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 maint ained in serum-free whole organ culture and that airway ligation accel erates lung development. In spite of extensive use of lung organ cultu re systems, the vasculature of the unperfused lung in organ culture ha s not been studied. The aim of the present study was to compare organ cultures of heart-lung blocks with continuous perfusion of the pulmona ry vasculature to those without perfusion, ie, whole lungs cultured wi thout the attached heart. Time-dated pregnant CD-1 mice were killed on gestational day (Gd) 14. The fetuses were removed via laparotomy. Hea rt-lung blocks and whole lungs without the heart were excised under st erile conditions and cultured in BGJb media. Some of the heart-lungs a nd whole lungs underwent tracheal ligation whereas others were left wi th the trachea unligated allowing free egress of airway fluid. After 7 days, the cultured organs were processed for histology, ultrastructur al analysis, and immunohistochemistry. (1) Lungs were fixed in 10% for malin, paraffin embedded, and processed for routine H&E staining. (2) Lungs were fixed in 2.5% glutaraldehyde in cacodylate buffer and proce ssed for transmission electron microscopy. (3) Lungs were embedded in CRYOform and flash frozen for immunohistochemical localization of PECA M-1 (CD31) (PECAM-1, Platelet endothelial cell adhesion molecule-1, a selective endothelial cell marker). Our daily observations of the cult ured 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 f etal Gd14 pseudoglandular lungs (time-zero) had a defined capillary ne twork, which was more centrally localized and peripherally less develo ped. The presence of more numerous lung capillaries in the cultured he art-lung blocks was noted when compared with cultured lungs alone. Ult rastructurally, endothelial cells with intact structural integrity wer e identified only in cultured whole lungs with hearts. Immunohistochem ical staining of the whole lungs with rat antimurine PECAM-1 monoclona l antibody performed on cryosections showed the presence of vasculatur e by specific PECAM-1 localization on endothelial cells. PECAM-1 label ing of capillaries was noted in Gd14 (time-zero) lungs. In addition, t he lungs cultured with hearts, ie, perfused lungs, showed more well de fined, distinct capillary networks stained with PECAM-1 antibody than unperfused lungs without hearts. Our results showed that microvasculat ure is present in murine fetal lungs at Gd14. After 7 days in organ cu lture, the maintenance of lung microvasculature was confirmed histolog ically, ultrastructurally, and immunohistochemically. The microvascula ture in whole lungs cultured as perfused/ beating heart-lung blocks wa s better maintained than the microvasculature of unperfused whole lung s cultured without hearts. A perfused whole lung organ culture model i s attractive because the lung architecture is better maintained and ma y be useful in lung developmental studies as it mimics the in situ hea rt-lung functional physiological relationship. Copyright (C) 1997 by W .B. Saunders Company.