Journal: Lab on a chip

Article Title: Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

doi: 10.1039/d3lc00719g

Figure Lengend Snippet: Fig. 1 Microvascular network formation on a microfluidic device. (A) Schematic illustration of blood vessels forming an intricate network of arteriole- and venule-like vessels formed by endothelial cells (HUAECs and HUVECs, respectively) with pericytes (PCs) residing along the vessels. T cells circulate in blood vessels and extravasate into the perivascular space upon being activated, i.e., in the presence of the protein ICAM-1 during the endothelial activation state. To emulate the microvasculature, we chose a microfluidic device with a central, fibrin-hydrogel filled chamber for cell co-culture and side channels, separated by an array of pillars, for culturing arteriole-like and venule-like vessels separately. The timeline of cell culture is depicted in the right illustration. (B) Photograph of the microfluidic device depicting the hydrogel region (filled with red dye) and side channels (blue dye). (C) Experimental setup during peristaltic flow condition. Syringes connected to the inlets deliver medium to the chip. Outlets are connected to a peristaltic pump which deposits the medium into tubes located outside of the pump. Up to 8 devices can be handled in parallel on one peristaltic pump. We used up to 16 devices in parallel. (D) Schematic overview of flow conditions used in the study: (1) peristaltic flow with either parallel flow or counterflow, (2) hydrostatic pressure-driven interstitial flow, and (3) static condition. Figure partially made with https://BioRender.com.

Article Snippet: Red fluorescent protein (RFP)-expressing human umbilical vein endothelial cells and green fluorescent proteinexpressing human umbilical artery endothelial cells (RFPHUVEC/GFP-HUAEC, Angio Proteomie) were subcultured in flasks coated with 0.2% gelatin in vascular medium (Vasculife) with vascular endothelial growth factor (VEGF, Medium Complete Kit, iCell media supplement, CDI).

Techniques: Activation Assay, Co-Culture Assay, Cell Culture

Journal: Lab on a chip

Article Title: Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

doi: 10.1039/d3lc00719g

Figure Lengend Snippet: Fig. 2 Peristaltic and interstitial flow promote the formation of microvascular networks. (A) Fluorescence image displaying NG2- stained PCs (red) residing on the endothelial barrier formed by HUAECs (green) and HUVECs (yellow) on day 6. Scale bar: 20 μm. (B) Cross- sectional micrographs and illustrative visualization capturing PC- enveloped vessels. Scale bar: 20 μm. (C) Fluorescence images (10× and 20× magnification) of the vessels consisting of HUAECs (green), HUVECs (yellow), and PCs (stained for actin) for different flow conditions. Scale bar: 50 μm. Fig. 2B partially made with https:// BioRender.com.

Article Snippet: Red fluorescent protein (RFP)-expressing human umbilical vein endothelial cells and green fluorescent proteinexpressing human umbilical artery endothelial cells (RFPHUVEC/GFP-HUAEC, Angio Proteomie) were subcultured in flasks coated with 0.2% gelatin in vascular medium (Vasculife) with vascular endothelial growth factor (VEGF, Medium Complete Kit, iCell media supplement, CDI).

Techniques: Fluorescence, Staining

Journal: Lab on a chip

Article Title: Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

doi: 10.1039/d3lc00719g

Figure Lengend Snippet: Fig. 6 T cells extravasate out of the lumen into the perivascular space when the endothelium is activated during inflammation. (A) ICAM-1 expression in the endothelium with and without TNF-α treatment, determined by immunostaining with human anti-ICAM-1. The graph depicts the fluorescence intensity per selected region of interest, ROI (*p ≤0.05, N = 1 for each condition, n ≥6). Scale bar: 50 μm. (B) IL-2-activated primary T cells were introduced to observe T cell adhesion, crawling and extravasation across the endothelial barrier (green and yellow: ECs, red: T cells). The white line indicates the movement of a T cell over 30 min, see also Video SI2†). The dashed line indicates cross section in the bottom micrographs. (C) Percentage of T cells perfused into the networks that have adhered to or transmigrated across the endothelial lumen after an overnight incubation for TNF-α-treated and untreated vessels (N = 6 and 8, ****p ≤0.0001). (D) Percentage of T cells perfused into the networks that have adhered to or transmigrated across the endothelial lumen. Data for TNF-α-treated and untreated vessels, and for TNF-α-treated vessels where T cells were pre-incubated with human antibodies against PSGL-1 and LFA1 (anti-PSGL-1 and anti-LFA1, respectively) (N = 11, N = 10 and N = 4, *p ≤0.05, ****p ≤0.0001). (E) Analysis of selected cytokines and chemokines in the cell culture supernatant, collected during the whole TNF-α treatment from the side channels (top) and the central channel (bottom), compared to controls without TNF-α treatment (N = 8, *p ≤0.05 and ***p ≤0.001).

Article Snippet: Red fluorescent protein (RFP)-expressing human umbilical vein endothelial cells and green fluorescent proteinexpressing human umbilical artery endothelial cells (RFPHUVEC/GFP-HUAEC, Angio Proteomie) were subcultured in flasks coated with 0.2% gelatin in vascular medium (Vasculife) with vascular endothelial growth factor (VEGF, Medium Complete Kit, iCell media supplement, CDI).

