(628f) On-Chip Analysis of Neutrophil Isolation and Migration In Complex Chemotactic Environments

Chemotaxis is one of the primary immunological responses against bacterial infection and plays a significant role in several physiological processes such as inflammation, wound healing, metastasis, atherosclerosis, arthritis etc. In vivo, the effective directed migration involves sequential events of intricate signaling and feedback mechanisms to guide the circulating neutrophils from blood stream to the site of injury. Recent studies suggest that neutrophil responses to chemoattractants following thermal injury are affected by a variety of cytokines/chemokines secreted by the injured tissue or the bacteria itself. Despite the fact that many of the involved signaling molecules have already been characterized, the global mechanisms through which neutrophils respond to heterogeneous microenvironment are not well understood. Several traditional approaches have been suggested to perform in vitro chemotaxis studies in artificially created chemotactic gradients. However, most of these approaches have limitations of gradient nonlinearity, spatial and temporal instability that are hard to replicate under similar conditions. Moreover, they involve extensive processes to isolate neutrophils (e.g. ficol separation) from large volumes of whole blood (of the order of milliliters) allowing their prolonged exposure to unfavorable conditions that could adversely affect their overall migration performances.

We hypothesize that the sepsis complications in burn patients occur due to the improper activation of neutrophils ultimately causing multiple organ failure. We study their migration behavior using microfluidic tools capable of performing fast switching between different chemokines in the gradient chamber (within ~ 5 seconds). An advanced switching gradient device incorporating microstructured valves has been developed that can be used to directly isolate primary neutrophils from just a drop (<10µL) of whole blood as well as provides a robust platform to perform chemotaxis assays in the competing environment of different chemokines. A drop of blood obtained from finger prick mixed with heparin solution is loaded in the flow chamber individually pre-coated with specific cell adhesion molecules (in this case P-selectin or E-selectin) that facilitate cell adhesion and capturing. We observe low affinity binding and rolling action of leukocytes in the absence of a stimulus while the rolling action turned into firm adhesion inside chemokine-pretreated channels. Observations from neutrophil capture characterization suggest that the P-selectin and E-selectin substrates demonstrate optimum capture efficiencies of 30-40 cells/mm² at concentrations 25µg/mL and 50µg/mL respectively. We also performed migration experiments on these substrates under individual gradients of chemoattractants fMLP and IL-8 and the observed average velocities indicate comparable migration patterns. Similar experiments will be performed (both pre-burn and post-burn) in an overlapping environment of different chemokines (IL8, C5a, GRO-α, fMLP and LTB4) and the migration behaviors will be compared. This study could open the possibility of using simplified and inexpensive point-of-care diagnostic methods based on microfluidic devices for predicting clinical outcome in burn patients.