Design and Application of Microfluidic Platforms for Integrated Adipocyte Perfusion and Secretion Analysis by Enzyme Assay and Mass Spectrometry.

Abstract

Microfluidic chips enable integration of many components onto one miniaturized platform, creating highly automated devices with the potential for increased throughput of chemical analysis processes. Chips can be adapted to load and perfuse cells; the continuous flow principle of these devices is more biomimetic to the in vivo cellular environment, and allows collection and analysis of secreted metabolites with greater temporal resolution. To learn more about their cellular function, polydimethyl siloxane (PDMS) microfluidic devices were developed to perfuse 3T3-L1 adipocytes and measure secreted lipolysis products. A chip was constructed that contained a cell perfusion chamber with integrated fluorescent enzyme assay reaction channels to simultaneously monitor adipocyte release of non-esterified fatty acids (NEFAs) and glycerol. This multiplexed analysis was measured with laser-induced fluorescence (LIF) detection. The NEFA assay had a limit of detection (LOD) of 6 µM and the glycerol assay had a LOD of 5 µM; both assays had sufficient linear dynamic ranges to measure the secreted products at a flow rate of 0.75 µL/min. Initial studies monitored the secretion of lipolysis products under basal and stimulated conditions. With simultaneous detection, NEFA recycling could be inferred, based on the ratio of secreted NEFAs to glycerol. Further studies compared on-line perfusion and analysis to conventional off-line techniques in terms of NEFA recycling; initial findings suggest there is less NEFA recycling with on-chip cell perfusion. Additionally, pilot studies investigated the kinetics of lipolysis regulation by applying brief pulses of chemical stimuli. A second chip was developed to couple on-line adipocyte perfusion with mass spectrometry (MS) detection to learn more about the identities of secreted NEFAs. As the enzyme assay only permits measurement of total NEFA concentration, MS allows multiplexed analyte detection. An injection loop was integrated downstream of a cell perfusion chamber, operated with multilayer pneumatically-actuated valves. The injection loop acted to isolate the cells from the back pressures generated from the solid phase extraction (SPE) bed coupled to the chip, which was necessary to improve electrospray ionization (ESI)-MS efficiency. Eight NEFAs secreted from adipocytes were monitored under basal and lipolysis-stimulated conditions using this chip.