In HeMiBio, the aim was to create a bioreactor culture system of hepatocytes alone or in combination with the non-parenchymal fraction of the liver (hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs)) to allow repeated toxicity testing of cosmetics and chemicals for up to 2-3 weeks in vitro.
In the project we made the following advances beyond the state of the art:
1. CELLS: We characterized for the first time human primary HSCs and LSECs at the functional and transcriptional level; characterized transcriptomic as well as epigenomic processes that cause activation of HSCs, and developed methods to counteract this activation; while we demonstrated that LSECs very quickly de-differentiate in culture and developed medium that can delay this event for ± 2 passages; we developed progressively improving methods to create hepatocytes from PSCs, yielding cells that can be used to study toxicants. However, despite the significant improvement in hepatocyte progeny, the cells remain less mature than primary hepatocytes. Therefore, transcriptome, epigenome and metabolome studies were performed to understand hurdles in the differentiation process, insights which were and are continuously used to improve creation of mature hepatocytes. Likewise, cells with HSC-like properties were created from hPSC. Here too the progeny is not fully similar to quiescent HSCs from the liver. Transcriptome studies confirmed this and are now being used to further improve differentiation. A similar set of studies was also done to create LSECs from either PSCs or from blood outgrowth endothelial cells. In a third set of studies, the transiently immortalized UpCyte hepatocytes were fully characterized, and were shown to have functional properties that approach those of primary hepatocytes. These UpCyte hepatocytes (generated from 5 different donors) were suitable for toxicity testing.
SENSORS: In a second major set of studies, sensors to be incorporated in cells or in the bioreactor were created and tested. For cellular sensors, a genome edited set of stem cells was generated that now allows very fast recombination of any incoming cassette such as for instance the NFkB reporter. For sensors within the bioreactor, fully biocompatible microbeads equipped with an oxygen-sensitive, phosphorescent dye were incorporated within the bioreactor allowing real time detection of oxygen consumption. In addition, a novel ALT sensor was created, which can assess with high sensitivity the excretion of ALT from diseased liver cells. These sensors, combined with additional standard pH and glucose sensors were incorporated in two different switch boards/liquid handling units to allow intermittent sampling of culture fluid enabling assessment of the health of cells within the bio-reactor for protracted times.
BIOREACTORS: Three different bioreactor designs were generated: (1) An antibody-based, microfluidic system: capable of patterning any biotin-conjugated set of antibodies using streptavidin-based surface chemistry, allowing the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. (2) A flow-over bioreactor: this stainless steel bioreactor protects hepatocytes from shear forces while creating stable oxygen and nutrient gradients mimicking the in vivo zonated liver. We demonstrated that HepG2/C3A cells could be maintained for over 28 days in vitro, while displaying over 98% viability and high expression of liver specific markers including CYP450 enzymes. (3) A flow-through bioreactor: although very challenging, significant progress was made towards producing a 3D COC-based bioreactor for liver cell culture, and most technological hurdles in producing prototype reactors were overcome. Further testing will be needed to ensure cell viability.
TOXICITY TESTING: The ultimate goal was to exploit the technologies, in toxicity studies. UpCyte hepatocytes and PSC hepatocytes were shown to be suitable for testing molecules shortlisted by the SEURAT-1 consortium on the gold compound list. A significant amount of work has also gone to develop an in vitro model for liver fibrosis, using co-cultures of HSCs and hepatocytes. These cocultures can identify fibrosis inducing drugs, as deposition of cross linked collagens can be detected following e.g. repeat dose exposure to methotrexate, a premier liver fibrosis inducing drug, among others.
In conclusion: HeMiBio has developed numerous tools towards the creation of innovative bioreactors, including cells, sensors and the reactors themselves to allow 2-3 week culture of hepatocytes with or without non-parenchymal cells to study the effect of drugs from the gold-compound list from SEURAT-1 and to for the first time allow assessment of liver fibrosis inducing drugs.
Project Context and Objectives:
Refinement, Reduction and Replacement of the use of animals in toxicity tests is of particular importance for the implementation of relevant EU policies, such as the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation or the Cosmetics Directive (76/768/EEC). Although multiple projects had been funded by the EC aimed at decreasing the need for animals in toxicity testing before the start of the HeMiBio project, the assessment of toxic effects of chronic exposure still required and requires a relatively high consumption of animals. Moreover, aside from these ethical considerations, there was also a great need for suitable human cells to be used in toxicity testing, due to the often poor concordance seen between animal models and toxic effects in humans.
