BBBoid: A spheroid-based model for investigating blood-brain barrier permeability

Abstract

The blood-brain barrier (BBB) is a specialized structure that controls the movement of substances between the bloodstream and the brain. Investigating BBB permeability is crucial for developing effective treatments for various central nervous system (CNS) disorders. In vitro BBB models offer several advantages over animal models, including high-throughput analysis, accessibility, and ethical considerations. Spheroid-based models have been proposed as an in vitro model for the BBB, but they require a specialized culture medium containing growth factors, making them cost-prohibitive. In this study, a novel method for constructing spheroid-based BBB models (BBBoids) is presented that does not require a specialized medium or growth factors, increasing affordability and cost-effectiveness. BBBoids comprise primary murine astrocytes which form the spheroid core, and the bEnd.3 endothelial cell line, which encases the spheroid surface, acting as a barrier. BBBoids form within 6 days and exhibit barrier properties and proper morphology for up to 2 weeks. This model is suitable for investigating the effects of various substances on BBB integrity and testing the permeability of different therapeutic agents. The BBBoid platform is a reliable, adaptable, and economical in vitro instrument that may expedite the study of potential therapeutics for a range of neuropathologies.

Keywords

BBB Blood-brain-barrier in vitro model spheroid

Introduction

The majority of microvessels in the brain constitute the blood-brain barrier (BBB), which is a specialized structure that plays a critical role in controlling the movement of substances and cells between the bloodstream and the brain.¹ The anatomical basis of the BBB is the neurovascular unit (NVU), which comprises endothelial cells, pericytes, and astrocytes, surrounded by basement membranes.

Study of the BBB permeability has great medical significance as it restricts the transportation of therapeutic agents to the brain.^2,3 This restriction poses a significant challenge in delivering drugs to the central nervous system (CNS), which impedes the effective treatment of various CNS disorders. Presently, considerable efforts are being directed toward developing techniques for bypassing the BBB to enable the delivery of therapeutic agents.⁴

An important method for investigating the permeability of the BBB to various compounds is through in vitro modeling.⁵ In recent years, numerous in vitro BBB models have been proposed. These models vary in spatial configuration, static or shear flow conditions, scaffold materials used, as well as sources and types of cells.⁶ In vitro BBB models have several advantages over animal models. They enable high-throughput analysis, are relatively inexpensive and easy to use, and their construction does not require significant time investment, making them accessible to a wide range of researchers.⁶ Furthermore, the use of in vitro models contributes to the reduction of experimental animals, which is an important ethical consideration.

One variant of an in vitro BBB model based on spheroids. The BBB spheroid platform is a reliable, adaptable, and economical in vitro instrument. It can be readily expanded to a high-throughput configuration, thereby providing a means for modeling the BBB that may expedite the identification of potential therapeutics for a range of neuropathologies. Several methodologies exist for the development of spheroid-based BBB models, allowing for customization to specific research needs.^7–12 An essential feature of the BBB organoid model is the capacity for cellular interactions among various cell types within the spheroid, which is imperative for maintaining the integrity and functionality of the BBB.¹³ Nonetheless, these protocols require a specialized culture medium containing a combination of growth factors, rendering them cost-prohibitive and inaccessible to a broad range of researchers.

In this study, we present a method for construction of spheroid-based BBB models (BBBoids) that does not require a specialized medium or growth factors, thus increasing affordability and cost-effectiveness (Figure 1). Our model is suitable for investigating the effects of various substances on BBB integrity and potentially can be used to test the permeability of different therapeutic agents.

Figure 1.

Scheme of construction of spheroid-based BBB model (BBBoid) and testing the influence of substances on their permeability.

Materials and methods

Cultural medium, antibodies, peptides, and fluorescent dyes

Full DMEM: DMEM (Gibco, Cat#31966-021) with 100 IU/mL Penicillin/Streptomycin (PanEco, Cat#А065п), 2 mM GlutaMAX (Thermo Fisher Scientific, Cat#35050061), and 10% fetal bovine serum (Gibco, Cat#16000044).

