Osteoblastlike cells in a serum-free methylcellulose medium form colonies: Effects of insulin and insulinlike growth factor I

Osteoblastlike (OB) cells obtained from a heterogeneous primary cell population by enzymatic cell digestion of calvaria of newborn rats are grown in a serum-free viscous α-MEM/F-12 medium containing 0.8% methylcellulose. In contrast to cell monolayers in conventional tissue cultures OB cells proliferate into colonies of rounded-up cells to form morulalike spherical cell clusters containing up to 100 cells. These colonies, with different cell numbers, are clearly not fibroblastlike since fibroblasts from the same rats always grow as a cell monolayer. Alkaline phosphatase activity and cAMP responsivness to PTH are expressed more markedly (70% and 250% respectively) by OB cells in the described culture system than in conventional tissue cultures. Rounded-up OB cells sediment and colonies stick to the dish; proliferation of OB cells is favored and starts 3–4 days after inoculation. Increasing concentrations of insulinlike growth factor (IGF) I (0.4–35 nM) and insulin (20–660 nM), as well as increasing initial cell density, enhances mitogenic activity of these cells in a dose-dependent way. On a molar ratio IGF I (physiological concentrations) is 10 times as potent as insulin (pharmacological concentrations) with respect to proliferation. If less than 105 cells/ml are inoculated, there exists an apparent relationship between initial cell density and major onset of replication, indicating the presence and accumulation of local growth factors. For OB cells, the described culture system (1) comes closer to thein vivo situation (2) leads to a clear morphological difference between OB cells and fibroblasts (3) provides an excellent system to study hormone actions on OB cell proliferation, and (4) allows inoculation at a wide variety of initial cell densities.