Establish Engineered Liver Lobe Based On Decellularized Liver Scaffold

Background: The shortage of donor livers has severely limited the clinical applicationof liver transplantation to acute liver failure and end of chronic liver diseases. Livertissue engineering is thought to be one of the methods to solve this difficult problem.The core content of tissue engineering is scaffolds, seed cells, extracellular matrix.Researchers face great challenges on building the whole frame of liver and tube systemfor substantive organs like liver with complicated structure and function. Researchersthink that the skill of decellularizing the whole organ is the most convenient method tobe used. Although acquired the whole frame and tube, it is difficult to fulfill the wholeliver with the liver parenchyma cells and mesenchymal cells. There is no report aboutthat it is possible to rebuild the liver lobe taking advantage of the whole old framethinking about parts of function in sick liver. There are mature liver cells, fetal livercells, bone mesenchymal stem cells as seed cells. But it is difficult to survive, if it cannot connected with blood circulation. Gel biological materials show good applicationprospect in many kinds of extracellular matrix. It is conducive to transport nutrition andmetabolite and target cells because it is easy to mix with cell factors and tissue cellcomplex and it has the advantage of mobility and plasticity. But it is not reported how tomix gel material with good biocompatibility with target cells and cell factors by thesuitable concentration and to integrate the outsourcing organic membrane and the tubesystem to form liver tissue. We assume that seed cells can be uniformly and stablyplanted in the whole liver matrix under the support of gel containing cytokinesis, so it isin favor of cell adhesion, growth and nutrition transportation avoiding lack of liner.According to above, we attempt to construct high imitation of engineering lobe to copewith the deficiency of liver during liver transplantation by getting the membrane ofpartial liver and internal duct system with decellurizing method and complex with gel.Objectives: To establish the method of partial liver lobe decellularization in vitro and in vivo, and construct the tube system of both the whole liver lobe and the duct system. Todevelop the gel mixture of hepatic cells and growth factors, and irrigate it into thebio-tube system to simulate the engineering liver lobe in vitro and in vivo.Methods:1. Preparation of partial liver lobe tube system with rat’s liver: remove theliver from the rats, and block the left lobe, left lateral lobe, middle lobe, and irrigatepartial liver lobe in vitro with the bio-detergent of Triton and SDS, and obtaindecellularizing tube of partial hepatic lobe. Block the portal vein, the superior inferiorvena cava, the inferior inferior vena cava, and dispose a tube in the breaking point ofinferior inferior vena cava as the efferent tract. Irrigate in situ partial liver lobe by portalvein puncturing with scalp needle in vivo to the tube system of liver decellurization.2.Prepare the hepatic cells: extract the neonatal hepatic cells of SD rats with the digestionof trypsin, and make the morphological identification with light microscope.3. Preparethe gel and screen the concentration: prepare the rat tail collagen of differentconcentration and culture with neonatal rat hepatic cells and detect the proliferationability of the co-culture cells.4. Construct high immitation engineering liver lobe invitro: rat tail collagen mixed with neonatal rat hepatic cells to get highly biologicalconstructed and injectable compound material, and irrigate it into the decellularizedliver tube to simulate the liver lobe, and culture for a short time in vitro.5.Morphological analysis of constructed liver lobe including HE staining and scanningelectron telescope.6. Construct and evaluate the engineering liver lobe in vivo and insitu: the mixture of gel, cells and growth factors was irrigated to decellularized livertube and perfused with blood for24h for evaluation.Results: The gross morphology of partially decellularized liver lobe containedtransparent tube system with integrated membrane and clear tube structure. HE stainingand scanning electron microscope demonstrated that these tube was membraneintegrated, tract cleared, cell depleted and lipid depleted.2. The extraction quantity ofneonatal rat hepatic cells was about2×106per rat with a survival rate of95percent andround smaller shape and big nucleus and full cytoplasma compared to mature hepaticcells in inverted microscope.3. The result of MTT demonstrated that the proliferation activity of neonatal hepatic cells was proportional to the concentration of rat tailcollagen, namely3mg/ml>2mg/ml>1mg/ml.4. After the culture of engineering liverlobe for a short time, HE staining displayed that the collagen would degradation in vitro,accompanying with the death of the hepatic cells. However, the gel distributed along thevessels or other ducts showed a slower speed of degradation and the cells there grewwell. Besides, the electron microscope depicted that the hepatic cells and the gel werewell integrated with homogeneous distribution.5. The engineering liver lobe which wasconstructed in vivo compared to in vitro showed a much slower degradation speed ofthe gel with homogeneous distribution and survival of cells, according to the results ofHE staining of blood reperfusion in the live rats.Conclusion: This research has successed to construct partial liver lobe with wholemembrane and tube system. And it has formed simulation engineered liver tissue afterperfused with mixture of cells/gel/growth factors. The gel material degraded quickly,and minor cells survived in vitro under static culture conditions in vitro engineeringlobe, while majority of cells survive in vivo engineering lobe because of establishingblood circulation in time.