This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices.
Reduced glutathione (GSH) has been determined by fluorescence detection after derivatization together with a variety of separations. The reactions between GSH and fluorescent reagents usually carry out during the sample pretreatment and require minutes to hours for complete reactions. For continuous monitoring of GSH, it would be very convenient to have an integrated microdevice that could perform online precolumn derivatization, separation, and detection. Heretofore, thiol-specific fluorogenic reagents require fairly long reaction times, preventing effective online precolumn derivatization. We demonstrate here that the fluorogenic, thiol-specific reagent, ThioGlo-1, reacts rapidly enough for efficient precolumn derivatization. The second order rate constant for the reaction of GSH and reagent (pH 7.5, room temperature) is 2.1 × 104 M−1s−1. The microchip integrates this precolumn derivatization, continuous flow gated sampling, separation and detection on a single device. We have validated this device for monitoring GSH concentration continuously by studying the kinetics of glutathione reductase (EC 1.8.1.7), an enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to GSH in the presence of β-NADPH (β-Nicotinamide adenine dinucleotide phosphate, reduced form) as a reducing cofactor. During the experiment, GSH being generated in the enzymatic reaction was labeled with ThioGlo-1 as it passed through a mixing channel on the microfluidic chip. Derivatization reaction products were introduced into the analysis channel every 10 s using flow gated injections of 0.1 s. Baseline separation of the internal standard, ThioGlo-1, and the fluorescently labeled GSH was successfully achieved within 4.5 s in a 9 mm separation channel. Relative standard deviations of the peak area, peak height, and full width at half maximum (FWHM) for the internal standard were 2.5%, 2.0%, 1.0%, respectively with migration time reproducibility for the internal standard of less than 0.1% RSD in any experiment. The GSH concentration and mass detection limit were 4.2 nM and ~10−18 mol, respectively. The Michaelis constants (Km) for GSSG and β-NADPH were found to be 40 ± 11 μM and 4.4 ± 0.6 μM, respectively, comparable with those obtained from UV/Vis spectrophotometric measurements. These results show that this system is capable of integrating derivatization, injection, separation and detection for continuous GSH determinations.
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