Human precision-cut lung slices as a screening tool: increased throughput, cryopreservation, and detection of DNA adducts for next generation tobacco product evaluation
Human-relevant, in vitro/ex vivo assays are considered an ethical and economically viable manner by which to screen the many chemicals requiring hazard assessment, such as next generation tobacco product (NGTP) ingredients. Human precision-cut lung slices (PCLS) are often considered the most physiologically relevant pulmonary test system, but low throughput and difficulties in cryopreservation have hampered PCLS use. Our objective was to increase PCLS production, optimize cryopreservation, and demonstrate 3R4F-induced DNA adduct formation using PCLS.
A tissue slicer modification allowed simultaneous slicing of three tissue cores. Increased production was quantified using agarose and tissue cores over 15 minutes. We tested five PCLS cryopreservation formulations against PCLS maintained in culture. Viability in each of the groups was assessed with the WST-8 viability assay, prior to fixation and histological evaluation. For mass-spectrometer analysis of DNA adduct formation, PCLS were exposed to ten 3R4F cigarettes (10 puffs each) using the Health Canada Intense regime or exposed to air.
The slicer modification resulted in 2.8-fold and 2.4-fold more slices from agarose cores, and lung cores, respectively. Cryopreservation efforts indicated freezing after slicing yields better average viability (48-73 % of fresh, non-frozen control) than culturing overnight and freezing (13-54 % of control) when assessing health over 4 days, post-thaw. Cryopreservation buffers containing University of Wisconsin preservation solution preserved viability the best (54 %-90 % of non-frozen control). Histological findings concurred with WST-8 viability results (> 75 % of normal healthy lung tissue features), post-thaw. DNA-adduct evaluation indicated every 3R4F exposed (N = 6) but no air-exposed, (N = 6) PCLS yielded acrolein DNA adducts.
The increased PCLS production indicates larger screening studies can be initiated from one lung and cryopreservation results suggest slices can be banked. Further, utilizing human PCLS for DNA adduct analysis demonstrates a relevant screening endpoint to evaluate NGTP safety.