Generating a Hepatic Microfluidic Bioreactor for Toxicity Testing

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Programs & Policies - EU

Generating a Hepatic Microfluidic Bioreactor for Toxicity Testing

Mar Coll (Institut d’Investigacions Biomèdiques August Pi i Sunyer [IDIBAPS], Barcelona, Spain) and Pau Sancho-Bru (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas  [CIBERehd], Barcelona, Spain)

Published: May 17, 2014

About the Author(s)

Mar Coll received her PhD in 2010 from the University Autonomous of Barcelona. The PhD project was based on understanding molecular pathways involved in the pathophysiology of portal hypertension and was performed in the Liver Disease Laboratory of Vall d’Hebron Research Institute in Barcelona. In 2012 she joined the group of Pau Sancho-Bru in the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) to investigate new molecular mechanisms that play a role in hepatic stellate cell activation and the potential use of this cell type in toxicity testing.

Pau Sancho-Bru received his Ph.D in 2006 from the University of Barcelona. He worked on the molecular and cellular mechanisms of liver fibrosis in the Liver Unit of Hospital Clínic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). In 2007 he joined the group of Catherine Verfaillie at the Stem Cell Institute Leuven at the KULeuven, Belgium as a postdoctoral researcher and worked on pluripotent stem cell differentiation to hepatic lineages. In January of 2010 he moved to IDIBAPS as a researcher. His laboratory is focused on understanding the role of hepatic stellate cells and liver progenitor cells in liver disease and assessing the potential of stem cells for biomedical and biotechnological applications.

Correspondence to:
Pau Sancho-Bru, PhD
Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS)
Centre Esther Koplowitz
C/ Roselló, 149-153, Third floor,
08036 Barcelona, Spain
psancho@clinic.ub.es
Tel. +34 932275400 Ext. 3371

The liver is the hub of drug metabolism in which hepatocytes carry out clearance and modifications of chemical entities from the circulation (Kidambi et al., 2009). However, this fact also makes the liver vulnerable to toxicological effects. During early stages of drug development or toxicity assessment, potential hepatotoxicity is an important issue to be addressed. Long-term exposure or repeated dose toxicity studies are of key importance to evaluate potential liver toxicity of the compound of interest or its metabolites. Animals are still required to evaluate long-term toxicity and repeated dose toxicity studies. Besides obvious ethical issues, Refinement, Reduction and Replacement of the use of animals in toxicity tests is of particular importance for the implementation of relevant European Union policies (Lilienblum et al., 2008; Schechtman, 2002). Moreover, one of the main drawbacks of animal testing is the interspecies difference between rodents and humans in terms of metabolization, sensitivity or compartmentalization (Langsch et al., 2009; Martignoni et al., 2006). These differences can make the translation of toxicity studies from animals to humans very difficult to interpret and often result in poor concordance between toxic effects in animals and in humans (Langsch et al., 2009; Martignoni et al., 2006). In this context, the possibility to use human cells or tissue increases the predictive value of in vitro models.

During the assessment of long-term toxicity testing, adverse outcomes are frequently related to liver toxicity often as a result of complex hepatocellular stress or injury, i.e. cholestasis, steatosis or fibrosis. Currently, there are no in vitro technologies able to fully replace long-term toxicity testing, and existing technologies for culturing liver cells do not fulfill the basic requirements to mimic in vivo repeated-dose toxicity assessment (Khetani and Bhatia, 2008). In vitro monoculture of primary hepatocytes for toxicity testing does not reveal hepatotoxicity potentially caused by the complex interplay of the different liver cell types (Chen et al., 1998). Moreover, under standard conditions primary hepatocytes rapidly lose their polarization and liver-specific functions, accumulate stress fibers and die within days (bu-Absi et al., 2002; Dunn et al., 1991). In vitro maintenance of hepatocyte functions and characteristics is extended when hepatocytes are cultured with supporting matrixes, or with cells such as endothelial cells or fibroblasts (Bhatia et al., 1997). As a general rule, with increasing culture complexity in terms of complex matrices and co-culture systems, hepatocyte function is better maintained. Moreover, it is plausible to think that complex systems comprising different liver cell types are more suitable to assess complex toxicological adverse outcomes such as liver fibrosis, which will certainly require the presence of non-parenchymal cell types (Liu et al., 2014). In this respect, culture systems that incorporate hepatocytes as well as non-parenchymal cellular components of the liver will have to be created to provide clinically relevant information not only on short-term but also mid/long-term drug clearance and drug toxicity of chemicals.

