the cells of the invention may be administered as a cell suspension or applied to a solid support such as a matrix.

According to another aspect, the present invention provides a composition, a mixture or combination of one or more regulatory cell populations (either freshly isolated cell populations or "enriched form") and one or more chondrogenic cell populations selected from the group consisting of: freshly isolated chondrocytes, passaged mature chondrocytes, mesenchymal stem cells or progenitor cells belonging to the osteochondral cell line. As indicated above, the chondroblasts used in combination with the regulatory cells of the invention are characterized either by a stable chondrocyte phenotype or by an unstable phenotype. These mixtures or compositions are useful as medicaments. The invention also provides pharmaceutical compositions comprising the mixtures and compositions of the invention.

Thus, the present invention provides methods for the treatment of cartilage defects by administering the compositions or mixtures of the invention, and the corresponding use of the compositions or mixtures of the invention in the manufacture of a medicament for the treatment of cartilage defects.

Methods of treatment involving administration of the regulatory cells of the invention, either alone or in combination or admixture with other cell populations, include, but are not limited to: a method for treating articular cartilage and meniscus cartilage of joint (such as knee joint), and other treatments of elastic cartilage, fibrocartilage or articular cartilage at other parts in body (such as intervertebral disc repair, and treatment of osteoarthritis or rheumatoid arthritis). Typically, when used to treat a joint defect, the cell population or composition of the invention is injected under a periosteal flap that is sutured to cover the cartilage defect. Alternatively, the cell populations or compositions of the invention are used to impregnate a synthetic carrier matrix, or seeded onto a natural biopolymer and then implanted into a cartilage defect. Various biopolymers have been used to date, including three-dimensional collagen gels (e.g., U.S. Pat. No. 4,846,835), reconstituted fibrin-thrombin gels (e.g., U.S. Pat. Nos. 4,642,120; 5,053,050 and 4,904,259), synthetic polymer matrices including polyanhydrides, polyorthoesters, polyglycolic acid and copolymers thereof (U.S. Pat. No. 5,041,138), and hyaluronic acid-based polymers.

In addition to one or more cell populations, the pharmaceutical compositions described herein often comprise at least one pharmaceutically acceptable carrier, which is well known to those skilled in the art, for example selected from proteins such as collagen and gelatin, carbohydrates such as starch, polysaccharides, sugars (dextrose, glucose and sucrose), cellulose derivatives such as sodium or calcium hydroxymethylcellulose, hydroxypropylcellulose or hydroxypropylmethylcellulose, pregelatinized starch, pectin agar, carrageenan, clays, hydrophilic gums (gum arabic, guar gum, acacia gum and xanthan gum), alginic acid, alginates, polyglycolic acid and polylactic acid, dextran, pectin, synthetic polymers such as water-soluble acrylic acid polymers or polyvinylpyrrolidone, proteoglycans, calcium sulfate and the like.

Additionally or alternatively, the pharmaceutical composition comprises a population of regulatory cells or a population of combination cells of the invention further comprising a factor, such as a chemotactic factor or differentiation factor.

Brief Description of Drawings

The following examples, which are not intended to limit the invention to the specific embodiments described, are to be understood in combination with the accompanying drawings, the contents of which are incorporated herein by reference, wherein:

fig. 1 shows a marker chart (QS score) for freshly isolated chondrocytes (FI), expanded monolayers of chondrocytes (P0) and a population of regulatory cells of the invention (NAC) from different individual biopsies. QS scores were defined by quantitative assessment of the expression levels of the unique genes detected by RT-PCR. High scores correlate with increased chondrocyte phenotypic stability.

Figure 2 shows the changes in expression of some markers in monolayer expanded chondrocytes (P0) and the non-adherent regulatory cell population (NAC) of the invention from different individual biopsies compared to freshly isolated chondrocytes. The markers were type II collagen (panel A), aggrecan (panel B) and BMP-2 (panel C).

FIG. 3 shows the in vitro modulation of chondrogenesis of a cell population.

FIG. 4 shows in vitro chondrogenesis of mixed cell mass cultures of chondrocytes mixed with varying amounts of a regulatory cell population of the invention (FIG. A). Panel a shows the cell mass formation of dedifferentiated chondrocyte samples containing 10, 20 or 30% (enriched) non-adherent cell populations (NAC), demonstrating a significant increase in the size and mechanical stability of cell mass formation with increasing numbers of regulatory cells of the invention. Panel B demonstrates by positive safranin O staining that TGF-beta stimulated cell mass containing P4 chondrocytes in admixture with 50% of the conditioned chondrocytes of the present invention are effective in forming extracellular matrix.

