Precision cut lung slices

Precision cut lung slices or PCLS refer to thin sections of lung tissue that are prepared with high precision and are typically used for experimental purposes in the field of respiratory research. These slices are utilized to study various aspects of lung physiology, pathology, and pharmacology, providing researchers with a valuable tool for investigating lung diseases and testing the effects of drugs on lung tissue.

Precision cut lung slices are prepared using specialized equipment called Vibratomes, ensuring that the tissue remains viable and retains its structural and functional characteristics, making them ideal for a wide range of experimental applications.

History
The history of Precision-cut Lung Slices (PCLS) dates back to the 1920s when scientists first explored tissue slices for studying organ metabolism and toxicology. Initially, manual slicing of tissues, such as the liver, led to significant variability in thickness and limited viability.

A critical advancement occurred in the 1940s when Stadie and Riggs introduced a microtome equipped with a thin razor blade, reducing thickness variability to about 5%. These improved slices became known as precision-cut tissue slices.

Creating PCLS posed unique challenges due to the lung's intricate structure. In the 1980s, Placke and Fisher achieved a breakthrough by infusing heated liquid agarose into the airways of hamster and rat lungs, preventing airway and alveolar collapse during slicing.

Basic preparation
Creating Precision-cut Lung Slices (PCLS) is a meticulous process that involves several essential steps. The use of vibratomes is crucial in ensuring the production of precise and high-quality lung slices for research purposes.

Use of vibratomes
The basic steps involved in preparing PCLS using vibratomes include:


 * Tissue Selection
 * Start by carefully selecting lung tissue from the desired species, such as rodents or humans, ensuring the tissue is of high quality and health.


 * Tissue Embedding
 * To facilitate slicing and maintain tissue structure, the lung tissue is typically embedded in a suitable medium, such as agarose or gelatin, into the specimen holder of the vibratome.


 * Slicing Process
 * The vibratome operates by oscillating a blade vertically or horizontally at high frequencies while the tissue is submerged in a cutting solution. This mechanical oscillation creates thin and precise slices of lung tissue. Researchers can adjust cutting parameters, such as slice thickness, to meet specific experimental requirements. Typically, PCLS have thicknesses ranging from 200-500 μm.


 * Post-processing
 * Depending on the research objectives, PCLS may undergo additional steps such as washing, culturing, or treatment with substances of interest, such as drugs or stimuli.

Maintenance of PCLS
Ensuring the viability of Precision-cut Lung Slices (PCLS) during ex vivo maintenance presents several challenges. Typically, PCLS are submerged in culture medium within multi-well plates, simulating tissue culture conditions at 37 °C, 5% CO2, and 95–100% air humidity. The culture medium is refreshed daily and optimized with essential nutrients, enabling viable PCLS to be maintained for up to 14 days, a significant improvement compared to previous reports of only 3–5 days. Additionally, the inclusion of antibiotics like penicillin and streptomycin helps prevent pathogen contamination from the outset of culture.

While in culture, PCLS retain their viability, normal metabolic activity, tissue integrity, and responsiveness to stimuli such as lipopolysaccharide (LPS). However, it's important to note that extended culture periods may lead to some changes in PCLS function. For instance, although human PCLS can contract in response to methacholine, the secretion of LPS-induced TNF-α, while maintained, may diminish over time. Furthermore, long-term cultivation can result in the loss of certain cell populations, such as pneumocytes and lymphocytes, as well as the degradation of connective tissue fibers. These changes may contribute to decreased sensitivity of cultured PCLS to external stimuli.

In practice, PCLS can maintain comparable viability and tissue homeostasis for 1 to 3 days, though extended periods can be achieved with optimized culture conditions.

Experimental applications
Precision-cut lung slices find extensive use in a variety of experimental applications in the field of respiratory research. Some of the key areas where PCLS are employed include:

Asthma
In the pursuit of understanding and developing treatments for asthma, researchers have explored various models, including animal models like mice and rats, to mimic different aspects of the condition. While these animal models have contributed to our knowledge, they come with limitations, particularly in terms of translatability to humans.

To address these limitations and enhance our understanding of asthma, researchers have turned to human Precision-cut Lung Slices (PCLS) obtained from both healthy and diseased individuals as a valuable ex vivo tool. PCLS derived from healthy and asthmatic lungs exhibit altered responses to various stimuli, including bronchoconstriction and hyperresponsiveness, which closely resemble those observed in patients and various animal models.

