Reuse of Cell Culture Inserts for in vitro Human Primary Airway Epithelial Cell Studies.

Human primary bronchial epithelial (HBE) cells have a long history in respiratory research.With the establishment of protocols for expansion of primary airway epithelial cells with conditionally reprogrammed cell methods (1, 2), cellular redifferentiation using air–liquid interface (ALI) conditions on porous membranes, and lentiviral infection and CRISPR/Cas9-mediated transduction and gene knockdown technologies (3, 4), the use of HBE cell cultures for respiratory research has greatly expanded. Because of the worldwide coronavirus disease (COVID-19) pandemic in 2020, a shortage of clinical supplies—for example, masks and hand sanitizers—emerged as a serious issue (5). Likewise, supplies for basic research have been limited. In the case of cell culture, Transwell (3460; Corning) and Millicell (PIH P01250; Millipore) inserts have been in exceptionally short supply. Owing to their limited supply and high cost, insert reuse is a rational approach to maintain timely and cost-efficient research. The only studies reporting insert reuse focused on reuse for cell line applications (6). In this study, we investigated the reuse of Transwell andMillicell inserts for primary HBE cell culture research. All experiments were conducted using cells from three to five different donor lung specimens. Detailed protocols are found in the data supplement. Protocols to remove primary airway cells from inserts were developed first. We tested four reagents (0.25% Trypsin-EDTA [25200056; Gibco], Accutase [A6964; Sigma], 6M urea in PBS, and RIPA buffer) to dissociate/solubilize and remove well-differentiated HBE cells from Transwell inserts. Dissociation solutions were applied to the apical surface for between 5 and 20 minutes, followed by pipetting or scraping with a cell lifter. Trypsin-EDTA, 6M urea, and RIPA buffer effectively removed cells. Accutase was less effective. Following cell dissociation, an overnight incubation with 0.25% Trypsin-EDTA at 37 C completed removal of residual cells and debris from inserts. Apical and basolateral surfaces of the inserts were then washed twice with PBS and twice with pure water. After air-drying in a hood, the inserts were coated with collagen (5005; Advanced BioMatrix), UV irradiated, and stored at 4 C until reuse. The reused inserts were tested in a series of HBE cell culture studies. HBE cells from new donors were seeded and cell differentiation was monitored over 4 weeks in ALI culture (7). None of the 110 second-use (U2) Transwell inserts exhibited contamination or a loss of integrity. Preservation of cellular differentiation in U1 (first-use) versus U2 cultures was tested by three approaches. First, mRNA expression of cell type markers was measured by qRT-PCR and was similar for U1 versus U2 cultures (Figure 1A) (8). Second, MUC5B protein in apical washes was measured byWestern blotting and was found to be similar between U1 versus U2 cultures (Figure 1B). Third, hematoxylin and eosin and whole mount immunofluorescent staining for ciliated (tubulin), secretory (CCSP), and goblet (MUC5B) cell markers confirmed similar differentiation between U1 and U2 inserts (Figures 1C and 1D). Measurements of HBE cell function on U1 versus U2 inserts were made for ion transport in Ussing chambers (Figure 1E) and cilia beat frequency with video microscopy (Figure 1F) (9–11). Both assays revealed virtually identical cellular functional activities of HBE cells on U1 versus U2 inserts. Importantly, donor-to-donor variability in all parameters was reproduced in assays comparing HBE cells on U1 versus U2 inserts, suggesting that insert reuse did not affect donorspecific cellular function. We then tested whether a low number of residual cells escaped the reuse protocol and caused DNA contamination of subsequent cultures. ACE2 knockout cells (GenBankMT505392, generated by introduction of gRNA/Cas9 complexes with electroporation [3]), were cultured on U2 inserts and, after 28 days in ALI, the HBE gene–modified cells were removed from the insert and the insert reconditioned for third use (U3). Wild-type (WT) donor cells were plated onU3 inserts. After attachment, expansion, and 7-day ALI culture, DNAwas extracted from the U3 culture and the gRNA target region was amplified by PCR and sequenced. Gene-editing efficiency analyses (Inference of CRISPR Edits [ICE] analysis; Synthego) (12) demonstrated that the gene-edited U2 insert cells did not contaminate U3 culturedWT cells (Figure 1G). HBE cells exposed to inflammatorymediators exhibit changes in epithelial structure/function that maymodify the produced basement membranematrix and affect subsequent culture properties. Accordingly, U2 HBE cells were treated with IL-1b (1 ng/ml, 201-LB; R&D Systems) for 7 days. IL-1b treatment resulted in induction of MUC5B and reduction of SCGB1A1mRNA (Figure 1H). Apical washes confirmedMUC5B protein induction by IL-1b (Figure 1I) (8). During IL-1b treatment, 8 of 27 U2 inserts exhibited leakage of media onto the apical surface. U2 inserts containing IL-1b–treated versus vehicle-treated HBE cells were prepared for reuse and plated with naive HBE cells (U3). HBE cells on U3 inserts from IL-1b–treated versus vehicle-treated HBE cells exhibited similarMUC5B and SCGB1A1 gene expression, similar MUC5B protein in apical washes, and absence of media leakage, suggesting residual IL-1b effects were not transmitted to subsequent HBE cultures with insert reuse (Figures 1J and 1K). We directly investigated whether the porous membrane structure of inserts was affected by reuse using scanning EM. Significant damage to or obstruction of the pores was not observed after any of the cell dissociation/solubilization protocols (Figure 1L). Finally, multiple reuses of Transwell andMillicell inserts were tested. HBE cells were grown and differentiated on U2 to U4 (fourthuse) Transwell and U2 to U3Millicell inserts (Figure 1M). Insert reuse four times with a cumulative longevity of use for 185 days was feasible. No bacterial contamination was observed at any time in these studies. In these experiments, insert reuse was limited to inserts maintained under sterile conditions at all times to minimize bacterial and/or endotoxin contamination. Therefore, it remains unknown Author Contributions: T.K. conceived and designed the study, performed experiments, analyzed data, and wrote the initial and the revised manuscript. Y.M. , L.S. , T.D. R, B.R. G, and C.B. M. performed experiments and analyzed data. P.R. S and L.E. O supervised cilia beat frequency assays. C.E. supervised scanning microscopy imaging. S.H. R provided human airway epithelial cells and guidance. R.C.B supervised the study, analyzed data, and revised the manuscript. All authors reviewed and approved the final version of the manuscript.