Introduction to Precision Cut Tissue Slices (PCTS):

Introduction to Precision Cut Tissue Slices (PCTS)

Precision cut tissue slices (PCTS) are viable explants of tissue that are prepared with a well-defined thickness and diameter and can be cultured ex vivo. They retain the structural and functional heterogeneity of tissues, including cell-matrix interactions and intercellular interactions. PCTS can be prepared from animal and human tissue, allowing for interspecies comparisons regarding biological effects.ref.11.2 ref.12.15 ref.11.3 They can be quickly prepared with specialized equipment that maintains tissue viability during the slicing process. Multiple experiments can be performed per animal as multiple PCTS can be prepared from a single piece of tissue. PCTS can be incubated for up to 48-120 hours, depending on the tissue type and species, making them suitable for studying endogenous metabolism, drug effects, and disease mechanisms.ref.43.12 ref.43.12 ref.43.12

Preparation and Culture of PCTS

The preparation of PCTS involves slicing the tissue into thin sections using specialized equipment. For example, precision-cut lung slices (PCLS) can be prepared by filling the lung lobes with low-melting point agarose and then slicing the lung tissue into thin slices. The slices are then incubated in a suitable culture medium to maintain their viability.ref.60.3 ref.29.30 ref.60.17 PCLS can be cultured for up to 8 days and can be used for various applications, including imaging, RNA isolation, flow cytometry, and exposure to viruses.ref.60.17 ref.9.10 ref.60.17

Viability and Integrity of PCTS

It is worth noting that PCTS require careful handling and maintenance to ensure their viability and integrity. Factors such as atmospheric oxygen levels, culture medium composition, and support materials are important for maintaining tissue morphology and function.ref.11.2 ref.11.2

Retention of Structural and Functional Heterogeneity

The slicing process of Precision Cut Tissue Slices (PCTS) ensures the retention of structural and functional heterogeneity of tissues by preserving the cell-matrix interactions and intercellular interactions. PCTS maintain the three-dimensional architecture and full heterogeneity of the tumor, including the presence of immune, vascular, and mesenchymal cells. The slices are prepared from viable explants of tissue and can be cultured ex vivo.ref.11.2 ref.11.3 ref.12.15 The slices capture the complexity and diversity of cell types found in the tissue, allowing for the study of organ-specific features of fibrogenesis and the evaluation of drug metabolism and toxicology. The slicing process itself has minimal stress on the tissue and does not compromise viability or epithelial integrity. The slices can be cultured for several days, and the medium-exposed section of each slice maintains viability.ref.51.12 ref.21.13 ref.12.15 The use of PCTS as an in vitro model for studying innate immunity to viral infections has been demonstrated, with the slices remaining viable for up to 8 days in culture. siRNA-mediated RNA interference can also be achieved in PCTS, with significant mRNA silencing observed without compromising slice viability and morphology.ref.12.15 ref.11.3 ref.51.12

Advantages of PCTS in Studying Organ-Specific Diseases

Precision-cut tissue slices (PCTS) are ex vivo models that mimic the structural and functional characteristics of various organs, including the lung, liver, and kidney. PCTS offer several advantages over conventional in vitro models, such as maintaining the cellular architecture and heterogeneity of tissues, including cell-matrix interactions and intercellular interactions.ref.11.2 ref.43.12 ref.60.3

In the study of liver diseases, PCTS have been used to assess hepatobiliary toxicity and the effects of drug candidates on liver tissue. PCTS maintain the relative abundance of each cell type in the liver, making them an excellent model for evaluating drug-induced hepatobiliary toxicity. PCTS can also be used to compare compound effects across multiple organs and species, allowing for the evaluation of target organ toxicity and species-specific toxicity.ref.43.12 ref.43.12 ref.43.12

In the study of lung diseases, PCTS, specifically precision-cut lung slices (PCLS), have been widely used to visualize interactions between lung structure and function. PCLS retain the presence, differentiation state, and localization of the more than 40 cell types found in the lung, including the right localization and patterning of extracellular matrix elements. PCLS have been used to study smooth muscle physiology, airway pharmacology, toxicology, and infection responses.ref.29.12 ref.60.3 ref.29.30

In the study of kidney diseases, precision-cut kidney slices (PCKS) have been used to study renal fibrosis and the efficacy of antifibrotic drugs. PCKS have been used to investigate the involvement of the TGFβ pathway in fibrogenesis and to test the effects of antifibrotic compounds.ref.36.3 ref.35.3 ref.35.5

Overall, PCTS, including PCLS and PCKS, provide valuable ex vivo models for studying liver, lung, and kidney diseases. They allow for the assessment of tissue-specific responses, the evaluation of drug toxicity and efficacy, and the investigation of disease mechanisms.ref.21.13 ref.43.12 ref.11.2

Applications of PCTS in Lung Diseases

Precision Cut Tissue Slices (PCTS) have been used to study various aspects of lung diseases, including smooth muscle physiology, airway pharmacology, toxicology, and infection responses. PCTS provide a model that closely resembles the physiological situation found in lung tissue, including the localization and patterning of extracellular matrix elements. They have been used to visualize interactions between lung structure and function, and to study the cells of the lower respiratory tract.ref.29.30 ref.60.3 ref.60.3 PCTS have been utilized in toxicology studies to assess the safety and efficacy of therapeutic targets for asthma treatment. They have also been used to evaluate allergic responses, vascular responses, early fibrosis, chronic obstructive pulmonary disease (COPD), and alveologenesis. Additionally, PCTS have been used as a model to study respiratory infections, such as infections with adenoviruses and influenza virus.ref.60.3 ref.43.12 ref.11.2 PCTS offer the advantage of retaining the complex architecture of the lung, allowing for the study of cell-matrix interactions and intercellular interactions. They can be prepared from various mammalian species, including humans. PCTS have been used to assess hepatic drug metabolism and toxicology, and to test pharmacological agents in liver diseases.ref.43.12 ref.43.12 ref.11.2 However, it should be noted that PCTS have some limitations, such as large wounded surfaces and the absence of ventilation and perfusion, which may affect their applicability in certain experimental settings. Overall, PCTS provide a valuable ex vivo model for studying lung diseases and have been used in a wide range of research applications.ref.11.2 ref.34.30 ref.34.30

