University of Groningen
Regenerative Pharmacology for COPD
Wu, Xinhui
DOI:
10.33612/diss.157528459
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XINHUI
WU
Chapter 2
Mouse Lung Tissue Slice Culture
Xinhui Wu
1,2, Eline M. van Dijk
1,2, I. Sophie T. Bos1,2, Loes E. M.
Kistemaker
1,2, Reinoud Gosens
1,2*1Department of Molecular Pharmacology, Faculty of Science and Engineering, University of Groningen, Antonius
Deusinglaan 1, 9713AV, Groningen, Netherlands, 2Groningen Research Institute for Asthma and COPD, University
Medical Center Groningen, University of Groningen, Groningen, Netherlands, * Correspondence
(2019) Mouse Lung Tissue Slice Culture. In: Bertoncello I. (eds) Mouse Cell Culture. Methods in Molecular Biology,l 1940: 297-311.
Abstract
Precision-cut lung slices (PCLS) represent an ex vivo model widely used in visualizing interactions between lung structure and function. The major advantage of this technique is that the presence, differentiation state and localization of the more than 40 cell types that make up the lung are in accordance with the physiological situation found in lung tissue, including the right localization and patterning of extracellular matrix elements. Here we describe the methodology involved in preparing and culturing PCLS followed by detailed practical information about their possible applications.
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1 Introduction
The lung slice represents a lung tissue preparation suitable for a great variety of studies ranging from airway pharmacology to toxicology1. Lung slices are prepared
from whole lung by filling the lung lobes with a low-melting point agarose to be made suitable for slicing. After slicing, the slices can either be used directly or are cultured before experimental endpoints are determined. Whereas this paper will focus on the preparation and applications of mouse lung slices (Fig. 1), lung slices can be prepared from all mammalian species. We will first briefly review the main strengths, weaknesses and applications of the lung slices, followed by detailed practical information into the methodologies used.
Figure 1. Procedure of precision-cut lung slices (PCLS). This flowchart briefly shows the whole
procedure of precision-cut lung slices.
1.1 Main methodological strengths and weaknesses of lung slices
Probably the main strength of working with lung slices is that the presence, differentiation state and localization of the more than 40 cell types that make up the lung is in accordance with the physiological situation found in lung, including the right localization and patterning of extracellular matrix elements. Despite considerable efforts and progress in the area of 3D cell culture and 3D printing, recapitulation of the complexity of lung tissue in such models is currently not possible. This makes the slice extremely suitable for studies into the relationships among lung, airway or pulmonary vascular structure and function2. Moreover, it
smooth muscle cells in culture3 and our understanding of smooth muscle physiology
(either airway or pulmonary vascular) is already restricted in cell culture studies for that reason alone. Not surprisingly, the first studies conducted with lung slices were smooth muscle physiology studies4. In laboratories working in that area, the
slices have taken center stage, mostly due to the pioneering work of the groups of Christian Martin4 and Michael Sanderson2. Finally, a significant advantage of the
lung slice model is that multiple slices (up to around 50 in mouse) can be taken from a single animal, reducing the use of animals as an entire experiment including all treatments and controls can be done on the same animal.
However, the lung slice model has weaknesses that should be taken into account when designing studies utilizing PCLS. One of the most significant weaknesses is that it has proven extremely difficult to culture slices for prolonged periods of time. This may be due to the diversity of cell types in one lung slice, each requiring specific culturing media, making it difficult to find one medium composition that fits all. Research groups working with cultured slices report their use for several days, with increases in ATP and LDH release, and the presence of dead cells appearing around day 3 to 75,6. Application of microfluidics or specific cell culture ingredients
may help to prolong this longevity, although virtually no in-depth studies in this area have been reported.
Another significant disadvantage of the lung slice model is that the tissue is taken out of its context, removing blood supply and neural network connections to the central nervous system. Accordingly, studies into inflammatory cell recruitment to the lung or studies into neural reflex control of lung physiology are not possible in the slice. Having said that, lung slices do contain structural cells and tissue embedded inflammatory cells such as macrophages and mast cells whose function can be studied6,7.
