Name: Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor (Video) | JoVE Journal
Uploaded: 2022-08-01T13:00:00.000+00:00
Duration: 10 min 56 s
Description: Learn perfusion decellularization of vascularized porcine flaps using a customized bioreactor setup. This versatile technique enables tissue engineering applications in reconstructive surgery.

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<li>Kueckelhaus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularized+composite+allotransplantation%3A+Current+standards+and+novel+approaches+to+prevent+acute+rejection+and+chronic+allograft+deterioration%5BTitle%5D)+AND+%22Transplant+International%22%5BJournal%5D)">Vascularized composite allotransplantation: Current standards and novel approaches to prevent acute rejection and chronic allograft deterioration.</a> Transplant International. 29 (6), 655-662 (2016).</li>
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<li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+tissue+and+whole+organ+decellularization+processes%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li>
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<li>Rouwkema, J., Rivron, N. C., van Blitterswijk, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularization+in+tissue+engineering%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">Vascularization in tissue engineering.</a> Trends in Biotechnology. 26 (8), 434-441 (2008).</li>
<li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularized+skin%2Fadipose+tissue+flap+matrix+for+engineering+vascularized+composite+soft+tissue+flaps%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps.</a> Acta Biomaterialia. 35, 166-184 (2016).</li>
<li>Jank, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creation+of+a+bioengineered+skin+flap+scaffold+with+a+perfusable+vascular+pedicle%5BTitle%5D)+AND+%22Tissue+Engineering+-+Part+A%22%5BJournal%5D)">Creation of a bioengineered skin flap scaffold with a perfusable vascular pedicle.</a> Tissue Engineering - Part A. 23 (13-14), 696-707 (2017).</li>
<li>Giatsidis, G., Guyette, J. P., Ott, H. C., Orgill, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+large-volume+human-derived+adipose+acellular+allogenic+flap+by+perfusion+decellularization%5BTitle%5D)+AND+%22Wound+Repair+and+Regeneration%22%5BJournal%5D)">Development of a large-volume human-derived adipose acellular allogenic flap by perfusion decellularization.</a> Wound Repair and Regeneration. 26 (2), 245-250 (2018).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfusion-decellularized+skeletal+muscle+as+a+three-dimensional+scaffold+with+a+vascular+network+template%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Perfusion-decellularized skeletal muscle as a three-dimensional scaffold with a vascular network template.</a> Biomaterials. 89, 114-126 (2016).</li>
<li>Duisit, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularization+of+the+porcine+ear+generates+a+biocompatible%2C+nonimmunogenic+extracellular+matrix+platform+for+face+subunit+bioengineering%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">Decellularization of the porcine ear generates a biocompatible, nonimmunogenic extracellular matrix platform for face subunit bioengineering.</a> Annals of Surgery. 267 (6), 1191-1201 (2018).</li>
<li>Duisit, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfusion-decellularization+of+human+ear+grafts+enables+ECM-based+scaffolds+for+auricular+vascularized+composite+tissue+engineering%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.</a> Acta Biomaterialia. 73, 339-354 (2018).</li>
<li>Duisit, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioengineering+a+human+face+graft%3A+The+matrix+of+identity%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">Bioengineering a human face graft: The matrix of identity.</a> Annals of Surgery. 266 (5), 754-764 (2017).</li>
<li>Haughey, B. H., Panje, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+porcine+model+for+multiple+musculocutaneous+flaps%5BTitle%5D)+AND+%22The+Laryngoscope%22%5BJournal%5D)">A porcine model for multiple musculocutaneous flaps.</a> The Laryngoscope. 99 (2), 204-212 (1989).</li>
<li>Khachatryan, A., et al. Radial Forearm Flap. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKhachatryan%2C+A+%22Microsurgery+Manual+for+Medical+Students+and+Residents%3A+A+Step-by-Step+Approach%22">Microsurgery Manual for Medical Students and Residents: A Step-by-Step Approach</a>. , Springer. Denmark. 177-181 (2021).</li>