Challenges and Limitations of PCTS

The challenges and limitations of using Precision Cut Tissue Slices (PCTS) include the decrease in viability after a certain period of incubation and changes observed on the protein expression level. PCTS have been found to decrease in viability after 48 hours of incubation. Additionally, changes in protein expression have been observed during the incubation period, such as the expression of HIF-1α.ref.15.31 ref.43.12 ref.11.22 These limitations can impact the reliability and applicability of PCTS in research and drug development as they may affect the accuracy and consistency of experimental results. It is important to consider these limitations when using PCTS as a model system and to interpret the results accordingly.ref.15.31 ref.43.12 ref.43.12

Specialized Equipment for PCTS Preparation and Maintenance

The specialized equipment used for preparing Precision Cut Tissue Slices (PCTS) includes a tissue slicer, such as the Krumdieck tissue slicer or the Leica VT 1000S tissue slicer. These tissue slicers are used to obtain slices of tissue with a specific thickness and diameter. The slices are typically around 250-300 μm thick. The tissue slicer is used in combination with a core holder to hold the tissue in place during slicing.

In addition to the tissue slicer, other equipment and materials are required for the preparation and maintenance of PCTS. These include low-melting point agarose for filling the lung lobes or other organs to be sliced, ice-cold solutions such as Krebs-Henseleit buffer or University of Wisconsin organ preservation solution for preserving the tissue before slicing, and culture medium such as Williams' medium E with GlutaMAX or serum-free minimal essential medium for incubating the slices.ref.13.7 ref.55.8 ref.29.38

During the slicing process, it is important to minimize tissue stress and maintain tissue viability. Mechanical slicing using the tissue slicer has been shown to induce minimal stress. To maintain tissue viability, the slices require organotypic support materials and atmospheric oxygen.ref.47.10 ref.46.3 ref.42.8 Atmospheric oxygen levels and filter supports are necessary for retaining tissue morphology in the slices. The slices are typically incubated in culture medium at 37 °C in an 80% O2/5% CO2 atmosphere.ref.42.31 ref.42.8 ref.46.3

Overall, the specialized equipment used for preparing PCTS includes a tissue slicer, core holder, low-melting point agarose, ice-cold solutions, culture medium, and organotypic support materials. These equipment and materials are essential for maintaining tissue viability and integrity during the slicing process and subsequent incubation of the slices.

In conclusion, Precision Cut Tissue Slices (PCTS) are valuable ex vivo models that retain the structural and functional heterogeneity of tissues, including cell-matrix interactions and intercellular interactions. They provide a platform for studying organ-specific diseases, such as liver, lung, and kidney diseases, and offer advantages over conventional in vitro models. PCTS can be prepared from various mammalian species, including humans, and can be used for multiple experiments per animal.ref.11.2 ref.43.12 ref.12.15 However, PCTS have limitations in terms of viability and protein expression changes over time. Specialized equipment and materials are required for the preparation and maintenance of PCTS. Overall, PCTS are a powerful tool for studying tissue-specific responses, evaluating drug toxicity and efficacy, and investigating disease mechanisms.ref.43.12 ref.43.12 ref.11.2

Application of PCTS in Disease Research:

The Versatility of PCTS in Disease Research

Precision-Cut Tissue Slices (PCTS) have proven to be a valuable tool in studying various diseases beyond fibrosis. Examples include intestinal fibrosis, liver fibrosis, renal fibrosis, and the development of fibrosis in the kidney (ref.20, ref.21, ref.22, ref.23, ref.24). PCTS have been utilized to investigate the mechanisms of fibrosis and evaluate the efficacy of antifibrotic drugs.ref.13.6 ref.35.3 ref.36.3 They have also been used to explore the shared and organ-specific features of fibrogenesis in different organs. These studies have shed light on the regulation of extracellular matrix (ECM) production, the involvement of signaling pathways such as TGFβ, and the effects of drug candidates on fibrosis-related gene expression.ref.13.3 ref.13.3 ref.13.6

One of the major advantages of using PCTS is the preservation of the 3D structure and cellular organization of tissues. This allows researchers to study the effects of drugs during the early onset and end-stage fibrosis. It also enables the detection of adverse events and the conduct of pharmacokinetic studies.ref.15.30 ref.15.31 ref.36.5 PCTS have been particularly useful in preclinical studies, providing insights into disease mechanisms and the development of new therapies.ref.15.31 ref.15.30 ref.43.12

For instance, PCTS have been used to study the mechanisms of intestinal fibrosis, liver fibrosis, renal fibrosis, and the efficacy of antifibrotic drugs. In the case of intestinal fibrosis, PCTS have helped elucidate the involvement of the TGFβ pathway and the impact of compounds on ATP levels, gene expression, and morphology. In liver fibrosis research, PCTS have been used to assess hepatobiliary toxicity and drug-induced toxicity.ref.13.6 ref.15.30 ref.15.30 Furthermore, PCTS have been employed to evaluate the impact of compounds on ECM regulation and the TGFβ pathway in organs such as the intestine, liver, and kidney. These studies have provided valuable insights into the development of fibrosis and have allowed for the screening of potential anti-fibrotic treatments, including pirfenidone, galunisertib, and imatinib.ref.13.6 ref.36.5 ref.35.5

