title: Controlled processing of a full-sized porcine liver to a decellularized matrix in 24 h.
abstract: The generation of full-sized humanized organs based on animal matrix scaffolds is a promising approach to overcome the shortage of transplant organs. Recent decellularization methods are mostly time-consuming and associated with large rinsing volumes and poorly standardized procedures. In this study we developed an optimized rapid and standardized decellularization method to obtain a functional porcine liver matrix within 24 h. Full porcine livers (n = 10) were decellularized by flushing with 3 L of an isotonic sodium chloride solution and controlled portal perfusion (20 mmHg) with 2 × 10 L of a 1% sodium dodecyl sulphate (SDS) solution at 37°C and a final perfusion with DNase (n = 5). Protein concentrations were continuously monitored by optical density (280 nm). DNA, glycosaminoglycans, and collagen contents were assessed and a haematoxylin and eosin (H&E) staining was performed. After 24 h of perfusion, the liver had a white and translucent appearance, and no further protein was eluted. Histological staining showed an intact extracellular matrix with no nuclear residuals. Moreover, only trace amounts of DNA were detectable in the decellularized tissue (p < 0.001), while glycosaminoglycans and about 60% of collagen levels could be preserved. Thus, we demonstrate that human-scale porcine livers can be successfully decellularized with small volumes of an SDS solution and DNase in a standardized process within 24 h to obtain a clinically relevant organ scaffold suitable for further tissue engineering.
authors.affiliation: Department of General, Visceral, and Transplant Surgery, University of Tübingen, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany. Electronic address: Nicola.buehler@med.uni-tuebingen.de.
authors.forename: Nicola Elena Maria
authors.initials: NE
authors.lastname: Bühler
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Straße 4, 72076 Tübingen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulze-Osthoff
authors.affiliation: Department of General, Visceral, and Transplant Surgery, University of Tübingen, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Königsrainer
authors.affiliation: Department of General, Visceral, and Transplant Surgery, University of Tübingen, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schenk
mesh_terms: D000818:Animals; D021522:Bioartificial Organs; D002469:Cell Separation / Q000379:methods*; D003094:Collagen / Q000032:analysis; D004247:DNA / Q000032:analysis / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry; D005260:Female; D006025:Glycosaminoglycans / Q000032:analysis; D006801:Humans; D007552:Isotonic Solutions / Q000494:pharmacology; D008099:Liver / Q000737:chemistry* / Q000166:cytology*; D010477:Perfusion; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013552:Swine; D013556:Swine, Miniature; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D006025:Glycosaminoglycans; D007552:Isotonic Solutions; D012967:Sodium Dodecyl Sulfate; D003094:Collagen; D004247:DNA; D003851:Deoxyribonucleases
keywords: Bioartificial liver; Controlled process; Decellularization; Extracellular matrix; Organ engineering; Porcine liver