A Hybrid Nanofiber/Paper Cell Culture Platform for Building a 3D Blood-brain Barrier Model. A Hybrid Nanofiber/Paper Cell Culture Platform for Building a 3D Blood-brain Barrier Model

The blood brain barrier (BBB) protects the central nervous system from toxins and pathogens in the blood by regulating permeation of molecules through the barrier interface. In vitro BBB models described to date reproduce some aspects of BBB functionality, but also suffer from incomplete phenotypic expression of brain endothelial traits, difficulty in reproducibility and fabrication, or overall cost. To address these limitations, we describe a three-dimensional (3D) BBB model based on a hybrid paper/nanofiber scaffold. The cell culture platform utilizes lens paper as a framework to accommodate 3D culture of astrocytes. An electrospun nanofiber layer is coated onto one face of the paper to mimic the basement membrane and support growth of an organized two-dimensional layer of endothelial cells (ECs). Human induced pluripotent stem cell-derived ECs and astrocytes are co-cultured to develop a human BBB model. Morphological and spatial organization of model are validated with confocal microscopy. Measurements of transendothelial resistance and permeability demonstrate the BBB model develops a high-quality barrier and responds to hyperosmolar treatments. RNA-sequencing shows introduction of astrocytes both regulates EC tight junction proteins and improves endothelial phenotypes related to vasculogenesis. This model shows promise as a model platform for future in vitro studies of the BBB. Overall design: The samples for RNA sequencing include stem cell-derived endothelial cells in triplicates, stem cell-derived astrocytes in triplicates, and the assembled BBB models in triplicates. 9 samples in total were analyzed.

血脑屏障（blood brain barrier, BBB）通过调控分子跨越屏障界面的渗透过程，保护中枢神经系统免受血液中的毒素与病原体侵害。目前已报道的体外血脑屏障模型虽可复现BBB功能的部分特征，但普遍存在脑内皮细胞表型表达不完全、可重复性与制备难度较高，或整体成本过高等局限。为解决上述局限，本研究报道了一种基于纸基/纳米纤维混合支架的三维（3D）血脑屏障模型。该细胞培养平台以镜头纸作为支架框架，用于支撑星形胶质细胞的三维培养；研究人员将电纺纳米纤维层涂布于纸张的单面，以模拟基底膜并支撑内皮细胞（endothelial cells, ECs）形成有序的二维单层培养结构。本研究将人诱导多能干细胞（induced pluripotent stem cell）衍生的内皮细胞与星形胶质细胞共培养，构建人源血脑屏障模型。通过共聚焦显微镜（confocal microscopy）验证了该模型的形态学特征与空间组织结构。跨内皮电阻（transendothelial resistance）与通透性检测结果表明，该血脑屏障模型可形成高质量的屏障结构，并对高渗处理产生特异性响应。RNA测序（RNA-sequencing）分析显示，星形胶质细胞的引入不仅调控内皮细胞紧密连接蛋白的表达，还改善了与血管生成相关的内皮细胞表型。该模型有望成为未来血脑屏障体外研究的优质平台。整体实验设计：本次RNA测序的样本包括三组重复的干细胞衍生内皮细胞、三组重复的干细胞衍生星形胶质细胞，以及三组重复的组装完成的血脑屏障模型，总计9个样本用于分析。