Additionally, PCTS have been used as a model to study respiratory infections, such as adenoviruses, influenza virus, rhinovirus, and respiratory syncytial virus. The versatility of PCTS extends to their ability to assess the effects of siRNA on gene expression in animal and human tissue, allowing for interspecies comparisons. Overall, these examples demonstrate the wide range of applications for PCTS in disease research, encompassing the study of various diseases, drug effects, gene expression, and tissue viability.ref.11.4 ref.11.3 ref.11.5

Limitations and Challenges of PCTS in Disease Research

While PCTS offer numerous advantages, there are also limitations and challenges associated with their use in disease research. One limitation is the short-term viability of PCTS. After 48 hours of incubation, there is a decrease in viability, which limits the duration of experiments.ref.15.31 ref.15.31 ref.15.31 This constraint hinders the ability to conduct long-term studies using PCTS alone.ref.15.31 ref.15.31 ref.15.31

Another limitation of PCTS is their focus on observing major effects on the gene expression level. While PCTS provide valuable insights into gene expression changes, it is important to note that changes at the gene expression level do not always directly correlate with changes at the protein expression level or morphological changes. Therefore, validation of the findings from PCTS with in vivo data is crucial to ensure their relevance and reliability.ref.15.31 ref.11.3 ref.15.31

Additionally, PCTS lack blood-derived immune cells, which are essential components of the immune response and crucial for studying interactions with the immune system. The absence of these immune cells in PCTS limits the ability to fully investigate immune responses and immune-related mechanisms in disease research.

Despite these limitations, the advantages of using PCTS outweigh the disadvantages. The preservation of tissue architecture and cellular organization, reproducibility, and the ability to test different drug concentrations in the same tissue make PCTS a valuable tool for preclinical studies of fibrosis and organ toxicity. However, researchers may need to consider using a combination of different models and approaches to fully study immune responses and interactions with the immune system in disease research.ref.15.31 ref.15.30 ref.43.12 One approach is the co-culture of PCTS with peripheral blood mononuclear cells, which introduces immune cells into the system and allows for the study of immune responses. Additionally, other in vitro models, such as synthetic network assays, can be utilized to study immune responses and interactions with the immune system.ref.34.30 ref.15.31 ref.43.12

To address the short-term viability limitation of PCTS, alternative methods or techniques can be employed. One approach is to combine several in vitro systems, including slices, to extend the viable incubation time. Another approach is the use of cryopreservation techniques to preserve the slices and recover them with a high degree of viability.ref.43.12 ref.43.12 ref.11.2 These methods can help overcome the short-term viability limitation of PCTS and enable long-term studies in disease research.ref.43.12 ref.43.12 ref.11.2

Conclusion

Precision-Cut Tissue Slices (PCTS) have proven to be a versatile tool in disease research, extending beyond the study of fibrosis to various diseases such as intestinal fibrosis, liver fibrosis, and renal fibrosis. PCTS have allowed for the investigation of disease mechanisms, evaluation of drug efficacy, and exploration of shared and organ-specific features of fibrogenesis. The preservation of tissue architecture and cellular organization, reproducibility, and the ability to test different drug concentrations in the same tissue make PCTS a valuable tool for preclinical studies.ref.13.6 ref.15.30 ref.35.3

However, PCTS do have limitations, including short-term viability, a focus on gene expression changes, and the lack of blood-derived immune cells. These limitations can be addressed by combining different models and approaches, such as co-culturing PCTS with immune cells or utilizing other in vitro models. Moreover, alternative methods such as cryopreservation can be employed to extend the viable incubation time of PCTS.ref.15.31 ref.11.2 ref.43.12

In conclusion, despite the challenges and limitations, PCTS offer valuable insights into disease mechanisms, drug effects, and tissue viability. Ongoing validation and comparison with in vivo data will further establish PCTS as a valuable tool in disease research, contributing to our understanding of disease progression and the development of new therapies.ref.15.31 ref.11.2 ref.43.12

Role of PCTS in Drug Discovery and Development:

Precision-cut tissue slices (PCTS) in the study of fibrosis and antifibrotic drugs

Precision-cut tissue slices (PCTS) have emerged as a valuable tool for studying the mechanisms of fibrosis in various organs, including the intestine, liver, and kidney. They provide a more physiologically relevant model compared to isolated cell culture by retaining the native three-dimensional architecture of the organs and preserving cell-cell signaling pathways that are lost in traditional cell culture systems. PCTS have been particularly useful in investigating the efficacy of potential antifibrotic drugs.ref.13.6 ref.35.42 ref.36.5

One of the key applications of PCTS in the study of fibrosis is evaluating the impact of potential antifibrotic drugs on gene expression levels. For example, the downregulation of fibrosis markers, such as COL1A1, ACTA2, SERPINH1, FN1, and PLOD2, can be assessed through gene expression analysis. PCTS allow for the testing of different drug concentrations in the same tissue under the same conditions, providing a reliable means of evaluating the dose-dependent effects of antifibrotic compounds.ref.13.6 ref.13.19 ref.15.30 Moreover, PCTS enable the exploration of drug effects during early onset and end-stage fibrosis, further enhancing their utility in preclinical studies.ref.15.30 ref.15.31 ref.13.6

In addition to gene expression analysis, PCTS can also be used to evaluate the antifibrotic effect of drugs through the measurement of collagen production and the expression of fibrosis-related genes. By quantifying the reduction in collagen production and the downregulation of fibrosis-related genes, researchers can assess the effectiveness of potential antifibrotic treatments. PCTS have been particularly useful in studying fibrosis in the kidney, where they accurately reflect the renal architecture and can be prepared directly from human tissues, minimizing cross-species heterogeneity in tissue responses to injury and drug intervention.ref.13.6 ref.13.19 ref.15.30

