Development of a new decellularization protocol for the whole porcine heart
Background: Cardiovascular diseases are the leading cause of death in many countries. Advances in technology have been promoted in this regard, especially in Tissue Engineering, to meet the need for tissue or organ grafts. In this way, the porcine model has been used due to his morphophysiological similarity between the human species, mainly regarding the cardiovascular system. Tissue engineering is employed using biological scaffolds that are currently derived from porcine. These scaffolds are produced by decellularization, a process to remove cells aiming to maintain only its three-dimensional structure, formed by extracellular matrix. Its main objective is to produce organs with recellularized scaffolds that could eventually substitute the ones with impaired functions.
Aim: In this way, the present study aimed to establish a new protocol for porcine heart decellularization with potential application on tissue engineering.
Methods: A porcine heart aorta was cannulated with a silicon tube, and the organ was washed in phosphate-saline buffer 0.1% solution through a peristaltic pump (Harvard Peristaltic Pump - Harvard Apparatus). After that, deionized water was perfused in the same system. The decellularization was carried out by perfusing four liters of 4% Sodium Dodecyl Sulfate were through myocardial circulation, at 400 mL / min for 24 hours, for 6 days; perfusion of Triton X-100 1% followed by phosphate saline buffer 0.1M and deionized water for 24 hours. The heart volume was measured before and after the recellularization. Macroscopic and microscopic analyses were performed on the cardiac tissue, and Hematoxylin & Eosin, Masson’s Trichrome, Weigert-Van Giesson, Alcian Blue and Picrosirius Red staining were evaluated. To observe the cell adhesion, the recellularized was provided in this scaffold, which was analyzed under Immunofluorescence and Scanning Electronic Microscopy.
Results: The protocol provided cells remotion, with adequate concentration of remaining DNA. Extracellular matrix components as collagen type I, elastin, glycosaminoglycans were successfully maintained. The scaffold showed a high cells adherence and proliferation in the recellularization process.
Conclusions: According to results, the protocol described in this work preserved the extracellular matrix and architecture of the organ, minimizing extracellular matrix loss and being possible to state that it is a promising approach to tissue bioengineering.
Relevance for patients: This study provides a protocol for whole porcine heart decellularization, which will ultimately contribute to heart bioengineering and may support further studies on biocompatibility relationship of new cells with recellularized scaffolds.
[1] Kawecki M, Łabuś W, Klama-Baryla A, Kitala D, Kraut M, Glik J, et al. A review of Decellurization Methods Caused by an Urgent Need for Quality Control of Cell-Free Extracellular Matrix’ Scaffolds and their Role in Regenerative Medicine. J Biomed Mater Res B Appl Biomater 2018;106:909-23.
[2] Crapo PM, Gilbert TW, Badylak SF. An Overview of Tissue and Whole Organ Decellularization Processes. Biomaterials 2011;32:3233-43.
[3] Keane TJ, Swinehart IT, Badylak SF. Methods of Tissue Decellularization used for Preparation of Biologic Scaffolds and In Vivo Relevance. Methods 2015;84:25-34.
[4] Sakina R, Llucià-Valldeperas A, Henriques Lourenço A, Harichandan A, Gelsomino S, Wieringa P, et al. Decellularization of Porcine Heart Tissue to Obtain Extracellular Matrix Based on Hydrogels. Methods Cell Biol 2020;157:3-21.
[5] Luo Y, Lou D, Ma L, Gao C. Optimizing Detergent Concentration and Processing Time to Balance the Decellularization Efficiency and Properties of Bioprosthetic Heart Valves. J Biomed Mater Res A 2019;107:2235-43.
[6] Taylor DA, Lee PF, Barac Y, Hochman-Mendez C, Sampaio LC. Decellularization of Whole Hearts for Cardiac Regeneration. In: Emerging Technologies for Heart Diseases. United States: Academic Press; 2020. p. 291-310.
