Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Abstract

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver [1][2][3][4][5] . Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment [6][7][8] . However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications. Video LinkThe video component of this article can be found at

Urinary bladder matrix promotes site appropriate tissue formation following right ventricle outflow tract repair

“…In addition to the commercially available products, recent efforts have sought to develop organ-specific scaffolds, including C-ECM, which are hypothesized to be preferred scaffold sources since the ECM was organs, the structure of the scaffold can be preserved through perfusion decellularization. 34,43 This hypothesis is supported by studies that have shown that liver ECM preserves the phenotype of liver specific cells better than heterotopic ECM, and studies with decellularized lung that show the ECM can promote siteappropriate differentiation of mouse embryonic stem cells. 28,44 However, there are other examples that show that heterotopic ECM from the urinary bladder and small intestine can promote the formation of site appropriate tissue, sometimes with greater efficacy than the organ-specific ECM.…”

“…Secondary antibodies were added to blocking solution at the following concentrations; donkey anti-goat AlexaFluor 488 and decellularization. 39,43 Following decellularization, the ventricular walls were separated and a small portion of the right ventricle near the apex was removed. This portion of the ventricle is much thinner than the surrounding myocardium and is most suitable for rat heart reconstruction.…”

“…It has recently been shown that an intact porcine heart can be fully decellularized to generate C-ECM patches. 39,40 C-ECM patches generated in this manner have shown the ability to support cardiac function as well as the infiltration of small areas of cardiac specific cells. 33 UBM and C-ECM have each been investigated individually as scaffolds for myocardial repair, but have shown limited success.…”

Acellular porcine heart matrices: whole organ decellularization with 3D-Bioscaffold & vascular preservation

“…At a larger scale using porcine hearts via retrograde aortic perfusion, successive perfusates of 0.02% trypsin/0.05% EDTA, 3% Triton X-100, and 4% deoxycholate with PBS rinses between reagents also resulted in successful decellularization [25]. Other porcine heart decellularization protocols implemented similar methods [25,26], with a few additional detergents such as CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) [27] used in heart valves and other organs [28,29]. All detergents have the potential to inflict damage on the ECM and disrupt the ultrastructure, by damaging the collagen and glycosaminoglycans for example [29].…”

Strategies and Processes to Decellularize and Recellularize Hearts to Generate Functional Organs and Reduce the Risk of Thrombosis

“…Of these citations, 13 articles were identified as most relevant to the design of an automated system for decellularizing whole hearts. [12][13][14][15][16][17][18][19][20][21][22][23][24] The types of detergents, concentrations of solutions, flow rates, pressures, incubation temperatures, total number of steps, and organ exposure times were identified as major factors for consideration. These parameters are summarized in Table 1.…”