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Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers

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Summary

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was employed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi, and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.

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Authors and Affiliations

  1. Division of Biosciences, University of Hertfordshire, AL10 9AB, Hatfield, Hertfordshire, UK

    Geraldine A. Hamilton

  2. Sofety Assesment, GlaxoSmithKline, SG12 0DP, Ware, Hertfordshire, UK

    Carl Westmoreland & Elisabeth George

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  1. Geraldine A. Hamilton
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  2. Carl Westmoreland
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  3. Elisabeth George
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Correspondence to Elisabeth George.

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Hamilton, G.A., Westmoreland, C. & George, E. Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers. In Vitro Cell.Dev.Biol.-Animal 37, 656–667 (2001). https://doi.org/10.1290/1071-2690(2001)037<0656:EOMCOT>2.0.CO;2

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  • DOI: https://doi.org/10.1290/1071-2690(2001)037<0656:EOMCOT>2.0.CO;2

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