cryopreserved passage 1 human liver sinusoidal endothelial cells (lsec)  (ScienCell)

Journal: Journal of Inflammation (London, England)

Article Title: Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

doi: 10.1186/s12950-024-00387-w

Figure Lengend Snippet: Consequence of IFNγ for microcirculatory disturbance in acute liver injury. A Serum TNFα and IFNγ levels in the SMD and NSMD groups. The data were expressed as mean and SD. ** P < 0.01 when compared to the NSMD group. B RT-qPCR analysis of TNFα and IFNγ gene expression in Con A (15mg/kg) and TNF/GalN (700 mg/kg GalN +15 µg/kg TNFα) livers. The gene expression levels were normalized to those in the untreated mice as NC. The data were presented as mean and SE ( n = 3–5 in each group). ** P < 0.01 vs. NC groups and †† P < 0.01 vs. TNF/GalN group. C Serum ALT levels after 0, 1, 3, 6, and 12 hours of Con A administration. The data were expressed as mean and SD ( n = 6–13 in each group). D Hematoxylin and eosin staining of the livers after 0, 1, 3, 6, and 12 hours of Con A administration. The dashed area indicates necrotic features and arrowheads indicate hemostasis. Scale bars = 50 μm. E RT-qPCR analysis of the genes associated with the inflammatory cytokines, coagulation, and hypoxia in Con A livers (25mg/kg Con A). The gene expression levels were normalized to those at 0 hour. The data were expressed as mean and SE ( n = 3–8 in each group). ALT, alanine aminotransferase; Con A, concanavalin A; GalN, galactosamine; HO-1, heme oxygenase 1. IFNγ, interferon-gamma; LDH, lactate dehydrogenase; NC, normal control; NSMD, non-sinusoidal microcirculatory disturbance; RT-qPCR, quantitative reverse transcription polymerase chain reactions; SMD, sinusoidal microcirculatory disturbance; TNFα, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor

Article Snippet: Human liver sinusoidal endothelial cells (LSECs) were purchased from LONZA (Walkersville, MD) and cultured on EGMTM-2 BulletKitTM media at 37°C and 5% CO 2 .

Techniques: Quantitative RT-PCR, Gene Expression, Staining, Coagulation, Control, Reverse Transcription

Journal: Journal of Inflammation (London, England)

Article Title: Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

doi: 10.1186/s12950-024-00387-w

Figure Lengend Snippet: Requirement of IFNγ in microcirculatory disturbance. A Serum ALT levels 0, 1, 3, 6, and 12 hours after Con A administration in the WT , Tnf –/– , and Ifng –/– mice. The results were expressed as mean and SD ( n = 6–13 in each group). ** P < 0.01 when compared to the WT mice. B Hematoxylin and eosin staining and PTAH staining of the liver for the WT , Tnf –/– , and Ifng –/– mice. The dashed area indicates necrotic features and arrowheads indicate fibrin depositions in sinusoids. Scale bars = 50 μm. C RT-qPCR analysis of the genes of the inflammatory cytokines, coagulation, and hypoxia in the liver of WT , Tnf –/– , and Ifng –/– mice treated with Con A. The gene expression levels were normalized to those of the WT at 0 hours. The data were expressed as mean and SE. ** P < 0.01 vs. the WT mice ( n = 3–8 in each group). D RT-qPCR analysis of CD40 and Tissue factor in sorted liver sinusoidal endothelial cells. The gene expression levels were normalized to those of the WT at 0 hours. The data were presented as mean and SE ( n = 3 in each group). * P < 0.05, ** P < 0.01 vs. the WT mice. E The distribution of total nonparenchymal cells with c3 re-clustered is presented on the UMAP plot. The feature plots showed the expression of CD40 and CD40L. The heatmap presented the relative strength of the CD40-CD40L signaling network for each cluster. ALT, alanine aminotransferase; CD40L, CD40 ligand; Con A, concanavalin A; IFNγ, interferon-gamma; ILC, innate lymphoid cell; LDH, lactate dehydrogenase; NK cell, natural killer cell; NKT cell, natural killer T cell; PTAH, phosphotungstic acid-hematoxylin; RT-qPCR, quantitative reverse transcription polymerase chain reactions; Tgd cell, gamma delta T cell; TNFα, tumor necrosis factor-alpha; UMAP, uniform manifold approximation and projection; VEGF, vascular endothelial growth factor; WT, wild type

Article Snippet: Human liver sinusoidal endothelial cells (LSECs) were purchased from LONZA (Walkersville, MD) and cultured on EGMTM-2 BulletKitTM media at 37°C and 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Coagulation, Gene Expression, Expressing, Reverse Transcription

Journal: Journal of Inflammation (London, England)

Article Title: Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

doi: 10.1186/s12950-024-00387-w

Figure Lengend Snippet: Induction of tissue factor by IFNγ and CD40 ligand in liver endothelial cells. A The LSEC were treated with 1,000 U/ml IFNγ for 12 hours and then incubated for 6 hours with Jurkat cells fixed with 1% paraformaldehyde. B Cell-surface expression of CD40L protein in untreated Jurkat cells by flow cytometry. The histograms depict isotype control (dashed line), unstained control (solid blue line), and CD40L expression (solid red line). The qualitatively identical results were obtained with at least three further batches of cells. C RT-qPCR analysis of CD40 and tissue factor in LSEC cells after IFNγ (1,000 U/ml) and Jurkat cell treatment. The gene expression levels were normalized to those of the IFNγ and Jurkat cell free group. The data were expressed as mean and SE ( n = 5–10 in each group). ** P < 0.01 vs. the IFNγ and Jurkat cell free group and †† P < 0.01 vs. the IFNγ group. CD40L, CD40 ligand; IFNγ, interferon-gamma; LSEC, liver sinusoidal endothelial cell; RT-qPCR, quantitative reverse transcription polymerase chain reactions

Article Snippet: Human liver sinusoidal endothelial cells (LSECs) were purchased from LONZA (Walkersville, MD) and cultured on EGMTM-2 BulletKitTM media at 37°C and 5% CO 2 .

