br Generation of the functional BBB br A previous study
3.2. Generation of the functional BBB
A previous study suggested that an integral BBB can be formed by both human microvascular endothelial cells (hBMVECs) that
line cerebral capillaries and the perivascular end-feet of astrocytes under capillary flow (0.1 dyne/cm2). To establish a functional BBB, hBMVECs were infused into the vascular channels and adhered to the bottom and side walls of the channel (Fig. 2 A(i), B, Fig. S1A) after incubating the channels with extracellular matrix (ECM), including collagen I and fibronectin, which mimicked the natural ECM of the brain. Once the endothelial cells formed a con-tinuous cell layer on the ECM, human astrocytes (HA) were intro-duced into the Pimozide parenchyma chamber (Fig. 2A(ii)) to be co-cultured with the endothelial cells. The co-cultured cells were exposed to a continuous fluidic shear (0.1 ll/min) through the vas-cular channel, which mimicked the blood flow in the brain (Fig. 2A (iii)). Under these conditions, hBMVECs grew well (Fig. S1B) and formed a complete barrier 48 h after dynamic co-culture with astrocytes.
We then evaluated the constructed BBB for both structure integrity and barrier function. The structure integrity was assessed by detecting TJ and TEER. TJs were confirmed by immunostaining for zonal occludin-1 (ZO-1) and vascular endothelial -cadherin (VE-Cad), which are known proteins signifying BBB tightness [38,39]. TEER, which is widely used to assess barrier integrity
[19,22], was measured every 12 h for 4 days. When hBMVECs were cultured in the bionic BBB state where the endothelial cells were cultured in the presence of astrocytes under fluidic flow for 48 h, both ZO-1 and VE-Cad were more broadly and highly expressed, while the TEER increased more than 2-fold, compared to the state where the endothelial cells were grown in isolation (Fig. 2C, D). Another important characteristic of an in vitro functional BBB is its impermeability to small molecules. To determine the perme-ability of the constructed barrier, we introduced FITC-conjugated 40 kDa dextran into the vascular channels and recorded time-lapse images of fluorescein diffusion when the dye penetrated into the brain parenchyma chamber under the dynamic states of co-cultured astrocytes or endothelial cells alone. Consistent with the expression of the TJ proteins, the outward flux of the dye was slower in the dynamic BBB barrier with co-cultured astrocytes (Fig. 2E). Therefore, the BBB on the chip we built was competent in terms of both structure and function.
3.3. Extravasation of lung cancer cells through the BBB on the multi-organ chip
To better visualize BBB penetration of lung cancer cells, which invaded the blood vessels in the upstream site and reached the downstream site following the flow medium, we used the trans-fected lung tumor cell line PC9 that stably expressed green fluores-cent protein (GFP), and stained the endothelial cells with red live Cell TrackerTM CM-Dil. The trajectories of tumor cells were moni-tored in real time using fluorescence microscopy. Two days after introducing the tumor cells into the upstream bionic culture, tumor cells were observed approaching the downstream site with the fluid, and we set the time when the first tumor cell arrived at the downstream site as the observation starting point (0 h). As shown in Fig. 3A, some of the PC9 cells began adhering to the endothelial layer at 12 h after reaching the downstream vascular channel. Notably, one of them extravasated through the BBB at 24 h, and invaded the brain parenchyma at 36 h. At 48 h after reaching the downstream vascular channel, the metastatic cell began to proliferate and colonize. We captured the whole process from a tumor cell adhering to the blood vessels, to breaking through the BBB, and finally reaching the brain parenchyma (Fig. 3A, the white arrow; Movie S1).
To determine if the TJ structure was destroyed during the extravasation process, we compared the expression of ZO-1 and VE-Cad in endothelial cells after infiltration of the tumor cells through the barrier at 48 h in the tumor-interacting channel to that
Fig. 3. Extravasation of PC9 lung cancer cells through the BBB on the chip. (A) Time-lapse images of PC9 tumor cell (green) extravasation through the BBB (red) in the same ROI (region of interest). White arrow heads indicate the migration route of the same cell; scale bar, 50 lm. (B) Expression of the TJ proteins ZO-1 (green) and VE-cadherin (red) in the control BBB (left) and in the BBB after extravasation of tumor cells (right); Images were captured with a confocal microscope. Scale bar, 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)