Influence of cell isolation, cell culture density, and cell nutrition on differentiation of rat calvarial osteoblast-like cells in vitro.

The effects of various cell isolation procedures, growth media and the cell culture density on the in vitro differentiation of neonatal rat calvarial osteoblast-like cells were investigated. Cells were isolated by enzymatic treatment, or after explant culture and inoculated as a monolayer or micromass in serum containing BGJb, or Dulbecco's Modified Eagle Medium (DMEM). The cells were kept for up to 3 weeks in culture and were then characterized, both morphologically and biochemically. The isolation technique appeared to have no effect on the differentiation process. The calvaria could be used several times as explant cultures for a reliable source of differentiating osteoblast-like cells. The cultures kept in DMEM had a significantly higher DNA content, but significantly less alkaline phosphatase activity (ALP) per DNA and protein per DNA content than the BGJb cultures. Monolayer cultures had a significantly higher DNA content than the micromass cultures, in both growth media. Furthermore, the micromass culture had a significantly higher ALP per DNA than monolayer cultures at 1 week. The morphology of all cell cultures at 3 weeks reflected the biochemical results. Only the cells grown in BGJb formed abundant ALP positive and mineralized nodules in monolayer cultures. In contrast, cells grown as micromasses formed a dense calcified area, independently of the growth medium used. DMEM promoted the proliferation, whereas BGJb stimulated the differentiation of osteoblast-like cells in monolayer cultures. Micromass cultures were less sensitive to nutritional conditions than monolayer cultures and promoted the differentiation of osteoblast-like cells.