Abstract
Cell culture-based blood–brain barrier (BBB) models are useful experimental tools for developing central nervous system drugs. Several endothelial cell sources exist for BBB models, including primary cultured brain endothelial cells and immortalized cell lines. Among them, primary cell-based models are considered suitable for the functional analysis of the BBB; however, little is known about the utility of low-passage brain endothelial cells for this purpose. In this study, we investigated the effect of passage on brain endothelial cells from human, mouse and rat brain tissue as BBB models. We established in vitro BBB models using primary brain endothelial cells (Passage 1–Passage 4) from humans, mice, and rats. To analyze the effect of cell type on BBB function, we evaluated transendothelial electrical resistance (TEER) and performed immunofluorescence staining of tight junction proteins. Among the brain endothelial cell models, TEER was highest in the Passage 1 (P1) cell-based BBB model. There was no adequate increase in TEER in other low-passage cultures (P2–P4). A confluent, non-overlapping, uniform monolayer of cells in all P1 cell-based models was visible on immunostaining of tight junction proteins, whereas it was weak or undetectable in more passaged cultures. Increasing passages cultured of brain endothelial cells did not exhibit restrictive BBB function regardless of the cell source and despite culturing with pericytes and astrocytes. Among the tested culture models, only the lowest cultured cell-based models are suitable for functional analysis of the BBB.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Appelt-Menzel A et al (2017) Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells. Stem Cell Rep 8:894–906. https://doi.org/10.1016/j.stemcr.2017.02.021
Benson K, Cramer S, Galla HJ (2013) Impedance-based cell monitoring: barrier properties and beyond. Fluids Barriers CNS 10(1):5. https://doi.org/10.1186/2045-8118-10-5
Bernas MJ et al (2010) Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier. Nat Protoc 5:1265–1272. https://doi.org/10.1038/nprot.2010.76
Bouis D, Hospers GA, Meijer C, Molema G, Mulder NH (2001) Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis 4:91–102
Canfield SG, Stebbins MJ, Faubion MG, Gastfriend BD, Palecek SP, Shusta EV (2019) An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons. Fluids Barriers CNS 16:25. https://doi.org/10.1186/s12987-019-0145-6
Coisne C et al (2005) Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium. Lab Invest 85:734–746. https://doi.org/10.1038/labinvest.3700281
Deli MA, Abraham CS, Kataoka Y, Niwa M (2005) Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol 25:59–127. https://doi.org/10.1007/s10571-004-1377-8
Delsing L et al (2018) Barrier properties and transcriptome expression in human iPSC-derived models of the blood-brain barrier. Stem Cells 36:1816–1827. https://doi.org/10.1002/stem.2908
Franke H, Galla H, Beuckmann CT (2000) Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood-brain barrier in vitro. Brain Res Brain Res Protoc 5:248–256. https://doi.org/10.1016/s1385-299x(00)00020-9
Gaillard PJ, de Boer AG (2000) Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug. Eur J Pharm Sci 12:95–102. https://doi.org/10.1016/s0928-0987(00)00152-4
Gumbleton M, Audus KL (2001) Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier. J Pharm Sci 90:1681–1698. https://doi.org/10.1002/jps.1119
Helms HC et al (2016) vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use. J Cereb Blood Flow Metab 36:862–890. https://doi.org/10.1177/0271678X16630991
Hoheisel D, Nitz T, Franke H, Wegener J, Hakvoort A, Tilling T, Galla HJ (1998) Hydrocortisone reinforces the blood-brain properties in a serum free cell culture system. Biochem Biophys Res Commun 247:312–315
Hupe M et al (2017) Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels. Sci Signal. https://doi.org/10.1126/scisignal.aag2476
Lippmann ES et al (2012) Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30:783–791. https://doi.org/10.1038/nbt.2247
Nakagawa S et al (2009) A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int 54:253–263. https://doi.org/10.1016/j.neuint.2008.12.002
Neal EH et al (2019) A simplified, fully defined differentiation scheme for producing blood-brain barrier endothelial cells from human. IPSCS Stem Cell Rep 12:1380–1388. https://doi.org/10.1016/j.stemcr.2019.05.008
Omidi Y, Campbell L, Barar J, Connell D, Akhtar S, Gumbleton M (2003) Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. Brain Res 990:95–112. https://doi.org/10.1016/s0006-8993(03)03443-7
Perriere N et al (2005) Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties. J Neurochem 93:279–289. https://doi.org/10.1111/j.1471-4159.2004.03020.x
Rahman NA, Rasil A, Meyding-Lamade U, Craemer EM, Diah S, Tuah AA, Muharram SH (2016) Immortalized endothelial cell lines for in vitro blood-brain barrier models: a systematic review. Brain Res 1642:532–545. https://doi.org/10.1016/j.brainres.2016.04.024
Sabbagh MF, Nathans J (2020) A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture. Elife. https://doi.org/10.7554/eLife.51276
Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ (2015) TEER measurement techniques for in vitro barrier model systems. J Lab Autom 20:107–126. https://doi.org/10.1177/2211068214561025
Strazza M, Maubert ME, Pirrone V, Wigdahl B, Nonnemacher MR (2016) Co-culture model consisting of human brain microvascular endothelial and peripheral blood mononuclear cells. J Neurosci Methods 269:39–45. https://doi.org/10.1016/j.jneumeth.2016.05.016
Veszelka S et al (2018) Comparison of a rat primary cell-based blood-brain barrier model with epithelial and brain endothelial cell lines: gene expression and drug transport. Front Mol Neurosci 22(11):166. https://doi.org/10.3389/fnmol.2018.00166
Weksler BB et al (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19:1872–1874. https://doi.org/10.1096/fj.04-3458fje
Funding
This study was partially funded by JSPS and HAS under the Japan-Hungary Research Cooperative Program (to Y.M.), Grants-in-Aid for Scientific Research (Fostering Joint International Research) 15KK0349 (to Y.M.), Grants-in-Aid for Scientific Research (C) 17K10840 (to Y.M.), (C) 17K10838 (to S.N.), (C) 18K08973 to (T.I. and Y.M.).
Author information
Authors and Affiliations
Contributions
TF and YM designed the study and wrote the initial draft of the manuscript. SN and AK contributed to data analysis and interpretation. MN and WB were involved in planning and supervision of the work. All other authors contributed to data collection and interpretation, and they critically reviewed the manuscript. All authors have approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Corresponding author
Ethics declarations
Conflict of interest
All authors have no conflict of interest.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Fujimoto, T., Morofuji, Y., Nakagawa, S. et al. Comparison of the rate of dedifferentiation with increasing passages among cell sources for an in vitro model of the blood–brain barrier . J Neural Transm 127, 1117–1124 (2020). https://doi.org/10.1007/s00702-020-02202-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00702-020-02202-1