[7] Massari CH, Miglino MA. Anatomia Cardíaca Aplicada à Medicina Veterinária. Portal de Livros Abertos da UPS; 2019. Available from: http://www.livrosabertos.sibi.usp. br/portaldelivrosusp/catalog/book/403. [Last accessed on 2021 Aug 24].
[8] Momtahan N, Poornejad N, Struk JA, Castleton AA, Herrod BJ, Vance BR, et al. Automation of Pressure Control Improves Whole Porcine Heart Decellularization. Tissue Eng Part C Methods 2015;21:1148-61.
[9] Ye X, Wang H, Gong W, Li S, Li H, Wang Z, et al. Impact of Decellularization on Porcine Myocardium as Scaffold for Tissue Engineered Heart Tissue. J Mater Sci Mater Med 2016;27:70.
[10] Haupt J, Lutter G, Gorb SN, Simionescu DT, Frank D, Seiler J, et al. Detergent-Based Decellularization Strategy Preserves Macro-and Microstructure of Heart Valves. Interact Cardiovasc Thorac Surg 2018;26:230-6.
[11] Lee PF, Chau E, Cabello R, Yeh AT, Sampaio LC, GobinAS, et al. Inverted Orientation Improves Decellularization of Whole Porcine Hearts. Acta Biomater 2017;49:181-91.
[12] Ferng AS, Connell AM, Marsh KM, Qu N, Medina AO, Bajaj N, et al. Acellular Porcine Heart Matrices: Whole Organ Decellularization with 3D-Bioscaffold and Vascular Preservation. J Clin Transl Res 2017;3:260-70.
[13] Roosens A, Somers P, de Somer F, Carriel V, van Nooten G, Cornelissen R. Impact of Detergent-Based Decellularization Methods on Porcine Tissues for Heart Valve Engineering. Ann Biomed Eng 2016;44:2827-39.
[14] Godehardt AW, Tönjes RR. Xenotransplantation of Decellularized Pig Heart Valves-Regulatory Aspects in Europe. Xenotransplantation 2020;27:e12609.
[15] Nyengaard JR, Gundersen HJ. The Disector: A Simple and Direct Method for Generating Isotropic, Uniform Random Sections from Small Specimens. J Microscopy 1992;165:427-31.
[16] Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusion-Decellularized Matrix: Using Nature’s Platform to Engineer a Bioartificial Heart. Nat Med 2008;14:213-21.
[17] Wainwright JM, Czajka CA, Patel UB, Freytes DO, Tobita K, Gilbert TW, et al. Preparation of Cardiac Extracellular Matrix from an Intact Porcine Heart. Tissue Eng Part C Methods 2010;16:525-32.
[18] Remlinger NT, Wearden PD, Gilbert TW. Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion. J Vis Exp 2012;70:e50059.
[19] Yang B, Zhang Y, Zhou L, Sun Z, Zheng J, Chen Y, et al. Development of a Porcine Bladder Acellular Matrix with Well-Preserved Extracellular Bioactive Factors for Tissue Engineering. Tissue Eng Part C Methods 2010;16:1201-11.
[20] Gilbert TW. Strategies for Tissue and Organ Decellularization. J Cell Biochem 2012;113:2217-22.
[21] Hodgson MJ, Knutson CC, Momtahan N, Cook AD. Extracellular Matrix from Whole Porcine Heart Decellularization for Cardiac Tissue Engineering. Methods Mol Biol 2018;1577:95-102.
[22] Heath DE. AReview of Decellularized Extracellular Matrix Biomaterials for Regenerative Engineering Applications. Regen Eng Transl Med 2019;5:155-66.
[23] Robertson MJ, Dries-Devlin JL, Kren SM, Burchfield JS, Taylor DA. Optimizing Recellularization of Whole Decellularized Heart Extracellular Matrix. PLoS One 2014;9:e90406.
[24] Hillebrandt KH, Everwien H, Haep N, Keshi E, Pratschke J, Sauer IM. Strategies Based on Organ Decellularization and Recellularization. Transpl Int 2019;32:571-85.