HeMiBio proposed to generate a liver-simulating device mimicking the complex structure and function of the human liver. The device should reproduce the interactions between parenchymal (hepatocytes) and non-parenchymal (hepatic stellate cells (HSC), and hepatic sinusoidal endothelial cells (HSEC) cells of the liver for over 1 month in vitro in a high-throughput format. Such a Hepatic Microfluidic Bioreactor should serve to test the effects of chronic exposure to chemicals, including cosmetic ingredients.
We postulated that to recreate a liver-simulating device suitable for long-term toxicity testing, (1) the cellular components of the liver need to be viable for extended periods of time (more than 1 month), with appropriate metabolic and transport function, and physiology that is comparable to the in vivo liver; (2) the device should allow fluid to flow over or even through the mixed cells that recreate a small liver section; (3) recreate the differences in function of hepatocytes and non-parenchymal liver cells depending on where they are localised near the artery or vein of in the liver; (4) assess the role of the non-parenchymal cells on the function and downstream toxicity of the hepatocytes, such as is the case in liver fibrosis. Such a device should be able to (iv) screen drug-drug interactions as well as long-term toxicity of chemical entities.
WHAT WAS KNOWN AT THE START OF HeMiBio:
It was/is believed that culture systems that incorporate hepatocytes as well as the non-parenchymal cellular components of the liver could be created to provide clinically relevant information on not only short-term but also mid/long-term drug clearance and drug toxicity of chemicals, i.e. over a period of at least one month, which can be used in the cosmetics industry. However, no reactor has yet been created that can indeed fulfil all criteria set forth above. Several culture methods have been evaluated, from simple cultures of hepatocytes in 2D- on extra-cellular matrix (ECM), to sandwich cultures where hepatocytes are cultured between two layers of ECM and 2D-flat membrane bioreactors, to co-cultures of different liver cell components in 2D-culture systems, and ultimately more complex 3D-culture and bioreactor systems, consisting of multiple or single compartments wherein the different liver cell components are co-cultured together. With increasing complexity, hepatocyte function is maintained better, whereas the less complex culture systems are more amenable to studying the mechanisms that control maintenance of cellular function.
These studies demonstrated that (1) co-culture of the different cellular compartments of the liver improves the long-term stability of hepatocytes as well as the non-parenchymal cells in vitro, which may ultimately lead to (2) the ability to use such devices for testing the effect of toxins directly on hepatocytes, or via their effects on the non-parenchymal component of the liver on the function and clearance ability of hepatocytes. Features of a liver-simulating device that play a role in the functionality of the device include (a) the matrix whereupon cells are maintained, (b) oxygenation, (c) shear flow, (d) transport phenomena, (e) the combination of cells included, (f) distance between the different cellular components, among others.
THE UNDERLYING HYPOTHESIS
The hypothesis for the successful creation of a 3D liver -simulating device suitable to test repeated toxicity was that
(1) parenchymal (hepatocytes) and non-parenchymal cells (LSEC, HSC) need to be combined.
(2) cellular interactions between the different components are required to maintain the functional, differentiated and quiescent state of both the parenchymal and non-parenchymal cell component.
(3) matrix whereupon cells are maintained, oxygenation, nutrient transport needs to be optimized to support long-term maintenance of parenchymal and non-parenchymal function
(4) the system needs to be built such that repeated on-line assessment of cellular integrity, as well as metabolic and transport function, and physiology of the different cellular components is possible.
To achieve the creation of a bioreactor taking into account these hypotheses, the specific objectives were therefore:
1. Engineer the different cellular components required for the development of a 3D-bioreactor (WP1-2) to:
(1) allow specific and spatially defined enrichment of the different cell components, using specific antibodies/ligands.
(2) allow non-invasive detection by fluorescence read-outs of the cellular state (
To accomplish this the following tools would have to be generated:
a. Use PSC-derived progeny (hepatocytes, LSEC or HSC)
b. We would also assess if cells other than pluripotent stem cells could be used to populate the liver device, including primary stellate cells, primary liver sinusoidal endothelial cells, or cells that have been transiently immortalized using the Medicyte (UpCyte) technology.
c. Use micropatterned cell-specific antibodies that would allow spatially specific immobilization of the different cell components in 2D-laminar flow in microfluidic devices and, if needed, also 3D-bioreactors.
d. We would knock in targeting constructs that code for fluorescent proteins and 3’ from selected genes characteristically expressed in terminally differentiated cells, to assess in vivo and in real-time cell fate and allow reselection of cells from reactors.
e. We would knock in cell damage-specific expression cassettes (e.g. based on the activation of NF-κβ, ...) in the AAVS1 region, to assess in vivo and in real-time cell damage.
