Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation (original) (raw)
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Primary culture and phenotyping of murine chondrocytes
Nature Protocols, 2008
The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.
Tissue Engineering, 2006
Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.
Culture and Phenotyping of Chondrocytes in Primary Culture
Cartilage and Osteoarthritis
The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. This chapter describes several protocols for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes) from various species (humans, mice, rabbits, and cattle). All these protocols involve primary cultures in order to limit dedifferentiation. This chapter also describes a new protocol for culturing mouse articular chondrocytes.
Redifferentiation of dedifferentiated human chondrocytes in high-density cultures
Cell and Tissue Research, 2002
High-density cultures are widely used as an in vitro model for studies of embryonic cartilage formation. In the present study we investigated the suitability of high-density cultures for the redifferentiation of dedifferentiated chondrocytes. When primary human chondrocytes were cultured in alginate beads, some cells emigrated into Petri dishes. These cells were cultured for one to eight passages (each passage lasting
Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes
Journal of Tissue …, 2010
A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co-culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non-diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co-cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co-culture with cryopreserved passaged cells for functional analysis. The co-culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline-like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co-culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair.
Behavior of Human Articular Chondrocytes During In Vivo Culture in Closed, Permeable Chambers
Cell Medicine, 2012
The exact contribution of transplanted chondrocytes for cartilage tissue repair prior expansion in monolayer cultures remains undetermined. At our laboratory, we have created a new permeable chamber to study the chondrogenesis of dedifferentiated cells implanted ectopically in a closed and controlled environment. The behavior of chondrocytes has been studied in settings frequently used in clinical approaches during transplantation, namely injection of autologous chondrocyte cells in suspension (ACI), cells soaked in collagen membranes (MACI), and cells applied in a polymer gel (fibrin). As controls, we have tested the redifferentiation of chondrocytes in cell aggregates, and we have checked the proper functionality of chambers both in vitro and in vivo. After retrieval, firmed tissue-like shapes were recovered only from chambers containing cells seeded in membranes. Histomorphological, immunohistochemical, and ultrastructural analyses revealed synthesis of fibrous-like tissue, characterized by low-density collagen fibers, low collagen type II, abundant collagen type I, and low amounts of proteoglycans. Additionally, neither the collagen membranes nor the fibrin gel was reabsorbed by cells. In summary, our results show that the newly developed permeable chambers function correctly, allowing proper cell feeding and preventing cell leakage or host cell invasion. Additionally, our results suggest that, under these circumstances, chondrocytes are not able to orchestrate formation of hyaline cartilage and have little capacity to degrade artificial membranes or carrier gels such as fibrin. These are interesting observations that should be considered for understanding what role the transplanted chondrocytes play during restoration of articular cartilage after implantation.
Extracellular matrix formation by chondrocytes in monolayer culture
The Journal of Cell Biology, 1981
In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from...
Journal of Cellular Physiology, 2005
Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as “dedifferentiation.” To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications. © 2004 Wiley-Liss, Inc.