The use of perfluorocarbons in encapsulated cell systems: Their effect on cell viability and function and their use in noninvasively monitoring the cellular microenvironment

Oxygen is a key parameter in maintaining cell viability and functionality. This is especially true in encapsulated cell systems, as the cells within the constructs rely solely on diffusion for the transport of dissolved oxygen. In order to develop a long-term functional implant, finding a means to enhance and monitor construct oxygenation in vivo is crucial. The addition of a perfluorocarbon (PFC) emulsion improves the overall construct oxygenation by increasing the effective diffusivity of dissolved oxygen into the constructs. Whether this enhancement is sufficient to significantly improve cell viability and function is still a subject of debate, as there have been conflicting reports in literature regarding the beneficial effects of PFCs. The work in this thesis investigated this question as it relates to encapsulated cell systems.;Besides having a high solubility of oxygen, the high fluorine content of PFCs makes them good oxygen concentration markers for 19F NMR. As the relaxation rates (R1 = 1/T1) of excited fluorine nuclei increase in the presence of oxygen, DO within an implant can be measured through 19F NMR when PFCs are incorporated. The ability to noninvasively monitor DO allows for an assessment of the metabolic activity of the cells within the construct, and for important correlations to be established between these in vivo measurements and construct function. The work in the second part of this thesis involved the development of a dual PFC noninvasive method of monitoring DO in a tissue engineered pancreatic construct using 19F NMR. The dual PFC method consists of the incorporation of each of two different PFCs with proximal chemical shifts, either in the experimental tissue implant (betaTC-tet cells encapsulated in alginate beads) or in cell-free alginate beads; the cell-free beads are used to monitor DO in the surrounding medium or implantation milieu, as equilibration of the intrabead and external DO is assumed to be rapid.;The feasibility of this method was tested in vitro where the encapsulated cells were cultured under perfused and static conditions in an NMR-compatible bioreactor. T1 relaxation measurements were acquired over time at different encapsulated cell densities. Using this method, T1 relaxations within the cell-containing beads and cell-free beads can be obtained simultaneously; therefore DO within the constructs containing metabolically active cells can be measured relative to the surrounding medium. Steady-state DO measurements acquired under medium perfusion showed minimal DO difference between the cell-containing beads and the surrounding medium; a considerable difference was only detected at a high encapsulated cell density of 7x107 cells/ml. Under static conditions, however, significantly lower DO levels were measured within the cellcontaining beads when compared to the surrounding medium, which demonstrated the capability of the method to track the oxygenation state of beads containing metabolically active cells. The DO profiles obtained under static conditions were also supported by mathematical simulations of the system, indicating that the observed trends were not measurement artifacts.;The dual PFC method of monitoring was implemented in vivo using murine models. The betaTC-tet cell-containing alginate beads were implanted in the peritoneal cavity of normal and streptozotocin-induced diabetic mice. Cell-free alginate beads were co-implanted to track the DO in the implantation milieu. T1 measurements were acquired from the two bead populations over time, and construct physiology and therapeutic function were assessed through post-explantation studies and blood glucose measurements, respectively. Through 19F NMR, the capability of acquiring real-time in vivo DO measurements using the dual PFC method was demonstrated. In both normal and diabetic mice, measurements showed the peritoneal environment to be hypoxic and variable. DO within cell-containing beads decreased over time and correlated with relative changes in the number of viable encapsulated cells and/or the extent of host cell attachment. The presence of the viable cell implant also caused a significant decrease in the surrounding peritoneal DO. Diabetic mice receiving betaTC-tet cell constructs remained normoglycemic for the duration of the experiment; the reduction in construct DO due to the metabolic activity of the encapsulated cells was observed to be compatible with the implant therapeutic function. (Abstract shortened by UMI.)...