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Isolating articular chondrocytes from cartilage for cell culture

Articular cartilage is located on bone surfaces within vertebral joints, and ensures a smooth and almost-frictionless surface for the movement of the bones within the joint. In osteoarthritis, the destruction of cartilage is the primary cause of pain and discomfort. For these reasons, understanding the molecular mechanisms of chondrocytes, the only cell type within cartilage, and proteins of the surrounding extracellular matrix (ECM) are key areas of research. Studies using isolated chondrocytes in vitro are helping to advance the understanding of this important tissue. This article is an introduction to the process of isolating chondrocytes from articular cartilage.

 

Cartilage Dissection:
You can obtain articular chondrocytes from animal or human sources. This must be done in accordance with institutional and regulatory guidelines. Chondrocytes are typically isolated from bovine metacarpophalangeal joints or human knee joints. Samples should be fresh because frozen cartilage does not yield viable cells. Cartilage from bovine sources that are 2 to 6 months of age will yield more chondrocytes than other animal sources such as mouse, rat, rabbit and porcine. The sample to be dissected should be washed thoroughly before dissection and extracted cartilage specimens should be placed into a tube containing Dulbecco’s modified Eagle medium (DMEM)/antibiotic-antifungal mixture. All work should be carried out in a sterile flow hood.

 

Enzymatic Digestion to Isolate Chondrocytes:
Chondrocytes in the cartilage sample are bound within a tight extracellular matrix (ECM) composed of collagen, aggrecan, and other proteins. The most well known method to isolate the chondrocytes from this matrix is to incubate your sample with enzymes that can hydrolyze the proteins, polysaccharides and lipids of the ECM. There are also multiple enzymatic digestions described in the literature, but using a combination of pronase and collagenase in DMEM is the most popular. Crude collagenase prepared from extracellular Clostridium Histolyticum culture filtrates is a common source of collagenase because it contains the proteases, polysacharidases and lipases needed to break down the components of the ECM. Pronase is used as a non-specific protease to aid in the digestion process. The digestion procedure is usually carried out in a 37°C, 5% CO2 humidified cell culture incubator and all reagents should be sterile filtered before use. An important point to consider, often optimized by each individual lab, is the digestion time and concentration of collagenase and pronase. These will vary depending on the animal and tissue source, but the below references will provide you with an idea for each of these parameters. Once the digestion process is complete, you can use a nylon filter to separate the chondrocytes from undigested ECM. The isolated chondrocytes can now be used for culturing.

ELISA Kits for use with chondrocyte cultures:

References:
Primary culture and phenotyping of murine chondrocytes.
Gosset M, et al. Nat Protoc. 2008; 3(8):1253-60.
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Chondrocyte culture & assay.
Liebman J, Goldberg RL. Current Protocols in Pharmacology. 2001: 12.2.1 - 12.2.18.
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