This should ultimately make it possible to determine which of the cell types in the liver-simulating device undergoes cell stress/death due to a given toxin.
OBJECTIVE 1: HeMiBio aimed to develop tools to engineer the cellular components for the bioreactor: to allow specific and spatially defined enrichment of the different cell components; to non-invasively and in real-time assess the differentiation state of the parenchymal and non-parenchymal cells as well as cell damage.
2. Generate innovative sensing tools that would allow for non-invasive detection of the cell state/fate: this would include biological fluorescent sensors for the dynamic readout of cell function and health (see objective 1, above). In addition, we would generate a combination of electro-chemical sensors to be embedded in the bioreactor to provide continuous measurement of liver-specific function and cellular health. These might include glucose, oxygen, potassium, ammonium, and ALT sensors. Such sensors should be able to dynamically assess the health/activation state of the different cellular compartments in a high-throughput format and provide critical information regarding long-term cellular function as well as repeated dose toxicity screening.
OBJECTIVE 2: HeMiBio aimed to incorporate molecular sensors to dynamically measure cellular function and toxicity in a high-throughput format. High-resolution fluorescent markers will be developed and integrated in a targeted fashion into the host cell genome to detect early inflammatory and pro-apoptotic effects (see also Objective 1). In addition, innovative electro-chemical sensors, such as ion-selective electrodes, would be integrated in the 3D-bioreactors to allow assessment of liver function (e.g. oxygen uptake, ammonium, and glucose concentrations), and also the continuous assessment of cell integrity (e.g. by measurement of potassium, and ALT release due to cell death).
3. As a rapid intermediary to the complex 3D-bioreactor, 2D-isolation-patterning bioreactors would be created that allow for the efficient transition from a mixed iPSC culture to a spatially controlled organ-simulating device. In addition, a 2D flow over bioreactor would be created, wherein sensors developed are integrated in the bioreactor, providing critical insight into the evolving design of the 3D-bioreactor. In addition, this platform would also serve for the optimization of culture medium formulation for maintenance of long-term function.
OBJECTIVE 3: HeMiBio would develop a 2D-bioreactor for the efficient isolation of differentiated iPSC mixtures by trapping different cell types on micropatterned surfaces.
4. The Hepatic Microfluidic Bioreactor (HeMiBio) would be developed and enhanced with the development of integrated sensors and their characterization under microfluidics. Based on an established packed-bed bioreactor design previously shown to support the function of primary hepatocytes for over a week, HeMiBio would support physiological-level perfusion of self-assembled hepatic organoids in an individually addressable microfluidic array for high-throughput screening. Similar self-assembled hepatic organoids were shown to support liver-specific function of primary hepatocytes for over 40 days under static conditions. The device will be further fitted with our biological and chemo-electrical sensors that provide dynamic high-throughput assessment of cellular function and health.
OBJECTIVE 4: HeMiBio would generate a liver-simulating device mimicking the human liver, which reproduces the function of the parenchymal (hepatocytes) and non-parenchymal (HSC and HSEC) liver cells over 1 month in culture. This will be accomplished by combining engineered cells and the electrophysical sensors. The liver-simulating device created in HeMiBio will thus allow for the dynamic monitoring of cellular function and health in a high-throughput format under numerous conditions.
5. During the final years of HeMiBio, proof-of-concept studies would be performed to assess the effect of known chemical entities on the cellular components of the liver-simulating device. The choice of compounds to be tested would be adapted throughout the tenure of HeMiBio based on studies from the SEURAT-1 cluster. The HeMiBio platform was composed of not only hepatocytes but also non-parenchymal cells, and should therefore be very well suited to study toxicants that cause liver fibrosis, which is due to the activation of HSCs that form scars, and then lead to the death of hepatocytes.
OBJECTIVE 5: HeMiBio will 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 over a minimum of 1 month, with specific emphasis on liver fibrosis
6. Throughout WPs 1, 3, 4 and 5, we planned to assess the phenotype of the different cellular components using a combination of transcriptomics, epigenomics and metabolomics. At all stages of reactor assembly, the functional properties of the different cell populations would be assessed using state-of-the-art functional assays. We hypothesized that the phenotype of the parenchymal and non-parenchymal cell components would be more similar to that of primary liver-derived cells when cells were cultured in 2D-cultures, compared with cells isolated from the liver/blood and cultured separately or cells generated under the established iPSC-liver differentiation cultures; and that the phenotype of the two cell compartments of the liver maintained in 3D-cultures would approach that of primary liver isolates even further.
