In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.
Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), and interleukin 1 alpha (IL-1α) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.
Respiratory diseases such as allergic asthma, occupational asthma, chronic obstructive lung disease (COPD), emphysema, and infections of the upper and lower airways are on the rise and represent a worldwide health burden1,2. Suitable test systems are required, in order to identify some of the basic mechanisms underlying these diseases, in addition to the development and testing of appropriate substances. Basic research as well as preclinical drug development are focused on results gained in in vitro or in vivo assays. These assays, however, have their limitations3. Firstly, in vitro assays utilize human cells that were isolated and removed from adjacent tissue or organs and are thus no longer able to interact with or be protected by other cells3. Secondly, animal models are often not translatable to humans due to differences in physiology and biochemical divergences4. In order to minimize these limitations, and in the context of the 3Rs (replacement, refinement, reduction) principle5, new alternative models are constantly evolving.
Alternative human 3D tissue models, such as PCLS, are a link between human-based in vitro and complex animal in vivo models. PCLS reflect the functional heterogeneity with all relevant cell types present in the respiratory tract6. Additionally, the PCLS technique has the advantage of reproducible preparation of several thin slices of precise thickness from a single animal or human tissue donor. This allows an internal control as well as different concentrations or drugs to be tested.
Since the first introduction of agar-filled human lung slices in 1994 by Fisher et al.7 the technique for slicing and culturing of lung tissue has been substantially improved. Christian Martin et al. have improved this technique for further chemical and pharmacological applications8. Our group was introduced to this technique by Christian Martin in 2007. Since then, the application of PCLS in research has broadened from testing of functional responses, such as airway9,10 and vasoconstriction11, to immunological, pharmacological12,13, and toxicological7 testing in various species in several laboratories. For instance, Schlepütz et al.14 investigated airway responses for species differences and compared peripheral neuron activation either by electrical field stimulation (EFS) or by capsaicin in mice, rats, guinea pigs, sheep, marmosets, and humans. They found the various species to have distinct but different patterns of nerve-mediated bronchoconstriction and concluded that the commonly used laboratory animals (mice and rats) do not always reflect the human response. For lung toxicity testing and to reduce the number of animals in this context, Hess et al.15 pre-validated rat PCLS as an ex vivo alternative for inhalation toxicity studies. This multicentric pre-validation study resulted in the development of two prediction models using PCLS with promising results.
Moreover, in basic research, PCLS have been used to elucidate calcium signaling16, early allergic responses17, and viral infection responses18,19. Technical progress is ongoing and further advances are being explored. For example, the field is increasing the benefit of human tissue by the storage and reuse of frozen tissue. Rosner et al. described a technique of freezing and thawing murine PCLS that preserves the ability of the airways to contract upon stimulation: therefore, the technique prolongs the limited time window within which tissues remain viable, and so further assays can be applied over time to the same donor20. In addition to these research advances, Lauenstein et al.21 recently investigated risk assessment of various chemicals that might act as potential sensitizers for occupational asthma in human PCLS.
Occupational asthma, whose symptoms are airflow obstruction and airway hyperresponsiveness, similar to allergic asthma, is induced by exposure to high-molecular-weight (HMW)22 or low-molecular-weight (LMW) substances23 (e.g., platinum compounds) in the workplace environment. LMW agents have a high sensitization potential when forming haptens and bind to carrier proteins21. Registration of new HMW or LMW chemicals requires in vitro and in vivo risk assessment regarding their putative sensitization potential (e.g., OECD Guideline 429)24. The tests used to determine the putative sensitization potential, however, were originally not designed for risk assessment of respiratory sensitizers, but for contact sensitizers, though there seemed to be some congruence for a small subset of substances25. The work by Lauenstein et al. was designed as part of the European Union project Sens-it-iv, to develop alternative testing strategies for risk assessment of putative contact or lung sensitizers21. For this project, we focused on testing the usability of human PCLS as an alternative testing tool. Therefore, a set of immunomodulatory endpoints (e.g., viability and cytokine secretion) was selected to determine the irritant or inflammatory potential of chemicals, such as LMW platinum compounds. Lauenstein et al. found no general pattern that could be applied to all respiratory sensitizers; however, their work provided the foundation for recently published protocols26.
