An in Situ Measurement of Extracellular Cysteamine, Homocysteine, and Cysteine Concentrations in Organotypic Hippocampal Slice Cultures by Integration of Electroosmotic Sampling and Microfluidic Analysis

Abstract

We demonstrate an all-electric sampling/derivatization/separation/detection system for the quantitation of thiols in tissue cultures. Extracellular fluid collected from rat organotypic hippocampal slice cultures (OHSCs) by electroosmotic flow through an11 cm (length) × 50 μm (ID) sampling capillary is introduced to a simple microfluidic chip for derivatization, continuous flow-gated injection, separation and detection.With the help of a fluorogenic, thiol-specific reagent, ThioGlo-1, we have successfully separated and detected the extracellular levels of free reduced cysteamine, homocysteineand cysteinefrom OHSCs within 25 s in a 23 mm separation channel with a confocal laser induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections.When the sample conductivity is much higher than the run buffer conductivities, the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow from the gated channel. This process may completely shut down the gated injection. Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine, homocysteine and cysteine in the extracellular space of OHSCs are10.6±1.0 nM (n=70), 0.18±0.01 μM (n=53) and 11.1±1.2 μM (n=70), respectively. This is the first in situquantitative estimation of endogenous cysteamine in brain. Extracellular levels of homocysteine and cysteine are comparable with other reported values.

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

“…In marked contrast to the intracellular case, cell surface PDI [29] is likely to encounter a much lower concentration of competing thiols (e.g. ~10 μM glutathione and cysteine [28,30,31]). Here, the susceptibility of PDI to inhibition by thiol directed reagents is expected to be strongly influenced by the presence of additional exofacial reductive pathways and thiol-containing proteins [32,33].…”

Integrated Electroosmotic Perfusion of Tissue with Online Microfluidic Analysis to Track the Metabolism of Cystamine, Pantethine, and Coenzyme A

“…20 It was fabricated using standard lithographic techniques as described earlier 29 with a 30 μm mask width for all channels. The etching depth is 20 μm and the channel length (solid line) is drawn to scale.…”

“…1, 3 We have reported integrating electroosmotic perfusion with a microfluidic chip for online analysis of fluid perfused through the extracellular space of organotypic hippocampal slice cultures (OHSCs). 20 OHSCs have the well-organized 3D architecture of the hippocampus in vivo. They provide a means to study biochemical and neurochemical events in the hippocampus.…”

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

“…Using this set-up we successfully measured the basal concentrations of cysteamine, homocysteine, and cysteine in the extracellular space of the OHSCs (Fig. 8), which were 10.6±1.0 nmol L −1 , 0.18± 0.01 µmol L −1 , and 11.1±1.2 µmol L −1 , respectively [107]. It is important to point out that GG is a substrate for γ-glutamyl transpeptidase (γ-GT), another ectoenzyme of interest in the regulation thiols (specifically in the regulation of glutathione, GSH), and is consequently a poor choice as a buffer component for studies involving that specific enzyme and determination of extracellular GSH concentrations.…”