Unbiased transcriptomic analysis of chondrocyte differentiation in a high-density cell culture model

ABSTRACTIn the developing vertebrate skeleton, cartilage is an important precursor to the formation of bones. Cartilage is produced by chondrocytes, which derive from embryonic mesoderm and undergo a stereotypical program of differentiation and maturation. Here we modeled this process in vitro, using primary fetal mouse rib chondrocytes in a high-density cell culture model of cartilage differentiation, and performed genome-wide gene expression profiling over the course of culture. The overarching goal of this study was to characterize the molecular pathways involved in cartilage differentiation and maturation. Our results also enable a comprehensive appraisal of distinctions between common in vitro models for cartilage differentiation, and of differences in their molecular resemblance to cartilage formation in vivo.

Expression of recombinant herpes simplex virus type 2 glycoprotein D by high-density cell culture of Spodoptera frugiperda

Herpes simplex virus type 2 (HSV-2) is the major cause of genital herpes in humans. The glycoprotein D of HSV-2 (gD2) is a promising subunit vaccine candidate for the treatment of genital herpes. The aim of the present study was to express a biologically active recombinant gD2 in eukaryotic baculovirus system in quantities sufficient for further studies. Human cDNA encoding a gD2 protein with 393 amino acids was subcloned into the pFastBac HTb vector and the recombinant protein was expressed in Spodoptera frugiperda (Sf9) cells by high-density cell culture. In a stirred bioreactor, the key limiting factors including glucose concentration, glutamine concentration and dissolved oxygen (DO) were optimized for high-density cell growth. The Sf9 cell density could reach 9.6×106 cells/mL and the yield of recombinant gD2 protein was up to 192 mg/L in cell culture under the optimal conditions of 15 mM glucose, 0.4 g/L glutamine and 40% DO. Production of significant amounts of pure, full-length gD2 opened up the possibility to investigate novel functions of gD2. Moreover, the purified recombinant gD2 protein revealed a partial prophylactic immune function in genital herpes of guinea pigs infected with HSV-2.