In summary, the protocol presented here for preparation and subsequent exposure of human PCLS provides a helpful method for evaluation of potentially lung-toxic and/or immunomodulatory substances that might be involved in the development of respiratory diseases, such as occupational asthma.
Experiments with human PCLS must be performed according to The Code of Ethics of the World Medical Association and approved by the local ethics committee (the exemplary PCLS experiments shown here were approved by the local ethics committee of the Hannover Medical School; number 2701-2015). Written informed consent to the use of human lung material is required from all donor patients or their next of kin. The results described in this manuscript were obtained with tissue slices from donors of different ages, genders, medical history, and cause of resection, though most of the tissue received was from patients suffering from lung cancer. Only tumor-free tissue was used for experiments.
1. General Preparation of Human PCLS and Subsequent Exposure to Chemicals
NOTE: Two persons are required to fill a lung. An up-to-date vaccination record for hepatitis A and B is recommended. Patients are routinely screened for HCV and HIV prior to lung transplantation. If an active infection with Mycobacterium tuberculosis is diagnosed or suspected, the lung should be rejected. Nevertheless, all fresh human lung tissue and samples derived from it must be treated as potentially infectious and corresponding protective measures must be taken (particle filter masks (FFP2), protective eyewear, gloves) to ensure occupational safety of the staff. The procedure takes 60 – 90 min.
2. Histological Analysis of PCLS
3. Preparation of Solutions for Substances
NOTE: Prepare working solutions and controls immediately before use.
Caution: Handle substances according to safety instructions or, if unknown, as potentially harmful and follow routine safety precautions.
4. Positive and Negative References for Cytotoxicity Assays
5. Measurement of Chemically-induced Cytotoxicity in Human PCLS by WST-1 Assay
NOTE: The WST-1 assay is performed in a 24-well plate with two PCLS per well. Preferably, use duplicates for each parameter and pool the results of these duplicates after measurement.
6. LDH Assay
Note: The LDH assay is performed in a 96-well plate with 100 µL culture supernatant in total after the post-incubation period.
7. Microscopic Viability Assay with Confocal Laser Scanning Microscope (cLSM)
Note: For the microscopic viability assay, two of the normal 250-µm-thick PCLS are stained in 250 µL/well of a 24-well plate. As each slice is imaged separately, no further duplicates are required.
8. Image Rendering
Note: The computer recommendation of the image rendering software must be taken into account, as this program requires the appropriate hardware.
9. Measurement of Cytokines and Chemokines in Human PCLS
Note: The cytokine and chemokine immune response can be measured extracellularly in the culture supernatant and intracellularly in tissue lysates after the incubation time. ELISA is measured in duplicates in a 96-well plate with 100 µL/well.
10. Measurement of Total Protein Content in Human PCLS
Note: The PCLS need to be lysed to measure total protein concentration. The tissue lysates from step 9.3 are used for this step. The assay is performed in a flat 96-well plate with 25 µL/well of tissue lysate, pipetted in duplicates.
With the present research method, human PCLS were prepared and exposed to five concentrations of industrial substances to assess chemically induced immunomodulatory effects in human lung material. Lung tissue was exposed under submerged conditions as permanent culture for 24 h. Toxicity was assessed by measurement of released LDH, mitochondrial activity, and microscopic viability staining. In addition, total protein content for normalization and pro-inflammatory cytokines were measured. Whenever possible, inhibitory concentration (IC) 75 values were calculated by non-linear sigmoidal curve fitting. The positive reference substances, detergent for cytotoxicity and LPS for the innate cytokine response, were used for validation of each run. The logarithmically transformed IC75 [µM] values (log IC50) were used as "irritant" concentrations for subsequent cytokine release measurements.
