Biotechnology Based Fetal Pulmonary Tissue Engineering

描述（由申请人提供）：肺发育不全或肺不成熟在所有新生儿尸检中发现的比例高达15-20%。从培养的细胞中培养出功能三维肺组织用于最终移植的能力对于防止新生儿因肺发育不全而死亡是非常宝贵的。该项目的长期目标是通过体外工程肺组织构建体增加受损肺来减轻新生儿肺发育不全。为了实现这一目标，我们将分离小鼠胎儿肺细胞，并利用工程纳米支架、复合凝胶、生长因子增强培养基和优化的微灌注系统，形成三维肺结构。该项目的假设是，在工程微环境中，培养的细胞将能够实现细胞间的接触、粘附、增殖，并分化成宏观的三维组织结构。该项目的短期目标是系统地确定具有适当形态和细胞分化的工程肺组织系统的参数。本项目的具体目的是：1-优化方法，分离和培养异型群体的肺胎儿细胞使用强化培养基含有组织特异性生长因子。2-通过在由复合凝胶或由细胞外基质衍生分子静电纺成的纳米纤维组成的器官型三维基质上生长，促进培养的胎儿肺细胞在长时间内的粘附、存活、生长和分化。3-利用微灌注系统，使这些3- d结构与营养物质连续沐浴，并使它们受到脉动流诱导的剪切力。随后的3-D结构的形态和细胞分化将通过组织学，免疫组织化学，电子显微镜和生化/分子生物学技术进行评估。本R-21申请的目标是探索和优化该方法的可行性，并为提交传统R01申请生成初步数据。

DESCRIPTION (provided by applicant): Pulmonary hypoplasia or lung immaturity is found in as many as 15-20% of all neonatal autopsies. The ability to grow functional 3-dimensional lung tissue from cultured cells for eventual transplantation would be invaluable in preventing the neonatal mortality associated with pulmonary hypoplasia. The long term goal of this project is to alleviate neonatal pulmonary hypoplasia by augmenting damaged lung with in vitro engineered pulmonary tissue constructs. In order to achieve this goal, murine fetal pulmonary cells will be isolated, and utilizing engineered nano-based scaffolds, composite gels, growth factor enhanced media and an optimized microperfusion system, 3-dimensional lung constructs will be formed. The hypothesis of this project is that in an engineered micro-environment, cultured cells will be able to achieve cell to cell contact, adhere, proliferate, and differentiate into macroscopic 3-dimensional tissue constructs. The short term goal of this project is to systematically determine the parameters of a system for engineering lung tissue with appropriate morphology and cytodifferentiation. The specific aims of this project are to: 1- Optimize methods for isolating and culturing heterotypic populations of pulmonary fetal cells using enhanced culture media containing tissue-specific growth factors. 2- To facilitate adhesion, survival, growth and differentiation of cultured fetal lung cells over a prolonged period of time by growing these cells on organotypic 3-dimensional matrices composed of either composite gels or nanofibers electrospun from extracellular matrix derived molecules. 3- Utilize a microperfusion system to enable the continuous bathing of these 3-D constructs with nutrients as well as subject them to pulsatile flow induced shear forces. Morphology and cytodifferentiation of the ensuing 3-D contructs will be evaluated by histology, immunohistochemistry, electron microscopy and biochemical/ molecular biological techniques. The goal of this R-21 application is to explore and optimize the feasibility of this approach and to generate preliminary data for submission of a traditional R01 application.