Multiplication of human Chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity

To treat a cartilage defect with tissue-engineering techniques, multiplication of donor cells is essential. However, during this multiplication in monolayer expansion culture chondrocytes will lose their phenotype and produce matrix of inferior quality (dedifferentiation). Dedifferentiation occurs more extensively with low seeding densities and passaging. To obtain cartilage of good quality it is important that the multiplicated cells regain their cartilaginous phenotype (redifferentiation capacity). A "gold standard" for the multiplication of chondrocytes in monolayer, with respect to seeding density and passaging, is lacking. In numerous available studies, various cell densities have been used, making comparison of the results of these studies difficult. Therefore, we performed a comparative study to gain insight concerning the effect of seeding density and passaging on the capacity of cells to redifferentiate. From the resulting data we deduced the seeding density in monolayer culture for which cell expansion is both sufficient and fast, while the cells retain a capacity to redifferentiate. As a guideline we calculated that, at minimum, 20-fold multiplication is needed to fill an average cartilage defect of 4 cm(2) with the amount of donor chondrocytes we obtained. For this study we used isolated ear chondrocytes from five children. Four different seeding densities in monolayer culture were used, ranging from 3500 to 30,000 cells/cm(2). The cells were cultured for four passages. The capacity of the expanded chondrocytes to redifferentiate (redifferentiation capacity) was studied after an additional 3-week culture in alginate beads and was assessed by glycosaminoglycan production and immunohistochemical stainings for collagen type I, collagen type II, elastin, and a fibroblast marker (11-fibrau). In general, we found that both passaging and decreasing seeding density yielded an increase in expanded chondrocytes, but at the same time decreased the dedifferentiation capacity. In further analyzing our data according to the proposed guidelines we found that with lower seeding densities sufficient multiplication (20 times) was reached in less time and with less passaging than at higher seeding densities. Importantly, the redifferentiation capacity of these chondrocytes was preserved. It was equal to or even surpassed that of chondrocytes multiplied 20 times at higher seeding densities, which required more time and more passages in monolayer culture. Thus, for cartilage tissue-engineering purposes we propose that expansion culture with low seeding densities is preferable.