Long-term cultivation of human osteoblasts.

Primary cultures of osteoblastic cells were obtained from human bone tissue after corrective surgery. Osteoblasts were isolated with a combined enzymatic and cell migration method and characterized by a high expression of alkaline phosphatase (AP), the marker enzyme for osteoblasts. For quantification of AP a new sensitive method has been developed. Also the secretion of osteocalcin measured by radioimmunoassay (RIA) which was stimulated by calcitriol, characterized isolated cells as osteoblasts. Osteoblasts isolated by the enzymatic and migration method formed 3-dimensional structures during cultivation in a mixed basal medium containing serum substitute, vitamin C and glycerophosphate. They maintained their specific cell characteristics over a period of 50 generations and did not dedifferentiate in contrast to their cultivation in medium containing fetal calf serum. Therefore a great amount of differentiated human osteoblasts could be obtained after cultivation of the primary cultures from different donors and this allows the examination of the influence of age and sex on cellular activity.