| 1. |
- Gineste, Charlotte, et al.
(författare)
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Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control
- 2022
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Ingår i: iScience. - : Elsevier. - 2589-0042. ; 25:12
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Tidskriftsartikel (refereegranskat)abstract
- Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.
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| 2. |
- Kemas, Aurino M., et al.
(författare)
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Insulin-dependent glucose consumption dynamics in 3D primary human liver cultures measured by a sensitive and specific glucose sensor with nanoliter input volume
- 2021
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 35:3
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Tidskriftsartikel (refereegranskat)abstract
- The liver plays a central role in glucose homeostasis and hepatic insulin resistance constitutes a key feature of type 2 diabetes. However, platforms that accurately mimic human hepatic glucose disposition and allow for rapid and scalable quantification of glucose consumption dynamics are lacking. Here, we developed and optimized a colorimetric glucose assay based on the glucose oxidase-peroxidase system and demonstrate that the system can monitor glucose consumption in 3D primary human liver cell cultures over multiple days. The system was highly sensitive (limit of detection of 3.5 mu M) and exceptionally accurate (R-2 = 0.999) while requiring only nanoliter input volumes (250 nL), enabling longitudinal profiling of individual liver microtissues. By utilizing a novel polymer, off-stoichiometric thiol-ene (OSTE), and click-chemistry based on thiol-Michael additions, we furthermore show that the assay can be covalently bound to custom-build chips, facilitating the integration of the sensor into microfluidic devices. Using this system, we find that glucose uptake of our 3D human liver cultures closely resembles human hepatic glucose uptake in vivo as measured by euglycemic-hyperinsulinemic clamp. By comparing isogenic insulin-resistant and insulin-sensitive liver cultures we furthermore show that insulin and extracellular glucose levels account for 55% and 45% of hepatic glucose consumption, respectively. In conclusion, the presented data show that the integration of accurate and scalable nanoliter glucose sensors with physiologically relevant organotypic human liver models enables longitudinal profiling of hepatic glucose consumption dynamics that will facilitate studies into the biology and pathobiology of glycemic control, as well as antidiabetic drug screening.
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| 3. |
- Shafagh, Reza Zandi, et al.
(författare)
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Bioengineered Pancreas–Liver Crosstalk in a Microfluidic Coculture Chip Identifies Human Metabolic Response Signatures in Prediabetic Hyperglycemia
- 2022
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Ingår i: Advanced Science. - : Wiley. - 2198-3844. ; , s. 2203368-2203368
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Tidskriftsartikel (refereegranskat)abstract
- Aberrant glucose homeostasis is the most common metabolic disturbance affecting one in ten adults worldwide. Prediabetic hyperglycemia due to dysfunctional interactions between different human tissues, including pancreas and liver, constitutes the largest risk factor for the development of type 2 diabetes. However, this early stage of metabolic disease has received relatively little attention. Microphysiological tissue models that emulate tissue crosstalk offer emerging opportunities to study metabolic interactions. Here, a novel modular multitissue organ-on-a-chip device is presented that allows for integrated and reciprocal communication between different 3D primary human tissue cultures. Precisely controlled heterologous perfusion of each tissue chamber is achieved through a microfluidic single “synthetic heart” pneumatic actuation unit connected to multiple tissue chambers via specific configuration of microchannel resistances. On-chip coculture experiments of organotypic primary human liver spheroids and intact primary human islets demonstrate insulin secretion and hepatic insulin response dynamics at physiological timescales upon glucose challenge. Integration of transcriptomic analyses with promoter motif activity data of 503 transcription factors reveals tissue-specific interacting molecular networks that underlie β-cell stress in prediabetic hyperglycemia. Interestingly, liver and islet cultures show surprising counter-regulation of transcriptional programs, emphasizing the power of microphysiological coculture to elucidate the systems biology of metabolic crosstalk.
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| 4. |
- Shafagh, Reza Zandi, et al.
(författare)
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Facile Nanoimprinting of Robust High-Aspect-Ratio Nanostructures for Human Cell Biomechanics
- 2020
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Ingår i: ACS Applied Bio Materials. - : American Chemical Society (ACS). - 2576-6422. ; 3:12, s. 8757-8767
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Tidskriftsartikel (refereegranskat)abstract
- High-aspect-ratio and hierarchically nanostructured surfaces are common in nature. Synthetic variants are of interest for their specific chemical, mechanic, electric, photonic, or biologic properties but are cumbersome in fabrication or suffer from structural collapse. Here, we replicated and directly biofunctionalized robust, large-area, and high-aspect-ratio nanostructures by nanoimprint lithography of an off-stoichiometric thiol–ene-epoxy polymer. We structured—in a single-step process—dense arrays of pillars with a diameter as low as 100 nm and an aspect ratio of 7.2; holes with a diameter of 70 nm and an aspect ratio of >20; and complex hierarchically layered structures, all with minimal collapse and defectivity. We show that the nanopillar arrays alter mechanosensing of human hepatic cells and provide precise spatial control of cell attachment. We speculate that our results can enable the widespread use of high-aspect-ratio nanotopograhy applications in mechanics, optics, and biomedicine.
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