Decellularized extracellular matrix of blood vessels, cardiac valves, bladder and intestine has been used for facilitating cell transplantation (17
). An in vitro
study of using decellularized liver extracellular matrix for hepatocyte culture has been reported (21
). It was shown that human hepatocytes cultured between two layers of porcine liver decellularized matrix in vitro
for 10 days exhibited liver-specific function similar to those cells grown in a Matrigel sandwich (21
), and that rat hepatocytes seeded between the sheets of decellularized liver matrix showed good viability and function in vitro
). Some of these previous studies employed pieces of decellularized liver matrices, and the decellularized matrix tissue was lyophilized into a powder form, and was rehydrated to generate a gel-like carrier. Our study started with whole liver decellularization and cells were infused into the DLM immediately after decellularization. Our decellularization procedure which employed a much shorter period (6 hrs instead of 3 days) was as effective as a long decellularization protocol in terms of residual DNA content in the DLM (24
). At the same time, the structure of DLM was extremely well preserved as demonstrated by full preservation of extracellular matrix and vasculature (). Moreover, our in vitro
and in vivo
data clearly demonstrated that the DLM facilitated both survival and function of human primary hepatocytes and fetal hepatocytes for up to 6–8 weeks after implantation as evidenced by bioluminescent imaging, immunohistochemical staining and quantitative RT-PCR assays.
Splenic injection has been widely used as a route for transplantation of hepatocytes in rodents (25
). We compared cell survival between using the DLM as a carrier and splenic injection, and found that fetal hepatocytes reconstituted in the DLM survived much longer than those with splenic injection. It appears that fetal hepatocytes migrated to the liver within a fewer days after splenic injection as demonstrated in our bioluminescent imaging study (data not shown). With this route of cell transplantation, the luciferase signal strength rapidly declined within 3 weeks after cell transplantation, which was similar to the findings we previously reported when NOD-SCID mice were not pre-treated with methylcholanthrene and monocrotaline (4
). We added an additional control group by the direct injection of HF-hTERT into the omentum after Matrigel encapsulation. The CCD camera imaging showed a trend of decline in bioluminescent intensity similar to that of splenic injection. In contrast, bioluminescent signal strength from HF-hTERT reconstituted into the DLM was sustained for up to 8 weeks. Presumably, the engraftment of HF-hTERT would be easier in DLM than in mouse liver because there is a vast space available, and intact extracellular matrix components in their original configuration remain after the completion of the decellularization. The result appeared to be better than when Matrigel was used to encapsulate HF-hTERT and encapsulated cells were implanted into the omentum (26
). Human primary hepatocytes via either splenic injection or implantation in DLM survived in mice, and expressed liver-specific genes, such as albumin and CYP2C9. Moreover, primary hepatocytes in DLM expressed key mature markers, CYP3A4 and CYP1A1. Our data indicate that DLM is superior to splenic injection for maintaining the function of primary human hepatocytes.
The establishment of a proper vascular system in the reconstituted DLM may be a critical issue for the survival of the transplanted cells. Bioluminescent imaging of FH-hTERT and primary hepatocytes with lentiviral LUX-PGK-EGFP transduction reconstituted in DLM revealed that the luciferase signals were sustained for a period of 8 weeks after implantation in NOD/SCID/IL2rγ−/−
mice, a strain of mouse which is to date the most immunodeficient, although the strength of the signals declined after the first week. These data indicate that the reconstituted cells may be able to access some, but not sufficient, blood supply as indicated by the presence of mouse cells in the implanted DLM. We employed small pieces (0.5×0.5×0.1 cm3
) of reconstituted DLM which were implanted in vascular-rich omentum in our experiments. This may have contributed to the prolonged survival and improved function of primary hepatocytes because the omentum has been a favorable site for engraftment of hepatocyte polymer tissue-engineered constructs in comparison to subcutaneous compartments (26
). However, when a larger size of DLM is needed for human cell transplantation, adequate blood supply with existing vasculature will be essential. Infusion of vascular endothelial cells or their precursor cells together with hepatocytes may facilitate the revascularization of the DLM. Linke et al.
reported that pre-seeding a decellularized porcine jejunal segment with macrovascular endothelial cells before seeding porcine hepatocytes led to the maintenance of liver-specific function for 3 weeks in vitro
). In our previous studies, we demonstrated that human bone marrow or umbilical cord blood-derived precursor endothelial cells or endothelial cells isolated from placenta and other stem cell types rapidly improved vascularization of ischemic tissues (28
). We are currently investigating the potential benefits of co-seeding hepatocytes with these cells in DLM to promote more rapid and robust revascularization. Another option would be vessel anastomosis to the recipient’s systemic or portal circulation (24
). Although the recent study reported by Uygun et al
. demonstrated the feasibility of the transplantation of a re-grown liver lobe from DLM with rat hepatocytes, the duration of the graft survival in rat recipients still requires improvement (24
). In the present study we have examined the long-term survival of human hepatocytes in an engineered liver graft.
Our data suggest that DLM is an excellent carrier for transplantation of primary hepatocytes. However, the mechanism underlying this benefit is yet to be investigated. Integrins are major mediators of cell adhesion. ECM components including collagen and fibronectin bind to the RGD domain of integrins, and activate not only focal adhesion molecules but also cell survival signals, for instance, via the phosphoinositol-3, Akt or MAPK signaling pathways (31
). In a study by Gupta and colleagues, infusion of collagen or fibronectin-like polymer through the portal vein prior to hepatocyte transplantation enhanced the engraftment of transplanted cells (32
), which suggests a crucial role of extracellular matrix components in the integrity and function of transplanted hepatocytes. The decellularized liver matrix with the natural extracellular matrix components in a three-dimensional configuration appears to be responsible for prolonged survival and function of hepatocytes.
In conclusion, the findings in the present study demonstrate that decellularized liver matrix allows human fetal hepatocytes to survive longer than splenic or omentum injection in mice after transplantation. Moreover, the decellularized liver matrix maintains the liver-specific function of primary hepatocytes after implantation. Taken together, these data suggest the possibility that decellularized liver matrix may be developed as an alterative carrier for hepatocyte transplantation, when a large number of viable hepatocytes are required to functionally replace a failing liver.