Figure 1 and Figure 2 show the procedure of filling human lung material and exemplary H&E staining of human PCLS. Routine hygienic procedures have to be followed to avoid adverse effects on health and ensure safety of the staff handling the human lung material. The technique requires personnel training and practice. Experienced pathologists who distinguish different regions (upper/lower lobes and subpleural versus more central) and diseased versus healthy areas are needed to assess the pathological status of the human lung material. The generated human PCLS should be used for preparation of H&E-stained thin sections, which can be re-evaluated by a pathologist. This procedure helps users gain practice in discriminating different diseased areas and locations within the lungs.
Figure 3 presents the measurement results for LDH and WST-1 activity in human PCLS after 24 h incubation with SLS. The untreated medium control without SLS, shown at 0 µg/mL SLS, is an important quality control. Values should be below 20% for LDH compared with detergent-lysed tissue and >0.7 absorbance units for the WST-1 assay. These quality controls also serve as indicators of insufficient tissue viability. Responsiveness of the human tissue towards the detergent solution, as an effective toxic substance, must be included as positive control. It should result in an increase by 300 – 500% (>2.0 absorbance units) of LDH compared with untreated tissue and in values <0.1 absorbance units for the WST-1 assay. SLS resulted in a dose-dependent reduction of cell viability as measured by an increase in LDH and a decrease of metabolic activity in the WST-1 assay at concentrations >87 µg/mL. A sigmoid concentration-response model was fitted to the data, so that it is possible to calculate IC75 or IC50 values. These values can subsequently be used to select concentrations for cytokine and chemokine levels: at highly toxic concentrations, usually no or only diminished cytokines and chemokines can be detected. Therefore, non-toxic or only slightly toxic concentrations (IC75) are recommended. It is important to note here that the expected increase in LDH activity in the cell culture supernatant is often influenced by the applied substance21. Frequently, interferences occur between the substance and enzyme activity, leading to misinterpretation of results if the LDH assay was the only cytotoxicity assay used. Thus, it is important and necessary to perform several cytotoxicity assays to obtain reliable and valid results. Moreover, it should be mentioned that sensitivity of the LDH and WST-1 assays is highly comparable.
In Figure 4, microscopic viability images of PCLS demonstrate responsiveness of the human tissue to detergent as an effective toxic substance. The results are very useful to confirm other viability data, but this requires additional equipment. Toxic effects are visually assessed by evaluation of calcein-positive tissue compared to EthD-1-positive cell nuclei. Randomly chosen segments within parenchyma generally result in comparable data, whereas airways or blood vessels should be avoided, as bigger cavities may shift the results. From our experience and with the microscopic settings described above, an average volume between 1.5 x 106 and 5 x 106 µm3 of the control tissue is measured. The values depend on lung material quality, PCLS quality, and also on the disease of the lung tissue donor. Even though the viability of human lung tissue varies among donors, the number of dead cell nuclei compared with viable tissue should not exceed 15% of the tissue control. If dead cell nuclei are predominantly present in the tissue control, however, the experiment should be abandoned.
