In the ever-evolving field of respiratory research, precision-cut lung slices (PCLS) have emerged as a versatile and physiologically relevant ex vivo model system. Their ability to maintain the complex multicellular architecture and functionality of the lung makes them an invaluable tool for studying respiratory diseases, pharmacological responses, and toxicological effects. This article delves into the preparation, utility, and potential of PCLS, offering insights for researchers eager to incorporate this method into their studies.
What Are Precision-Cut Lung Slices?
PCLS are thin (typically 100-400 μm) cross-sections of lung tissue that preserve the native cellular composition and architecture of the lung. They provide a 3D environment in which cells interact in a manner that closely mimics in vivo conditions. These slices can be prepared from human or animal lungs, enabling researchers to study species-specific and human-relevant respiratory mechanisms.
Why Use PCLS in Respiratory Research?
PCLS offer several advantages:
Preservation of Lung Architecture: Unlike 2D cultures, PCLS retain the spatial organization of airways, alveoli, and vasculature, enabling a more accurate study of physiological and pathological processes.
Reproducibility and Standardization: The consistent thickness and uniformity of PCLS allow for highly reproducible experimental results.
Multifunctional Applications: PCLS can be used to investigate airway reactivity, inflammation, infection, fibrosis, and even cancer progression. They also serve as a valuable tool for drug screening and toxicological assessments.
Ethical and Practical Benefits: By using tissue from a single lung to generate multiple slices, PCLS reduce the number of animals required for research and maximize the utility of human donor tissue.
Preparation of Precision-Cut Lung Slices
The preparation of high-quality PCLS requires meticulous technique and specialized equipment. Here’s a step-by-step guide:
Tissue Procurement
Freshly excised lungs are essential to preserve tissue viability. Lungs can be obtained from rodents, larger mammals, or human donors, depending on the research focus.
Inflation of the Lung
The lung is inflated with a low-melting-point agarose solution to stabilize the tissue. This ensures that the airways and alveoli maintain their native architecture during slicing.
Typically, a concentration of 2% agarose is used, and the lung is inflated via the trachea using a syringe or a pump system.
Cooling and Solidification
The inflated lung is placed on ice or refrigerated to allow the agarose to solidify. This step is critical for achieving uniform slices.
Preparation of the Lung Core
Once solidified, cylindrical cores of tissue are extracted from specific regions of the lung (e.g., proximal airways or distal alveoli) using biopsy punches or similar tools.
Precision Cutting
Lung cores are mounted onto a vibratome, a specialized cutting device that produces slices of uniform thickness (usually 200-400 μm; see 7000smz-2 or 5100mz).
Slice Recovery and Maintenance
Slices are transferred into a culture medium to recover and equilibrate. Commonly used media include DMEM or RPMI, often supplemented with antibiotics and foetal bovine serum (FBS) to maintain viability and prevent contamination.
Slices can be maintained for several days in air-liquid interface or submerged culture conditions.
Credit: Rachel Blomberg, University of Colorado | Anschutz
Campden Instruments vibratomes are widely recognized for their reliability and precision in PCLS preparation. These instruments feature advanced vibration mechanisms and customizable settings, such as amplitude and frequency, which allow for minimal tissue damage during slicing. Campden's vibratomes are designed with user-friendly interfaces and robust build quality, ensuring reproducibility and consistency across experiments.
Proper calibration of the vibratome and the use of a sharp blade are critical for producing high-quality slices that preserve the structural and cellular integrity of the lung tissue.
Applications of PCLS
PCLS have been successfully employed in a wide range of respiratory research applications:
Drug Development: Screening novel compounds for efficacy and toxicity in a physiologically relevant lung model.
Disease Modeling: Investigating the pathophysiology of diseases such as asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF).
Infection Studies: Studying host-pathogen interactions, including viral and bacterial infections, in a controlled environment.
Toxicology: Evaluating the impact of environmental pollutants, chemicals, and nanoparticles on lung health.
Mechanistic Research: Exploring signaling pathways, cellular interactions, and immune responses in the lung.
Challenges and Limitations
Despite their numerous advantages, PCLS are not without challenges:
Tissue Availability: Access to fresh human lung tissue can be limited, necessitating reliance on animal models.
Short Viability: Slices typically remain viable for only a few days to weeks, depending on experimental conditions.
Complex Preparation: The process requires expertise and specialized equipment, which may limit accessibility for some laboratories.
Species Differences: Results from animal-derived PCLS may not always translate to human physiology.
Future Perspectives
Advances in PCLS technology, such as co-culturing with immune cells or incorporating organ-on-chip systems, are likely to enhance their utility further. Innovations in imaging and molecular analysis techniques will also enable deeper insights into lung biology at the cellular and subcellular levels.
By integrating PCLS into their experimental toolkit, researchers can bridge the gap between traditional cell culture and in vivo models, paving the way for more accurate and translational discoveries in respiratory science.