Moreover, PCLS from individuals with asthma have been shown to display significantly increased airway inflammation and hyperresponsiveness when stimulated by factors such as rhinovirus. These PCLS also exhibit elevated gene expression related to asthma pathogenesis, including genes like Il25, Tslp, and Il13. These findings align with observations in asthmatic patients, indicating that PCLS models provide a promising platform for asthma research.

COPD (Chronic Obstructive Pulmonary Disease)
While no in vivo models fully encompass all aspects of clinical COPD pathology, certain animal models, such as those involving cigarette smoke exposure, elastase-induced emphysema, and LPS challenge, have yielded valuable insights. For instance, exposing guinea pigs or mice to cigarette smoke can reproduce key features of human COPD, including emphysema, small airway remodeling, and pulmonary hypertension. However, this model typically manifests mild emphysema and requires months to develop. In contrast, delivering elastase to the lungs of mice rapidly induces an emphysematous phenotype, allowing for controlled disease severity by adjusting elastase dose, administration route, and duration. It's worth noting that the physiological relevance of elastase and LPS models is debatable due to differences in underlying mechanisms.

The use of Precision-cut Lung Slices (PCLS) from in vivo models has proven particularly valuable in modeling COPD. For instance, PCLS obtained from smoke-exposed mice have shown elevated expression of chemokines when stimulated with viral mimics or influenza A virus. Murine PCLS have also demonstrated that Influenza A infection and cigarette smoke can impair bronchodilator responsiveness to β2-adrenoceptor agonists. Future studies employing PCLS from COPD patients hold the potential to enable both functional and phenotypic immune cell characterization, facilitating a more comprehensive understanding of molecular mechanisms underlying disease heterogeneity.

Idiopathic Pulmonary Fibrosis (IPF)
Precision-cut Lung Slices (PCLS) have proven effective in studying the early stages of lung fibrosis in IPF. When exposed to TGF-β1 and cadmium chloride, both human and rat PCLS have displayed relevant pathohistological changes commonly observed in the early phases of lung fibrosis. These changes include the upregulation of critical pro-fibrotic genes, increased thickness of alveolar septa, and abnormal activation of pulmonary cells.

Recent advancements in research have led to the establishment of an ex vivo human PCLS model specifically focused on early-stage fibrosis. This model involves exposing PCLS to a combination of pro-fibrotic growth factors and signaling molecules, including TGF-β1, TNF-α, platelet-derived growth factor-AB, and lysophosphatidic acid. This approach offers a pathway to investigate the underlying mechanisms of early IPF and assess novel therapies.

Researchers are actively evaluating novel treatments for IPF using PCLS. For example, caffeine, which inhibits TGF-β-induced increases in pro-fibrotic gene expression, has shown promise by significantly reducing fibrosis in PCLS from bleomycin-treated mice. Additionally, targeting PI3K signaling has emerged as a promising anti-fibrotic treatment strategy, as demonstrated using PCLS derived from IPF patients.

The use of PCLS in IPF research holds great potential for understanding the disease's early stages, testing innovative therapies, and uncovering novel treatment strategies.

Infection and Inflammation
Precision-cut Lung Slices (PCLS) have been instrumental in studying the body's innate responses to viral and, to a lesser extent, bacterial challenges. This system has shed light on which cells become infected within the intact lung, offering insights distinct from in vitro air-liquid interface cultures.

Studies by Goris et al. have revealed variations in the infectability of different cell types within the lung. For instance, bovine parainfluenza virus infection was observed primarily in cells beneath the lung epithelium within the PCLS system. Importantly, this suggests that the epithelium, when in its natural physiological structure, resists infection. Similar findings were reported by Kirchhoff et al. These studies emphasize the significance of studying cells within their physiological environment, considering cell associations and structural architecture. Such interactions not only affect infectability but also shape the system's response to infection.

The PCLS system serves as a valuable tool for understanding inflammatory responses. It has been employed to investigate the innate response to bacterial wall components like LPS and to conduct complex mixed infection studies involving multiple viruses or viral and bacterial co-infections. This approach enables precise analysis of immune responses to each stimulus. In simpler models, PCLS have been used to assess the impact of LPS on the innate immune response, testing the effects of various immunomodulators on innate signaling.