Primary antibodies: Anti-ZO-1 rabbit polyclonal antibody (Affinity Biosciences, Cat#AF5145) and Anti-GFAP mouse monoclonal antibody (HyTest LTD, Cat#4G25).

Secondary antibodies: Alexa Fluor 568 rabbit anti-mouse antibody (Thermo Fisher Scientific, Cat#A-11061) and Alexa Fluor 488 goat anti-rabbit antibody (Cat#A-11034).

Fluorescent dyes: Lucifer Yellow (Thermo Fisher Scientific, Cat#L453), DiO (Thermo Fisher Scientific, Cat#V22886), and DiI (Thermo Fisher Scientific, Cat#D-3911).

Recombinant protein: interleukin 17A (IL17A) (Cloud-Clone Corp., Cat#RPA063Hu01).

Isolation of primary murine astrocytes

All manipulations with animals were conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123) and approved by the Institutional Animal Care and Use Committee of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (approval no. 331).

Murine astrocytes were obtained as described previously.¹⁴ Briefly, 3 mouse pups (age 2 days) were subjected to inhalation with CO2 and were decapitated using scissors in one cut. The brain was removed from the skull and placed in a Petri dish containing ice-cold PBS. The meninges and cerebellum were removed, and the cortex was cut into pieces about 2-3 mm in size. The cortex pieces were incubated with Trypsin-EDTA and DNase I at 37°C for 30–45 minutes. Next, 4 mL of full DMEM was added to stop the digestion. The tissue was triturated with a pipette to obtain a single-cell suspension, which was then filtered through a 70 μm cell strainer into a new 50 mL conical tube. The tube was centrifuged at 300 g for 5 minutes. The pellet was suspended in full DMEM and plated in a T-75 cell culture flask at a rate of 3 brains per one flask. The next day, all medium was removed, and the cells were washed with PBS to remove debris. Astrocytes were maintained in full DMEM. Cells were passaged when confluence reached 70–80%.

Culturing of bEnd.3 cells

Healthy, exponentially growing bEnd.3 cells were maintained in full DMEM in CO2 incubator at 37°C, 5% CO2, 80% humidity. Cells were passaged when confluence reached 100%.

Construction of BBBoids

Coating of cultural dish

To provide low adhesion conditions, a culture dish was coated with 1% agarose in PBS. 100 µl of the thawed agarose was added to each well of a 96-well plate. The agarose was allowed to solidify for 10 minutes until it became cloudy (Figure 2). Incorrectly agarose-covered wells were not used for plating BBBoids. To ensure optimal spheroid formation, only freshly coated culture dishes were utilized as the agarose gel could dry out when stored, leading to potential negative effects on well coating.

Figure 2.

Correctly (left) and incorrectly (right) agarose-covered wells. Arrow indicates defect in coating.

Astrocytic spheroids formation

For BBBoid construction astrocytes only of passages 2–5 were used. Optionally, astrocytes were pre-stained with the fluorescent lipophilic dye DiO, which allowed for monitoring the step-by-step formation of BBBoids without time-consuming immunostaining. This step was skipped if immunostaining or testing the barrier function was to perform, as staining with dye interferes with these analyses. The cells were stained with the dye as recommended by the manufacturer. All medium was removed from the cells and cells were rinsed with PBS. Cells were then stained with DIO (final concentration 5 µg/mL) for 20 minutes. Staining effectiveness was examined under a fluorescent microscope.

For forming astrocytic spheroids, astrocytes were detached using the standard trypsinization protocol, and the concentration of viable cells was counted using the trypan blue exclusion method. 100 µl of the cell suspension containing 3000 astrocytes was placed in each well of the agarose-coated 96-well plate. The formation of spheroids was monitored under an inverted microscope. Only the wells where astrocytic spheroids had formed were used for further construction of the BBBoids.