References
Acharya, A. P., Tang, Y., Bertero, T., Tai, Y.-Y., Harvey, L. D., Woodcock, C.-S. C., Sun, W., Pineda, R., Mitash, N., Königshoff, M., Little, S. R., & Chan, S. Y. (2021). Simultaneous Pharmacologic Inhibition of Yes‐Associated Protein 1 and Glutaminase 1 via Inhaled Poly (Lactic‐co‐Glycolic) Acid–Encapsulated Microparticles. Am Heart Assoc, 10(12), 19091. https://doi.org/10.1161/JAHA.120.019091
Blomberg, R., Sompel, K., Hauer, C., Peña, B., Driscoll, J., Hume, P. S., Merrick, D. T., Tennis, M. A., & Magin, C. M. (2023). Tissue-engineered models of lung cancer premalignancy. BioRxiv, 2023.03.15.532835. https://doi.org/10.1101/2023.03.15.532835
Blomberg, R., Sompel, K., Hauer, C., Smith, A. J., Peña, B., Driscoll, J., Hume, P. S., Merrick, D. T., Tennis, M. A., & Magin, C. M. (2024). Hydrogel-Embedded Precision-Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo. Advanced Healthcare Materials, 13(4), 2302246. https://doi.org/10.1002/ADHM.202302246
Cheong, S. S., Luis, T. C., Hind, M., & Dean, C. H. (2024). A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS). Bio-Protocol, 14(8), e4980. https://doi.org/10.21769/BIOPROTOC.4980
Cramer, N., Nawrot, M. L., Wege, L., Dorda, M., Sommer, C., Danov, O., Wronski, S., Braun, A., Jonigk, D., Fischer, S., Munder, A., & Tümmler, B. (2022). Competitive fitness of Pseudomonas aeruginosa isolates in human and murine precision-cut lung slices. Frontiers in Cellular and Infection Microbiology, 12. https://doi.org/10.3389/FCIMB.2022.992214/FULL
Edström, D., Niroomand, A., Stenlo, M., Uvebrant, K., Bölükbas, D. A., Hirdman, G., Broberg, E., Lim, H. C., Hyllén, S., Lundgren-Åkerlund, E., Pierre, L., Olm, F., & Lindstedt, S. (2023). Integrin α10β1-selected mesenchymal stem cells reduced hypercoagulopathy in a porcine model of acute respiratory distress syndrome. Respiratory Research, 24(1). https://doi.org/10.1186/S12931-023-02459-6
Fercoq, F., Cairns, G. S., Donatis, M. de, Mackey, J. B. G., Floerchinger, A., McFarlane, A., Raffo-Iraolagoitia, X. L., Whyte, D., Arnott, L. W. G., Nixon, C., Wiesheu, R., Kilbey, A., Brown, L., Al-Khalidi, S., Norman, J. C., Roberts, E. W., Blyth, K., Coffelt, S. B., & Carlin, L. M. (2024a). Integrin conformation-dependent neutrophil slowing obstructs the capillaries of the pre-metastatic lung in a model of breast cancer. BioRxiv, 2024.03.19.585724. https://doi.org/10.1101/2024.03.19.585724
Fercoq, F., Cairns, G. S., Donatis, M. de, Mackey, J. B. G., Floerchinger, A., McFarlane, A., Raffo-Iraolagoitia, X. L., Whyte, D., Arnott, L. W. G., Nixon, C., Wiesheu, R., Kilbey, A., Brown, L., Al-Khalidi, S., Norman, J. C., Roberts, E. W., Blyth, K., Coffelt, S. B., & Carlin, L. M. (2024b). Integrin inactivation slows down neutrophils congesting the pre-metastatic lung in a model of breast cancer. BioRxiv, 2024.03.19.585724. https://doi.org/10.1101/2024.03.19.585724
Gonzales-Huerta, L., Williams, T., Aljohani, R., Robertson, B., Evans, C., & Armstrong-James, D. (2024). Precision-cut lung slices in air-liquid interface (PCLS-ALI): A novel ex-vivo model for the study of Pulmonary Aspergillosis. BioRxiv, 2024.11.15.615211. https://doi.org/10.1101/2024.11.15.615211
Gvazava, N., Konings, S. C., Cepeda-Prado, E., Skoryk, V., Umeano, C. H., Dong, J., Silva, I. A. N., Ottosson, D. R., Leigh, N. D., Wagner, D. E., & Klementieva, O. (2023). Label-Free High-Resolution Photothermal Optical Infrared Spectroscopy for Spatiotemporal Chemical Analysis in Fresh, Hydrated Living Tissues and Embryos. Journal of the American Chemical Society, 145. https://doi.org/10.1021/JACS.3C08854
Hargrave, K. E., Worrell, J. C., Pirillo, C., Brennan, E., Garriga, A. M., Gray, J. I., Purnell, T., Roberts, E. W., & MacLeod, M. K. (2023). Lung influenza virus specific memory CD4 T cell location and optimal cytokine production are dependent on interactions with lung antigen-presenting cells. BioRxiv, 2023.09.19.558387. https://doi.org/10.1101/2023.09.19.558387
