Perfusion decellularization of human and porcine lungs: bringing the matrix to clinical scale.

Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale. Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs. Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture. SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture.

Gilpin SE ，Guyette JP ，Gonzalez G ，Ren X ，Asara JM ，Mathisen DJ ，Vacanti JP ，Ott HC ... -  《-》

Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts.

Whole-organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole-organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human-sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale. Whole porcine liver and kidney were decellularized by perfusion using different detergents (1% SDS, 1% Triton X-100, 1% peracetic acid (PAA), and 1% NaDOC) via the portal vein and renal artery of the liver and kidney, respectively. After rinsing with PBS to remove the detergents, the obtained liver and kidney extracellular matrix (ECM) were processed for histology, residual cellular content analysis, and ECM components evaluation to investigate decellularization efficiency, xenoantigens removal, and ECM preservation. The resulting liver and kidney scaffolds in the SDS-treated group showed the most efficient clearance of cellular components and xenoantigens, including DNA and protein, and preservation of the extracellular matrix composition. In comparison, cell debris was observed in the other decellularized groups that were generated using Triton X-100, PAA, and NaDOC. Special staining and immunochemistry of the porcine liver and kidney ECMs further confirmed the disrupted three-dimension ultrastructure of the ECM in the Triton X-100 and NaDOC groups. Additionally, Triton X-100 effectively eliminated the residual SDS in the SDS-treated group, which ensured the scaffolds were not cytotoxic to cells. Thus, we have developed an optimal method that can be scaled up for use with other solid whole organs. Our SDS-perfusion protocol can be used for porcine liver and kidney decellularization to obtain organ scaffolds cleared of cellular material, xenoimmunogens, and preserved vital ECM components.

Wang Y ，Bao J ，Wu Q ，Zhou Y ，Li Y ，Wu X ，Shi Y ，Li L ，Bu H ... -  《-》

Residual Detergent Detection Method for Nondestructive Cytocompatibility Evaluation of Decellularized Whole Lung Scaffolds.

Zvarova B ，Uhl FE ，Uriarte JJ ，Borg ZD ，Coffey AL ，Bonenfant NR ，Weiss DJ ，Wagner DE ... -  《-》

Perfusion-Decellularization of Porcine Lung and Trachea for Respiratory Bioengineering.

Decellularization of native organs may provide an acellular tissue platform for organ regeneration. However, decellularization involves a trade-off between removal of immunogenic cellular elements and preservation of biomechanical integrity. We sought to develop a bioartificial scaffold for respiratory tissue engineering by decellularization of porcine lungs and trachea while preserving organ architecture and vasculature. Lung-trachea preparations from 25 German Landrace pigs were perfused in a modified Langendorff circuit and decellularized by an SDC (sodium deoxycholate)-based perfusion protocol. Decellularization was evaluated by histology and fluorescence microscopy, and residual DNA quantified spectrophotometrically and compared with controls. Airway compliance was evaluated by endotracheal intubation and mechanical ventilation to simulate physiological breathing-induced stretch. Structural integrity was evaluated by bronchoscopy and biomechanical stress/strain analysis by measuring passive tensile strength, all compared with controls. Decellularized lungs and trachea lacked intracellular components but retained specific collagen fibers and elastin. Quantitative DNA analysis demonstrated a significant reduction of DNA compared with controls (32.8 ± 12.4 μg DNA/mg tissue vs. 179.7 ± 35.8 μg DNA/mg tissue, P < 0.05). Lungs and trachea decellularized by our perfusion protocol demonstrated increased airway compliance but preserved biomechanical integrity as compared with native tissue. Whole porcine lungs-tracheae can be successfully decellularized to create an acellular scaffold that preserves extracellular matrix and retains structral integrity and three-dimensional architecture to provide a bioartifical platform for respiratory tissue engineering.

Weymann A ，Patil NP ，Sabashnikov A ，Korkmaz S ，Li S ，Soos P ，Ishtok R ，Chaimow N ，Pätzold I ，Czerny N ，Schmack B ，Popov AF ，Simon AR ，Karck M ，Szabo G ... -  《-》

Decellularization of porcine whole lung to obtain a clinical-scale bioengineered scaffold.

Whole-organ engineering is emerging as an alternative source for xenotransplantation in end-stage diseases. Utilization of decellularized whole lung scaffolds created by detergent perfusion is an effective strategy for organ replacement. In the current study, we attempted to decellularize porcine whole lungs to generate an optimal and reproducible decellularized matrix for future clinical use. Porcine whole lungs were decellularized via perfusion of various detergents (sodium dodecyl sulfate (SDS)/Triton X-100, sodium lauryl ether sulfate (SLES)/Triton X-100, dextrose/SDS/Triton X-100 and dextrose/SLES/Triton X-100) through the pulmonary artery and bronchus of the lung. The decellularized scaffolds were evaluated for decellularization efficiency, extracellular matrix (ECM) component preservation, xenoantigen removal and compatibility. The resulting lung scaffolds obtained from treatment with the dextrose/SLES/Triton X-100 cocktail showed minimal residual cellular components and xenoantigens, including DNA and protein, and good preservation of ECM composition. Evaluation of the porcine lung ECM by specific staining and immunofluorescence confirmed that the three-dimensional ultrastructure of the ECM was noticeably preserved in the SLES-treated groups. In addition, the decellularized lung scaffolds originating from the dextrose/SLES/Triton X-100 cocktail supported cell adhesion and growth. In summary, the novel detergent SLES alleviated the damage to retain a better-preserved ECM than SDS. Sequential Triton X-100 perfusion eliminated SLES. Moreover, performing dextrose perfusion in advance further protected scaffold components, especially collagen. We developed an optimal dextrose/SLES/Triton X-100 cocktail method that can be used for the decellularization of porcine whole lung to obtain a clinical-scale bioengineered scaffold.

Li Y ，Wu Q ，Li L ，Chen F ，Bao J ，Li W ... -  《-》