Name: procurement and perfusion-decellularization of porcine vascularized flaps in a customized perfusion bioreactor
Uploaded: 2022-08-01T13:00:00.000+00:00
Duration: 10 min 56 s
Description: Watch this Scientific Journal Video about Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor at JoVE.com

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	<li>Richardson, D., Fisher, S. E., Vaughan, D. E., Brown, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+Forearm+Flap+Donor-Site+Complications+and+Morbidity:+A+Prospective+Study.">Radial Forearm Flap Donor-Site Complications and Morbidity: A Prospective Study.</a> Plastic and Reconstructive Surgery. 99 (1), 109-115 (1997).</li><li>Edsander-Nord, &. #. 1. 9. 7. ;., Jurell, G., Wickman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-site+morbidity+after+pedicled+or+free+TRAM+flap+surgery:+A+prospective+and+objective+study.">Donor-site morbidity after pedicled or free TRAM flap surgery: A prospective and objective study.</a> Plastic and Reconstructive Surgery. 102 (5), 1508-1516 (1998).</li><li>Qian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+and+meta-analysis+of+free-style+flaps:+Risk+analysis+of+complications.">A systematic review and meta-analysis of free-style flaps: Risk analysis of complications.</a> Plastic and Reconstructive Surgery. Global Open. 6 (2), 1651 (2018).</li><li>Issa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+composite+allograft-specific+characteristics+of+immune+responses.">Vascularized composite allograft-specific characteristics of immune responses.</a> Transplant International. 29 (6), 672-681 (2016).</li><li>Kueckelhaus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+composite+allotransplantation:+Current+standards+and+novel+approaches+to+prevent+acute+rejection+and+chronic+allograft+deterioration.">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><li>Iske, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+tissue+allotransplantation:+Opportunities+and+challenges.">Composite tissue allotransplantation: Opportunities and challenges.</a> Cellular and Molecular Immunology. 16 (4), 343-349 (2019).</li><li>Londono, R., Gorantla, V. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+implications+for+extracellular+matrix-based+technologies+in+vascularized+composite+allotransplantation.">Emerging implications for extracellular matrix-based technologies in vascularized composite allotransplantation.</a> Stem Cells International. 2016, 1541823 (2016).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Hussey, G. S., Dziki, J. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-based+materials+for+regenerative+medicine.">Extracellular matrix-based materials for regenerative medicine.</a> Nature Reviews Materials. 3, 159-173 (2018).</li><li>Colazo, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applied+bioengineering+in+tissue+reconstruction,+replacement,+and+regeneration.">Applied bioengineering in tissue reconstruction, replacement, and regeneration.</a> Tissue Engineering. Part B Reviews. 25 (4), 259-290 (2019).</li><li>Rouwkema, J., Rivron, N. C., van Blitterswijk, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+in+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+skin/adipose+tissue+flap+matrix+for+engineering+vascularized+composite+soft+tissue+flaps.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+bioengineered+skin+flap+scaffold+with+a+perfusable+vascular+pedicle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+large-volume+human-derived+adipose+acellular+allogenic+flap+by+perfusion+decellularization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+skeletal+muscle+as+a+three-dimensional+scaffold+with+a+vascular+network+template.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+the+porcine+ear+generates+a+biocompatible,+nonimmunogenic+extracellular+matrix+platform+for+face+subunit+bioengineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularization+of+human+ear+grafts+enables+ECM-based+scaffolds+for+auricular+vascularized+composite+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+a+human+face+graft:+The+matrix+of+identity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+for+multiple+musculocutaneous+flaps.">A porcine model for multiple musculocutaneous flaps.</a> The Laryngoscope. 99 (2), 204-212 (1989).</li><li>Khachatryan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+Forearm+Flap.">Radial Forearm Flap.</a> Microsurgery Manual for Medical Students and Residents: A Step-by-Step Approach. , 177-181 (2021).</li><li>Hammouda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+the+nanostructure+of+SDS+micelles+in+water.