Introduction The development of regenerative therapies holds promise for the future treatment of parenchymal lung diseases. However, encouraging preclinical data from animal models have translated poorly in clinical trials. The cellular and molecular response to lung injury is difficult to study in man. To address this fundamental question, we have developed a novel in vitro human model. Precision cut lung slice (PCLS) culture is a wellestablished tool in airway biology and pharmacology. Here, we demonstrate lung parenchyma can be maintained and manipulated in vitro generating a tractable model, which allows study of lung injury and repair in man. Methods PCLS (500 mm) were generated from agarose-inflated lung lobes from human lungs maintained ex-vivo by perfusion and ventilation (EVLP). The slices were cultured in serum-free medium in a rotating incubator (37ºC, 5% CO 2 ) and analysed at days 1, 3 and 7. Cell specific immunofluorescence markers were used to identify smooth muscle, type I and type II alveolar epithelial cells (AT1, AT2), vascular endothelial cells and proliferating cells (using aSMA, Aquaporin5, ProSPC, PECAM1 and Phospho-histone H3 respectively). Slice viability was confirmed using MitoTracker, LDH and Live/Dead assays. Results All of the expected cell types were identified in PCLS by immunofluorescence demonstrating that human PCLS maintained cellular differentiation in culture. Pro-SPC was predominant in the alveolar wall cells, particularly in the alveolar septal junctions, corresponding to known location of AT2 cells; AQ5 was distributed in thin bands lining the alveolar walls suggestive of the apical membrane of AT1 cells; aSMA was positive around airways, the known location of smooth muscle cells (SMCs); PECAM-1 was positive within alveolar walls corresponding to microvascular capillaries within alveolar septae. There was no significant cell proliferation during culture under basal conditions. Finally, cell viability studies demonstrated that PCLS can be maintained for up to 1 week in serum-free culture. Conclusion PCLS of human lung parenchyma remain differentiated and viable for up to 7 days in serum-free culture. In future, human PCLS derived from normal and injured regions of lung from the EVLP model may provide a novel means of studying alveolar repair in human lung in vitro.
Introduction The development of regenerative therapies holds promise for the future treatment of parenchymal lung diseases. However, encouraging preclinical data from animal models have translated poorly in clinical trials. The cellular and molecular response to lung injury is difficult to study in man. To address this fundamental question, we have developed a novel in vitro human model. Precision cut lung slice (PCLS) culture is a wellestablished tool in airway biology and pharmacology. Here, we demonstrate lung parenchyma can be maintained and manipulated in vitro generating a tractable model, which allows study of lung injury and repair in man. Methods PCLS (500 mm) were generated from agarose-inflated lung lobes from human lungs maintained ex-vivo by perfusion and ventilation (EVLP). The slices were cultured in serum-free medium in a rotating incubator (37ºC, 5% CO 2 ) and analysed at days 1, 3 and 7. Cell specific immunofluorescence markers were used to identify smooth muscle, type I and type II alveolar epithelial cells (AT1, AT2), vascular endothelial cells and proliferating cells (using aSMA, Aquaporin5, ProSPC, PECAM1 and Phospho-histone H3 respectively). Slice viability was confirmed using MitoTracker, LDH and Live/Dead assays. Results All of the expected cell types were identified in PCLS by immunofluorescence demonstrating that human PCLS maintained cellular differentiation in culture. Pro-SPC was predominant in the alveolar wall cells, particularly in the alveolar septal junctions, corresponding to known location of AT2 cells; AQ5 was distributed in thin bands lining the alveolar walls suggestive of the apical membrane of AT1 cells; aSMA was positive around airways, the known location of smooth muscle cells (SMCs); PECAM-1 was positive within alveolar walls corresponding to microvascular capillaries within alveolar septae. There was no significant cell proliferation during culture under basal conditions. Finally, cell viability studies demonstrated that PCLS can be maintained for up to 1 week in serum-free culture. Conclusion PCLS of human lung parenchyma remain differentiated and viable for up to 7 days in serum-free culture. In future, human PCLS derived from normal and injured regions of lung from the EVLP model may provide a novel means of studying alveolar repair in human lung in vitro.
P19 Figure 1 A549 cells plated on an iCelligence 8-well gold electrode coated plate were incubated with TNFR1 dAb™, a dummy dAb or Adalimumab™ for 1 h then exposed to exogenous TNF or vehicle control. Electrical impedance was measured continuously over 50 h. Trough normalised impedance was measured over 50 h post treatment (n = 3-5).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.