Hasanaj, E., Beaulieu, D., Wang, C., Hu, Q., Bueno, M., Sembrat, J. C., Pineda, R. H., Melo-Narvaez, M. C., Cardenes, N., Yanwu, Z., Yingze, Z., Lafyatis, R., Morris, A., Mora, A., Rojas, M., Li, D., Rahman, I., Pryhuber, G. S., Lehmann, M., … Königshoff, M. (2024). SenSet, a novel human lung senescence cell gene signature, identifies cell-specific senescence mechanisms. BioRxiv, 2024.12.21.629928. https://doi.org/10.1101/2024.12.21.629928
Hauer, C., Blomberg, R., Sompel, K., Magin, C. M., Ten-Nis, M. A., & Tennis, M. A. (2024). Hydrogel-embedded precision-cut lung slices support ex vivo culture of in vivo-induced premalignant lung lesions. BioRxiv, 2024.04.29.591698. https://doi.org/10.1101/2024.04.29.591698
Jiang, M., Roth, M. G., Chun-On, P., Sullivan, D. I., & Alder, J. K. (2020). Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery. American Journal of Respiratory Cell and Molecular Biology, 62(6), 692–698. https://doi.org/10.1165/RCMB.2019-0276TR
Kim, S. Y., Mongey, R., Griffiths, M., Hind, M., & Dean, C. H. (2020). An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision-Cut Lung Slices to Understand Lung Injury and Repair. Current Protocols in Mouse Biology, 10(4). https://doi.org/10.1002/CPMO.85
Mondoñedo, J. R., Bartolák-Suki, E., Bou Jawde, S., Nelson, K., Cao, K., Sonnenberg, A., Obrochta, W. P., Imsirovic, J., Ram-Mohan, S., Krishnan, R., & Suki, B. (2020). A High-Throughput System for Cyclic Stretching of Precision-Cut Lung Slices During Acute Cigarette Smoke Extract Exposure. Frontiers in Physiology, 11. https://doi.org/10.3389/FPHYS.2020.00566/FULL
Nowakowska, J., Gvazava, N., Langwi nski, W., Ziarniak, K., Augusto da Silva, I. N., Stegmayr, J., Wagner, D. E., & Szczepankiewicz, A. (2024). Optimizing miRNA transfection for screening in precision cut lung slices. American Journal of Physiology-Lung Cellular and Molecular Physiology. https://doi.org/10.1152/AJPLUNG.00138.2024
Pirillo, C., al Khalidi, S., Sims, A., Devlin, R., Zhao, H., Pinto, R., Jasim, S., Shearer, P. A., Shergold, A. L., Donnelly, H., Bravo-Blas, A., Loney, C., Perona-Wright, G., Hutchinson, E., & Roberts, E. W. (2023). Cotransfer of antigen and contextual information harmonizes peripheral and lymph node conventional dendritic cell activation. Science Immunology, 8(85). https://doi.org/10.1126/sciimmunol.adg8249
Raffo-Iraolagoitia, X. L., McFarlane, A. J., Kruspig, B., Fercoq, F., Secklehner, J., Donatis, M. de, Mackey, J. B. G., Wiesheu, R., Laing, S., Hsieh, Y.-C., Shaw, R., Corbyn, R., Nixon, C., Miller, C., Kirschner, K., Bain, C. C., Murphy, D. J., Coffelt, S. B., & Carlin, L. M. (2023). γδ T cells impair airway macrophage differentiation in lung adenocarcinoma. BioRxiv, 2023.09.14.557344. https://doi.org/10.1101/2023.09.14.557344
Remion, E., Frohberger, S. J., Vallarino-Lhermitte, N., Hoerauf, A., le Quesne, J., dé ric Landmann, F., Hü bner, M. P., Carlin, L. M., & MartinID, C. (2019). IL-4 receptor dependent expansion of lung CD169+ macrophages in microfilaria-driven inflammation. Journals.Plos.Org, 13(8). https://doi.org/10.1371/journal.pntd.0007691
Rodriguez, A. D., Horowitz, L. F., Castro, K., Kenerson, H., Bhattacharjee, N., Gandhe, G., Raman, A., Monnat, R. J., Yeung, R., Rostomily, R. C., & Folch, A. (2020). A microfluidic platform for functional testing of cancer drugs on intact tumor slices. Lab on a Chip, 20(9), 1658–1675. https://doi.org/10.1039/C9LC00811J
Stegmayr, J., Alsafadi, H. N., Langwiński, W., Niroomand, A., Lindstedt, S., Leigh, N. D., & Wagner, D. E. (2021). Isolation of high-yield and -quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts. American Journal of Physiology - Lung Cellular and Molecular Physiology, 320(2), L232–L240. https://doi.org/10.1152/ajplung.00401.2020
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