Design, construction and testing of a COC 3D flow-over flow-through bioreactor for hepatic cell culture

In this poster, we present the joint development efforts for a 3D microfluidic bioreactor for hepatic cell cultures. Cyclic Olefin Copolymer (COC) was selected for constructing the bioreactor, since the material has good chemical resistance, low adsorption and good optical properties, including low auto-fluorescence. A downside of COC is that it is much more difficult to structure than more traditional microfluidic materials, such as PDMS, PMMA, … Two parallel approaches were developed for structuring the COC. In a first approach, mechanical micro-milling of the channels allows for extremely fast manufacturing of new design variations, at the expense of difficulties in scalability to mass-production and a channel surface that requires post-processing to achieve sufficient optical quality. In a second approach, hot embossing using epoxy molds allows for direct structuring of optical grade channels and is scalable to mass production, at the expense of longer cycle time in the development of new channel designs. To facilitate the handling of the bioreactor, a holder was designed to provide the fluidic connections to a pump,ensuring medium exchange and sampling to down-stream sensors connected to the outlets. The design of the bioreactor was intended to maintain and expose pre-formed hepatic co-culture spheroids to toxicants for more than a week. Once seeded, spheroids rest on a polycarbonate membrane with 12 µm pore size, allowing the medium to flow-through, while flow-over is maintained to avoid an excess pressure on the cells. In a single bioreactor, 9 wells are connected to a common inlet to provide the cells with fresh culture medium or test compounds. On a first cell culture trial, it was possible to visually detect the spheroids in the wells after seeding, however, after 1 week of culture there was no possibility to accurately detect the presence and viability of the cells. In the framework of HeMiBio, significant progress has been made towards producing a 3D COC-based bioreactor for hepatic cell culture, and most technological hurdles in producing prototype reactors have been overcome. Further testing is needed to see which improvements to the reactor or the flow conditions should be made to ensure cell viability

Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro

Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis