Abstract
Human precision-cut lung slices (PCLS) have proven to be an invaluable tool for numerous toxicologic, pharmacologic, and immunologic studies. Although a cultivation period of <1 week is sufficient for most studies, modeling of complex disease mechanisms and investigating effects of long-term exposure to certain substances require cultivation periods that are much longer. So far, data regarding tissue integrity of long-term cultivated PCLS are incomplete. More than 1500 human PCLS from 16 different donors were cultivated under standardized, serum-free conditions for up to 28 days and the viability, tissue integrity, and the transcriptome was assessed in great detail. Even though viability of PCLS was well preserved during long-term cultivation, a continuous loss of cells was observed. Although the bronchial epithelium was well preserved throughout cultivation, the alveolar integrity was preserved for about 2 weeks, and the vasculatory system experienced significant loss of integrity within the first week. Furthermore, ciliary beat in the small airways gradually decreased after 1 week. Interestingly, keratinizing squamous metaplasia of the alveolar epithelium with significantly increasing manifestation were found over time. Transcriptome analysis revealed a significantly increased immune response and significantly decreased metabolic activity within the first 24 hours after PCLS generation. Overall, this study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of PCLS.
Highlights
Information of the Human Donors Used for the Generation of Precision-Cut Lung Slices Donor no
This study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of precision-cut lung slices (PCLSs)
Trimmed reads were aligned to the reference genome using the open source short read aligner STAR with settings according to the log file.[39]
Summary
Human PCLSs were generated from seven fibrotic lung explants The WST-1 and LDH assays were performed in accordance to the manufacturer’s instructions and as described by Neuhaus et al.[38] As a cell death control, two PCLSs per well were previously treated with 1% Triton X100 for 45 minutes at 37 C, 5% CO2, and 100% humidity for each LDH and WST-1 assay. Trimmed reads were aligned to the reference genome using the open source short read aligner STAR (https://code.google.com/p/rna-star, last accessed February 10, 2021) with settings according to the log file.[39] Feature counts were determined using the R package Rsubread.[40] Only genes showing counts >5 at least two times across all samples were considered for further analysis (data cleansing). All reported P values for parameters are calculated from the t statistics from this type of models, with the selection of samples and parameters depending on the question at hand
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