Name: Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells (Video) | JoVE Journal
Uploaded: 2025-03-28T13:25:00.000+00:00
Duration: 10 min 13 s
Description: Learn a standardized protocol for bioengineering mouse lungs using decellularization and recellularization with primary human endothelial cells. This method enables scalable organ transplantation research.

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5 minutes

This study was financially supported by the Grant-in-Aid for Scientific Research / KAKENHI (C) #20K09174, #23K08308, the Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)) #22KK0132 for TS, JSPS KAKENHI Grant Number 21K08877 for TW, Leave a Nest Grant Ikeda-Rika award for FT, and the Grant-in-Aid for JSPS Fellows #21J21515 for FT. We greatly appreciate Ms. Maiko Ueda, technical staff in the Biomedical Research Core of Tohoku University Graduate School of Medicine, for her intensive work in histological observation. We also appreciate the technical advice of Ms. Yumi Yoshida and Mr. Koji Kaji in the Center of Research Instruments at IDAC, Tohoku University, for their image processing support.

<ol><li>van der Mark, S. C., Hoek, R. A. S., Hellemons, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developments+in+lung+transplantation+over+the+past+decade%5BTitle%5D)+AND+%22Eur+Respir+Rev%22%5BJournal%5D)">Developments in lung transplantation over the past decade.</a> Eur Respir Rev. 29 (157), 190132(2020).</li>
<li>Valapour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OPTN%2FSRTR+2022+Annual+Data+Report%3A+Lung%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">OPTN/SRTR 2022 Annual Data Report: Lung.</a> Am J Transplant. 24 (2S1), S394-S456 (2024).</li>
<li>Hoffman, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Waiting+list+dynamics+and+lung+transplantation+outcomes+after+introduction+of+the+lung+allocation+score+in+the+Netherlands%5BTitle%5D)+AND+%22Transplant+Direct%22%5BJournal%5D)">Waiting list dynamics and lung transplantation outcomes after introduction of the lung allocation score in the Netherlands.</a> Transplant Direct. 7 (10), e760(2021).</li>
<li>Wilk, A. R., Edwards, L. B., Edwards, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+augmenting+OPTN+data+with+external+death+data+on+calculating+patient+survival+rates+after+organ+transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">The effect of augmenting OPTN data with external death data on calculating patient survival rates after organ transplantation.</a> Transplantation. 101 (4), 836-843 (2017).</li>
<li>Neizer, H., Singh, G. B., Gupta, S., Singh, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Addressing+donor-organ+shortages+using+extended+criteria+in+lung+transplantation%5BTitle%5D)+AND+%22Ann+Cardiothorac+Surg%22%5BJournal%5D)">Addressing donor-organ shortages using extended criteria in lung transplantation.</a> Ann Cardiothorac Surg. 9 (1), 49-50 (2020).</li>
<li>Oliveira, P., Yamanashi, K., Wang, A., Cypel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+an+ex+vivo+lung+perfusion+rat+model+for+translational+insights+in+lung+transplantation%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Establishment of an ex vivo lung perfusion rat model for translational insights in lung transplantation.</a> J Vis Exp. (199), e65981(2023).</li>
<li>Anand, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+testing+of+a+humanized+porcine+donor+for+xenotransplantation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Design and testing of a humanized porcine donor for xenotransplantation.</a> Nature. 622 (7982), 393-401 (2023).</li>
<li>Shakir, S., Hackett, T. L., Mostaco-Guidolin, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioengineering+lungs%3A+An+overview+of+current+methods%2C+requirements%2C+and+challenges+for+constructing+scaffolds%5BTitle%5D)+AND+%22Front+Bioeng+Biotechnol%22%5BJournal%5D)">Bioengineering lungs: An overview of current methods, requirements, and challenges for constructing scaffolds.</a> Front Bioeng Biotechnol. 10, 1011800(2022).</li>
<li>Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat Med. 16 (8), 927-933 (2010).</li>
<li>Petersen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+lungs+for+in+vivo+implantation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Tissue-engineered lungs for in vivo implantation.</a> Science. 329 (5991), 538-541 (2010).</li>
<li>Leiby, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+engineering+of+lung+alveolar+epithelium%5BTitle%5D)+AND+%22NPJ+Regen+Med%22%5BJournal%5D)">Rational engineering of lung alveolar epithelium.</a> NPJ Regen Med. 8 (1), 22(2023).</li>
<li>Kitano, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+transplantation+of+human+bioartificial+lung+grafts+in+a+porcine+model%3A+A+feasibility+study%5BTitle%5D)+AND+%22Semin+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Orthotopic transplantation of human bioartificial lung grafts in a porcine model: A feasibility study.</a> Semin Thorac Cardiovasc Surg. 34 (2), 752-759 (2022).</li>
