A study of bioreactor surfaces for cell interactions and sensing.

Abstract

Polymeric hydrogels were used to create bio-smart hydrogels serving multifunctional roles interfacing with cells and enzyme substrates. Their value lies in their use as: i) Stimuli- responsive membranes that directly transmute chemical potential energy into proportionate electrical signals, ii) as biomimetically inspired biocompatible coatings on stents and other implantable bionic devices, iii) as bio receptor hosting membranes for enzyme-based implantable biosensors. Biosensors use oxidoreductase enzymes such as glucose oxidase (GOx) and lactate oxidase (LOx) to confer specificity. Such enzymes may initiate more complex in vivo inflammatory response. In this thesis individual and combined effects of different enzymes (GOx, Superoxide dismutase (SOD), and catalase) were studied to achieve hydrogelenzyme systems, which in theory may mitigate against adverse cell outcomes. The incorporation of enzymes into bioactive hydrogels was investigated, and revealed effects on the growth, viability and attachment of surface dependant RMS13 human muscle fibroblasts and B50 rat neuronal cells. Agarose and p(HEMA)-based hydrogels were prepared with fibrinogen 5% (w/v) to promote integrin-mediated cellular attachment and also with different combinations of glucose oxidase (GOx), catalase (CAT) and superoxide dismutase (SOD). Cell viability was maintained best on catalase hydrogels. The presence of GOx within hydrogels membrane compromised cell viability in both hydrogel types, presumably due to accumulation of H2O2 confirmed by amperometric detection using fabricated platinum needle electrodes. Hydrogels prepared with GOx and CAT showed improved cell viability, further suggesting the negative influence of H2O2. High temperature treatment of the enzyme-hydrogel membranes, resulting in enzyme denaturation, returned all constructs to control levels of viability, confirming the relationship of cell viability with enzyme activity. An additional study was undertaken into the viability and growth of B50 cells on crosslinked protein membranes of fibrinogen and albumin as a potential bioreactor surface. The use of crosslinked fibrinogen to facilitate cell growth within microfluidic channels appears to have been realized. Fabrication and use of miniaturized gold-filled silica recess and inlaid disc electrodes, compared with the use of agarose gels in the recesses was investigated to improve stabilization of an amperometric H2O2 electrode. From this, a microfluidic device with an integrated inner diameter working and counter / reference electrode was fabricated which showed feasibility of more rapid amperometric detection of H2O2 in miniature flow channels.