Covering of astrocytic spheroids with endothelial cells

On the 3rd day, bEnd.3 cells were added to the astrocyte spheroids. Optionally, bEnd.3 cells were pre-stained with the fluorescent lipophilic dye DiI (final concentration 5 µg/mL). This step should be skipped if immunostaining or testing the barrier function will take place, as staining with dye interferes with these analyses.

50 µl of cell suspension containing 300 bEnd.3 cells was added to wells with spheroids formed by astrocytes. On the 3rd day after adding bEnd.3 cells, BBBoids were gently resuspended to remove any excess attached cells and underwent further analysis.

Immunostaining of BBBoids

The immunofluorescence staining was performed using antibodies against the astrocytic marker GFAP and endothelial marker ZO-1 to confirm the correct formation of BBBoids. The BBBoids were collected in a 2 mL tube and allowed to sink to the bottom of the tube without centrifugation. They were fixed in PBS with 4% PFA for 1 hour at room temperature on an orbital rotator, then rinsed twice with 1.5 mL of PBS. Blocking and permeabilization were performed in 1.5 mL of blocking solution (PBS with 5% BSA and 0.05% Tween) for 16 hours at room temperature on an orbital rotator. The fixed and blocked BBBoids were incubated with primary antibodies in the blocking buffer for 16 hours at room temperature on an orbital rotator. Anti-ZO-1 and anti-GFAP primary antibodies were used at a dilution of 1:100. The BBBoids were then rinsed twice with PBS to remove non-bound antibodies. Secondary antibodies were added at a dilution of 1:100 in the blocking buffer, and the BBBoids were incubated for 3 hours at room temperature on an orbital rotator. After rinsing twice with PBS, the stained BBBoids were transferred to a confocal dish, and z-stacks of each BBBoid were acquired using confocal microscopy with a 1 μm interval. The ImageJ software was used for 3D reconstructions of the Z stack.

Test of barrier properties of BBBoids

To test the barrier properties, the BBBoids were placed in a fluorescent dye Lucifer Yellow, and the dye penetration rate was monitored under a confocal microscope. Only correctly formed spherical BBBoids without visible defects were used for testing barrier properties.

To test the effect of substances on barrier properties, BBBoids could be pre-incubated with tested substances, or substances could be added during imaging. To test the effect of H₂O₂ on BBB permeability, H₂O₂ was added during the imaging to final concentration 100 μM. To test the effect of IL17A on permeability, BBBoids were pre-incubated with 100 ng/mL IL17A for 3 hours. BBBoid which have not been subjected to any influence were used as control.

Imaging was performed in the microscope incubation chamber pre-warmed to 37°C. For imaging, BBBoids were transferred to confocal dishes, and Lucifer Yellow was added to a final concentration of 1 mM. The FITC fluorescent channel was used for imaging (excitation 490 nm, emission 530 nm). Imaging settings were adjusted using the first BBBoid as a reference. Imaging was started immediately after adding Lucifer Yellow solution to BBBoids.

Quantification and statistical analysis of barrier properties

Obtained images were analyzed using ImageJ software (version 1.53k.). To calculate the fluorescent signal of Lucifer Yellow penetrating into BBBoids, the entire area inside the BBBoid was selected as the region of interest (ROI). The intensity profile and data table were obtained using the “Plot Z-axis profile” command. To calculate the fluorescent signal from the background, the background was selected as the ROI and the Z-axis profile was plotted. The resulting data were processed using Prism GraphPad software (version 10.0.3.) by dividing the signal from inside the BBBoid by the signal from the background at each time point. The data are presented as the mean value and standard error of the mean (SEM).

Results and discussion

In this study, we provide a method for constructing an in vitro spheroid-based BBB model called BBBoids and describe an approach to test the influence of different substances on its permeability. BBBoids do not require specialized growth factors, supplements, or cultural dishes, which makes it more affordable compared to other spheroid-based assays. BBBoids are cultured in full DMEM in a 96-well plate coated with agarose. BBBoids form within 6 days and exhibit barrier properties and proper morphology (Figure 3).