Despite the advantages of PCTS in studying fibrosis and assessing the efficacy of antifibrotic drugs, there are limitations to consider. PCTS have a relatively short-term viability, typically up to 48 hours of incubation, which may restrict the duration of experiments. Additionally, PCTS primarily focus on gene expression analysis and may not fully capture protein expression or morphological changes associated with fibrosis.ref.15.31 ref.15.30 ref.13.6 Nonetheless, the advantages of PCTS, including their preservation of tissue architecture and cell-cell signaling pathways, outweigh these limitations. Ongoing validation and comparison with in vivo data further confirm the value of PCTS in preclinical studies of fibrosis and organ toxicity.ref.15.31 ref.15.30 ref.13.6

Precision-cut tissue slices (PCTS) for drug metabolism and pharmacokinetics

PCTS also find application in the field of drug metabolism and pharmacokinetics. In vitro biotransformation studies using PCTS guide researchers in selecting the appropriate animal model to validate newly developed drug candidates. PCTS offer advantages over conventional in vitro models by maintaining the structural and functional heterogeneity of tissues, including cell-matrix interactions and intercellular interactions.ref.43.12 ref.43.12 ref.43.12 This makes PCTS a more physiologically relevant model for studying drug metabolism.ref.43.12 ref.43.11 ref.43.12

The viability of PCTS can be assessed using ATP measurement, which provides an indication of tissue health and metabolic activity. Gene expression analysis can also be performed to evaluate fibrosis-related markers, such as COL1A1, ACTA2, SERPINH1, FN1, and PLOD2. These markers are involved in extracellular matrix regulation and TGFβ pathway signaling.ref.13.19 ref.13.6 ref.13.19 By quantifying the expression levels of these markers, researchers can gain insights into the impact of test compounds on gene expression and fibrosis-related pathways.ref.36.27 ref.35.27 ref.35.27

Liquid chromatography and mass spectrometry (LC-MS) are commonly used in in vitro biotransformation studies using PCTS to identify and characterize the metabolites formed during drug discovery and development. LC-MS allows for the separation and detection of drugs and metabolites based on their polarity and mass. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) enable faster analysis of samples, while sensitive mass spectrometers facilitate the detection of drugs and metabolites at very low quantities.ref.40.11 ref.40.11 ref.40.11

The identification and characterization of metabolites formed during in vitro biotransformation studies are crucial for understanding the fate of new chemical entities (NCEs) and optimizing their structure for pharmaceutical development. LC-MS plays a significant role in this process by identifying metabolic softspots, which involve common biotransformation pathways such as oxidation, reduction, hydroxylation, and dehydroxylation. Phase-I metabolites resulting from these biotransformation pathways can be separated and detected by LC-MS.ref.40.12 ref.40.1 ref.40.11 Additionally, LC-MS can identify phase-II metabolites, which result from conjugation reactions such as glucuronidation and sulfation.ref.40.11 ref.40.4 ref.40.8

LC-MS is also used for the quantitation of drugs and metabolites in in vitro biotransformation studies. Sample preparation methods, such as liquid-liquid extraction and solid-liquid extraction, are employed to purify samples and remove unwanted impurities. Internal standards with different retention times and molecular weights compared to the drugs and metabolites are used for accurate quantitation.ref.40.11 ref.40.12 ref.40.11 The quantitation of drugs and metabolites is essential for understanding their pharmacokinetic properties and optimizing their dosing regimens.ref.40.11 ref.40.11 ref.40.12

In summary, PCTS provide a more physiologically relevant model for studying drug metabolism and pharmacokinetics compared to traditional in vitro models. The use of ATP measurement and gene expression analysis allows for the assessment of tissue viability and the evaluation of fibrosis-related markers. LC-MS is a powerful technique employed in in vitro biotransformation studies using PCTS to identify, characterize, and quantitate drugs and metabolites.ref.15.31 ref.43.12 ref.43.12 The information obtained from these studies is crucial for understanding the fate of new chemical entities and optimizing their development for pharmaceutical use.ref.40.1 ref.40.1 ref.40.11

Interpreting in vitro toxicity data obtained through Precision Cut Tissue Slices (PCTS)

Interpreting in vitro toxicity data obtained through the use of Precision Cut Tissue Slices (PCTS) requires careful consideration and a tiered approach to data collection and interpretation. The goal is to accurately predict adverse effects in vivo based on in vitro toxicity data.ref.43.12 ref.43.11 ref.43.12

Validation of in vitro models for predicting in vivo toxicity in a prospective manner is often challenging due to time and cost constraints. Instead, retrospective studies can be conducted by testing large numbers of approved drugs in a blinded manner to reduce bias. It is crucial to link the in vitro data to in vivo parameters to predict adverse effects.ref.43.32 ref.43.2 ref.43.32 This can be achieved by establishing a clear link between the in vitro data and an in vivo parameter, such as plasma concentration, which is closely associated with toxicity. The ability of the in vitro data to estimate what will occur in vivo is essential for considering the results valuable in terms of predicting in vivo toxicity.ref.43.7 ref.43.7 ref.43.32

To effectively link in vitro toxicity data to in vivo parameters, a tiered approach to data collection and interpretation is recommended. Multiple endpoints should be analyzed, including concentration-response curves, temporal relationships, metabolic stability, and metabolic activation. By considering various endpoints, researchers can build a robust screening program that incorporates physical-chemical properties, ADME (absorption, distribution, metabolism, and excretion) parameters, potency, protein binding, and toxicity parameters.ref.43.40 ref.43.2 ref.43.36 In vitro toxicity data should not be viewed in isolation but should be combined with other key parameters and endpoints to make informed decisions about the potential adverse effects of drug candidates.ref.43.40 ref.43.40 ref.43.34

