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Development of a Novel Three-Dimensional, Automatable and Integrated Bioprocess for the Differentiation of Embryonic Stem Cells into Pulmonary Alveolar Cells in a Rotating Vessel Bioreactor System

Siti-Ismail, N., et al. (2012). "Development of a Novel Three-Dimensional, Automatable and Integrated Bioprocess for the Differentiation of Embryonic Stem Cells into Pulmonary Alveolar Cells in a Rotating Vessel Bioreactor System." Tissue Engineering Part C-Methods 18 4: 263-272

Application of stem cells for cell therapy of respiratory diseases is a developing field. We have previously established several protocols for the differentiation of embryonic stem cells (ESC) into alveolar epithelial cells, which require a high degree of operator interference and result in a low yield of target cells. Herein, we have shown that, by provision of a medium conditioned using A549 cells and by integration of classic steps of ESC differentiation into a single step through encapsulation in hydrogels (three-dimensional) and culture in a rotary bioreactor, murine ESC (mESC) could be directed to differentiate into distal respiratory epithelial cells. Type I and II pneumocytes (with a yield of 50% for type II) and Clara cells were demonstrated by the expression of aquaporin 5, surfactant protein C, and Clara cell secretory protein, respectively. We identified target cells as early as day 5 of culture and stably maintained our differentiated cells in vitro for 100 days. Electron microscopy demonstrated microvilli and intracellular lamellar bodies (LB), and fluorescent staining confirmed the active process of exocytosis of these LB in differentiated type II cells. When these cells were decapsulated and cultured in static conditions in flask cultures (two-dimensional), they retained their characteristic type II phenotype and morphology. In conclusion, our protocol offers integrated bioprocessing, shorter time of differentiation, lower cost, no use of growth factors, high reproducibility, and high phenotypic and functional stability, as well as being amenable to automation and being scalable, which would move this field closer to future clinical applications.

Related URLs:
<Go to ISI>://WOS:000302136300003

ISSN: 1937-3384

DOI: 10.1089/ten.tec.2011.0299

Accession Number: WOS:000302136300003

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Tags: Animals, Antibodies/immunology, Automation, Bioreactors, Cell Aggregation/drug effects, Cell Culture Techniques/instrumentation/*methods, Cell Differentiation/drug effects, Cell Line, Cells, Conditioned/pharmacology, Culture Media, Embryonic Stem Cells/*cytology/drug effects/metabolism/ultrastructure, Humans, Immobilized/cytology/drug effects/metabolism, Mice, Pulmonary Alveoli/*cytology/drug effects/metabolism/ultrastructure, Reverse Transcriptase Polymerase Chain Reaction, Rotation