Figure 3.

Formation of BBBoids. (a) 2 hours after plating of astrocytes, (b) 3 days after plating of astrocytes, (c) 1 day after adding of bEnd.3 cells, and (d) 3 days after adding of bEnd.3 cells, сorrectly formed BBBoid. Scale bar—150 µm.

Applying the described method, we obtain a large percentage (approximately 80%) of correctly formed BBBoids with a diameter about 150 μm. Correctly formed BBBoids are smooth spheres and do not have any protrusions or indentations (Figure 3(d)).

We confirm the correctness of the formation of BBBoids using two approaches: (1) pre-staining bEnd.3 cells and astrocytes with lipophilic dyes DiI and DiO, respectively, or (2) immunofluorescent staining with antibodies against astrocytic marker GFAP and endothelial marker ZO-1. These stainings demonstrate whether the cells are properly arranged in the spheroid, with astrocytes inside and endothelial cells on the surface. If cells were pre-stained with DiO and DiI, it is possible to monitor the process of BBBoid formation in real-time. In correctly formed BBBoids, bEnd.3 cells cover the core from astrocytes (Figure 4 and Figure 5), and astrocytes are in direct contact with endothelial cells.

Figure 4.

BBBoid formed with pre-stained astrocytes (DiO, green) and bEnd.3 (DiI, orange). (a) cCorrectly formed BBBoid and (b) defective BBBoid. Scale bar—50 µm.

Figure 5.

BBBoids stained against ZO-1 (yellow) and GFAP (green). Scale bar—50 µm.

Immunofluorescent staining also demonstrates whether tight junctions are formed at the borders between endothelial cells. BBBoids express the endothelial tight junction marker ZO-1. ZO-1 is localized at the borders between endothelial cells, giving the stained spheroid a characteristic pattern (Figure 5). Localization of ZO-1 in the cell borders related to the maturity of tight junctions.¹⁵ Consequently, this indicates proper formation of tight junctions, which provide the BBB functions.^16,17

To test barrier properties, we monitor the diffusion of Lucifer Yellow inside the BBBoids under a confocal microscope. The diffusion of Lucifer Yellow occurs through the paracellular pathway and serves as a marker for the establishment of proper tight junctions.¹⁸ This method is widely used to study the barrier functions of the BBB in in vitro models, and we have adapted it to study the barrier properties of BBBoids.¹⁸ BBBoids demonstrate low permeability to Lucifer Yellow (Figure 6). During 1 hour of incubation with Lucifer Yellow, the signal ratio inside/background changed slightly, indicating functionality of tight junctions and that BBBoids indeed demonstrate barrier functions. Thus, BBBoids reproduce such BBB features as the expression of tight junction proteins and impermeability to Lucifer Yellow and hence can be used to model the BBB.

Figure 6.

BBBoid permeability for Lucifer Yellow. The blue line represents BBBoids without treatment, the red line represents the addition of H₂O₂ (final concentration 100 μM) at the 20th minute, the black line represents BBBoids pretreated with 100 ng/mL IL17A for 3 hours. The data is presented as the mean and +/− SEM, N = 5.

We tested the effect of exposure to H₂O₂ and IL17A on BBBoid permeability (Figure 6). We have chosen to test these substances because they are considered to be negative regulators of BBB permeability in vivo. In the case of H₂O₂, it was added directly to BBBoids during imaging on a confocal microscope. In the case of IL17A, BBBoids were pre-incubated with it for 3 hours.