Techniques: Expressing, Immunostaining, Fluorescence, Incubation, Cell Culture

Journal: Theranostics

Article Title: Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors

doi: 10.7150/thno.45973

Figure Lengend Snippet: Antibody binding and metabolism in living cells in vitro . Representative immunofluorescence images show the binding of selected anti-mouse (A) and anti-human (B) mAb clones to living endothelial cells. (C, D) EC 50 values of anti-mouse (C) and anti-human (D) mAb clones after binding to living cells for 15 min, n=3. (E, F) Quantitative assessment of the disappearance (due to capture, uptake, and elimination) of anti-mouse (E) and anti-human (F) mAb clones in cell cultures, n=3. (G-I) Cytotoxic effects on living cells in vitro , after different incubation times for anti-mouse mAbs at concentrations of 1-1000 ng/mL (G) or 1-2000 ng/mL (H), or (I) anti-human mAbs at 1-1000 ng/mL, n=3. n.s. no significant difference. mAb: monoclonal antibody; HUVEC: human umbilical vein endothelial cells; HDMEC: human dermal microvascular cells; # P <0.01.

Article Snippet: Human endothelial cell lines (passage 7-10), human umbilical vein endothelial cells (HUVEC,) and human dermal microvascular endothelial cells (HDMEC) (PromoCell, Heidelberg, Germany), were cultured in endothelial cell growth medium and endothelial cell growth medium MV2 (PromoCell) respectively.

Techniques: Binding Assay, In Vitro, Immunofluorescence, Clone Assay, Incubation

Journal: Communications Biology

Article Title: CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

doi: 10.1038/s42003-021-02293-w

Figure Lengend Snippet: a Signalling bias plots were calculated as ∆∆Log(τ/K A ) for CGRP in the three cell lines, HUVECs, RAMP1 expressing HUVECs and HCMs for each pathway. Values have been normalised to a reference agonist (AM2) and the reference pathway (cAMP) for all three cell lines. b As for ( a ) except the calculated values are for AM. c Log potency ratios (as measured by the accumulation of cAMP) calculated as Log (EC 50 AM2/EC 50 agonist). Data are compiled from , . HUVECs and HUAECs are shown in red and green respectively, HCMs in cyan and RAMP1-HUVECs in blue. d – f Schematic representation of the signalling bias produced by CGPR ( d ), AM ( e ) and AM2 ( f ), and the intracellular ‘signalling codes’ they bring about based on the potencies recorded at individual pathways in HUVECs, RAMP1-HUVECs, and HCMs.

Article Snippet: HUVECs and HUAECs (were both sourced from PromoCell, Germany; C-12250 and C-12252 respectfully) were cultured in Endothelial Cell Growth Media (ECGM) (PromoCell).

Techniques: Expressing, Produced

Journal: Aging (Albany NY)

Article Title: Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells

doi: 10.18632/aging.203931

Figure Lengend Snippet: Isolation and enrichment strategy of primary NK cells from human PBMC. ( A ) Experimental design of the NK cell enrichment strategy. PBMCs were collected from multiple donors (ages 20-42 years old), and NK cells were isolated and enriched. ( B ) Flow cytometry analysis of CD56 and CD16 expression in NK cells before and after enrichment for a representative donor. ( B-i ) Before enrichment, 1.64% of NK cells (0.18% of PBMCs) were CD56 Bright CD16 - and 46.34% of NK cells (2.8% of PBMCs) were CD56 Dim CD16 + NK cells. ( B-ii ) After enrichment, 2.29% of NK cells (0.47% of PBMCs) were CD56 Bright CD16 - and 66.18% of NK cells (12.6% of PBMCs) were CD56 Dim CD16 + NK cells. ( C-i ) Average percentage of CD56 Dim CD16 + NK cell population in PBMCs from five donors. ( C-ii ) Average percentage of CD56 Bright CD16 - NK cell population in PBMCs from five donors. Donor sex and age are indicated in the figure. Statistical analysis performed using paired t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Primary human arterial endothelial cells purchased from Coriell Institute for medical research (AG10770) were maintained in promo cell basal medium MV2 (PromoCell; Cat# C-22221) supplemented with Growth Medium MV 2 Supplement Pack (PromoCell; Cat# C-39221) and assayed within 10 or less passages.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Aging (Albany NY)

Article Title: Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells

doi: 10.18632/aging.203931

Figure Lengend Snippet: Activated primary NK cells selectively eliminate senescent cells. ( A ) Quantitative Realtime PCR was performed to detect the mRNA levels of CCL5 , CXCL9 , and CXCL11 in non-senescent and senescent IMR-90 fibroblasts. The results are presented as mean fold change in NS compared to S samples from two independent experiments performed in triplicate, and error bars represent ±SEM. Statistical analysis performed using unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001. ( B ) NS or S IMR-90 fibroblasts were co-incubated with NK cells for 16 h at T:E ratios of 1:1, 1:2 and 1:3 and cytotoxicity was evaluated by LDH release. The graphs show the mean and S.E. of % LDH release. NS or S ( C-i ) doxorubicin-treated (n=6), ( C-ii ) irradiated (n=3) or ( C-iii ) etoposide-treated (n=3) IMR-90 fibroblasts or ( C-iv ) doxorubicin-treated endothelial cells (n=2) were overlayed with NK cells for 16 hours at T:E ratio of 1:1, and cytotoxicity was evaluated by LDH release. The results are plotted as mean % cytotoxicity for NS and S cells with each experiment performed in at least triplicate. The graphs show mean % LDH release. ( D ) NK cells isolated and enriched from three different individuals were co-cultured with NS or S IMR-90 cells at T:E ratio of 1:1 and cytotoxicity was evaluated by LDH release after 16 hours of co-culture. Experiments were performed in triplicate and the results are plotted as mean % cytotoxicity for NS and S. Donor sex and age are indicated in the figure. Statistical analysis performed using unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Primary human arterial endothelial cells purchased from Coriell Institute for medical research (AG10770) were maintained in promo cell basal medium MV2 (PromoCell; Cat# C-22221) supplemented with Growth Medium MV 2 Supplement Pack (PromoCell; Cat# C-39221) and assayed within 10 or less passages.

Techniques: Incubation, Irradiation, Isolation, Cell Culture, Co-Culture Assay