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Precision cut lung slices (PCLS) are complex three-dimensional (3-D) lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules [e.g., microRNA (miRNA)]. The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. PCLS of 4 mm diameter were generated using agarose-filled rodent lungs and a vibratome. TYE665-labeled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualized using live high-content fluorescence microscopy, followed by higher-resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2, we established that cellular membranes in PCLS are permeable in the initial 24 h of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 h after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared with other LNPs, with detection possible as low as 25 nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs. NEW & NOTEWORTHY RNA-based therapeutics hold significant promise for disease treatment; however, limited research exists on miRNA transfection specifically within PCLS. miRNA transfection has thus far required custom functionalization for stabilization and internalization. We aimed to optimize a transfection protocol for rapid screening approaches of miRNA sequences. We show that transfecting miRNA in PCLS is possible using lipid nanoparticles. In addition, we show that 25 nM of TYE665-miRNA is sufficient for detection in a high-content imaging system.

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared with animal models. Several experimental readouts have been established for use with PCLS, but obtaining high-yield and -quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for nonbiased and high-throughput analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high-quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis. We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.