Autologous human articular chondrocytes for clinical use were first propagated in culture medium composed of Ham's F-12 medium supplemented with 15% (v/v) autologous serum . Cells from each individual patient were cultured in medium supplemented with their own serum. The use of multiple lots of individual patient serum for culture of articular chondrocytes from each patient creates a potential for variability in the production of high quality cells, given the variable levels of growth and differentiation observed for cells in different lots of serum .
The colony-forming efficiency of ten different strains of human articular chondrocytes cultured in a single lot of FBS was compared to the colony-forming efficiency of the same strains cultured in donor-matched autologous human serum (AHS). Human articular chondrocytes were isolated and cultured by two distinct methods [5, 7]. Biopsies of human articular cartilage were harvested from a minor load-bearing surface of the articular cartilage of the affected knee, and then trimmed, bisected, and transported to the laboratory for cell isolation. Isolated chondrocytes were propagated in monolayer tissue culture in vessels containing either: Ham s F-12 (F-12; Gibco, Grand Island, NY) supplemented with 15% AHS (Method 1; Brittberg et al., 1994); or Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10% FBS (Hyclone, Logan, UT) (Method 2; Binette et al., 1998). All cultures were incubated at 37°C in a humidified 5% CO2 environment, with medium changes every 2-3 days. Cells were passaged at 80-90% confluence using 0.05% trypsin-EDTA and were diluted ten-fold for subculture. Trypsin was neutralized by addition of the appropriate serum (10% v/v).
Following approximately three weeks of monolayer culture, chondrocytes cultured by both methods were tested for their ability to re-differentiate using the agarose suspension culture assay, as described previously . The colony-forming efficiencies of chondrocytes cultured in medium supplemented with the single lot of FBS were at least as good as, if not better than the colony-forming efficiencies observed for the same patient strains grown in their own AHS (Figure 2).
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