Currently, there are several efforts in developing 3-D liver mimics for different applications such as mechanistic models of human processes or drug safety and efficacy studies (Griffith et al., 2014). Bathia and collaborators have developed a perfused hepatocyte bioreactor which imposes a physiologic oxygen gradient on rat hepatocytes and non-parenchymal cells, producing an in vitro model of liver zonation (Allen et al., 2005). Also, the Griffith laboratory has developed a bioreactor that fosters maintenance of 3D tissue cultures and integrated multiple bioreactors into an array in a multiwell plate format (Domansky et al., 2010). Other good examples are those generated by companies such as  HuREL or Hepragen.

Besides the few attempts to recreate the liver structure, most existing culture technologies have not been shown to fully reproduce the whole complexity of the liver since they are lacking three-dimensional organization, cell polarization, the influence of highly specified liver cells, and adequate extracellular matrix (Andersson and van den, 2004). An additional downside of some of these technologies is the use of primary liver cells, which implies a high inter-donor variability between donors and primary cell cultures. For that reason, renewable sources of well-characterized liver cells such as stem cells have been suggested as one of the most promising alternatives.

The requirements for efficient development of new technologies able to mimic liver function and assess long-term toxicity are diverse. It will be important to develop or standardize: (1) robust and stable sources of liver cell types; (2) three-dimensional culture conditions to maintain functional liver cells; (3) in vitro culture systems able to recreate the complexity of the liver structure; and (4) sensors or methodologies to monitor cell function and to assess the effects of toxicity.

Hepatic Microfluidic Bioreactor (HeMiBio)

HeMiBio is one of the seven projects funded under the overarching SEURAT-1 (Safety Evaluation Ultimately Replacing Animal Testing) project “Towards the Replacement of in vivo Repeated Dose Systemic Toxicity Testing.” The goal of SEURAT-1 is to develop long-term research strategies for future research and development work leading to pathway-based human safety assessment in the field of repeated dose systemic toxicity testing of chemicals.

The aim of HeMiBio is to generate a liver-simulating device (Hepatic Microfluidic Bioreactor) mimicking the complex structure and function of the human liver to substantially reduce the use of animals for toxicity testing. The device should reproduce the heterotypic interactions between the parenchymal (hepatocytes) and non-parenchymal cells (hepatic stellate cells (HSC) and hepatic sinusoidal endothelial cells (HSEC) of the liver, with in vivo-like metabolic and transport function. The HeMiBio reactor could then serve to test the effects of chronic exposure to chemicals, including cosmetic ingredients, thus limiting the need for animal models. The cultures generated will allow induction and maintenance of mature hepatocyte, HSC, and HSEC cell function, while creating a bioreactor that can provide clinically relevant information on drug and chemical clearance and toxicity.

To achieve the creation of a liver-bioreactor, HeMiBio is focusing on the following objectives:

  • Develop tools to engineer three different liver cells (hepatocytes, hepatic stellate cells, and hepatic sinusoidal endothelial cells) generated from induced pluripotent stem cells (iPSC) (or expanded using the UpCyte® technology) to be used in the hepatic bioreactor.
  • Generate a three-dimensional liver-simulating device mimicking the human liver, which reproduces the function of the hepatocyte and non-parenchymal liver cells over one month in culture. This will be accomplished by combining engineered cells and sensors under three-dimensional conditions.
  • Incorporate molecular sensors to dynamically measure cell function and toxicity in a high-throughput format.
  • Provide proof-of-principle that a liver-simulating device can recreate the toxicity profile in vitro of toxins with a known in vivo toxicity profile.
  • Assess the molecular, functional and metabolic phenotype of hepatocellular, hepatic stellate, and hepatic sinusoidal endothelial cell components at all stages of bioreactor development and compare this with that of cells isolated fresh from human livers.

