(HFM) bioreactor made of alternating polyethersulphone (PES) HFMs. Spheroids were also seeded on PES HFMs in batch system to compare between the static ...
European Cells and Materials Vol. 31. Suppl. 1, 2016 (page 22)
Liver microtissues in hollow-fiber membrane systems HMM Ahmed1,2, S Salerno1, L De Bartolo1 1
Institute on Membrane Technology, ITM-CNR, Italy. 2 Department of Environmental and Chemical Engineering, University of Calabria, Italy
INTRODUCTION: Liver failure, due to various etiologies, is a life-threatening medical condition leading to over 220,000 deaths per year in Europe . The only proven therapy is orthotopic liver transplantation; however, due to the limited number of donors, it is crucial to consider alternative therapies. Bioartificial liver (BAL) devices present themselves as a promising alternative that can act as a temporary support for liver failure patients. The BAL device should be able to maintain hepatocytes functional for long periods; since the liver-specific functions of primary hepatocytes, such as albumin secretion or drug-metabolizing activity, are rapidly downregulated during in vitro cultures, limiting their use in BAL devices. METHODS: Primary human hepatocyte spheroids were realised utilizing an agarose mold with 400µm wide wells. Hepatocyte spheroids were seeded in a perfused crossed hollow-fiber membrane (HFM) bioreactor made of alternating polyethersulphone (PES) HFMs. Spheroids were also seeded on PES HFMs in batch system to compare between the static and perfused conditions. Liver-specific functions were assessed in terms of diazepam metabolism, urea and albumin synthesis. Confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) analysis of the spheroids was carried out to study the interaction of cells within the spheroids as well as the interaction of spheroids with the HFMs. The cultures were maintained for more than 3 weeks. RESULTS: Hepatocyte spheroids fused over time forming human liver microtissues around the hollow-fiber membrane as observed by light microscope. CLSM image (Fig. 1) confirms the fusion of multiple spheroids. The formation of tight cell-cell interactions, as well as the formation of bile canaliculi was observed in CLSM images, thus confirming the maintenance of polarity of hepatocytes which is important for their survival. SEM image (Fig. 2) shows the weak interaction of the spheroids with the PES HFMs that allow spheroids to be attached but prevents them from spreading and losing their 3D structures. Liver-
specific functions were maintained in the perfused bioreactor throughout the whole period of the culture at levels higher than those in static conditions.
Fig. 1: CLSM image of fusing human hepatocyte spheroids cultured on crossed PES/PES hollowfiber membranes.
Fig. 2: SEM image showing the interaction of human hepatocyte spheroids with the PES hollowfiber membranes. Magnification 1500X DISCUSSION & CONCLUSIONS: A hollowfiber membrane bioreactor was realised that allows the long term maintenance of functional human hepatocytes through the formation of liver microtissues via fusion of hepatocyte spheroids, paving the way for the development of a BAL device. REFERENCES: 1 M. Blachier, et al. (2013). The burden of liver disease in Europe: a review of available epidemiological data. Journal of hepatology 58(3): 593-608. ACKNOWLEDGEMENTS: This work has been funded by a grant from the European Commission through the BIOART project within the framework of Marie Curie Initial Training Network, Contract No. FP7-PEOPLE-2012-ITN-316690. The authors declare that they have no conflict of interest. BIOART/KIDNEY-LIVER http://www.ecmjournal.org