Abstract PURPOSE: The aims of this study were to investigate the effects of sterilization with peracetic acid (PAA) and ethanol on the biological activity of porcine liver scaffolds and to develop a new technique for sterilization using slightly acidic electrolyzed water (SAEW). METHODS: Decellularization of liver slices was performed using 0.1% sodium-dodecyl-sulfate, then evaluated by histological and polymerase chain reaction analyses. Decellularized slices were treated with either PAA or ethanol or SAEW, and then DNA content was quantified. We determined sterilization efficiency by culturing scaffolds in culture medium and on blood agar. We next analyzed the glycosaminoglycan and collagen contents of the scaffolds. Finally, we tested the cytotoxicity of the scaffolds as well as the effects of sterilization on host cell attachment and proliferation. RESULTS: Complete cell and antigenic epitopes removal emphasized the decellularization efficiency. PAA and SAEW treatments achieved the highest efficiency of sterilization compared to that of the ethanol treated scaffolds, and were able to remove a considerable fraction of DNA from decellularized livers. The retained glycosaminoglycan content decreased in all treatments in the following order: SAEW, ethanol, and PAA. Ethanol caused a significant loss in collagen content compared to the other groups. A cytotoxicity evaluation revealed that all scaffolds were nontoxic. SAEW-treated scaffolds supported cell attachment and proliferation at a significantly higher rate than other groups. CONCLUSIONS: These data suggest that SAEW is highly efficient for sterilizing scaffolds and allowed the scaffolds to retain their bioactivity in addition to its high efficiency for cell remnant removal.

A better understanding of the organ specific factors that regulate the migration of mesenchymal stem cells (MSCs) into the target organ is essential for optimization of strategies to improve the repair after injury. In the present study, we showed that the kidney injury molecule-1 (KIM-1), a well-known kidney-specific biomarker, enhanced the in vitro migration capacity of MSCs as a potent kidney-specific chemo-attractant or an inducer. The in vitro roles were verified by migration assay using KIM1-PK1 cell lines, the mouse proximal tubular epithelial cells (mPTEs) and recombinant human KIM-1 proteins (rhKIM-1). Immunofluorescence staining displayed specific ectodomain binding of KIM-1 on the surface of MSCs. Upregulation of chemokine receptor type 4 (CXCR4) protein when treated with tumor necrosis factor alpha (TNF-α) was shown. The effect of KIM-1 on migration of MSCs was augmented by TNF-α pretreatment in a dose-dependent manner, and reduced by AMD3100, an antagonist of CXCR4. These results suggest that KIM-1 is a potential chemo-ligand of CXCR4 and may play an important role in kidney-specific migration of MSCs via interaction between KIM-1 and CXCR4.

Three-dimensional in vitro tumor models are needed to obtain more information about drug behavior in tumors. The aim of this study is to establish a new model for hepatocellular carcinoma (HCC) using decellularized rat livers. After generating the rat liver scaffolds, HepG2 liver cancer cells were perfused via the portal vein and placed in a bioreactor for 10 days. Histology was performed to analyze cell distribution within the scaffolds. Function and tumor-related gene expression were examined by polymerase chain reaction (PCR). We evaluated the function of HepG2 cells grown on scaffolds in the presence of a well-known anti-cancer drug to investigate the potential application of our system for drug screening. The scaffolds were devoid of cellular materials and preserved extracellular matrix components. HepG2 cells grew well on the scaffolds. The PCR results showed that the cells maintained function and invasion ability at significantly higher levels than cells grown on two-dimensional (2-D) dishes or spheroids on Matrigel. Unlike the 2-D cultures, albumin secretion and alpha-fetoprotein expression in three-dimensional cultures were less susceptible to lower concentrations of the drug. Cells grown in scaffolds seemed to respond to the drug in an analogous manner to its known activity in vivo. These findings strengthen the potential use of rat liver scaffolds for screening HCC drugs.

Objective: One of the major obstacles in applying decellularized organs for clinical use is the recellularization step, during which huge numbers of cells are required to develop whole livers. We established a simple protocol for constructing a bioartificial hepatic lobe and investigated its biocompatibility. Methods: The right lateral lobe of porcine liver was decellularized using 0.1% sodium dodecyl sulfate through the right branch of the portal vein. Decellularized lobes were evaluated by histological and biochemical analyses. DNA content was quantified to validate the decellularization protocol. The presence of immunogenic and pathogenic antigens was checked to exclude potential rejection and thrombosis after xenotransplantation. Xeno-reactivity of decellularized tissue against human peripheral blood mononuclear cells was examined. Cytotoxicity was evaluated against hepatocarcinoma cells. Finally, scaffolds were incubated in collagenase for biodegradation testing. Results: The decellularized lobe preserved the three-dimensional architecture, ultrastructure, extracellular matrix components, and vasculature. Scaffolds were almost depleted of DNA in addition to antigenic and pathogenic antigens, which are considered barriers to xenotransplantation. The human immune response against scaffolds was considered non-significant. Our matrices were biocompatible and biodegradable. Conclusions: We successfully developed a non-cytotoxic, non-immunogenic, and biodegradable porcine hepatic lobe for future liver regeneration and bioengineering.