HeMiBio Liver Cells. As mentioned above, to mimic hepatocyte liver function increasingly complex strategies are currently under investigation. However, these approaches are frequently based in primary cell types, which demonstrate the limitation of the shortage of human livers, as well as the fact that primary hepatocytes rapidly de-differentiate under standard conditions. Hence, there is a need for innovative culture systems that incorporate hepatocytes as well as non-parenchymal liver cells, derived from expandable/renewable cell sources such as stem cells. HeMiBio seeks to address this unmet need by developing new methods to differentiate liver cell types. Moreover, HeMiBio also aims to compare the characteristics of primary liver cell types with those used at each step of the development of the bioreactor. Generated cells should behave as closely as possible to primary counterparts and be stable in their phenotype and function along the period in which they are co-cultured in vitro. To develop a successful liver-simulating device, different hepatic cells, hepatocytes, HSCs, and HSECs should be combined in order to mimic the different interactions between these cells in vivo.

HeMiBio seeks to incorporate both non-parenchymal and parenchymal cells in combination in a co-culture system. Both components are required to generate fully functioning bioartificial liver-devices, and therefore, a cell type with high renewable capacity and high plasticity is required. In this context, pluripotent stem cells are the cell type with the greatest potential to meet all the requirements of the HeMiBio. iPSCs can be expanded almost indefinitely and have the potential to differentiate to all liver cell types. Moreover, they can be obtained from any adult cell type, originally by overexpressing key factors involved in the maintenance of the pluripontent state (Takahashi et al., 2007; Yu et al., 2007), but recently also by non-integrating factors or even with synthetic compounds (Yoshioka et al., 2013; Miyoshi et al., 2011; Okita et al., 2011; Cheng et al., 2012).  The use of iPSCs will eventually allow the possibility to generate patient-specific liver cells or cells derived from populations with specific polymorphisms of interest.

Within HeMiBio, protocols for the differentiation of iPSCs towards hepatocyte-, HSCs-, and HSEC-like cells are being developed. Moreover, an important effort is underway to compare the phenotype and the functionality of iPSC-derived cells with primary cells in order to evaluate that bona-fide liver cell types that are being generated.

Using flow-cytometry sorting, primary hepatocytes, HSCs, and HSEC have been isolated from human livers. Transcriptome, microRNA, and epigenetic profile analyses have been performed from isolated HSC (unpublished information). The generation of iPSCs implies an almost complete change in the epigenetic marks from the adult cell used for reprogramming (Fouse et al., 2008; Meissner et al., 2008). Differentiated cells will have to acquire the epigenetic marks characteristic of the different liver cell types.  Epigenetic status will determine cell phenotype and function and most probably their response to stress or injury. For that reason, it is important to determine the epigenetic status of primary non-parenchylmal liver cells to be able to understand the epigenetic changes required for a stem cell to acquire a liver cell phenotype.

In addition, cells will be genetically modified in such a way that the mature progeny in the bioreactors can be (re)selected using immunobeads or fluorescence-activated cell sorting (FACS), to allow examination of changes in the transcriptome and epigenome and for reseeding in more complex bioreactors. For this purpose, fluorescent markers under the control of lineage-specific promoters will be integrated into predefined loci using the zinc finger nuclease (ZFN) technology for repeated assessment of maturation of cells in three-dimensional cultures.

An alternative cell source for the generation of the bioreactor is parenchymal and non-parenchymal cells derived from primary liver that have been genetically modified using Upcyte® technology. This technology is able to expand hepatocyte populations for 30-40 doublings, and might also be applied to other liver cell types such as HSC and HSEC (Burkard et al., 2012).

