Cartilage was cut from the subchondral bone using a sterile sharp scalpel blade and placed in serum free dul beccos modified eagles medium dmem. Conclusionhuman chondrocytes proliferate in this culture system, show an outgrowth into the surrounding fibrin, and synthesise a cartilagelike matrix for up to eight weeks. Remove one cryovial of imef cells from the liquid nitrogen cell storage dewar and place on ice for transfer to the cell culture suite. The concentration of free active transforming growth factor tgf. The cell suspension was added back into the old dishes and. Here we describe the isolation, shortterm and longterm culture, and analysis of primary costal chondrocytes from the mouse.
Chondral and osteochondral defects affect a large number of. Kinetics of the ph value of 3d chondrocyte cultures of a days cultivation within 24well hydrodishes. Isolation and culture of murine primary chondrocytes. This present study demonstrated the differences or similarities in the cytoskeletal morphologyorganization of chondrocytes in different modes of culture i. Such dedifferentiated chondrocytes reexpress the differentiated phenotype during suspension culture in firm gels of 0.
These cells are directly received from source human or animal and started culturing. University of manchester chondrocyte protocol susan kimber lab. For example, in monolayer culture chondrocytes do not retain all of their specific functions. Chondrogenic redifferentiation potential of chondrocytes. Articular chondrocytes were cultureexpanded in media supplemented with either low 1.
Dna free rna from normal and osteosteoarthritic human articular cartilage. Wash the chondrocyte spheroids twice with distilled water. It is composed solely of chondrocytes, which are specialized cells that produce and maintain the extracellular matrix of cartilage including collagen mostly type ii and proteoglycans. The design includes internal loop wavy riser airlift reactor for chondrocyte culture with 5% co 2 sparging which gives a good yield of chondrocyte after 28 days. Primary human chondrocytes in culture and chondrogenesis in. Isolating articular chondrocytes from cartilage for cell culture. An important aspect of any prolonged culturing period is the maintenance of cell viability. Primary culture is the in vitro establishment of the cell growth. Aci has long been regarded as the youngest and the most complicated new procedure in a round of three aci, osteochondral cylinders and microfracture, and has been frowned upon as too technological and expensive by many. Actin was seen to form stress fibres only in 2d culture conditions, but not in 3d culture and in situ.
Briefly, hac and msc were harvested, washed once with phosphate buffered saline pbs and resuspended in serum free medium consisting of dulbeccos minimal essential medium dmem high glucose 4. Chondrocyte phenotype and cell survival are regulated by. Chondrocyte sheets applied to cartilage regeneration have been prepared with the cellsheet technique using temperatureresponsive culture dishes. Chondrocyte culture in alginate hydrogel constitutes the most frequently. Critical factors in chondrogenic differentiation of mscs in vitro appear to be the. Redifferentiation of dedifferentiated human articular chondrocytes. Morphology of the bovine chondrocyte and of its cytoskeleton.
Chondrocyte isolation samples of articular cartilage were taken to the culture laboratory and submitted to enzyme digestion process to isolate cells. The cultures were stimulated with triiodothyronine t3 to promote chondrocyte hypertrophy. Three methods are presented in this unit for culturing chondrocytes, and two assays are described that characterize proteoglycan synthesis, a key measure of chondrocyte function. The serum free 50 mm and 1 mm calcium media provide good alternatives to classical media for monolayer culture since both growth and chondrocyte phenotype were maintained.
Chondral and osteochondral defects affect a large number of people in. Passage cells just before they reach confluence using 0. The present results show that, as with primary chondrocytes, the expression of these extracellular matrix genes is differentially modulated by specific cytokines. Human serum for culture of articular chondrocytes sage journals. An attempt has been made to design a novel airlift reactor for chondrocyte culture, and the reactor has been evaluated for its performance.
Dedifferentiated chondrocytes reexpress the differentiated. During serial monolayer culture this phenotype is lost and replaced by a complex collagen phenotype consisting predominately of type i collagen and a low level of proteoglycan synthesis. Original article primary culture and adenoviral infection of. Flow cytometric analysis of the human articular chondrocyte. Culture chondrocytes an overview sciencedirect topics. Transfer cells to new culture vessels at the same concentration as in step 3 x 10 5 per cm2. Interaction between zonal populations of articular. Many types of culture exist and have their own advantages and drawbacks. Pdf chondrocyte is a unique cell type in articular cartilage tissue and is. Tissue specific differentiation of human chondrocytes. Decontaminate the bottle with 70% alcohol in a sterile hood. Enhanced chondrocyte culture and growth on biologically inspired nanofibrous cell culture dishes garima bhardwaj,1 thomas j webster1,2 1department of chemical engineering, northeastern university, boston, ma, usa. Articular chondrocytes produce factors that inhibit. Arrows indicate the change of culture medium, stars the short removal of the culture from incubator.