<li>Ohata, K., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human-scale+lung+regeneration+based+on+decellularized+matrix+scaffolds+as+a+biologic+platform%5BTitle%5D)+AND+%22Surg+Today%22%5BJournal%5D)">Human-scale lung regeneration based on decellularized matrix scaffolds as a biologic platform.</a> Surg Today. 50 (7), 633-643 (2020).</li>
<li>Ren, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+pulmonary+vasculature+in+decellularized+rat+and+human+lungs%5BTitle%5D)+AND+%22Nat+Biotech%22%5BJournal%5D)">Engineering pulmonary vasculature in decellularized rat and human lungs.</a> Nat Biotech. 33 (10), 1097-1102 (2015).</li>
<li>Doi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+bioengineered+rat+lungs+recellularized+with+endothelial+and+adipose-derived+stromal+cells%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Transplantation of bioengineered rat lungs recellularized with endothelial and adipose-derived stromal cells.</a> Sci Rep. 7 (1), 8447(2017).</li>
<li>Stone, K. C., Mercer, R. R., Gehr, P., Stockstill, B., Crapo, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allometric+relationships+of+cell+numbers+and+size+in+the+mammalian+lung%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">Allometric relationships of cell numbers and size in the mammalian lung.</a> Am J Respir Cell Mol Biol. 6 (2), 235-243 (1992).</li>
<li>Basil, M. C., Morrisey, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+regeneration%3A+a+tale+of+mice+and+men%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Lung regeneration: a tale of mice and men.</a> Semin Cell Dev Biol. 100, 88-100 (2020).</li>
<li>Hsia, C. C., Hyde, D. M., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+structure+and+the+intrinsic+challenges+of+gas+exchange%5BTitle%5D)+AND+%22Compr+Physiol%22%5BJournal%5D)">Lung structure and the intrinsic challenges of gas exchange.</a> Compr Physiol. 6 (2), 827-895 (2016).</li>
<li>Tomiyama, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+transplantation+of+the+bioengineered+lung+using+a+mouse-scale+perfusion-based+bioreactor+and+human+primary+endothelial+cells%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Orthotopic transplantation of the bioengineered lung using a mouse-scale perfusion-based bioreactor and human primary endothelial cells.</a> Sci Rep. 14 (1), 7040(2024).</li>
<li>Stoian, A., Adil, A., Biniazan, F., Haykal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+decades+of+advances+and+limitations+in+organ+recellularization%5BTitle%5D)+AND+%22Curr+Issues+Mol+Biol%22%5BJournal%5D)">Two decades of advances and limitations in organ recellularization.</a> Curr Issues Mol Biol. 46 (8), 9179-9214 (2024).</li>
<li>Crabbe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recellularization+of+decellularized+lung+scaffolds+is+enhanced+by+dynamic+suspension+culture%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Recellularization of decellularized lung scaffolds is enhanced by dynamic suspension culture.</a> PLoS One. 10 (5), e0126846(2015).</li>
<li>Daly, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initial+binding+and+recellularization+of+decellularized+mouse+lung+scaffolds+with+bone+marrow-derived+mesenchymal+stromal+cells%5BTitle%5D)+AND+%22Tissue+Eng+Part+A%22%5BJournal%5D)">Initial binding and recellularization of decellularized mouse lung scaffolds with bone marrow-derived mesenchymal stromal cells.</a> Tissue Eng Part A. 18 (1-2), 1-16 (2012).</li>
<li>Regulations for Animal Experiments and Related Activities. , Tohoku University. at
 <a target="_blank" href=" https://www.clag.med.tohoku.ac.jp/clar/en/"> https://www.clag.med.tohoku.ac.jp/clar/en/</a> (2024).</li>
<li>Sokocevic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+age+and+emphysematous+and+fibrotic+injury+on+the+re-cellularization+of+de-cellularized+lungs%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs.</a> Biomaterials. 34 (13), 3256-3269 (2013).</li>
<li>Ren, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+non-invasive+assessment+of+cell+viability+and+proliferation+in+bio-engineered+whole+organ+constructs%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs.</a> Biomaterials. 52, 103-112 (2015).</li>
<li>Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+attenuate+ischemia-reperfusion+injury+after+prolonged+cold+ischemia+in+a+mouse+model+of+lung+transplantation%3A+a+preliminary+study%5BTitle%5D)+AND+%22Surg+Today%22%5BJournal%5D)">Mesenchymal stem cells attenuate ischemia-reperfusion injury after prolonged cold ischemia in a mouse model of lung transplantation: a preliminary study.</a> Surg Today. 47 (4), 425-431 (2017).</li>
<li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Donor+IL-17+receptor+A+regulates+LPS-potentiated+acute+and+chronic+murine+lung+allograft+rejection%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Donor IL-17 receptor A regulates LPS-potentiated acute and chronic murine lung allograft rejection.</a> JCI Insight. 8 (21), e158002(2023).</li>
</ol>

The authors do not have any conflicts of interest regarding this manuscript.

