Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Materials and Reagents
2.3. Primary Cell Cultures
2.4. Construction of the BBB Model
2.5. Immunohistochemistry
2.6. Reverse Transcription Polymerase CHAIN Reaction
2.7. Evaluation of the Barrier Integrity of the BBB Models
2.8. Functional Assay for Efflux Transport
2.9. Drug Permeability Experiments
2.10. Western Blotting
2.11. Statistical Analysis
3. Results
3.1. Characterization of Monkey Brain Endothelial Cells
3.2. Expression of Transporters in Monkey Brain Endothelial Cells
3.3. Effect of Astrocytes on Barrier Function of Monkey Brain Endothelial Cells
3.4. Barrier Integrity of Different BBB Models
3.5. Expression of Transporters on Different BBB Models
3.6. Correlation between Permeability of Drugs Tested in Rat and Monkey BBB Co-Culture Models, and Mouse In Vivo Model
3.7. Effect of TGF-β1 on Barrier Integrity in the Monkey Co-Culture BBB Model
4. Discussion
4.1. Characterization of the Primary Triple-Co-Culture Primate BBB Model
4.2. Application of the Primary Triple-Co-Culture Primate BBB Model
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Primer Sequence | Size (bp) |
---|---|---|
ABCB1 (P-gp) | F: GGCCTAAAGCCGAACACATTG R: CTGAAGCACTGGGATGTCCA | 90 |
ABCG2 (BCRP) | F: GCCACGGAGATCATAGAGCC R: TCACCCCCGGAAAGTTGATG | 125 |
ABCC1 (MRP-1) | F: CAAGGGATTGCCGTGTTTGG R: AAGAAGCTCATGGGTGACCG | 116 |
ABCC2 (MRP-2) | F: GCACAAGCAACTGCTCAACA R: CCGTGGAAATATCACCGGCA | 104 |
ABCC4 (MRP-4) | F: TCGCAATACCCTTGGTTCCC R: CACTGGGCTCCGAGTTGTAG | 110 |
ABCC5 (MRP-5) | F: CTTTGTCAAGGGCACACTGC R: CCTGTGGGGGTTGTGTCAAA | 102 |
SLC2A1 (GLUT1) | F: GAACTCTTCAGCCAGGGTCC R: GGACCACATAGTTGCTCCAC | 116 |
SLC16A1 (MCT1) | F: ACAAGTAAACGAGGCAGCGA R: ACAAATATCGTTATAAGCGCGGA | 123 |
SLC7A5 (LAT1) | F: CGTGAACTGCTACAGCGTGA R: TTGGACACATCACCCTTCCC | 126 |
MFSD2a | F: GCCCAGGTGAAGAAAGAACC R: CACAGCCTGTCACCTGGTAG | 103 |
GAPDH | F: CTCAAGATCGTCAGCAACGC R: TCTTCTGGGTGGCAGTGATG | 130 |
Compound | Primary Transport Mechanism | Monkey BBB | Rat BBB * | Mouse In Vivo * | ||||
---|---|---|---|---|---|---|---|---|
Papp A-B (×10−6 cm/s) | Papp B-A (×10−6 cm/s) | EffluxRatio | Papp A-B (×10−6 cm/s) | Papp B-A (×10−6 cm/s) | EffluxRatio | Papp (×10−6 cm/s) | ||
Caffeine | PT/CMT | 60.53 | 65.25 | 1.08 | 64.95 | 59.91 | 0.92 | 11.11 |
Antipyrin | PT | 48.06 | 47.07 | 0.98 | 51.78 | 45.38 | 0.88 | 10.75 |
Indomethacin | PT | 32.70 | 39.11 | 1.20 | 33.63 | 44.07 | 1.31 | 0.13 |
Verapamil | PT/E | 20.10 | 29.86 | 1.49 | 23.38 | 31.82 | 1.38 | 5.69 |
Loperamide | PT/E | 9.16 | 16.26 | 1.78 | 15.57 | 21.76 | 1.47 | 1.05 |
Quinidine | PT/E | 6.01 | 10.33 | 1.74 | 6.28 | 12.11 | 1.93 | 1.69 |
Cimetidine | E | 3.00 | 2.88 | 0.97 | 3.86 | 2.80 | 0.75 | 0.20 |
Digoxin | E | 2.25 | 3.34 | 1.49 | 1.25 | 3.07 | 2.47 | 0.10 |
Vinblastine | E | 1.47 | 2.14 | 1.46 | 2.42 | 1.39 | 0.69 | 1.43 |
Atenolol | PP/E | 4.60 | 3.43 | 0.83 | 1.36 | 2.26 | 1.71 | 0.23 |
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Watanabe, D.; Nakagawa, S.; Morofuji, Y.; Tóth, A.E.; Vastag, M.; Aruga, J.; Niwa, M.; Deli, M.A. Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes. Pharmaceutics 2021, 13, 1484. https://doi.org/10.3390/pharmaceutics13091484
Watanabe D, Nakagawa S, Morofuji Y, Tóth AE, Vastag M, Aruga J, Niwa M, Deli MA. Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes. Pharmaceutics. 2021; 13(9):1484. https://doi.org/10.3390/pharmaceutics13091484
Chicago/Turabian StyleWatanabe, Daisuke, Shinsuke Nakagawa, Yoichi Morofuji, Andrea E. Tóth, Monika Vastag, Jun Aruga, Masami Niwa, and Mária A. Deli. 2021. "Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes" Pharmaceutics 13, no. 9: 1484. https://doi.org/10.3390/pharmaceutics13091484
APA StyleWatanabe, D., Nakagawa, S., Morofuji, Y., Tóth, A. E., Vastag, M., Aruga, J., Niwa, M., & Deli, M. A. (2021). Characterization of a Primate Blood-Brain Barrier Co-Culture Model Prepared from Primary Brain Endothelial Cells, Pericytes and Astrocytes. Pharmaceutics, 13(9), 1484. https://doi.org/10.3390/pharmaceutics13091484