| AIMCurrently, liver transplantation in an orthotopic location has been the only life-saving therapy for end-stage liver failure. In the United States, there are approximately 27,000 deaths annually due to end-stage liver disease; however, the number of people needing new livers far exceeds the number of donor livers. In addition, patients receiving donor livers often suffer from organ rejection. Therefore, innovative efforts in regenerative medicine have attempted to create available organs for transplantation. Recently, some significant progress has occurred in the field of tissue engineering and regenerative medicine; some functional tissues and organs have been created in vitro, and successfully transplanted into recipients, such as blood vessels, the bladder, corneas, and the trachea. However, whole organ structures, such as the lung, heart, kidney, and liver, are more complex, and artificial material-based scaffolds can hardly mimic the structure of these large organs. These scaffolds cannot provide intact vascular networks to exchange nutrients and oxygen. Moreover, it is difficult to simulate the microenvironment maintaining the growth and function of the cells in vivo.In order to solve these problems, one needs to make use of the natural organs to obtain integral structures containing the vascular systems. An acellular organ scaffold preserving the native extracellular matrix (ECM) and vascular networks can be achieved by decellularizing natural organs. Currently, perfusion-based decellularization protocols have been used in solid organs, such as the lungs, heart, kidneys, and liver, to remove all native cells and DNA while maintaining the natural ECMs, which are crucial to cell phenotype and functionality.In recent years, many decellularization protocols have been applied for obtaining natural bioscaffolds in different species of liver. Two kinds of detergents have commonly been used (successfully) in many studies. As a non-ionic detergent, Triton X-100 has been used in different concentrations, from 1% to 3%, and the ionic detergent, SDS, has been used in concentrations ranging from 0.25% to 4%. Due to the loss of certain cardinal matrix components, the effects of different decellularization methods are not accordant. In addition, there have been rare studies about the perfusion rate of decellularization agents in dynamic decellularization techniques. The effects of different perfusion rates in the decellularization procedure are indefinite.Therefore, a simple and effective decellularization protocol is required to clear the residual cytoplasmic and nuclear materials and minimize the damage to the extracellular matrix. In this study, we evaluated the effectiveness of different concentrations of SDS (0.25%,0.5%, and 0.75%) and Triton X-100 (1%,2%, and 3%) with a flow rate of 5ml/min. After obtaining the optimal decellularization procedure, we assessed the effectiveness of different flow rates (3 ml/min,5 ml/min, 7 ml/min, and 10 ml/min) of the most proper decellularization protocol.METHODS1.Animals and liver harvestFifty specifically pathogen-free (SPF) male or female Sprague-Dawley (SD) rats, weighing 200-300 g,.All rats were anesthetized using an intraperitoneal injection of chloral hydrate (1 ml/100 g) and 2 ml heparin sodium, followed by topical skin disinfection. To achieve the most proper decellularization procedure, the isolated livers were divided into six experimental groups (n=5), based on six different treatment protocols, and a control group (n=5). After obtaining the most appropriate decellularization procedure, the group treated with that procedure was divided into four subgroups (n=5), including four different perfusion rates of 3 ml/min,5 ml/min, 7 ml/min, and 10 ml/min. All rats were anesthetized using an intraperitoneal injection of chloral hydrate (1 ml/100 g) and 2 ml heparin sodium, followed by topical skin disinfection. After anesthesia, we made a transverse incision in the abdomen, and exposed the portal vein, the bile duct, and the inferior vena cava. The branches of the portal vein were ligated, and the portal vein was then cut with a side hole for intubation and double-fixed with number 1 silk sutures. The hepatic superior and inferior venae cavae were isolated, and both were cut with a side hole for intubation. The inferior