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...

Graphical abstract

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.

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