<li>Hammouda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature+effect+on+the+nanostructure+of+SDS+micelles+in+water%5BTitle%5D)+AND+%22Journal+of+Research+of+the+National+Institute+of+Standards+and+Technology%22%5BJournal%5D)">Temperature effect on the nanostructure of SDS micelles in water.</a> Journal of Research of the National Institute of Standards and Technology. 118, 151-167 (2013).</li>
<li>Qu, J., Van Hogezand, R. M., Zhao, C., Kuo, B. J., Carlsen, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularization+of+a+fasciocutaneous+flap+for+use+as+a+perfusable+scaffold%5BTitle%5D)+AND+%22Annals+of+Plastic+Surgery%22%5BJournal%5D)">Decellularization of a fasciocutaneous flap for use as a perfusable scaffold.</a> Annals of Plastic Surgery. 75 (1), 112-116 (2015).</li>
<li>Keane, T. J., Swinehart, I. T., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+of+tissue+decellularization+used+for+preparation+of+biologic+scaffolds+and+in+vivo+relevance%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance.</a> Methods. 84, 25-34 (2015).</li>
<li>Mendibil, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-specific+decellularization+methods%3A+Rationale+and+strategies+to+achieve+regenerative+compounds%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Tissue-specific decellularization methods: Rationale and strategies to achieve regenerative compounds.</a> International Journal of Molecular Sciences. 21 (15), 5447(2020).</li>
<li>Lupon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+vascularized+composite+allografts+using+natural+scaffolds%3A+A+systematic+review%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+B+Reviews%22%5BJournal%5D)">Engineering vascularized composite allografts using natural scaffolds: A systematic review.</a> Tissue Engineering. Part B Reviews. 28 (3), 677-693 (2022).</li>
<li>Duisit, J., Maistriaux, L., Bertheuil, N., Lellouch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+vascularized+composite+tissues+by+perfusion+decellularization%2Frecellularization%3A+Review%5BTitle%5D)+AND+%22Current+Transplantation+Reports%22%5BJournal%5D)">Engineering vascularized composite tissues by perfusion decellularization/recellularization: Review.</a> Current Transplantation Reports. 8, 44-56 (2021).</li>
<li>Adil, A., Xu, M., Haykal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recellularization+of+bioengineered+scaffolds+for+vascular+composite+allotransplantation%5BTitle%5D)+AND+%22Frontiers+in+Surgery%22%5BJournal%5D)">Recellularization of bioengineered scaffolds for vascular composite allotransplantation.</a> Frontiers in Surgery. 9, 843677(2022).</li>
<li>Phelps, E. A., García, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+more+than+a+cell%3A+Vascularization+strategies+in+tissue+engineering%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">Engineering more than a cell: Vascularization strategies in tissue engineering.</a> Current Opinion in Biotechnology. 21 (5), 704-709 (2010).</li>
<li>Pozzo, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+reliable+porcine+fascio-cutaneous+flap+model+for+vascularized+composite+allografts+bioengineering+studies%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A reliable porcine fascio-cutaneous flap model for vascularized composite allografts bioengineering studies.</a> Journal of Visualized Experiments. (181), e63557(2022).</li>
<li>Uygun, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularization+and+recellularization+of+whole+livers%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Decellularization and recellularization of whole livers.</a> Journal of Visualized Experiments. (48), e2394(2011).</li>
<li>Uzarski, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial+cell+repopulation+and+preparation+of+rodent+extracellular+matrix+scaffolds+for+renal+tissue+development%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development.</a> Journal of Visualized Experiments. (102), e53271(2015).</li>
<li>Sullivan, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.</a> Biomaterials. 33 (31), 7756-7764 (2012).</li>
<li>Choudhury, D., Yee, M., Sheng, Z. L. J., Amirul, A., Naing, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decellularization+systems+and+devices%3A+State-of-the-art%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Decellularization systems and devices: State-of-the-art.</a> Acta Biomaterialia. 115, 51-59 (2020).</li>
<li>Schilling, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+fabrication+of+an+automatable%2C+3D+printed+perfusion+device+for+tissue+infusion+and+perfusion+engineering%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+A%22%5BJournal%5D)">Design and fabrication of an automatable, 3D printed perfusion device for tissue infusion and perfusion engineering.</a> Tissue Engineering. Part A. 26 (5-6), 253-264 (2020).</li>
</ol>