Figure 5 displays representative data for the immunomodulatory effect of HClPt and SLS on human lung tissue. The pro-inflammatory cytokines IL-1α and TNF-α are biomarkers of inflammation, indicating the beginning of the inflammatory processes after exposure to stimulating substances. Both cytokines were measured by ELISA. Extracellular IL-1α is usually low in human PCLS, whereas intracellular IL-1α reaches levels of 1,000 pg/mL and above. A decrease in intracellular IL-1α indicates ongoing cytotoxic processes – and is mostly observed after exposure of human PCLS to chemicals. If tissue is stimulated with LPS, an activator of the innate immune system and as such the positive treatment control, then most of the induced IL-1α can only be detected intracellularly after 24 h. In contrast, TNF-α secretion induced by LPS stimulation can mainly be measured extracellularly. The use of a suitable positive control, such as LPS, demonstrates the validity of a method. With the present research method, human PCLS were exposed to three concentrations of HClPt (32, 64, and 125 µg/mL) or two concentrations of SLS (1 and 10 µg/mL) for 24 h. Cytokine secretion was determined as a sum of the extracellular and intracellular cytokines normalized to total protein concentrations as depicted in Figure 5. It is worth mentioning here that the BCA assay is generally used for the determination of total protein content, however, it also reflects substance-induced cytotoxicity. Hence, at highly toxic concentrations the total protein content cannot be used for normalization of cytokine data. Secretion of IL-1α increased significantly from 263 ± 38 pg/mg to 887 ± 216 pg/mg and for TNF-α from 263 ± 38 pg/mg to 1,160 ± 286 pg/mg (Figure 5A, B). Moreover, LPS-induced IL-1α and TNF-α secretion is presented in comparison to an unstimulated tissue control. Induction of pro-inflammatory cytokines by pathogen-associated molecular patterns, such as LPS, demonstrates the validity of a method and is highly recommended to be used when working with human PCLS to investigate immunomodulatory effects. For more details on HClPt and SLS data, please refer to the study by Lauenstein et al.21
Figure 1: Procedure for filling human lung material. The human lung is prepared immediately after resection. The lung should be stored at room temperature for consistent filling with agarose and to avoid sudden agarose polymerization during filling. The lung is positioned horizontally, with the lobes spread out for an optimal view on the main bronchus or bronchi (A & B for closeup on bronchus). The bronchi are cannulated with a flexible silicone tube and fixed with clamps (C). After the filling procedure and agarose polymerization in the tissue, trained pathologists cut the lung into slices of about 3 – 5 cm thickness (D). From these slices cylindrical tissue cores (Ø e.g., 8 mm) are prepared with a coring tool (E). These tissue cores are cut with a tissue slicer into PCLS, which are immediately transferred into medium-filled Petri dishes for incubation under normal cell culture conditions (E). After several washing steps the PCLS are transferred into cell culture plates (e.g., 24-well plate with two PCLS per well and 500 µL medium) to remove residual cell debris and released cell mediators (F). Please click here to view a larger version of this figure.
Figure 2: Determination of the health status of human PCLS by H&E staining. The PCLS in this image, prepared from a donor with a lung tumor, shows intact lung tissue with alveolar parenchyma, peripheral airways (***), and blood vessels (arrowheads) in the overview (A). At higher magnification, conducting airways with intact ciliated respiratory epithelium (arrowheads) (B), an intact alveolar structure lined with viable pneumocytes, including a capillary with numerous erythrocytes (arrowheads) (C), alveolar spaces containing alveolar macrophages (CD68 immunohistochemistry) (D), as well as blood vessels with intact endothelium (CD31 immunohistochemistry) (E) can be observed. Only tumor-free tissue was used for the PCLS, which is why no macroscopically diseased areas are visible. In contrast to PCLS prepared from donors with other lung diseases, such as emphysema or fibrosis, the alveolar structure is not disrupted and the PCLS is only slightly frayed at the outer boarder, most likely due to preparation steps. Scale bars indicate 1,000 µm (A) and 50 µm (B–E), respectively. Please click here to view a larger version of this figure.
Figure 3: Determination of cytotoxicity induced by SLS in human PCLS, measured by leakage of the enzyme LDH into culture medium (A) and loss of metabolic enzyme activity assessed with the dye WST-1 (B). Human PCLS were exposed to increasing concentrations of SLS. A sigmoid concentration-response model was subsequently fitted to the data, so that IC75 or IC50 values (inhibitory concentration at 25% and 50% reduction of cell viability) can be calculated (right figures). Data are presented as mean ± SEM, n = 3 – 5. Absorbance of LDH assay was measured at 492 nm (reference at 630 nm) and of WST-1 assay at 450 nm (reference at 630 nm). Data were adapted and modified from Lauenstein et al.21 Please click here to view a larger version of this figure.
