Name: engineered lung tissues prepared from decellularized lung slices
Uploaded: 2022-01-21T13:30:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about Engineered Lung Tissues Prepared from Decellularized Lung Slices at JoVE.com

The authors would like to thank Lorenzo Sewanan and Jorge Nunez for their work developing the tissue culture cassette design used in this protocol, the Kaminski lab for use of their vibratome, Maurizio Chioccioli and Jessica Nouws for assistance with lung slicing, Allie LaRocco for assistance with initial pilot experiments, and Hong Qian for careful reading of the protocol. This work was supported by NIH grants F30HL143880 (K.L.L.), the Medical Scientist Training Program Training Grant T32GM136651 (K.L.L.), and U01HL145567 (L.E.N.); and by an unrestricted research gift from Humacyte Inc. (L.E.N.).

L.E.N. is a founder and shareholder in Humacyte, Inc, which is a regenerative medicine company. Humacyte produces engineered blood vessels from allogeneic smooth muscle cells for vascular surgery. L.E.N.'s spouse has equity in Humacyte, and L.E.N. serves on Humacyte's Board of Directors. L.E.N. is an inventor on patents that are licensed to Humacyte and that produce royalties for L.E.N. L.E.N. has received an unrestricted research gift to support research in her laboratory at Yale. Humacyte did not influence the conduct, description or interpretation of the findings in this report.

This paper describes the use of decellularized precision-cut lung slices as a platform to generate engineered lung tissues in vitro, which contain multi-lineage alveolar-like structures. By combining strategies that we previously developed to repopulate high-fidelity acellular ECM lung scaffolds for whole lung engineering22,31, with our robust system for culturing small-scale engineered heart tissues30, this protocol enables the use of physiologically relevant lung ECM as a tissue culture substrate, in a repeatable and moderate-throughput manner.
The methods presented here detail ELT scaffold preparation from rat lungs, which are readily attainable, may be extracted en bloc with direct access to intact airways for agarose inflation, and are of larger size than mouse lung. However, any lung tissue that can be inflated with agarose and yield slices at least 9 mm in length may be used within this system. Regardless of tissue source, uniform inflation of the lung tissue with agarose is the most critical step for ensuring success of downstream tissue slicing, clipping, and tissue handling. Underinflated lung tissue tends not to slice cleanly, while overinflated tissue may tear during clipping. Following agarose gelation, appropriately inflated tissue regions are firm but provide a little give when gently pressed with forceps. For intact rat lungs, we found that pre-inflating the extracted lungs with air several times, followed by agarose inflation as soon as possible after extraction, results in the best slicing outcomes and best quality of resulting tissue scaffolds. The appropriate volume of agarose needs to be optimized empirically; for a rat lung the volume required to inflate the lung to total lung capacity is approximately 30 mL/kg animal mass (e.g., 10.5 mL agarose for lungs from a 350 g rat). For larger resected lung tissues with less straightforward airway access (such as those from human donors), some additional troubleshooting may be necessary to inflate the tissue via a bronchus32. During subsequent lung slicing, the selection and orientation of the tissue on the plunger is another important step to 1) ensure that the slices are large enough to generate tissue strips that can be clipped into tissue culture cassettes and 2) maximize parenchymal (alveolar) tissue area, excluding large airways or vessels.
Clipping the PCLS into tissue culture cassettes can be a challenging step initially, but the cassettes greatly simplify tissue handling during decellularization and seeding. Two potential problems that may arise are tissue tearing (either during the process of clipping, or during decellularization), or tissue positioning in the clips that results in poor downstream seeding (e.g., no seeding, or seeding just at the ends). Tearing may be the result of agarose overinflation, overstretching of the tissue during tab insertion, or leaving too little overhang to provide adequate tissue grip when inserting the tabs. Note that slices that tear at one clip end may be successfully seeded, however, they are difficult to visualize under the microscope during culture as the tissue is not flat. Poor tissue seeding (such as that in Figure 6C) is likely the result of the slice not lying flat between the two clips, and thus making poor contact with the base of the seeding bath well when flipped upside-down. Another possible cause is improper seating of the cassette in the bottom of the seeding bath well. In terms of clipping, apply slightly more tension in the tissue when placing the second clip to help it lie flat. Some slices have a slight concavity; in these cases clip the slice with the convex side up. With practice, we typically experience failed seeding with fewer than 2% of slices.
