Precision Cut Lung Slice (PCLS) Resource Hub

1. Background and Significance

Precision Cut Lung Slices (PCLS) have emerged as a valuable ex vivo model for studying lung biology and disease due to their capacity to retain the native tissue architecture, cellular composition, and microenvironment of the lung. PCLS provides a middle ground between in vitro cell cultures and in vivo models, offering a platform for controlled experimental conditions while preserving the complexity of the lung tissue. This model has proven particularly useful for studying respiratory diseases, pharmacology, toxicology, and infectious disease dynamics, as well as for investigating physiological processes such as inflammation, immune response, and tissue remodeling.

The importance of PCLS as a research tool is further underscored by the growing need to develop alternatives to animal testing. The demand for reduction, refinement, and replacement (3Rs) of animal use in scientific research is increasingly addressed through PCLS, which allows repeated studies on the same lung sample, effectively minimizing animal usage. Additionally, PCLS provides insights into human lung physiology by allowing direct experimentation on human-derived tissue, making it a powerful tool for translational and personalized research.

2. Principles of PCLS

The principle behind PCLS is to obtain thin, uniform slices of lung tissue that maintain viability and functionality over a period of time in a controlled laboratory environment. By preserving the complex cellular architecture and natural interactions within the lung tissue, PCLS allows researchers to study cellular responses, structural changes, and tissue-specific reactions in a way that closely resembles in vivo conditions.

To achieve this, lung tissue is first prepared in a way that maintains its integrity. The lungs are usually inflated to ensure that the alveolar structure is fully expanded, which is critical for accurately replicating the lung’s natural state. The tissue is then embedded in a medium, often agarose, which provides structural support to maintain the shape and architecture during slicing. Precision-cutting instruments, such as vibrating microtomes, are used to obtain thin slices, typically ranging between 100 to 500 microns in thickness. This uniformity in slice thickness ensures consistent exposure of the cells to media or test compounds, enabling reproducibility across experiments.

3. Preparing PCLS

The preparation of PCLS involves several critical steps to ensure that the slices are viable, structurally intact, and functionally representative of the native lung.

• Harvesting: For animal-derived PCLS, lungs are typically harvested from euthanized animals, usually rodents, under aseptic conditions. Human PCLS can be derived from surgical biopsies, explants, or post-mortem specimens with consent.
• Inflation: After excision, the lungs are inflated with a physiological saline solution or culture medium, often mixed with a supportive substance such as low-melting-point agarose. Inflation stabilizes the alveolar spaces and bronchial structures, ensuring that the slices reflect the lung’s functional state.
• Embedding: Once inflated, the lung tissue is embedded in an agarose matrix to further stabilize the tissue during slicing. The lung-agarose block is cooled to solidify the matrix, creating a semi-rigid structure.
• Sectioning: Precision cutting instruments, like a vibrating microtome or tissue slicer, are used to cut thin, uniform slices from the embedded lung tissue. The thickness of the slices is carefully controlled, as it impacts tissue viability, diffusion of oxygen and nutrients, and exposure to test agents.
• Culture Medium: After slicing, PCLS is cultured in media that provides nutrients and mimics the lung’s environment. Common culture mediums are supplemented with essential nutrients, antibiotics, and, if necessary, specific growth factors or signaling molecules to maintain tissue viability.
• Oxygenation and Temperature Control: Proper oxygenation and temperature control are critical, as lung tissue is sensitive to hypoxia and temperature fluctuations. A typical culture condition involves maintaining the slices in an incubator set at 37°C with 5% CO₂.
• Viability Testing: The metabolic activity and integrity of the lung slices are often evaluated using assays such as the MTT or LDH assays. These tests assess mitochondrial activity and membrane integrity, respectively, indicating the viability of the cells within the slices.
• Functional Assays: Functional assays, including cytokine production, contractility measurements, and imaging of immune cell responses, are employed to verify that PCLS retains expected physiological responses over the culture period.

Here are two excellent recent review articles here to get you started:

• Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery. (2024). Koziol-White, C., et al. Respiratory Research, 25(231)
• Perspectives on precision cut lung slices—powerful tools for investigation of mechanisms and therapeutic targets in lung diseases. (2023). Lam, M., et al. Frontiers in Pharmacology, 14: 1162889