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.
"Direct cell injection such as encapsulation is a method to deliver cells into tissues for the treatment or restoration of diseased/damaged liver. Many kinds of biomaterials including collagen, hyaluronic acid and peptides as well as anionic polysaccharides from algae have been used as encapsulation materials    . These materials should have various characteristics including biocompatibility, biodegradation, physical properties, mechanical stability, permeability, and morphology  . "
[Show abstract][Hide abstract] ABSTRACT: Abstract
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.
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.
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.
DCLS can be used to make an encapsulation cell delivery system.
"While the established 3-D tumor models have been successfully used for the evaluation of the effects of drugs (10–12), they are not suitable for the screening of immune cells. Our previous study used a tissue engineering approach and type I collagen hydrogel as a matrix to successfully create hepatic tissue in vitro(13). However, whether the engineered 3-D tumor tissues cultured with a similar approach may be used for evaluating the efficacy of antitumor immune cells has not been explored. "
[Show abstract][Hide abstract] ABSTRACT: Monolayer tumor culture models have been used for evaluating the antitumor activity of immune cells in vitro. However, their value in this research is limited. We used human gastric cancer cells (BGC823) and collagen hydrogel as a matrix to establish an engineered three-dimensional (3-D) tumor culture model in vitro. Tumor cells grew in 3-D culture and formed spheroids in the collagen matrix. Evaluation of the antitumor activity of cytokine-induced killer (CIK) cells revealed that, compared with the 2-D cell culture models, CIK cells migrated towards the tumor cells and destroyed the spheroids and tumor cells in the engineered 3-D tumor culture model. The cytotoxicity of CIK cells against the tumor cells in the engineered 3-D tumor culture model was lower than that in 2-D tumor culture models at 12-36 h post-interaction, but there was no significant difference in the cytotoxicity at later time points. Further analysis indicated that dendritic cell-activated CIK cells had a significantly higher level of cytotoxicity against tumor cells, compared with CIK and anti-CEA/CD3-treated CIK cells, in the engineered 3-D tumor culture model. Our data suggest that the engineered 3-D gastric tumor culture model may better mimic the interaction of immune cells with tumor cells in vivo than the 2-D tumor culture models, and may be used for evaluating the antitumor activity of immune cells in vitro.
"α-SMA (A, tissue: 7-day cutaneous rat wound) and desmin (B, tissue: 7-day rat cutaneous wound) are used to identify vascular smooth muscle cells, whereas NG-2 is often used to label the pericytes in the blood vessels as seen here in a tissue construct harvested from a mouse tissue engineering chamber (C). through graded alcohols and clearance in methyl salicylate or cedar wood oil enables visualisation of tissue neovascularisation (Hickey et al., 1998), vascularisation of implanted scaffolds (Andrade et al., 2007) and inosculation of the recipient vascular bed and implanted tissue engineering construct vasculature (Zhao et al., 2010). India Ink/gelatine is relatively inexpensive and easily processed into paraffin and methacrylate blocks for sectioning of thin and thick sections. "
[Show abstract][Hide abstract] ABSTRACT: The physiological processes involved in tissue development and regeneration also include the parallel formation of blood and lymphatic vessel circulations which involves their growth, maturation and remodelling. Both vascular systems are also frequently involved in the development and progression of pathological conditions in tissues and organs. The blood vascular system circulates oxygenated blood and nutrients at appropriate physiological levels for tissue survival, and efficiently removes all waste products including carbon dioxide. This continuous network consists of the heart, aorta, arteries, arterioles, capillaries, post-capillary venules, venules, veins and vena cava. This system exists in an interstitial environment together with the lymphatic vascular system, including lymph nodes, which aids maintenance of body fluid balance and immune surveillance. To understand the process of vascular development, vascular network stability, remodelling and/or regression in any research model under any experimental conditions, it is necessary to clearly and unequivocally identify and quantify all elements of the vascular network. By utilising stereological methods in combination with cellular markers for different vascular cell components, it is possible to estimate parameters such as surface density and surface area of blood vessels, length density and length of blood vessels as well as absolute vascular volume. This review examines the current strategies used to visualise blood vessels and lymphatic vessels in two- and three-dimensions and the basic principles of vascular stereology used to quantify vascular network parameters.
Histology and histopathology 06/2011; 26(6):781-96. · 2.10 Impact Factor
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