Maintenance of fetal murine pulmonary microvasculature in heart-lung en bloc whole organ culture.

The authors have previously shown that murine fetal lungs can be maintained in serum-free whole organ culture and that airway ligation accelerates lung development. In spite of extensive use of lung organ culture systems, the vasculature of the unperfused lung in organ culture has not been studied. The aim of the present study was to compare organ cultures of heart-lung blocks with continuous perfusion of the pulmonary vasculature to those without perfusion, ie, whole lungs cultured without the attached heart. Time-dated pregnant CD-1 mice were killed on gestational day (Gd) 14. The fetuses were removed via laparotomy. Heart-lung blocks and whole lungs without the heart were excised under sterile conditions and cultured in BGJb media. Some of the heart-lungs and whole lungs underwent tracheal ligation whereas others were left with the trachea unligated allowing free egress of airway fluid. After 7 days, the cultured organs were processed for histology, ultrastructural analysis, and immunohistochemistry. (1) Lungs were fixed in 10% formalin, paraffin embedded, and processed for routine H&E staining. (2) Lungs were fixed in 2.5% glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy. (3) Lungs were embedded in CRYOform and flash frozen for immunohistochemical localization of PECAM-1 (CD31) (PECAM-1, Platelet endothelial cell adhesion molecule-1, a selective endothelial cell marker). Our daily observations of the cultured organs showed that the heart maintained synchronized beating for all 7 days in culture. Perfusion of the pulmonary microvasculature was demonstrated. Light microscopically, H&E sections showed that fresh fetal Gd14 pseudoglandular lungs (time-zero) had a defined capillary network, which was more centrally localized and peripherally less developed. The presence of more numerous lung capillaries in the cultured heart-lung blocks was noted when compared with cultured lungs alone. Ultrastructurally, endothelial cells with intact structural integrity were identified only in cultured whole lungs with hearts. Immunohistochemical staining of the whole lungs with rat antimurine PECAM-1 monoclonal antibody performed on cryosections showed the presence of vasculature by specific PECAM-1 localization on endothelial cells. PECAM-1 labeling of capillaries was noted in Gd14 (time-zero) lungs. In addition, the lungs cultured with hearts, ie, perfused lungs, showed more well defined, distinct capillary networks stained with PECAM-1 antibody than unperfused lungs without hearts. Our results showed that microvasculature is present in murine fetal lungs at Gd14. After 7 days in organ culture, the maintenance of lung microvasculature was confirmed histologically, ultrastructurally, and immunohistochemically. The microvasculature in whole lungs cultured as perfused/beating heart-lung blocks was better maintained than the microvasculature of unperfused whole lungs cultured without hearts. A perfused whole lung organ culture model is attractive because the lung architecture is better maintained and may be useful in lung developmental studies as it mimics the in situ heart-lung functional physiological relationship.