Nove lMethod sfo rDeliver yo fCell-Base dTherapies

Background. Pulmonary hypoplasia (PH) is found in 15% to 20% of all neonatal autopsies, accounting for 2850 deaths yearly. Development of engineered tissue substitutes that could functionally restore damaged tissue remains a unique opportunity for biotechnol- ogy. Recently, we isolated and characterized murine fetal pulmonary cells (FPC) and engineered 3-D pul- monary tissue constructs in vitro. Our goal is to devise a reliable and reproducible method for delivering FPC into a live animal model of PH. Materials and methods. Three methods of delivery were explored: intraoral, intratracheal, and intrapul- monary injection. Adult Swiss Webster mice were anesthetized and fluorescent labeled microspheres (20 m diameter) were delivered by intraoral and intra- tracheal injection. Subsequently, labeled FPC (Cell Tracker, CMTPX; Molecular Probes, Eugene, OR) were delivered by the same methods. In addition, direct transpleural intrapulmonary injection of FPC was performed. Outcome analysis included survival, reproducibility, diffuse versus confined location of the injected substance, and adequacy of delivery. Routine histological examination, fluorescent mi- croscopy, and immunostaining were performed. Results. Microspheres: We demonstrated reproduc- ible, diffuse instillation via tracheotomy into the distal alveoli. Intraoral delivery appeared less reliable com- pared to direct intratracheal injection. FPC: Intratra- cheal injection was a reliable method of delivery. La- beled FPC showed transepithelial migration after 7 d of in vivo culture. Intrapulmonary injection led to lo- cal accumulation of cells in sites of injection. Conclusions. We demonstrate that delivery of FPC is feasible with intratracheal injection giving the most reliable, diffuse delivery throughout the lung. This represents the first step toward translational research with site-specific delivery for a cell-based therapeutic approach toward PH and similar pulmo-