One limitation of this protocol is the requirement for some specialized equipment - a laser cutter and a 3D printer - to generate the initial materials for ELT preparation. However, once the tissue culture cassettes and seeding baths are created, no additional special materials are required. The lung slicing and decellularization steps of ELT scaffold preparation are moderately time-consuming; however, these steps may be performed in advance, or in numbers sufficient to prepare for multiple experiments at the same time. Many PCLS (&#62;100 if optimizing for parenchymal regions) can be cut from a single lung and snap-frozen for later use. While a single freeze-thaw cycle may cause minor ultrastructural damage to the ECM46, even multiple freeze-thaw cycles have been demonstrated not to cause a significant loss in ECM23,47. PCLS may also be clipped and decellularized in advance of an experiment, to be used within one month. (Notably, the described decellularization protocol can be accomplished in approximately 6 hours, which represents a significant advantage over previously described methods that require a day or more27,28.) Once the scaffolds are prepared, the cell seeding process is simple and fast, and the culture of ELTs does not require specialized techniques.
A caveat of the described ELT method is the lack of region-specific seeding, i.e., the delivery of AEC2s specifically to the alveolar space, or endothelial cells specifically to the vascular space. Nevertheless, although cells are simply seeded on top of the tissue scaffolds, the pattern of recellularization is non-random, with some semblance of alveolar-like organization, including epithelial rings. We suspect that cell-cell interactions, as well as local differences in ECM composition and geometry20,21, likely contribute to the observed recellularization patterns. In support of this hypothesis, a previously published study, in which fibroblasts were seeded non-specifically onto decellularized lung slices, demonstrated that the pattern of tissue repopulation and associated cellular phenotypes varied significantly by microscopic tissue region and ECM scaffold source (e.g., healthy versus diseased)27. Fibroblasts were also observed to invade into the interstitium - the location in which they reside in native lung tissue1,27. The primary alternative method that we can imagine to culture cells on lung slices in a truly region-specific manner would entail seeding intact decellularized lungs via the airway31,48 and vascular compartments49,50, and then slicing the recellularized tissue. However, this alternative 1) is significantly more cost-, time-, and resource-intensive; 2) is lower throughput; 3) requires increased numbers of animals; and 4) is associated with an increased risk of contamination due to the challenges of whole lung culture and subsequent slicing of the seeded lung. While not recapitulating all aspects of native cellular organization, the ELT platform enables lung cell culture on a physiologically relevant ECM substrate, in a manner that is accessible to many more labs.
The flexibility of the ELT system is a major advantage of this platform, and should allow small-scale lung tissue culture with any number of tissue scaffolds, cells, or culture media of interest. The use of scaffolds derived from diseased tissue or from injury models may permit the study of cell-cell or cell-matrix interactions in the setting of disease-altered ECM27,29,51. However, note that the decellularization protocol may need to be adapted to account for matrix differences among species52. The described seeding strategy can be used for any cell type, and the culture timeline adapted to fit the needs of the researcher. As a starting point, 1 x 106 cells per scaffold should yield a highly cellular tissue within 7 days of culture, whereas 1 x 105 total cells result in poor cellularity. In any adaptation of the timeline, the tissue culture cassettes should be removed from the seeding bath 24 h following the last tissue seeding. Here, with the goal of modeling some of the cellular complexity of the lung alveolus, we describe a tri-culture recellularization strategy that supports the maintenance of well-differentiated neonatal AEC2s in alveolar-like structures for at least 7 days. Our results also demonstrate the successful engraftment of both fibroblasts and endothelial cells within ELTs, emphasizing the broad applicability of the culture substrate and its suitability for co-culture studies. Seeding adult cells in ELTs may facilitate the modeling of more quiescent alveolar structures, while seeding human PSC-derived AEC2s, including those with genetic modifications, could facilitate translational studies of human disease13,53. In general, the bottom-up approach enabled by the ELT platform presents the opportunity to investigate the contributions of particular cell types to readouts of interest - such as AEC2 proliferation or differentiation state.
