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In Vitro Assessment of Acute Neuro‐inflammation in a Model of the Blood Brain Barrier
Author(s) -
Phillips Cassandra,
Ortega-Alvarez Ramon,
Ojuka Dak,
Victor Anne-Deyssa,
Fama Kathryn,
Toth Charles
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05894
Subject(s) - blood–brain barrier , barrier function , in vitro , microbiology and biotechnology , tight junction , induced pluripotent stem cell , inflammation , chemistry , fluorescein , biology , immunology , neuroscience , biochemistry , central nervous system , embryonic stem cell , physics , quantum mechanics , gene , fluorescence
The purpose of this experiment was to develop an in vitro model of the blood brain barrier that incorporated brain endothelial microvascular cells (BMECs) and neuronal co‐cultures composed of neurons and astrocytes. All cell types were derived from human induced pluripotent stem cells (iPSCs) in accordance to the differentiation protocols utilized. The neurons and astrocytes were differentiated separately, and then seeded together when both cultures reached maturity. A 12‐well transwell plate format was used for the barrier model; the BMECs were seeded in the transwell inserts and the co‐cultures were seeded in the bottom wells. The inserts were coated with collagen and fibronectin, and a poly‐L‐ornithine and laminin ECM was used for the bottom wells. A fluorescein assay was used as a preliminary investigation of barrier function with only the BMECs in the transwell inserts. Barrier function will also be evaluated with immunofluorescence by staining for the ZO‐1 tight junction marker, which is associated with endothelial monolayers. In addition to assessing barrier function, the model will be used to investigate acute neuro‐inflammatory responses. Inflammation will be induced with small molecules such as poly (I:C) and LPS, and then barrier integrity will be evaluated using a fluorescein assay. Tryptophan metabolites will be utilized to analyze the role of the microbiome in neuro‐inflammation. An in vitro model of the blood brain barrier will be a valuable tool to observe and assess neuro‐inflammatory responses because a human stem cell model is more physiological relevant. Support or Funding Information Gift from Dr. John Mullen