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+a+fasciocutaneous+flap+for+use+as+a+perfusable+scaffold.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+tissue+decellularization+used+for+preparation+of+biologic+scaffolds+and+in+vivo+relevance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+decellularization+methods:+Rationale+and+strategies+to+achieve+regenerative+compounds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+composite+allografts+using+natural+scaffolds:+A+systematic+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+composite+tissues+by+perfusion+decellularization/recellularization:+Review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recellularization+of+bioengineered+scaffolds+for+vascular+composite+allotransplantation.">Recellularization of bioengineered scaffolds for vascular composite allotransplantation.</a> Frontiers in Surgery. 9, 843677 (2022).</li><li>Phelps, E. A., Garc&#237;a, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+more+than+a+cell:+Vascularization+strategies+in+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+porcine+fascio-cutaneous+flap+model+for+vascularized+composite+allografts+bioengineering+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+and+recellularization+of+whole+livers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+repopulation+and+preparation+of+rodent+extracellular+matrix+scaffolds+for+renal+tissue+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+systems+and+devices:+State-of-the-art.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+an+automatable,+3D+printed+perfusion+device+for+tissue+infusion+and+perfusion+engineering.">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 with histological techniques (Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3). This method offers a straightforward approach that achieves decellularization within a short time frame and at a reasonable cost. In our experience, the decellularization and sterilization of porcine flaps take between 2-5 days, with longer duration decellularization needed for tissues with greater density (e.g., forearm skin at 5 days vs. omentum at 2 days). Furthermore, the low concentration of SDS used in this protocol was selected based on the rationale that a low concentration of SDS at 0.05% falls below the critical micellar concentration of SDS (approximately at 0.2%21) and, thus, allows the surfactant to effectively solubilize cell membranes while leaving important bioactive ECM components intact. The use of low concentration SDS in this protocol contrasts with the use of relatively higher concentrations of SDS (e.g., 1%) for perfusion-decellularization of vascularized fascio-cutaneous flaps as reported by previous authors13,22. High concentrations of SDS, while effective at decellularization, come at the cost of incurring detrimental effects on the ECM scaffold, such as GAG and growth factor depletion, as well as the denaturation of proteins such as collagen and vimentin23,24. Indeed, future work in the perfusion-decellularization of porcine vascularized flaps will benefit from additional characterization studies to examine the effects of the decellularization agent on the ECM at the structural and molecular levels and their implications for downstream recellularization25,26,27. Assays for ECM components such as collagen, elastin, or GAG content can be used to quantitatively assess changes to the ECM following decellularization. Furthermore, mechanical strength testing is a useful modality to study changes to the biomechanical properties after decellularization. Finally, assessment of the vascularity using techniques such as angiography or microCT scanning will also help to characterize the vascular architecture at a systemic level as a requisite to generating perfusable vascularized flaps28.
Several technical considerations should be noted with this protocol. Firstly, extreme care should be taken during flap handling and bioreactor assembly to prevent accidental decannulation, as recannulation in an ex vivo setting is comparatively more difficult. Secondly, during flap procurement, obtaining an intact flap with a perfusable and intact vascular pedicle is critical to successful decellularization. Care should be taken during procurement to ensure distal leak points in the scaffold are either clipped or hand-tied. This will help reduce leakage, which can cause incomplete perfusion of solutions through the flap vasculature. An initial period of perfusion with heparinized saline after procurement prevents the retention