Figure 4: Representative images of the three-dimensional microscopic staining and image rendering of human PCLS. Live tissue control and responsiveness of tissue to detergent, as an effective toxic substance, are shown after staining of human PCLS with calcein AM (yellow) and EthD-1 (red). Stacks of 30 µm were taken with a 10X objective (A) and rendered (B). The scale bar indicates 100 µm. Excitation wavelengths 488 nm and 543 nm, emission filters BP 505-550 nm and LP 560 nm. Please click here to view a larger version of this figure.
Figure 5: Ammonium hexachloroplatinate (HClPt)-induced IL-1α and TNF-α secretion in human PCLS after 24 h exposure. PCLS were exposed to three concentrations (32 µg/mL, 64 µg/mL, and 125 µg/mL) of HClPt and two different concentrations (1 µg/mL and 10 µg/mL) of SLS. Extracellular and intracellular IL-1α (A) and TNF-α (B) were determined by ELISA and normalized to the total protein content. To show the validity of the method a positive activator of the innate immune system, LPS, is shown as a reference for intracellular IL-1α (A) and for extracellular TNF-α (B) release, normalized to total protein content. Data are presented as mean ± SEM, *p <0.05 and ***p <0.001; HClPt and SLS: n = 9, IL-1αLPS: n = 5, TNF-αLPS: n = 4 in technical duplicates (Mann-Whitney test). This figure has been modified from Lauenstein et al.21 Please click here to view a larger version of this figure.
Supplementary Figure 1: Detailed computational processing for images of microscopic viability staining using rendering software. Step-by-step operating procedure for image analysis of uploaded LSM images for surface rendering of calcein-stained viable tissue (A–G, I) and spot detection of ethidium homodimer-stained cell nuclei (H, J–M). Please click here to view a larger version of this figure.
Supplementary Figure 2: Overview of computational image analysis of microscopic viability staining of human lung tissue. Viable tissue (yellow) was analyzed by surface rendering (B–I) and dead cell nuclei (red) were detected by spot detection (J–P). The snapshot mode was used to export the image (Q). Please click here to view a larger version of this figure.
The human PCLS technique is well established in our laboratory. The present paper gives a description of this technique and its use for toxicity testing of substances in lung tissue ex vivo. In general, any laboratory using this technique should seek to set up an assay related definition of quantifiable ranges, estimation of variabilities, and quality controls guaranteeing validity of the experiment. Possible standard procedures could be, for example, to repeat each endpoint, e.g., the cytotoxicity assay, in a minimum of three biological donors (individual runs) with a minimum of two to three technical replicates per sample including positive and negative references. Laboratory personnel should be trained to increase assay consistency and minimize assay variability.
Endpoints frequently used to assess immunomodulatory effects of substances on lung tissue include cytotoxicity measurements using different assays (e.g., LDH assay, WST-1 assay, microscopic staining assay), cytokine release assays, as well as changes in expression profile and characterization of changes in cellular populations by immunohistopathological methods6. Furthermore, there are techniques describing the visualization of cells, such as pulmonary dendritic cells, in murine PCLS28 that can also be transferred to human PCLS and might thus provide more detailed insight into the cellular composition before and after treatment with putative cytotoxic substances.
This basic protocol and technique for preparation of human lung tissue sections is comparable to techniques that have been well described in publications29. In short, the organ material is obtained from patients suffering from live-threatening chronic diseases such as lung cancer, who have to undergo surgery for resection or transplantation. The studies must be approved by the local ethics committee. Patients' informed written consent is required. Setting up a workflow between clinic and laboratory is a critical issue and requires communication, and definition of interfaces and infrastructure between both sites. Human lung material has to be processed directly after resection to preserve viability of the tissue. It is worth mentioning that the technique described here for human PCLS can be applied to both young and old lungs and to healthy and diseased lungs. In the US, for example, it is possible to obtain lungs from healthy organ donors who died in accidents or whose organs have been rejected for transplantation.
