Background Culture of embryonic lung buds has provided insight into airway branching morphogenesis; however, similar insights into pulmonary vascular morphogenesis have not been possible in lung bud explants because the pulmonary vasculature collapses and withers. We developed a method to isolate and culture the pulmonary arterial tree from chick embryos (Albertine, PAS 2005;57:3219 [abstract #1994]). One of the remaining uncertainties is whether the PA cultures are lined by endothelial cells.
Objective We hypothesized that luminal support and hypoxia will facilitate retention of endothelial cells lining embryonic pulmonary vascular explants.
Methods Chick embryos at 16-18 d gestation (hatching is 21 d) were used. The pulmonary arterial (PA) tree was injected with 0.75% low gelling temperature agarose dissolved in Waymouth's media containing Evan's blue dye (Van Winkle, AJRCMB 1996). The injected embryos were placed on ice to gel the agarose. The PA tree was dissected from the lung tissue (5-6 arterial generations). Arterial trees without agarose were controls. Agarose-filled or unfilled arterial trees (> 6 each) were cultured at 37°C in 12% O2 for 21 d, in F12 + 6 media with retinol and bovine serum albumin. The PA trees were fixed and analyzed by transmission electron microscopy (TEM) weekly.
Results The PA trees retained the dissected generations. Electron microscopy revealed that the PA vessels were lined by endothelial cells and vascular smooth muscle cells for at least 6 d. By 12 d, however, few cultured PA trees had endothelial cells lining the lumen.
Conclusion Embryonic PA trees, with intact endothelial and smooth muscle cells, can be cultured for at least 6 d, thereby providing a model system to study embryonic pulmonary vascular biology.
Supported by CHRC, HL62875, HL56401, and T35 HL07744.
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