Name: generation of human 3d lung tissue cultures (3d-ltcs) for disease modeling
Uploaded: 2019-02-12T13:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling at JoVE.com

The authors are grateful to Marisa Neumann for expert technical assistance. All lung tissues were kindly provided by the CPC-M Bio-Archive. This work was supported by the German Center of Lung Research (DZL), the Helmholtz Association and CPC Research School grants.

The use of human tissue was approved by the ethics committee of the Ludwig-Maximillian University [Munich, Germany (project number 455-12)]. Tumor-free human lung resections were provided by the Asklepios Biobank for Lung Diseases (Gauting, Germany, project number 333-10).
NOTE: All procedures of human PCLS production (Figure 1) are done under a sterile laminar flow hood.
1. Preparation of Instruments and Materials
<ol>
	<li>Prepare all materials for the inflation of lung tissue with agarose as described below.
	<ol>
		<li>Prepare the cultivation medium: Dulbecco&#8217;s Modified Eagle Medium (DMEM) F-12 supplemented with L-Glutamine, HEPES, 10,000 IE Penicillin, 10,000 IE Streptomycin and 0.1% (v/v) fetal bovine serum. 
		NOTE: Medium is used at 37 &#176;C.</li>
		<li>Prepare a sterile metal tray covered with tissue paper. Place a sterile 15 cm cell culture dish on the tray.</li>
		<li>Fill the cell culture dish with the 15 mL of cultivation medium.</li>
		<li>Prepare a 3% (w/v) agarose solution by dissolving the appropriate amount of low-melting point agarose in a minimum of 30 mL of cultivation medium.</li>
		<li>Heat the solution in a microwave until boiling. Cool the agarose solution to 42 &#176;C in a water bath. Keep the liquid agarose solution stored in the water bath.</li>
		<li>Prepare several 50 mL conical tubes filled with the liquid agarose.</li>
	</ol>
	</li>
</ol>
2. Resected Lung Tissue
<ol>
	<li>Store fresh tumor free lung tissue of lobectomy resectates immediately after resection in DMEM F-12 medium at 4 &#176;C until step 3.</li>
	<li>Do not exceed cold ischemia time of 4-8 h prior to processing.</li>
</ol>
3. Inspection and Selection of Resected Tissue Prior to Agarose Filling
<ol>
	<li>Lift the tissue from the medium with tweezers. In order to avoid any damage to the tissue, especially to the pleura, handle the tissue with tweezers at the airways only.</li>
	<li>Score the tissue quality by criteria of the Lung Agarose Filling Score defined in Table 1.</li>
	<li>Proceed to step 4 if the tissue&#8217;s quality is scored above or equal to 72. If the tissue&#8217;s quality is scored below 60, do not continue further with agarose filling. 
	NOTE: If the tissue score is between 60 and 68, the agarose-filling and tissue slicing still might produce reasonable results, and a final decision for prolongation of the experiment has to be made case to case. However, lung tissue that did not meet the above-mentioned requirements, mostly fails in agarose filling.</li>
</ol>
4. Lung Tissue Inflation by Agarose Filling
<ol>
	<li>Lift the tissue from the storage medium and drain excess media from the tissue. Transfer the lung tissue into the 15 cm culture dish prepared in 1.1.2.</li>
	<li>Fill a 30 mL syringe with the low-melting point agarose from 1.1.3.</li>
	<li>Prepare a peripheral venous catheter by removing the obturator and attach it to a 30 mL syringe</li>
	<li>Identify a bronchus (0.5-3 mm in diameter) in the tissue that is ventilating an intact section of the tissue (see Figure 2).</li>
	<li>Insert the cannula into the selected bronchus (0.5-3 mm diameter).</li>
	<li>Gently push the cannula gently forward as far as possible.</li>
	<li>Seal the bronchus around the cannula by compressing the bronchial wall around the cannula with forceps, ideally clamping any adjacent pulmonary artery at the same time.</li>
	<li>Occlude other additional airways with a surgical clamp to prevent agarose leaking through these airways.</li>
	<li>Lift the tissue with the forceps from the culture dish.</li>
	<li>Manually pour the agarose with the syringe no faster than 0.3 mL/s. The speed of agarose filling might vary between approximately 0.05 and 0.3 mL/s due to the heterogeneous resistance of airways and/or atelectasis.</li>
	<li>If high resistance while filling or agarose leaking from the tissue is observed, retry the whole procedure with a different bronchus from step 4.4. Perform troubleshooting as described below. 
	NOTE: The degree of agarose filling is highly dependent on the position of the catheter in the tissue and deep penetration of the catheter results in agarose filling of small cone like regions (*) of the lung tissue (Figure 2C).