Organ bioengineering is a demanding enterprise. The costly screening process has been hindering this field&#39;s research and development cycle. By using mice as an experimental platform, space, cells, and media are significantly reduced compared to the previously used rat platform. Although measuring detailed physical parameters such as gas exchange, vascular resistance, or lung compliance has not been achieved yet, the mouse lung model allows for accelerated research timelines as it enables rapid iteration of experimen...

Lung transplantation has been the decisive cure for patients having end-stage lung disease1 such as idiopathic pulmonary fibrosis, where drug treatment is ineffective to stop the deterioration of respiratory function. More eligible patients add up to the waiting list every year; however, the number of organ donations from deceased donors has been trailing the increasing number of waiting patients2,3. Even after undergoing lung transplantation, quite a few problems would degrade the function of transplanted lungs, including primary organ dysfunction, reactive allogenic syndrome, and infections, which significantly lower the 5-year survival of the lung transplantation recipients4.
Several options exist to counter the current problems in organ transplantation, including the utilization of marginal donors5, recovery of donor lungs in an ex vivo lung perfusion system6, and xenotransplantation using gene-edited swine7. These alternatives can expand the pool of donor organs; however, none can entirely address the donor organs&#39; scarcity, immunogenicity, and functional heterogeneity.
It is far from reality but bioengineered artificial organs where patient-specific cells are integrated into the decellularized animal organ scaffold are a fascinating potential source of solid organ transplantation8. Several pioneering studies that demonstrated the potential utility of bioengineered lungs have been reported since 20109,10. In these studies, lungs from rats or swine were decellularized by detergents, animal or human cells were injected from the trachea or pulmonary vasculature to regenerate the lung tissue in the perfusion-based bioreactor, and some of them were transplanted orthotopically into the animal thoracic cavities11,12,13,14,15. However, the function and structure of the bioengineered lungs were premature, presumably because of the inadequate number of cells cultured in the bioreactor or less integrated intercellular junctions.
One obstacle to advancing research in organ bioengineering is the lack of a small-scale experimental platform. While rats or swine are the commonly used animals in this field, they require &#62;108 lung cells per lung16, which is highly costly to academic labs. If mice are available for organ bioengineering research, we could dramatically reduce the cost of each experiment and speed up the research program. Although there exist anatomical differences between mouse and human lungs17, the basic architecture of the lung is similar across mammals18. Therefore, the results of mouse-scale experiments can apply to larger animals by simply multiplying the number according to the body size.
This protocol aims to describe the detailed experimental procedure of lung bioengineering using mouse heart-lung blocks and human primary cells19. We adopted previously reported and widely used mouse lung decellularization protocol for this study20,21,22. The challenging part of lung bioengineering is the recellularization of the decellularized capillary vasculature20; therefore, human umbilical cord vein endothelial cells will be used in this protocol.

<table><tbody><tr><td>DECELLULARIZATION</td><td></td><td></td><td></td></tr><tr><td>27 G x 1/2 in. BD PrecisionGlide Needle</td><td>BD</td><td>305109</td><td>Or equivalent 27 G injection needle</td></tr><tr><td>BD Insyte IV Catheter 20 GA X 1.8 8IN</td><td>BD</td><td>381237</td><td>Or equivalent 20 G IV catheter</td></tr><tr><td>Blade silk suture (4-0)</td><td>Nesco</td><td>GA04SB</td><td>Or equivalent</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Catheter for rat jugular vein, PU 2Fr 10 cm</td><td>Instech</td><td>C20PU-MJV1301</td><td>Recommended for mice weighs 30 g and under.</td></tr><tr><td>Catheter for rat jugular vein, PU 3Fr 10 cm</td><td>Instech</td><td>C30PU-RJV1307</td><td>Recommended for mice weighs over 30 g.</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>PinPort injectors</td><td>Instech</td><td>PNP3M</td><td></td></tr><tr><td>PinPorts, 22 G</td><td>Instech</td><td>PNP3F22-50</td><td>Fits C30PU-RJV1307</td></tr><tr><td>PinPorts, 25 G</td><td>Instech</td><td>PNP3F25-50</td><td>Fits C20PU-MJV1301</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Sterile syringe, 5 mL</td><td>Generic</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>9036-19-5</td><td></td></tr><tr><td>CELL CULTURE</td><td></td><td></td><td></td></tr><tr><td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>HUVEC &amp;ndash; Human Umbilical Vein Endothelial Cells</td><td>Lonza</td><td>C2519A</td><td></td></tr><tr><td>PERFUSION-BASED BIOREACTOR</td><td></td><td></td><td></td></tr><tr><td>20 G needle</td><td>Generic</td><td></td><td></td></tr><tr><td>3-way stopcock</td><td>Generic</td><td></td><td></td></tr><tr><td>Cork borer</td><td>Generic</td><td></td><td>Boring size, 6-10 mm</td></tr><tr><td>EasyLoad III pump head</td><td>Cole-Parmer</td><td>243934</td><td></td></tr><tr><td>Glass canister</td><td>Hario</td><td>SCN-200T</td><td>Inner diameter: 80 mm</td></tr><tr><td>Heating magnetic stirrer</td><td>Generic</td><td></td><td></td></tr><tr><td>Lure fitting, PVDF, For Soft Tube</td><td>Nordson Medical</td><td>2-9965-01</td><td>Female, fits tubing with I.D. 1.5 mm (L/S 14)</td></tr><tr><td>Lure fitting, PVDF, For Soft Tube</td><td>Nordson Medical</td><td>2-9964-01</td><td>Male, fits tubing with I.D. 1.5 mm (L/S 14)</td></tr><tr><td>Lure fitting, PVDF, For Soft Tube</td><td>Nordson Medical</td><td>2-9965-03</td><td>Female, fits tubing with I.D. 3 mm (L/S 16)</td></tr><tr><td>Lure fitting, PVDF, For Soft Tube</td><td>Nordson Medical</td><td>2-9964-03</td><td>Male, fits tubing with I.D. 3 mm (L/S 16)</td></tr><tr><td>Magnetic stirring bar</td><td>Generic</td><td></td><td></td></tr><tr><td>Masterflex L/S Digital Precision Modular Drive with Remote I/O and Benchtop Controller</td><td>Cole-Parmer</td><td>07557-00</td><td></td></tr><tr><td>Masterflex L/S Precision Pump Tubing, PharMed BPT, L/S 16</td><td>Cole-Parmer</td><td>06508-16</td><td></td></tr><tr><td>Masterflex L/S Pricision Pump Tubing, Platinum-Cured Silicone, L/S 14</td><td>Cole-Parmer</td><td>96410-14</td><td></td></tr><tr><td>Millex-GP Syringe Filter Unit, 0.22 &amp;micro;m, polyethersulfone, 33 mm, gamma sterilized</td><td>Millipore</td><td>SLGPR33RS</td><td></td></tr><tr><td>Pyrex 250 mL grass bottle, GL-45 screw cap</td><td>Corning</td><td>1395-250</td><td></td></tr><tr><td>Silicon Septa for GL45 Open Top PBT Screw Cap</td><td>Corning</td><td>1395-455S</td><td></td></tr><tr><td>Silicone Light Stopper</td><td>IMG</td><td>07763-18</td><td>Upper diameter: 87 mm, Lower diameter: 75 mm</td></tr><tr><td>Sterile syringe, 10 mL, 50 mL</td><td>Generic</td><td></td><td></td></tr><tr><td>MOUSE SURGERY (Isolation of the heart-lung block | Lung transplantation)</td><td></td><td></td><td></td></tr><tr><td>10-0 Nylon ties</td><td>Kono Seisakusho</td><td>N/A</td><td></td></tr><tr><td>10-0 Silk ties</td><td>Kono Seisakusho</td><td>N/A</td><td></td></tr><tr><td>4-0 Silk ties</td><td>Kono Seisakusho</td><td>N/A</td><td></td></tr><tr><td>Arterial clamp, 45 mm curved, grooved</td><td>Natsume seisakusyo</td><td>C-17-45</td><td></td></tr><tr><td>BD Insyte IV Catheter 24GA</td><td>BD</td><td>381512</td><td>Or equivalent 24G i.v. catheter</td></tr><tr><td>Bulldog Vascular Forceps 45mm curved</td><td>Natsume seisakusyo</td><td>M2</td><td></td></tr><tr><td>Butorphanol tartrate&amp;nbsp;</td><td>Meiji Seika Pharma</td><td>N/A</td><td></td></tr><tr><td>Cefazolin Sodium</td><td>Otsuka Pharmaceutical</td><td>N/A</td><td></td></tr><tr><td>Dumont