Furthermore, the ability to obtain slices from diseased lungs, such as those affected by conditions like COPD and asthma, provides a robust model for studying how respiratory diseases influence infectivity and host responses. This is particularly relevant for diseases like COPD and asthma, which have links to pathogen-induced exacerbations.

PCLS research in infection and inflammation enhances our understanding of immune responses, paving the way for insights into disease mechanisms and potential therapeutic strategies.

Drug Testing
Precision-cut Lung Slices (PCLS) play a crucial role in evaluating novel therapeutic targets for asthma, especially as tolerance to conventional treatments like glucocorticoids and β2-receptor agonists becomes more common. Researchers have increasingly focused on targets relevant to asthma pathogenesis, and PCLS have become a valuable tool for evaluating these targets as potential therapeutics.

For instance, studies have shown that inhibiting histone deacetylase with trichostatin A can alleviate airway constriction in human PCLS and simultaneously reduce airway hyperresponsiveness in antigen-challenged mice. Additionally, activating soluble guanylate cyclase in airway smooth muscle using riociguat and cinaciguat analogs has been found to induce bronchodilation in normal human PCLS and reverse airway hyperresponsiveness in allergic asthmatic mice, restoring normal lung function.

The use of PCLS in drug development is expanding further, with specific agonists or inhibitors targeting bitter-taste receptors, peroxisome proliferator-activated receptor (PPAR) γ, phosphoinositide-3 kinase (PI3K), BK channels, and spleen tyrosine kinase (Syk) all undergoing investigation within this context.

PCLS research contributes significantly to the development of innovative therapeutic strategies for asthma, addressing the evolving challenges of treatment resistance.

Advantages of PCLS
Precision-cut Lung Slices (PCLS) offer several distinct advantages that make them invaluable tools in respiratory research. They excel in preserving the intricate lung architecture, maintaining essential tissue structures like small airways, respiratory parenchyma, structural and immune cell populations, and connective tissue. The cellular composition within PCLS closely mirrors that of intact lungs, retaining the organization of structural and immune cells. However, it's important to note that specific cell types' distribution may vary among slices due to regional variability within the lung, especially in the presence of non-uniform disease-related changes.

In certain contexts, PCLS can be considered as "mini" lungs. While lacking a recruitable immune system, PCLS provide a unique opportunity to correlate cell-specific functions with organ physiology. They exhibit complex responses to challenges and stimuli, such as airway contraction and immune responses, shedding light on disease mechanisms and treatment evaluations.

PCLS have found applications in a wide range of respiratory research areas, including asthma, COPD, idiopathic pulmonary fibrosis, allergies, infections, and toxicology studies. Researchers have harnessed the advantages of PCLS to model and study these prominent respiratory diseases, facilitating insights and translational relevance to human disease.

Limitations of PCLS
Precision-cut Lung Slices (PCLS) provide valuable insights into lung physiology and pathology, but they do have limitations. Firstly, PCLS represent a static "snapshot" of lung tissue at the time of excision, lacking access to the dynamic, recruitable immune system present in living organisms. This limitation hinders the full understanding of immune responses within the lung.

Furthermore, lung tissue is inherently heterogeneous, with variations in epithelial integrity, immune cell populations, and responses to stimulation across different lung regions. When studying diseases, such as respiratory conditions, this heterogeneity can complicate data interpretation, requiring careful statistical analysis to account for variability between slices.

PCLS have a limited ability to fully replicate the intricate and dynamic immune responses observed in living organisms. They cannot recruit non-resident immune cells, and their viability is restricted to approximately two weeks. While they can capture initial signals induced by pathogens, they cannot fully mimic the complex immune responses seen in a living lung.

Another limitation is that PCLS are typically used as static systems and do not replicate the natural breathing motion of the lung. This is particularly relevant when studying diseases like ventilator-induced lung injury, where mechanical stress from ventilation plays a crucial role. Attempts to stretch or deform PCLS have been made to simulate mechanical dynamics, but accurately replicating these processes remains a challenge.

Administering treatments in PCLS can be challenging because the entire slice is bathed in the compound or stimulant of interest. This poses difficulties in translating findings to inhaled or systemic applications in vivo, making dosing and translation

Administering treatments in PCLS can be challenging because the entire slice is bathed in the compound or stimulant of interest. This poses difficulties in translating findings to inhaled or systemic applications in vivo, making dosing and translation complex.