Research Program
Nanoscale Cell Biology

Project #
NCB13

Participating Faculty:	Tracy Stokol, Michael L. Shuler, Chris Schaffer
NBTC Students/Postdocs:	Mandy B. Esch
Other Students/Postdocs:	Nozomi Nishimura

Objectives
Our goal is to develop microfluidic PDMS devices with which we will be able to stimulate a single human pulmonary endothelial cell with calcium ionophore or histamine and monitor the progression of calcium signals as well as histamine-induced endothelial leakiness from a single cell to neighboring endothelial cells under physiological flow conditions. Using this method our objective is to determine to what extent physical parameters play a role in the propagation of calcium signals and in the propagation of endothelial leaks. Changing the physical factors involved, both propagation events might be altered. Our long-term goal is to use the developed system to identify molecules that compromise the lung endothelium and lead to breast cancer cell attachment thereby causing the invasion of the lung by breast cancer cells.

Methods
We will fabricate microdevices in PDMS consisting of two parallel channels alongside each other. Using a femto-second laser we will optically ablate several micro-pores, 2 mm in diameter, at various locations between the two channels. While conventional PDMS fabrication is capable of producing channels with 2 mm dimensions, only the femtosecond laser method allows us to direct the pores to the bottom of the channels in an easy way (figure 1). Within one of the channels we will then grow human pulmonary endothelial cells so that they are entirely lined with cells. A single cell then covers each ablated micropore through which we will supply calcium ionophore and histamine. The propagation of Ca²⁺ signals and the propagation of histamine-induced increased vascular permeability in response to the stimulation will be mapped under changing physical conditions to determine to what degree physical factors play a role in both events.

Summary

Microfluidic polymer vessels confluently lined with human pulmonary endothelial cells provide physiologically correct replica of the lining of the lung microvasculature - a place at which metastasizing breast cancer cells attach and invade the lung. A single cell of this in vitro endothelial lining can be addressed with calcium-ionophore and histamine through a 2µm delivery channel. Comparing the pattern of propagation of generated calcium signals and histamine-induced vascular permeability under static and under physiological flow conditions will let us gain an understanding to what degree physical factors play a role in these two processes that may be involved in cancer cell attachment.

Goals/Accomplishments

• We will fabricate microfluidic PDMS devices that contains two channels connected via micropores.
• We will map the propagation of Ca²⁺ signals in response to calcium and histamine under varying physical conditions.
• We will map the propagation of histamine-induced vascular permeability propagation under varying physical conditions.