Journal: Frontiers in immunology

Article Title: Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

doi: 10.3389/fimmu.2023.1118624

Figure Lengend Snippet: FIGURE 1 A screening platform for automated seeding of an organ-on-a-chip endothelial model combined with high throughput TEER measurements. (A) A OrganoPlate 3-lane 64 tissues in a standardized 384-titerplate format. Here, the Organoplate 3-lane 64 seeded on a Biomek i5 automated liquid handling platform. The expanded portion of the plate highlights the top view of one individual chip covering six microtiter wells. One microfluidic chip is enlarged to show the 3 individual microchannels. The perfusion channels used for vessel formation are filled with red dye and the the gel channel is filled with blue dye. (B) The bottom view of the OrganoPlate 3-lane 64 highlighting the microfluidic channels, making up 64 individual chips attached to the bottom of the microtiter plate, allowing for high content imaging of each individual chip. One microfluidic chip is enlarged to highlight the 3 individual microchannels. The perfusion channels used for vessel formation are highlighted in red and the gel channel is highlighted in blue. (C) The OrganoTEER, a commercially available automated TEER measurement system compatible with previous OrganoPlate 3-lane 40 based tubular models (28). (D) OrganoTEER software used to perform TEER measurements and perform automated analysis of the results on an OrganoPlate 3-lane 64.

Article Snippet: This paper shows the use of scalable, automatable OrganoPlate based model of vascular inflammation comprised of up to 64 HUVEC endothelial tubules per plate including cytokine treatment and/or to human Peripheral Blood Mononuclear Cells (PBMC) in combination with continuous TEER measurements.

Techniques: High Throughput Screening Assay, Imaging, Software

Journal: Frontiers in immunology

Article Title: Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

doi: 10.3389/fimmu.2023.1118624

Figure Lengend Snippet: FIGURE 2 Immunofluorescent staining and TEER measurements of HUVEC endothelial tubules in OrganoPlate 3-lane 64. (A) Montage of Immunofluorescent images (VE-cadherin (green) and nucleus (blue)) of 64 HUVEC endothelial tubes (right perfusion channel) cultured against a collagen I ECM layer (center channel) in the OrganoPlate 3-lane 64 (scale bar 1000 µm). Bottom three rows were Staurosporine treated. One chip from the montage is blown up to illustrate how the chip looks (scale bar 150 µm). Another zoom of the endothelial tube is shown to demonstrate the cell morphology (scale bar 50 µm). (B) Confocal reconstruction of a HUVEC tubule using VE-Cadherin (green) and DAPI (blue) staining. (C) Schematic illustration of how a microfluidic chip and the positioning of the TEER electrodes. Both current carrying and voltage sensing loops are formed across the gel and perfusion channel via four pairs of electrodes, shorted pairwise in the inlets and outlets of their perfusion channels. The bottom schematic depicts a side view of the chip in the X-Z plane showing how the endothelial cells will grow to form a tubule against the gel, while the left channel remains empty. (D) values of the HUVEC tube at 0, 24, and 48 hours with the addition of Staurosporine to interrupt the barrier (n= 4-6). Scale bars are 100µm.

Article Snippet: This paper shows the use of scalable, automatable OrganoPlate based model of vascular inflammation comprised of up to 64 HUVEC endothelial tubules per plate including cytokine treatment and/or to human Peripheral Blood Mononuclear Cells (PBMC) in combination with continuous TEER measurements.

Techniques: Staining, Cell Culture

Journal: Frontiers in immunology

Article Title: Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

doi: 10.3389/fimmu.2023.1118624

Figure Lengend Snippet: FIGURE 3 Cytokine response of HUVEC endothelial tubes in the OrganoPlate 3-lane 64. (A) Representative phase contrast images of zoom in of endothelial vessel exposed to inflammatory triggers for 44hrs. Concentration of TNFa and INF-g in ng/ml. Scale bar is 100µm. (B–D) Relative TEER timelapse of 44 hours of exposure to increasing concentrations of (B) INF-g, (C) TNFa and (D) combination of TNFa and INFg (N=3, n=3-5 per experiment).

Article Snippet: This paper shows the use of scalable, automatable OrganoPlate based model of vascular inflammation comprised of up to 64 HUVEC endothelial tubules per plate including cytokine treatment and/or to human Peripheral Blood Mononuclear Cells (PBMC) in combination with continuous TEER measurements.

Techniques: Concentration Assay

Journal: Frontiers in immunology

Article Title: Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

doi: 10.3389/fimmu.2023.1118624

Figure Lengend Snippet: FIGURE 5 Perfusion and extravasation of peripheral blood mononuclear cells (PBMCs) through a HUVEC tubule into an ECM gel in an OrganoPlate 64. (A) Schematic of TEER electrodes with endothelial tubule and PBMCs adhered to the endothelial tube (B) Schematic of TEER electrodes with endothelial tubule and PBMCs migrating from the endothelial tube into gel channel due to addition of chemokine (purple) in basal lateral channel. (C) Relative change in barrier of PBMCs or 10 ng/ml of TNFa + INF-g can be observed in the 48h timelapse. (D) TEER timelapse comparing conditions with and without CLCX12. No difference was observed between comparable conditions with +/- CLCX12. (E) Number of PBMCs adhered to the HUVEC tubules 48 hour after addition. Significant difference unstimulated and stimulated PBMCs (****p < 0.0001) was analyzed using Welch’s T tests. (F) PBMC migration out of endothelial vessel into gel channel. Significant difference between PBMC and PBMCs with cytokines and CLCX12 (* p=0.04, ** p < 0.0018) was analyzed using Brown-Forsythe and Welch ANOVA tests and Dunnett’s T3 multiple comparison test. 3way ANOVA showed no significant (ns) difference between unstimulated and stimulated PBMCs in migration of PBMCs into the ECM. (G) Fluorescent based image of immune cells perfused through a HUVEC tubule and migrated into ECM. (H) Area of interest from (G) to highlight PBMC migration and staining. Shown are nucleus (blue), CellTracker™Orange stain (yellow) and CD45 (red). (I) ICAM-1 quantification after exposure and addition of PBMCs. Significant difference due to addition of cytokines (****p < 0.0001) as well an effect of addition of stimulated PBMCs without cytokines (****p < 0.0001) Data was analyzed using Two-way ANOVA tests with Šı́dák’s multiple comparisons test. (J) Quantification of cell roundness determined from VE-Cadherin staining. Significant difference due to addition of cytokines (****p < 0.0001) as well a significant different between medium and stimulated PBMC and unstimulated PBMCs and stimulated PBMCs (****p < 0.0001). Data was analyzed using Two-way ANOVA tests with Tukey’s multiple comparisons test. (N=2, n=3-6).

Article Snippet: This paper shows the use of scalable, automatable OrganoPlate based model of vascular inflammation comprised of up to 64 HUVEC endothelial tubules per plate including cytokine treatment and/or to human Peripheral Blood Mononuclear Cells (PBMC) in combination with continuous TEER measurements.

Techniques: Migration, Comparison, Staining