Three-dimensional co-culture of blood-brain barrier-composing cells in a culture insert with a collagen vitrigel membran
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Three-dimensional co-culture of blood-brain barrier-composing cells in a culture insert with a collagen vitrigel membrane Ai Shima 1 & Shogo Nagata 2 & Shoji Takeuchi 1,2,3 Received: 9 June 2020 / Accepted: 26 July 2020 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2020
Abstract The blood-brain barrier (BBB) is a structure located in brain capillaries that protects the brain from toxic substances in blood due to its high barrier function. The brain capillaries form a layered structure with pericytes, neurons, glial cells, and extracellular matrix proteins that is called neurovascular unit, and the structure is important to express the high barrier function of BBB. Here, we propose a method to construct a three-dimensional BBB tissue using three human BBB-composing cells, including brain endothelial cells, pericytes, and astrocytes, that mimics the in vivo BBB-like layered structure. Primary human brain endothelial cells were plated on the back side (outside) of the collagen vitrigel membrane of a culture insert, pericytes were plated on the upper side (inside), and astrocytes mixed in Matrigel were plated on the pericyte layer. The layered structure was maintained for at least 2 wk. The BBB tissue-loaded collagen vitrigel membrane can be detached from the insert frame using acetone with the tissue fixed intact and used for vertical cryosectioning to analyze the tissue interior. We also measured transendothelial electrical resistance (TEER) in the three-dimensional BBB co-culture to investigate barrier function of the brain endothelial cells. We believe that our co-culture method is useful to study engineered BBB tissues and develop reliable in vitro human BBB models in the future. Keywords Neurovascular unit . Transendothelial electrical resistance (TEER) . Brain endothelial cell . Organ-on-a-chip . Tissue engineering
The blood-brain barrier (BBB) is a structure located in brain capillaries that prevents toxic substances in the blood from invading into the brain. The basic elements of the BBB are brain endothelial cells (BECs), pericytes, and astrocytes, and together, they form a layered structure called neurovascular unit (Abbott et al. (2006)). Since both pericytes and astrocytes have been reported to contribute to the high barrier function of the BBB (Brown et al. (2015), Wang et al. (2016), Wang et al.
* Shoji Takeuchi [email protected] 1
Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, Tokyo, Japan
2
Institute of Industrial Science, The University of Tokyo, Tokyo, Japan
3
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo Institutes for Advanced Study (UTIAS), The University of Tokyo, Tokyo, Japan
(2017)), which is achieved through tight junctions between intercellular spaces of BECs, the triplet cell co-culture is highly expected to mimic BBB function in vitro. The extremely high barrier function of the BBB, which protects human brain tissue from external toxins,
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