Three-dimensional expansion: in suspension culture of SD rat's osteoblasts in a rotating wall vessel bioreactor.

Objective: To study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB).

Methods: The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation.

Results: The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones.

Conclusions: The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D) interactions among cells and is suitable for osteopath expansion in vitro.