	<ol>
		<li>In case of high resistance, try positioning of the catheter leads to proper filling of most regions of the tissue (#) (Figure 2D).</li>
		<li>As plugs of early solidified agarose in the proximal bronchi or other airway obstructions (arrow) can lead to an incomplete filling of the tissue (Figure 2E), do not force agarose filling as this might lead to defects in the filled area, but not in a filling of the obstructed tissue parts.</li>
		<li>If the respiratory tree derived from the cannulated bronchus is damaged during resection and the agarose filling results in a constant leaking of the liquid agarose (arrow in Figure 2F), insert the catheter into a more peripheral part of the airway system to fill at least a minor part of the tissue (*) (Figure 2G). Additionally, seal the damaged peripheral airway with a surgical clamp (arrow) (Figure 2H).</li>
	</ol>
	</li>
	<li>Apply agarose until the lung tissue is filled completely. Do not over-inflate the tissue as this may cause irreversible damage to the tissue structure and its cells.</li>
	<li>Clamp the bronchus that was used for the filling immediately. Remove the cannula prior to clamping.</li>
	<li>Incubate the tissue in the culture medium at 4 &#176;C for 30 min to ensure agarose solidification.</li>
	<li>If the resected tissue has multiple bronchial entries, repeat step 4.2 to 4.13 until all parts of tissue are filled with agarose.</li>
	<li>Store the agarose filled lung tissue sections in 4 &#176;C cold medium until slicing.</li>
</ol>
5. Precision-cut Lung Slicing
<ol>
	<li>Identify regions in the lung tissue that are solidly filled with the agarose. Solidly filled regions will not collapse when they are gently pressed with tweezers against the bottom of the cell culture dish.
	<ol>
		<li>Excise a 1-1.5 cm3 block of regions described in 5.1, whereas one side still should be covered with pleura.</li>
	</ol>
	</li>
	<li>Attach each individual tissue block with the pleural side contacting the holder of the vibratome by using a cyanoacrylate glue. 
	NOTE: The pleura is slightly elastic and therefore impedes the cutting with the vibratome blade. Placed on the tissue holder, the pleura will not interfere with the cutting and, importantly, forms a natural barrier between the cyanoacrylate glue and the tissue&#8217;s parenchyma allowing for minimal diffusion of the glue.</li>
	<li>Slice the lung tissue with the vibratome with the following settings: thickness: 500 &#181;m, frequency: 100 Hz, amplitude of the knife: 1.2 mm, forward speed of the blade of 3-12 &#181;m/s, which depends on tissue stiffness. Reduce the protrusion-speed of the blade if the slice is not cut properly, or if the tissue block itself starts to vibrate.</li>
	<li>Gently transfer the slice by lifting it with forceps from the vibratome tray into a well of a 12-well plate filled with cultivation medium. Finally, incubate the lung slices in an incubator under standard cell culture conditions.</li>
	<li>Stop slicing when 2-3 mm of the tissue block are left unsliced since the cyanoacrylate glue may have compromised the tissue integrity of this region.</li>
</ol>
6. Generation of PCLS Punches
<ol>
	<li>Transfer the lung slices from a single well to an empty 10 cm dish.</li>
	<li>Place a 4 mm biopsy puncher orthogonally to the upper surface of a PCLS and start to move the puncher in clockwise and counterclockwise rotations.</li>
	<li>Fill cell culture medium into the wells of a 96-well plate. Lift the tissue punches with forceps and transfer the punches into the wells of a 96-well plate. Finally, incubate the lung punches submerged in the medium prepared in 1.1.1. in a cell culture incubator under standard conditions (21% (v/v) oxygen, 5% (v/v) carbon dioxide and 95% humidity, at 37 &#176;C).</li>
</ol>
7. Tissue Culture and Sample Harvesting
<ol>
	<li>Culture the PCLS and punches overnight in an incubator under standard cell culture conditions.</li>
	<li>Culture PCLS and punches under outlined condition for a maximum of 120 hours after their generation to ensure cellular viability and function.</li>
	<li>For harvesting protein as well as RNA, wash PCLS and punches three times in phosphate buffered saline (PBS), transfer them into cryovials and snap-freeze in liquid nitrogen.</li>
	<li>Sample medium supernatant of cultured PCLS punches for the analysis of secreted proteins.
	<ol>
		<li>For histological analyses, wash the PCLS and punches three times with PBS and fix them with 4% paraformaldehyde by incubating for 30 min at 37 &#176;C. Finally, store the PCLS in PBS at 4 &#176;C for further downstream staining.</li>
	</ol>
	</li>
</ol>