Background. The dog has served as an important experimental model for biomedical research such as transplantation and developing immunosuppressive agents. Although major histocompatibility complex (MHC) in dogs is a dominant factor of graft rejection, it has not been well investigated in dogs compared with human. For that reason, imprecise cross-matching or time-consuming sequence-based typing methods have generally been used to choose specific donor and recipient pairs. Investigation of matching distribution of MHC in dogs with the use of simple and accurate methods would be beneficial for biomedical researchers. The aim of this study was to identify the diversity of dog leukocyte antigen (DLA) types in genetically unrelated dogs by means of microsatellite markers. Methods. Thirty-three Beagle and Shih-Tzu dogs, which were negative in cross-matching, were chosen. The genomic DNA was isolated from peripheral blood leukocytes, and highly polymorphic short tandem repeats located in MHC class I and II were amplified with the use of specific primers. Results. Among all of the dogs, MHC matching groups, including class I full matcheclass II full match (M-M), class I full matcheclass II haplo match (M-H), class I haplo matcheclass II full match (H-M), class I haplo matcheclass II haplo match (H-H) groups, were w1.55%, 0.39%, 1.94%, and 6.59%, respectively. MHC class I nonmatcheclass II nonmatch (U-U) groups were 58.14% of the total dogs. Conclusions. Because differences of histocompatibility between donor and recipient leads to various allograft rejections, knowledge of the distribution of MHC matching in unrelated dogs would be helpful in designing studies and to get more accurate results from experiments using dog transplantation models.

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

ABSTRACT: The dog has served as an important experimental model for biomedical research such as transplantation and developing immunosuppressive agents. Although major histocompatibility complex (MHC) in dogs is a dominant factor of graft rejection, it has not been well investigated in dogs compared with human. For that reason, imprecise cross-matching or time-consuming sequence-based typing methods have generally been used to choose specific donor and recipient pairs. Investigation of matching distribution of MHC in dogs with the use of simple and accurate methods would be beneficial for biomedical researchers. The aim of this study was to identify the diversity of dog leukocyte antigen (DLA) types in genetically unrelated dogs by means of microsatellite markers. Thirty-three Beagle and Shih-Tzu dogs, which were negative in cross-matching, were chosen. The genomic DNA was isolated from peripheral blood leukocytes, and highly polymorphic short tandem repeats located in MHC class I and II were amplified with the use of specific primers. Among all of the dogs, MHC matching groups, including class I full match-class II full match (M-M), class I full match-class II haplo match (M-H), class I haplo match-class II full match (H-M), class I haplo match-class II haplo match (H-H) groups, were ∼1.55%, 0.39%, 1.94%, and 6.59%, respectively. MHC class I nonmatch-class II nonmatch (U-U) groups were 58.14% of the total dogs. Because differences of histocompatibility between donor and recipient leads to various allograft rejections, knowledge of the distribution of MHC matching in unrelated dogs would be helpful in designing studies and to get more accurate results from experiments using dog transplantation models.

Purpose To improve effect of liver disease treatment, tissue engineering approach such as direct hepatocyte injection has been investigated. Encapsulation, mixing cells and biomaterials to enclose cells within a biomaterial capsule, is commonly used to deliver cells into the body. Many kinds of biomaterials including natural and artificial materials have been used. The capsule must have biocompatibility and microstructure for cell culture, survival and proliferation as well as cell function and therapeutic effects. However, most biomaterials used for encapsulation have low biocompatibility, insufficient constituents and an unsuitable 3-dimensional structure. To solve these problems, we performed encapsulation using a decellularized liver scaffold (DCLS) with a native extracellular matrix (ECM) and natural porous microstructure including vasculature. Methods DCLS was prepared with 0.1% sodium dodecyl sulfate under agitation and 2 mm2 sized DCLS pieces were sterilized with peracetic acid (25.6 µl/10 ml) for 24 hours. Histological analysis showed that the DCLS had native ECM, liver specific major architecture and blood vessel structure but no cells. For cell encapsulation, hepG2 cells were injected into DCLS pieces with a syringe and cultured for 5 days. Results The cells survived and formed a cell mass with a liver ECM microstructure inside the DCLS capsules. The encapsulation status was similar to capsules formed by current encapsulation techniques. Conclusions DCLS can be used to make an encapsulation cell delivery system.