In summary, this protocol outlines a robust system for generating engineered lung tissues for co-culture studies of AEC2s, fibroblasts, and endothelial cells within acellular ECM lung slice scaffolds. ELTs represent a novel 3D culture strategy for primary AEC2s, which to date have typically relied upon less-physiologic gel-type matrices to maintain a well-differentiated phenotype6,11,12. The current platform builds upon previous work in the repopulation of decellularized lung slices24,25,26,27,28,29, but offers several advantages: 1) a tissue culture cassette system to facilitate ELT handling during decellularization, seeding, and culture; 2) a customized seeding bath to precisely seed a known number of cells on each slice scaffold; and 3) a tri-culture reseeding strategy that enables alveolar tissue repopulation with epithelial, mesenchymal, and endothelial cells. Thus, ELTs represent an important step forward toward creating reproducible in vitro models that capture the cellular and substrate complexity of the native alveolus and the AEC2 stem cell niche.

Alveoli are the functional units of the distal lung, comprising a meshwork of gas-exchanging airspaces lined by alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s). Underlying the epithelium is a dense network of capillaries as well as supporting mesenchyme, all buttressed by an extracellular matrix (ECM) scaffold that provides both strength and flexibility to these delicate air sacs1. The alveoli are also the site of injury in numerous lung pathologies, including idiopathic pulmonary fibrosis2, acute respiratory distress syndrome3, and severe coronavirus disease-19 (COVID-19)4. Although work over the past decade has uncovered a remarkable plasticity within the lung epithelium, the mechanisms that enable distal lung repair in some settings - and that preclude repair in others - remain an area of intense investigation5. The development of improved in vitro platforms to model the alveolus would facilitate studies of alveolar biology, regeneration, and therapeutics.
AEC2s self-renew and differentiate into AEC1s, and thus are considered the primary stem cell of the distal lung6,7,8. However, these cells pose a particular challenge to in vitro study given the difficulties associated with culturing primary AEC2s without a loss of phenotype9. In conventional 2-dimensional (2D) culture, AEC2s flatten and adopt some features of AEC1-like cells10. In contrast, 3D culture strategies, most commonly organoids, support the maintenance of differentiated features in primary AEC2s6,11,12 and permit long-term culture of pluripotent stem cell (PSC)-derived AEC2s13,14. Organoids have been used to model distal lung development15, viral infection11,15, and AEC2-related genetic disease13,16,17, enabling important insights into AEC2 biology and regeneration. However, these culture models typically comprise just one or two cellular lineages, and embed the cells in gel-type matrices that fail to recapitulate either the architecture or the ECM substrate of the native lung alveolus.
The ECM is a critical regulator of cell phenotype and behavior via molecular, topological, and mechanical cues; comprises a key component of tissue-specific niches regulating stem cell fate; and serves as a reservoir that modulates the availability of locally-secreted growth factors18,19,20,21. Culturing cells on native ECM may thus increase the predictive capacity of in vitro systems to model the biology of in vivo tissues. Decellularization, a process that removes cellular material from tissues via detergents, enzymes, or physical or other methods, can preserve in large part the ECM scaffolding of a native organ, when carefully performed22,23. Such scaffolds can be repopulated with cells for 3D biomimetic culture. However, while decellularized scaffolds are widely used for tissue engineering applications, their use for routine cell culture has been limited. Several previous studies have reported the decellularization and recellularization of lung slices or small lung tissue segments. In addition to proof-of-concept studies24,25,26, repopulated lung slices have been used to study fibroblast-matrix adhesion27,28 and to investigate the effect of diseased lung matrices on fibroblast phenotype27,29. With improved technologies available for generating precision-cut tissue slices, decellularized lung slices could offer a convenient and small-scale platform with which to culture cells, while preserving alveolar, airway, and vascular substructures. Incorporating multiple cell types would enable studies of cell-cell interactions within a physiologically relevant 3D environment. However, improved strategies are needed to facilitate the handling of tissues throughout the culture process, and to ensure controlled and reproducible seeding of tissues with known numbers of cells.
