Characterization of Human Osteoblastic Cells: Influence of the Culture Conditions

• Published in: In vitro cellular & developmental biology. Animal 1997-11, Vol.33 (10), p.757-762
• Main Authors: A. Rattner, O. Sabido, C. Massoubre, F. Rascle, Frey, J.
• Format: Article
• Language: English
• Subjects: Adult; Alkaline phosphatase; Alkaline Phosphatase - analysis; Alkaline Phosphatase - metabolism; Animal cells; Biochemical markers; Biomedical materials; Bones; Calcification, Physiologic - drug effects; Calcitriol - pharmacology; Cancellous bone; Cell culture; Cell culture techniques; Cell growth; Cell lines; Cells, Cultured; Cellular Models; Collagen; Collagen (type I); Collagen - biosynthesis; Collagens; Crystals; Culture; Culture Media; Cultured cells; Cytology; Fibroblasts; Flow Cytometry; Fluorescein; Glycerophosphates - pharmacology; Humans; Hyaluronan Receptors - analysis; Hydroxyapatite; Microscopy, Electron, Scanning; Mineralization; Morphology; Multiplication; Osteoblasts; Osteoblasts - chemistry; Osteoblasts - cytology; Osteoblasts - metabolism; Osteocalcin; Osteocalcin - biosynthesis; Osteocytes; Phosphatases; Physical characteristics; Vitamin D3
• ISSN: 1071-2690; 1543-706X
• DOI: 10.1007/s11626-997-0154-7

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)2vitamin D3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D3. 10 mM$\beta GP$induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.