In vivo generation of thick, vascularized hepatic tissue from collagen hydrogel-based hepatic units.
ABSTRACT In vivo engineering of hepatic tissue based on primary hepatocytes offers new perspectives for the treatment of liver diseases. However, generation of thick, three-dimensional liver tissue has been limited by the lack of vasculature in the tissue-engineered constructs. Here, we used collagen hydrogel as a matrix to generate engineered hepatic units to reconstitute three-dimensional, vascularized hepatic tissue in vivo. Hepatocytes harvested from Sprague-Dawley rats were mixed with liquid type I collagen, concentrated Dulbecco's modified Eagle's medium (2 x), and hepatocyte maintenance medium to create hepatocyte/collagen hydrogel constructs. The constructs were then dissociated into cylindrical hepatic units (diameter/height: 2000-4000 microm/500-1000 microm). Stacking of hepatic units under the subcutaneous space resulted in significant cell engraftment, with the formation of large fused hepatic system (more than 0.5 cm thickness) containing blood vessels. In contrast, only less cell engraftment could be achieved when hepatocytes were transplanted in a manner of whole constructs. Functional maintenance of the engineered hepatic tissue was confirmed by the expression of liver-specific mRNA and proteins. The engineered hepatic tissue has the ability to respond to the regenerative stimulus. In conclusion, large hepatic tissue containing blood vessels could be engineered in vivo by merging small hepatic units. This approach for tissue engineering is simple and represents an efficient way to engineer hepatic tissue in vivo.
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ABSTRACT: Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro.PLoS ONE 03/2014; 9(3):e92397. · 3.53 Impact Factor
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ABSTRACT: Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix, which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 hours and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank= 0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases (AST) and alanine aminotransferases (ALT) serum levels as compared with the 2-to-20-fold increase in control groups (p<0.0001) accompanied with improved liver histology. Sustained normal serum albumin levels were observed in alginate-scaffold-treated mice 48 hours after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in alginate scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3 hours post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.Acta biomaterialia 03/2014; · 5.68 Impact Factor
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ABSTRACT: ABSTRACT Introduction: The liver is the natural microenvironment for hepatocytes transplantation but unfortunately engraftment efficiency is low. Cell-laden microhydrogels made of fibrinogen attached to poly(ethylene glycol) (PEG)-diacrylate side chains, were used as a cell carrier, for intravascular transplantation. This approach may reduce shear stress and immediate immunological pressure after intravascular transplantation and provide biomatrix for environmental support. Aims: In vitro assessment of HuH-7 viability and function after polymerization within PEGylated fibrinogen-hydrogel. In vivo assessment of intraportal transplantation of cell-laden microhydrogels with rat adult parenchymal cells. Methods: 1. In vitro assessment of HuH-7 cell viability and function, after cell-laden hydrogel (hydrogel volume 30μl) fabrication, by propidium iodide (PI)/fluorescein diacetate (FDA), and MTT assays, albumin concentration and CYP1A activity. 2. Fabrication of cell-laden microhydrogels and their intraportal transplantion. Engraftment efficiency in vivo was evaluated by real time qPCR of Y chromosome (SRY gene) and histology. Results: The viability of cells in hydrogels in culture was comparable to viability of not embedded cells during the first 48 hours. However. the viability of cells in hydrogels was reduced after 72 hours as compared to not embedded cells. Activity of CYP1A in hydrogel was comparable to that of not embedded cells (4.33±1 pmole/μg DNA/4h vs. 5.13±1 pmole/μg DNA/4h, respectively). Albumin concentration increased at day 3 in hydrogels to 1.4±0.6μg/10<sup>4</sup>/24h and was greater to that of free cells, 0.3±0.1 μg/10<sup>4</sup>/24h. Cell-laden microhydrogels at a size of 150-150-600 μm (6x10<sup>6</sup> cells/rat) showed better engraftment efficiency at 21 days post transplantation, compared to isolated cell transplantation (54.6±5% vs. 1.8±1.2%, P<0.001). Conclusions: The in vitro HuH-7 viability and function after polymerization in PEGylated fibrinogen hydrogel was comparable to cells without the hydrogel. Long term survival and engraftment efficiency of intravascular transplanted adult hepatocytes is much better in within cell-laden microhydrogels as compared to isolated cells. The overall efficiency of the procedure needs to be improved.Tissue Engineering Part A 05/2014; · 4.64 Impact Factor