Techniques: Incubation, Expressing, Flow Cytometry, Control, Quantitative RT-PCR, Gene Expression, Reverse Transcription

Journal: Frontiers in Genetics

Article Title: The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression

doi: 10.3389/fgene.2024.1302685

Figure Lengend Snippet: microRNAs targeting F8 gene. (A) Relative expression of F8 gene (Probe ID: ILMN_1675083) in the used ECs and LSECs samples. (B) Left panel: Unsupervised, 3D-PCA displaying different endothelial cells for 25 expressed microRNAs that bind to F8 gene according to IPA. Middle panel: heat map showing the expression of these 25 microRNAs in three LSEC samples. Right panel: Correlation between F8 expression in different endothelial cells and PCA-1, X-axis represents F8 expression and y-axis represents PCA-1. (C) A depiction of the 1808-base pair long 3′ mRNA sequence of the F8 gene along with the 25 expressed microRNAs known to bind to this sequence. (D) Box plots illustrating the expression levels of four microRNAs in different endothelial cells, potentially binding to the F8 gene, as well as the transcription factors binding to the F8 gene promoter. (E) Top Panel: An IPA-generated figure showing the relationships between potentially F8 -binding microRNAs and transcription factors identified using TRANSFAC, which bind to the F8 promoter. Sequence logos of individual transcription factors are displayed on the right. Bottom Panel: Representation of the F8 promoter, covering 1,200 base pairs with 1 kb before the transcription start site and 200 base pairs after the transcription start site. The numbers represent the transcription factors binding sites identified using TRANSFAC (shown and numbered in top panel above).

Article Snippet: In this study, Primary adult Human Liver Sinusoidal Endothelial Cells (LSECs) were procured from Biozol Diagnostica Vertrieb GmbH (product lot No. HEC03020491) and cultured in a specialized endothelial cell culture medium, denoted as ENDO-Growth Medium MED001, supplemented with growth factors and antibiotics.

Techniques: Expressing, Sequencing, Binding Assay, Generated

Journal: Nature Communications

Article Title: Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function

doi: 10.1038/s41467-024-45122-4

Figure Lengend Snippet: a Representative maximum projection of vessels in ACE-liver spheroid acquired by in vivo imaging by intravenous injection of DyLight-649-conjugated lectin (black) after binary conversion and thresholding at 1-week, 1 and 2-months post-tx. Spheroid area limit: pink dashed line. b Immunofluorescence vasculature network (CD31, orange) staining of whole-mount ACE-liver spheroid at 2-months post-tx. Max. projection image. c Vascular density of ACE-liver spheroids. Whiskers represent min/max values, with the mean shown as ‘+’. **** p < 0.0001 by one-way ANOVA test, n = 9 in 3 recipient mice. d Vessel network assessed by number of branch points within the spheroids. Whiskers represent min/max values, with the mean shown as ‘+’. * p < 0.05 by Mann–Whitney test, n = 4 in 3 recipient mice. e Average diameter of iris and intra-spheroid vessels at 1 and 2-months post-tx. Whiskers represent the min/max values, with the mean shown as ‘+’. **** p < 0.0001 by Kruskal–Wallis test, n = 8 measurements per spheroid, 5 spheroids in 3 recipient mice. f In vivo imaging of labeled red blood cells (red) through intra-spheroid vessels (lectin, white) and their trajectory (red-dashed line). Spheroid mass is delimited by a white-dashed line, scale bar = 50 µm. Chronological images taken from Supplementary Movie . g Immunofluorescence staining of whole-mount ACE-liver spheroids and surrounding iris tissue. Sympathetic (TH, red) and parasympathetic (VACHT, green) nerves at 2-months post-tx. Max. projection images. h Immunofluorescence staining within the ACE-liver spheroid mass. Sympathetic (TH, red) and parasympathetic nerves (VACHT, yellow); vessels (CD31, white). Arrowheads indicate nerves alongside vessels and spheroid mass is delimited by white-dashed line. Single plane image. i Immunofluorescence staining of macrophages (F4/80, yellow) within the ACE-liver spheroid. Single plane image. j Experimental outline of human liver spheroids generated from primary human hepatocytes (PHH) and primary human liver sinusoidal endothelial cells (LSECs) transplanted into the ACE of immunodeficient mice. k In vivo imaging of vascularization in engrafted human liver spheroids at 1-month post-tx, visualized by intravenous injection of DyLight-649-conjugated lectin (red). l Vasculature network (CD31, orange) and nuclei (DAPI) staining of ACE-human liver spheroid at 1-month post-tx. Max. projection image. a , b , g , h , i , k , l scale bars = 100 µm. Source data are provided as a Source Data file.

Article Snippet: Cryopreserved purified primary human hepatocytes (PHH) and liver sinusoidal endothelial cells (LSECs) were obtained from suppliers BioIVT (New York, US) and Lonza (Basel, Switzerland), respectively (Supplementary Table ).

Techniques: In Vivo Imaging, Injection, Immunofluorescence, Staining, MANN-WHITNEY, Labeling, Generated