H₂O₂ is the primary reactive oxygen species (ROS) found in cells.^19,20 There is evidence that excessive generation of H₂O₂ negatively affects the BBB and is associated with multiple sclerosis, stroke, brain injuries, and other neurological disorders.^21–28 However, it remains unclear whether hyperproduction of H₂O₂ in BBB cells is the primary cause of its dysfunction leading to the development of neurological disorders or a secondary process that develops as a result of the disease. Thus, studying the effect of H₂O₂ on the function of the BBB has important medical significance, and in vitro models are used for this purpose.²⁹ The addition of H₂O₂ disrupts the integrity of the BBBoids within a few minutes, accelerating Lucifer Yellow penetration into the BBBoids (Figures 6 and 7). We also observed an accumulation of Lucifer Yellow inside the cells, indicating cell death due to oxidative stress (Figure 7(b)). Thus, adding H₂O₂ at a concentration of 100 μM may lead to an increase in BBBoid permeability by causing cell death. Our observations are consistent with data obtained from other in vitro models.²⁹

Figure 7.

Testing of barrier function of BBBoids. (a) BBBoid at the start of the analysis, (b) after 10 min of incubation with 100 μM of H₂O₂. Arrows indicate dead cells. Scale bar—50 µm.

IL17A is proinflammatory cytokine, which is associated with impairment of BBB functions, multiple sclerosis, and neuroinflammation.³⁰ The negative effect of IL-17A on the BBB permeability may be attributed to its influence on the expression and localization of tight junction proteins, including claudin-5 and occludin.³¹ BBBoids pre-incubated with IL17A demonstrate lower barrier properties and accumulate Lucifer Yellow inside faster in comparison with untreated BBBoids (Figure 6). These observations are also consistent with data on the influence of IL17A obtained in other in vitro models.³²

Therefore, substances that are in vivo negative regulators of BBB permeability demonstrate the negative effect on BBBoids. The influence of IL17A and H₂O₂ that we observed on BBBoids is consistent with what is observed in other in vitro models. Using the same approach, influence of other substances on BBBoid permeability can be studied.

Despite the fact that we developed a cost-effective and affordable method to construct an organoid-based BBB model, it should be noted that it has some limitations. Advantages and limitations of BBBoids are summarized in Table 1. BBBoids present an “inside-out” cellular architecture compared to that found in vivo. Indeed, vascularized spheroids represent the most physiologically relevant model of the BBB, which reproduces the architecture of brain vessels. However, at present, obtaining vascularized spheroids presents difficulties and is the subject of active development.^33–36 As cerebral endothelial cells, we used the bEnd.3 cell line. BEnd.3 cells are widely used in BBB modeling, but they are transformed cells and exhibit phenotypic variations from primary endothelial cells.^37,38 Therefore, this should be taken into consideration as in some cases these cells may not precisely replicate the impact of specific factors on the BBB endothelium.³⁸ Moreover, due to the fact that this model utilizes murine cells, its translation to human BBB physiology may be restricted.³⁹ In this configuration, BBBoids lack pericytes, resulting in the absence of pericytic factors that may have an effect on the physiology of the BBB. But the “minimal” BBB model can be considered one that contains endothelial cells and astrocytes. Indeed, many in vitro BBB models only contain these two cell types.^40–42 Moreover, pericytes require a special medium for their cultivation.⁴³ The absence of pericytes allows this protocol to be performed without special media or supplements, making it inexpensive and accessible.

Table 1.

Advantages and limitations of BBBoids.

Advantages	Limitations
Economically efficient	“Inside-out” architecture
Not labor-intensive	BEnd.3 are transformed cells
Not time-consuming	Murine cells
Does not require special cultural medium, growth factors, or supplements	No pericytes
Does not require special culture dishes
Astrocytes and endothelial cells are in direct contact with each other
No artificial scaffolds or membranes
Scalability
Real-time monitoring of barrier properties
Potential for studying the permeability of therapeutic agents
Reduction the need for laboratory animals

Conclusion

In conclusion, despite some potential limitations, BBBoids is a promising in vitro BBB model for testing the effects of various substances on BBB permeability. This model allows for high-throughput analysis and reduces the need for laboratory animals. Its cost-effectiveness, low time consumption, and ease of construction make it attractive to a wide range of researchers.

