American Institute of Chemical Engineers Annnual Meeting, October 28 – November 2, 2012, Pittsburgh, PA
Abstract:
Introduction
The ability of cells to signal one another is an important aspect in tissue function, activation of immune functions, and other bodily functions. Long range cell communication occurs via paracrine and endocrine signaling factors. Understanding how different cell types communicate in response to pathogens or neighboring cells is essential for developing new therapies and drugs. Additionally, cellular communication networks hold information for understanding cancer and disease states, and can help lead to effective culture and modulation of cell and stem cell maintenance and function. The goal of this work is to to culture an engineered model system in a microfluidic device. This model system allows for real time monitoring of cellular communication via fluorescent reporter systems. Lipopolysaccharide released by E. coli stimulates mouse macrophage (RAW) cells, which then secrete TNFα. The TNFα activates cellular signaling cascades in nearby human embryonic kidney (HEK) cells to fluoresce. To control the cell signaling, cells were encapsulated within a Matrigel scaffold and seeded in a microfluidic device. The system was then monitored over 16 hours to evaluate the cellular communication.
Materials and Methods
A stable HEK NFκB cell line was developed by transfecting the HEK cells with the NFκB-GFP lentiviral vector. The E. coli, RAW, and HEK cells were encapsulated in Matrigel and individually patterned in a microfluidic platform comprised of a molded poly(dimethylsiloxane) slab bonded to a glass substrate. The microfluidic platform was incubated for 14 hours and the cells imaged via confocal microscopy.
Results and Discussion
To determine if the HEK cell fluorescent intensity can be modulated by LPS concentration, varying concentration of LPS were introduced in the microfluidic platform as shown in Figure 1. The 0 ng/mL LPS and No RAW cell conditions are negative controls. In the 0 ng/mL LPS case, no LPS was introduced into the microfluidic platform. In the No RAW cell case, both HEK cells and 5 ng/mL LPS were introduced into the microfluidic platform without any RAW cells. In both cases, there is a small amount of background fluorescence. When LPS, RAW cells, and HEK cells are all introduced into the microfluidic platform an increase in fluorescent signal can be noted. In addition, compared to the negative control conditions 0.5, 5, and 50 ng/mL of LPS cause a significantly higher fluorescent intensity (p-value of 0.05).
Conclusions
In vivo, a cell is not an isolated entity; it is part of a three-dimensional tissue where it is surrounded by other cells, extracellular matrix, and gradients of signaling molecules and soluble factors. In this work, we developed a microfluidic platform to individually pattern cells in three-dimensional microenvironments and allow the cells to communicate via diffusible molecules. A model one way cell-cell communication system with fluorescent reporters was developed to monitor the presentation of soluble molecules to the cells. This microfluidic platform was validated through the real-time monitoring of cellular signaling events. By varying the concentration of the initial diffusible molecule (LPS), a change in fluorescent intensity was noted.