Articular chondrocytes were isolated according to a published procedure
[36 (link)]. Briefly, untreated C57BL/6J
mice were sacrificed and articular cartilages from both knee joints were
collected, rinsed in PBS, and incubated with 2.5% trypsin (Atlanta Biologicals,
Flowery Branch, GA) in 3 mL of PBS at 37°C for an hour. The trypsin was
removed and the tissue was digested with Pronase (2 mg/mL) in Minimal Essential
Medium α Medium (α-MEM) containing penicillin and streptomycin at
37 °C for 1 hr. The Pronase was removed and the exposed articular
cartilage layer was then digested overnight with 0.25 mg/mL of collagenase IV in
α-MEM containing 5% FBS and antibiotics. The digestion was terminated by
the addition of α-MEM containing 10% FBS. The released articular
chondrocytes were filtered through a 70-μ sterile filter, collected by
centrifugation, and expanded in α-MEM supplemented with 10% FBS. The
isolated cells expressed predominantly type II collagen (a marker for
proliferative chondrocytes) with 400- to 500-fold lower levels of type X
collagen (a marker for hypertrophic chondrocytes) or type I collagen (a marker
for fibroblasts and osteoblasts) (Fig. 1C ),
confirming that these are bona fide chondrocytes. These cells also expressed
substantial amounts of Sox9 and Rho-associated protein kinase (ROCK), two of the
genes that are essential for cartilage matrix production [37 (link)].
[36 (link)]. Briefly, untreated C57BL/6J
mice were sacrificed and articular cartilages from both knee joints were
collected, rinsed in PBS, and incubated with 2.5% trypsin (Atlanta Biologicals,
Flowery Branch, GA) in 3 mL of PBS at 37°C for an hour. The trypsin was
removed and the tissue was digested with Pronase (2 mg/mL) in Minimal Essential
Medium α Medium (α-MEM) containing penicillin and streptomycin at
37 °C for 1 hr. The Pronase was removed and the exposed articular
cartilage layer was then digested overnight with 0.25 mg/mL of collagenase IV in
α-MEM containing 5% FBS and antibiotics. The digestion was terminated by
the addition of α-MEM containing 10% FBS. The released articular
chondrocytes were filtered through a 70-μ sterile filter, collected by
centrifugation, and expanded in α-MEM supplemented with 10% FBS. The
isolated cells expressed predominantly type II collagen (a marker for
proliferative chondrocytes) with 400- to 500-fold lower levels of type X
collagen (a marker for hypertrophic chondrocytes) or type I collagen (a marker
for fibroblasts and osteoblasts) (
confirming that these are bona fide chondrocytes. These cells also expressed
substantial amounts of Sox9 and Rho-associated protein kinase (ROCK), two of the
genes that are essential for cartilage matrix production [37 (link)].
