| Summary: | 碩士 === 臺灣大學 === 醫學工程學研究所 === 98 === In our research, we demonstrated that the pulmonary stem/progenitor cells within monodisperse gelatin-microbubble scaffold could create alveoli-like construct in vitro and in vivo. In previous study, we have reported a serum-free primary culture system to generate pulmonary stem/progenitor cells from neonatal mouse. The cells expressed stem cell markers, such as Oct-4, Nanog, Sox-2 and CC10, and also Clara cell secretion protein indicating they represented a sub-population of Clara cells which have long been implicated as pulmonary stem/progenitor cells in lung injury models. These stem cells could be kept for weeks in the primary cultures and underwent terminal differentiation to alveolar type II (AT2) and type I (AT1) like cells sequentially in in vitro differentiation. The results displayed that the stem/progenitor cells hold the potential promise to grow pulmonary tissue in vitro. In the study, the microfluidic method was applied to production of monodisperse gelatin-microbubble scaffold. Under the stable formation of bubble regiem, monodisperse gelatin-microbubbles modulated by varying the flow rate, gas pressure, and channel size. The microbubble scaffolds, the size ranging from 50μm to 200μm, were seeded with pulmonary stem/progenitor cells to explore the application potential for stem cell-based tissue engineering. After 10 to 14 days’ culture, the scaffold showed porous and spongy structures similar to alveolar units. The structure for the scaffold and the cells were examined by RT-PCR, immunohistochemistry and electromicroscopy. Cells forming “alveolar-like structures” in the inner sites of the microbubbles were stained positive for alveolar AT2 and AT1 cells by immunohistochemical analysis and RT-PCR, and there were still a few stem/progenitor cells keeping their phenotype in the structure. The results indicated that the pulmonary stem/progenitor cells could be induced to the terminal differentiation for AT2-like and AT1-like cells as well as the promotion of three-dimensional alveolar tissue growth via the gelatin-microbubble scaffold. In animal model, we utilized the subcutaneous transplantation model on in vivo generation of vascularized pulmonary tissue constructs. The constructs were transplated subcutaneously into the dorsal body of adult C.B17/Icr-scid mice to facilitate in vivo pulmonary tissue construct formation. After transplantation, the constructs showed “alveolar-like structures” in scaffold with positive immunohistochemical staining for epithelial and endothelial cells. The pulmonary cells of GFP transgenic mice in the constructs were able to survive in the subcutaneous tissue of adult mice for at least 28 days. Angiogenesis of the constructs was demonstrated with tail vein injection of fluorescein isothiocyanate–conjugated dextran. In the scaffold-only controls, some level of host infiltrate, but no measurable vascularization, was detected. This study suggests that the three dimensional cell culture system provided an appropriate microenvironment for pulmonary cells, and demonstrates that the cell-based tissue engineering possessed the potential promise to regenerate alveolar-like structures for the development of engineering lung tissue for clinical applications.
|
|---|
