Department of Bioengineering, Northeastern University, Boston, MA, USA.
Department of Chemical Engineering, Northeastern University, 360 Huntington Avenue, 129 Interdisciplinary Science and Engineering Complex, Boston, MA, 02115, USA.
Lab Chip. 2022 Nov 22;22(23):4603-4620. doi: 10.1039/d2lc00657j.
Blood-brain barrier (BBB) endothelial cell (EC) function depends on flow conditions and on supportive cells, like pericytes and astrocytes, which have been shown to be both beneficial and detrimental for brain EC function. Most studies investigating BBB EC function lack physiological relevance, using sub-physiological shear stress magnitudes and/or omitting pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no-flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular EC (HBMEC) barrier integrity. Astrocytes, pericytes, and a 1-to-1 ratio of both cell types increased HBMEC barrier integrity reduced 3 and 40 kDa fluorescent dextran permeability and increased claudin-5 expression. There were differing levels of low 3 kDa permeability in HBMEC-pericyte, HBMEC-astrocyte, and HBMEC-astrocyte-pericyte co-cultures, while levels of low 40 kDa permeability were consistent across co-cultures. The 3 kDa findings suggest that pericytes provide more barrier support to the BBB model compared to astrocytes, although both supportive cell types are permeability reducers. Incorporation of 24-hour 12 dynes per cm flow significantly reduced dextran permeability in HBMEC monolayers, but not in the tri-culture model. These results indicate that tri-culture may exert more pronounced impact on overall BBB permeability than flow exposure. In both cases, monolayer and tri-culture, flow exposure interestingly reduced HBMEC expression of both claudin-5 and occludin. ZO-1 expression, and localization at cell-cell junctions increased in the tri-culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed HBMEC alignment in the tri-culture but not in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain EC alignment . Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating BBB EC function. Consideration of the roles of shear stress and supportive cells within the BBB is critical for elucidating the physiology of the neurovascular unit.
血脑屏障（BBB）内皮细胞（EC）的功能取决于流动条件和支持细胞，如周细胞和星形胶质细胞，它们对脑 EC 功能既有有益的影响，也有有害的影响。大多数研究 BBB EC 功能的研究都缺乏生理相关性，使用的是亚生理剪切应力大小，并且/或者没有周细胞和星形胶质细胞。在这项研究中，我们开发了一种与标准 Transwell 插入物兼容的毫流控装置来研究 BBB 功能。与标准的聚二甲基硅氧烷（PDMS）微流控装置相比，这种模型允许在无需 PDMS 装置常见局限性（如营养扩散不足和气泡形成）的情况下，轻松、可重复地暴露于剪切应力下。在无流动条件下，我们首先使用该装置研究了原代人周细胞和星形胶质细胞对人脑微血管内皮细胞（HBMEC）屏障完整性的影响。星形胶质细胞、周细胞和这两种细胞类型的 1:1 比例均增加了 HBMEC 屏障完整性，降低了 3 kDa 和 40 kDa 荧光葡聚糖的通透性，并增加了紧密连接蛋白-5 的表达。在 HBMEC-周细胞、HBMEC-星形胶质细胞和 HBMEC-星形胶质细胞-周细胞共培养物中，3 kDa 的低通透性水平存在差异，而在共培养物中，40 kDa 的低通透性水平是一致的。3 kDa 的发现表明，与星形胶质细胞相比，周细胞为 BBB 模型提供了更多的屏障支持，尽管这两种支持细胞类型都是通透性降低剂。在 HBMEC 单层中，24 小时 12 达因/厘米的流量显著降低了葡聚糖的通透性，但在三细胞培养模型中没有降低。这些结果表明，三细胞培养物对整体 BBB 通透性的影响可能比流动暴露更为显著。在这两种情况下，即单层和三细胞培养物，流动暴露都有趣地降低了 HBMEC 紧密连接蛋白-5 和闭合蛋白的表达。紧密连接蛋白-1 的表达和在细胞-细胞连接处的定位在三细胞培养物中增加，但在 HBMEC 单层中没有明显变化。在流动条件下，我们还观察到三细胞培养物中 HBMEC 的排列，但在 HBMEC 单层中没有观察到，表明支持细胞和流动对观察脑 EC 排列都是必不可少的。综上所述，这些结果支持在研究 BBB EC 功能时使用生理相关的多细胞 BBB 模型的必要性。考虑剪切应力和支持细胞在 BBB 中的作用对于阐明神经血管单元的生理学至关重要。
