Development of a defined medium for articular chondrocyte culture

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Human articular chondrocytes were initially seeded at 3-4000 cells per cm2 and allowed to attach to tissue culture plastic for one day in DMEM supplemented with 10% FBS. Following overnight incubation, culture medium was removed and replaced with DMEM/FBS as control, or the defined medium, as described below.

Chondrocyte Culture

Figure 2. Comparison of chondrocyte culture in autologous human serum versus fetal bovine serum. Human articular chondrocytes were cultured in medium supplemented with fetal bovine serum or donor-matched autologous human serum. Re-differentiation of chondrocytes was assessed by counting colonies in agarose suspension culture as described in Section 3.

Figure 2. Comparison of chondrocyte culture in autologous human serum versus fetal bovine serum. Human articular chondrocytes were cultured in medium supplemented with fetal bovine serum or donor-matched autologous human serum. Re-differentiation of chondrocytes was assessed by counting colonies in agarose suspension culture as described in Section 3.

Figure 3. Effect of patient age on the ability of chondrocytes to form colonies in suspension culture following proliferative expansion in monolayer. The colony-forming ability of human articular chondrocytes derived from the articular cartilage of 22 donors ranging in age from 5 to 65 years was assessed following monolayer culture into the third passage (approximately 7-10 population doublings).

Figure 3. Effect of patient age on the ability of chondrocytes to form colonies in suspension culture following proliferative expansion in monolayer. The colony-forming ability of human articular chondrocytes derived from the articular cartilage of 22 donors ranging in age from 5 to 65 years was assessed following monolayer culture into the third passage (approximately 7-10 population doublings).

Figure 4. Figure 4: Cell yield following culture of human articular chondrocytes in different formulations of defined medium. Cell yields were compared following culture of normal human articular chondrocytes in (A) serum containing DMEM/FBS; or serum-free DRF medium supplemented with (B) insulin, transferrin, and selenium (ITS+); (C) ITS+ and insulin-like growth factor-1 (IGF-1); (D) ITS+, IGF-1, and basic fibroblast growth factor (bFGF); (E); ITS+, IGF, bFGF, and fibronectin (FN); or (F) ITS+, IGF, bFGF, FN, and hydrocortisone (HC).

Culture Condition

Figure 4. Figure 4: Cell yield following culture of human articular chondrocytes in different formulations of defined medium. Cell yields were compared following culture of normal human articular chondrocytes in (A) serum containing DMEM/FBS; or serum-free DRF medium supplemented with (B) insulin, transferrin, and selenium (ITS+); (C) ITS+ and insulin-like growth factor-1 (IGF-1); (D) ITS+, IGF-1, and basic fibroblast growth factor (bFGF); (E); ITS+, IGF, bFGF, and fibronectin (FN); or (F) ITS+, IGF, bFGF, FN, and hydrocortisone (HC).

Defined medium was composed of a 1:1:1 mixture of three basal media: DMEM, RPMI-1640, and Ham's F12 + HEPES (Gibco), referred to hereinafter as DRF. DRF was then supplemented with (a) ITS+ (insulin at 10 |g/ml, transferrin at 5.5|g/ml, and selenium at 7ng/ml), linoleic acid (5|g/ml), ethanolamine (2|g/ml), and bovine serum albumin (1mg/ml); (b) ITS+, with and without insulin-like growth factor-I (IGF-I); (c) ITS+, IGF-I, with and without basic fibroblast growth factor (bFGF); (d) ITS+, IGF-I, bFGF, with and without fibronectin; or (e) ITS+, IGF-I, bFGF, fibronectin, with and without hydrocortisone. The culture medium in serum-containing cultures was completely replenished every 2-3 days, whereas cultures containing defined medium were not replenished. The cell yields obtained using complete defined medium were equivalent to those obtained in control cultures (Figure 4).

Articular chondrocytes cultured in DMEM supplemented with 10% FBS grew to confluence within 7-9 days (Figure 5A). The morphology of cells cultured in DRF with ITS+, and with ITS+ supplemented with IGF-I, was characterized by large cells having thin processes (Figures 5B and 5C). The addition of bFGF and fibronectin enhanced chondrocyte morphology, with smaller, more numerous, refractile cells (Figures 5D and 5E). Finally, the addition of hydrocortisone to DRF supplemented with ITS+, IGF-I, bFGF, and fibronectin resulted in the achievement of culture confluence and a morphology which was indistinguishable from control, DMEM with 10% FBS (Figure 5F).

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Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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