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Bioactive sugar surfaces for hepatocyte cell culture
[Thesis]. Manchester, UK: The University of Manchester; 2010.
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Abstract
The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.