Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue

Definitions

• Cartilage shows a very limited capacity for tissue regeneration. Therefore in medical transplantation engineering, there has been an interest in substituting cartilage with autologous cells, including autologous cells having the potential for differentiation to cartilage. This interest has contributed to increased study into tissue culture systems, where tissues can be controlled and monitored during their differentiation states as they form cartilage.
• chondrocytes Of particular interest is the optimization of expansion and differentiation of cultured human and animal chondrocytes, since cartilage tissue engineering and newly developed cell therapeutic methods such as autologous chondrocyte transplantation (ACT) rely on chondrocytes or chondrocyte-like cells obtaining expanded chondrogenic potency in tissue culture systems.
• ACT autologous chondrocyte transplantation
• chondrocytes embedded in the extracellular matrix of cartilage tissue are released by enzymatic digestion of the matrix. After digestion, the chondrocytes obtained are expanded in a cell culture medium, including grown stimulation factors in order to obtain a high cell number count for transplantation.
• mesenchymal stem cells which are also autologous, fall into the category of chondrocyte-like cells.
• Mesenchymal stem cells are an attractive alternative cell source from which to obtain chondrocytes for an ACT process, since they can be isolated from diverse tissues without irreversible tissue damage.
• De-differentiation of chondrocytes and a low rate of proliferation during cell culture results in a poor transplantation success rate. Since monolayer culturing increases chondrocyte proliferation capacity and decreases de-differentiation, it is the chosen method of tissue engineering for generating chondrocytes used in ACT. In addition to the native effects of monolayer culturing, growth factors are used to initiate re-differentiation of chondrocytes or for chondrogenic differentiation of MSC in vitro.
• the present invention provides a method for measuring SERPINA1 and/or
• MMP3 as secreted marker molecules from cells in monolayer culture systems or from cells embedded in three dimensional scaffolds for correlation with a concentration of these marker molecules with a specific level of cell potency.
• the present invention provides a method for measuring the concentration of SERPINA1 or MMP3 or a combination of concentrations of these markers in the supernatant of the cultured chondrocytes or chondrocyte-like cells in monolayer culture systems or from cells embedded in three dimensional scaffolds.
• the invention further establishes a method for analyzing chondrogenic potency in cells re-differentiated from chondrocytes losing their chondrogenic potency or which are differentiated from cells to receive chondrogenic potency by measuring SERPINA1 and/or MMP3 as secreted marker molecules from cells in a growth factor treated culture.
• the method also comprises establishing a condrogenic re-differentiation protocol for returning chondrogenic potency to chrondocytes as well as establishing a chondrogenic differentiation protocol for cells achieving chondrogenic potency.
• the present invention comprises a method for measuring SERPINA1 and/or MMP3 as secreted marker molecules from cells in monolayer culture for correlation of the concentration of these markers with a specific cell potency.
• the culture supernatant is isolated from a cultivation of cell cultures in a liquid medium.
• the SERPINA1 gene provides instructions for making a protein called alpha-1 antitrypsin, which is a type of serpin protein. Serpin proteins help control several types of chemical reactions by inhibiting certain enzymes.
• Alpha-1 antitrypsin is named for inhibiting properties against the digestive enzyme trypsin. It also inhibits other enzymes including neutrophil elastase, a powerful enzyme found in white blood cells. Therefore, the marker molecule SERPINA1 measured in the cell culture supernatant is the aforementioned serpin protein or a part of this protein secreted from the cultured cells into the culture supernatant.
• MMP3 is known as Matrix metalloproteinase 3 (stromelysin 1, progelatinase). This protein is involved in the breakdown of extracellular matrix in normal physiological processes, such as reproduction and tissue remodeling. The protein is secreted as inactive proproteins which are activated when cleaved by extracellular proteinases.
• the MMP3 gene encodes an enzyme which degrades fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans. Therefore, the marker molecule measured in the cell culture supernatant is the aforementioned enzyme or a part of this enzyme.
• secreted marker molecules refers to molecules secreted from cells in liquid medium cultivation.
• the secreted marker molecules described in this invention are SERPINA1 and MMP3 that allow monitoring of expansion and differentiation in vitro and thus comparison and optimization of cell cultures for in vivo applications
• correlating used with reference to the concentration of the marker molecules indicates a correlation between the measured marker molecules secreted from the cells into the supernatant of the liquid culture medium and the chondrogenic potency of the cells.
• chondrocytes described herein are mammal chondrocytes found in human or in animal cartilage which produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans.
• Chondrocyte-like cells are cells which can terminally differentiate into chondrocytes.
• CFU-F colony-forming unit-fibroblasts
• mesenchymal stem cells are chondrocyte-like cells for purposes of this invention.
• the term "monolayer culture systems" refers to a layer of cells in which no cell is growing on top of another, but all are growing side by side and often touching each other on the same growth surface.
• cells embedded in three dimensional scaffolds refers to cells seeded in a three dimensional structure or seeded in a gel or gel-like matrix to form a transplantation body.