The first critical step in the protocol is inflation of the airways and surrounding parenchyma with agarose solution. This step is necessary to solidify the very soft tissue for the subsequent slicing procedure. The quality of human lung material, based on the disease background, is critical here. Only lobes with intact pleura can be filled. End-stage tumors near the bronchus sometimes prevent the filling process. Before inflating the human material, the temperature of the agarose solution has to be thoroughly checked. Too much blood (or other fluids, exudates) inside the human lung tissue will result in undesired dilution of agarose and will influence the polymerization process. After inflation of the lung tissue and gelling of agarose on ice, lungs are cut into sections of 200 to 300 µm thickness. Consistency of the tissue is a very critical issue. If the tissue is too soft, slicing of equal sections is difficult. Even if the same microtome parameters are set for each donor, the thickness of the slices between donors may vary, due to individual conditions and changes during the inflating process for each lung. Inhomogeneous filling of the tissue will result in different slice thicknesses. Instead of measuring and standardizing the thickness of slices, measurement of total protein content can be used to indirectly monitor lung slice thicknesses. Several end-stage diseases hamper the slicing process; e.g., blood vessels are extremely thickened in pulmonary hypertension, and fibrotic tissue can be so stiff that slicing of tissue cylinders is hardly possible and the microtome blade needs to be replaced very often.
After preparation of human PCLS and intensive washing steps, which are necessary to remove cell debris and released enzymes, tissue sections can be used for experiments29. Human PCLS are cultured under normal cell culture conditions and exposed, for example, to chemicals, drugs, or lipopolysaccharides. Occasional contamination of PCLS due to (unknown) infections is a special issue in the culture of human lung material. Tissue cultures showing infections must be discarded and the equipment must be thoroughly disinfected. Spatial separation between laboratory places used for preparation on the one hand and culturing on the other hand may help to avoid cross-infections. With regard to the equipment, the microtome may leak, and loose hex screws may lead to motor damage and stop of blade movement. Not every part of the slicer is made of stainless steel, and so it will oxidize if not dried immediately alter cleaning. To overcome equipment issues, it may be necessary to have at least one backup device.
In previous publications, agarose has been reported to be washed out and removed during intensive washing steps after preparation. In fact, this is not possible, the agarose cannot be removed. For complete removal of the agarose, it needs to be re-melted at high temperatures, which would destroy the tissue. The agarose in alveoli and airways does not interfere with the described endpoints. Other endpoints might be influenced by the presence of agarose (see also limitations). The need to prepare very fresh tissue sections has to be emphasized, as tissue viability is a critical issue in culture. Bronchoconstriction is not a valid parameter for viability. We recommend using at least two or three independent cytotoxicity assays to check viability of the surrounding parenchyma; This has to be checked in every experiment30. Quality controls in cytotoxicity assays serve as indicators of insufficient tissue viability. Therefore, it is recommended to assess the responsiveness of the tissue, for example, to an effective toxic substance such as a detergent in all cytotoxicity assays. Based on dose-response curves, minimum and maximum values of absorption need to be defined for cytotoxicity assays and met for subsequent experiments. Further modifications to the protocol mostly depend on the applied chemicals and the endpoints of interest. The applicability of insoluble or highly reactive chemicals is limited. The highest solvent concentration for DMSO is limited to 1%. Higher concentrations can be used but may result in pronounced release of pro-inflammatory cytokines, such as IL-8. On the other hand, the stimulus used might be relatively weak. In this case, the amount of tissue can be increased from two to four slices per well. This approach limits the viability to 24 h.
