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.
Song
Ke-Dong
KD
Stem Cell and Tissue Engineering Laboratory, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, Liaoning, China.
Liu
Tian-Qing
TQ
Li
Xiang-Qin
XQ
Cui
Zhan-Feng
ZF
Sun
Xiang-Yu
XY
Ma
Xue-Hu
XH
D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation; D049108:Cell Enlargement; D003470:Culture Media; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000378:metabolism; D009994:Osmolar Concentration; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley
D003470:Culture Media; D019344:Lactic Acid; D005947:Glucose