Reversibly sealed multilayer microfluidic device for integrated cell perfusion and on-line chemical analysis of cultured adipocyte secretions.

A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that allowed cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from approximately 6,200 cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 +/- 2.4 microM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 x 10(-5) nmol cell(-1) min(-1). Upon pharmacological treatment with a beta-adrenergic agonist to stimulate lipolysis, a 6.9 +/- 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions in near real-time.