The authors have no conflicts of interest to disclose.

The proposed protocol uses the perfusion of low concentration SDS to decellularize a range of porcine-derived flaps. With this procedure, acellular omentum, tensor fascia lata, and radial forearm flaps can be successfully decellularized using a protocol that favors low concentration SDS. Preliminary optimization experiments have determined that SDS at a low concentration (0.05%) between 2 days to 5 days is capable of removing cellular material for the omentum, tensor fascia lata, and radial forearm flap when analyzed wit...

Traumatic injury and tumor removal can lead to large and complex soft tissue defects. These defects can impair patient quality of life, cause loss of function, and result in permanent disability. While techniques such as autologous tissue flap transfer have been commonly practiced, issues with flap availability and donor site morbidity are major limitations1,2,3. Vascularized composite allotransplantation (VCA) is a promising alternative that transfers composite tissues, e.g., muscle, skin, vasculature, as a single unit to recipients. However, VCA requires long-term immunosuppression, which leads to drug toxicity, opportunistic infections, and malignancies4,5,6.
Tissue-engineered acellular scaffolds are a potential solution to these limitations7. Acellular tissue scaffolds can be obtained using decellularization methods, which remove cellular material from native tissues while preserving the underlying extracellular matrix (ECM) microarchitecture. In contrast to the use of synthetic materials in tissue engineering, the use of biologically derived scaffolds offers a biomimetic ECM substrate that allows biocompatibility and the potential for clinical translation8. Following decellularization, the subsequent recellularization of scaffolds with recipient-specific cells can then generate functional, vascularized tissues with little to no immunogenicity9,10,11. By developing an effective protocol to obtain acellular tissues using perfusion decellularization techniques, a broad range of tissue types can be engineered. In turn, building on this technique allows the application to more complex tissues. To date, perfusion decellularization of vascularized soft tissues has been investigated using simple vascularized tissues such as a full thickness fasciocutaneous flap in rodent12, porcine13, and human models14, as well as porcine rectus abdominis skeletal muscle15. Additionally, complex vascularized tissues have also been perfusion decellularized as demonstrated in porcine and human ear16,17 models and human full-face graft models18.
Here, the protocol describes the decellularization of vascularized free flaps using biologically derived ECM scaffolds. We present the decellularization of three clinically relevant flaps: 1) the omentum, 2) the tensor fascia lata, and 3) the radial forearm, all of which are representative of workhorse flaps used routinely in reconstructive surgery and have not been previously examined in animal studies within the context of tissue decellularization. These bioengineered flaps offer a versatile and readily available platform that has the potential for clinical applications for use in the field of large soft tissue defect repair and reconstruction.