Here, we present a protocol to generate engineered lung tissues (ELTs) by repopulating decellularized precision-cut lung slices (PCLS) with primary endothelial cells, AEC2s, and fibroblasts. In an adaptation of our previously described engineered heart tissue system30 and whole lung decellularization-recellularization strategies22,31, we describe procedures to cut PCLS from rat lungs and to clip the slices into reusable tissue culture cassettes that simplify and standardize downstream manipulations. Clipped slices are decellularized to form acellular ECM scaffolds, which are repopulated in customized seeding baths. Lung slice scaffolds preserve critical ECM components and architecture, and support the growth of AEC2s within multi-lineage alveolar-like structures for at least 7 days. ELTs represent a novel alveolar co-culture system within a physiologically relevant 3D matrix, which should support the development of lung tissue engineering strategies, while facilitating basic biological studies of AEC2s and the alveolus.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Printer: Form 2</td>
			<td>Formlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Bromo cAMP</td>
			<td>Sigma</td>
			<td>B7880</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, UltraPure LMP</td>
			<td>Invitrogen</td>
			<td>15517-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Barbed reducer fitting, 3/8 inch x 1/4 inch</td>
			<td>McMaster-Carr</td>
			<td>5121K271</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Sigma</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) Fraction V</td>
			<td>Gemini</td>
			<td>700-104P</td>
			<td>For AEC2 growth medium</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), standard grade</td>
			<td>Gemini</td>
			<td>700-100P</td>
			<td>For benzonase buffer</td>
		</tr>
		<tr>
			<td>Check valve, polypropylene, 1/8 inch hose barb</td>
			<td>Cole-Parmer</td>
			<td>SK-98553-10</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>PeproTech</td>
			<td>2520691</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Resin, 1 L</td>
			<td>Formlabs</td>
			<td>RS-F2-GPCL-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue, such as Krazy Maximum Bond Permanent Glue</td>
			<td>Any hardware, craft, or drug store</td>
			<td>KG483 or similar</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (low glucose)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA assay (Quant-iT PicoGreen dsDNA Assay Kit)</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M, pH 8.0</td>
			<td>AmericanBio</td>
			<td>AB00502-01000</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU kit (Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647)</td>
			<td>Invitrogen</td>
			<td>C10340</td>
			<td>Used according to manufacturer&#39;s directions</td>
		</tr>
		<tr>
			<td>Elbow fitting, 3/8 inch</td>
			<td>McMaster-Carr</td>
			<td>5121K907</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS), characterized</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>Gemini</td>
			<td>400-100P</td>
			<td>Reconstituted in diH2O for a stock solution at 50 mg/mL</td>
		</tr>
		<tr>
			<td>Hair clippers</td>
			<td>Wahl</td>
			<td>MiniArco</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS), Phenol Red Free</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium injection, USP, 1000 U/mL</td>
			<td>Sagent</td>
			<td>NDC: 25021-400-30</td>
			<td>For intraperitoneal and intracardiac injection</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td>For pulmonary artery perfusion; prepare stock solution at 100 U/mL in PBS</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Corning</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline tee fitting, 3/8 inch x 1/8 inch</td>
			<td>McMaster-Carr</td>
			<td>5121K851</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculating loop, disposable</td>
			<td>Fisherbrand</td>
			<td>22-363-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine injection, 100 mg/mL</td>
			<td>Covetrus (Butler Animal Health)</td>
			<td>010177</td>
			<td></td>
		</tr>
		<tr>
			<td>KGF, recombinant human</td>
			<td>PeproTech</td>
			<td>100-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser cutter, VLS 3.50 30 watt</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-lock, female, 3/32 inch</td>
			<td>Cole-Parmer</td>
			<td>45508-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-lock, male, 1/8 inch</td>
			<td>Cole-Parmer</td>
			<td>30800-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-lock, male, 1/4 inch</td>
			<td>McMaster-Carr</td>
			<td>51525K146</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB-131 Complete without serum</td>
			<td>VEC Technologies</td>
			<td>MCDB-131 WOFBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2), 1 M</td>
			<td>AmericanBio</td>
			<td>AB09006-00100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>American Bioananalytical</td>
			<td>AB01915</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), without Ca2+ and Mg2+, 10X</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>Reconstitute to 1X with diH2O</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), with Ca2+ and Mg2+</td>