1.2 Applications
As discussed above, one of the first applications for the lung slices was the study of small airway physiology in its natural context of parenchymal connections. Until lung slices became available, studies into airway physiology were mostly done in tracheal or large bronchial preparations in traditional organ bath settings. The lung slices made it possible to study small airway and pulmonary vascular physiology in response to agonists or electrical field stimulation (to release endogenous neurotransmitters)8–10, which is of great importance as these anatomical regions
2
play a major role in asthma, COPD and PAH. Thus, understanding of the receptor populations and excitation contraction coupling mechanisms in the distal airways and vessels has provided new insights with important therapeutic implications. These include the findings that the distal airways have significant neuronal innervation and functional control and the finding that distal airways have completely different pharmacology, with large differences in the sensitivity and maximal response to bronchoconstrictor agents such as methacholine (together with own unpublished observations)8,9,11.
The connectivity to the parenchymal structure has additional benefits as it allows studies into the impact of changes in parenchymal mechanics (e.g. In COPD) on airway mechanics. Such studies have revealed that the elastin/collagen network in the parenchyma significantly impacts on airway reactivity and airway re-opening, which is distorted in COPD and in lung slices exposed to elastase12–14. The elastin and
collagen fibers can be visualized in the slices using two-photon confocal microscopy as outlined below. Strain mapping during bronchoconstriction15 provides further
insight into these airway-parenchymal interactions and can be used to map how deep strain penetrates into the lung tissue following bronchoconstriction.
Moreover, additional applications for the lung slices have been considered. These include studies into bronchoconstriction-induced or growth-factor-induced airway and lung remodeling. We have shown that prolonged exposure to bronchoconstrictors leads to changes in smooth muscle content in the airways5,16, although an important
side note here is that this was more readily measurable in the guinea pig and less so in the mouse. This is possibly the result of differences in bronchoconstriction to methacholine between these species, as methacholine challenge leads to complete airway closure in the guinea pig but only partial closure in the mouse13,16. Exposure
to TGF-β or a mix of growth factors and mediators involved in lung fibrosis does induce changes in gene and protein expression of matrix elements and contractile protein in the mouse and the guinea pig, showing the suitability of the lung slices for early studies of remodeling and fibrosis as well17.
The slices may also offer opportunities to study the regenerative capacities of the lung. Alveolar epithelial type II cells (AT II cells) are difficult to culture in vitro but abundantly expressed in the lung slices. We have shown that disruption of elastin fibers reduces the expression of the AT II cell marker pro-SPC in the lung slices13,
airway functions in lung slices. Progenitor cell populations and their response to treatment can also be studied in the lung slices, as has been done by Uhl et al, who mapped WNT-active cells in TCF/LEF-H2B:GFP mice, and showing their response to GSK-3 inhibition6. Another intriguing application of the lung slices in this area is to
decellularize the slices for subsequent repopulation with progenitor cells to study the impact of local matrix-derived cues on cell fate decision and differentiation18.
This is an active area of research that will likely be expanded more in the future. Studies into the immunomodulatory properties of structural cells can also be done in the lung slices19, although this method does not offer the possibility to pinpoint
the cellular source of secreted factors such as cytokines in the medium. The lack of inflammatory cell recruitment into the lung as would be seen in vivo also limits the use of the slice for such studies. Studies of drug metabolism and toxicology can also be done in the lung slices given the presence of metabolic enzymes such as P450 in the lung tissue20–22.
2 Materials
2.1 PCLS Preparation
Prepare all surgery tools (including scissors, tweezers, suture line), cannula, syringes, cotton pad, ethanol 70%, ice, a 10 cm-diameter dish, and anesthesia (ketamine and dexdomitor), agarose medium before going to the animal facility. Slicing medium, incubation and washing medium should be prepared in advance. A tissue slicer machine (Leica VT 1000 S Vibrating blade microtome, Leica Biosystems B.V., Amsterdam, the Netherlands) is used in this protocol.