Figure 5 shows the expression characteristics of type II collagen (panel a) and aggrecan (panel B), TGF β -stimulated cell mass culture of P6 chondrocytes mixed with 30% regulatory cell population (NAC) (left) and TGF β -stimulated cell mass culture of P6 chondrocytes mixed with 30% P0 chondrocytes (right), respectively, in control of unstimulated cell mass culture of the same composition.

Figure 6 shows the type II/I collagen ratio of P4 chondrocytes (left), P4 chondrocytes to TGF β -stimulated cell mass cultures of 30% (middle) or 50% regulatory cell population (NAC) (right).

FIG. 7 shows ectopic chondrogenesis of dedifferentiated P6 chondrocytes from a 60 year old donor mixed with 10% of the regulatory cell population.

FIG. 8 shows type II/I collagen ratios under TGF-beta stimulated and non-stimulated conditions for cell mass cultures comprising Human Synovial Membrane (HSM) -derived Mesenchymal Stem Cells (MSCs) and mesenchymal stem cells with different concentrations of regulatory cells (the numbers for each sample indicate the percentage of MSCs in the MSC and regulatory cell population mixture).

Fig. 9 shows a graph comparing type I collagen and type II collagen expression of a fresh isolated cell population obtained from meniscal cartilage, adherent mature chondrocytes of P0, and a regulatory cell population (NAC), according to an embodiment of the invention.

FIG. 10 shows fibrogenesis in cell mass cultures of regulatory cells (NAC) obtained from meniscal cartilage after TGF-beta treatment, compared to synovial-derived stem cells (HSM) according to embodiments of the present invention. A: a cell pellet comprising 100% synovial-derived stem cells; b: a cell pellet comprising 100% of a population of meniscal chondrocyte regulatory cells (NAC); c: a cell pellet comprising 70% of a population of meniscal cartilage regulating cells (NAC) and 30% of synovial-derived stem cells; d: a cell pellet comprising 50% of a meniscal chondrocyte regulatory cell population (NAC) and 50% mesenchymal stem cells (HSM).

Examples

Example 1: isolation and characterization of regulatory cells

The joint/semilunar biopsies were minced in serum-free DMEM medium and antibiotics. Serum-free DMEM medium containing 0.1% collagenase a and antibiotic solution were then added to the tissue. The enzyme reaction was performed in tubes in an incubator rotor at 37 ℃ for about 12 hours to release individual cells or clusters of cells from the biopsy. The cells were collected by centrifugation and then transferred to DMEM medium containing serum and antibiotics. Cell culture at 37 ℃ with 5% CO₂The process is carried out as follows.

After 4 days from the start of cell culture, the medium was changed as follows: culture supernatants were harvested and fresh medium was added to the adherent chondrocyte fraction. Loosely adherent and non-adherent cells were collected from the cell supernatant by centrifugation at 1500rpm for 10 minutes. These phenotypically stable cells are then frozen or stored in culture flasks or specially treated plates, or used directly in various experimental procedures/clinical applications (as described below).

This round regulatory cell population is found in humans in digested cartilage biopsies of different tissue origin, including articular cartilage and meniscal cartilage, regardless of age or disease. The chondrocyte subpopulation with regulator cells found in articular chondrocytosis was present at a concentration of 8-20% of the seeded cell mass (table 1). In a meniscal cartilage biopsy, the non-adherent cell population was found to be about 5-20% of the total number of cells.

Table 1: non-adherent cells obtained from the chondro digest account for the percentage of the total cell population.

Articular chondrocyte biopsy	Age (age)	Sex	Cell viability	% non-adhesive
					CS228	81	For male	85％	14％
CC0039	25	Is not indicated by	91％	20％
					CC0009	23	For male	86％	8.5％
CC0044			89％	14％

Immunohistochemical analysis of the population of non-adherent cells confirmed that at least a portion of the non-adherent cells had an extracellular matrix comprising VI. Flow cytometric analysis showed expression of CD29, CD49e, CD51, CD54, and CD 56.

The regulatory cell population demonstrates an aberrant expression profile of markers associated with cartilage phenotypic stability.