Footnotes

ORCID iD

Margarita Shuvalova

Author contributions

Conceptualization, M.S., A.M., E.S., G.N., and V.B.; investigation, M.S.; writing – original draft, M.S.; writing – review & editing, M.S., G.N., A.M., E.S., and V.B.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by the Russian Science Foundation (RSF), grant number 23-75-30023.

Conflicting interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

This protocol did not involve generation of new datasets or code.

References

Daneman

Prat

. The blood-brain barrier. Cold Spring Harb Perspect Biol 2015; 7: a020412.

Geldenhuys

Mohammad

Adkins

, et al. Molecular determinants of blood-brain barrier permeation. Ther Deliv 2015; 6: 961–971.

Zhang

Yuan

, et al. CircRBM33 induces endothelial dysfunction by targeting the miR-6838-5p/PDCD4 axis affecting blood-brain barrier in mice with cerebral ischemia-reperfusion injury. Clin Hemorheol Microcirc 2023; 85: 355–370.

Sánchez-Dengra

González-Álvarez

Bermejo

, et al. Access to the CNS: strategies to overcome the BBB. Int J Pharm 2023; 636: 122759.

Bagchi

Chhibber

Lahooti

, et al. In-vitro blood-brain barrier models for drug screening and permeation studies: an overview. Drug Des Dev Ther 2019; 13: 3591–3605.

Williams-Medina

Deblock

Janigro

. In vitro models of the blood-brain barrier: tools in translational medicine. Front Med Technol 2020; 2: 623950.

Bergmann

Lawler

, et al. Blood-brain-barrier organoids for investigating the permeability of CNS therapeutics. Nat Protoc 2018; 13: 2827–2843.

Cho

C-F

Wolfe

Fadzen

, et al. Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents. Nat Commun 2017; 8: 15623.

Kassianidou

Simonneau

Gavrilov

, et al. High throughput blood-brain barrier organoid generation and assessment of receptor-mediated antibody transcytosis. Bio Protoc 2022; 12: e4399.

10.

Urich

Patsch

Aigner

, et al. Multicellular self-assembled spheroidal model of the blood brain barrier. Sci Rep 2013; 3: 1500.

11.

Adams

Clausen

Jensen

PØ

, et al. 3D blood-brain barrier-organoids as a model for Lyme neuroborreliosis highlighting genospecies dependent organotropism. iScience 2023; 26: 105838.

12.

Chen

Sun

Tian

, et al. Modeling sporadic Alzheimer’s disease in human brain organoids under serum exposure. Adv Sci 2021; 8: e2101462.

13.

Cecchelli

Berezowski

Lundquist

, et al. Modelling of the blood-brain barrier in drug discovery and development. Nat Rev Drug Discov 2007; 6: 650–661.

14.

Protocol for isolation of primary murine astrocytes. https://bio-protocol.org/exchange/preprintdetail?id=2327&type=3

15.

Gottardi

Arpin

Fanning

, et al. The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts. Proc Natl Acad Sci U S A 1996; 93: 10779–10784.

16.

Watson

Anderson

Vanltallie

, et al. The tight-junction-specific protein ZO-1 is a component of the human and rat blood-brain barriers. Neurosci Lett 1991; 129: 6–10.

17.

Branca

JJV

Maresca

Morucci

, et al. Effects of cadmium on ZO-1 tight junction integrity of the blood brain barrier. Int J Mol Sci 2019; 20.

18.

Zhao

Han

Bae

, et al. Lucifer yellow - a robust paracellular permeability marker in a cell model of the human blood-brain barrier. J Vis Exp 2019; 10: 3791–58900.

19.

Boveris

Oshino

Chance

. The cellular production of hydrogen peroxide. Biochem J 1972; 128: 617–630.

20.

Commoner

Townsend

Pake

. Free radicals in biological materials. Nature 1954; 174: 689–691.

21.

