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The restoration of the functions of serially passaged calf hepatocytes by spheroid formation

  • Growth, Differentiation and Senescence
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Summary

A serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers—including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate hydrogenase, and ammonia-metabolizing activities—have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.

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  1. Department of Biochemistry, National Defense Medical College, 3-2, Namiki, Tokorozawa, 359, Saitama, Japan

    Fumiko Karikusa & Yoshio Sawasaki

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  1. Fumiko Karikusa
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  2. Yoshio Sawasaki
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Karikusa, F., Sawasaki, Y. The restoration of the functions of serially passaged calf hepatocytes by spheroid formation. In Vitro Cell.Dev.Biol.-Animal 32, 30–37 (1996). https://doi.org/10.1007/BF02722991

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  • DOI: https://doi.org/10.1007/BF02722991

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