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All authors declare no competing financial interests.

The protocol described in this manuscript covers the generation of PCLS from human lung tissue resectates by filling it with liquid agarose and subsequent vibratome slicing. Generation of tissue slices was demonstrated before for a couple of organs, like liver and brain, whereas the inherent stiffness of these organs allowed direct slicing without any modification of the tissue. Of note, the initial proper preparation of the lung tissue is the most crucial step in generating PCLS. Agarose filling of the lung is the method of choice to stabilize its soft and elastic nature, and to ensure a homogenous and reproducible PCLS generation. Large airways of the resected lung tissue are cannulated to provide access to the small airways, as well as to the intact lung parenchyma. The lack of an intact pleura, which makes agarose filling nearly impossible, is a major reason why lung tissue is mostly not usable for lung slicing. Prospectively, a synthetic pleura originally designed to perform functional experiments on decellularized scaffolds could potentially be applied to achieve successful agarose filling of explants that lack an intact pleura31. Resections resulting in a human lung tissue piece with intact pleura are essential for generating tissue blocks for slicing. Resected tissue is more available due to tumor-free tissue from cancer resections than fully intact lobes or whole-lung explants of patients who underwent lung transplantation.
Commonly, two systems are used to produce PCLS: the Krumdieck tissue slicer15 and vibratory microtomes (vibratomes). Tissue slicers generate slices by passing a tissue block through a metal vessel, which cuts the PCLS at 90&#176; at the end of this vessel. Vibratomes generate PCLS by moving a vibrating knife horizontally over an anchored block of tissue that is submerged in a cooled medium bath, which compared to the Krumdieck slicer exerts less shear force on the tissue. This results in less harsh treatment of the tissue before cultivation. On the other hand, the vibratome cutting is more time and work consuming. In our hands, vibratome slicing enabled the production of a maximum of 100 PCLS or 500 PCLS punches in one day, sufficient for most experimental studies. PCLS can be cultured in various ways: (a) attached to Trans-wells, thus generating an air liquid interface (ALI) system, (b) as dynamic organ culture (DOC), or (c) submerged in cell culture medium at standard cell culture conditions. The in-detail cultivation of PCLS was previously described22,23,25; however, a common standard of cultivation conditions between their usage in various labs around the world is still missing. In particular, the culture time might be critical: as in murine PCLS, a loss of SFTPC positive alveolar type 2 cells is observed after 144 h, but not after 120 h22. In addition, metabolic activity seems to remain stable in murine22 and human PCLS25 for 120 h.
There are a couple of technical limitations for the generation of PCLS: the number and size of the resectates fluctuates over time; the efficiency of the agarose filling, which depends on the presence of intact pleura within the obtained tissue, determines the final success of the PCLS generation; and tissue destruction caused by pathological changes within the obtained (diseased) lung tissue might interfere with the PCLS preparation. Airway obstructions and fibrotic tissue lacking intact alveolar space impede with agarose filling and thus make slicing of the fibrotic tissue a demanding task. Emphysematous tissues as found in diseases such as COPD or alpha-1-anti-trypsin deficiency might not withstand the pressure of agarose filling, and will result in rupture of the alveoli and architectural artefacts. In these cases, the usage of low agarose concentration, e.g., 1% (w/v), might be useful to decrease pressure and speed during agarose filling. Overall, the disease state of the tissue can dramatically limit the use of the tissue for PCLS generation. All these parameters determine the amount of PCLS that can be generated from lung tissue, and also the amount of time it takes to produce the PCLS. Further limitations of PCLS are inconsistencies in between different lung slices with respect to size or tissue content, which requires further normalization steps for experiments. To overcome this, biopsy punches of similar regions of the same slice can be generated. This procedure is apt to reduce the tissue variability and, as an additional benefit, increase the number of PCLS samples that can be used for experiments.
In conclusion, human 3D lung tissue cultures from agarose filled PCLS provide a complex human model for studying lung physiology and diseases. The protocol provides a detailed description of the preparation of PCLS from resected lung tissue and their cultivation, and moreover addresses challenges in agarose filling of human lung resections and how to overcome them.

