constructing the 3d human-stem-cell-derived in vitro blood brain barrier

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

The blood-brain barrier (BBB) is a key microvascular network separating the central nervous system (CNS) from the periphery to maintain an ideal environment for proper neuronal function. It has a critical role in regulating the influx and efflux of substances into the CNS by maintaining metabolic homeostasis1,2,3,4, clearing waste4,5,6, and protecting the brain from pathogens and toxins7,8.
The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane10,11,12,13,14 while expressing transporters to facilitate the movement of nutrients into and out of the CNS1,4,12,14. Microvascular ECs are encircled by pericytes (PCs)-mural cells that regulate microvascular function and homeostasis and are critical for regulating the permeability of the BBB to molecules and immune cells15,16,17. The astrocyte, a major glial cell type, is the final cell type comprising the BBB. Astrocyte end-feet wrap around the EC-PC vascular tubes while the cell bodies extend into the brain parenchyma, forming a connection between neurons and vasculature1. Distinct solute and substrate transporters are localized on astrocyte&#160;end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function18,19,20,21.
The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer&#39;s disease (AD)22,23,24,25, multiple sclerosis7,26,27,28, epilepsy29,30, and stroke31,32. It is increasingly recognized that cerebrovascular abnormalities play a central role in neurodegeneration, contributing to increased susceptibility to ischemic and hemorrhagic events. For example, more than 90% of AD patients have cerebral amyloid angiopathy (CAA), a condition characterized by the deposition of amyloid &#946; (A&#946;) along the cerebral vasculature. CAA increases BBB permeability and decreases BBB function, leaving the CNS vulnerable to ischemia, hemorrhagic events, and accelerated cognitive decline33.
We recently developed an in vitro model of the human BBB, derived from&#160;patient-induced pluripotent stem cells, which incorporates ECs, PCs, and astrocytes encapsulated in a 3D matrix (Figure 1A). The iBBB recapitulates physiologically relevant interactions, including vascular tube formation and localization of astrocyte end-feet with vasculature24. We applied the iBBB to model the susceptibility of CAA mediated by APOE4 (Figure 1B). This enabled us to identify the causal cellular and molecular mechanisms by which APOE4 promotes CAA, and leverage these insights to develop therapeutic strategies that reduce CAA pathology and improve learning and memory in vivo in APOE4 mice24. Here, we provide a detailed protocol and video tutorial for reconstructing the BBB from human iPSCs and modeling CAA in vitro.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer&#039;s Disease Using an In Vitro Blood-Brain Barrier Model 

体外血脑屏障重建，模拟和治疗神经系统疾病

Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier  

构建3D人干细胞来源的体外血脑屏障

使用解离的内皮细胞、寄生虫和星形胶质细胞组装人多能干细胞衍生的血脑屏障或 iBBB，将三种类型的细胞组合在一个锥形管中，同时包括比计算的细胞数量多 10% 以考虑移液错误。将细胞混合物以 300 g 旋转三分钟。吸出上清液，使细胞沉淀中含有约50μL的培养基。使用P200移液管，轻轻地将细胞沉淀重悬于残留培养基中，以产生单细胞浆液。将含有所得细胞浆液的试管放在冰上，并根据所需数量的 iBBB 向其添加足量的还原 GF 基底膜基质。均匀混合细胞，同时不引入任何气泡。将 50 μL 所得细胞基质混合物移液到 48 孔或 96 孔玻璃底培养皿的孔中，并将其均匀分布在整个培养皿底部。将培养皿在 37 摄氏度下孵育 30 至 40 分钟，以聚合基质，同时封装细胞。最后，向孔中加入 500 微升补充的星形胶质细胞培养基，并在培养物中维持 iBBB。对于 96 孔板，向孔中加入 100 至 150 μL 培养基，直到它们充分显影用于下游测定，这通常需要两周时间。正确形成的iBBB在明场显微镜下显示为凝固的单个半透明圆盘。24小时后，鉴定出均匀分布的单细胞。两周后，虽然难以定义，但可以看到更明显的结构。

constructing-3d-human-stem-cell-derived-vitro-blood-brain

Research

JoVE Journal

Neuroscience

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

119 次观看.  Icahn School of Medicine at Mount Sinai. 使用解离的内皮细胞、寄生虫和星形胶质细胞组装人多能干细胞衍生的血脑屏障或 iBBB，将三种类型的细胞组合在一个锥形管中，同时包括比计算的细胞数量多 10% 以考虑移液错误。将细胞混合物以 300 g 旋转三分钟。吸出上清液，使细胞沉淀中含有约50μL的培养基。使用P200移液管，轻轻地将细胞沉淀重悬于残留培养基中，以产生单细胞浆液。将含有所得细胞浆液的试管?...

视频: Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier  

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

科研

神经科学

Preparing Cells to Assemble a 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

Begin by preparing the differentiated endothelial cells, parasites, and astrocytes for assembling the iBBB model. After aspirating the cell culture medium from the differentiated endothelial cells, wash the cells with PBS once, then add a protease collagenase mixture to the washed cells and dissociate the cells at 37 degrees Celsius for five minutes. Transfer the protease collagenase mix with the dissociated cells into a conical tube containing hECSR, maintaining a 1:3 ratio of the mix to hECSR, then centrifuge the cells at 300 G for three minutes and aspirate the supernatant before resuspending the resultant cell pellet in the hECSR.Dissociate the differentiated parasites with the protease collagenase mixture at 37 degrees Celsius for five minutes. Add the appropriate amount of N2B27 in a conical tube and transfer the dissociated cells in the protease collagenase mix to it, maintaining a 1:3 ratio of the mix to N2B27. Spin down the cells at 300 G for three minutes.After aspirating the supernatant, resuspend the cell pellet in N2B27. Dissociate the differentiated astrocytes with the protease collagenase mixture at 37 degrees Celsius for five minutes. Transfer the dissociated cells in the protease collagenase mix to a conical tube containing complete astrocyte medium at a 1:3 ratio of the mix to the medium.After centrifuging the cells at 300 G for three minutes, aspirate the supernatant and resuspend the cell pellet in complete astrocyte medium. The cells are now ready for assembling the iBBB.

准备细胞组装 3D 人干细胞来源的体外血脑屏障

Assemble the human pluripotent stem cell derived blood-brain barrier, or iBBB, using the dissociated endothelial cells, parasites, and astrocytes, Combine the three types of cells in a conical tube while including 10%more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 g for three minutes. Aspirate the supernatant, leaving the cell pellet with approximately 50 microliters of the medium.Using a P200 pipette, gently resuspend the cell pellet in the residual medium to create a single cell slurry. Place the tube containing the resultant cell slurry on ice and add a sufficient amount of reduced GF Basement Membrane Matrix to it per the desired number of iBBBs. Mix the cells homogenously while not introducing any air bubbles.Pipette 50 microliters of the resultant cell matrix mixture into a well of a 48-well or 96-well glass-bottom culture dish and distribute it evenly throughout the bottom of the dish. Incubate the dish at 37 degrees Celsius for 30 to 40 minutes to polymerize the matrix while encapsulating the cells. Finally, add 500 microliters of supplemented astrocyte medium to the well and maintain the iBBBs in culture.For a 96-well plate, add 100 to 150 microliters of medium to the well until they are sufficiently developed for downstream assays, which typically takes two weeks. The properly formed iBBB appeared as a solidified, single translucent disk under a brightfield microscope. After 24 hours, evenly distributed single cells were identified.After two weeks, more distinct structures, although difficult to define, were visible.