The Development of an Animal Product-free, Precision-cut Lung Slice Cryopreservation and Post-thaw Culture Method

As new approach methodologies (NAMs) are increasingly explored to identify dependable and accurate non-animal alternatives to predict human toxicities, several 3-D test systems have emerged as excellent models of the human respiratory tract. Among these, human precision-cut lung slices (hPCLS) are considered highly relevant, as they contain many cell types (including key immune cells), feature small airway structures and boast native respiratory parenchymal architecture. However, a lack of long-term preservation methods has hampered the use of the hPCLS model for repeat and mainstream testing. In the current study, a range of potential methods were progressively evaluated for the optimum recovery of hPCLS after thawing and multi-week culturing. These methods featured: five different cryopreservation buffer (CB) recipes; freezing either before or after culture initiation; two culture media (based on E-199 and DMEM/F12); and two culture maintenance methods (submerged and air-liquid interface (ALI)). Endpoints used for the assessment of hPCLS culture health included the WST-8 viability assay, protein content and H&E histology of slice sections. Two of the CBs and immediate cryopreservation after slicing produced hPCLS with higher post-thaw viability. While both media recipes and culture methods maintained high slice viability for approximately 2 weeks, the use of DMEM-F12-based medium in ALI culture was superior for the 3-week cultures. Applying due diligence to hPCLS cryopreservation and post-thaw method development provides researchers with an underutilised human respiratory model. Studies making use of cryopreserved banks of normal or diseased tissues (from a diverse demographic pool of donors) can now be initiated as desired, repeated, or expanded upon to interrogate numerous aspects of physiology, toxicology and drug efficacy. These can be applied as routine screening applications or complex evaluations, including those benefitting a regulatory setting.