ABSTRACT: The lung epithelium contains multiple distinct cell types which are maintained by regionally specific progenitor cell populations in both the airways and in the alveolar compartments. Each of the progenitor populations are known to have varying roles during homeostasis and have increasingly been shown to be deranged in lung diseases. The progenitor cells of the proximal lung epithelium are the basal cells (Krt5+/Krt14+/p63+) which sit at the basement membrane of the trachea, referred to as, proximal progenitors in this study. On the other hand, the distal alveolar epithelium is maintained by alveolar type II cells (Sftpc+), referred to as distal progenitors in this study. Several cell isolation methods are available to isolate each of these populations of proximal and distal progenitors but are traditionally performed separately on mice which is especially important in the context of disease. Methods which allow the isolation of the entire epithelial population from murine lungs, while retaining the capability to perform downstream studies such as organoid assays and air-liquid-interface, would open new possibilities for studying interactions between the proximal and distal airways in lung disease. However, an easy and reproducible method that allows isolation of both cell types simultaneously from an individual mouse has not been previously described. This is in part due to the technical and surgical challenges associated with each isolation method as well as, in part, due to a lack of cell surface markers that are specific and unique to each cell type that could allow their isolation from a single cell suspension of the entire epithelium. Additionally, transgenic models have been shown to be leaky and mark populations other than the target cells. Thus, classical methods that rely on positive or negative selection, coupled by surgical isolation remain the state of the art. In order to facilitate isolation of proximal and distal progenitors from an individual mouse, we developed a 3D printable Lobe Divider (3DLD) that allows for the reproducible separation of the trachea and lung lobes while allowing for controllable and temporal instillation of cell dissociation buffers for both cell isolation procedures. The efficacy of the 3DLD in isolating the two populations and the functionality of the cells isolated with the 3DLD was validated through multiple differentiation assays. Proximal progenitors were differentiated in 2D at air-liquid interface (ALI) culture for 28 days after monolayer formation for 7days. Distal progenitors isolated with the 3DLD or with the equivalent classical tracheal ligation method were differentiated in organoid culture in Matrigel for 14 days while at ALI. Total RNA was isolated from cell pellets of all cell types before differentiation, from ALI cultures of proximal cells, and organoids of distal cells isolated with 3DLD or classic method. Qiagen RNeasy micro kit (74004, QIAGEN, Sweden) was used to isolate total RNA following the manufacturer’s protocol. RNA concentrations were measured using NanoDrop 1000 and Qubit™ RNA HS Assay Kit (Q32852, Fisher Scientific). RNA quality was evaluated by the Agilent 2100 BioAnalyzer. Library preparation was done using the Illumina Stranded mRNA Prep Ligation (20040534, Illumina) with 14 cycles under final PCR amplification and samples were indexed with IDT for Illumina RNA UD Indexes Set B, Ligation (20040554, Illumina) for multiplexing. Clean up steps were automated using the King Fisher FLEX (18-5400620, Thermo Scientific). Library concentration was checked using the QuantIT 1X dsDNA HS Assay Kit (Q33232) and library size and quality were checked using the 5200 Fragment Analyzer™ 12-capillary (M3510AA, Agilent) using the DNF-930 dsDNA Reagent Kit, 75 bp – 20,000 bp(500 Samples) (Part # DNF-930-K0500, Agilent). A library input of 1.25 nM was sequenced using NovaSeq 6000 Sequencing System (20012850, Illumina).
