Precision Cut Lung Slices (PCLS) Thickness has Limited Impact on Tissue Viability

Abstract

As an ex-vivo model, PCLS offers numerous benefits over classical in vitro techniques whilst retaining similar levels of flexibility. PCLS maintains cellular architecture, structure and function, thus facilitating responses more akin to the in vivo environment. Numerous variations of PCLS preparation exist, with limited consensus on optimum thickness and culture duration, here these variations will be considered in an attempt to determine optimum conditions. Lung tissue was inflated with 3% agarose, embedded and sliced at 300-1000µm thickness using a Leica Vibratome. Slices were incubated with media changes 2h post-slicing and every 24-36h thereafter for a maximum of 168h. Slice viability was assessed at 24, 120 and 168h post-slicing via LDH release, trypan blue exclusion and tissue morphology. Microscopy images showed limited change to gross or cellular morphology over the culture period for any thickness PCLS. Furthermore, LDH cytotoxicity data suggested PCLS remained viable for 168h at thicknesses between 300-700µm (P>0.05) however, LDH release from 1000µm PCLS was significantly reduced compared to all other thicknesses (P<0.005). Similarly, trypan blue exclusion indicated all thicknesses maintain a >70% viability for 168h however, viability of 300µm is lower than PCLS between 500-1000µm (P<0.05). Given the observation regarding tissue morphology, alongside LDH cytotoxicity and trypan blue exclusion, the data suggests experimental periods of up to 168h are feasible without impact on tissue viability and that there is little difference between PCLS thickness, thus rather than a specific optimum, PCLS thickness and culture duration can be based on experimental need.