Extended primary culture of human hepatocytes in a collagen gel sandwich system |
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Authors: | Yumi Kono Suyun Yang Eve A Roberts |
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Institution: | (1) Division of Gastroenterology and Nutrition, Room 8415, University Wing, The Hospital for Sick Children Research Institute, 555 University Avenue, M5G 1X8 Toronto, Ontario, Canada |
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Abstract: | Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of
collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that
of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells.
Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period
of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin
secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion
could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer
of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture.
These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes
in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves
differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system
is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro. |
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Keywords: | human hepatocyte culture collagen gel sandwich extracellular matrix albumin cytochromes P450 |
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