Precision cut lung slices #2: Preparing and maintaining PCLS

The concept of using tissue slices to study organ metabolism and disease emerged in the 1920s, marking a significant milestone in the field of biomedical research. Researchers recognized the immense value of preserving the structural integrity and functional properties of tissues by carefully slicing them into thin sections. This breakthrough technique allowed investigators to study specific organs, such as the liver, kidney, and lung, ex vivo, enabling the assessment of metabolic processes, drug metabolism, and the effects of diseases on tissue function. Over the years, tissue slice preparations have continued to evolve, paving the way for advancements in understanding organ physiology, drug discovery, and personalized medicine. In this article, we will explore the historical foundations, methodologies, and the remarkable contributions of tissue slice techniques in unraveling the complexities of organ metabolism and disease.

Making precision cut lung slices (PCLS)

Creating precision-cut lung slices (PCLS) is a meticulous process that involves several steps to ensure the preservation of tissue architecture and functionality. The following is a detailed overview of how PCLS are made:

1. Tissue Harvesting: The process begins with the careful extraction of lung tissue from an appropriate source, such as human lung biopsies or animal models. Special care is taken to minimize ischemia time, ensuring the tissue’s viability.
2. Tissue Preparation: Once the lung tissue is obtained, it is rinsed with a physiological buffer solution to remove blood and debris. This step helps maintain tissue integrity and minimizes potential contamination.
3. Slicing: The lung tissue is then sliced into thin sections using a precision-cutting instrument, typically a vibratome or a microtome. These instruments provide the necessary control and precision to generate slices with consistent thickness, usually ranging from 150 to 400 micrometers.
4. Incubation: After slicing, the lung slices are carefully transferred into an incubation chamber containing a suitable culture medium. This medium maintains the viability and metabolic activity of the slices while providing necessary nutrients and oxygenation.
5. Tissue Stabilization: The slices are allowed to equilibrate in the incubation chamber under controlled temperature and humidity conditions. This step allows the tissue to stabilize and recover from any potential stress caused during the slicing process.
6. Experimental Manipulation: Once the PCLS have stabilized, they can be subjected to various experimental manipulations based on the research objectives. This may involve exposure to specific substances, such as drugs, toxins, or pathogens, to study their effects on the lung tissue. PCLS can also be used to investigate physiological processes, such as inflammation, tissue remodeling, or gas exchange.

Throughout the entire process, maintaining the viability and physiological relevance of the PCLS is crucial. The use of appropriate culture media, temperature control, and careful handling helps ensure the slices retain their native characteristics and functional properties, allowing for accurate representation of the lung microenvironment.

Importance of agarose infusion & embedding for PCLS

In the process of creating precision-cut lung slices (PCLS), agarose infusion and agarose embedding play a crucial role in obtaining healthy and viable slices that closely resemble the native lung tissue. Here is a detailed explanation of agarose infusion and its importance in PCLS preparation:

Agarose infusion involves impregnating the lung tissue with a low-melting-point agarose solution before slicing. This process serves multiple purposes. Firstly, it provides structural support and stability to the delicate lung tissue during the slicing procedure, minimizing damage and maintaining the integrity of the slices. The infused agarose acts as a solid matrix, preventing tissue deformation or distortion that may occur during slicing.

Secondly, agarose infusion aids in obtaining consistent and reproducible slice thickness. The agarose solution serves as a guide during the slicing process, ensuring precise and uniform thickness across the entire slice. This uniformity is crucial for accurate data analysis and comparison between experiments.

Agarose embedding further enhances the stability and viability of the PCLS. After slicing, the individual lung slices are carefully transferred onto a supporting matrix, such as a porous membrane or filter paper, and embedded in a solidified agarose gel. This embedding step provides mechanical support and prevents the slices from moving or detaching during subsequent handling or experimental manipulations.

The agarose gel surrounding the PCLS creates a microenvironment that closely mimics the physiological conditions of the lung tissue. It helps maintain proper hydration, oxygenation, and nutrient supply to the slices, promoting their viability and metabolic activity. Additionally, the agarose gel acts as a barrier, protecting the slices from potential shear forces and maintaining their structural integrity throughout the experimental procedures.

The use of agarose infusion and agarose embedding is essential for obtaining healthy and functional PCLS. It ensures that the slices maintain their native architecture, cellular interactions, and physiological functions, making them a representative ex vivo model for studying respiratory diseases and conducting various experimental assays.

Using PCLS for further experiments

The survival time of precision-cut lung slices (PCLS) can vary depending on several factors, including the species from which the lung tissue is derived, the specific experimental conditions, and the preservation techniques employed. Generally, PCLS can remain viable and functional for a limited period, typically ranging from a few hours to a few days.

Several factors influence the viability and longevity of PCLS. The oxygen and nutrient supply provided by the culture medium, the maintenance of proper temperature and humidity, and the prevention of microbial contamination are critical for sustaining the viability of the slices. Additionally, the age and health of the tissue source, as well as the use of appropriate preservation techniques, can impact the survival time of PCLS.

To extend the survival time of PCLS, researchers often employ specialized culture media supplemented with nutrients, growth factors, and antibiotics to support cellular viability and function. Maintaining the slices in controlled incubation conditions, including appropriate temperature and gas exchange, also contributes to their longevity.

It is important to note that while PCLS can retain viability for a limited period, their functional properties and responses to experimental manipulations may gradually decline over time. Hence, researchers often perform time-dependent analyses or plan experiments within the optimal survival window of the slices to ensure accurate and reliable results.

Ultimately, the survival time of PCLS is influenced by multiple factors, and careful attention to experimental conditions and preservation techniques can help maximize their viability and functionality. Researchers must consider these factors to plan their experiments effectively and obtain meaningful insights into respiratory diseases and related research areas.

Next week: PCLS in specific diseases

In our forthcoming article next week, we will delve into the fascinating utilization of precision-cut lung slices (PCLS) as a powerful tool in studying specific respiratory diseases. We will explore how PCLS have contributed to advancements in understanding the pathogenesis, mechanisms, and potential therapeutic interventions for these respiratory conditions, providing valuable insights for researchers and clinicians alike.