Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro

Sodja, Caroline; Callaghan, Deborah; Haqqani, Arsalan S.; Stanimirovic, Danica; Costain, Willard J.; Jezierski, Anna

doi:10.1007/7651_2021_456

Cited by 1 publication

(1 citation statement)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples were cooled to room temperature, vortexed to mix and centrifuged in a Mandel mini microfuge. Biotinylated ladder, samples, primary and secondary antibodies, and luminol were loaded on the plate and Wes was run, as previously described [ 18 ]. Primary antibodies were mouse anti-transferrin receptor (Thermo Fisher, 13–6800, 1:10; Thermo Fisher Scientific, Waltham, Massachusetts), rabbit anti-LRP1 (Abcam, ab92544, 1:200; Waltham, Massachusetts), rabbit anti-TMEM30A (Abcam, Ab105062, 1:10; Waltham, Massachusetts), rabbit anti-insulin receptor (Cell Signalling, 3025 T, 1:100; Danvers, Massachusetts), and anti-actin-HRP (Sigma, A3854, 1:200; Sigma-Aldrich, St. Louis, Missouri).…”

Section: Methodsmentioning

confidence: 99%

Mouse embryonic stem cell-derived blood–brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis

Jezierski

Huang

Haqqani

et al. 2023

Fluids Barriers CNS

Self Cite

View full text Add to dashboard Cite

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10–5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10–3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10–5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.

show abstract

Section: Methodsmentioning

confidence: 99%