			<td>Gibco</td>
			<td>21300-058</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS - SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Dow Corning Corporation</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10,000 U/mL penicillin/10,000 &#956;g/mL streptomycin)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 150 mm</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic film (parafilm)</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmed BPT tubing, LS 16</td>
			<td>Masterflex</td>
			<td>06508-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmed BPT tubing, LS 17</td>
			<td>Masterflex</td>
			<td>06508-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum-cured silicone tubing, LS 14</td>
			<td>Masterflex</td>
			<td>96420-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum-cured silicone tubing, LS 16</td>
			<td>Masterflex</td>
			<td>96420-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum-cured silicone tubing, LS 36</td>
			<td>Masterflex</td>
			<td>96410-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Poloxamer 407 (Pluronic F-127)</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone/iodine prep pads, 10%</td>
			<td>Dynarex Corporation</td>
			<td>1108</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE sheet, 0.060 inch (1/16 inch) thick</td>
			<td>ePlastics</td>
			<td>PTFENAT0.060X12X12</td>
			<td>For tissue culture cassette tabs</td>
		</tr>
		<tr>
			<td>PTFE sheet, 0.093 inch (3/32 inch) thick</td>
			<td>ePlastics</td>
			<td>PTFENAT0.093X12X12</td>
			<td>For tissue culture cassette frames and clips</td>
		</tr>
		<tr>
			<td>Peristaltic pump drive: Masterflex L/S Variable-Speed Digital Drive</td>
			<td>Cole-Parmer</td>
			<td>ZM-07528-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump head: Masterflex L/S Easy-Load II Pump Head</td>
			<td>Cole-Parmer</td>
			<td>EW-77202-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat, Sprague Dawley</td>
			<td>Charles River</td>
			<td>Strain Code: 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Any hardware or craft store</td>
			<td>Personna 94-120-71 or similar</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary blades, 28 mm</td>
			<td>Omnigrid</td>
			<td>2046</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary cutter, 28 mm</td>
			<td>Olfa</td>
			<td>Model 9551</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate (SDC)</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrorusside (SNP)</td>
			<td>Sigma</td>
			<td>71778</td>
			<td></td>
		</tr>
		<tr>
			<td>Stopcock, 4-way</td>
			<td>Edwards</td>
			<td>594WSC</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 4-0 monofilament polypropylene</td>
			<td>Covidien</td>
			<td>VP-557-X</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 50 mL</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish, 35 mm</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish, 100 mm</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 6-well</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate 12-well</td>
			<td>Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin human</td>
			<td>Sigma</td>
			<td>T8158</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris, 1 M solution, pH 8.0</td>
			<td>AmericanBio</td>
			<td>AB14043-01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>American Bioanalytical</td>
			<td>AB02025-00500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome, Compresstome VF-300-0Z</td>
			<td>Precisionary Instruments LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, 100 mg/mL</td>
			<td>Henry Schein</td>
			<td>NDC: 11695-4022-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-connector, 1/16 inch barbed</td>
			<td>Cole-Parmer</td>
			<td>30614-43</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

An overview of the process to generate ELTs - comprising lung slicing, slice clipping and decellularization, and scaffold repopulation - is presented in Figure 3. The ELTs presented here were cultured using primary rat lung microvascular endothelial cells (see Table of Materials), neonatal rat AEC2s, and lipofibroblast-enriched neonatal rat lung fibroblasts36. AEC2s were freshly isolated via magnetic bead-based sorting as previously described37; alternative isolation protocols have been detailed and discussed elsewhere38,39,40. Purity of isolated rat AEC2s can be assessed via flow cytometry for rat-specific AEC2 surface marker RTII-7041, or via staining of a cytocentrifuged cell sample for RTII-70 or pro-surfactant protein C (pSPC). Rat lung fibroblasts were isolated from postnatal day 7-9 rat pups according to an adaptation of a published protocol42 and used at passage 1-2; alternative isolation protocols have been described elsewhere43,44. Purity of isolated fibroblasts can be assessed via staining of cultured or cytocentrifuged cells for mesenchymal marker vimentin, and lipofibroblast enrichment can be assessed via staining for Oil Red O45.
