C20-06 A Novel In Vitro Model of Neonatal Cyclic Lung Stretch Using Precision Cut Lung Slices

Rationale Mechanical ventilation (MV) is used to treat respiratory failure but leads to ventilator-induced lung injury (VILI) from repeated cyclic stretch. Current in vitro precision cut lung slice (PCLS) stretch models are hindered by critical technical limitations including agarose retention (which impacts physiologic relaxation and biochemical assays), damaging or toxic substrate anchoring methods, short exposure times (∼1 hour), and short culture viability. Our objective, using several technical innovations, was to create and validate an in vivo PCLS system that would overcome these barriers and be broadly applicable to preclinical and translational lung tissues across the lifespan. Methods Lungs from neonatal (postnatal day 0-1) mice were exposed to room air or 100% oxygen for 24 hours, euthanized, and inflated with ultra-low melting point agarose. After gelation, PCLS were generated from right lung lobes on a vibratome at 300 μm thickness. After resting overnight slices were treated with 50U/ml agarase enzyme and tissue retraction was quantified over 6 hours compared to untreated controls. Tissue viability was confirmed using calcein AM (live) and ethidium homodimer (dead) staining. Agarose-free PCLS were embedded in poly (ethylene-glycol) (PEG) hydrogels that were incorporated into the foam matrix of FlexCell™ TissueTrain™ plates. Neonatal PCLS were subjected to 15% elongation at 0.25 Hz for 24 hours and immediately preserved for transcriptomic analysis. Results Agarase treatment resulted in a 39% reduction in tissue area (Fig. 1A) within 2 hours, stabilizing thereafter (Fig. 1B). PEG Hydrogels remained intact, permitting CMS of at least 21% elongation at 0.25Hz for 24 hours (Fig. 1C). RNA-seq derived PCA plots of stretched and unstretched RA and oxygen treated controls showed good separation by oxygen and stretch condition. Examination of ECM and stretch-related gene expression based on matrisomeDB genes showed altered expression profiles of numerous genes in stretched tissues relative to non-stretched, PEG-embedded controls (Fig. 1D) indicating that PCLS are stretch-responsive, viable, and demonstrate altered gene expression can be observed after 24 hours of exposure. Further, fibroblast growth was evident in transwell controls (data not shown), confirming the need for supportive PEG hydrogels. Conclusions Numerous technical innovations were developed to permit longer-term CMS of PCLS and increased physiological relevance. These developments enable novel in vitro testing of CMS in human tissues and multiple stretch paradigms (ongoing). This abstract is funded by: University of Rochester University Research Award, NIH K08HL155491