The Application Of The Decellularized Liver Matrix In Cell Transplantation

Successful organ transplantation is the ultimate treatment for end-stage organ failure.However, the demand for suitable organs for transplantation far exceeds the available donororgans. Especially in some Asian country such as China, Japan, their organ transplantationsurgery is limited because of the serve shortage of donors due to the religion and otherfactors. Thus, there is an urgent need to develop solid organs for transplantation using tissueengineering and regenerative medicine-based strategies. Cell transplantation combined withtissue engineering is a very attractive solution to resolve the shortage of donors. Here, wewill discuss the application of liver progenitor cells and islets cells transplantation withdecelluarized liver matrix.Hepatocyte transplantation has been proposed as an attractive therapeutic approach toa variety of liver diseases. Progress, however, in several areas is needed, before this form oftherapy is broadly accepted and applied to patients with liver disease. Clinical applicationof hepatocyte transplantation is limited by good quality donor livers for the isolation ofcells. Recent publications thus focus on stem cells as suitable sources for hepatocytes andliver repopulation strategies that could reduce the number of transplanted cells. How toovercome host immune responses against allogeneic cells can potentially be learned from anew tolerance protocol. Recently discovered technologies for reprogramming of postnatalcells into pluripotent stem cells may pave the way towards the generation of patient-specificautologous cells. The current focus of research aims to reduce the shortage of transplantablecells by the application of stem cell sources or by the conditioning of recipient livers.Therapies for severe chronic liver diseases based on adult (stem) cells are already beginningto move into clinical trials. However, many questions about safety and efficacy need to beanswered, before fetal liver progenitor cells, embryonic stem cells and induced pluripotentstem cells can be applied in humans. Islet transplantation is also the effective therapy which adjusts the normal insulinsecretion based on the blood sugar to achieve control the hyperglycemia in long term.Because of the small injury, low complications, islet transplantation showed therepeatability to replace the endogenous damaged islets. There are several studies focused onthe enhancement of islet function. Since Edmonton protocol was applied in2000, the hugeprogress of islet transplant was made. But the insulin independence rate of5years is stilllow because of immunorejection. Moreover, the technique of islet transplant is based on theportal vein injection to the liver, which has the risk of portal vein embolism and liverdysfunction. So, it is needed to develop a new transplant site which can reduce thecomplications and also can quickly be accepted in the clinic application.Cell transplantation can be an alternative for the organ transplantation, but thedifferentiation and proliferation of most cells need special microenvironment and threedimension structure. The tissue engineering methods can achieve this requirement. Theultimate goal of tissue engineering is to reconstruct tissues or organs in order to physicallyand/or functionally replace damaged and injured organs. A successful tissue engineeringendeavor consists of at least three essential components, including tissue-specific cells,scaffolding biomaterials and an appropriate environment for promoting tissue formation.Significant advances have been made in the development of engineered tissues, such asblood vessels, urinary bladder and trachea. However, whole organ construction for largeorgans, such as the heart, lung, liver and kidney, requires immediate access to the bloodsupply following transplantation, which requires extensive vascular networks for nutrientand gas exchange in order to obtain nutrients and oxygen.Lots of cells lose their typical morphology and function in culture within a few daysvia dedifferentiation or epithelial-mesenchymal transition. This underscores the importanceof the microenvironment in maintaining cell function. The extracellular matrix (ECM) notonly provides a scaffold to house cells but also regulates the adhesion, migration,differentiation, proliferation, and survival of cells as well as the interactions amongdifferent cell types. Recent advances in organ and tissue decellularization make it possibleto obtain tissue-specific ECM from whole organs through the perfusion of the organs withvarious detergents. In contrast to the traditional method of decellularization, in which thinlysliced tissues are immersed in various solutions for decellularization, the whole organ decellularized matrix maintains entire vascular network beds. These vascular network bedsprovide not only a convenient route for the infusion of desired cell types but also a3-dimensional environment for the infused cells in contrast to the2-dimensionalenvironment provided by thin layers of decellularized matrix. Hence, we hypothesized thatdecellularized liver matrix (DLM) might provide an excellent microenvironment andscaffold for cell transplantation.In this study, we explored the feasibility and potential benefits of using decellularizedliver matrix as a carrier for mouse fetal hepatic progenitor cell transplantation and islettransplantation.Part one Decellularization of mouse liverObjective: After several attempt, using the perfusion method through the portal veinto decellurize mouse liver. Observe the morphology and structure of decellularized livermatrix; analysis the DNA content of decellularized liver matrix.Method: Using the BABL/c adult mice, following the portal vein perfusion protocolto do the liver decellularization after anesthesia. After getting the decellularized liver matrix,we stained the liver matrix with HE and immunohistochemistry. At last we implant the apiece of liver matrix under the mouse kidney capsule to check the revascularization.Result: we can effectively get decellularized liver matrix using portal vein perfusionmethods; the DNA content of decellularized liver matrix has almost nothing; after staining,the decellularized liver matrix maintained the extracellular matrix structure includingvascular system and collagen but has no cells and nuclei; revascularization can be observedafter implanting the decellularized liver matrix pieces under the kidney capsule.Conclusion: Using perfusion method can effectively get whole liver matrix withoutcell component. Decellularized liver matrix can offer the nutrition and oxygen due to thevascular system and extracellular matrix was preserved.Part two The application of the decellularized liver matrix in mouse fetal hepaticprogenitor cell transplantationObejective: to discovery if decellularized liver matrix can potentially represent idealscaffolds for the hepatocytes transplantation.Method: we investigate whether decellularized liver scaffolds provide cell-friendlybiocompatible three-dimensional environment to support the proliferation and differen- tiation of hepatic progenitor cells. Mouse liver tissues are efficiently decellularized throughportal vein perfusion. Using the reversibly immortalized mouse fetal hepatic progenitorcells (iHPCs), we are able to effectively recellularize the decellularized liver scaffolds.Result:The perfused iHPCs survive and proliferate in the three-dimensional scaffoldsin vitro for2weeks. When the recellularized scaffolds are implanted into the kidneycapsule of athymic nude mice, cell survival and proliferation of the implanted scaffolds arereadily detected by whole body imaging for2weeks. Furthermore, EGF is shown tosignificantly promote the proliferation and differentiation of the implanted iHPCs.Histologic and immunochemical analyses indicate that iHPCs are able to proliferate anddifferentiate to mature hepatocytes upon EGF stimulation in the scaffolds. Therecellularization of the biomaterial scaffolds is accompanied with vascularization.Conclusion: these results indicate that decullarized liver scaffolds effectively supportthe proliferation and differentiation of iHPCs, suggesting that decellularized liver matrixmay be used as ideal biocompatible scaffolds for hepatocyte transplantation.Part three The application of the decellularized liver matrix in islet transplan-tationObjective: To get the islet cells and evaluate the function. After transplantation,observe the growth of islet in the decellularized liver matrix.Method: We isolated adult mice islets using collagenase digestion method. Afterinfusion islets cell into decellularized liver matrix, we observe the islet under kidneycapsule by microscope.Result: We can get150-200islet per mouse using collagenase digestion method. In theSTZ induced diabetic mice, the islets can survive in the decellularized liver matrix underthe kidney capsule.Conclusion: Collagenase digestion method can be used for islet isolation.Decellularized liver matrix can offer the nutrition and oxygen and three dimension structureto the survival of islets. Decellularized liver matrix has the potential for new transplant sitein islet transplantation.