It is important to consider species-specific toxicity and potential differences in metabolism and metabolite profiles when interpreting in vitro toxicity data. In vitro systems that allow direct species comparisons, such as primary hepatocytes and liver microsomes from different species, can help address species-specific toxicity. By understanding the mechanisms underlying species-specific toxicity, researchers can make more accurate predictions about the potential effects of drugs in humans.ref.43.7 ref.16.5 ref.16.5

In conclusion, the use of PCTS and the interpretation of in vitro toxicity data are crucial for improving the selection process of new drug candidates, reducing compound risk, and increasing the probability of success in preclinical and clinical safety studies. By incorporating a tiered approach, considering multiple endpoints, and linking the in vitro data to in vivo parameters, researchers can make more informed decisions about the potential adverse effects of drug candidates.ref.43.5 ref.43.5 ref.43.36

Conclusion

Precision-cut tissue slices (PCTS) have proven to be a valuable tool in the study of fibrosis, the assessment of the efficacy of antifibrotic drugs, and the investigation of drug metabolism and pharmacokinetics. PCTS retain the native three-dimensional architecture of organs, preserve cell-cell signaling pathways, and provide a more physiologically relevant model compared to isolated cell culture. They allow for the evaluation of drug effects on gene expression levels, collagen production, and fibrosis-related markers.ref.36.5 ref.35.5 ref.15.30 PCTS can be prepared from both animal and human tissues, enabling interspecies and interorgan comparisons.ref.43.12 ref.11.2 ref.15.31

Interpreting in vitro toxicity data obtained through PCTS requires careful validation and a tiered approach to data collection and interpretation. Linking in vitro data to in vivo parameters and considering species-specific toxicity are important factors in accurately predicting adverse effects and reducing compound risk. By incorporating multiple endpoints and considering key parameters, researchers can make more informed decisions about the potential adverse effects of drug candidates.ref.43.7 ref.43.40 ref.43.36

Overall, PCTS offer a valuable tool for preclinical studies of fibrosis, organ toxicity, drug metabolism, and pharmacokinetics. Ongoing validation and comparison with in vivo data further confirm their value in scientific research and drug development.ref.15.31 ref.15.30 ref.13.6

Techniques and Tools for PCTS Analysis:

Advantages and Limitations of Imaging Techniques for PCTS Analysis

Imaging techniques offer several advantages for the analysis of Precision Cut Lung Slices (PCTS). One key advantage is the ability to capture dynamic and spatial biomechanical data, such as tissue deformation and strain profiles. These techniques allow for the visualization and quantification of changes in airway caliber and the mechanical interdependence between the constricting airway and the surrounding parenchyma.ref.32.3 ref.30.22 ref.29.30 By providing a non-invasive and non-destructive method, imaging techniques enable the study of airway reactivity and the assessment of the effects of contractile and relaxing drugs on PCTS.ref.32.26 ref.32.26 ref.32.27

However, there are limitations to using imaging techniques for PCTS analysis. One limitation is the difficulty in culturing slices for prolonged periods of time. PCTS are often cultured for several hours to assess the effects of drug treatments or to study tissue responses over time.ref.15.31 ref.29.30 ref.43.12 Maintaining the viability of the slices during prolonged culture can be challenging, and optimizing culture conditions is necessary to ensure reliable results.ref.51.12 ref.29.30 ref.43.12

Another limitation is the need for accurate segmentation of the cardiac boundaries in order to calculate structural and functional indices. Accurate segmentation is essential for obtaining reliable measurements and quantifying changes in tissue properties. The choice of segmentation technique should be based on the specific application and desired segmentation accuracy.ref.62.46 ref.62.72 ref.62.72 However, the accuracy of segmentation techniques can vary, and it is important to carefully evaluate and validate the chosen technique.ref.62.69 ref.62.70 ref.62.46

Additionally, the evaluation of segmentation techniques can be challenging due to differences in error metrics and datasets used in different studies. Comparing the performance of different segmentation techniques can be difficult when different error metrics are used or when datasets with different characteristics are employed. It is important to standardize evaluation methods and datasets to facilitate comparisons and improve the reliability of segmentation results.

Overall, imaging techniques provide valuable insights into the biomechanics of PCTS. However, careful consideration should be given to the limitations and challenges associated with these techniques, such as optimizing culture conditions and selecting appropriate segmentation methods.

Molecular and Cellular Analysis using PCTS

PCTS can be used for molecular and cellular analysis, allowing researchers to study the structural and functional heterogeneity of tissues and assess the effects of drugs and potential treatments. PCTS provide a unique platform for investigating cell-matrix interactions, intercellular interactions, and biological effects across different species.ref.11.3 ref.11.2 ref.43.12

In terms of molecular analysis, PCTS can be used for RNA interference (RNAi) studies. RNAi is a powerful tool for gene silencing, and PCTS offer a suitable model for assessing the diffusion of siRNA into slices and determining the efficacy of RNAi induction. Successful RNAi in PCTS requires the delivery of siRNA into the cytoplasm of cells.ref.11.5 ref.11.5 ref.11.17 This can be achieved using cationic nanocomplexes, such as polyplexes, lipoplexes, or lipopolyplexes, or through the use of self-deliverable siRNA. Cationic nanocomplexes facilitate the delivery of siRNA into cells by overcoming the negative charge of siRNA and promoting its internalization. On the other hand, self-deliverable siRNA, which is conjugated with cell-penetrating peptides or sterols, does not require complexation with polymers or lipids for transport into cells.ref.11.5 ref.11.5 ref.11.5 PCTS provide a valuable model for assessing the diffusion of siRNA and evaluating the success of RNAi induction.ref.11.5 ref.11.5 ref.11.24