OBJECTIVE 6: HeMiBio planned to assess the molecular, functional and metabolic phenotype of the hepatocellular, HSEC and HSC components at all stages of bioreactor development, and compare this with that of cells isolated fresh from human livers.
Overall strategy of the work plan
HeMiBio wished to create a prototype microfluidic device containing parenchymal (hepatocyte) and non-parenchymal (LSEC and HSC) liver cells for repeated toxicity testing of cosmetic ingredients and pharmaceuticals, to ultimately eliminate the need for laboratory animals in toxicology testing.
hiPSC, cultured under standard differentiation protocols, yielding immature hepatocytes and cells with some features of HSC and LSEC, would be used, as they can be generated from individuals with different metabolic and toxicity profiles, and expanded for more than 100 PDs, thus representing a steady source of cells for liver-simulating devices. As an alternative, we planned to test if primary hepatocytes, HSC and LSEC can be expanded using Medicyte’s proprietary technique for 30-40 PDs, without losing important cellular functions. If the expansion procedure were to cause irreversible de-differentiation, Upcyte® hepatocytes, HSC and HSEC would still be very useful for the initial development of the bioreactors to be built in WP3 and WP4. In addition, we would consider primary liver derived cells, such as stellate cells and endothelial cells, provided that they could be cultured for some time in vitro.
iPSC would be genome engineered to incorporate (1) selection cassettes (fluorophore) downstream of either a gene expressed specifically in mature hepatocytes, HSC or LSEC (WP1); (2) or cell damage-specific expression cassettes (p53, NF-κβ) incorporated, using Flp between FRT sites to be recombined in the AAVS1 region (WP2). This combination would allow precise detection of toxic effects on any of the three cell components to be incorporated in the device.
As a second means of following the health of the cells, we would create several electronic microsensors to be located in the direct vicinity of the cells to measure in real-time or at specific interrogation times (e.g. after a toxic insult) relevant parameters of the health of the cells (WP2). Microsensors would also be generated that can monitor cell culture conditions to aid in optimization of the culture medium (WP2).
In WP3 and 4, sequentially more complex bioreactors would be built to culture hepatocytes, HSC and HSEC for >28 days. We hypothesized that this would lead to further maturation of immature cells derived from iPSC AND assure their persistent differentiated and quiescent state for lengthy periods of time. In WP3, we would test if hepatocytes, HSEC and HSC can be captured from mixed iPSC cultures by microfluidic isolation on hepatocyte, HSEC and HSC-specific antibody-micropatterned surfaces. This would also allow testing if such micro-patterned co-cultures support differentiation and long-term maintenance of liver-specific functions. In a second step, we would also create a 2D flow over bioreactor, that could be used to test the effect of bioreactor mediated culture on maturation of iPSC derived cells, or initial studies of toxicants on hepatocytes, as well as testing of the sensors created in WP3.
The function of the different electronic microsensors from WP2 and the molecular sensors introduced in the cells in WP1/WP2, would be tested first in the 2D-bioreactors in WP3, and if validated subsequently incorporated in the 3D-bioreactors in WP4. In WP3 and WP4 we planned to also ensure that the molecular and electronic sensors provide the information aimed for, i.e. the continuous (intermittent) in vivo assessment of the differentiated state of the three cellular components, their overall state of health and ultimate function and survival of the liver-like tissue in vitro.
As the ultimate goal of HeMiBio was to use the to-be-created device as an alternative to rodent toxicology studies, it will be of the utmost importance to evaluate if the 3D-bioreactors reveal the toxicity expected from a number of prototypical hepatotoxic compounds known to trigger clear-cut liver injuries in vivo. As proof-of-concept, the 3D-bioartificial liver-device would be treated in WP5 with cosmetic ingredients with suspected toxicity. A prerequisite to accomplish this critical task was the establishment of a set of function and toxicity screening assays as well as a list of test compounds, which will be done in close collaboration with investigators in call HEALTH-2010-4.2.9-6.
Throughout WP1, 3, 4 and 5, we planned to use classical “-omics” and functional studies to assure that the cellular components are liver-like, and to assess the effect of the toxic compounds on that state.. This was planned to also be done in collaboration with the team responsible for “-omics” for the consortium as a whole (Call HEALTH-2010-4.2.9-5).
In WP6 (Training and Education) we planned a series of education and training opportunities to train young scientists within HeMiBio and beyond, in the highly innovative and technically challenging concepts embedded in the proposal. Finally, we employed a professional management group to ensure the smooth and professional administration and implementation of the above strategy described in WP7 (Management and Coordination).