Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution.

Jonas Tigges,Florian Eggerbauer,Franz Worek,Horst Thiermann,Timo Wille,Ursula Rauen

doi:10.1152/ajplung.00076.2021

Abstract

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Highlights

Precision-cut lung slices (PCLS) are a widely used tool in medical research to study lung biology in an ex vivo tissue system [1]
Metabolic activity was significantly decreased in DMEM/F-12, solution 1, and solution 2 (Table 1) with or without the iron chelators deferoxamine and LK 614 and ranged between 4 ± 1% and 6 ± 1% (P < 0.001 vs. incubator stored PCLS)
To extend storage duration of PCLS without medium change, PCLS were cold-stored in DMEM/F-12 or two designed preservation solutions (Table 1) with and without iron chelators at 4C for 3–28 days and were subsequently rewarmed in DMEM/F-12 for 3 h

Summary

Introduction

Precision-cut lung slices (PCLS) are a widely used tool in medical research to study lung biology in an ex vivo tissue system [1]. PCLS offer the opportunity to study structural and physiological alterations upon treatment in specific airway sections or distal lung, which is of increasing interest for the use in preclinical validation. Addition of the neurotransmitter acetylcholine (ACh) to PCLS medium results in constriction of airway smooth muscle cells and decrease of the airway cross-section area. This bronchoconstriction is spontaneously reversible due to lungresident active acetylcholinesterase [6, 9]. As human lung tissue specimens are rare and samples of diseased patients are even more precious, an optimized storage protocol to process high numbers of PCLS is highly desirable.

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Conclusion