Kono Y, Yang S, Roberts E A
Division of Gastroenterology and Nutrition, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
In Vitro Cell Dev Biol Anim. 1997 Jun;33(6):467-72. doi: 10.1007/s11626-997-0065-7.
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 cocultured 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.
为了制定人肝细胞延长原代培养的策略，我们将人肝细胞置于两层胶原凝胶之间，即所谓的“胶原凝胶三明治”。将该系统中肝细胞功能的维持情况与在干胶原包被培养皿上培养的相同肝细胞制剂或与大鼠肝上皮细胞共培养的情况进行了比较。胶原凝胶三明治中的人肝细胞（5个独立培养物）存活超过4周，最长培养期为78天。在整个培养期间，它们保持多角形形态，具有胆小管样结构，并分泌高水平的白蛋白。相比之下，干胶原上的肝细胞变得没有特征，培养14天后检测不到白蛋白分泌。通过覆盖一层胶原凝胶，白蛋白分泌的这种丧失得到部分恢复。与细胞色素P450 1A2相关的乙氧异吩唑酮O-脱乙基酶活性在胶原凝胶三明治培养中在第29天之前一直可以检测到。用二苯并(a,h)蒽诱导后，这些活性增加了4至8倍。共培养物在第29天之前也保持基础活性。然而，它们的诱导性低于胶原凝胶三明治中的肝细胞。在干胶原上培养7天的肝细胞中未检测到乙氧异吩唑酮O-脱乙基酶活性。因此，胶原凝胶三明治系统在原代培养中长时间保留了人肝细胞的分化形态和功能。该系统是研究人肝细胞功能（包括体外蛋白质合成和药物代谢）的一个有前景的模型。
