Utility Of The System

Halki Diabetes Remedy

New Diabetes Cure

Get Instant Access

The clinical application of autologous chondrocyte implantation to the treatment of articular cartilage defects has stimulated the development of highly reproducible, standardized methods for human articular chondrocyte isolation and culture. The autologous nature of the chondrocyte implantation procedure requires that the cell culture medium must promote rapid proliferation and maintenance of re-differentiation potential of adult human articular chondrocytes in patients of varied health and age.

The articular chondrocyte culture system developed by Brittberg et al. (1994) used serum derived from each individual patient as a culture supplement. The inconsistent maintenance of articular chondrocyte redifferentiation potential, measured by suspension culture analysis, in these matched donor "lots" of AHS is the likely result of varying levels of growth and differentiation factors present in different lots of serum [12, 28]. This variability was reduced when the same human articular chondrocyte strains were cultured in a single lot of FBS (Figure 2).

Figure 5. Phase contrast photomicrographs of normal human articular chondrocytes cultured 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).

Figure 5. Phase contrast photomicrographs of normal human articular chondrocytes cultured 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).

Although serum has been widely used for mammalian cell culture, there are several potential problems associated with its use: 1) serum contains many unidentified or non-quantified components and therefore is not "defined"; 2) the composition of serum varies from lot to lot, making standardization difficult for experimentation or other uses of cell culture; 3) because many of these components affect cell attachment, proliferation, and differentiation, controlling these parameters, or studying the specific requirements of cells with respect to these parameters, is precluded by the use of serum; 4) some components of serum are inhibitory to the proliferation of specific cell types, and to some degree may counteract its proliferative effect resulting in sub-optimal growth; and 5) serum may contain adventitious agents that could affect the outcome of experiments or provide a potential health hazard if the cultured cells are intended for implantation in humans [12].

The potential problems associated with culturing chondrocytes in serum-containing medium can be addressed by using serum that is extensively screened for safety and efficacy, as is currently done, or using a defined serum-free medium. The development of defined medium must be tailored to the cell type of interest since requirements for specific nutrients, growth and attachment factors, hormones and other components vary from one cell type to another.

Chondrocytes produce and secrete factors that promote their own attachment and proliferation [24]. Examples include bFGF [14], insulin-like growth factors [13], transforming growth factor-P [26], vitronectin, and possibly some unidentified factors that promote their attachment and proliferation. Maximal cell proliferation and maintenance of redifferentiation capacity were achieved when serum-free, chemically defined medium (DRF) was supplemented with IGF-I, bFGF, hydrocortisone, and fibronectin (Figures 4 and 5).

Previous attempts to culture articular chondrocytes in defined medium have been partially successful [1, 17]. However, exposure to undefined bovine serum was necessary in these systems for 24-48 hours for chondrocyte attachment. While the addition of purified human fibronectin to the defined medium facilitated chondrocyte attachment, the use of undefined serum was slightly superior. Finally, there was no difference in the ability of human articular chondrocytes expanded in defined medium to re-differentiate, as compared to the same human articular chondrocyte strains expanded in FBS-containing medium (Table 1).

Was this article helpful?

0 0
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...

Get My Free Ebook


Post a comment