M. Fujii1, K. Yamanouchi1, Y. Sakai1, A. Kinoshita1, M. Hidaka1, A. Soyama1, K. Kobayashi1, M. Takatsuki1, K. Kanetaka1, F. Fujita1, T. Kuroki1, S. Eguchi1 1Department Of Surgery, Nagasaki University Graduate School Of Biomedical Sciences,Nagasaki, NAGASAKI, Japan
Introduction:
Subcutaneous hepatocyte transplantation using cell sheet technology could be one of the attractive therapeutic options for inherited liver metabolic disease. However, subcutaneous hepatocytes reportedly do not survive for even 2 weeks without pre-vascularization. On the other hand, adipose-derived mesenchymal stem cells (ADSCs) are known to produce various humoral factors, such as VEGF and HGF, which should induce vascularization. Liver tissue is comprised of 70% hepatocytes, 30% non-parenchymal cells (NPCs), including endothelial cells, biliary epithelial cells, and small hepatocytes, etc. The small hepatocytes have the capacity to proliferate more effectively than hepatocytes, and can differentiate into mature hepatocytes. To construct liver tissue by transplanting co-cultured cell sheets consisting of NPCs/ADSCs subcutaneously without pre-vascularization.
Methods:
Male 5- to 7-week-old Fischer rats were used. ADSCs were separated from the inguinal adipose tissue of the donor by collagenase-digestion and ADSCs sheets were created by culturing them on temperature-responsive culture-dishes (TRCDs). The donor liver was digested by the collagenase perfusion method and we thereby separated hepatic cells into NPCs and hepatocytes employing centrifugations.
Ⅰ) In vitro evaluation of NPCs and hepatocytes: We spread 1.0 × 106 NPCs or hepatocytes each on 35-mm collagen-coated dishes and evaluated their structures and functions.
Ⅱ) Transplantation of co-cultured cell sheets and the corresponding evaluations: We spread NPCs or hepatocytes onto ADSCs cultured beforehand on TRCDs for 2 or 3 days to create co-cultured cell sheets. We then transplanted these sheets into subcutaneous tissues of the recipient rats 2 days after dissemination of the NPCs or hepatocytes. After 2 and 4 weeks, animals were sacrificed and samples were examined histologically.
Results:
Ⅰ) Cultured NPCs showed hepatocyte-like structures. They formed functioning bile canaliculi and produced glycogen. In addition, their albumin secretion capacity was equal to that of hepatocytes (3-day: 98.0 ± 8.4, 90.2 ± 5.8; 7-day: 67.4 ± 10.3, 59.5 ± 3.4μg/mL/dish/2-day).
II) The 1 to 3 layered cell sheets of NPCs/ADSCs survived subcutaneously for 4 weeks while producing glycogen and albumin and expressing CD26, the marker of bile canaliculi. Moreover, vascularization around and in the cell sheets was confirmed by CD31 immunohistochemistry.
Conclusion:
The co-cultured sheets of NPCs/ADSCs had liver-specific functions and survived subcutaneously without pre-vascularization. These results suggest NPCs to be a potential cell source for liver disease therapy and that the technique of creating co-cultured cell sheets might be useful for regenerative medicine by promotion of vascularization.