preparing cells to assemble a 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 

Nörolojik Hastalığı Modellemek ve Terapötik Olarak Hedeflemek için İn Vitro Kan-Beyin Bariyerinin Yeniden Yapılandırılması

Watch this Scientific Journal Video about Preparing Cells to Assemble a 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

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

3D İnsan Kök Hücre Kaynaklı İn Vitro Kan Beyin Bariyerini Birleştirmek İçin Hücrelerin Hazırlanması

iBBB modelini monte etmek için farklılaşmış endotel hücrelerini, parazitleri ve astrositleri hazırlayarak başlayın. Hücre kültürü ortamını farklılaşmış endotel hücrelerinden aspire ettikten sonra, hücreleri bir kez PBS ile yıkayın, ardından yıkanan hücrelere bir proteaz kollajenaz karışımı ekleyin ve hücreleri beş dakika boyunca 37 santigrat derecede ayırın. Proteaz kollajenaz karışımını, ayrışmış hücrelerle birlikte, karışımın hECSR'ye 1:3 oranını koruyarak hECSR içeren konik bir tüpe aktarın, ardından hücreleri 300 G'de üç dakika santrifüjleyin ve elde edilen hücre peletini hECSR'de yeniden süspanse etmeden önce süpernatanı aspire edin.Farklılaşmış parazitleri proteaz kollajenaz karışımı ile 37 santigrat derecede beş dakika boyunca ayırın. Uygun miktarda N2B27'yi konik bir tüpe ekleyin ve proteaz kollajenaz karışımındaki ayrışmış hücreleri, karışımın N2B27'ye 1: 3 oranını koruyarak ona aktarın. Hücreleri üç dakika boyunca 300 G'de döndürün.Süpernatanı aspire ettikten sonra, hücre peletini N2B27'de yeniden süspanse edin. Farklılaşmış astrositleri proteaz kollajenaz karışımı ile 37 santigrat derecede beş dakika boyunca ayırın. Proteaz kollajenaz karışımındaki ayrışmış hücreleri, karışımın ortama 1:3 oranında tam astrosit ortamı içeren konik bir tüpe aktarın.Hücreleri üç dakika boyunca 300 G'de santrifüjledikten sonra, süpernatanı aspire edin ve hücre peletini tam astrosit ortamında yeniden süspanse edin. Hücreler artık iBBB'yi monte etmeye hazırdır.

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Research

JoVE Journal

Neuroscience

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

116 Görüntüleme.  Icahn School of Medicine at Mount Sinai. iBBB modelini monte etmek için farklılaşmış endotel hücrelerini, parazitleri ve astrositleri hazırlayarak başlayın. Hücre kültürü ortamını farklılaşmış endotel hücrelerinden aspire ettikten sonra, hücreleri bir kez PBS ile yıkayın, ardından yıkanan hücrelere bir proteaz kollajenaz karışımı ekleyin ve hücreleri beş dakika boyunca 37 santigrat derecede ayırın. Proteaz kollajenaz karışımını, ayrışmış hücrelerle birlikte, karışımın hECSR'ye 1:3 oran...

Video: Preparing Cells to Assemble a 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 ...

Nörobilim

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.

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

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.