vena cava was then transected and the entire liver was isolated.2. paraffin-embedded tissue sectionsThe native/decellularized livers were cut into small pieces, followed by fixation using a 4%paraformaldehyde solution. After fixation, the embedded samples were cut into 6μm sections, which were dewaxed using xylene, followed by a graded series of ethanol-water solutions (100%,95%,85%, and 70%)3. Immunofluorescence analysisAn immunofluorescence analysis was performed by using specific antibodies to verify the preservation of the liver ECM components. All sections were incubated with primary antibodies overnight at 4℃, followed by washing in PBS. Then, the sections were incubated with anti-rabbit IgG fluorescence labeling and anti-mouse IgG fluorescence labeling for 1 h at room temperature. All second antibodies were diluted to 1:200. DAPI was used to stain the nuclei.4. DNA assayThe DNA was extracted from the samples with the DNeasy Tissue Kit according to the manufacturer’s instructions. The residual DNA content was quantified using the Quant-iT PicoGreen dsDNA assay according to the manufacturer’s instructions.5. GAGs assayThe GAGs were extracted by incubation with a papain extraction reagent overnight, and this was followed by centrifugation and incubation with a Blyscan reagent for quantification via absorbance readings at 656 nm.6. Scanning electron microscopy (SEM)The morphology and structural integrity of these samples were observed using scanning electron microscopy.7. In vitro cytotoxicity testEach sample of the lyophilized scaffold (5 mg) was placed in a 5 ml,1:1 mixture of Dulbecco’s minimal essential medium and Ham’s F12 medium containing 10% fetal bovine serum for 24 h at 37℃ for extraction of the liquid. C3A cells(human hepatocyte cell line, ATCC)were seeded into each well of a 96-well plate and cultured with extracts at a density of 2×103 cells/well (experimental group, n=5) or normal medium (control group, n=5). The proliferation activity of the cells was quantitatively determined at 1,3, and 5days by an MTT assay.8. Perfusion rate optimizationAfter obtaining the most appropriate decellularization protocol, the decellularization agent was perfused into the rat livers at different flow rates (n=5 per subgroup):3 ml/min,5 ml/min,7 ml/min, and 10 ml/min. The other procedures in this protocol did not change. The quantifications of the DNA and GAGs were conducted to assess the effect of decellularization at the different perfusion rates of the decellularization agent.9. Statistical analysisThe results were expressed as the means plus/minus standard deviation (mean±SD), and the statistical analysis was performed by SPSS (Statistical Package for the Social Sciences, version 13.0). The significance of the differences between the native and decellularized samples was determined by a one-way ANOVA (analysis of variance). SNK (Student-Newman-Keuls) tests were used for comparisons between individual groups, and a P-value of less than 0.05 was considered to be significant.RESULTS1 Perfusion decellularization of whole rat liversAfter the 2 h decellularization, all of the livers appeared to be devoid of native cells. Therefore, acellular liver scaffolds with intact Glisson’s capsules were obtained. The 0.25% SDS-treated,0.5% SDS-treated, and 0.75% SDS-treated livers were translucent, but the 1% Triton X-100-treated,2% Triton X-100-treated, and 3% Triton X-100-treated livers were less translucent after the 2 h decellularization. The quantification of the residual DNA, an indicator for cellular removal, showed that the decellularization agents and DNase treatment significantly reduced the residual DNA levels in all decellularization groups, compared with the native rat livers (P<0.05). No significant differences in the residual DNA quantities were observed among the six decellularization groups (P>0.05). However, the residual DNA levels in the 0.25% SDS treatment,0.5% SDS treatment, and 0.75% SDS treatment were 46.14±6.08,40.89±6.73, and 35.35±5.31 ng/mg of dry tissue weight, respectively, which were below 50 ng/mg of dry tissue weight.2. MorphologyThe immunostaining of five ECM proteins showed the preservation of collagen-â… , collagen-â…£, laminin α-2, fibronectin, and elastin, which indicated that the structural components of the ECM were similar to those of native