Decellularization and Recellularization of Rat Livers WithHepatocytes and Endothelial Progenitor Cells
*Pengcheng Zhou, *Yan Huang, Yibing Guo, *Lei Wang, *Changchun Ling,*Qingsong Guo, *Yao Wang, *Shajun Zhu, *Xiangjun Fan, *Mingyan Zhu, Hua Huang,
*Yuhua Lu, and *Zhiwei Wang
*Department of General Surgery; Department of Emergency Surgery; Surgical Comprehensive Laboratory; andDepartment of Pathology, Affiliated Hospital of Nantong University, Nantong, China
Abstract: Whole-organ decellularization has been identi-fied as a promising choice for tissue engineering. The aimof the present study was to engineer intact whole rat liverscaffolds and repopulate them with hepatocytes andendothelial progenitor cells (EPCs) in a bioreactor.Decellularized liver scaffolds were obtained by perfusingTriton X-100 with ammonium hydroxide. The architectureand composition of the original extracellular matrix werepreserved, as confirmed by morphologic, histological, andimmunolabeling methods. To determine biocompatibility,the scaffold was embedded in the subcutaneous adiposelayer of the back of a heterologous animal to observe the
infiltration of inflammatory cells. Hepatocytes werereseeded using a parenchymal injection method and cul-tured by continuous perfusion. EPCs were reseeded usinga portal vein infusion method. Morphologic and functionalexamination showed that the hepatocytes and EPCs grewwell in the scaffold. The present study describes an effec-tive method of decellularization and recellularization ofrat livers, providing the foundation for liver engineeringand the development of bioartificial livers. KeyWords: DecellularizationExtracellular matrixLiverTissue EngineeringEndothelial progenitor cellsRecellularization.
Liver transplantation is currently regarded as theonly definitive and curative therapy for end-stage liverdisease. However, the increasing demand for trans-plantable livers far exceeds the availability of donorlivers (1). In addition, surgical complications, chronicrejection, and high expenses limit the wide applicationof liver transplantation. Hepatocyte transplantationoffers an alternative method to treat patients withliver diseases (2,3). Although clinical studies haveshown the efficacy of hepatocyte transplantation(36), it is associated with problems predominantlyrelated to the limited cell supply and low engraftmentefficiency (79). Bioartificial livers (BALs) are a tem-porary alternative to liver transplantation; they can beused to sustain the patient until a suitable donor organ
becomes available (10). However, BAL cannot sub-stitute liver transplantation permanently. Using theconcept of tissue engineering, artificial three-dimensional scaffolds have been generated and shownto successfully enhance the attachment and survival ofhepatocytes (1114). However, the liver is a complexorgan that requires a constant delivery of nutrientsand oxygen, and the removal of metabolic products.In addition, the artificial scaffolds are not tissue-specific because of the lack of specific cell bindingfactors for cell functions. In recent years, with thedevelopment of regenerative medicine, a promisingapproach for organ replacement has emerged.Bioscaffolds derived from decellularized organs havebeen used to create materials for tissue engineeringapplications. Using this technology, organs such as theheart (1519), lung (2028), liver (2937), and kidney(3845) have been decellularized and recellularizedsuccessfully. The decellularized scaffolds, consistingof extracellular matrix (ECM), show good biocompat-ibility, provide tissue microarchitecture and intactvascular systems, and maintain biological factors thatpromote cell attachment, migration, and proliferation(46). With these advantages, decellularized scaffolds
Received February 2015; revised August 2015.Address correspondence and reprint requests to Dr. Zhiwei
Wang, Department of General Surgery, Affiliated Hospital ofNantong University, No. 20, XISI Road, Nantong, Jiangsu Prov-ince 226001, China. E-mail: [email protected] or Dr. Yuhua Lu,Departments of General Surgery, Affiliated Hospital of NantongUniversity, No. 20, XISI Road, Nantong, Jiangsu Province 226001,China. E-mail: [email protected]
Copyright 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Artificial Organs 2015, ():
are regarded as a promising choice for liver tissueengineering.
The ultimate goal of the decellularization processis to remove cellular material while minimizing thedamage to the ECM. Commonly used methods ofdecellularization include a combination of physical,chemical, and enzymatic approaches (47). Because ofthe thickness and complex intrinsic structures of theliver, perfusion decellularization is the best choice forthe construction of liver scaffolds. In the presentstudy, we used Triton X-100 (Amresco, Solon, OH,USA), which is a non-ionic detergent, as the maindetergent to remove cellular components. After thedecellularization process, we examined thedecellularization efficiency, ultrastructure and ECMcomponents, and the biocompatibility of the scaffold.
