A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0±0.9 x 10−6 cm/s to 2.9±1.0 x 10−6 cm/s or 1.1±0.4 x 10−6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

新生儿神经病理学研究以及适配性治疗方案的开发，长期受限于缺乏相关的新生儿体外血脑屏障（blood-brain barrier, BBB）模型。为构建此类模型，本团队研发了一款新型芯片级血脑屏障模型（blood-brain barrier on a chip, B3C），该模型将组织腔室与血管通道并排设置，可精准模拟体内微血管的三维形态、尺寸与流动特性。研究人员从新生大鼠体内分离得到大鼠脑微血管内皮细胞（rat brain endothelial cells, RBEC），将其接种于B3C的血管通道中，并在剪切流环境下进行培养；同时将新生大鼠星形胶质细胞静态培养于B3C的组织腔室内。RBEC可在B3C血管通道的全长形成带有中央管腔的连续内皮单层，并可形成紧密连接，这一特征可通过紧密连接蛋白-1（zonula occludens-1, ZO-1）的表达水平得以验证。血管通道内的剪切流环境，以及组织腔室内添加的星形胶质细胞条件培养基（astrocyte conditioned medium, ACM）或共培养的星形胶质细胞，均可显著上调ZO-1的表达水平。与体内血脑屏障的生理特征一致，B3C通过多孔界面实现了类似终足结构的星形胶质细胞-内皮细胞相互作用，该界面可将培养星形胶质细胞的组织腔室与血管通道内的RBEC分隔开。当RBEC在星形胶质细胞或ACM共培养条件下培养时，荧光标记的40 kDa右旋糖酐从血管通道向组织腔室的通透性显著降低，分别从41.0±0.9 × 10−6 cm/s降至2.9±1.0 × 10−6 cm/s与1.1±0.4 × 10−6 cm/s。对B3C内电阻的检测进一步证实，ACM的添加可显著改善新生大鼠RBEC构成的屏障功能。此外，相较于Transwell模型，B3C的屏障特性得到显著优化；且B3C的通透性与新生大鼠体内血脑屏障的通透性无显著差异。综上，本团队开发了首款动态体外新生大鼠血脑屏障芯片模型（B3C），该模型可精准模拟体内微环境，支持实时灵活分析，适用于血脑屏障功能研究以及新型治疗手段的筛选。