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Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model

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Abstract

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17–70 × 10−6 cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3–5 × 10−6 cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10−6 cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.

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Acknowledgments

The authors are grateful to Profs. Kwang Sik Kim, Dennis Grab, Reto Brun, and Tanja Wenzler for provision of the hBMEC cell line. Thanks go to Orlando Fertig for technical assistance and to the Swiss National Science Foundation (SNSF) for financial support (grant 05320_126888/1 to MH).

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Authors and Affiliations

  1. Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland

    Daniela Elisabeth Eigenmann, Evelyn Andrea Jähne, Matthias Hamburger & Mouhssin Oufir

  2. Molecular Modeling, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland

    Martin Smieško

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  1. Daniela Elisabeth Eigenmann
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Correspondence to Mouhssin Oufir.

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ESM 1

Fig. S1. TEER values recorded real time by the CellZscope system for hBMEC monolayers cultured with media containing different concentrations of (a) retinoic acid (RA) and (b) puromycin (n = 1). Table S1. Optimized UHPLC parameters for compounds. Table S2. Calibrators and calibration curve parameters for analytes. Response: A x Conc.2 + B x Conc. + C, quadratic regression, weighting factor 1/X (exception caffeine and cimetidine: 1/X 2), origins: included. Table S3. Carryover assessment for both analytes and IS (n = 7–10). Table S4. Selectivity test at the LLOQ, based on 3 different RHB batches (n = 6). Table S5. Within-run imprecision (CV %) and inaccuracy (RE %) of QCs (n = 6). Table S6. Dilution test (n = 6). Table S7. Absolute extraction yield of analytes and IS (n = 6). Table S8. Short-term stabilities during storage at various conditions expressed as CV % and RE % (n = 6). Table S9. Long-term stabilities expressed as difference (%) between t = 0 and t = last and slopes (n = 3). Table S10. Stock solution stability of compounds. (PDF 143 kb)

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Eigenmann, D.E., Jähne, E.A., Smieško, M. et al. Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model. Anal Bioanal Chem 408, 2095–2107 (2016). https://doi.org/10.1007/s00216-016-9313-6

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