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Precision-cut lung slices as alternative to mimic inflammation, lung injury and bronchoconstriction after short and long-term maintenance in culture

: Neuhaus, Vanessa; Schaudien, Dirk; Sewald, Katherina; Braun, Armin

American Journal of Respiratory and Critical Care Medicine 189 (2014), Art. A5737
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2014, San Diego/Calif.>
Fraunhofer ITEM ()

Rationale: Precision-cut lung slices (PCLS) reflect the relevant microanatomy of the respiratory tract and have been proven to be a valuable alternative method for adverse health outcomes such as inflammation and lung injury. However, PCLS have been mainly used for short-term cultivation less than 72h.This study focus on maintenance and functionality of lung tissue in long-term cultured (>= 14 days) rat PCLS.
Methods: Rat PCLS were cultured for 15 days under optimized cell culture conditions with daily medium exchanges. Functional and histopathological endpoints were assessed at different time points. Vitality was measured by live/dead staining with subsequent confocal image analysis, and LDH assay. Total protein content was measured by BCA assay. The ability to react with bronchoconstriction after addition of methacholine was used as marker for functionality. Lipopolysaccharide (LPS)-induced cytokine release was assessed for inflammation. Additionally, hematoxylin and eosin stained slices were analyzed histopathologically.
Results: Quantitative analysis of the live/dead stained lung sections after culture for 15 days showed a stable ratio of nuclei of dead cell to volume of living cells. Total protein content markedly decreased from day 1 to day 5 (248 ± 40 pg/mL vs. 139 ± 21 pg/mL). LDH assay showed a high level of LDH in supernatant of PCLS on the first day (OD 2.1 ± 0.2), which decreased in the following days (OD7d 0.86 ± 0.35 to OD15d 0.54 ± 0.25). Bronchoconstriction by methacholine was inducible at any time including after 15 days of culture. However, maximum reduction of the initial airway area increased from 28.5% on day 1 to 56.5% on day 15. Further, sensitivity to methacholine also decreased (EC50 1d=8x10 -8M vs. EC50 15d=5x10 -7M). LPS induced release of Tumor necrosis factor alpha until day 2. Histophathological examination showed a decreased cellular content. Moreover, alveolar structure retained up to day 15 and bronchiolar epithelium and smooth musculature exhibited a moderate degree of preservation. Furthermore, no overgrowth of fibroblasts was detected.
Conclusion: Overall these results show that in general long-term cultivation of rat PCLS is feasible. Microanatomical structure of lung tissue and integrity of cells is maintained for 15 days. However, alterations on cellular level take place over time. Cells that are more sensitive and fade away during the long-term culture need to be analyzed. Appropriate alterations of the culture conditions might increase the cellular vitality and condition in order to use long-term rat PCLS.