Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture

Abstract

Auricular cartilage is an attractive potential source of cells for many tissue engineering applications. However, there are several requirements that have to be fulfilled in order to develop a suitable tissue engineered implant. Animal experiments serve as important tools for validating novel concepts of cartilage regeneration; therefore rabbit auricular chondrocytes were studied. Various parameters including isolation procedures, passage number, rate of proliferation and gene expression profile for major extracellular matrix components were evaluated in order to assess the potential use of elastic chondrocytes for tissue engineering. Chondrocytes were isolated from rabbit ear cartilage and grown in monolayer cultures over four passages. Yields of harvested cells and proliferation were analysed from the digestion step to the fourth passage, and changes in phenotype were monitored. The proliferation capacity of cell cultures decreased during cultivation and was accompanied by enlargement of cells, this phenomenon being especially evident in the third and fourth passages. The expression of cartilage specific genes for collagen type II, aggrecan and cartilage non-specific collagen type I was determined. The mRNA levels for all three genes were obviously lower in the primo culture than immediately after isolation. During subsequent cultivation the expression of collagen type II decreased further, while there were only slight changes in expression of aggrecan and collagen type I. This study provides a valuable basis for testing of different tissue engineering applications in rabbit model, where auricular chondrocytes are considered as cell source.

Stem cell genes are poorly expressed in chondrocytes from microtic cartilage

“…But, as the passage increases, the cells starts to changed and exhibit spindle shape appearance or fibroblastic appearances. This phenomenon and morphological changes has been reported before in many studies [10,18,19]. It was reported that this changes happen because the cell's cytoskeleton structures is remodified in vitro.…”

Catalase Enhances Viability of Human Chondrocytes in Culture by Reducing Reactive Oxygen Species and Counteracting Tumor Necrosis Factor-α-Induced Apoptosis

“…Accumulated evidence demonstrated that passaged chondrocytes change their metabolic behavior, especially altering their gene expression profiles and dedifferentiating to fibroblastic status of decreased collagen type II and aggrecan, and increased collagen type I [48,49]. Since activities of cells in culture are largely under the influence of media and complemented bioactive agents, the differentiation phenotype of in vitro expanded chondrocytes can be achieved experimentally [34,50].…”

Effects of bromelain on cellular characteristics and expression of selected genes in canine in vitro chondrocyte culture

“…The characteristics of the primary chondrocyte in this study were most similar to those used by Yang et al (2000) and Frohlich et al (2007). The process of isolation from cartilage tissue is an important step, because at this point it must be ensured that there are sufficient viable cells for further cultivation and analysis.…”

“…The patients had suffered a car accident or disease at the time of limb amputation (for example, osteosarcoma). To isolate chondrocytes by enzymatic treatment according to the method of Frohlich et al (2007), cartilage was cut into pieces (approximately 1 mm 3 ) and digested with 10% collagenase type II (Sigma, USA) at 37 °C for 21 h. After that, cartilage pieces were washed in PBS (phosphate buffer saline) and incubated in a culture medium containing DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 50 µg/ml gentamicin. Samples were cultured at 5% CO 2 and 37 °C.…”