Typically, articular chondrocytes lose their characteristic phenotype when they are cultured in a monolayer. Concomitant with the shift towards polygonal and even fibroblast morphologies is a dramatic decrease in cartilage-specific extracellular matrix gene expression. The non-adherent regulatory cell population not only maintained its circular morphology during the first expansion, but the expression profile of these markers of phenotypically stable cartilage was improved (analyzed at the end of the first expansion) compared to the adherent P0 mature chondrocytes (fig. 1). Since the appearance of regulatory cells in freshly isolated chondrocytes (FI) ensured that the expression profile of phenotypically stable chondrocyte markers, whose expression was lower than that of the enriched population of regulatory cells, was improved over the adherent P0 mature chondrocytes (fig. 1).

All analyses confirmed that the population of regulatory cells of the invention showed a higher QC score compared to P0 chondrocytes or even Freshly Isolated (FI) chondrocytes, regardless of the age of the patient from which the cartilage was obtained.

All but one sample (donor 81 years old, with severe OA) had a higher collagen type II expression rate than freshly isolated cells (data not shown) and significantly exceeded P0 adherent mature chondrocytes (fig. 2A). The expression rate of aggrecan significantly exceeded P0 adherent mature chondrocytes (fig. 2B) and freshly isolated chondrocytes (data not shown). BMP-2 expression was higher than P0 adherent mature chondrocytes (fig. 2C) but lower than freshly isolated chondrocytes (data not shown).

Example 2: the invention modulates the chondrogenic capacity of cells

A non-adherent conditioned cell population of about 200,000 cells (biopsy CS225) was centrifuged at 1500rpm in 15ml conical tubes and cultured in clumps under chondrogenesis inducing conditions. The formula of the culture medium is as follows: DMEM with 1:100 antibiotics, 1:100 sodium pyruvate, 1:100ITS + [ BD Biotech (BD Biosciences) ]]，10^-7M of dexamethasone and 1:100037.5mg/ml ascorbic acid. The tube cap was then gently unscrewed and cell culture was performed at 37 ℃ with 5% CO 2. The cell culture cycle was 2 weeks with medium changes every 2-3 days. TGF-. beta.was also added to some cell masses. After 2 weeks, the cell pellet was used for histological analysis or RNA extraction for real-time quantitative PCR.

TGF β stimulation led to mechanically stable, abnormally textured chondrogenic tissue, as evidenced by metachromatic staining of toluidine blue and safranin O of the extracellular matrix of paraffin-embedded cartilage sections (figure 3).

These data demonstrate that the regulatory cell populations of the present invention are themselves phenotypically stable populations of chondrocytes capable of producing high quality cartilage.

Example 3: modulating cell populations capable of improving chondrocyte phenotypic stabilization of mature chondrocytes Property of (2)

The cultivation of the mixed cell mass is similar to the procedure described in example 2 above, but using a combination of regulatory cells and mature chondrocytes.

Observations after two weeks of repeated passaging of chondrocytes and TGF β -stimulated mixed cell pellet cultures of different numbers (10%, 20%, 30%) of the regulatory cell population (NAC) of the invention showed a greater chondrogenic capacity than single dedifferentiated chondrocytes, an increase in the size of the pellet (fig. 4A) and safranin O metachromatic staining (fig. 4B) could be confirmed.

The combination of the regulatory cell population and 70% P6 chondrocytes of the present invention induces high quality extracellular matrix. A significant increase in type II collagen and aggrecan expression was observed in the mixed cell pellet culture containing the conditioned chondrocytes compared to the mixed cell pellet culture using the combination of P0 chondrocytes and P6 chondrocytes (fig. 5).

Type II collagen: a significant increase in type I collagen ratio also reflected an improved chondrogenic capacity (fig. 6). A decrease in collagen I expression was observed after an increase in the collagen II expression rate (fig. 6, middle, right panel). In contrast, cell mass cultures of pure dedifferentiated P4 chondrocytes did not form mechanically stable cartilage and showed only limited type II collagen re-expression rate in 3D culture (fig. 6, left).

Example 4: production of epitypic stable cartilage in muscle tissue of nude mice

The ability of the present invention to modulate the re-differentiation of cell populations was demonstrated by ectopic cartilage formation following intramuscular injection in nude mice. In this method approximately 3 to 4 million cells were mixed with HBSS medium and injected into the thigh of a nude mouse in a volume of 50 microliters. Animals were sacrificed after 2-3 weeks, and their thigh muscles were fixed with formalin, embedded in wax and sectioned for histological studies.

