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A Novel Method for Faster Formation of Rat Liver Cell Spheroids
Author(s) -
Okubo Hisashi,
Matsushita Michiaki,
Kamachi Hirofumi,
Kawai Tomoaki,
Takahashi Manabu,
Fujimoto Tetsuya,
Nishikawa Kazutaka,
Todo Satoru
Publication year - 2002
Publication title -
artificial organs
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.684
H-Index - 76
eISSN - 1525-1594
pISSN - 0160-564X
DOI - 10.1046/j.1525-1594.2002.06836.x
Subject(s) - spheroid , hepatocyte , cell culture , chemistry , cell , biophysics , confocal , confocal microscopy , bioreactor , microbiology and biotechnology , in vitro , biology , biochemistry , genetics , geometry , mathematics , organic chemistry
Hepatocyte spheroids are expected to be the main component of the artificial liver bioreactor for their higher function. The preparation of hepatocyte spheroids, however, can require as many as 24 to 96 h. To reduce this time, we investigated a method employing a new technique of rat hepatocyte preparation and a dynamic culture. The modified Seglen's method for standard hepatocyte isolation was altered by elimination of ethyleneglycol bis(aminoethylether) tetraacetate from the first perfusate and calcium from the second perfusate. Isolated hepatocytes were cultured in a spinner flask by spinning at 120 rpm. The modified Seglen's method was used as a control. Cells obtained by the new method were more cohesive and formed a higher proportion of cell aggregates than control cells. In the spinning culture, hepatocytes had a tendency to aggregate and 80% of cells formed spheroids within 6 h of culturing. The mean size of spheroids was 68.5 ± 18.5 μm. Confocal laser scanning microscopy revealed that individual spheroids contained approximately 30% of nonparenchymal cells over their surface. Using the new hepatocyte preparation method followed by a spinning culture, we were able to produce hepatocyte spheroids in as few as 6 h.