A new bone banking technique to maintain osteoblast viability in frozen human iliac cancellous bone

• Published in: Cryobiology 2002-06, Vol.44 (3), p.279-287
• Main Authors: Oh, Jung-Hwan, Zöller, Joachim E, Kübler, Alexander
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - metabolism; Biological and medical sciences; Bone Matrix - cytology; Bone Matrix - metabolism; Cell Division; Cell Survival; Cells, Cultured; Collagen Type I - metabolism; Cryopreservation; Cryopreservation - methods; Cryoprotective Agents; Dimethyl Sulfoxide; Fundamental and applied biological sciences. Psychology; Humans; Iliac cancellous bone; Ilium - cytology; Ilium - metabolism; In Vitro Techniques; Osteoblast; Osteoblasts - cytology; Osteoblasts - metabolism; Osteocalcin - metabolism; Skeleton and joints; Tissue Banks; Tissue Preservation - methods; Vertebrates: osteoarticular system, musculoskeletal system
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/S0011-2240(02)00034-2

The aim of this study was to develop a new cryopreservation technique to maintain the osteoblast viability in frozen iliac bone and to prove cell viability using cell culture techniques. Human iliac cancellous bones were frozen with and without 10% Me 2SO at −80 °C. The tubes were kept in a −80 °C freezer for at least 2 days. After the storage period, the frozen bone was thawed by placing the tube in a 37 °C water bath. A serial enzymatic digestion technique using 0.2% collagenase was employed to isolate osteoblast-like cells from the bone. The cells that were released were inoculated into tissue culture flasks containing DMEM supplemented with 10% FCS. They were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO 2. Cells of the second passage were plated at a density of 5×10 3 cells/cm 2 in a 24-well plate and used for characterization. For characterization, WST-1 assay, determination of alkaline phosphatase, Type I collagen assay, osteocalcin assay, and von Kossa staining were used. The assays were performed at 3, 6, 9, and 12 days after plating the cells. Based on the results of this study, we conclude that the osteoblast-like cells in the frozen bone can survive, only when the bone is frozen with cryoprotectants to prevent injury during freezing and thawing.