A major limitation of human PCLS is that in Germany they can only be prepared from diseased human lung material. Patients who undergo surgery are normally older than 50 years and 80% of patients suffering from lung cancer are or used to be smokers. Medication of patients, such as glucocorticoids, can also influence the outcome of experiments using human tissue. Therefore, it is essential to: i) validate each experiment by positive references verifying viability, functionality, and sensitivity of the individual tissue, and ii) cross-validate the results using healthy, non-diseased, middle-age lung tissue from laboratory animals (non-human primates such as cynomolgus and, if possible, mouse, rat, guinea pig). The better the pathology score of the diseased tissue, the better the experimental outcomes. Heavily diseased tissue can hardly be used and very often shows limited viability, inadequately low or extremely high cytokine levels, bacterial or fungal infections, and less bronchoconstriction. Donor-to-donor variation is higher compared with results obtained from laboratory animals, reflecting the individual variability of humans. This is, however, not a limitation in general; as mentioned above, in other countries (e.g., the U.S.) it is possible to obtain healthy lungs from deceased organ donors rejected for transplantation.Responsiveness of the tissue has been well described for the first up to 48 h in acute exposure experiments. Viability and functionality of the tissue is reduced after many days of culture or after storage at -80 °C. It is possible to culture human lung tissue for up to about 14 days. Viability continues during this time; however, an increase in variability, as well as a loss of functionality of several cell populations, such as macrophages, in the tissue is observed, resulting in limited cytokine release in response to mitogens. Another limitation for some endpoints is the presence of agarose in the tissue, hindering, for example, the isolation of high-quality and sufficient amounts of RNA31 or the preparation of single-cell suspensions for subsequent flow cytometry and phenotyping of cells. The possibility to gain mechanistic insights into single-cell responses and functionality is thus limited.
Organotypic tissue models, such as human PCLS, are considered to have a high impact on basic and non-clinical research. Human lung material has a biological composition which closely reflects the normal organ architecture. It contains, for example, residential alveolar and bronchial epithelial cells, smooth muscle cells, fibroblasts, endothelial cells, nerve fibers, and macrophages. The tissue is viable and cells respond to several stimuli. Nerve fibers, although cut, can be locally activated, leading to terminal reflex responses14. Consequently, this ex vivo model offers the possibility to study cellular innate immune responses, defense responses, cytokine signaling, and induction of cell surface markers. Several improvements in technique, culturing, and validation of endpoints allow the use of human PCLS in translational science. Examples of future approaches are: i) validation of new targets in human lung tissue, ii) assessment of immune responses after exposure, for example, to chemicals, drugs, nanoparticles, etc., iii) supplementation of lung tissue with immune cells, such as T-lymphocytes, iv) identification and modification of molecular patterns, for example, after exposure to respiratory sensitizers, disease-inducing substances, or active compounds inhibiting pathways; furthermore, v) airway remodeling and vi) neuronal regulation32. The scientific field is interested in these present and future approaches with PCLS. In addition, there are a variety of different developments that will help to improve the PCLS technique, such as cryo-preservation20, and tissue stretching33 to mimic the natural movement of the tissue during breathing or mechanical ventilation.
The major advantage of human PCLS compared with other 3D models is the presence of immune cells and nerve fibers. Experiments can also be performed in mouse, rat, and non-human primates, which are the animal species that are still used most often in pharmacology and toxicology. The complexity of human lung tissue supports the translation of results from animal to human and from in vitro à in vivo. In the context of existing alternative assays for the identification of respiratory sensitizers, human PCLS are very complex and do not allow insights into single cell responses. Yet, microscopy and flow cytometry might give information about cellular responses, if the right cellular marker is used in combination, for example, with apoptosis, necrosis, or intracellular markers. There are published assays which have been validated and reported to have been used for the identification of respiratory sensitizers. However, the advances in the use of PCLS with all their advantages over single-cell assays are making a valuable contribution in the sense that the technique can be used for high-throughput screening, as described by Watson et al.34 They developed a high-throughput screening assay to predict airway toxicity in murine cryo-preserved PCLS. With their miniaturized 96-well PCLS format, they detected similar readouts in murine lavage fluid, making PCLS a feasible high-throughput assay.
The authors have nothing to disclose.