Ferretti

Bacchetti

Principi

, et al. Increased levels of lipid hydroperoxides in plasma of patients with multiple sclerosis: a relationship with paraoxonase activity. Mult Scler 2005; 11: 677–682.

22.

Greco

Minghetti

Sette

, et al. Cerebrospinal fluid isoprostane shows oxidative stress in patients with multiple sclerosis. Neurology 1999; 53: 1876–1879.

23.

Yang

Xiu

, et al. Reactive oxygen species play a biphasic role in brain ischemia. J Invest Surg 2019; 32: 97–102.

24.

Khatri

Thakur

Pareek

, et al. Oxidative stress: major threat in traumatic brain injury. CNS Neurol Disord: Drug Targets 2018; 17: 689–695.

25.

Hergenroeder

Redell

Moore

, et al. Biomarkers in the clinical diagnosis and management of traumatic brain injury. Mol Diagn Ther 2008; 12: 345–358.

26.

Zhao

. Oxidative stress and the pathogenesis of Alzheimer’s disease. Oxid Med Cell Longev 2013; 2013: 316523.

27.

Choi

D-H

Hwang

Lee

K-H

, et al. DJ-1 cleavage by matrix metalloproteinase 3 mediates oxidative stress-induced dopaminergic cell death. Antioxid Redox Signal 2011; 14: 2137–2150.

28.

Sahil

Bodh

Verma

. Spirulina platensis: a comprehensive review of its nutritional value, antioxidant activity and functional food potential. J Cell Biotechnol 2024; 10: 159–172.

29.

Anasooya Shaji

Robinson

Yeager

, et al. The tri-phasic role of hydrogen peroxide in blood-brain barrier endothelial cells. Sci Rep 2019; 9: 133.

30.

Milovanovic

Arsenijevic

Stojanovic

, et al. Interleukin-17 in chronic inflammatory neurological diseases. Front Immunol 2020; 11: 947.

31.

Dong

Wang

, et al. IL-17A contributes to perioperative neurocognitive disorders through blood-brain barrier disruption in aged mice. J Neuroinflammation 2018; 15: 332.

32.

Huppert

Closhen

Croxford

, et al. Cellular mechanisms of IL-17-induced blood-brain barrier disruption. FASEB J 2010; 24: 1023–1034.

33.

Cakir

Xiang

Tanaka

, et al. Engineering of human brain organoids with a functional vascular-like system. Nat Methods 2019; 16: 1169–1175.

34.

Ham

Jin

Kim

, et al. Blood vessel formation in cerebral organoids formed from human embryonic stem cells. Biochem Biophys Res Commun 2020; 521: 84–90.

35.

Shi

Sun

Wang

, et al. Vascularized human cortical organoids (vOrganoids) model cortical development in vivo. PLoS Biol 2020; 18: e3000705.

36.

Adams

Jensen

. Cerebral malaria - modelling interactions at the blood-brain barrier in vitro. Dis Model Mech 2022; 15: dmm049410.

37.

Yin

Peng

, et al. Immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells. Zhong Guo Dang Dai Er Ke Za Zhi 2010; 12: 474–478.

38.

Williams

Risau

Zerwes

, et al. Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment. Cell 1989; 57: 1053–1063.

39.

Song

Foreman

Gastfriend

, et al. Transcriptomic comparison of human and mouse brain microvessels. Sci Rep 2020; 10: 12358.

40.

Lepak

Hussain

, et al. An endothelial and astrocyte co-culture model of the blood–brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane. Lab Chip 2005; 5: 74–85.

41.

Wang

Cai

, et al. In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells. Neural Regen Res 2015; 10: 2011–2017.

42.

Takeshita

Obermeier

Cotleur

, et al. An in vitro blood-brain barrier model combining shear stress and endothelial cell/astrocyte co-culture. J Neurosci Methods 2014; 232: 165–172.

43.

Tigges

Welser-Alves

Boroujerdi

, et al. A novel and simple method for culturing pericytes from mouse brain. Microvasc Res 2012; 84: 74–80.