Chronic and acute lung diseases are a major cause of morbidity and mortality worldwide1. For patients with chronic lung diseases like obstructive pulmonary disease (COPD)2, severe asthma3, lung cancer4 and diffuse parenchymal lung diseases5, curative therapies are currently not available. Although studies in animal models for lung diseases have deepened the understanding of disease pathomechanisms6 and have led to the identification of potential novel therapeutic targets7,8,9, these models exhibit relevant biological and physiological differences when compared to humans10. To overcome these discrepancies between murine and human biology as well as anatomy, human ex vivo 3D lung tissue culture (3D-LTC) systems are used in various areas of biomedical research. These 3D-LTC culture systems are based on precision-cut lung slices (PCLS). The generation of PCLS ex vivo allows analysis of a third spatial dimensionality, which allows for the investigation of the spatial and functional relationships of cells in entire alveoli and airways11, as well as the interstitium, vasculature and mesothelium. Notably, PCLS ex vivo models are multicellular, meaning that they contain most functional cells of in situ lungs, thus closely representing the cells&#39; native biological environment and thus overcoming the limited cell-cell and cell-matrix interaction in most 2D cell culture approaches. Up to now, ex vivo murine PCLS were used to model pulmonary diseases, like COPD12, lung fibrosis13, lung cancer14, viral infection15,16, bronchopulmonary dysplasia17, and asthma18. However, a considerable proportion of novel drug therapies in human lung diseases that were investigated in clinical trials do not translate to the clinic due to their lack of efficacy or safety, assumingly due to yet considerable differences between human and murine biology and disease19,20,21.
Over several years, human PCLS have been largely used to assess lung toxicity of chemicals and drugs. Only recently, human lung tissue has been used from patients with COPD22,23, asthma24, and lung fibrosis25, to pursue pathophysiological and pharmacological studies. By using resected patient organ material and generating PCLS thereof, one can recapitulate major disease hallmarks in a complex 3D tissue environment22 representing and maintaining most of the native cellular diversity of the organ. Moreover, diseased tissue applied in a variety of experimental setups was shown to mimic disease-like changes in liver, intestine and kidney26,27,28,29.
However, processing of lung tissue remains challenging for several reasons. Unlike solid tissue, native lung parenchyma tends to collapse without ventilation and exhibits lower tissue stiffness. These properties impede the slicing of the tissue. Thus, filling of airways and the alveolar space with low-melting point agarose preserves the native lung structure and provides the stiffness needed for precision-cut slicing of murine and human lungs30. Human lung resectates donated for research purposes are by their nature anatomically, genetically and physiologically highly diverse, thus often presenting a high inter-patient variability when performing experiments25. In contrast to the whole lobe or whole lung explants, lung samples resected by means of thoracic surgery do not necessarily follow the anatomical segments and, therefore, require special preparation. In this article, we provide a detailed and optimized protocol for the generation of human PCLS from resected lung tissue and their subsequent cultivation and experimental use to model lung disease.

