Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes.
A novel method for the preparation of spheroids containing two types of cells (hetero-spheroid) has been successfully developed by utilizing a collagen-conjugated thermo-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), as a cell substratum. PNIPAAm solidifies above its lower critical solution temperature (LCST, about 30 degrees C), and instantly dissolves into the culture medium below its LCST. We firstly seeded and cultured human dermal fibroblasts on the substratum up to a confluent state and then seeded rat primary hepatocytes onto the fibroblast monolayer. The heterospheroid was prepared by detaching the hepatocyte-attached fibroblast monolayer at a temperature below LCST and culturing it on the non-adhesive substratum. The surface area of the substratum and the seeding population ratio of each cell precisely and reproducibly regulated the size and the cell composition of the resulting hetero-spheroid, respectively. Histological and immuno-cytochemical observations of spheroids revealed characteristic organizations of fibroblasts and hepatocytes within a spheroid because the latter cells expressed albumin for up to at least 3 weeks. TEM study of the hetero-spheroid showed the presence of structures morphologically similar to the Disse's space and the bile canaliculus, which are features characteristic of liver. These findings suggest that the method described above is useful for making a hetero-spheroid that morphologically and functionally resembles tissues or organs in vivo, i.e. an organoid.
Takezawa
T
T
Japan Research Center, W.R. Grace & Co. Conn., Kanagawa, Japan.
Yamazaki
M
M
Mori
Y
Y
Yonaha
T
T
Yoshizato
K
K
D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D013102:Spherocytes / Q000166:cytology* / Q000648:ultrastructure