Research Program
Biomolecular Devices and Analysis

Objectives
Our goal is to couple micro and nanofabrication techniques with cell cultures to predict toxicology and efficacy of pharmaceuticals. Our CCA (cell culture analog) systems are microfluidic devices with interconnected cell-containing compartments to mimic pharmacokinetic response of humans to drugs or environmental chemicals. The systemic CCA can be coupled with modules representing barrier tissues such as the gastro-intestinal (GI) tract and the blood brain barrier (BBB). We are particularly interested in three-dimensional (3-D) cell culture models for each compartment and models with multiple cell types. Applications are to cancer treatment and response to environmental toxicants.

Methods
Real time response in each compartment can be monitored by automated optical systems in development. Effective models of tissue response are critical and three-dimensional hydrogel cultures, especially with multiple cell types, are particularly promising. For the BBB alginate functionalized with cell adhesion peptides (RGD, IKVAV) and proteins (laminin) have been developed with neural cell immobilization and endothelial cell co-culture. Electrochemical impedance microscopy has been used to characterize these constructs. For the GI tract co-cultures of epithelial, mucus-producing, and immune cells self-assemble in a microfluidic device to form an effective GI model. For the liver, cancer, and bone marrow models using Matrigel as a hydrogel is effective. The GI tract model has been integrated with a systemic CCA. Reporter cell lines that respond to endocrine disruptors have been constructed. Applications to combination drug treatment for cancer, exposure to multiple environmental chemicals or nanoparticles, or to neural probes have been used to illustrate the value of these approaches.

Summary
A cell culture model has been developed that permits interactions between neural cells and endothelial cells. Alginate was found to exhibit mechanical properties similar to brain tissue and provided a porous aqueous environment to permit diffusion of oxygen and nutrients. Neural cells remain metabolically active within the alginate maxrix and were found to extend processes in 3-D. Alginate constructs were developed that permitted cell culture at densities as high as 1x10⁵ cells/microliter, a density similar to that of rat brain cortex. Endothelial cells attached directly to the alginate surface and interacted with neural cells. TEER measurements indicated that endothelial cells formed a confluence monolayer on the alginate surface, occluding the flow of electrical current. Using a GI-tract model we found that carboxylated nanoparticles, 50 nm in size, lower the transmembrane resistance of the barrier and are able to pass from the apical to the basolateral side of the cell layer. Preliminary results with a GI tract/liver µCCA suggest that a concentration of 2.5x10⁶ of aminated 200 nm sized particles alter the morphology of liver cells.  Our results may be useful to estimate exposure limits for nanoparticles.

In the systemic CCA we demonstrated successful operation with 3-D hydrogel cell cultures in the chamber for three days. The cytotoxic effect of Tegafur, a prodrug, used in chemotherapy on colon cancer cells was tested. As expected, liver cells are necessary to observe toxicity; dosing strategies using Tegafur and uracil were tested. We have demonstrated the successful integration of the GI tract module with the systemic CCA. Reporter constructs allowed the direct comparison of response of mammary and endometrial cancer cell lines to estrogen receptor agonists or antagonists as a basis for future experiments in a CCA on response to chemicals that may be endocrine disruptors.

Accomplishments

• 3-D neural cell/endothelial cell co-cultures established as BBB model
• CCA system with 3-D culture elements used to probe colon cancer treatment
• GI tract models used to characterize possible mechanisms of toxicity of nanoparticles