<table><tbody><tr><td>Vibratome Hyrax V50</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Hyrax CU 65</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Vasofix Braun&amp;uuml;le 18 G</td><td>B. Braun Melsungen AG</td><td>4268130B</td><td></td></tr><tr><td>30 mL NORM-INJECT</td><td>Henke Sass Wolf</td><td>4830001000</td><td></td></tr><tr><td>Guarded disposable scalpels, sterile</td><td>Swann-Morton</td><td></td><td></td></tr><tr><td>Loctite 406</td><td>Henkel</td><td>LOCTITE 406</td><td></td></tr><tr><td>Synthetic Single Crystal Sapphire</td><td>Delaware Diamond Knives</td><td>-</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium F-12 Nutreient Mixture (Ham) + L-Glutamine + 15mM HEPES</td><td>Gibco</td><td>31330-038</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco by Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Special process fetal bovine serum (Sera Plus)</td><td>Pan Biotech</td><td>P30-3702</td><td></td></tr><tr><td>Disposable Biopsy Punch</td><td>pfm medical</td><td>48401</td><td></td></tr><tr><td>96 Well, Black/Clear, Tissue Culture Treated Plate, Flat Bottom with Lid, sterile</td><td>Falcon / Corning</td><td>353219</td><td></td></tr><tr><td>Agarose, low geling temperature</td><td>Sigma</td><td>A9414-100G</td><td></td></tr></tbody></table>

generation of human 3d lung tissue cultures (3d-ltcs) for disease modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