We would like to thank Lan Lauenstein for her pre-work regarding alternative testing strategies for risk assessment with human PCLS. Some of the research was supported by a grant from the European Commission within the 6th framework program SENS-IT-IV “Novel Testing Strategies for In Vitro Assessment of Allergens”.
Silicon hose 3.0 x 5.0 mm | A. Hartenstein (Leipzig, Germany) | SS04 | |
Syringe | Faust Lab Science (Klettgau, Germany) | 9.410 050 | |
Coring tools | custom-made | custom-made | |
Trimming Blade Handle (FEATHER) | pfm medical ag (Cologne, Germany) | 205530001 | |
Trimming Blades (FEATHER) | pfm medical ag (Cologne, Germany) | 205500000 | |
Microtome: Tissue Slicer | Alabama R&D (Bad Homburg, Germany) | 303400-ADPT | Krumdieck Tissue Slicer (MD6000) |
Microtome blade | Wilkinson Sword (Solingen, Germany) | ENR-4027800011506 | |
Tissue culture dishes | Sigma (München, Germany) | Z666246-420EA | |
Cell strainer filter (100 µm Nylon) | Becton Dickinson (Heidelberg, Germany) | BD352360 | |
Inoculation loop | Copan Diagnostics (Murrieta, USA) | CD176S01 | |
TPP Tissue culture plates 24wells | Sigma (München, Germany) | Z707791-126EA | |
Cassette | Thermo Scientific (Schwerte, Germany) | 1000957 | |
Nunc MaxiSorp flat-bottom | Fisher Scientific GmbH (Hannover, Germany) | 44-2404-21 | |
Nunc MicroWell 96-Well | Thermo Scientific (Schwerte, Germany) | 260836 | |
Confocal Microscope | Zeiss (Jena, Germany) | Confocal LSM Meta 510 | |
Image rendering software | Bitplane AG (Zürich, Switzerland) | IMARIS 7.6 | |
LSM Image Browser | Zeiss (Jena, Germany) | ||
Multiwell-Reader | Tecan Group Ltd. (Männedorf, Switzerland) | Tecan infinite F200Pro | Plate Reader |
Plate shaker | Edmund Buehler GmbH (Hechingen, Germany) | KM-2 Akku | |
BenchMark ULTRA | Ventana Medical Systems (Tucson, USA) | Automated IHC/ISH slide staining system | |
Assays | |||
Cell Proliferation Reagent WST-1 | Roche (Basel, Switzerland) | 11644807001 | |
Cytotoxicity Detection Kit (LDH) | Roche (Basel, Switzerland) | 11644793001 | |
Microscopical vitality staining | Invitrogen (Carlsbad, USA) | L-3224 | LIVE/DEAD Viability/Cytotoxicity Kit |
BCA Protein Assay | Thermo Scientific (Schwerte, Germany) | 23225 | |
ELISA assay kit | R & D systems | various, e.g. DY210 (TNF-α) or DY200 (IL-1α) | DuoSet |
Reagents | |||
Agarose, low gelling temperature | Sigma (München, Germany) | A9414-100G | |
Balanced Salt Mixtures and Solutions, Cell Culture, Classic Media and Salts, Earle’s Balanced Salts (EBSS) | Sigma (München, Germany) | E2888-500ML | |
Penicillin and streptomycin | Lonza (Verviers, Belgium) | 17-602E | |
Dulbecco´s Modified Eagle´s Medium Nutrient Mixture F-12 Ham (DMEM F-12) | Gibco (Darmstadt, Germany) | 11039-047 | Culture medium |
Dulbecco´s Phosphate with Ca and Mg (DPBS) | Lonza (Verviers, Belgium) | BE17-513F | Buffer solution |
Albumin from bovine serum (BSA) | Sigma (München, Germany) | A9647-500G | |
Detergent | Sigma (Saint Louis, USA) | X100-100ML | Triton X-100 |
Washing buffer | Merck (Darmstadt Germany) | 524653 | 0.05% Tween 20 in PBS |