Anesthetics: Ketamine (40 mg/kg) and Dexdomitor (0.5 mg/kg) are used in the
experiments.
Agarose medium: Agarose powder was dissolved in a solution composed of CaCl2
(0.9 mM), MgSO4 (0.4 mM), KCl (2.7 mM), NaCl (58.2 mM), NaH2PO4 (0.6 mM), glucose (8.4 mM), NaHCO3 (13 mM), HEPES (12.6 mM), sodium pyruvate (0.5 mM), glutamine (1 mM), MEM-amino acids mixture (1:50), and MEM-vitamins mixture (1:100, pH = 7.2) within ultra-pure water (UP). A final concentration of 1.5 % agarose is n used to fill in the mouse lung (see Note 1).
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Slicing medium: Medium composed of CaCl2 (1.8 mM), MgSO4 (0.8 mM), KCl (5.4 mM), NaCl (116.4 mM), NaH2PO4 (1.2 mM), glucose (16.7 mM), NaHCO3 (26.1 mM), HEPES (25.2 mM), pH = 7.2 within ultra-pure water (UP).
Incubation medium: Medium composed of CaCl2 (1.8 mM), MgSO4 (0.8 mM), KCl (5.4 mM), NaCl (116.4 mM), NaH2PO4 (1.2 mM), glucose (16.7 mM), NaHCO3 (26.1 mM), HEPES (25.2 mM), pH = 7.2 within ultra-pure water (UP). Add 1 % Na-Pyruvate (100 mM), 2 % Non-essential Amino Acids (100 ×), 1 % MEM-Vitamin Solution (100 ×), 1% L-Glutamine (100 ×) and 1% Penicillin Streptomycin (Pen Strep, 5,000 Units/ mL penicillin and 5,000 μg/mL streptomycin, Gibco® by Life Technologies) to the
medium before use.
Culture medium: DMEM supplemented with sodium pyruvate (1 mM), MEM
non-essential amino acid mixture, gentamycin, Penicillin Streptomycin (Pen Strep, 5,000 Units/mL penicillin and 5,000 μg/mL streptomycin, Gibco® by Life Technologies),
and amphotericin B (1.5 μg/mL; Gibco® by Life Technologies).
2.2 PCLS Decellularization
Washing solution: Solution composed of sterile ultra-pure water (UP) with 5 %
Penicillin-Streptomycin (Pen Strep, 5,000 Units/mL penicillin and 5,000 μg/mL streptomycin, Gibco® by Life Technologies).
Triton solution: 0.1 % triton-x100 in 5 % Penicillin-Streptomycin in UP. C solution: 2 % Sodium deoxycholate in 5 % Penicillin-Streptomycin in UP. NaCl solution: 1M NaCl solution with 5 % Penicillin-Streptomycin in UP.
Dnase I solution: Solution composed of 30 μg/mL Dnase I, 2 mM CaCl2, 1.3 mM MgSO4.
Peracetic acid solution: 0.1 % Peracetic acid in 40 % Ethanol.
Storage solution: PBS supplemented with 5 % Penicillin-Streptomycin, 0.1 mg/mL
Cytoskeleton Buffer: CB Buffer, MES (10 mM), NaCl (150 mM), EGTA (5 mM), and
glucose (5 mM), pH = 6.1.
Cyto-TBS: Tris-base (20mM), NaCl (154 mM), EGTA (2.0 mM) and MgCl2 (2.0 mM), pH = 7.2.
Cyto-TBST: cyto-TBS containing 0.1 % Tween-20.
3 Methods
3.1 Isolation of murine lung
1. Weigh the animal first by using a scale.
2. Euthanize the mouse following subcutaneous injection of ketamine and dexodomitor (see materials). Observe the mouse and check the depth of anaesthesia by pressing the feet and eye reflexes (blink eyes can be checked by approaching a cotton stick to the eyes). Once the mouse did not show feet and eye reflexes anymore, pin the animal to a base. Open the abdominal cavity with scissors by cutting the skin and peritoneum from the middle of the abdomen up to the jaw. Pull the intestines aside with forceps and cut the inferior vena cava and aorta abdominalis to exsanguinate the animal. Puncture the thoracic diaphragm with the sharp tip of the scissors to allow expansion of the rib cage, being careful not to cut the lung23,24 to open the thoracic cavity.
