title: Porcine liver decellularization under oscillating pressure conditions: a technical refinement to improve the homogeneity of the decellularization process.
abstract: Decellularization and recellularization of parenchymal organs may facilitate the generation of autologous functional liver organoids by repopulation of decellularized porcine liver matrices with induced liver cells. We present an accelerated (7 h overall perfusion time) and effective protocol for human-scale liver decellularization by pressure-controlled perfusion with 1% Triton X-100 and 1% sodium dodecyl sulfate via the hepatic artery (120 mmHg) and portal vein (60 mmHg). In addition, we analyzed the effect of oscillating pressure conditions on pig liver decellularization (n=19). The proprietary perfusion device used to generate these pressure conditions mimics intra-abdominal conditions during respiration to optimize microperfusion within livers and thus optimize the homogeneity of the decellularization process. The efficiency of perfusion decellularization was analyzed by macroscopic observation, histological staining (hematoxylin and eosin [H&E], Sirius red, and alcian blue), immunohistochemical staining (collagen IV, laminin, and fibronectin), and biochemical assessment (DNA, collagen, and glycosaminoglycans) of decellularized liver matrices. The integrity of the extracellular matrix (ECM) postdecellularization was visualized by corrosion casting and three-dimensional computed tomography scanning. We found that livers perfused under oscillating pressure conditions (P(+)) showed a more homogenous course of decellularization and contained less DNA compared with livers perfused without oscillating pressure conditions (P(-)). Microscopically, livers from the (P(-)) group showed remnant cell clusters, while no cells were found in livers from the (P(+)) group. The grade of disruption of the ECM was higher in livers from the (P(-)) group, although the perfusion rates and pressure did not significantly differ. Immunohistochemical staining revealed that important matrix components were still present after decellularization. Corrosion casting showed an intact vascular (portal vein and hepatic artery) and biliary framework. In summary, the presented protocol for pig liver decellularization is quick (7 h) and effective. The application of oscillating pressure conditions improves the homogeneity of perfusion and thus the outcome of the decellularization process.
authors.affiliation: 1 Department of General, Visceral, and Transplantation Surgery, Charité-Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Benjamin
authors.initials: B
authors.lastname: Struecker
authors.forename: Karl Herbert
authors.initials: KH
authors.lastname: Hillebrandt
authors.forename: Robert
authors.initials: R
authors.lastname: Voitl
authors.forename: Antje
authors.initials: A
authors.lastname: Butter
authors.forename: Rosa B
authors.initials: RB
authors.lastname: Schmuck
authors.forename: Anja
authors.initials: A
authors.lastname: Reutzel-Selke
authors.forename: Dominik
authors.initials: D
authors.lastname: Geisel
authors.forename: Korinna
authors.initials: K
authors.lastname: Joehrens
authors.forename: Philipp A
authors.initials: PA
authors.lastname: Pickerodt
authors.forename: Nathanael
authors.initials: N
authors.lastname: Raschzok
authors.forename: Gero
authors.initials: G
authors.lastname: Puhl
authors.forename: Peter
authors.initials: P
authors.lastname: Neuhaus
authors.forename: Johann
authors.initials: J
authors.lastname: Pratschke
authors.forename: Igor M
authors.initials: IM
authors.lastname: Sauer
mesh_terms: D000818:Animals; D057785:Catheters; D024141:Collagen Type IV / Q000378:metabolism; D003343:Corrosion; D004247:DNA / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D007797:Laminin / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000000981:diagnostic imaging; D008297:Male; D009929:Organ Size; D010477:Perfusion; D011312:Pressure; D013194:Staining and Labeling; D034421:Sus scrofa; D023822:Tissue Engineering / Q000379:methods*; D014057:Tomography, X-Ray Computed
chemical_list: D024141:Collagen Type IV; D005353:Fibronectins; D006025:Glycosaminoglycans; D007797:Laminin; D004247:DNA