forceps #5/45</td><td>Fine Science Tools</td><td>1251-35</td><td></td></tr><tr><td>Fine vannas style spring scissors</td><td>Fine Science Tools</td><td>15403-08</td><td>45&amp;deg; tip, 0.01 x 0.06 mm</td></tr><tr><td>Gemini Cautery Kit</td><td>Harvard Apparatus</td><td>RS-300</td><td></td></tr><tr><td>Halsted-Mosquito clamp curved tip, 125 mm</td><td>Bioresearch center</td><td>16181670</td><td></td></tr><tr><td>Hegar needle holder, 150 mm</td><td>B Braun/Aesculap</td><td>BM065R</td><td></td></tr><tr><td>Heparine solution</td><td>Mochida Seiyaku</td><td>N/A</td><td></td></tr><tr><td>Medetomidine</td><td>Nippon Zenyaku Kogyo</td><td>N/A</td><td></td></tr><tr><td>Micro forceps straight</td><td>B Braun/Aesculap</td><td>BD33R</td><td></td></tr><tr><td>Midazolam</td><td>Sandoz</td><td>N/A</td><td></td></tr><tr><td>Mouse Ventilator</td><td>Harvard Apparatus</td><td>Model 687&amp;trade;</td><td></td></tr><tr><td>Normal Saline, Clinical grade</td><td>Otsuka Pharmaceutical</td><td>N/A</td><td></td></tr><tr><td>Petri dish, 60 x 15 mm</td><td>BD</td><td>351007</td><td></td></tr><tr><td>Safelet Cath PU 20 gauge polyurethan catheter</td><td>Nipro</td><td>09-031</td><td></td></tr><tr><td>Sakaki stainless scissors curved 14 cm</td><td>Bioresearch center</td><td>64152034</td><td></td></tr><tr><td>Scalpel holder</td><td>Bioresearch center</td><td>16101040</td><td></td></tr><tr><td>Small animal retraction system</td><td>Fine Science Tools</td><td>18200-20</td><td></td></tr><tr><td>Spare blade scalpel #11</td><td>Muranaka Medical Instruments</td><td>567-001-03</td><td></td></tr><tr><td>Spring scissors, 15 cm</td><td>Bioresearch center</td><td>PRI13-3736</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>M525</td><td>Clinical-grade surgical microscope with a flexible arm system is preferable.</td></tr><tr><td>Sugita titanium aneurysm clip curved slim, No.98</td><td>Mizuho medical</td><td>17-001-98</td><td></td></tr><tr><td>Sugita titanium clip applier, 110 mm</td><td>Mizuho medical</td><td>17-013-53</td><td></td></tr><tr><td>Temperature-adjustable electric warmer</td><td>Generic</td><td></td><td></td></tr><tr><td>Ultrafine cotton swab</td><td>Generic</td><td></td><td></td></tr><tr><td>VASCULAR AND BRONCHIAL CUFF</td><td></td><td></td><td></td></tr><tr><td>Fine sandpaper</td><td>Generic</td><td></td><td></td></tr><tr><td>Venula 20 gauge Teflon angiocatheter</td><td>Top</td><td>1160</td><td></td></tr><tr><td>Venula 22 gauge Teflon angiocatheter</td><td>Top</td><td>1161</td><td></td></tr><tr><td>Venula 24 gauge Teflon angiocatheter</td><td>Top</td><td>1124</td><td></td></tr></tbody></table>

transplantation of bioengineered lung using decellularized mouse lungs and primary human endothelial cells

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.

Following the decellularization protocol, mouse lungs are visibly white and translucent (Figure 6A). Cellular components should be entirely removed, but the alveolar structure remains intact in the histological observation (Figure 6B,C). Recellularized mouse lungs using 3 × 107 HUVECs with 2 day perfusion-based bioreactor culture show a homogeneous distribution of HUVECs (Figure 7A). HUVECs migrate into t...

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 ...

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Research

JoVE Journal

Bioengineering

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.

Procedure for Lung Engineering

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.

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

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Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

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Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

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Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

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Isolation of Primary Mouse Lung Endothelial Cells

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.

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Learn a standardized protocol for bioengineering mouse lungs using decellularization and recellularization with primary human endothelial cells. This method enables scalable organ transplantation research.

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

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