3. Clear the muscle tissue away from the trachea by grabbing the tissue and manually pulling it away from the underlying trachea with forceps. Make a small incision in the trachea on the anterior side of the thickest band of cartilage using fine forceps or scissors, being careful not to cut off the trachea. Insert the cannula into the trachea through the incision and use the suture line to tie the cannula firmly in place.
4. Inflate the lung by injecting approximately 1.5 mL of low melting-point agarose solution through the cannula making sure that the distal tips of the lung are also filled with agarose medium (see Note 2).
After agarose injection, tie off the cannulated trachea with the suture line to prevent the agarose medium from flowing out of the lung (see Note 3).
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5. Keep the cannula inserted in the trachea. Cover the lung with ice after agarose injection and place it in the fridge (4 ˚C), for 20 min to let the agarose solidify within the lung (see Note 4).
6. Once the agarose has solidified, remove the cannula from the trachea, carefully excising the agarose-inflated lung. Cut off the trachea and remove the front ribs around the heart, then remove the connective tissue in the back and take out the lung. Put the lung in a dish and keep it on ice.
3.2 Preparation of PCLS
1. Separate the lung into individual lobes and remove the connective tissue between each lobe. Then use each lobe as a resource to obtain lung slices. 2. A tissue slicer, Leica VT 1000 S Vibrating blade microtome (Leica Biosystems
B.V., Amsterdam, the Netherlands) is used to cut lung slices in this protocol (Fig. 1). Follow the instruction of this slicer machine to cut lung slices (see Note 5) 3. Before cutting, a higher-dose of agarose medium (2 – 5 %) is recommended to
make a gel column around the lung lobe to facilitate slicing.
4. 250 μm-thick lung slices are cut in slicing medium at 4 ˚C and collected in incubation medium at 37 ˚C.
5. The lung slices are incubated in a humidified incubator in atmosphere of 5 % CO2 / 95 % air at 37˚C. Lung slices are washed in every 30 min, four times in total, using the incubation medium.
6. Lung slices are placed in incubation medium and cultured at 37˚C in 12-well culture plates, using three to four slices per well.
Lung slices prepared using this procedure can be used in a number of experimental applications. Previous work from our lab (unpublished) demonstrates that murine lung slices viability is preserved for 72 h of culturing, as mitochondrial activity did not change during this time window. This indicates that the lung slices are viable for at least 3 days.
3.3 Airway narrowing studies
Airway narrowing can be studied by fixating the lung slices into a 3-well cluster (Fig. 2). These slices are then exposed to a contractile stimulus following which airway contraction is recorded using a microscope.
Figure 2 Representative images of airway narrowing studies. (a) Device used in the study of airway
narrowing. (b) Two adjacent airways before treatment, 40 ×. (c) Airway narrowing in response to methacholine (MCh), 40 ×. (d) Airway reopening in response to chloroquine (Cq), 40 ×.
1. 12-well cell culture plates can be cut into small 3-well clusters which will fit under a microscope. These 3-well clusters can be used for the airway contraction studies.
2. Select slices with airways of the desired size. To do so, fill the wells of the custom-made 3-well clusters with 1 mL of warm (37 °C) incubation medium per well. Put one lung slice to each well, and inspect these slices using a microscope (we used Eclipse, TS100; Nikon). It is recommended to inspect a few slices as not every slice will contain the desired airway size. Only use slices in which the airways are cut in cross-sectional manner. Determine the airway size by using the image acquisition software (NIS-elements; Nikon, see Note 6)
3. Following medium removal, use a nylon mesh and metal washer to fix the lung slice, as described previously25. The nylon mesh (which is also washer shaped)
should be slightly bigger than the metal washer to make sure the PCLS tissue does not come in direct contact with the metal washer. First place the nylon mesh on top of the slice, then place the metal washer. The slice is now fixated while the airway of interest is still visible through the hole in the middle of the nylon mesh and metal washer. For ease of preparation, one could carefully remove the medium from the well using a pipette, leaving the slice in the well. Following fixation, 1 mL of warm (37 °C) incubation medium has to be added again. Put the plate back under the microscope, on top of the heating pad, and fix the plate. Choose the desired magnification and focus the microscope (see fig. 2).
