A Unique Triculture Model to Study Osteoblasts, Osteoclasts, and Endothelial Cells

Many studies have shown that bone is a dynamic organ, in which cell communication plays a central role. A production of relevant in vitro models appears essential to better understand the cellular interactions. The conventional in vitro monocultures are hindered by oversimplification and are unable to reproduce the complex features of the processes investigated. Here, our aim was to establish an original triculture model allowing the retention of the phenotype of bone formation, bone resorbing, and vasculature forming cells. Human bone marrow stromal cells (HBMSCs) were used to obtain osteoblasts (OBs). Mononuclear cells (MNCs) were isolated from human umbilical cord blood and were used to obtain both osteoclast (OC)-derived MNCs and endothelial progenitor cell (EPC)-derived MNCs. First, results led to the establishment of a new cell culture medium: EGM2*/alpha MEM (alpha minimum essential medium). In separated monocultures, the tested medium did not affect the retention of EPC, OB, and OC phenotypes nor the OC resorptive activity. Similarly, both HBMSCs and EPC-derived MNC phenotypes were preserved in the coculture. Then, we demonstrated that our medium, made of EGM2*/alpha MEM, was compatible with the retention of OB, OC, and EPC phenotypes, as well as OC resorptive activity in an original and unique direct triculture model. Finally, we investigated the homeostasis of our system through the stimulation of osteoclastogenesis. Thus, this study was a mandatory first step to establish an in vitro microenvironment suitable to study bone cell communication. These promising results will permit further investigations to study cell-to-cell cross talk, which will be of relevance to the process of bone regeneration and bone diseases. Impact Statement In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.