Similar changes may occur in osteoarthrosis muir, 1977. In suspension culture, the serum free 1 mm calcium medium also possesses the bene. Cell therapy, such as autologous chondrocyte implantation aci, is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. It is of note that the various studies on the morphology and on the responses of the chondrocyte, and of its cytoskeleton, to mechanical strain have been primarily conducted on chondrocytes isolated from their extra cellular matrix ecmtheir natural habitat, and cultured either in monolayer or in agarose gels. Cells directly isolated from the living tissue are known as primary cells. The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. Human chondrocytes hc culture protocol sigmaaldrich. Studies on various culture systems for chondrocytes and. Applications of chondrocytebased cartilage engineering. Cell culture studies of excess vitamin a inhibits the synthesis of chondroitin sulfate by chondrocytes and causes the inhibition of chondrogenesis in the developing embryo which may result in limb malformations. Human chondrocytes osteoarthritis hc oa are derived from human articular cartilage of donors with oa. Culture and phenotyping of chondrocytes in primary culture. Proliferation medium in threedimensional culture of auricular. Aci is primarily used to treat fullthickness cartilaginous defects of the distal femur by arthroscopically extracting healthy chondrocyte cells from the patients knee, culturing the cells through a process.
Keywords human articular chondrocytes, total knee arthroplasty. Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. The gelatinbased haemostyptic compound spongostan was tested as a threedimensional 3d chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. Primary human chondrocytes and chondrocyte growth media. Human chondrocytes worldwide provider of cell culture. This study is to investigate the efficacy of frs in the prevention of estradiolinduced chondrocyte damage, using a chondrocyte culture system materials and methodsisolation and culture of rabbit chondrocytes inbred new zealand white rabbits of 2 53 0 kg were used to obtain the cartilage for chondrocyte culture. Original article primary culture and adenoviral infection. Isolating articular chondrocytes from cartilage for cell culture articular cartilage is located on bone surfaces within vertebral joints, and ensures a smooth and almostfrictionless surface for the movement of the bones within the joint. Materials for chondrocyte isolation and culture, cartilage explant preparation, and micromanipulation studies were obtained as described previously. Rinse the chondrocyte spheroids with 3% glacial acetic acid solution.
The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. In 3d environment, igf1 induce matrix synthesis in explant culture of cartilage 27,28 and a scaffold free 3d culture of chondrocytes using. Chondrocytes seeded in monolayer may be the least relevant in. Pro teogiycans released into culture medium and pg present in chondrocyte aggregate were measured according to the radioimmunoassay described by gysen et al.
The cell suspension was pipetted and spun, and then resuspended in escsupportive medium. Gag content in the chondrocyte culture in alr was assayed in triplicate, using ml,dimethyl methylene blue solution and aliquots of ltrypsindigest solution. Research article studies on a novel bioreactor design for. Chondrocyte dedifferentiation after extensive in vitro propagation is another drawback cooke et al. Co culture models were established to evaluate the interaction of chondrocytes derived from the surface, middle and deep zones of articular cartilage. Threedimensional, scaffoldfree, autologous chondrocyte. Ultrastructure typical chondrocytes are ovoid cells ranging in maximum diameter from about 10 tm in articular cartilage to about 30,tm in other hyaline cartilages stockwell and meachim, 1973. Pluripotent hesc, are established as feeder free cultures. Pdf gelatinbased haemostyptic spongostan as a possible. Chondrocyte behaviour within different types of collagen gel in vitro. Colorimetric cell viability kit i wst8 colorimetric cell viability and cytotoxicity detection. Different culture methods are being developed to overcome such issues.
Primary culture and phenotyping of murine chondrocytes. The purpose of this study was to compare the chondrogenic redifferentiation potential of chondrocytes, from osteoarthritis oa patients and young adult patients, after monolayer culture. Pdf catalase prevents estradiolinduced chondrocyte. Pipette 15 ml of chondrocyte growth medium to a t75 flask. Culture of chondrocytes in alginate surrounded by fibrin.
