Title page for ETD etd-10062011-154327

Type of Document Master's Thesis
Author Awwad, Yousef Ahmad
Author's Email Address awwad@vt.edu
URN etd-10062011-154327
Title The Effect of Interleukin-1 (IL-1) Concentration on Single Cell NF-kappaB Activation in a Gradient-Generating Microfluidic Device
Degree Master of Science
Department Biomedical Engineering
Advisory Committee
Advisor Name Title
Lu, Chang-Tien Committee Chair
Davalos, Rafael V. Committee Member
Li, Liwu Committee Member
  • Microfluidics
  • Single-Cell
  • NF-kappaB
  • Gradient
Date of Defense 2011-09-27
Availability unrestricted
Interleukin-1 (IL-1) is a multifunctional cytokine produced primarily by activated monocytes/macrophages and by a variety of other cell types. IL-1 plays an integral role in the immuno-inflammatory response of the body to a variety of stimuli including infection, trauma and other bodily injuries. Once IL-1 is released from the synthesizing cell, it acts as a hormone, initializing a variety of responses in different cells and tissues. These responses are believed to be crucial to survival and are termed acute-phase responses. NF-κB is a family of dimeric transcription factors that control the expression of hundreds of genes which regulate cellular stress responses, cell division, apoptosis, and inflammation. NF-κB dwells in the cytoplasm of the cell until activation in response to a wide range of extracellular stimuli including signaling molecules such as cytokines. NF-κB regulates transcription and gene expression through nucleocytoplasmic transport. Most previous studies on NF-κB activation have been performed using bulk assays to look at populations of cells. Determining cell variance at a single-cell level is crucial in understanding the full mechanisms of drug response. The goal of this study is to analyze the effects of variant concentrations of IL-1β on the activation of NF-κB in individual cells through use of a microfluidic gradient generator.

The gradient generator was adopted from Jeon et al and used principles of diffusive mixing and splitting of flows in order create a solute concentration gradient. A soft lithography procedure was used. Briefly, the design was printed on a transparency using a high resolution printer. A master of the design is then created using an SU-8 photoresist and UV light to imprint the design on a silicon wafer. The master is then used to create a Polydimethylsiloxane (PDMS) mold of the design which can be irreversibly attached to a glass slide through oxidation in order to close off the microfluidic channels.

FITC-conjugated β-Casein (a protein with similar molecular weight to IL-1β) was used in order to verify the gradient generated by the design. The concentration gradient was analyzed by measuring fluorescent intensity of images taken under a UV light microscope and found to agree with microfluidic simulations run on COMSOL. A procedure for culturing cells in a microfluidic device was then adapted from Jeon that is explained in detail in Chapter 3.

Two main trends were revealed; firstly, as IL-1β concentration decreased, the percent of cells activated also decreased. Secondly, as IL-1β concentration decreased, the activation time of the responding cells increased. Cells were observed to act in a single-cell manner; in which multiple cells subjected to the same concentration would not all respond in the same fashion. No major activation threshold was observed but two minor thresholds were; the first at 0.02 ng/mL IL-1β where activation levels drop from 20% to around 5%. The second around 1 ng/mL, in which all greater concentrations show nearly complete activation of all cells exposed.

Of the cells that activated, the activation times were recorded and analyzed as well. In general, a decrease in IL-1β concentration caused cells to take longer to activate. Concentrations greater than 5 ng/mL responded on average in 30 minutes with a significant amount of variation. Between 5 ng/mL and 0.1 ng/mL, activation time increased as IL-1β concentration decreased in a linear fashion when concentration was plotted on a base-10 log scale. Below 0.1 ng/mL, the trend disappears and an average activation time of around 95 minutes is observed that no longer depended on concentration. This is interesting because fewer and fewer cells are activating in this concentration range but activation time follows no trend and remains partially stochastic with times ranging from 80 to 105 minutes.

The previous results were all observed with a continuous flow and stimulation of the cells. Experiments were also run by only exposing the cells to the IL-1β for 10 minutes and then replacing the flow with a buffer. These studies yielded interesting results; the fraction of activated cells reported the same trends and values as those that were continuously stimulated. The activation times, however, were delayed between 10 and 20 minutes but otherwise followed the same trend as the continuous stimulation. These results suggest that a brief exposure to an external stimulant is all it takes for the cascade of intercellular events to take place and cause NF-κB translocation.

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