livers (Fig.2). In the native livers, immunolabeling of collagen I was found in the fibrous connective tissues; however, the collagen I fibers were decreased after the six decellularization method treatments. Fibronectin was also found in the fibrous connective tissues, but it was mostly removed in the applied protocols of group 2, group 3, and group 6, compared to the other groups. Collagen IV was observed within the basement membrane of the bile ducts and blood vessels in the native livers, while positive staining for collagen â…£ was mainly found in the remaining blood vessels, after the different decellularization treatments, and was slightly decreased when compared to the native livers. Positive labeling for laminin a-2 was observed in the basement membrane of the blood vessels in the native and decellularized livers, while laminin α-2 was significantly removed in the applied protocols of group 2, group 3, and group 6. Positive staining for elastin was predominantly found in the fibrous connective tissues of the native and decellularized livers.3. Scanning electron microscopy (SEM)The 3D ECM microstructure of the decellularized liver scaffolds was shown in SEM, and the vascular structures were visible. The cell remnants were not observed in any sample. The arrangement of the microstructures in the decellularized liver scaffolds of group 4, group 5, and group 6 were loosely organized, while they were more organized in group 1, group 2, and group 3.4. Quantitative biochemical assaysThe GAG content in the decellularized scaffolds treated with six decellularized protocols was significantly different from that in native rat livers (8.16±0.28μg/mg dry weight) (P<0.05, n=5). The total GAG content of the decellularized scaffolds treated with 0.25% SDS and 1% Triton X-100 (4.05±0.77μg/mg dry weight and 4.25±0.54μg/mg dry weight, respectively) was higher than that in the decellularized scaffolds prepared using the other protocols (P<0.05, n=5). However, there was no significant difference between the 0.25% SDS-treatment and 1% Triton X-100-treatment (P>0.05,n=5).5. Cytotoxicity of DLBSThere were no significant differences in the proliferation of C3A cells among the 0.25% SDS group,0.5% SDS group,0.75% SDS group,1% Triton X-100 group, and the control group, as determined by the MTT assay (P>0.05, n=5) (Fig.4), which means that the decellularized scaffolds in these groups had a lower cytotoxicity for C3A. However, the OD values of the 2% Triton X-100 group and 3% Triton X-100 group showed a significant decrease from day 1 to day 5, and were 0.071±0.008 and 0.069±0.006, respectively (n=5) on day 5. This means that the 2% Triton X-100 and 3% Triton X-100 were cytotoxic to C3A.6. Perfusion rate optimizationThe volumes of 0.25% SDS used in the 3 ml/min,5 ml/min,7 ml/min, and 10 ml/min subgroups were 500ml,600ml,600ml, and 600ml, respectively. The quantification of the residual DNA, GAG, and collagen was to evaluate the effect of the decellularization at the different perfusion rates of 0.25% SDS. The residual DNA in the 10 ml/min subgroup (62.14±5.82 ng/mg dry weight) was higher than in the 3 ml/min,5 ml/min, and 7 ml/min subgroups (49.16±5.98 ng/mg dry weight, 46.14±6.08 ng/mg dry weight,43.68±7.82 ng/mg dry weight, respectively) (P<0.05, n=5), which means that the clearance of the residual material in the 10 ml/min subgroup was not incomplete, but there were no significant differences among the 3 ml/min,5 ml/min, and 7 ml/min subgroups. The total GAG content showed no significant differences among the 3 ml/min (3.66±0.57μg/mg dry weight),5 ml/min (4.05±0.77μg/mg dry weight),7 ml/min (4.82±0.47μg/mg dry weight), and 10 ml/min subgroups (5.18±0.54μg/mg dry weight).CONCLUSION1.0.25% SDS showed some advantages in preparing the decellularized rat liver scaffolds when compared to the other detergents2. The scaffolds treated with SDS or 1%Triton X-100were not toxic to the growth of C3 A cells3. The decellularization time in the 7 ml/min subgroup was the shortest time among the subgroups.7 ml/min subgroup. The decellularization protocol based on 0.25% SDS, with a perfusion rate of 7 ml/min obtains not only complete decellularization, but also the preservation of the structure and native proteins of the extracellular matrix... |
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