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><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&#039;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&#039;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 &amp;mu;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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.

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

engineered-lung-tissues-prepared-from-decellularized-lung-slices

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.3K Views.  Yale University. 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.

Video: Engineered Lung Tissues Prepared from Decellularized Lung Slices

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 ...

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

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 ...

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.

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.

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

Medicine

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of suitable donor lungs and both acute and chronic rejection after transplantation. Ascertaining novel bioengineering approaches for the replacement of diseased lungs is imperative for improving patient survival and avoiding complications associated with current transplantation methodologies. An alternative approach involves the use of decellularized whole lungs lacking cellular constituents that are typically the cause of acute and chronic rejection. Since the lung is such a complex organ, it is of interest to examine the extracellular matrix components of specific regions, including the vasculature, airways, and alveolar tissue. The purpose of this approach is to establish simple and reproducible methods by which researchers may dissect and isolate region-specific tissue from fully decellularized lungs. The current protocol has been devised for pig and human lungs, but may be applied to other species as well. For this protocol, four regions of the tissue were specified: airway, vasculature, alveoli, and bulk lung tissue. This procedure allows for the procurement of samples of tissue that more accurately represent the contents of the decellularized lung tissue as opposed to traditional bulk analysis methods.

Author Spotlight: Dissection and Isolation of Region-Specific Decellularized Lung Tissue  

Presented here is a protocol for the isolation of regional decellularized lung tissue. This protocol provides a powerful tool for studying complexities in the extracellular matrix and cell-matrix interactions.

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...