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. 2023 Sep 13;10(9):1080.
doi: 10.3390/bioengineering10091080.

A Rapid-Patterning 3D Vessel-on-Chip for Imaging and Quantitatively Analyzing Cell-Cell Junction Phenotypes

Li Yan  1 , Cole W Dwiggins  1 , Udit Gupta  1 , Kimberly M Stroka  1   2   3   4
Affiliations

A Rapid-Patterning 3D Vessel-on-Chip for Imaging and Quantitatively Analyzing Cell-Cell Junction Phenotypes

Li Yan et al. Bioengineering (Basel). .

Abstract

The blood-brain barrier (BBB) is a dynamic interface that regulates the molecular exchanges between the brain and peripheral blood. The permeability of the BBB is primarily regulated by the junction proteins on the brain endothelial cells. In vitro BBB models have shown great potential for the investigation of the mechanisms of physiological function, pathologies, and drug delivery in the brain. However, few studies have demonstrated the ability to monitor and evaluate the barrier integrity by quantitatively analyzing the junction presentation in 3D microvessels. This study aimed to fabricate a simple vessel-on-chip, which allows for a rigorous quantitative investigation of junction presentation in 3D microvessels. To this end, we developed a rapid protocol that creates 3D microvessels with polydimethylsiloxane and microneedles. We established a simple vessel-on-chip model lined with human iPSC-derived brain microvascular endothelial-like cells (iBMEC-like cells). The 3D image of the vessel structure can then be "unwrapped" and converted to 2D images for quantitative analysis of cell-cell junction phenotypes. Our findings revealed that 3D cylindrical structures altered the phenotype of tight junction proteins, along with the morphology of cells. Additionally, the cell-cell junction integrity in our 3D models was disrupted by the tumor necrosis factor α. This work presents a "quick and easy" 3D vessel-on-chip model and analysis pipeline, together allowing for the capability of screening and evaluating the cell-cell junction integrity of endothelial cells under various microenvironment conditions and treatments.

Keywords: 3D vessel-on-chip; blood-brain barrier; cell morphology; tight junctions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic overviewing the fabrication process of our in vitro 3D vessel-on-chip model. (a) PDMS is polymerized on a blank silicon wafer and diced into chips. (b) A rectangle section is cut in the middle of the chips. (c) Two slits are made in the bottom layer and then a microneedle is inserted through it. (d) Space-filling with PDMS. (e) PDMS device baking. (f) Removal of microneedles. (g) Punching of inlets and outlets. (h) Preparation of top layer and bonding. (i) Bonding reservoir to chip (j) Multiple chips in one coverslip. (k) The iPSCs were cultured on Matrigel-coated 6-well plates and differentiation was initiated on day 0. The iBMEC-like cells were subcultured into the ECM-coated microchannels on day 6. On day 8, vessels were well formed in the channels and treatments were introduced into system. Figure was generated by BioRender.
Figure 2
Figure 2
Overview of fabrication process. (a) For the fabrication process, PDMS is polymerized on top of a blank silicon wafer, diced, and extracted into 20 mm × 25 mm chips. (b) A 5 mm × 15 mm section is then cut from the middle to allow the microneedle to pass through. (c) Two slits are made along the rectangular section bisection line. (d) The microneedle is inserted within the slits. (e) The chips are flipped over, microneedle side down, and placed back on the wafer, where PDMS is poured into the cut section and polymerized. (f) The microneedle is then pulled and two ø 1.5 mm holes are punched along the channel path to make a channel inlet and outlet. (g) The reservoir layers are made by punching two 7 mm holes in blank 20 mm × 25 mm PDMS chips. (h) Channel layer and reservoir layer are bonded to a glass coverslip. (i) Brightfield image of microchannel with iBMEC-like cells.
Figure 3
Figure 3
Unwrapping 3D immunostained image stacks into 2D surfaces. (a) 3D image of BBB channel reconstructed by the UNWRAP program. (b) A vertical section of the BBB channel. (c) A semicircular cross-section of the BBB channel. (d) A circle is then fitted to the channel circumference based on user-specified waypoints along the channel’s path. (e) The channel is then “unwrapped” into a 2D surface. Scale bar represents 33 μm.
Figure 4
Figure 4
Analysis of cell–cell junctions and cell morphology in unwrapped images. (a) An unwrapped 2D image. The dotted outline box in the left image is shown zoomed in the right image of this panel. This zoomed image also corresponds to the image in panels (b–d). (b) Cell is identified by the JAnaP when the user “waypoints” a cell along its border. Once all the cells have been waypointed, the JAnaP then processes each cell. (c) In a particular cell of interest, the JAnaP will then apply a filter along the user-specified cell border to eliminate background. (d) Along the cell border, cell junctions are then classified according to the scheme indicated. (e) The classified cell junctions are displayed, and their phenotype data are saved for analysis. (f) The presentation of continuous, punctate, and perpendicular junctions for ZO-1 are shown, respectively, for 3D vessel-on-chip devices (3D) and 2D PDMS surfaces (2D). (g) The total junction coverage of ZO-1. (h–k) Cell shape factors based on ZO-1 expression. Note: 203 ≤ N ≤ 297, where N is the number of cells pooled from three biological replicates. In dot plots, each dot represents the value for one cell. Bars represent mean and error bars represent standard deviation. Statistical significance was indicated as **** p < 0.0001.
Figure 5
Figure 5
Analysis of cell–cell junctions in 3D vessels with TNF-α treatment. (a) The presentation of continuous, punctate, and perpendicular junctions for ZO-1 are shown, respectively. (b) The total junction coverage of ZO-1. (c) The presentation of continuous, punctate, and perpendicular junctions for Occludin are shown, respectively. (d) The total junction coverage of Occludin. Note: 119 ≤ N ≤ 211, where N is the number of cells pooled from three biological replicates. In dot plots, each dot represents the value for one cell. Bars represent mean and error bars represent standard deviation. Statistical significance was indicated as **** p < 0.0001.
Figure 6
Figure 6
Analysis of cell morphology in 3D vessels with TNF-α treatment. Morphologies analyzed include (a) perimeter, (b) area, (c) circularity, and (d) solidity. Note: 119 ≤ N ≤ 211, where N is the number of cells pooled from three biological replicates. In dot plots, each dot represents the value for one cell. Bars represent mean and error bars represent standard deviation. Statistical significance was indicated as ** p < 0.01, *** p < 0.001.
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