Primary rat LSECs preserve their characteristic phenotype after cryopreservation

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 the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target

Muslime in Europa zwischen Globalisierung und Lokalisierung. Gesellschaftspolitische und theologische Perspektiven im Anschluss an Enes Karic und Tariq Ramadan

). Culture media was collected from hyperoxic conditions (open bars) or normoxic conditions (filled bars) at 24 hours intervals. Final concentrations were estimated from individual standard curves. Generation of endogenous HOwas monitored in separate experiments at the indicated time-points in LSEC cultures by HO-mediated oxidation of DCFH-DA into DFC during 6 h (b). Values are total fluorescence emitted at 545 nm.<p><b>Copyright information:</b></p><p>Taken from "The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells"</p><p>http://www.comparative-hepatology.com/content/7/1/4</p><p>Comparative Hepatology 2008;7():4-4.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2408922.</p><p></p

Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy

The dimensions of mitochondria are close to the diffraction limit of conventional light microscopy techniques, making the complex internal structures of mitochondria unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for optical imaging below the conventional limit, enabling super-resolution (SR). Possibly the most promising SR and diffraction-limited microscopy techniques for live-cell imaging are structured illumination microscopy (SIM) and deconvolution microscopy (DV), respectively. Both SIM and DV are widefield techniques and therefore provide fast-imaging speed as compared to scanning based microscopy techniques. We have exploited the capabilities of three-dimensional (3D) SIM and 3D DV to investigate different sub-mitochondrial structures in living cells: the outer membrane, the intermembrane space, and the matrix. Using different mitochondrial probes, each of these sub-structures was first investigated individually and then in combination. We describe the challenges associated with simultaneous labeling and SR imaging and the optimized labeling protocol and imaging conditions to obtain simultaneous three-color SR imaging of multiple mitochondrial regions in living cells. To investigate both mitochondrial dynamics and structural details in the same cell, the combined usage of DV for long-term time-lapse imaging and 3D SIM for detailed, selected time point analysis was a useful strategy

New ways of looking at very small holes – using optical nanoscopy to visualize liver sinusoidal endothelial cell fenestrations

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, the study of sub-cellular nanoscale biological structures in living cells, which in the past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy has particular utility in the study of “fenestrations” – phospholipid transmembrane nanopores of 50–150 nm in diameter through liver sinusoidal endothelial cells (LSECs) that facilitate the passage of plasma, but (usually) not blood cells, to and from the surrounding hepatocytes. Previously, these fenestrations were only discernible with EM, but now they can be visualized in fixed and living cells using structured illumination microscopy (SIM) and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy. Importantly, both methods use wet samples, avoiding dehydration artifacts. The use of nanoscopy can be extended to the in vitro study of fenestration dynamics, to address questions such as the following: are they actually dynamic structures, and how do they respond to endogenous and exogenous agents? A logical further extension of these methodologies to liver research (including the liver endothelium) will be their application to liver tissue sections from animal models with different pathological manifestations and ultimately to patient biopsies. This review will cover the current state of the art of the use of nanoscopy in the study of liver endothelium and the liver in general. Potential future applications in cell biology and the clinical implications will be discussed

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells-0

at normoxia (d-f). The general morphology of the cultures was monitored by light microscopy at day 1 (a, d), day 3 (b, d) and day 5 (c, f) after isolation. Decline of LSECs cultures may be observed in dishes maintained at atmospheric oxygen levels (a-c) after several days of culture.<p><b>Copyright information:</b></p><p>Taken from "The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells"</p><p>http://www.comparative-hepatology.com/content/7/1/4</p><p>Comparative Hepatology 2008;7():4-4.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2408922.</p><p></p

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells-3

E excluded from the analysis. Porosity measurements are expressed as percentage of the total area covered by cells in each coverslip. Black columns: 20% oxygen. White columns: 5% oxygen.<p><b>Copyright information:</b></p><p>Taken from "The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells"</p><p>http://www.comparative-hepatology.com/content/7/1/4</p><p>Comparative Hepatology 2008;7():4-4.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2408922.</p><p></p

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells-1

maintained in either high (a) or low (b) oxygen tension. Separately, viability was determined at the indicated time points by MTT colorimetric assay (c). Freshly isolated LSECs cultures were established on 24 well-plates and incubated either at hyperoxia (open bars) or at normoxia (filled bars). The obtained results demonstrate a faster decay of loss of cells in cultures maintained at hyperoxic conditions. Statistical analyses by t-student test: *P < 0.05, **P < 0.001.<p><b>Copyright information:</b></p><p>Taken from "The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells"</p><p>http://www.comparative-hepatology.com/content/7/1/4</p><p>Comparative Hepatology 2008;7():4-4.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2408922.</p><p></p

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells-5

Nt time-points. Conditioned media were collected from LSECs cultured at hyperoxia (open bars) or normoxia (filled bars) at 24 hours intervals. Final concentrations were estimated from individual standard curves. Values are means of triplicate measurements. The results are representative data obtained from three independent experiments. Statistical analyses by t-student test: *P < 0.001.<p><b>Copyright information:</b></p><p>Taken from "The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells"</p><p>http://www.comparative-hepatology.com/content/7/1/4</p><p>Comparative Hepatology 2008;7():4-4.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2408922.</p><p></p

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells-4

Ncubation of cells at normoxic or hyperoxic conditions. Each column represents separate values of cell-associated (lower part) and degraded (upper part) I-FSA. Total endocytosis is the result of adding cell associated and degraded ligand (full column size), calculated as percentage of total I-FSA added to cultures. Values are means of triplicate measurements. The results are representative data obtained from three independent experiments. Statistical analyses by Student's -test: *P < 0.05, **P < 0.001.<p><b>Copyright information:</b></p><p>Taken from "The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells"</p><p>http://www.comparative-hepatology.com/content/7/1/4</p><p>Comparative Hepatology 2008;7():4-4.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2408922.</p><p></p