Precision Cut Lung Slices: Emerging Tools for Preclinical and Translational Lung Research. An Official American Thoracic Society Workshop Report.

The urgent need for effective treatments for acute and chronic lung diseases underscores the significance of developing innovative preclinical human research tools. The 2023 ATS Workshop on Precision Cut Lung Slices (PCLS) brought together 35 experts to discuss and address the role of human tissue-derived PCLS as a unique tool for target and drug discovery and validation in pulmonary medicine. With increasing interest and usage, along with advancements in methods and technology, there is a growing need for consensus on PCLS methodology and readouts.

New approach methodologies (NAMs) for the assessment of cleaning products for respiratory irritation: workshop report.

The use of new approach methodologies (NAMs) to assess respiratory irritation depends on several factors, including the specifics of exposure methods and cell/tissue-based test systems. This topic was examined in the context of human health risk assessment for cleaning products at a 1-day public workshop held on 2 March 2023, organized by the American Cleaning Institute (ACI). The goals of this workshop were to (1) review NAMs for evaluation of respiratory irritation, (2) examine different perspectives on current challenges and suggested solutions, and (3) publish a manuscript of the proceedings.

The Fibrotic Phenotype of Human Precision-Cut Lung Slices Is Maintained after Cryopreservation.

Human precision-cut lung slices (hPCLS) prepared from fibrotic lungs recapitulate the pathophysiological hallmarks of fibrosis. These hallmark features can also be induced by treating non-fibrotic hPCLS with a fibrotic cocktail (FC). As a result, the fibrotic and fibrosis-induced hPCLS are rapidly emerging as preferred models for disease modeling and drug discovery.

Multiscale stiffness of human emphysematous precision cut lung slices.

Emphysema is a debilitating disease that remodels the lung leading to reduced tissue stiffness. Thus, understanding emphysema progression requires assessing lung stiffness at both the tissue and alveolar scales. Here, we introduce an approach to determine multiscale tissue stiffness and apply it to precision-cut lung slices (PCLS).

Key Challenges and Recommendations for Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements.

Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of tobacco and other nicotine-containing products.

Regulatory frameworks on tobacco and other nicotine-containing products (TNCP) continue to evolve as novel products emerge, including electronic nicotine delivery systems (e.g., electronic cigarettes or vaping products), heated tobacco products, or certain smokeless products (e.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study.

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled "Dosimetry for conducting in vitro evaluations" and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.

Comparison of Long-term Human Precision-cut Lung Slice Culture Methodology and Response to Challenge: An Argument for Standardisation.

As non-animal alternatives gain acceptance, a need for harmonised testing strategies has emerged. Arguably the most physiologically-relevant model for assessing potential respiratory toxicants, that based on human precision-cut lung slices (hPCLS) has been utilised in many laboratories, but a variety of culture methodologies are employed. In this pilot study, combinations of three different hPCLS culture methods (dynamic organ roller culture (DOC), air-liquid interface (ALI) and submersion) and various media (based on E-199, DMEM/F12 and RPMI-1640) were compared.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP.

Editor's Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay.

Comparative Metabolism of Batracylin (NSC 320846) and N-acetylbatracylin (NSC 611001) Using Human, Dog, and Rat Preparations .

Background: Batracylin is a heterocyclic arylamine topoisomerase inhibitor with preclinical anticancer activity. Marked species differences in sensitivity to the toxicity of batracylin were observed and attributed to differential formation of N-acetylbatracylin by N-acetyltransferase. A Phase I trial of batracylin in cancer patients with slow acetylator genotypes identified a dose-limiting toxicity of hemorrhagic cystitis.

Assessment of in vitro COPD models for tobacco regulatory science: Workshop proceedings, conclusions and paths forward for in vitro model use.

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD).

Analyses of the combination of 6-MP and dasatinib in cell culture.

A major tenet of cancer therapeutics is that combinations of anticancer agents with different mechanisms of action and different toxicities may be effective treatment regimens. Evaluation of additivity/synergy in cell culture may be used to identify drug combination opportunities and to assess risk of additive/synergistic toxicity. The combination of 6-mercaptopurine and dasatinib was assessed for additivity/synergy using the combination index (CI) method and a response surface method in six human tumor cell lines including MCF-7 and MDA-MB‑468 breast cancer, NCI-H23 and NCI-H460 non‑small cell lung cancer, and A498 and 786-O renal cell cancer, based on two experimental end‑points: ATP content and colony formation.

In vitro exposure of precision-cut lung slices to 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole lysylamide dihydrochloride (NSC 710305, Phortress) increases inflammatory cytokine content and tissue damage.

The anticancer drug (2-[4-amino-3-methylphenyl]-5-fluorobenzothiazole lysylamide dihydrochloride) (NSC 710305, Phortress) is a metabolically activated prodrug that causes DNA adduct formation and subsequent toxicity. Preclinically, it was found that hepatic, bone marrow, and pulmonary toxicity presented challenges to developing this drug. An ex vivo precision-cut lung slice (PCLS) model was used to search for concentration dependent effects of NSC 710305 (10, 25, 50, and 100 µM) on cytokine content, protein content, and immuno/histological endpoints.

Prevalidation of the rat CFU-GM assay for in vitro toxicology applications.

In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies - however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations.

In vitro comparison of O4-benzylfolate modulated, BCNU-induced toxicity in human bone marrow using CFU-GM and tumor cell lines.

2-Amino-O4-benzylpteridine derivatives inactivate the human DNA repair protein O6-alkylguanine-DNA alkyltransferase and have been tested as modulators of alkylating agent chemotherapy. Recently, the therapeutic potential of O4-benzylfolate (O4BF) in modulating bis-chloroethylnitrosourea (BCNU) toxicity was demonstrated in vitro using human HT29 and KB tumor lines. The current studies replicated the previous findings in HT29 and KB cells using ATP as an endpoint.

In vitro detection of differential and cell-specific hepatobiliary toxicity induced by geldanamycin and 17-allylaminogeldanamycin using dog liver slices.

Experiments on rat liver slices demonstrated the differential hepatobiliary toxic potency of two anticancer agents, geldanamycin (GEL) and 17-allylaminogeldanamycin (17-AAG), over a 5-day period. This report describes the pattern of toxicity of these agents in dog liver tissue, using the in vitro liver slice culture model. Liver slices (200 microm thick) from male beagle dogs were cultured for 5 days in chemically defined culture medium containing a range of GEL and 17-AAG concentrations (0.

Mitogen-activated protein kinase mediates purinergic-enhanced nerve growth factor-induced neurite outgrowth in PC12 cells.

In 1999, we reported new observations that several compounds, including ATP, enhance neurite expression in PC12 cells when coapplied with nerve growth factor (NGF). Because purinergic and NGF signaling have several potential interfaces in PC12 cells, a series of experiments was conducted to elucidate the signal mediators contributing to the enhancement. Activities of selected kinases were measured and Western blots evaluated mitogen-activated protein kinase (MAPK) active and nonactive isoforms in lysates of the treated PC12 cells.

Extended rat liver slice survival and stability monitored using clinical biomarkers.

Precision-cut liver slices are reportedly limited as toxicity models by their short half-life in culture. We used traditional clinical chemistry biomarkers and histology to assess a newer procedure for improved liver slice maintenance. Slices from Sprague-Dawley rat livers were well maintained in a roller culture system for up to 10 days based on protein content (60-70% or higher of initial values) and biomarker retention and verified by histological examination of the tissues showing morphological integrity and viability of hepatocyte and biliary regions.