4. Capture the airway contraction in time-lapse (1 frame per 2 seconds) via the microscope using image acquisition software (NIS-elements; Nikon). Start the time-lapse and wait for 2 minutes to record the baseline airway luminal area. Following 2 minutes, add methacholine in increasing concentration (10 -9M – 10-3M final concentrations) to the well using a pipette. Be careful not to
touch the slice or chamber with the tip of the pipette. Use an interval of 7.5 minutes between each dose (see Note 7). Following the dose response curve for methacholine, it is also possible to dilate the airways again, e. g. using the
2
bitter taste receptor agonist chloroquine (10-3 M, Sigma-Aldrich, see Note 8).
One could also use other approaches including β-agonists but might be less successful.
1. Quantification airway luminal area. To quantify the airway luminal area, image
acquisition software (NIS-elements; Nikon) could be used.
3.4 Collagen and elastin imaging by Two-Photon and Multiphoton Microscopy
1. Wash the lung slices twice with PBS (500 µL/slice), leave the PBS in the well after the second wash. Using a small spatula with a flat end, scoop one slice out of the well and carefully place it onto a microscope slide. Make sure the slice does not fold. Using a piece of paper towel, carefully remove any excessive PBS surrounding the slice. Place a coverslip on top of the slice. Seal the edges of the coverslip using transparent nail polish (see Note 9). As the protocol mentioned above, the imaging has to be performed straight away if use fresh tissue. 2. 2-Photon and multiphoton excitation fluorescence (MPEF) imaging can be
used to visualize collagen and elastin polymers, respectively, as described previously26. Under excitation at 820 nm, the collagen bundles will naturally
emit a second harmonic generation signal which can be collected around 410 nm. Elastin can be visualized by using its endogenous fluorescence. Elastin images can be generated by using an infrared laser (excitation wavelength 880 nm). The broadband emission spectrum ranges from 455 to 650 nm with a peak at ~500 nm (Fig. 3).
Figure 3 Representative images from two0photon imaging and multiphoton imaging. (a)
two-photon and multitwo-photon excitation fluorescence imaging are used to visualize a-sm-actin (green) and collagen (red), (b) two-photon and multiphoton excitation fluorescence imaging is used to visualize collagen (green) and elastin (red) polymers in lung slices13.
The Mean linear intercept (Lmi) can be determined as a measurement of alveolar airspace size. This can be measured either by confocal microscopy or by light microscopy.
1. Lung slices used for contraction experiments or non-used slices are washed four times with incubation medium. Then they are transferred to an embedding cassette filled with a biopsy pad (see Note 10). The slice is placed on the pad and a second pad is placed on top of the slice before closing the cassette and placing it in a formalin solution (10 %) for 24 h. The slices are processed for paraffin embedding and embedded into paraffin blocks. Sections of 4 μm are cut with a microtome and stained by H&E staining. Twenty random photomicrographs of the parenchyma of lung (magnification 200 ×) tissue rather than airways or blood vessels are made, as airways or blood will interfere with the measurement of alveolar airspaces.