<table><tbody><tr><td>0.2 &amp;micro;m pore Acrodisk Filter</td><td>VWR</td><td>CA28143-310</td><td></td></tr><tr><td>0.9 % Sodium Chloride Solution (Normal Saline)</td><td>Baxter</td><td>JF7123</td><td></td></tr><tr><td>20 L Polypropylene Carboy</td><td>Cole-Parmer</td><td>RK-62507-20</td><td></td></tr><tr><td>3-0 Sofsilk Nonabsorbable Surgical Tie</td><td>Covidien</td><td>&amp;nbsp;LS639</td><td></td></tr><tr><td>3-way Stopcock</td><td>Cole-Parmer</td><td>UZ-30600-04</td><td></td></tr><tr><td>Adson Forceps</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution, 100X</td><td>Wisent</td><td>450-115-EL</td><td></td></tr><tr><td>Atropine Sulphate 15 mg/30ml</td><td>Rafter 8 Products</td><td>238481</td><td></td></tr><tr><td>BD Angiocath 20-Gauge</td><td>VWR</td><td>BD381134</td><td></td></tr><tr><td>BD Angiocath 22-Gauge</td><td>VWR</td><td>BD381123</td><td></td></tr><tr><td>BD Angiocath 24-Gauge</td><td>VWR</td><td>BD381112</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td>DNAse Co-factor</td></tr><tr><td>DNase I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</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>DPBS, 10X</td><td>Wisent</td><td>311-415-CL</td><td>&amp;nbsp;without Ca++/Mg++</td></tr><tr><td>Halsted-Mosquito Hemostat</td><td>Fine Science Tools</td><td>13008-12</td><td></td></tr><tr><td>Heparin, 1000 I.U./mL</td><td>Leo Pharma A/S</td><td>453811</td><td></td></tr><tr><td>Ketamine Hydrochloride&amp;nbsp; 5000 mg/50 ml</td><td>Bimeda-MTC Animal Health Inc.</td><td>612316</td><td></td></tr><tr><td>Ismatec Pump Tygon 3-Stop Tubing</td><td>Cole-Parmer</td><td>RK-96450-40</td><td>Internal Diameter:&amp;nbsp; 1.85 mm</td></tr><tr><td>Ismatec REGLO 4-Channel Pump</td><td>Cole-Parmer</td><td>78001-78</td><td></td></tr><tr><td>Ismatec Tubing Cassettes</td><td>Cole-Parmer</td><td>RK-78016-98</td><td></td></tr><tr><td>Isoflurane 99.9%, 250 ml</td><td>Pharmaceutical Partners of Canada Inc.</td><td>2231929</td><td></td></tr><tr><td>LB Agar Lennox</td><td>Bioshop Canada</td><td>LBL406.500</td><td>Sterility testing agar plates</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td>DNAse Co-factor</td></tr><tr><td>Masterflex L/S 16 Tubing</td><td>Cole-Parmer</td><td>RK-96410-16</td><td></td></tr><tr><td>Midazolam 50 mg/10 ml</td><td>Pharmaceutical Partners of Canada Inc.</td><td>2242905</td><td></td></tr><tr><td>Monopolar Cautery Pencil</td><td>Valleylab</td><td>E2100</td><td></td></tr><tr><td>Normal Buffered Formalin, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>N&amp;deg;11 scalpel blade</td><td>Swann Morton</td><td>303</td><td></td></tr><tr><td>Papain from papaya latex</td><td>Sigma-Aldrich</td><td>P3125</td><td></td></tr><tr><td>Peracetic Acid</td><td>Sigma-Aldrich</td><td>269336</td><td></td></tr><tr><td>Plastic Barbed Connector for 1/4&quot; to 1/8&quot; Tube ID</td><td>McMaster-Carr</td><td>5117K61</td><td></td></tr><tr><td>Plastic Barbed Tube 90&amp;deg; Elbow Connectors</td><td>McMaster-Carr</td><td>5117K76</td><td></td></tr><tr><td>Plastic Quick-Turn Tube Plugs</td><td>McMaster-Carr</td><td>51525K143</td><td>Male Luer</td></tr><tr><td>Plastic Quick-Turn Tube Sockets</td><td>McMaster-Carr</td><td>51525K293</td><td>Female Luer</td></tr><tr><td>Punch Biopsy Tool</td><td>Integra Miltex</td><td>3332</td><td></td></tr><tr><td>Potassium Chloride 40 mEq/20 ml</td><td>Hospira Healthcare Corporation</td><td>37869</td><td></td></tr><tr><td>Povidone-Iodine, 10%</td><td>Rougier</td><td>833133</td><td></td></tr><tr><td>Serological Pipet, 2mL</td><td>Fisher Science</td><td>13-678-27D</td><td></td></tr><tr><td>Snap Lid Airtight Containers</td><td>SnapLock</td><td>142-3941-4</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate Powder</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>Surgical Metal Ligation Clips, Small</td><td>Teleflex</td><td>001200</td><td></td></tr><tr><td>Stevens Tenotomy Scissors, 115 mm, straight</td><td>B. Braun</td><td>BC004R</td><td></td></tr><tr><td>TruWave Pressure Monitoring Set</td><td>Edwards Lifesciences</td><td>PX260</td><td></td></tr></tbody></table>

procurement and perfusion-decellularization of porcine vascularized flaps in a customized perfusion bioreactor

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

This protocol to decellularize vascularized porcine flaps relies on the perfusion of an ionic-based detergent, SDS, through the flap vasculature in a customized perfusion bioreactor. Prior to decellularization, three vascularized flaps in a porcine model were procured and cannulated according to their main supplying vessels. The flaps were immediately flushed after procurement in order to maintain a patent, perfusable vasculature to allow for successful decellularization. Using airtight snap-lid containers, a customized ...

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

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JoVE Journal

Bioengineering

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decellularization and Recellularization of Whole Livers

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

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

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

Engineered Vascularized Muscle Flap

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

This protocol describes the surgical technique for harvesting a human perforator flap model based on the deep inferior epigastric artery pedicle, intended for experimental research.

An Experimental Human DIEP Flap Model to Investigate Preservation Strategies for Vascularized Composite Allografts and Free Flaps

Learn perfusion decellularization of vascularized porcine flaps using a customized bioreactor setup. This versatile technique enables tissue engineering applications in reconstructive surgery.

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

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