Professor Johns Hopkins University BALTIMORE, Maryland, United States
Introduction:: The blood-brain barrier (BBB) is a specialized vascular structure composed of brain vascular endothelial cells, mural cells, basement membrane and astrocytes. BBB plays an important role in regulating brain homeostasis and dysfunction of BBB is associated with many brain diseases. The development of in vitro BBB models enables real-time, high-magnification imaging of biological and pathological processes relevant to human brain health. However, the lack of a perusable BBB model incorporating all the cell components is a major roadblock. Therefore, the goal of this project is to develop a perfusable isogenic BBB model incorporating endothelial cells, pericytes, and astrocytes
Materials and Methods:: Induced brain microvascular endothelial cells (iBMECs), induced brain pericytes (iPCs), and induced brain astrocytes (iACs) were derived from human induced pluripotent stem cells (iPSCs, WTC-11). Microfluidic chips with a hydrogel chamber of 1 × 2 × 15 mm (H × W × L) were fabricated using soft lithography. Rat tail type I collagen was loaded to the microfluidic device, followed by adding concentric hydrogel mixed with 2 million mL-1 iACs (Fig.1a). After the device was incubated on ice for 2 hours, the needle template was removed, and the device was cultured in astrocyte medium for two days. iPCs and iBMECs were sequentially seeded to the center channel and perfused with iBMEC medium for two days to allow the formation of astrocyte endfeet around iPCs and iBMECs. Fluorescence images of the multicellular BBB model were acquired on a fluorescence microscope (Nikon, Eclipse Ti-E) with a 10 × objective (Nikon).
Results, Conclusions, and Discussions:: We have successfully differentiated iBMECs, iPCs and iACs from WTC-iPSCs. The iBMECs were differentiated from GFP tagged ZO1 iPSCs, which allows the direct observation of tight junction. iPCs were differentiated from GFP tagged Actin iPSCs and iACs were differentiated from RFP-tagged iPSCs. Using the concentric microfluidic chips, we were able to colocalize the three brain vascular cells around a 150 µm channel. After iACs were seeded in the concentric region for two days, we observed the spreading of iACs in the concentric hydrogel. Following iPC and iBMEC seeding, a confluent endothelial layer was formed after two days. We can clearly see that the endothelial tube was surrounded by a layer of astrocytes (Fig.1b). Multiple astrocyte branches touching the endothelial and pericyte layers resemble the typical astrocyte endfoot structures in native brain (Fig.1c). Under confocal microscope, iBMECs showed strong ZO1 expression around the endothelial cell-cell junction, indicating the formation of tight junction. Most iPCs stayed adjacent to the iBMEC layer. 3D reconstructed view confirmed the astrocyte endfeet in direct contact with the endothelial cells or pericytes (Fig.1d).