PCLS generation 
The generation of PCLS can be separated into four essential steps: surgical lung tissue resection, agarose filling, vibratome-based PCLS generation, and culture of PCLS. The resected lung tissue is filled with low-melting point agarose, which adds the required stiffness to the lung tissue for slicing and preserves the native lung structure and architecture. Of note, PCLS generation is highly time consuming, thus often overnight storage of the filled lung tissue in DMEM F-12 medium can be included as an additional step and PCLS generation is started on the next day. Depending on the following experimental setup, generated PCLS can be incubated overnight in standard cell culture medium containing 0.1% (w/v) fetal bovine serum, before experimental conditions are applied. 3D-LTCs were viable and exhibited cellular functionality (such as surfactant protein secretion) for up to 120 h22 in the culture conditions outlined in this protocol (Figure 1) and might be optimized upon further improvement thereof.
Agarose filling 
For agarose filling of the tissue, a cannula of a peripheral venous catheter with a 1.3 mm diameter attached to the agarose-filled syringe was inserted into a bronchus at the surface of the cut tissue (Figure 2A). Bronchi are often localized near to a pulmonary artery. While the arteries have thinner walls and tend to collapse, bronchi exhibited a good visible lumen. Depending on the tissue's integrity, the catheter can be advanced through several generations of the respiratory tree into the periphery of the lung. The penetrated bronchus was sealed around the cannula by using tweezers (Figure 2B). The pulmonary artery can be clamped with the tweezers at the same time. Afterwards, the tissue is lifted up and liquid agarose is gently instilled into the airways.
Depending on the position of the catheter, a majority of the tissue can be filled with liquid agarose (Figure 2D). Optionally, cone like parts of the lung tissue, which reflect the lung's parenchyma ventilated by the penetrated bronchus, might get filled with the agarose (Figure 2C). In both scenarios, a characteristic pattern of solidly filled tissue regions can be observed: first, a major part of the tissue is filled in wedges (Figure 2D), or secondly, smaller protruding round areas of thoroughly filled tissue regions appear (Figure 2C). If parts of the airways obstruct due to agarose clots or other causes, parts of the tissue might not be properly filled with agarose. Thus, only parts of the tissue might be applicable for slicing. In case of leakages during the agarose filling procedure, parts of the filled respiratory tree might get perforated and filling of the lung tissue gets nearly impossible However, possible workarounds include the filling via a more peripheral bronchus, a deeper penetration of the cannula into the distal airways (Figure 2G), or potential clamping of the leakage area (Figure 2H).
Precision-cut lung slicing 
Tissue blocks at a length and width of 1-1.5 cm were excised from tissue regions, which were completely filled with solidified agarose (Figure 3A-3B). Next, the individual tissue blocks were glued on the tissue holder of the vibratome (Figure 3C). 500 µm thick PCLS were generated, whereas the tissue block at the vibratome was advancing forward with speeds between 3-12 µm/s. (Figure 3D-3F). Finally, the PCLS were submerged in cell culture medium containing 0.1% (w/v) fetal bovine serum and cultured at standard cell culture conditions, as outlined step 7.
Experimental readouts of human 3D-LTC after 48h of culturing 
A representative immunofluorescence staining, as previously described by Alsafadi et al.25, is shown in Figure 4A-4C. Immunolabeling of fibronectin (red) and cell nuclei (DAPI, blue), allowed for imaging of the preserved alveolar structure in the human 3D-LTC ex vivo. Treatment of the human PCLS punches with a profibrotic cytokine cocktail (including transforming growth factor beta 1, platelet derived growth factor AB, lipophosphatidyl acid, and tumor necrosis factor alpha) for 48 h resulted in fibrosis-like changes in human 3D-LTCs. By qPCR, a significant induction of the fibrosis-relevant extracellular matrix components collagen type 1 and fibronectin genes in 3D-LTC punches was observed upon treatment with the profibrotic cocktail (Figure 4D). Additionally, protein levels of the mesenchymal marker vimentin were found upregulated in 3 out of 4 patients after treatment of 3D-LTC punches (Figure 4E).
<img alt="Tissue resection to PCLS culture; steps: agarose filling, preparation; diagram shows process timeline." class="xfigimg" src="/files/ftp_upload/58437/58437fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Workflow of PCLS generation. Tumor-free areas of lung resections are thoroughly inspected due to their tissue integrity. If the tissue is scored suitable for further use (scoring is explained in detail in the material and methods section), it is next filled with liquid agarose. Tissue blocks filled with solidified agarose are subsequently sliced with a vibratome. Submerged in cell culture medium, 3D-LTC are cultured up to 120 h after their generation. Downstream analyses of the 3D-LTCs involve protein- or RNA-expression, live-tissue fluorescence imaging, as well as immunofluorescence staining after fixation of the tissue. