In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® are genetically engineered cell strains derived from primary by lenti-viral transduction genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating undergo a finite number divisions, i.e., 20 to 40 population doublings, but upon withdrawal stimulating factors, they regain most specific characteristics cells. When defined mixture...

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that sorting assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins p62/SQSTM1 act as a receptor for components SAM mitochondrial contact site cristae organizing system (MICOS) complexes. regulates architecture controlling formation MICOS decisive...

Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters 50 to 200 nm. The small sizes of fenestrae have so far prohibited any functional analysis standard advanced light microscopy techniques. Only advent super-resolution optical fluorescence now permits recording such cellular structures. Here, we demonstrate complementary use two different modalities, 3D structured...

// Adil El Taghdouini 1, * , Anita L. Sørensen 2, Andrew H. Reiner 2 Mar Coll 3 Stefaan Verhulst 1 Inge Mannaerts Cristina I. Øie 4 Bård Smedsrød Mustapha Najimi 5 Etienne Sokal Aernout Luttun 6 Pau Sancho-Bru Philippe Collas Leo A. van Grunsven Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium Department of Molecular medicine, Institute Basic Medical Sciences, Faculty Medicine, University Oslo, Norway Institut d’Investigacions...

Abstract The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection infection, while the few studies designed look into clearance mechanisms focused mainly on role macrophages. In recent years, attention directed towards sinusoidal endothelial cells (LSECs), which play a central innate immunity by their ability scavenge pathogen- damage-associated molecular patterns....

Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional lacks ability to generate throughput data, as imaged region is small. Photonic chip-based has demonstrated potential for imaging large areas, but a lateral of 130 nm. However, all existing super-resolution methods provide 100 nm or better. In this work, 75 over an...

Liver sinusoidal endothelial cells (LSECs) are the first liver to encounter waste macromolecules, pathogens, and toxins in blood. LSECs highly specialized mediate clearance of these substances via endocytic scavenger receptors equipped with fenestrae that passage macromolecules toward hepatocytes. Although some transcription factors (TFs) known play a role LSEC specialization, information about signature its transcriptional determinants remains incomplete.Based on comparison liver, heart,...

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered sinusoids is general considerably lower than air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions regard to would expect improve cell survival, structure function. In this study were from rats cultured either 5% (normoxic) or 20% (hyperoxic)...

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling deforming large cell populations along length of waveguide. Bright field microscopy employed with does not provide quantitative information about structural changes. Here, we combined phase techniques study RBC morphology planar transportation. By using interference...

Abstract Super-resolution fluorescence microscopy, also known as nanoscopy, has provided us with a glimpse of future impacts on cell biology. Far-field optical nanoscopy allows, for the first time, study sub-cellular nanoscale biological structures in living cells, which past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy particular utility “fenestrations” – phospholipid transmembrane nanopores 50–150 nm diameter through liver sinusoidal endothelial...

The mechanism of elimination blood borne heparin was studied. To this end unfractionated (UFH) tagged with FITC, which served as both a visual marker and site labeling (125)I-iodine. UFH labeled in manner did not alter the anticoagulant activity or binding specificity glycosaminoglycan. Labeled administered intravenously to rats (0.1 IU/kg) had circulatory t(1/2) 1.7 min, increased 16 min upon coinjection unlabeled (100 IU/kg). At 15 after injection, 71% recovered radioactivity found liver....

Abstract The dimensions of mitochondria are close to the diffraction limit conventional light microscopy techniques, making complex internal structures unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for below limit, enabling super-resolution (SR). Possibly most promising SR and diffraction-limited live-cell structured illumination (SIM) deconvolution (DV), respectively. Both SIM DV widefield therefore provide fast-imaging speed as...

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50-300 nm in diameter, which is below resolution limit of a conventional light microscopy. To date, there no standardized method fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, semi-automatic (threshold-based) method, an automatic based on user-friendly...

Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver non-parenchymal cells have gained increasing attention for their potential role in development disease. sinusoidal endothelial (LSECs), specialized type that unique morphology function, play fundamental maintaining homeostasis. Current protocols LSEC isolation cultivation rely on freshly isolated which can only be maintained differentiated culture few days. This creates limitation use LSECs research...

Abstract Correlative light and electron microscopy (CLEM) unifies the versatility of (LM) with high resolution (EM), allowing one to zoom into complex organization cells. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) over large field view precision localization target area interest within EM. The chips are used as a substrate hold, illuminate provide landmarking sample through specially designed grid-like numbering systems....

The liver as the largest organ in human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging particularly challenging because need cover length scales across 7 orders magnitude (from centimeter scale nanometer scale) order fully assess ultrastructure entire down subcellular probe function. This task becomes even more deeper within one hopes image, strong absorption scattering...

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view throughput. We present a novel generating imaging with waveguides, called chip-based nanoscopy. aim this protocol demonstrate how performed an already built...

In both septic and aseptic inflammation, N-formyl peptides may enter the circulation induce a systemic inflammatory response syndrome similar to that observed during shock. The is brought about by binding of peptide formyl receptors (FPRs), specific signaling expressed on myeloid as well non-myeloid cells involved in process. conjugated with fluorochromes, such fluorescein isothiocyanate (FITC) are increasingly experimentally used identify tissues inflammation. Hypothesizing process...

Super-resolution optical microscopy, commonly referred to as nanoscopy, has enabled imaging of biological samples with a resolution that was only achievable previously using electron microscopy. Optical nanoscopy is rapidly growing field, several different techniques and implementations overcome the diffraction limit light. However, common nanoscope continues be rather complex, expensive bulky instrument. Direct stochastic reconstruction microscopy (dSTORM) recently demonstrated waveguide...

Abstract Atherogenesis is associated with elevated plasma levels of oxidized low-density lipoproteins (oxLDL). In vivo, oxLDL causes liver endothelial swelling, and disrupts sinusoidal cell (LSECs) fenestrations. We mapped the nanoscale kinetics these changes in vitro isolated rat LSECs challenged monitored viability endocytosis cytotoxicity assays. OxLDL disrupted LSEC ultrastructure – increasing concentrations oxidation caused sieve plate loss, fenestration fusion, gap formation....

Photonic chips have the potential to cause a paradigm shift in super-resolution optical microscopy. Here we discuss geometry optimization of high refractive index waveguides for bioimaging and implementation methods.

The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates liver endothelial cells. In waveguide excitation, the dominant only near surface (~100-150 nm) providing a default sectioning by illuminating fluorophores in close proximity thus benefiting higher signal-to-noise ratio. sieve plates sinusoidal cells are present cell membrane, near-field chip-based microscopy configuration preferred over epi-fluorescence. chip compatible with fiber components...