Generation of rodent and human osteoblasts

Developers

Sarah E. B. Taylor, Mittal Shah and Isabel R. Orriss

Description of the technology

This technology provides the isolation, culture and staining of primary osteoblasts from samples collected from organism of neonatal rodents and human. The calvaria and long-bone assays allow direct measurement of bone matrix deposition and mineralisation, as well as producing osteoblasts at defined stages of differentiation for molecular and histological analysis. Culture of human osteoblasts enables cell function to be investigated in targeted patient groups. The described methods provide a step-by-step guide of what to expect at each stage of the culture and highlight the varied tissue culture conditions required to successfully grow osteoblasts from different sources. A special focus of this paper is the methods used for analysis of bone mineralisation and how to ensure that nonspecific mineral deposition or staining is not quantified.

Practical application

Human osteoblasts, taken from several anatomical sources according to this technology, can be used to study osteoblast physiology. Human cells obtained from explant bone tissues consisting of a heterogeneous cell population that predominantly includes early osteoblasts, can be differentiated in vitro into mature, bone-forming osteoblasts by the addition of glucocorticoids, vitamin D and bone morphogenetic proteins.

The use of human osteoblasts will allow cell function to be investigated in patients suffering from age-related bone diseases.

The technology proposes methods for the isolation of rat calvarial and long-bone osteoblasts from the same animal.

These methods could be also adapted to successfully culture mouse osteoblasts, which require more nutritional support in vitro.

Finally, the technology gives a protocol, describing how to culture primary human osteoblasts from bone samples.

Laboratories

• Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford (USA)
• Department of Surgery and Cancer, Imperial College London, London (UK)
• Department of Comparative Biomedical Sciences, Royal Veterinary College, London (UK)

Links

Publications

• Taylor, S.E.B. et al. «Generation of rodent and human osteoblasts." 3 BoneKEy Reports (2014): 585.
• Orriss, I.R., Taylor, S.E., Arnett, T.R. «Rat osteoblast cultures." 816 Methods Mol Biol (2012): 31–41.
• Orriss, I.R. et al. «Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats." 34 Int J Mol Med (2014): 1201–1208.