Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing

Abstract

Primary cultures of human hepatocyte spheroids are a promising in vitro model for longterm studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n 5 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4a, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture. Conclusion: Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format. (HEPATOLOGY 2012;55:1227-1236 T he liver-specific functions of hepatocytes, such as albumin secretion or drug-metabolizing activity, are rapidly down-regulated during in vitro primary cultures, limiting their use for drug development and toxicity tests. 1 For such assays, the current gold standard for long-term human hepatocyte culture is the collagen sandwich in vitro model. 2 The overlaying collagen layer increases cell-cell and cell-matrix contacts, providing a more three-dimensional (3D)-like architecture than a monolayer culture. For rat hepatocyte spheroids, where cell-cell interactions are maximized, liverspecific functions 3,4 and multicellular architecture 5,6 are increased, when compared to monolayer cultures.The use of microfluidic devices for primary cultures of hepatocytes is a promising approach to enable highthroughput screening in drug development. 7,8 However, the downscaling enabled by these technologies makes the culture environment harder to be controlled and limits the application of microfluidics for longterm primary cultures of hepatocytes. In fact, the most useful applications of microtechnologies for such cultures couple either microfluidic perfusion or coculture micropatterning to 12-9 or 24-well culture plates, 10Abbreviations: 2D, two-dimensional; 3D, three-dimensional; 7-EC, 7-ethoxycoumarin; 7-HC, 7-hydroxycoumarin; ANOVA, analysis of variance; aPKC, atypical protein ki...

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells

“…The ultrastructure did not reveal any necrosis despite the 100-150 mm size range. In previous studies with primary hepatocytes (19) and rat adult bone marrow derived stem cells (21), high viability was seen in spheroids with a similar size range. How the viability or ultrastructure is affected by different seeding cell densities is yet to be explored.…”

“…Three-dimensional culture systems have been shown to promote enhanced cellular structure and function in many cell and tissue types, including mammary epithelial cells [25], mesenchymal stem cells [26], neural cells [27], and hepatic cells [19,[28][29][30][31][32][33][34]. In those studies employing mouse, rat or pig primary hepatocytes, cells were allowed to adhere to moderate to low attachment surface to enhance spheroid formation [33,35].…”

Concise Review: 3D cell culture systems for anticancer drug screening

“…Given that 3D models not only mimic tumor architecture but mimic similar environmental challenges, these models are great and conservative systems to study candidate drug. Bioreactors are labor-intensive due to their ability to produce a large number of spheroids (Tostoes et al, 2012). However, the created spheroids are usually heterogeneous in size and cell population (Mehta et al, 2012).…”

Organ-on-a-chip platforms for studying drug delivery systems

“…With extensive research on developing 3D hepatic spheroids for drug testing [87, 88, 97, 98] and also with availability of high-throughput commercial platforms for formation of spheroids, liver spheroids represent a promising 3D construct for rapid clinically relevant evaluation of nanoparticles. Spheroids formed from primary human hepatocytes have been used for long-term drug studies [97].…”