Research Program
Cell Surface Interactions

Objectives
1)  Develop a microfluidic tumor model:  a 3-D, in vitro system in which microfluidic channels are embedded directly within a matrix seeded with tumor cells.  These channels allow for convective mass transfer with the volume of the culture for both delivery and extraction of soluble factors

2)  Develop methods to operate and observe tumor model in order to provide quantitative information on the spatial and temporal evolution of the soluble chemical environment within the scaffold in real time and at fixed endpoints

3)  Exploit microfluidic tumors to quantify the proangiogenic activity of tumors cells as a function of the characteristics of their microenvironment, as defined by the geometry of and flows through the scaffold.

Methods
We adapted a soft-lithographic process developed by Stroock and Bonasssar to be compatible with the 3-D culture methods of tumor cells developed by Fischbach (Fig. 1).  Oral squamous cell carcinoma cells (OSCC-3) were used as representative of a highly aggressive tumor cell line with up-regulated pro-angiogenic signaling.  Calcium alginate served as the scaffold material.  We operated the microfluidic system with growth medium delivered via a syringe pump; the entire system was placed within a CO₂-controlled incubator.  The oxygen tension within the scaffold was manipulated by controlling the tension within the growth medium that was continuously driven through the microchannels (Fig. 2).  To achieve real-time monitoring of oxygen tension within the scaffold, an oxygen-sensitive phosphor was encapsulated within latex colloidal beads during emulsion polymerization.  These beads were then seeded homogeneously within the scaffold along with the cells (Fig. 2).  Oxygen status was also measured in fixed samples via hypoxyprobe staining (Fig. 3b).  The levels of vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8) secreted by the cells was measured by ELISA in the media after passage through the device and in sections of the scaffold at the end of the experiment (Fig. 3c-3d).

Summary
Reduced tissue oxygen tension critically contributes to tumorigenesis. Understanding the underlying mechanisms requires accurate in vitro models of tumor development, but the molecular processes and mass transport phenomena may not be adequately represented with conventional 2-D cell culture or existing 3-D culture systems. In this project, we have developed and validated a new microfluidic, in vitro system to study the importance of spatiotemporal variations of oxygen tension and paracrine signaling in the angiogenic activity of tumors. We have developed a robust lithographic technique to micropattern alginate hydrogels pre-seeded with tumor cells (Fig. 1).  We have shown that perfusion of the embedded microfluidic channels can serve to modulate the soluble chemical environment experienced by the cells in the bulk.  To monitor oxygen status within the scaffold, we have developed a new technique based on colloid-encapsulated dyes to sense oxygen tension within a 3-D culture (Fig. 2). We have demonstrated that the convective mass transfer mediated by the microchannels can serve the dual purposes of modulating the concentrations of soluble factors within the scaffold (Fig. 3b) and monitoring the soluble factors generated by the cells in the scaffold (Fig. 3c-3d).  Our preliminary studies indicate that secretion of VEGF and IL-8 is significantly affected by rates of delivery of oxygenated medium; furthermore, the response appears to be non-monotonic (contrary to expectations in literature).  Based on these findings, we are proceeding to a more complete mapping and modeling of the coupled response of the cells to the multiplicity of soluble factors in the tumor (see continuation).

Accomplishments

• Development of a microfluidic tumor model in a 3-D scaffold of calcium alginate (Fig. 1)
• Development and validation of biocompatible, oxygen-sensing beads for real time monitoring of oxygen status in 3-D cell culture (Fig. 2).
• Validation of use of microfluidic scaffold to control and monitor the soluble chemical environment of tumor cells in 3-D (Fig. 3).