Chondrocyte culture and assay liebman 2001 current. At top, the dokinetics of cell free controls are shown for comparison. Here we describe the isolation, shortterm and longterm culture, and. Human chondrocyte cell culture in phase contrast close. During a 48hour culture period in the compressive cell strain system fig. The primary osteoblast chondrocyte co culture model developed in this study is based on the combined culture of a high density chondrocyte micromass and a mono. Culture of these cells in monolayer showed that at early passage these cells expressed chondrocyte. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. Cartilage, chondrocyte, srebp2, col2a1, aggrecan introduction the articular cartilage is composed of chon. Chondrocyte culture in 3d scaffold is based on culturing these cells in an artificial. Take the chondrocyte growth medium from the refrigerator. These methods are capable of limiting the number of cell divisions.
Cellbased therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. The effects of zonal chondrocyte interactions on chondrocyte mineralization were examined over. Full text enhanced chondrocyte culture and growth on. In a control experiment of monolayer cultures, the proliferation or dedifferentiation of bovine chondrocytes was either not or only marginally influenced by the presence of. Thermal containers were used to transport cartilage samples so that the temperature was kept between 48c. Isolation and characterization of human articular chondrocytes from. Culture expansion in lowglucose conditions preserves chondrocyte. Culture of chondrocytes in alginate and collagen carrier gels. Biosafety evaluation of cultureexpanded human chondrocytes with. Bmp4 and fgf2 have been experimentally shown to increase chondrocyte differentiation.
The culture and adenoviral infection of adult mouse knee articular chondrocyte described here was supposed to provide a powerful tool for deeper researches concerning knee articular chondrocytes. Osteoarthritis is an inflammatory disease characterized by increased degradation of cartilage tissue in the joint due to overproduction of enzymes degrading the extracellular matrix. Protein free, defined and animal component free cryopreservation medium. M e t h o d s i n m o l e c u l a r m e d i c i n e cartilage. Cells passages 1 and 4 were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Ijms free fulltext chondrocyte culture parameters for. On the other hand, mesenchymal stem cells msc, as an alternative autologous source, have excellent potential for chondrogenesis. Pdf cartilage tissue culture is a new biotechnological field that has been recently emerged to provide an.
Although the word chondroblast is commonly used to describe an immature chondrocyte, the term is imprecise, since the. Full text the full text of this article is available as a pdf 304k. We offer the means for a deeper understanding of authentic chondrocyte behavior. It is composed solely of chondrocytes, which are specialized cells that produce and maintain the extracellular matrix. Layered chondrocyte sheets were able to maintain the phenotype of cartilage, and could be attached to the sites of cartilage damage, which acted as a barrier to prevent a loss of proteoglycan from. We hypothesize that mrna expression of cytokines and proteases.
Autologous chondrocyte transplantation removal of patient own cells by biopsy. To identify factors that are necessary and sufficient for chondrocyte hypertrophic differentiation and cartilage matrix mineralization, primary chondrocyte culture models have been developed. Chondrogenic differentiation of human bone marrow stem cells in. They produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans. Add to culture flasks or wells at x 10 5 per cm 2. Pdf in serumfree culture thyroid hormones can induce full. Keep the medium to surface area ratio at 1ml per 5 cm 2. Human primary chondrocyte culture system in the human body, articular cartilage covers the bones in the major joints, such as knee and hip joints. Optimization studies on growth of human chondrocytes.
Culture of chondrocytes in alginate surrounded by fibrin gel. Centrifuge the chondrocyte spheroids at 400 x g for 5 minutes. Add 15 mltube of dpbs to wash the chondrocyte spheroids. Chondrocyte aggregate sections were observed with a fluorescent microscope. In the present study, the effects of an integrated threedimensional and dynamic culture on rabbit articular chondrocytes were investigated. Optimem invitrogen, carlsbad, ca or dmemf12 test for optimal transfection efficiency. Human chondrocyte cultures as models of cartilagespecific gene.
Studies on a novel bioreactor design for chondrocyte culture. Based on that, a new act product called spherox formerly called chondro. Studies on a novel bioreactor design for chondrocyte culture harshadpatil, 1 ishansauravchandel, 1 amitk. Chondrocyte culture is an efficient target system for evaluation of the pharmacological and toxicological effects of drugs acting on cartilaginous tissue.
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