2. This method is previously described by van der Strate27. In short: a sheet with
vertical lines in three horizontal rows (21 in total) is placed on the top of the photograph. Whenever an intercept crosses the parenchymal walls, two points will be given. When the intercept touches the parenchymal cells, one point will be given. Importantly, intercepts that cross or touch blood vessels or airways are not taken into account to prevent misjudgments. When more than 3 intercepts are crossing or touching blood vessels or airways, the picture should not be taken into account and a different field should be chosen. With the total scores the Lmi is calculated as: (n × l × 2)/m in which “n” represents the number of intercepts “l” represents the length of the individual lines (as calculated with the scale of microscopic photo), and “m” refers to the amount of points given. 3. Proteins could be visualized by immunofluorescence (described below). Fluorescence can be determined with a confocal laser scanning microscope (CLSM) equipped with true confocal scanner (TCS; SP8 Leica, Heidelberg, Germany), using a 200 × lens. To avoid bleed-through, sequential scans need to be performed. Alexa Fluor 488 can be excited using the 488 nm blue laser line, and Cy™3 can be excited using the 552 nm green laser line. Record all images in the linear range, at an image resolution of 1024 × 1024 pixels and with a pinhole size of 1 Airy unit, while avoiding local saturation28.
4. Single Z-stack images can be used to quantify the Lmi with the analysis method described above (see Note 11).
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3.3.4 Decellularization
1. Decellularization with detergents (Acellular scaffolds) maintains the architecture and proteins of extracellular matrix for use as scaffolds in the field of lung tissue engineering or progenitor cell biology. We decellularized the lung slices for subsequent repopulation with progenitor cells to study the impact of local matrix-derived cues on cell fate decision and differentiation (Fig. 4). Place 1 to 4 slices in each well of a 24-well plate and incubate the slices overnight in 1 % triton - ×100 medium with 5 % Penicillin-Streptomycin (1 mL medium per well), at 4 °C.
Figure 4 Representative images of naïve and decellularized lung slices. (a) Naïve lung slice, 40 ×. (b)
Decellularized lung slice, 40 ×. (c) Mason’s trichrome stain (see Note 14) on naïve lung slice, 40 ×. (d) Mason’s trichrome stain (see Note 14) on decellularized lung slice, 40 ×.
1. Wash the slices twice in washing solution for decellularization (see materials). 2. Incubate the slices in a 2 % sodium deoxycholate (SDC) solution for 3 h at room
temperature.
3. Wash the slices twice in washing solution for decellularization. 4. Incubate the slices in 1M NaCl solution for 1h at room temperature. 5. Wash the slices twice in washing solution for decellularization. 6. Incubate the slices in Dnase I solution for 1 h at room temperature. 7. Wash the slices twice in washing solution for decellularization. 8. Wash the slices in 0.1 % peracetic acid for 1 h at room temperature.
9. Store the slices in storage solution. Slices can be stored in the storage solution for short periods at 4 °C, place the slices at -20 °C in storage solution for long term.
10. Slices are now ready to be repopulated with progenitor cells.
3.6 mRNA isolation and real-time PCR
Total RNA is extracted from PCLS by using the Maxwell 16 instrument and corresponding Maxwell 16 LEV simply RNA tissue kit (Promega, Madison, USA) for automated purification according to manufacturer’s instructions. This is an optional method to extract RNA from PCLS, as the quality of RNA obtained with other methods including Trizol and kit extraction is too low to be used for experiments. The Reverse Transcription System (Promega, Madison, USA) is used to reverse transcribe total RNA (1 µg) into cDNA. 1 µL diluted cDNA (1:20) is subjected to the Illumina Eco Personal QPCR System (Westburg, Leusden, the Netherlands) using FastStart Universal SYBR Green Master (Rox) from Roche Applied Science (Mannheim, Germany). The cycle parameters used in real-time PCR system are denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s and extension at 72 °C for 30 s for 40 cycles followed by 5 min at 72 °C. The amount of target genes could be normalized to the housekeeping genes such as β-2 microglobulin (B2M), and ribosomal protein L13A (RPL13). LinRegPCR analysis software was used to analyze data.
3.7 Immunofluorescence Imaging
Immunofluorescence was performed as described below (Fig. 5). Primary antibodies and secondary antibodies could be obtained from various companies according to the research interests. Rabbit anti-Prosurfactant Protein C (proSP-C, EMD Millipore Corporation, CA, USA) and mouse anti E-cadherin (BD Biosciences, Bedford, MA, USA) were used in our study by this method.