• chondrogenic potency used in this application describes cells having the capacity to form substitute cartilage when implanted by injection or transplanted in a three dimensional scaffold into a point of defect in the cartilage.
• re-differentiation refers to molecular effects in chondrocytes that have lost their chondrogenic potency, assumed a fibroblast-like morphology and have ceased to synthesize cartilage-specific molecules like collagen 2 and aggrecan. However these effects are reversible and chondrocytes capable of re-differentiation can get back their chondrogenic potency with growth factor treatment.
• re- differentiation when used in the invention also refers to the acceleration of the redifferentiation process by up-regulation of chondrocyte-specific genes using specific growth factors.
• chondrocytes losing their chondrogenic potency refers to chondrocytes in monolayer or three-dimensional culture systems showing changes that include: loss of their rounded cell shape, loss of collagen type Il and aggrecan core protein expression, and loss of the capacity to induce stable cartilage implants after transplantation into a defective area of cartilage.
• the term "differentiated from cells to receive chondrogenic potency” refers to chondrocyte-like cells as explained above expanded in vitro in cell culture systems.
• mesenchymal stem cells MSC are commonly known as chondrogenic cells since MSC have shown the ability to differentiate into osteoblasts.
• differentiation of MSC in a non-vascularized area yields a chondrocyte.
• growth factor treated culture refers to cell cultures in monolayer or three dimensional culture system conditions treated with recombinant growth factors stimulating de-differentiation, re-differentiation or differentiation of cells into cells with chondrogenic potency by directed gene expression or gene inhibiting, for example the stimulation of proteoglycan synthesis by BMP-4.
• growth factor also refers to hormones and morphogenes.
• BMP bone morphogenic proteins
• TGF- ⁇ transforming growth factor beta
• IGF-1 insulin-like growth factor 1
• the term "protocol” refers to standardized data to determine the capacity of chondrocytes to proliferate, altered gene expression in chondrocytes or the chondrogenic potency of re-differentiated chondrocytes, or the differentiation state of chondrocyte-like cells with chondrogenic potency switched to chondrocytes.
• the practice of the invention involves culture models including monolayer, three dimensional culture systems such as alginate culture or high density pellet culture, and explant models. Tissue engineering techniques to achieve these culture models are known in the art. A wide variety of expansion protocols of different mammalian cell types such as by application of growth factors are also well-known to persons of skill.
• Cartilage samples from peripheral, less load bearing regions of the knee joints were obtained after informed consent from parties undergoing total joint replacement in an ethically approved study. Chondrocyte isolation and culture was performed by removing cartilage slices outside regions with macroscopic evident degeneration from the underlying bone, mincing the slices with a scalpel, and digesting with 1.5 mg/ml of collagenase B (Roche, Mannheim, Germany) and 0.1 mg/ml hyaluronidase (Serva, Heidelberg, Germany) overnight (18h) at 37 9 C.
• the released cells were plated in 80 cm 2 cell culture flasks in Dulbeccos modified Eagle's medium (DMEM (Life Technologies, Düsseldorf, Germany)) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and maintained in a humidified atmosphere at 6% CO 2 and 37 9 C.
• DMEM Dulbeccos modified Eagle's medium
• FCS fetal calf serum
• RNA and cDNA was isolated from cultured cells by the methods described in W. Richter et al. (2002) Biochemical and Biophysical Research Communications 293: 284-292.
• the isolated cells were encapsulated in alginate beads as three dimensional scaffolds at a density of 1 x 10 6 cells/ml of gel as described in E.J. Thonar et al. (1994) J. Cell Sci., 107 (Pt1): 17-27. Briefly, cells were suspended in sterile 0.15 M NaCI containing low viscosity alginate gel (1.2%) and then slowly pressed through a 22 gauge needle in a drop-wise fashion into a 102 mM CaCI 2 solution. [0033] After instantaneous gelation, the beads were maintained in a complete medium in a humidified atmosphere at 6% CO 2 and 37 9 C. The medium was replaced twice a week.
• RNA and cDNA were isolated from cultured cells by the methods described in W. Richter et al. (2002) Biochemical and Biophysical Research Communications 293: 284-292.
• adipose tissue-derived MSC were plated at a cell density of
• RNA and cDNA were also isolated from cultured cells by the methods described in W. Richter et al. (2002) Biochemical and Biophysical Research Communications 293: 284-292.
• the protein level of SERPINA1 and MMP3 obtained from the supernatant with an enzyme linked immunosorbent assay showed a reduction in the concentration of SERPINA1 and MMP3 in the supernatant of all culture systems.
• the availability of SERPINA1 and MMP3 from the supernatant in cell culture systems provides a method for measuring chondrogenic potency. Correlations of the reduction in SERPINA1 and MMP3 concentrations in the supernatant of a culture medium with datasets from the RNA and cDNA experiments showed that gene expression of chondrocyte relevant RNA, and signal intensities of chondrocyte relevant cDNA were lowered.
• chondrongenic potency can be reliably monitored without sacrificing cells to determine SERPINA1 and MMP3 levels secreted from cells in the medium; not only from cells suspended in a culture medium (see EP 873 236 A1 , filed Jun. 14, 2007), but also from monolayer cell culture systems and from three dimensional culture systems. Therefore with the method claimed by this invention, chondrongenic potency in three dimensional scaffold cultures can be monitored at the point of transplantation.

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