Compositions of chondrocytes, preparation and utilization

Details Compositions of chondrocytes, preparation and utilization Compositions of chondrocytes, preparation and utilization

2000-09-15

2002-04-02

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Primate cell, per se

C435S380000, C435S381000

Reexamination Certificate

active

06365405

ABSTRACT:

This invention concerns compositions of chondrocytic cells, particularly of human origin, and methods for preparing them. It also concerns the use of these compositions to implant chondrocytes in vivo, to treat various pathologies. More particularly, the invention describes the production of suspensions of autologous human chondrocytes, techniques for conserving them, and their use in vivo to restore cartilaginous structures affected, such as post-traumatic cartilaginous defects or the dissecting osteochondritis of the knee, or, more generally, to treat and repair clinically significant defects in cartilage, particularly in articular cartilage.
Chondral lesions, such as, for example, focal lesions of the knee's articular cartilage, in the load bearing zone, greatly expose the subjects to osteoarthritis. This type of lesion is frequent (from trauma, sports activity, dissecting osteochondritis, etc.) and produces a significant gene. The capacity for spontaneous repair of chondral lesions is modest. They can be characterized by the presence of fibrocartilaginous scars, through arthroscopic studies. Essentially these lesions are treated by washing, which temporarily heals the symptoms, but does not treat the origin of the defect and, in particular does not prevent progressive degradation of the cartilage. This osteoarthritic evolution of the cartilage in reality, it appears, can be treated effectively only by replacing the defective cartilage with healthy cartilage. In particular, the use of cells other than chondrocytes results in ineffective repair to the cartilage, because it changes the nature of the extracellular matrix.
This is why the literature describes the production and therapeutic use of human chondrocytes, specially the autologous ones, to reconstitute or compensate for defects in cartilage.
Thus, isolation of chondrocytes is practiced, starting from various types of cartilage or even from precursors drawn from bone marrow. For this purpose, a sample of healthy cartilage (or marrow) can be taken from a subject, mechanically dissected to form fragments of reduced size, then treated in the presence of one or several enzymes, generally trypsin and/or an extracted collagenase, in order to separate the chondrocytes contained in the cartilage. The chondrocytes can then be cultivated, generally in a monolayer, to obtain a sufficient number of cells (several passages generally are performed.) Although chondrocytes dedifferentiate in the form of fibroblasts during their culture and expansion phases, it has been shown that the chondrogenic cells obtained could differentiate themselves again into functional chondrogenic cells, either in situ or in vitro, through insemination in three-dimensional cultures.
Various approaches have been developed to grow in vitro chondrocyte cultures, and various treatment strategies have been developed. In particular, international application number W095/30742 proposes reconstituting artificial cartilage patches in vitro (that is, multilayer chondrocytes included in an endogenous extracellular matrix) of a predefined form and volume, and to implant these patches onto the site being treated. Other strategies are based on producing chondrocyte cultures (or chondrocyte cells) in vitro expanded, which are implanted directly to reconstitute the cartilage in situ, or after biocompatible synthetic matrices have been deposited. The use of factors that stimulate the growth of precursors to chondrocyte cells has also been suggested, in order to induce chondrocyte production in situ.
This invention presently describes new compositions and methods enabling the conditions of production and utilization of chondrocytes to be improved, for therapeutic applications. This invention describes, in particular, new methods which result in improved production of chondrocytes. Notably, this invention describes chondrocyte production techniques which enable pharmaceutical quality chondrocyte preparations to be generated. This invention also describes new techniques which allow chondrocytes to be conserved efficiently, notably in frozen form, which considerably promotes the conditions for implementing a clinical grafting program. This invention can be implemented to generate suspensions of autologous human chondrocytes which are intended to be implanted in vivo, or to be used in producing artificial cartilage patches, or also to be used in preparing synthetic matrices covered with chondrocytes. This invention also describes methods for implanting chondrocytes in the areas to be treated (notably, cartilage).
Thus, a first aspect of the invention resides in the production of chondrocytes from a biological sample, in particular of chondrocyte cultures from a biological sample comprising chondrocytes included in an cartilaginous extracellular matrix, and even more preferably of chondrocytes suspensions from a biopsy of cartilaginous tissue. The invention's processes include various stages of treatment and conditioning of the biological sample, then of chondrocyte culture and treatment, making it possible to obtain preparations of chondrocytes of pharmaceutical quality, that is, which can be used in human therapy.
To enable this invention to be more clearly understood, the following definitions are provided:
Cartilage (or cartilaginous tissue): The cartilage is essentially formed of chondrocytes, incorporated into an extracellular matrix. The extracellular matrix mainly includes collagen (essentially of type II) and proteoglucanes (essentially, proteins to which are connected glucosamine glucanes such as chondroitin sulfate and keratane sulfate). The extracellular matrix is serrated by the chondrocytes. There exist various types of cartilage, such as hyalin cartilage (notably present in joints, in particular the rib, nasal, bronchial cartilage, etc.), the elastic cartilage, which in addition encloses the elastin fibers (present, for example, in the ear) and the fibrous cartilage, which moreover encloses Type I collagen (present, notably, in intervertebral disks, the meniscus, etc.). In terms of the invention, cartilage designates all types of cartilage, notably including chondrocytes and an extracellular matrix.
Chondrocytes are cartilage cells which secrete the extracellular matrix. In terms of this invention, the term chondrocyte designates more specifically the cells which secrete the cartilaginous extracellular matrix, notably type II collagen and proteoglycanes such as “agrecannes”. In terms of this invention, the term chondrocyte also includes “chondrogenic” cells, that is, cells which are capable of producing chondrocytes. Thus, this consists of cells that are apt of producing the cartilaginous extracellular matrix, such as chondrocyte precursors, notably mesenchymatous precursors (or mesenchymatous stem cells), or dedifferentiated chondrocytes, for example, following cultures in monolayers. The term chondrocyte also designates primary cultures (freshly isolated from biopsies) as well as cells expanded in vitro, including genetically modified, immortalized, selected, conserved, etc.
According to a primary advantageous characteristic, the invention provides that the production method includes a stage of a) treatment of the biological sample in the presence of a recombinant enzyme, thus facilitating the dissociation of chondrocytes, in particular through hydrolysis (at least partial) of collagen molecules contained in the sample.
As it will be explained in detail further on, a primary characteristic of the invention's process consists of the employment of a recombinant enzyme to dissociate the biological sample, notably, the cartilage biopsy. In effect, this invention shows that the use of such an enzyme improves not only the quality of the product, but also the effectiveness of the process.
Thus, the objective of the invention is to develop a process for dissociation chondrocytes contained within a biological sample, including the in vitro treatment of this biological sample in the presence of a recombinant enzyme capable of hydrolyzing collagen molecules

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