2
1. Fixation
1) Wash lung slices twice with cold (4 °C) cytoskeleton buffer (CB buffer) 2) Incubate the slices for 15 min with 3 % paraformaldehyde (PFA) at room
temperature (400 µL/slice)
3) Incubate the slices for 5 min with 3 % PFA + 0.3 % triton-x100 at room temperature (400 µL/slice)
4) Wash the slices twice with cold (4 °C) CB buffer (see Note 12). 2. Blocking
1) Prepare blocking buffer (1 x cyto-TBS with 1 % BSA and 2 % normal donkey serum).
2) Incubate the lung slices with blocking buffer (250 µL/slice) for 1h on the shaker at room temperature.
3. Incubation
1) Dilute primary antibody in cyto-TBST solution.
2) Incubate the lung slices with primary antibody (Use 200 µL/slice) for 1.5 h at room temperature or overnight at 4 °C.
3) Wash the lung slices with cyto-TBST (500 µL/slice), for 15 min, repeat 3 times. (see Note 13).
4) Dilute the secondary antibody (1:50) in cyto-TBST (250 µL/slide) and incubate for 2-3 h at room temperature.
5) Wash the lung slices with cyto-TBST (500 µL/slide), for 15 min, repeat 3 times.
6) Wash the lung slices with UP water twice quickly.
7) Wash the lung slices with UP water for 2 min, repeat 4 times. 4. Anti-fade staining
1) Transfer the slices to glass slides.
2) Add 10 µL/slide anti-fade reagent (Invitrogen, Breda, The Netherlands) on the glass slide (cover the whole slice) and cover them with clean microscopic glass plate.
5. Use fluorescence microscope to make images (Fig. 5)
Figure 5. Representative immunofluorescence images of mouse PCLS.
(a) Blue signals are DAPI, which stained the nuclei, 63 ×. (b) Green signals represent the expression of surfactant protein c (SPC), which is an alveolar epithelial type 2 cell marker, 63 ×. (c) The merge picture of (a) and (b), 63 ×.
4 Notes:
1. The agarose medium must be kept warm in thermal bottle (around 37 °C) so
that it will not solidify prior to injection.
2. The volume of agarose is dependent on the size of lung and should not exceed
lung capacity. Since lung tissue is highly compliant and easily damaged injection pressure should also be minimized.
3. If this PCLS model is used to study the arteriole physiology, 6 % gelatin should
be used to perfuse the pulmonary arteries [2].
4. This step aims to use the agarose to maintain the shape of the lung, which
makes it easier to cut lung slices.
5. The cutting frequency of 90Hz, the amplitude of 1.0 mm, and the sectioning
speed of 2.25 mm/s are chosen in this protocol.
6. The microscope should have a see-through heating pad that can be kept at 37
°C on which the plate with slices can be placed. This is especially important during the contraction experiments as these can last up to 1 hour.
7. In the described system, medium is not washed away, and methacholine
accumulates in the well with each new dose.
8. In mouse lung slices, β-agonists are less effective in inducing airway relaxation
than chloroquine.
9. This will prevent the coverslip and slice from moving during the microscopy. 10. The pads are needed to keep the slices flat without wrinkles. Because the slice
is only 250 μm thick, when put in a paraffin block it should be very straight, otherwise it is impossible to make sections.
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11. Z-stacks imaging is optional, making it possible to access structures throughout
the slice. Image J 1.48d can be used to further process images.
12. Optional: store in 1 × cyto-TBS buffer (200 μL) in sealed chamber up to maximum
2 weeks.
13. Since the second antibody is labeled by fluophore, the experiments should be
performed in dark room.
14. Masson’s trichrome stain is a three-colour staining protocol used in histology.
Trichrome stain (Masson) kit (sigma-aldrich) is used to stain the lung slices in this study by using three dyes: hematoxyline (for nucleous), aniline blue (connective tissue), and biebrich scarlet (cytoplasm). Cytoplasm and muscle fibers stain red whereas collagen displays blue coloration.
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