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58437/58437fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lung perfusion setup diagram illustrating bronchus and artery injection process for tissue study." class="xfigimg" src="/files/ftp_upload/58437/58437fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Filling the lung tissue with low-melting point agarose. The lung tissue is cannulated with a peripheral venous catheter which is inserted into a bronchus adjacent to the pulmonary artery (Figure 2A). Tweezers are used to fix the cannula in the bronchus and to clamp the pulmonary artery to avoid leaking of the liquid agarose. Liquid agarose at 42 °C is poured into the lung tissue with a 30 mL syringe (Figure 2B). A distal positioning of the cannula during filling will result in small areas of filled tissue (Figure 2C), while proximal positioning will ensure the filling of a larger tissue volume (Figure 2D). Any obstructions of the airways will reduce the amount of tissue volume that can be filled (Figure 2E). In case of agarose leaking, a distal cannula positioning and/or clamping of the leakage side enables proper agarose filling of the lung tissue (Figure 2F-2H). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58437/58437fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tissue sectioning process: includes tissue block, angle diagram, microtome setup, and final tissue slice." class="xfigimg" src="/files/ftp_upload/58437/58437fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Precision-cut lung slicing. A successfully agarose-filled lung tissue is used to excise a piece of a tissue block (1 cm x 1.5 cm x 1 cm) with a scalpel (Figure 3A). Next the excised tissue block is glued to the tissue holder, scale bar indicates 1 cm (Figure 3B). Preferably, the tissue is glued with its pleural surface to the surface of the tissue holder as shown in Figure 3C. 500 µm thick slices are cut by the vibratome with a sapphire knife in a 10°-15° angle relative to the tissue (Figure 3D and 3E). The cutting procedure results in 2-3 cm3 large intact lung slices, Scale bar = 5 mm (Figure 3F). Additionally, by using a biopsy puncher, small reproducible punches with a diameter of 4 mm can be generated. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58437/58437fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Immunofluorescence and western blot analyses; fibrotic cocktail effect; protein expression levels." class="xfigimg" src="/files/ftp_upload/58437/58437fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Experimental readouts of human 3D-LTCs after 48h of culture. A human 3D-LTC punch of 4 mm diameter was immunostained for fibronectin (in red) and DAPI (in blue) (Figure 4A-4C). Scale bars = 1,000 µm. Figure 4C shows the merged image. RNA analysis of PCLS by quantitative RT-PCR shows significant increases of COL1A1 and FN1 gene expression by the profibrotic cocktail25. Figure 4E displays an immunoblot of whole protein lysates of PCLS, which were treated with a fibrotic cocktail25. Probing for Vimentin and β-Actin demonstrated an increased protein expression of the mesenchymal marker (vimentin) after treatment with profibrotic factors in patient samples 1, 3, and 4. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58437/58437fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Criterion</td>
			<td>Points</td>
		</tr>
		<tr>
			<td>The tissue sample has intact pleural surface.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>The tissue sample seems macroscopically intact, lacking incisions, squashing, ruptures and distortions.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>The tissue sample contains at least one bronchus with a diameter &gt;1mm.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>The tissue sample contains no or only little amounts of blood.</td>
			<td>4</td>
		</tr>
		<tr>
			<td>The tissue sample was stored fully in medium and shows no obvious sign of atelectasis.</td>
			<td>4</td>
		</tr>
		<tr>
			<td>The tissue sample was resected within the last four hours.</td>
			<td>4</td>
		</tr>
		<tr>
			<td>The tissue sample is larger than 5cm in its largest diameter.</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Score in sum:</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Lung Agarose Filling Score. The Lung Agarose Filling Score (LAFS) correlates with the success-rate to fill a tissue resection with agarose for its subsequent vibratome-based PCLS production. The score sums up all points of criteria met by the tissue. An LAFS equal or above 72 predicts good agarose filling properties, a score below 60 predicts a highly probable failure of agarose filling of the tissue.

Watch this Scientific Journal Video about Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling at JoVE.com

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

generation-human-3d-lung-tissue-cultures-3d-ltcs-for-disease

Research

JoVE Journal

Medicine

20.3K Views.  Ludwig-Maximilians-Universität and Helmholtz Zentrum Munich. Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Video: Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Bioengineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Immunology and Infection

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Developmental Biology

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device&#8217;s capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Summary

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Chapters in this video

Procedure for Lung Engineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

Engineered Lung Tissues Prepared from Decellularized Lung Slices

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture