Date of Award

1-1-2013

Document Type

Open Access Dissertation

Department

Biomedical Engineering

First Advisor

James Blanchette

Abstract

Cell transplantation can be considered a regenerative therapy, an intervention which attempts to replace or restore the function of compromised tissue by harnessing innate properties of cells that cannot be replicated artificially. For such therapies to succeed, it will be necessary to understand and closely match the physiological conditions that govern cell behaviors in vivo. One important factor is low oxygen tension, termed hypoxia, which is often overlooked in vitro. Because oxygen insufficiency can lead to cell death, hypoxia has traditionally been viewed as a negative condition. However, hypoxia can also serve as a potent regulator of crucial cell behaviors involved in normal development, adaptation, and regeneration through transcriptional activity of hypoxia-inducible factors (HIFs). Characterizing HIF activity, especially in three-dimensional tissues which can experience oxygen heterogeneities, will be useful in investigating how culture parameters impact local microenvironmental conditions to affect cell behavior.

In the present work, a novel fluorescent reporter system was used to detect cellular HIF activity in islet-like clusters of pancreatic &beta-cells and adipose-derived stem cell (ADSC) spheroids, two cell systems used in transplantation therapies. Fluorescent signaling was observed in cultures incubated in reduced oxygen conditions and in large cultures, indicating the development of tissue size-dependent oxygen gradients. HIF activity could be monitored in the same samples over time and could be correlated to other cell behaviors. In &beta-cells, signal was observed in all clusters incubated in 1% or 2% oxygen and in the center of large clusters (>300ìm) incubated in 20% oxygen. In clusters that exhibited HIF activity, insulin secretion and cluster morphology were severely impaired. In ADSC spheroids, both reduction of external oxygen concentration and an increase in spheroid size increased HIF activity. Moderate HIF activity corresponded with increased vascular endothelial growth factor (VEGF) secretion from spheroids, while intense HIF activity corresponded with regional reduction of cell viability; therefore, an optimal size for VEGF secretion could be demonstrated. Altogether, the HIF reporter system represents a useful tool in monitoring cellular hypoxia so that culture parameters such as external oxygenation and culture size can be properly considered in order to guide desired cell behaviors in cell transplantation.

Rights

© 2013, Matthew Lorincz Skiles

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