For cellular analysis, PCTS offer a unique opportunity to study the effects of drugs and test the efficacy of potential treatments. PCTS maintain a cellular architecture similar to that in vivo, allowing for the assessment of drug-induced toxicity and the evaluation of drug effects across multiple organs and species. PCTS can be used to evaluate hepatobiliary toxicity, toxicity resulting from the activation of Kupffer cells, and species-specific toxicity.ref.43.12 ref.43.12 ref.43.12 Viability of PCTS can be assessed by measuring the adenosine triphosphate (ATP) content, which is an indicator of cellular energy metabolism and overall viability. Morphological analyses, such as histological evaluation, can also be performed to evaluate the structural integrity of the slices.ref.15.8 ref.52.7 ref.11.3

Overall, PCTS provide a valuable tool for molecular and cellular analysis, enabling the study of tissue heterogeneity, drug effects, and the diffusion and efficacy of siRNA. These techniques have the potential to improve our understanding of various diseases and develop new therapies.ref.11.5 ref.11.4 ref.11.5

Tools and Technologies for PCTS Analysis

Several tools and technologies have been developed for the analysis of PCTS to enhance their utility and facilitate experimental procedures.

One such tool is the use of cationic nanocomplexes, such as polyplexes, lipoplexes, or lipopolyplexes, for the delivery of siRNA into the cytoplasm of cells in PCTS. These nanocomplexes are utilized because siRNA is negatively charged and does not readily pass cell membranes. Cationic nanocomplexes can promote the internalization of siRNA into cells, enabling the induction of RNA interference (RNAi) in PCTS.ref.11.5 ref.11.5 ref.11.5

Another tool is the use of self-deliverable siRNA, which refers to siRNA conjugated with cell-penetrating peptides or sterols. Self-deliverable siRNA does not require complexation with polymers or lipids for transport into cells and has a smaller size, allowing for greater diffusion through biological tissues. This enables efficient delivery of siRNA into cells within PCTS and facilitates RNAi induction.ref.11.5 ref.11.5 ref.11.5

Cryopreservation techniques have also been developed for PCTS, allowing for the preservation and recovery of tissue slices with a high degree of viability. Cryopreservation involves freezing the tissue slices at very low temperatures, typically below -80°C, to preserve their cellular structure and function. The slices are treated with a cryoprotectant solution, such as dimethyl sulfoxide (DMSO) or glycerol, to prevent ice crystal formation and damage to the cells during the freezing process.ref.13.7 ref.13.7 ref.13.7 Cryopreservation enables long-term storage of tissue slices, which can be thawed and used for experiments at a later time. This increases the availability and accessibility of tissue samples for analysis.ref.13.7 ref.13.7 ref.13.7

Viability of PCTS can be assessed using various methods, such as measuring the adenosine triphosphate (ATP) content. ATP is a molecule that serves as a universal energy source in cells, and its content can be measured using a bioluminescence kit. Measurement of ATP content provides an indicator of cellular energy levels and overall viability.ref.22.10 ref.13.9 ref.52.7 Additionally, gene expression analysis can be performed using quantitative reverse transcription polymerase chain reaction (qRT-PCR), which allows for the assessment of gene expression patterns in PCTS. Furthermore, histomorphology and immunohistochemistry techniques, such as hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining, and immunohistochemistry for specific markers, can be used to evaluate the morphology and specific features of PCTS.ref.11.3 ref.52.7 ref.53.12

In summary, several tools and technologies have been developed to enhance the analysis of PCTS. These include the use of cationic nanocomplexes and self-deliverable siRNA for siRNA delivery, cryopreservation techniques for long-term storage of tissue slices, and various methods for assessing tissue viability and performing gene expression analysis.ref.11.5 ref.11.4 ref.11.5

Cryopreservation Techniques for PCTS Viability

Cryopreservation techniques play a crucial role in ensuring a high degree of tissue slice viability in PCTS analysis. These techniques allow for the long-term storage of tissue slices and increase the availability and accessibility of tissue samples for analysis.ref.43.13

Cryopreservation involves freezing the tissue slices at very low temperatures, typically below -80°C, to preserve their cellular structure and function. The slices are treated with a cryoprotectant solution, such as dimethyl sulfoxide (DMSO) or glycerol, which helps to prevent ice crystal formation and damage to the cells during the freezing process. Cryoprotectants act by reducing the freezing point of water and by protecting cellular structures from damage caused by ice crystal formation.

When the tissue slices are thawed, the cryoprotectant solution is removed, and the slices are rehydrated and cultured in a suitable medium. The rehydration process involves gradually removing the cryoprotectant solution and replacing it with a culture medium. The choice of culture medium depends on the specific application and desired experimental outcomes. Suitable media should contain nutrients and supplements necessary for maintaining tissue viability and function.

The viability of the tissue slices can be assessed using various methods. One commonly used method is measuring the adenosine triphosphate (ATP) content, which provides an indicator of cellular energy levels and overall viability. ATP is a molecule that serves as a universal energy source in cells, and its content can be measured using a bioluminescence kit.ref.22.10 ref.36.10 ref.52.7 Other methods, such as histology and immunohistochemistry, can also be used to assess tissue damage and specific markers of viability. Histological evaluation allows for the assessment of cell morphology, including parameters such as cell swelling, necrosis, and nuclear pyknosis. Immunohistochemistry techniques, on the other hand, allow for the detection and localization of specific proteins or markers within the tissue slices.ref.47.10 ref.48.10 ref.52.7

It is important to note that cryopreservation techniques may need to be optimized for different tissues and species. Each laboratory may need to develop and validate methods that are specific to the tissues they are working with. Factors such as the thickness of the tissue slice, the diffusion of nutrients, and the type of incubator used for culture can also affect the viability of the tissue slices.ref.43.13 ref.43.13 ref.43.13 Therefore, it is essential to carefully optimize and validate cryopreservation methods for specific tissues and experimental conditions to ensure reliable and reproducible results.ref.43.13 ref.43.13 ref.43.13

In summary, cryopreservation techniques are crucial for maintaining a high degree of tissue slice viability in PCTS analysis. These techniques involve freezing the tissue slices with a cryoprotectant solution, thawing them when needed for experiments, and assessing tissue viability using methods such as measuring ATP content or performing histology and immunohistochemistry. Optimization and validation of cryopreservation methods are necessary for different tissues and species to ensure reliable and reproducible results.ref.43.13

Specialized Equipment and Culture Conditions for PCTS Analysis

Specialized equipment and optimized culture conditions play a crucial role in maintaining tissue viability during the slicing process and subsequent incubation of PCTS.