When the lung tissue is uniformly inflated with agarose, and tissue pieces strategically selected and oriented for slicing so as to maximize total and parenchymal tissue area, one rat lung may yield tissue for &#62;100 alveolar ELTs. Strips of PCLS exhibit sufficient mechanical integrity to be clipped into tissue cassettes with few (&#60;5%) instances of tearing (Figure 3B).
The protocol for decellularizing lung slices is closely based on our previously published whole lung decellularization protocol, which by quantitative proteomics was demonstrated to preserve many ECM components at levels not significantly different from those in native lung22. Decellularized slice scaffolds preserve the native architecture of the alveoli, as viewed by hematoxylin and eosin (H&#38;E) staining (Figure 4A,B) and by phase contrast microscopy (Figure 4C). We typically exclude scaffolds containing large airways or vessels (Figure 4D) or tears, although the former can be included if they are of interest to the researcher. Decellularization leads to a 96% reduction in tissue DNA content as measured by an assay for double-stranded DNA (see Table of Materials; 0.50 &#181;g/mg &#177; 0.073 &#181;g/mg vs 0.018 &#181;g/mg &#177; 0.0035 &#181;g/mg in native vs decellularized tissue, respectively, mean &#177; SEM) (Figure 5A), with no DNA visible by hematoxylin staining (Figure 4B). Histological and immunofluorescent staining of decellularized scaffolds reveals maintenance of ECM proteins collagen, elastin, collagen IV, and laminin with architecture and quantity similar to that in native lung slices (Figure 5B-E). Note that the nuclei of native tissues stain blue/black with trichome (for collagen) and EVG (for elastin) stains. Immunofluorescent staining was performed as described previously, using standard methods for staining tissues37. The antibodies used and their respective concentrations are listed in Table 4.
Successful scaffold repopulation leads to highly cellular ELTs after 7 days, with an alveolar-like repopulation pattern visible by light microscopy (Figure 6A-C). In some cases, with very high cellularity, organoid-type structures may be visible (Figure 6A,B). Unsuccessful tissue seeding can be visualized by phase-contrast microscopy during culture (Figure 6C). After culturing tissue scaffolds with AEC2s, fibroblasts, and endothelial cells, ELTs are densely repopulated with alveolar-like structures comprising all three cell lineages (Figure 6D,E). At day 7 or 8, AEC2s maintain cuboidal morphology and express surfactant protein-B (SPB) and lamellar body protein ABCA3, without evidence of significant differentiation to AEC1s (Figure 6E,F). AEC2s are highly proliferative in ELTs, as demonstrated by 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation following a 2 h pulse at 10 &#181;M (Figure 6G).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63151/63151fig01.jpg" /> 
Figure 1: Schematic of perfusion system for lung extraction and clearing. (A) The perfusion system comprises a gravity-driven limb, used for initial cannulation of the pulmonary artery under flow; and a pump-driven limb, used to clear the lungs efficiently after initial cannulation. The pump line includes a &#34;pulse dampener&#34; that dampens the spikes in pressure caused by the pump. The design of the tracheal and pulmonary arterial cannulas is detailed at left. SNP = sodium nitroprusside. (B) Details of pulse dampener assembly. BPT and silicone refer to types of tubing. (C) Positions of tracheal and pulmonary arterial cannulas placed during lung extraction. <a href="https://www.jove.com/files/ftp_upload/63151/63151fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63151/63151fig02.jpg" /> 
Figure 2: Culture timeline for tri-lineage recellularization. Proposed timeline for tri-lineage ELT seeding and culture, including timing of two-phase seeding. Cell numbers for seeding and culture medium for each phase are indicated. See culture media details in Table 3. AEC2 = alveolar epithelial type 2 cell. EC = endothelial cell. FB = fibroblast. <a href="https://www.jove.com/files/ftp_upload/63151/63151fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63151/63151fig03.jpg" /> 
Figure 3: Schematic of engineered lung tissue preparation. (A) Native lung tissue is cut into slices using a vibratome. (B) Precision-cut lung slices are cut into standardized 3 mm wide strips, clipped into polytetrafluorethylene (PTFE) tissue-culture cassettes, and detergent-decellularized to yield acellular extracellular matrix scaffolds. (C) Scaffolds are reseeded in specialized seeding baths that confine the seeding area to the area of the tissue, and then cultured in a standard well plate. <a href="https://www.jove.com/files/ftp_upload/63151/63151fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63151/63151fig04.jpg" /> 