HeMiBio Bioreactor. To assess repeated dose toxicity assessment, cells will have to be maintained in culture for a long period of time. For that purpose, it is required to culture these cells in a bioreactor in which the culture conditions can be controlled. In this context, HeMiBio has chosen microfluidic technology, which allows a better ratio between medium volume and cells, and thus, better control over the conditions in which cells are living. Moreover, microfluidic technology allows the generation of flexible designs with the possibility to set up high-throughput configurations.

The HeMiBio three-dimensional bioreactor is composed of four parts: (1) a high-throughput microfluidic addressable array, (2) a chamber for cell housing, (3) a sensor integrated multi-well plate, and (4) a filter matrix on which the cells sit. The dimensions of the bioreactor have been designed to support the seeding of 200,000 hepatocytes and equal numbers of non-parenchymal cells (about 25,000 cells per 0.07 cm2 well) under shear and normoxic physiological conditions. Cells are expected to be packed 250-500 μm high, the approximate length of the hepatic sinusoid, possibly giving rise to metabolic zonation. This design of the bioreactor provides flow-through for cell seeding and flow-over for perfusion of media.

HeMiBio Sensors. One of the major challenges in building a three-dimensional liver bioreactor is the lack of information on the complex environment present inside the chamber where the cells live, aggregate, and differentiate. This lack of information is even more important regarding microfluidic bioreactors, in which there is a low number of cells and a small volume of fluid are used, thereby making sampling and testing remarkably difficult. For that reason, the strategy of building sensors that can monitor cell culture conditions, cell viability, and response to stress in the vicinity or in the cells is of major importance. HeMiBio is considering two strategies: first, to generate micro-sensors able to test small volumes of medium in order to make measurements in either a real-time configuration or at a specific required moment. Innovative electro-chemical sensors, such as ion-selective electrodes, are being integrated into the three-dimensional bioreactor to monitor culture conditions and liver function (e.g. oxygen uptake, ammonium, and glucose concentrations) as well as continuous assessment of cell integrity (e.g. by measurement of potassium and alanine transaminase release due to cell death). The second strategy to integrate sensors within the bioreactor is to generate molecular sensors built into the cells. These molecular sensors are intended to dynamically measure cell differentiation status, cellular function, and toxicity in a high-throughput format. High-resolution fluorescent markers are being developed and integrated in a targeted fashion into the host cell genome to detect early cell stress and inflammatory and pro-apoptotic effects. The molecular sensors can be individually identified thanks to fluorescent probes incorporated behind lineage specific promoters that allow the assessment in the three cellular components that are incorporated in the bioreactor: hepatocytes, HSC, and HSEC.

HeMiBio has now been running for more than three years. HeMiBio has already provided important advances in areas that are key for the development of a complex liver simulating bioreactor. First, primary parenchymal and non-parenchymal cells have been characterized at a transcriptional and epigenetic level; second, we have developed and refined new protocols to differentiate pluripotent stem cells to liver cells; third, molecular engineered pluripotent stem cells have been generated with lineage tracing markers, inducible overexpression of liver specific transcription factors and toxicology readout cassettes; fourth, culture conditions for hepatocytes and non-parenchymal cells have been developed; fifth, HeMiBio has developed and characterized new sensors for the monitoring of the cell culture conditions as well as cell death and function, and long-term mechanical stability of this sensors is now under development; finally, flow-over bioreactor has been developed and validated for its suitability to sustain hepatocytes for several weeks. Moreover, several iterations of the three dimensional HeMiBio flow-through bioreactor have been generated, and are now ready for testing.

Overall, HeMiBio is developing new technologies and protocols to reach its final goal at its fifth year, to create a prototype of a hepatic microfluidic microreactor suitable for repeated dose toxicity testing.

Acknowledgements:

HeMiBio is jointly funded by the European Union’s Seventh Framework Programme for research, technological development and demonstration and Cosmetics Europe – The Personal Care Association, as part of the SEURAT cluster. Grant Agreement number HEALTH-F5-2010-266777.

©2014 Mar Coll & Pau Sancho-Bru

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