One key piece of equipment used in PCTS analysis is the tissue slicer. There are different types of tissue slicers available, such as the Krumdieck tissue slicer or the Brendel/Vitron tissue slicer. These tissue slicers are designed to obtain uniform slices of tissue with a specific thickness. For example, liver tissue cores can be made using a 6mm biopsy punch, and slices with a wet weight of 4-5mg and an estimated thickness of 250-300 μm can be prepared. The tissue slicer allows for precise and consistent slicing of tissue samples, which is essential for obtaining reliable and reproducible results.

To ensure tissue viability during the slicing process and subsequent incubation, various factors need to be considered and optimized. One important factor is the use of appropriate culture medium and supplements. The culture medium provides the necessary nutrients and support for maintaining tissue viability and function.ref.47.10 ref.48.10 ref.47.10 Supplements such as insulin, transferrin, and selenium (ITS), amino acids, and vitamins can be added to the culture medium to extend tissue survival. The addition of these supplements helps to provide the necessary growth factors and cofactors required for cell growth and function.ref.47.10 ref.48.10 ref.47.10

Maintaining proper oxygen tension is another critical factor in ensuring tissue viability. The use of an appropriate oxygen tension, such as 80% O2, helps ensure proper diffusion of oxygen to the inner cell layers of the tissue slices. This is particularly important for maintaining the viability of cells in the core of the tissue slices, which may have limited access to oxygen due to diffusion limitations.ref.42.23 ref.46.3 ref.46.3

Minimizing tissue damage during the slicing process is also essential for maintaining tissue viability. Careful handling of the tissue samples and precise slicing techniques help to minimize tissue trauma and preserve the structural integrity of the tissue slices. Additionally, maintaining a sterile environment and using proper aseptic techniques during the slicing process and subsequent incubation is crucial for preventing contamination and ensuring the reliability of the results.ref.42.4 ref.42.4 ref.42.4

Viability of the tissue slices can be assessed using various methods, such as measuring the adenosine triphosphate (ATP) content, which is an indicator of cellular energy levels and overall viability. Other viability assessments include histological evaluation of cell morphology, including parameters such as cell swelling, necrosis, and nuclear pyknosis.

In summary, specialized equipment such as tissue slicers and optimized culture conditions play a crucial role in maintaining tissue viability during the slicing process and subsequent incubation of PCTS. The use of appropriate culture medium, supplements, and oxygen tension, along with careful tissue handling and assessment of viability, are important considerations for obtaining reliable and reproducible results in PCTS analysis.ref.35.9 ref.36.10 ref.47.10

Future Directions and Challenges in PCTS Research:

Potential Areas of PCTS Research

PCTS (Precision-cut tissue slices) have the potential to drive future advancements in various areas of research. Some of these areas include studying the effects of siRNA on gene expression, investigating fibrosis and organ toxicity, assessing drug toxicity and metabolism, understanding the impact of tumor-intrinsic phenotypic variation, modeling liver diseases, and advancing knowledge in hepatology.ref.43.11 ref.11.4 ref.11.0

1. Studying the effects of siRNA: PCTS can be used to study the effects of siRNA (small interfering RNA) on gene expression in both animal and human tissue. siRNA is a powerful tool for gene silencing and has the potential to target specific genes involved in disease processes.ref.11.5 ref.11.5 ref.11.24 By delivering siRNA to PCTS and observing the resulting changes in gene expression, researchers can gain insights into the molecular mechanisms underlying diseases and identify potential therapeutic targets.ref.11.5 ref.11.5 ref.11.18

2. Investigating fibrosis and organ toxicity: PCTS can be used to study the mechanisms of fibrosis in various organs, such as the liver, kidney, and intestine. Fibrosis is a pathological process characterized by excessive deposition of extracellular matrix components, leading to tissue scarring and dysfunction.ref.13.6 ref.15.30 ref.43.12 PCTS allow researchers to investigate the cellular and molecular changes associated with fibrosis and test the efficacy of potential antifibrotic drugs. Furthermore, PCTS can be used to assess the organ toxicity of drugs and chemicals, providing valuable information for drug development and safety evaluation.ref.13.6 ref.15.30 ref.15.31

3. Assessing drug toxicity and metabolism: PCTS provide a more holistic representation of tissue structure and function compared to conventional in vitro models. They can be used to assess the toxicity and metabolism of novel drugs, allowing researchers to evaluate the potential adverse effects of drugs on specific organs or tissues.ref.43.12 ref.43.12 ref.43.12 By studying the effects of drugs on PCTS, researchers can identify potential drug candidates with improved safety profiles and optimize drug dosing regimens.ref.43.12 ref.43.12 ref.43.12

4. Understanding the impact of tumor-intrinsic phenotypic variation: PCTS derived from patient tumors can be used to study the impact of tumor-intrinsic phenotypic variation on drug responses. Tumors are known to exhibit significant heterogeneity, both between patients and within individual tumors. By using PCTS, researchers can investigate how different tumor phenotypes respond to various therapies and validate the efficacy of combination therapies. This information can guide personalized treatment strategies and improve patient outcomes.