Figure 4: Structure of decellularized lung scaffolds. H&#38;E staining of native (A) and decellularized (B) lung slices showing preservation of alveolar architecture after decellularization. (C,D) Examples of decellularized ECM scaffolds viewed at 5x magnification by phase contrast microscopy, comprising predominately alveolar tissue (C) or containing large branching airways and vessels (D, black and red arrowheads). Scale bars, 50 &#181;m (A,B); 500 &#181;m (C,D). <a href="https://www.jove.com/files/ftp_upload/63151/63151fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63151/63151fig05.jpg" /> 
Figure 5: DNA removal and matrix preservation in decellularized lung scaffolds. (A) Quantification of DNA in native and decellularized lung slices (mean &#177; SEM, n = 5). Welch&#39;s t test, **P &#60;&#160;0.01. Decell = decellularized. (B,C) Histological staining of native and decellularized lung slices for collagen (B) and elastin (C). Arrowheads, elastin preserved in alveolar entrance rings of decellularized tissue. (D,E) Immunofluorescent staining of native and decellularized lung slices for collagen IV (D) and laminin (E). Scale bars, 50 &#181;m. In all panels, dotted boxes outline the image region that is magnified to the right in each respective panel. <a href="https://www.jove.com/files/ftp_upload/63151/63151fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63151/63151fig06.jpg" /> 
Figure 6: Cellular repopulation of engineered lung tissues. (A-C) Examples of recellularized ELTs on day 7 of culture, as visualized during culture by phase contrast microscopy. The recellularization pattern mirrors the alveolar structure of the tissue. In some areas of high cellularity, organoid-like structures may form (arrowheads). (A) and (B) represent successful cell repopulation, whereas (C) represents a poor level of recellularization after 7 days of culture. (D-G) Staining of recellularized ELTs on day 7 or 8 of culture. (D) H&#38;E staining showing cellular repopulation of the alveolar septa. (E) Immunofluorescent staining labels engrafted proCollagenI&#945;1+ fibroblasts, ABCA3+ AEC2s, and CD31+ endothelial cells. (F) Tissues contain abundant SPB+ AEC2s but few RTI-40 (podoplanin)+ AEC1s under these conditions. (G) Many AEC2s are proliferating in ELTs, as measured by EdU incorporation. Scale bars, 500 &#181;m (A-C); 25 &#181;m (D-G). <a href="https://www.jove.com/files/ftp_upload/63151/63151fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Decellularization solutions. Preparation details for decellularization solutions. <a href="https://www.jove.com/files/ftp_upload/63151/Table 1 - Decellularization solutions_Revised.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Decellularization protocol. Details of protocol for decellularizing lung slices. <a href="https://www.jove.com/files/ftp_upload/63151/Table 2 - Decellularization protocol.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Culture media. Preparation details for endothelial and AEC2 growth media. <a href="https://www.jove.com/files/ftp_upload/63151/Table 3 - Culture media.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Antibodies used for immunostaining. Details of antibodies and their concentrations used for immunostaining. <a href="https://www.jove.com/files/ftp_upload/63151/Table 4 - Antibodies used for immunostaining.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Design for laser cutting tissue culture cassette frames. <a href="https://www.jove.com/files/ftp_upload/63151/Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Design for laser cutting tissue culture cassette clips. <a href="https://www.jove.com/files/ftp_upload/63151/Supplementary File 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Design for laser cutting tissue culture cassette tabs. <a href="https://www.jove.com/files/ftp_upload/63151/Supplementary File 3.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 4: CAD file for seeding bath mold base. <a href="https://www.jove.com/files/ftp_upload/63151/Supplementary File 4.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 5: CAD file for seeding bath mold ring. <a href="https://www.jove.com/files/ftp_upload/63151/Supplementary File 5.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Engineered Lung Tissues Prepared from Decellularized Lung Slices at JoVE.com

Engineered Lung Tissues Prepared from Decellularized Lung Slices

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.

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