Abstract
To make cultured cells express highly differentiated functions for a long time, a novel culture technique that cultured cells form a multicellular aggregate with tissue-like structure (organoid) has much attentions. It is important to control the thickness of the organoid within 100 µm to prevent growth of dead cells layer caused by oxygen exhaustion. Previously, we developed a culture technique to form a hepatocyte organoids with spherical (spheroid) and cylindrical shape (cylindroid) performed highly liver specific functions for development of hybrid artificial liver. In this study, we have developed a novel method to form an organoid having sheet shape (organoid-sheet). Hepatocytes were inoculated inside gap between parallel positioned porous membranes with spacer by centrifugation, and an organoid-sheet was formed with homogenous thickness and high cell density distribution. Using 150 µm thickness spacer, about 30-40 µm thickness dead cells layer appeared inside the organoid-sheet. On the other hand, using 100-µm thickness spacer, such a layer did not appear. Hepatocyte organoid-sheet using 100 µm thickness spacer could express liver specific functions such as ammonia metabolism and albumin secretion about 1.5 times higher than those of cylindroid and maintained them for at least 1 month. Moreover, we found myocardial cells also formed organoid-sheet, and it expressed spontaneous synchronized contraction to the whole sheet area at least 4 days. These results suggest that using organoid-sheet is expected to applying hybrid artificial organs and patches for graft.