5. Modeling liver diseases: PCTS can be used to model liver diseases, such as fibrosis and hepatobiliary toxicity. Liver diseases pose a significant global health burden, and effective models are needed to study their underlying mechanisms and develop new therapies.ref.43.12 ref.13.6 ref.15.30 PCTS allow for the study of hepatic response to new compounds and the assessment of drug effects on liver tissue. By using PCTS, researchers can gain insights into the pathogenesis of liver diseases and identify potential therapeutic targets.ref.43.12 ref.43.12 ref.54.5

6. Advancing knowledge in hepatology: PCTS can contribute to advancing personalized medicine in hepatology. They can be combined with other techniques, such as single-cell analysis and tissue cartography, to improve our understanding of liver diseases.ref.21.23 ref.43.12 ref.43.12 By characterizing the cellular and molecular changes in liver tissue using PCTS, researchers can identify novel disease biomarkers, develop new therapies, and optimize treatment strategies for individual patients.ref.21.23 ref.43.12 ref.15.30

Challenges and Limitations in Using PCTS

While PCTS offer several advantages for research, there are also challenges and limitations that researchers in the field must address.

1. Viability: PCTS have a limited viability after 48 hours of incubation, which restricts the duration of experiments. This limited viability poses a challenge for studying long-term effects and conducting experiments that require extended incubation periods.ref.58.28 ref.58.28 ref.58.28 To overcome this limitation, researchers are exploring strategies to optimize culture conditions and develop specialized culture media that can extend the viability of PCTS.ref.58.28 ref.58.28 ref.58.28

2. Gene expression vs. protein expression: PCTS primarily show major effects on the gene expression level, while changes in protein expression or morphology are often limited.ref.13.15 ref.11.3 ref.13.15 This limitation impacts the ability to study the functional consequences of gene expression changes and understand the downstream effects on protein function. To overcome this limitation, researchers are exploring techniques such as proteomic analysis and immunohistochemistry to assess protein expression and morphology in PCTS.ref.13.15 ref.11.3 ref.13.15

3. Lack of blood-derived immune cells: PCTS do not contain blood-derived immune cells, which may limit the ability to study immune responses. Immune cells play a crucial role in the body's defense against pathogens and in the development of immune-mediated diseases. To address this limitation, researchers are exploring strategies to incorporate immune cells into PCTS to study immune responses and immune-mediated diseases more comprehensively.

4. Limited number of slices: The relatively small number of slices that can be obtained from a single animal and tissue type makes high-volume screening less practical. High-throughput screening is a valuable approach for drug discovery and toxicity testing.ref.43.12 ref.43.12 ref.43.12 To address this limitation, researchers are exploring strategies to increase the yield of PCTS from a single tissue sample, such as optimizing the slicing technique or developing tissue engineering approaches to generate larger quantities of tissue slices.ref.43.12 ref.43.12 ref.43.12

5. Cryopreservation: Cryopreservation of PCTS has been challenging, but recent studies have shown progress in developing cryopreservation techniques. Cryopreservation allows for the long-term storage and transportation of tissue slices, increasing their availability for research purposes.ref.43.12 ref.43.12 ref.11.2 Researchers are exploring cryopreservation methods for various tissues, including the liver, kidney, lung, and intestine, to improve the availability and expand the applications of PCTS.ref.43.12 ref.43.12 ref.13.7

6. Diffusion of siRNA: Delivery of siRNA into the cytoplasm of cells in PCTS is challenging due to the limited penetration of cationic nanocomplexes and the aggregation of nanocomplexes. siRNA delivery is essential for studying gene function and developing RNA interference-based therapies.ref.11.5 ref.11.5 ref.11.5 To overcome this challenge, researchers are exploring the use of self-deliverable siRNA, which refers to siRNA conjugated with cell-penetrating peptides or sterols that require no complexation with polymers or lipids to enable transport into cells.ref.11.5 ref.11.5 ref.11.5

7. Short-term incubation: PCTS have a limitation to short-term incubation, which may restrict the study of long-term effects. Many biological processes, such as tissue remodeling and disease progression, occur over extended periods.ref.15.31 ref.11.2 ref.15.31 To overcome this limitation, researchers are optimizing culture conditions and developing specialized culture media that can support the long-term viability of PCTS.ref.15.31 ref.11.2 ref.15.31

8. Variability: Variability in PCTS models can be observed, especially in diseased tissues, which may affect the reproducibility of experiments. Variability can arise from differences in tissue quality, slicing technique, and experimental conditions. To address this limitation, researchers are working towards standardizing experimental variables, such as biomechanics and circulating components, to facilitate comparison of results across different research groups.

9. Standardization: Standardization of experimental variables is needed to facilitate comparison of results across different research groups. Variability in experimental conditions can introduce confounding factors and make it difficult to compare findings from different studies. Researchers are working towards establishing standardized protocols for PCTS preparation, culture conditions, and experimental procedures to improve reproducibility and comparability of results.

10. Cryo-banks: The absence of cryo-banks for PCTS limits material exchange and standardization. Cryo-banks provide a centralized repository of well-characterized tissue samples, allowing researchers to access a wide range of samples for their experiments. Cryo-banking of PCTS would facilitate material exchange, standardization, and collaboration among researchers in the field.

In conclusion, PCTS research has the potential to drive future advancements in various areas of study. However, researchers in the field must address the challenges and limitations associated with PCTS, such as viability, gene expression vs. protein expression, lack of blood-derived immune cells, limited number of slices, cryopreservation, diffusion of siRNA, short-term incubation, variability, standardization, and the absence of cryo-banks.ref.15.31 ref.11.4 ref.11.5 By developing strategies and technologies to overcome these challenges, researchers can further advance the field of PCTS and improve our understanding of tissue biology, disease mechanisms, and drug development.ref.15.31 ref.11.4 ref.11.3

Works Cited