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

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 analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction after acetylcholine stimulus. After 14 days 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 to 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 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.

Perspectives on precision cut lung slices—powerful tools for investigation of mechanisms and therapeutic targets in lung diseases

“…An alternative method to optimize PCLS utilization without cryopreservation evaluated long-term hypothermic (cold) storage at 4°C, in either DMEM/F-12 or two different preservation solutions ( Tigges et al, 2021 ). After 14 days of cold storage, a range of parameters were better preserved after storage in potassium and chloride-rich tissue preservation solution (solution 1) and a chloride-poor, lactobionate-rich version (solution 2) compared with DMEM/F-12.…”

“…PCLS stored in solution one responded substantially longer to inflammatory stimulation with LPS, resulting in higher TNF-α levels in conditioned media than in PCLS stored in DMEM/F-12 or solution 2. Notably, bronchoconstriction to ACh was observed after 14 days in both optimized solutions, but not DMEM/F-12 alone ( Tigges et al, 2021 ). This approach may reduce logistical challenges in shipment of PCLS between laboratories and provide opportunities for stockpiling of PCLS samples.…”

Prolonged airway explant culture enables study of health, disease, and viral pathogenesis

“…The ability to prepare viable airway explants from cryopreserved tissue presents another major advancement in the field. While cryopreservation of PCLS has been previously reported 31–33 , our study represents the first application of this methodology to generate viable airway tissue explants from cryopreserved tissue. Airway explants from cryopreserved tissue successfully replicated morphologic and electrophysiologic features of fresh tissue.…”

Application of Precision-Cut Lung Slices as an In Vitro Model for Research of Inflammatory Respiratory Diseases

“…Surprisingly, the airway contraction and relaxation abilities of specific agonists and antagonists were still retained in thawed PCLSs, and the contractile force of smooth muscle cells in the airway was still well regulated by Ca 2+ -dependent mechanisms [ 27 , 28 ]. In order to optimize the refrigeration method of PCLSs, Tigges et al refrigerated the standard medium DMEM/F-12 for 14 days after adding a high concentration of potassium chloride; they found that the response of PCLSs to inflammatory stimuli and their bronchial constriction ability were significantly improved compared with PCLSs cultured in DMEM/F-12 standard medium only [ 29 ]. In terms of PCLS cultures, due to the complex mechanisms of chronic lung disease, the use of PCLSs may be required in longer studies to observe irreversible ECM remodeling.…”