Revascularization remains the major challenge forcreating an artificial organ. All organs require a vascularnetwork to supply oxygen and nutrients, and the intactvascular structure of the artificial organ can be directlyconnected to the circulation of the recipient. The advan-tage of decellularized scaffolds over polymer scaffolds isthe presence of an intact vascular network. Researchershave used microvascular endothelial cells and humanumbilical vein endothelial cells for endothelialization ofdecellularized liver scaffolds (29,34,48,49). Endothelialprogenitor cells (EPCs) are a cell population that isreleased mainly from the bone marrow into the periph-eral blood circulation and have been confirmed to par-ticipate in vasculogenesis (5052). EPCs have been usedin the endothelialization of blood vessels (53,54) andheart valves (55,56); however, they have not been usedin the endothelialization of liver decellularized scaf-folds. In the present study, we cultured EPCs from ratbone marrow and repopulated them into decellularizedliver scaffolds.
The methods used for reseeding of the whole organinclude direct parenchymal injection and infusion. Inour study, we compared these two methods forhepatocyte reseeding. After the reseeding process,the scaffold was transferred to the chamber and cul-tured under continuous perfusion. During the cultureprocess, the specific function of the cells in the scaffoldwas monitored. At the end of the culture, the scaffoldwas harvested and histological examination was per-formed to observe the repopulation status.
MATERIALS AND METHODS
AnimalsC57BL/6 mice weighing 2025 g, Sprague Dawley
(SD) rats weighing 300350 g, and 4-week-old SDrats were purchased from the Experimental AnimalCenter of Nantong University, Nantong, China. All
the animals were kept under constant environmentalconditions with a 12-h light/dark cycle and free accessto water and food. All animal procedures wereperformed according to institutional and nationalguidelines and approved by the Animal Care EthicsCommittee of Nantong University.
Harvesting of livers from SD ratsSD rats were anesthetized by intraperitoneal injec-
tion of chloral hydrate (5%, 0.5 mL/100 g). Underdeep anesthesia, rats were treated with topical skindisinfectant, and a laparotomy extending from thepubis to the xyphoid was performed in the abdomen.The distal end of the portal vein (PV) was ligated, andthen the vein was cannulated with a 22-G cannula andfixed with 3-0 silk sutures. A total of 2 mL heparinsodium (100 U/mL) was injected through the vein foranticoagulation. Then, the infrahepatic inferior venacava was transected to allow the outflow of theperfusate. A total of 50 mL phosphate-buffered saline(PBS) was perfused slowly through the PV clear bloodfrom the liver. Then, the suprahepatic inferior venacava, the hepatic artery, and the common bile ductwere freed. Finally, the whole liver was isolated andtransferred to a cell culture dish.
Decellularization of the liverThe cannula in the PV was connected to the peri-
staltic pump (Masterflex Technical Hoses Ltd.,Oldham, UK), and the perfusion rate for each stepwas set at 4 mL/min. The decellularization processwas initiated by PV perfusion with PBS for 1 h, fol-lowed by distilled water for 30 min. Then, the liverwas perfused with 0.02% ethylenediaminetetraaceticacid (EDTA) (Sinopharm Chemical Reagent Co.Ltd., Shanghai, China) in PBS for 30 min. Then, asthe most important step of the decellularization, 1%(w/v) Triton X-100/0.1% ammonium hydroxide(Xilong Chemical Reagent Co. Ltd.) in distilled waterwas perfused for 20 h. Subsequently, distilled waterwas used to rinse cellular lysis and circuit debris for2 h. Finally, the liver was perfused with PBS for 4 h tomaintain isotonicity. The decellularized liver scaffoldwas preserved in PBS at 4C.
Analysis of ECM componentsTo examine the ECM components of the scaffolds,
tissues were randomly cut from fresh livers (n = 3) anddecellularized liver scaffolds (n = 3) and fixed with4% formaldehyde, dehydrated, and embedded in par-affin. Tissue sections were deparaffinized and stainedwith hematoxylin and eosin (H&E), Massonstrichrome, and Sirius red stain. Slides were visualizedand recorded under an Olympus microscope
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Artif Organs, Vol. , No. , 2015
(Olympus Microscope Corp., Tokyo, Japan) for H&Eand Massons trichrome stain and a Nikon E200(Nikon, Nanjing, China) for detection of the Sirius redstain. To determine whether collagen I, collagen IV,laminin, and fibronectin were retained in thedecellularized scaffolds, the slides were blockedagainst nonspecific binding with a solution consistingof 1% bo