Neuroendocrine tissue engineering in rotating wall vessel bioreactors under simulated microgravity conditions

The low-shear, microgravity-simulating cell culture environment in rotating wall vessel (RWV) bioreactors is well suited for generating differentiated 3-D tissue constructs (organoids). Biochemical, immunological and molecular biological techniques, including cDNA arrays, were used to analyze signal transduction, catecholamine contents, and gene expression in 3-D tissue-like constructs of PC12 pheochromocytoma cells grown for <30 d in RWVs. Vascularization of the organoids implanted s.c. into Matrigel plugs in B56 mice was evaluated by fluorescence microscopy. The unique culture environment of RWV bioreactors facilitates the generation of macroscopic, functional neuroendocrine tissue-like assemblies, as assessed by the enhanced production of catecholamines. Organoid formation is accompanied by prolonged activation of specific signaling pathways (erk, p38, jnk) and transcription factors, leading to a unique pattern of gene expression characteristic for the neuroendocrine phenotype. Thus in RWV, "neuronal" genes (e.g. GAP 43) were downregulated while "neuroendocrine" genes (e.g. chromogranin A) were upregulated. When implanted into Matrigel plugs in vivo, PC12 organoids generated in RWV bioreactors, but not 3-D aggregates formed under static conditions, became highly vascularized. This latter finding is in line with the enhanced expression of angiogenic growth factors, such as VEGF in RWVs but not in the controls. The unique cell culture environment in RWV bioreactors activates differentiative signaling pathways and hence facilitates engineering of functional neuroendocrine tissue constructs, which might be clinically useful as implants in the treatment of debilitating neurodegenerative diseases, such as Parkinson's disease.