Tissue injury triggers complex communication between cells via secreted signaling molecules such as cytokines and growth factors. Discerning when and where these signals begin and how they propagate over time is very challenging with existing cell culture and analysis tools. The goal of this study was to develop new tools in the form of microfluidic co-cultures with integrated biosensors for local and continuous monitoring of secreted signals. Specifically, we focused on how alcohol injury affects TGF-β signaling between two liver cell types, hepatocytes and stellate cells. Activation of stellate cells happens early during liver injury and is at the center of liver fibrosis. We demonstrated that alcohol injury to microfluidic co-cultures caused significantly higher levels of stellate cell activation compared to conditioned media and transwell injury experiments. This highlighted the advantage of the microfluidic co-culture: placement of two cell types in close proximity to ensure high local concentrations of injury-promoting secreted signals. Next, we developed a microsystem consisting of five chambers, two for co-culturing hepatocytes with stellate cells and three additional chambers containing miniature aptamer-modified electrodes for monitoring secreted TGF-β. Importantly, the walls separating microfluidic chambers were actuatable; they could be raised or lowered to create different configurations of the device. The use of reconfigurable microfluidics and miniature biosensors revealed that alcohol injury causes hepatocytes to secrete TGF-β molecules, which diffuse over to neighboring stellate cells and trigger production of additional TGF-β from stellate cells. Our results lend credence to the emerging view of hepatocytes as active participants of liver injury. Broadly speaking, our microsystem makes it possible to monitor paracrine crosstalk between two cell types communicating via the same signaling molecule (e.g. TGF-β).

Cytokines are produced by immune cells in response to viral or bacterial pathogens and therefore have significant diagnostic value. The goal of the present study was to develop a miniature device for detection of interleukin (IL)-2 and interferon (IFN)-gamma cytokines secreted by a small population of CD4 and CD8 T-cells. Microarrays of T-cell- and cytokine-specific Ab spots were printed onto poly(ethylene glycol) (PEG) hydrogel-coated glass slides and enclosed inside a microfluidic device, creating a miniature ( approximately 3 microL) immunoreaction chamber. Introduction of the red blood cell (RBC) depleted whole human blood into the microfluidic device followed by washing at a pre-defined shear stress resulted in isolation of pure CD4 and CD8 T-cells on their respective Ab spots. Importantly, the cells became localized next to anti-IL-2 and -IFN-gamma Ab spots. Mitogenic activation of the captured T-cells was followed by immunofluorescent staining (all steps carried out inside a microfluidic device), revealing concentration gradients of surface-bound cytokine molecules. A microarray scanner was then used to quantify the concentration of IFN-gamma and IL-2 near CD4 and CD8 T-cells. This study represents one of the first demonstrations of a microdevice for capturing desired T-cell subsets from a small blood volume and determining, on-chip, cytokine profiles of the isolated cells. Such a microdevice is envisioned as an immunology tool for multi-parametric analysis of T-cell function with direct applications in diagnosis/monitoring of HIV and other infectious diseases.