| Background: Liver transplantation is an effective treatment for end-stageliver diseases, however, the development of which has been limited owing toshortage of donor livers. Therefore, new therapeutic techniques were required tosolve the problems above. Liver tissue engineering is to build implantableliver-like tissue by recombining liver cells and carrier bracket; it has the potentialto alleviate shortage of organ donors. However, in research ways, abundant bloodsupply in the liver brings about great difficulty in orthotropic transplantation oflarge liver tissue. Thus, an effective and safe way for implantation has not beenfound yet. This paper, therefore, aims to establish basis for exploring newtreatment through building in vitro and orthotropic transplantation of tissueengineering.Objective: To solve the problem of larger scale hepatocytes orthotropictransplanted in liver are difficult to survive, we tried to build sheet engineeredpre-vascularized liver tissue in vitro, paste it on liver surface with artificial wound,and establish blood supply as soon as possible, then explore a new way tointegrate transplanted tissue and orthotropic liver easily and safely.Methods：1. Preparation of seed cells: cut the1d-old neonatal rat liver intopieces and digest with cold trypsin so as to obtain the neonate rat hepatocyteswhich will be cultured routinely; take out lung lobe of the rat and cut it into pieces,separate the rat pulmonary microvascular endothelial cells with lung tissueadherent method, and culture it as routine. Observe morphology of these two cellswith inverted microscope, evaluate the different activity of hepatocyticproliferation by MTT and detect the expression of hepatocyte albumin and endothelial cell CD31by immunofluorescence.2. Evaluation of the five kinds ofsheet scaffolds for cell compatibility in vitro: vessel-tree-like scaffold material,sponge-like collagenic material, film-like collagenic material, polylactic acid andchitosan. Assess their apparent morphology and internal structure by scanningelectron microscopy. Neonatal rat hepatocytes and endothelial cells compoundingwith the five scaffold material respectively are seeded in culture plates. Observethe cell material adhesion with the scanning electron microscope, measure cellgrowth on the stent with MTT assay, and detect the secretion of inoculated livercells with albumin and urea nitrogen testing kits.3. Two steps of building sheetengineered liver tissue in vitro: First endothelial cell/vascular growth factor werecultured on sheet stent which was over1cm3for2h, then compounded withhepatocytes for1h to construct sheet engineered liver tissue.4. Surfacetransplantation of the composites in livers of the fifteen rats: we first made a slightwound on liver epidermis, then transplanted sheet engineered liver tissue in liversof rats by surface posting, fixed with medical fibrin glue and four weeks later,take samples and observe tissue sections’ HE staining, albumin and CD31immunohistochemical staining.Results:1. Approximately1.5×106/rat liver hepatocytes are separatedfrom the neonatal rat, with a survival rate of over95%. The liver cells maintainnormal morphology in primary culture and are in cluster growth. MTT values for7d rose to a peak, and then decreased gradually during the experiment. Albuminstaining of new-born liver cells appeared to be positive; the adhesion rate of ratpulmonary microvascular endothelial cells was approximately80%, and survivalrate of primary cells was over98%. After adherence, the endothelial cellsarranged like paving stones with monolayer mosaic. MTT values increased inmultiples in2-4days, and then slowly grew four days later. Endothelial cellsCD31stained positive.2. The five kinds of materials showed different surfaceshapes. After liver cells and endothelial cells were compounded with severalstents, the distributions of cells on the scaffolds materials were most suitable for cell adhesion in SEM view. And cell proliferation by the MTT assay showed thatthe proliferative activity of liver cells was in the following order: sponge-likecollagenic material>simulate vascular bundle scaffolds>film-like collagenicmaterial> chitosan> polylactic acid. The proliferation of endothelial cells arrangedin the order of simulate vascular bundle scaffolds>sponge-like collagenicmaterial> film-like collagenic material> chitosan> polylactic acid. The contentdetermination of albumin and urea in the liver-cells culture supernatant showedthat the liver cells in the early stage of incubation in the five scaffolds maintaineda certain synthetic metabolic functions, of which the sponge-like collagenic materialwas best. After sorting with the above experiments, the sponge-like collagenicmaterials were chosen as the carrier brackets to construct engineered liver tissue.3.Size of sheet engineering liver tissue was1.5×1.5×0.6approximately, inmicroscopic view: after2h pre-cultured, vascular endothelial cells has adherentand gathered around the collagen filaments and achieve the purpose ofpre-vascularized. A large number of liver cells gathered around the collagenfilaments and almost covered all the pores.4.Sheet engineering liver tissue can beeasily and safely implanted into the liver surface, and fully integrated with the ratreceptor which formatted a volume of1cm3new tissues. HE staining showed thatliver cells in the new tissue were in good condition and connected with each otherin large pieces, and that endothelial cells formed various sizes of lumens withinthe tissue, with the red blood cells distributing in it. Immunohistochemicalstaining showed that the regenerated tissue expressed albumin, and the internallumens expressed CD31.Conclusion:1. Neonatal rat liver cells and lung microvascular endothelialcells can be conveniently and efficiently obtained through the cold trypsindigestion and tissue adherent method, and they possess value-added vitality andmaintain original secretion function in vitro culture conditions, thus can be usedfor liver tissue engineering study as seed cells of engineered liver tissue building.2. Considering well compatibility with liver cells, sponge-like collagenic materials can be used as the carrier brackets to construct engineered liver tissue.3.The method of compounding seed cells and liver-like tissue with scaffoldmaterials at different times was able to successfully build a sheet ofpre-vascularization of engineered liver tissue in vitro.4. This sponge-like sheetengineered liver tissue can be conveniently and safely transplanted into the ratorthotropic liver by liver surface paste, and it will form some functionalhepatocyte-like tissues with the volume of more than1cm3in the hepatic surface.This method has prospect for clinical application and provides feasible options forfollow-up orthotropic repair of the damaged liver. |
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