NuSerum, a synthetic serum replacement, supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Abstract

In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23-24 chick embryos and plated at various densities (1, 5, 10, or 20 x 10(6) cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [3H]leucine and [3H]thymidine. Chondrogenesis was determined by the incorporation of [35S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 x 10(6) cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 x 10(6) cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [35S]sulfate (2.6-fold), [3H]leucine (1.4-fold), and [3H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 x 10(6) cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV.(ABSTRACT TRUNCATED AT 250 WORDS)