Cartilage engraftment was not detected after injection of 4 million cells consisting of a combination of 90% P6 chondrocytes and 10% P0 mature adherent chondrocytes. However, large cartilage implants were formed when the same number of P6 chondrocytes were injected in combination with 10% regulatory non-adherent cells (CS 225-regulatory cells/CS 216P 6).

Metachromatic staining with toluidine blue and safranin O confirmed the quality of the hyaline cartilage-like implant (fig. 7).

Example 5: differentiation of mesenchymal stem cells towards chondrogenic lineage

Mesenchymal stem cells were combined with varying numbers of regulatory cells, placed in mixed cell pellet cultures stimulated with TGF β as described above, or used in nude mice in vivo assays as described above. Differentiation into chondrogenic lineages was dramatically promoted by culturing a mixture of 70% mesenchymal stem cells and 30% regulatory chondrocytes. The number of chondrocyte aggregates was assessed by measuring the expression of type II collagen and type I collagen (fig. 8B). The results show that regulatory cells are superior to the single TGF differentiation agent most commonly used in promoting differentiation into cartilage by enhancing collagen type II expression to a considerably high level.

Example 6: using autologous or allogeneic non-hematopoietic stem cells and autologous or allogeneic phenotypes Combination cell product for stabilizing chondrocyte composition to treat OA joint

Autologous or allogeneic non-adherent cells harvested as in example 1 are combined with expanded mature chondrocytes, human synovial-derived stem cells, or other sources of non-hematopoietic stem cells. The combination of non-adherent regulatory cells with chondrocyte precursor cells forms a cell product with combined therapeutic activity, wherein the regulatory cells direct the formation of stem cells into a chondrogenic lineage, and the stem cells modulate the inflammatory response. The combined cell product can generate cartilage and regulate inflammation, and has special significance in treating rheumatoid arthritis cartilage defect.

Example 7: isolation and characterization of meniscal cartilage regulatory cell populations

The conditioned cell population was enriched from the meniscal cartilage sample by collecting the non-adherent fraction as described in example 1. Determining an expression profile for the population of regulatory cells. The regulatory cell population derived from meniscal cartilage was characterized by a significantly higher collagen type II expression rate than the corresponding adherent P0 cartilage cell population derived from the meniscal cartilage sample (fig. 9). Type II collagen low expression was found in freshly isolated meniscal chondrocytes. In contrast, the expression rate of type I collagen in enriched regulatory cell populations derived from meniscal cartilage is decreased.

Example 8: fibrogenic capacity of regulatory cell populations derived from meniscal cartilage

Cell pellet cultures were obtained using enriched regulatory cell populations derived from meniscal cartilage as described in example 2. TGF β of the meniscal cartilage regulatory cell population was found to stimulate the cell mass culture to form collagen fibrils, suggesting that this cell population is capable of integrating into the surrounding tissue (figure 10, upper right panel). Mixed cell mass cultures of meniscal cartilage-derived regulatory cell populations and synovial membrane-derived stem cells were prepared and stimulated with TGF β. It was observed that the meniscal chondrocyte regulator cells had a variable relative number of collagen fibrils present (fig. 10, bottom panel), whereas single synovial-derived stem cell mass cultures did not form collagen fibrils (fig. 10, top left panel). This property of meniscal cartilage regulatory cells is particularly beneficial in repairing meniscal internal tears for which there is currently no treatment (see below).

Example 9: treatment of meniscal cartilage defects using meniscal cartilage-derived regulatory cell populations

Conditioned cell populations were enriched from the semilunar cartilage samples using the procedure described in example 1 and cell counts were counted. The meniscal cartilage-derived regulatory cell population is then mixed with the expanded human synovial-derived stem cells, optionally placed in a support, for the treatment of meniscal cartilage defects, even in the avascular region of the meniscus.

Claims (24)

1. A method of enriching a population of regulatory cells from an isolated cartilage sample, the method comprising the steps of:

a) mechanically and/or enzymatically treating the isolated cartilage sample to obtain individual cells,

b) transferring the isolated cells obtained according to step a) to a cell culture vessel to expand chondrocytes in a monolayer,

c) subjecting the vessel to suitable cell culture conditions to obtain a population of adherent cells and a supernatant comprising a population of non-adherent cells,

d) collecting the supernatant comprising the population of non-adherent cells from the container after at least 2 days and/or when at least about 30% are confluent,

e) collecting said population of non-adherent cells corresponding to said population of regulatory cells from said supernatant.

