Isolation, Purification, Generation, and Culture of Osteocytes.

Osteocytes reside within the bone matrix and produce both paracrine and endocrine factors that influence the skeleton and other tissues. Despite their abundance and physiological importance, osteocytes have been challenging to study in vitro because they are difficult to extract and purify and do not retain their phenotype in standard culture conditions. New techniques for this purpose are emerging. This chapter will describe two methods and adaptations we use to study osteocytes: (1) isolating and purifying primary osteocytes from murine bone, with and without hematopoietic lineage depletion and (2) differentiating cultured osteoblasts or osteoblast-lineage cell lines (including cell lines termed "osteocytic") until they reach a stage of osteocytic gene expression. We will also discuss the limitations of these methods and possible directions for future improvements.