Abstract
Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.
Highlights
Engineered cell sheets composed of cells and cell-produced extracellular matrix (ECM) have become attractive for researchers from various fields ranging from basic science to clinical applications for regenerative medicine
Most of the HepaRG cells quickly adhered onto the TIG118 cell monolayers compared to the fetal bovine serum (FBS)-coated temperature-responsive culture dish (TRCD)
It has been reported that the morphology and the functional expression levels of hepatocytes and stem cells are subject to anchorage-dependent control, and the HepaRG cell morphology on the TIG-118 cells was similar to that on a linear cell adhesion patterning substrate [14,15,16]
Summary
Engineered cell sheets composed of cells and cell-produced extracellular matrix (ECM) have become attractive for researchers from various fields ranging from basic science to clinical applications for regenerative medicine. Cell sheets have several advantages compared with biomaterials, such as bacterial cellulose and polymers, in the clinical field; abundant cell-cell and cellECM adhesions and high viability in vivo. Many researchers have already reported in vitro investigations of multi-layered cell sheets, such as fabrication methods using a temperature-responsive culture dish (TRCD) and a gelatin stamp or magnetic force system with a high cell density, vascularized sheet tissues using endothelial cells for the biomimesis and maintenance of cell survival, and cell distributions based on biology and computational chemistry [1,2,3,4]. The fabrication of multi-layered cell sheets is one of the hottest topics related to cell sheet engineering
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