2. The method of claim 1, wherein the cartilage sample is an articular cartilage sample.

3. The method of claim 1, wherein the cartilage sample is a half-moon cartilage sample.

4. The method of any one of claims 1-3, wherein step (a) comprises treating the isolated cartilage sample with collagenase A.

5. A composition comprising a combination of two or more different cell populations, the composition comprising:

a) a cell population comprising regulatory cells, which is (i) a freshly isolated chondrocyte population obtained from a cartilage biopsy, or (ii) an enriched regulatory cell population obtained by collecting non-adherent cells from a P0 culture supernatant of freshly isolated chondrocytes obtained from a cartilage biopsy

b) One or more cell populations selected from the group consisting of: the cell population of the passed cartilage cell, the mesenchymal stem cell population and the chondrocyte precursor cell population.

6. The composition of claim 5, comprising an enriched population of regulatory cells, wherein the relative amount of the enriched regulatory cells to the total number of cells in the composition is between 1-75%.

7. The composition of claim 5 or 6, wherein the population of cells comprises regulatory cells obtained from articular cartilage.

8. The composition of claim 5 or 6, wherein the population of cells comprises regulatory cells obtained from meniscal cartilage.

9. A pharmaceutical composition comprising a combination according to any one of claims 5 to 8.

10. A support comprising a combination of cells according to any one of claims 5 to 8.

11. Use of a combination according to any one of claims 5 to 8 in the manufacture of a cellular therapeutic agent for the treatment of cartilage defects.

12. A method of treating a cartilage defect comprising administering to a patient having said cartilage defect a combination according to any one of claims 5 to 8.

13. A non-adherent, non-passaged population of regulatory cells obtainable by the method of any one of claims 1 to 4 for use as a medicament.

14. A method of treating a cartilage defect comprising administering to a patient having said cartilage defect a population of regulatory cells obtained by the method of any one of claims 1 to 4.

15. A pharmaceutical composition comprising a population of non-adherent, non-passaged regulatory cells obtained by the method of any one of claims 1 to 4.

16. Use of a non-adherent, non-passaged population of regulatory cells for improving, maintaining or restoring the chondrocyte phenotypic stability of an expanded or passaged chondrocyte population in vitro.

17. Use of a population of non-adherent regulatory cells for differentiating mesenchymal stem cells into chondrogenic lineage cells in vitro.

18. A method of preparing a cell population for ACT transplantation, the method comprising the steps of:

a) mechanically and/or enzymatically treating the isolated cartilage sample to obtain individual cells,

b) transferring the isolated cells obtained according to step a) to a cell culture vessel to expand chondrocytes in a monolayer,

c) subjecting the vessel to suitable cell culture conditions to obtain a population of adherent cells and a supernatant comprising a population of non-adherent cells,

d) collecting the supernatant comprising the population of non-adherent cells from the container after at least 2 days and/or when at least about 30% confluence is reached in the container,

e) collecting the non-adherent cells from the supernatant.

f) Combining the non-adherent cell population obtained in step (e) with a population of chondroblasts that is not an enriched population of regulatory cells.

19. The method of claim 18, wherein said chondrogenic cell population is a mature chondrocyte population obtained from said isolated cartilage sample, wherein the method further comprises the steps of:

g) expanding and passaging the adherent cell population obtained in step (c),

h) collecting said expanded and passaged adherent cell population,

i) in step (f), combining the population of non-adherent cells obtained in step (e) with the expanded and passaged population of adherent cells obtained in step (h).

20. The method of claim 18 or 19, wherein said enzyme treatment in step (a) is effected with collagenase a.

21. The method of any one of claims 18 to 20, wherein the isolated cartilage sample is a half-moon cartilage sample.

22. The method of any one of claims 18 to 21, wherein the chondrogenic cell population is derived from a meniscal cartilage biopsy.

23. A method according to any one of claims 18 to 21, wherein the chondrogenic cell population is a mesenchymal stem cell population.

24. The method of claim 18 or 19, further comprising the steps of:

j) (ii) seeding the combined cell population obtained in step (f) or (i) onto a support.