of blood clots and verifies a perfusable vasculature as evidenced by venous perfusate outflow. Decellularized tissues can also be flushed again upon completion of decellularization to check for any potential intravascular obstructions (e.g., from cell debris/air emboli) that may preclude flap perfusability. In our experience, decellularized flaps showed no intravascular resistance to syringe flushing following this protocol, while venous outflow could again be observed from the decellularized flap after flushing (Supplementary Video 1, Supplementary Video 2, Supplementary Video 3). This was indicative of a patent arterial to venous vascular loop with connecting capillaries that can be perfused.
Another consideration is made regarding the perfusion parameters during decellularization. In this protocol, a constant flow rate perfusion was selected with a target flow rate of 2 mL/min, which was within the range of the operating flow rates used in previously published decellularization studies of porcine flap tissues13,29. Furthermore, our decellularization system incorporates a real-time in-line pressure monitoring capability in order to monitor potential fluctuations in intravascular resistance over the course of perfusion; this method can help avoid high perfusion pressures arising from possible intravascular occlusion that may damage the microvascular ECM.
Finally, sterilization of the resultant scaffold is another important technical consideration. We incorporated a sterilization phase as the last step of the decellularization protocol to address incidental environmental contamination that may occur during decellularization. Flap sterility is especially important so that flaps can be used in the future for recellularization. The perfusion of 0.1% peracetic acid/4% EtOH as a sterilant has been previously reported by groups for sterilizing acellular scaffolds30,31 and was also found to be effective with this protocol. Sterility was verified by examining flap swab cultures on agar plates and noting absent growth after 14 days of incubation.
The customized perfusion bioreactor designed for the experiments is relatively cost-effective as well as simple and quick to assemble. Furthermore, all the components of this perfusion bioreactor are autoclavable and adaptable for recellularization work while maintaining the sterility of the flaps. The customized bioreactor circuit can be easily modified to permit open-circuit perfusion that can circulate cell culture media through a recellularized scaffold following cell seeding experiments. Nonetheless, a few limitations of the bioreactor system are worth mentioning. First, the use of two commercial pumps, while necessary to prevent inadvertent overflow of the system, particularly when large volumes of perfusate are used, detracts from the otherwise low cost of the perfusion set-up. Additionally, the low throughput of the current perfusion bioreactor system can present logistical challenges when needing to run numerous experimental replicates in parallel; comparatively higher throughput systems developed for porcine kidney decellularization32 may one day be adapted for porcine flap decellularization in order to alleviate these logistical challenges. Finally, while the developed bioreactor is simple to set up, human supervision and manual operation are still needed regularly and frequently to ensure no malfunction of the system occurs. Modifications to the bioreactor incorporating automation capabilities that can both monitor and readjust bioreactor performance with minimal or no human intervention can be pursued in order to advance perfusion-decellularization bioreactor technology in the future33,34.
The main application of decellularized flaps is to permit the subsequent recellularization of these acellular scaffolds with cell populations that can regenerate the vasculature. While much future research is required to determine the appropriate cell numbers, populations, seeding strategies, and bioreactor conditions to regenerate functional and viable vascularized tissue, this protocol represents the initial foundational work toward this goal. Future methods of recellularization will help establish strategies to engineer soft tissue flaps with an intact perfusable vascular network and contribute to potentially regenerating more complex composite tissues, such as extremity and facial allografts. These scaffolds can one day circumvent donor site morbidity and immunosuppression for patients undergoing large-scale soft tissue reconstruction, thereby improving their clinical outcomes and quality of life.

