1. Dynamic sampling from ex vivo adipose tissue using droplet-based microfluidics supports separate mechanisms for glycerol and fatty acid secretion.
- Author
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Moniruzzaman M, Bezerra AB, Mohibullah M, Judd RL, Granneman JG, and Easley CJ
- Subjects
- Animals, Mice, Lab-On-A-Chip Devices, Microfluidic Analytical Techniques instrumentation, Fatty Acids, Nonesterified metabolism, Fatty Acids metabolism, Fatty Acids chemistry, Glycerol metabolism, Glycerol chemistry, Adipose Tissue metabolism
- Abstract
Pathologies in adipose (fat) tissue function are linked with human diseases such as diabetes, obesity, metabolic syndrome, and cancer. Dynamic, rapid release of metabolites has been observed in adipocyte cells and tissue, yet higher temporal resolution is needed to adequately study this process. In this work, a microfluidic device with precise and regular valve-automated droplet sampling, termed a microfluidic analog-to-digital converter (μADC), was used to sample secretions from ∼0.75 mm diameter adipose explants from mice, and on-chip salt water electrodes were used to merge sampled droplets with reagent droplets from two different fluorometric coupled enzyme assays. By integrating sampling and assays on-chip, either glycerol or non-esterified fatty acids (NEFA), or both, were quantified optically within merged 12 nanoliter droplets using a fluorescence microscope with as high as 20 second temporal resolution. Limits of detection were 6 μM for glycerol (70 fmol) and 0.9 μM for NEFA (10 fmol). Multiple ex vivo adipose tissue explants were analyzed with this system, all showing clear increases in lipolytic function after switching from feeding to fasting conditions. Enabled by high temporal resolution, lipolytic oscillations of both glycerol and NEFA were observed for the first time in the range of 0.2 to 1.6 min
-1 . Continuous wavelet transform (CWT) spectrograms and burst analyses (0.1 to 4.0 pmol bursts) revealed complex dynamics, with multiplexed assays (duplex for glycerol and NEFA) from the same explants showing mostly discordant bursts. These data support separate mechanisms of NEFA and glycerol release, although the connection to intracellular metabolic oscillations remains unknown. Overall, this device allowed automated and highly precise temporal sampling of tissue explants at high resolution and programmable downstream merging with multiple assay reagents, revealing unique biological information. Such device features should be applicable to various other tissue or spheroid types and to other assay formats.- Published
- 2024
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