Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells.

BACKGROUND AND AIMS: Decellularized liver matrix (DLM) hold great potential for reconstructing functional hepatic-like tissue (HLT) based on reseeding of hepatocytes or stem cells, but the shortage of liver donors is still an obstacle for potential application. Therefore, an appropriate alternative scaffold is needed to expand the donor pool. In this study, we explored the effectiveness of decellularized spleen matrix (DSM) for culturing of bone marrow mesenchymal stem cells (BMSCs), and promoting differentiation into hepatic-like cells. METHODS: Rats' spleen were harvested for DSM preparation by freezing/thawing and perfusion procedure. Then the mesenchymal stem cells derived from rat bone marrow were reseeded into DSM for dynamic culture and hepatic differentiation by a defined induction protocol. RESULTS: The research found that DSM preserved a 3-dimensional porous architecture, with native extracellular matrix and vascular network which was similar to DLM. The reseeded BMSCs in DSM differentiated into functional hepatocyte-like cells, evidenced by cytomorphology change, expression of hepatic-associated genes and protein markers, glycogen storage, and indocyanine green uptake. The albumin production (2.74±0.42 vs. 2.07±0.28 pg/cell/day) and urea concentration (75.92±15.64 vs. 52.07±11.46 pg/cell/day) in DSM group were remarkably higher than tissue culture flasks (TCF) group over the same differentiation period, P< 0.05. CONCLUSION: This present study demonstrated that DSM might have considerable potential in fabricating hepatic-like tissue, particularly because it can facilitate hepatic differentiation of BMSCs which exhibited higher level and more stable functions.

Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering.

BACKGROUND: The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS: After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing x 2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5 x 10(6) bone marrow mesenchymal stem cells (BMSCs) was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold. RESULTS: After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The porosity of scaffold, aperture of 45.36 +/- 4.87 µm and pore rate of 80.14% +/- 2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION: Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Using a decellularized splenic matrix as a 3D scaffold for hepatocyte cultivation in vitro: a preliminary trial.

Using a decellularized liver matrix (DLM) to reengineer liver tissue is a promising therapy for end-stage liver disease. However, the limited supply of donor organs still hampers its potential clinical application, while a xenogenic decellularized matrix may bring a risk of zoonosis and immunological rejection. Therefore, an appropriate alternative scaffold is needed. In this research, we established a decellularized splenic matrix (DSM) in a rodent model, which preserved the 3D ultrastructure, the components of the extracellular matrix (ECM) and the native vascular network. The DSM and DLM had similar components of ECM, and similar mechanical properties. Hepatocytes were seeded to the DSM and DLM for dynamic culturing up to 6 d, and distributed both in decellularized sinusoidal spaces and around the vessels. The TUNEL-positive cell percentage in a dynamic culturing decellularized splenic matrix (dDSM) was 10.7% ± 3.6% at 3d and 25.8% ± 5.6% at 5d, although 14.2% ± 4.5% and 24.8% ± 2.9%, respectively, in a dynamic culturing decellularized liver matrix (dDLM) at the same time point (p > 0.05). Primary hepatocytes in the dDSM and dDLM expressed albumin, G6pc and Ugt1a1. The gene expression of Cyp2b1, Cyp1a2 and HNF1α in the gene transcription level revealed hepatocytes had lower gene expression levels in the dDSM compared with the dDLM at 3d, but better than those in a sandwich culture. The cumulative albumin production at 6 d of culture was 80.7 ± 9.6 µg per million cells in the dDSM and 89.6 ± 4.6 µg per million cells in the dDLM (p > 0.05). In summary, the DSM is a promising 3D scaffold for hepatocyte cultivation in vitro.

[Preparation of a decellularized scaffold derived from human liver tissue].

OBJECTIVE: To develop a method for preparing a decellularized scaffold based on human liver tissue. METHODS: A surgical specimen of the left lateral lobe of the liver was obtained from a patients with hepatic hemangioma. The decellularization process was performed by repeated freezing-thawing, sequential perfusion with 0.01% SDS, 0.1% SDS and 1% Triton X-100 through the portal vein, and sterilization with peracetic acid. L-02 cells were then engrafted onto the decellularized liver scaffold. RESULTS: HE staining, DAPI staining and scanning electron microscopy all verified the absence of residual cellular components in the decellularized scaffold. The residual DNA content in the decellularized scaffolds was 25.3∓14.6 ng/mg (dry weight), which was less than 1% of the total DNA content in a fresh human liver. Immunohistochemistry demonstrated that type I and IV collagens, fibronectin and elastin were all retained in the scaffold. The engrafted L-02 cells survived well on the scaffold with active proliferation and expressed albumin and G6pc. CONCLUSION: It is feasible to prepare decellularized scaffolds using surgical specimens of human liver, which can be a new approach to constructing a tissue-engineered liver for clinical purposes.

Hepatocyte culture in autologous decellularized spleen matrix.

BACKGROUND AND AIMS: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. METHODS: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). RESULTS: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 ± 6.34 µg/10(7) cell/24h in dynamic culture in DSM compared to 62.43 ± 4.59 µg/10(7) cell/24h in sandwich culture (P < 0.01) and to 87.54 ± 5.25 µg/10(7) cell/24h in DLM culture (P > 0.05); urea release was 32.14 ± 8.62 µg/10(7) cell/24h in dynamic culture in DSM compared to 20.47 ± 4.98 µg/10(7) cell/24h in sandwich culture (P < 0.05) and to 37.38 ± 7.29 µg/10(7) cell/24h cultured in DLM (P > 0.05). CONCLUSION: The present study demonstrates that DSM can be prepared successfully using a tissue engineering approach. The DSM is an appropriate scaffold for primary hepatocytes culture.

Induced pluripotent stem cells model personalized variations in liver disease resulting from α1-antitrypsin deficiency.

UNLABELLED: In the classical form of α1-antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function, "proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without SLD could model these personalized variations in hepatic disease phenotypes. Patient-specific iPSCs were generated from ATD patients and a control and differentiated into hepatocyte-like cells (iHeps) having many characteristics of hepatocytes. Pulse-chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild-type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from SLD patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with SLD. CONCLUSION: iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that "proteostasis" mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies.

The fabrication and cell culture of three-dimensional rolled scaffolds with complex micro-architectures.

Cell cultures for tissue engineering are traditionally prepared on two-dimensional or three-dimensional scaffolds with simple pores; however, this limits mass transportation, which is necessary for cell viability and function. In this paper, an innovative method is proposed for fabricating porous scaffolds with designed complex micro-architectures. Channels devised by computer-aided design were used to simulate features of blood vessels in native rat liver. Rapid prototyping and microreplication were used to produce a negative polydimethylsiloxane mold, and then a planar porous scaffold with predefined microchannel parameters was obtained by freeze-drying a silk fibroin/gelatin solution of an optimized concentration. After seeding with rat primary hepatocytes, the planar scaffold was rolled up to build spatial channels. By reconstructing the three-dimensional channel model in the scaffold in the form of micro-computed topography data and observing the cross-sections of the scroll, we confirmed that the bent channels were still interconnected, with restricted deviations. A comparison of the primary hepatocyte culture in the scaffolds with and without the devised channels proved that our design influenced cell organization and improved cell survival and proliferation. This method can be used for the construction of complex tissues for implantation and for culturing cells in vitro for biological tests and observations.