Liver Tissue Engineering Utilizing Hepatocytes Propagated in Mouse Livers in Vivo

Abstract

Recent advances in tissue engineering technologies have highlighted the ability to create functional liver systems using isolated hepatocytes in vivo. Considering the serious shortage of donor livers that can be used for hepatocyte isolation, it has remained imperative to establish a hepatocyte propagation protocol to provide highly efficient cell recovery allowing for subsequent tissue engineering procedures. Donor primary hepatocytes were isolated from human α-1 antitrypsin (hA1AT) transgenic mice and were transplanted into the recipient liver of urokinase-type plasminogen activator-severe combined immunodeficiency (uPA/SCID) mice. Transplanted donor hepatocytes actively proliferated within the recipient liver of the uPA/SCID mice. At week 8 or later, full repopulation of the uPA/SCID livers with the transplanted hA1AT hepatocytes were confirmed by blood examination and histological assessment. Proliferated hA1AT hepatocytes were recovered from the recipient uPA/SCID mice, and we generated hepatocyte sheets using these recovered hepatocytes for subsequent transplantation into the subcutaneous space of mice. Stable persistency of the subcutaneously engineered liver tissues was confirmed for up to 90 days, which was the length of our present study. These new data demonstrate the feasibility in propagating murine hepatocytes prior to the development of hepatic cells and bioengineered liver systems. The ability to regenerate and expand hepatocytes has potential clinical value whereby procurement of small amounts of tissue could be expanded to sufficient quantities prior to their use in hepatocyte transplantation or other hepatocyte-based therapies.Key words: Liver tissue engineering; Cell proliferation; Cell sheet; Tissue engineering; Hepatocyte transplantation; Regenerative medicine INTRODUCTIONbeen largely predicated on two key factors. First, the creation of a vascularized platform using a growth factor-releasing device at the target subcutaneous site prior Liver tissue engineering using primary hepatocytes presents the opportunity to create a de novo liver systo the transplantation of the donor hepatocytes (30). The prevascularization process plays a pivotal role in the surtem, which can lead to a new therapeutic modality towards the treatment of various liver diseases (4,10, vival of the transplanted hepatocytes by providing an adequate blood supply to these transplanted cells, due to 15,18,28) and may have the potential to obviate the need for organ liver transplantation. In fact, hepatocyte transtheir high metabolic activities that require an active nutrient supply and waste exchanges (13,16). The secplantation into the liver of a recipient patient has proven to be effective in some experimental and clinical settings ond factor is the creation of uniform hepatocyte sheets using the isolated individual donor hepatocytes in cul- (3,10,14,20,21,24,26).We have recently developed a novel approach for ture (17). The in vitro created hepatocyte sheets maintain their intercellular communications (e.g., desmosomes a...

Transplantation of a human iPSC-derived hepatocyte sheet increases survival in mice with acute liver failure

“…Recently, a cell sheet-based tissue engineering approach has attracted much attention in cell transplantation therapy [13][14][15][16][17] because of its potential for delivering a large number of cells to the desired organ without loss of transplanted cells. A previous study has shown that the cell viability after islet cell sheet transplantation was higher than that after direct cell infusion [18].…”

“…Therefore, it might be reasonable to expect that a cell sheet engineering approach would realize improvements in the control of engraftment efficiency and the avoidance of unexpected site engraftment of human iPS-HLCs. To date, several researchers have conducted hepatocyte sheet transplantation into ectopic sites (e.g., the subcutaneous space) and succeeded in achieving therapeutic values in experimental animal models [14][15][16]. However, the feasibility and therapeutic values of human iPS-HLC sheet transplantation on the liver surface (orthotropic transplantation) have yet to be clarified.…”

Hepatocyte Transplantation: Cell Sheet Technology for Liver Cell Transplantation

“…In the experiments in which normal mouse hepatocytes are transplanted to uPA/SCID mice, we identified that the propagated and then isolated hepatocytes can be propagated again in the uPA/SCID mice liver. Furthermore, we have succeeded in fabricating cell sheets composed of the hepatocytes propagated in the uPA/SCID liver [ 89 ]. Although there are certainly concerns about an ethical issue and a contamination of small amount of mice-derived components, the proposed method utilizing uPA/SCID mice is advantageous in terms of enabling researchers to keep stocks of the hepatocytes and provide the fresh cells any time on demand.…”

Generation of Transplantable Three-Dimensional Hepatic-Patch to Improve the Functionality of Hepatic Cells In Vitro and In Vivo

“…Hepatocyte phenotype depends on 3D conditions, as well as cell-cell and cell-extracellular matrix (ECM) interactions [15]. Recently, a cell sheet-based tissue engineering (TE) approach that improves engraftment efficiency and avoids unexpected site cell engraftments has been introduced [14,16,17]. Furthermore, multilayered hepatocyte sheets containing endothelial cells [18] or fibroblasts [19] improved hepatocyte functions by cell-cell interactions.…”