Automation of Pressure Control Improves Whole Porcine Heart Decellularization

Abstract

Whole heart decellularization combined with patient-specific cells may prove to be an extremely valuable approach to engineer new hearts. Mild detergents are commonly used in the decellularization process, but are known to denature and solubilize key proteins and growth factors and can therefore be destructive to the extracellular matrix (ECM) during the decellularization process. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated. Glycosaminoglycans and collagen quantification both showed no significant differences in content after decellularization. The compression elastic modulus of the ECM after decellularization was lower than native at low strains, but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. Finally, the recellularization potential of the ECM samples was demonstrated by reseeding cardiac fibroblasts and endothelial cells on the myocardium and endocardium samples.

Using Hemolysis as a Novel Method for Assessment of Cytotoxicity and Blood Compatibility of Decellularized Heart Tissues

“…6,25,26 As stated by Taylor et al 27 as one of the pioneers in developing whole organ decellularization, the improvement of the decellularization process to produce ECM from deceased organs is one of the most notable achievements in the field of tissue engineering within the past 7 years. Acellular ECM can be obtained by decellularization of native tissue either by perfusing detergent solutions through the vasculature or diffusion of this solution through pieces of tissue.…”

“…To obtain cECM, the hearts were decellularized in an automated pressure and temperature-controlled apparatus designed and built by our group as described previously. 6 In the SDS group, hearts were rinsed by antegrade perfusion with 1X PBS and type 1 distilled water (DW) for 1 h each, followed by three sets of 0.5% (w/v) SDS solutions for 2 h each with 1 h of nonrecycled DW between each set. In the Triton X-100 (TX-100, fisher Scientific, fair Lawn, Nj) group, hearts were perfused with 1× PBS, and type 1 DW for 1 h each, followed by three sets of 0.5% SDS solutions for 2 h each with 1 h of nonrecycled DW between each set.…”

“…We have previously shown that our decellularization process results in more than 98% DNA removal from various sections of the hearts, and the produced cECM has very limited immunogenicity. 6 …”

“…To test this method, porcine hearts were harvested and then decellularized using an automated bioreactor. 6 After decellularization, samples were taken from the left ventricle (LV) and rinsed in phosphate-buffered saline (PBS). At various time points, samples were removed from the washes and tested for cytotoxicity.…”

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

“…The study of this topic is limited. It has been shown that automating pressure during decellularization with digital pressure sensors improves whole heart decellularization [23], however the effects of pressures have not been studied despite the variations in pressure between protocols. The same holds true for total decellularization time.…”