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

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 bioreactor was designed to allow for flap perfusion within an enclosed environment. Perfusion of flaps within the bioreactor was achieved in a single-pass fashion using two peristaltic pumps connected to the tissue chamber. The perfusion pressure was monitored with an in-line pressure sensor.
During decellularization, the duration of SDS exposure was dependent on the type of tissue being processed. With the described perfusion decellularization technique, the omentum, tensor fascia, and radial forearm flaps were decellularized with 0.05% SDS for 2 days, 3 days, and 5 days, respectively. &#65279;Successful sterilization following decellularization was demonstrated by the absence of microbial colony growth after swabbing the flaps and culturing the swabs on agar plates for 14 days. Perfusion pressures were monitored at a 2 mL/min flow rate and ranged between 20-60 mmHg during all stages of decellularization for all three flaps. Upon the conclusion of decellularization, the flaps were flushed under manual control and demonstrated evidence of outflow from a venous cannula left to free drainage (Supplementary Video 1, Supplementary Video 2, Supplementary Video 3).
A total of 15 flaps were decellularized, with five replicates for each of the three tissue types. On examination, the gross morphology of native tissues appeared pink-colored (Figure 4A,E,I), whereas decellularized tissues were characteristically white/opaque in appearance (Figure 4C,G, K). Histological examination of native tissues with H&#38;E shows the presence of blue nuclei (Figure 4B,F,J). In decellularized flaps, H&#38;E staining showed a loss of cellular material with the absence of blue nuclear staining (Figure 4D,H,L), indicating an acellular tissue scaffold. Additional quantification of the DNA content in the five replicates showed a statistically significant decrease in DNA in the acellular scaffolds compared to native tissues by a Student&#39;s t-test for each flap (Figure 5). In the omentum, &#65279;DNA decreased from 460 ng/mg &#177; 124 ng/mg dry tissue in the native flap to 25.8 ng/mg &#177; 5.90 ng/mg dry tissue in the decellularized flap (n = 5, p &#60; 0.05). In the tensor fascia lata, &#65279;DNA decreased from 297 ng/mg &#177; 68.2 ng/mg to 58.3 ng/mg &#177; 13.5 ng/mg between native and decellularized flaps, respectively (n = 5, p &#60; 0.05). The radial forearm flap showed &#65279;a DNA decrease from 1180 ng/mg &#177; 241 ng/mg in the native flap to 162 ng/mg &#177; 34.9 ng/mg in the decellularized flap (n = 5, p &#60; 0.05).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64068/64068fig04.jpg" /> 
Figure 4: Decellularization of three porcine flaps. Gross examination of the (A) native omentum, (E) tensor fascia lata, and (I) radial forearm flaps demonstrate the pink appearance of the flaps immediately after procurement. The histological staining of native tissues demonstrates clear hematoxylin staining of cellular nuclei with H&#38;E (B,F,J). After decellularization, the (C) omentum, (G) tensor fascia lata, and (K) radial forearm appear grossly white and opaque. Histologically, the three decellularized flaps show an absence of nuclear staining with H&#38;E (D,H,L). Scale bars: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/64068fig04large.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/64068/64068fig05.jpg" /> 
Figure 5: Quantification of DNA content in acellular scaffolds. Values are normalized to mg of dry scaffold mass. Fresh tissue samples from native, non-decellularized tissues (n = 5) and decellularized tissues (n = 5) were dried and weighed before overnight digestion in papain prior to quantification.&#160;Statistical testing used Student&#39;s t-tests with a significance (**) level defined as a p-value &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/64068fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. Time-course examination of the progression of perfusion-decellularization of omentum using 0.05% sodium dodecyl sulfate over 5 days by gross examination (scale bars = 3 cm) and H&#38;E histology (scale bars = 200 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/SuppFig1_dOmen_Timecoursetiff_RE.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2. Time-course examination of the progression of perfusion-decellularization of tensor fascia lata using 0.05% sodium dodecyl sulfate over 5 days by gross examination (scale bars = 3 cm) and H&#38;E histology (scale bars = 200 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/SuppFig2_dTFL_Timecoursetiff_RE.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3. Time-course examination of the progression of perfusion-decellularization of radial forearm flap using 0.05% sodium dodecyl sulfate over 5 days by gross examination (scale bars = 3 cm) and H&#38;E histology (scale bars = 200 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/SuppFig3_dRFF_Timecoursetiff_RE.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Video 1. Representative manual perfusion of the decellularized omentum flap via the arterial cannula (pink) demonstrating outflow from the freely draining venous cannula (yellow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/SupplVid1_dOmen_outflow.mov" target="_blank">Please click here to download this Video.</a>
Supplementary Video 2. Representative manual perfusion of the decellularized tensor fascia lata flap via the arterial cannula (pink) demonstrating outflow from the freely draining venous cannula (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/SupplVid2_dTFL_outflow.mov" target="_blank">Please click here to download this Video.</a>
Supplementary Video 3. Representative manual perfusion of the decellularized radial forearm flap via the arterial cannula (blue) demonstrating outflow from the freely draining venous cannula (yellow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64068/SupplVid3_dRFF_outflow.mov" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor at JoVE.com

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

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 ...

procurement-perfusion-decellularization-porcine-vascularized-flaps

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.7K Views.  Toronto General Hospital Research Institute, University Health Network. Deze methode beschrijft de chirurgische verkrijging van drie gevasculariseerde varkensflappen en hun daaropvolgende perfusiedecellularisatie. Deze methode kan helpen bij het regenereren van varkensflappen met behulp van een weefseldecellularisatie- en recellularisatiebenadering. Het belangrijkste voordeel van deze techniek is de veelzijdigheid om profusie-decellularisatie te bereiken over een verscheidenheid aan gevasculariseerde varkensflappen in een eenvoudig te monteren bioreactoropstellin...

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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

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 ...

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

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

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.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

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.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

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

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients&#39; quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports&#160;a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

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

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

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

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

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

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.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

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

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

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.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

Enhancing CVD Research with Modified Langendorff Perfusion

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.

Author Spotlight: Advancing Cardiovascular Research &#8212; Tailored Langendorff Perfusion Techniques for Improved Experimental Outcomes

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

Medicine

Development and Validation of a Porcine Ex Situ Isolated Liver Perfusion Model 

Porcine Normothermic Isolated Liver Perfusion

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost.
In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 &#176;C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine ...

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