This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.
The delivery of drugs to the brain remains a challenge due to the blood-brain barrier’s (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.
The BBB, localized at the level of brain capillary endothelial cells (ECs), controls and regulates access to the brain parenchyma, which is crucial for maintaining brain homeostasis and the function of neural cells1,2. However, in the case of brain pathology, the lack of access to the brain parenchyma represents a real obstacle to developing therapeutic strategies.
The BBB ECs possess a complex set of properties, including tight junction (TJ) proteins, which seal the intercellular space, associated with a system of efflux pumps, specific transporters, and receptors, which control the transcellular pathway1,2,3. Moreover, all these properties are induced and maintained, thanks to communications with the pericytes embedded in the BBB EC basement membrane and the astrocytes, whose end-feet surround the brain capillaries1,2,3. Hence, studying the BBB in vitro is a challenge considering the complexity of its architecture and the communications among the different cell types constituting the neurovascular unit (NVU)2. Moreover, the different cell types are crucial for the induction and maintenance of BBB properties and consequently impact the prediction of the crossing through the BBB. Different strategies for drug delivery to the brain were already tested using a large panel of tactics to bypass the BBB restricted properties4. More recently, with the progress of nanotechnologies, new materials are being developed for applications as drug carriers5,6. In addition to their higher load, reduced toxicity, and increased drugs' bioavailability, these new nanomaterials can be functionalized for a Trojan horse strategy to cross the BBB and specifically target cells in the parenchyma5,6. Among the different types of nanomaterials being evaluated, nanogels have attracted considerable attention, mainly due to their colloidal properties and ability to tailor the chemical structure to introduce stimuli-responsive properties7,8,9,10,11,12,13,14,15.
In vitro models are now developed for preclinical studies using human cells to predict brain penetration of drugs16. Different settings of these models are available, from monolayers of brain ECs to multiple cell systems16. Considering the importance of the NVU cells in the BBB induction and maintenance and the coordinated response to the pathological environment, BBB in vitro models need to consider all these protagonists to improve the relevance of the prediction2,17.
The current method describes setting up a triple culture in vitro model of the human BBB, which is fully developed with human cells to study specific cellular and human molecular mechanisms. To be physiologically relevant, the model consists of the main three cellular actors of the BBB (ECs, pericytes, and astrocytes) necessary to induce and maintain the BBB properties, without the use of tightening compounds and displaying a set of properties required to be considered as an in vitro BBB model16,18. The model is set up in a configuration delimiting the blood and brain compartment, suitable for preclinical studies of drug and particle transport to predict brain penetration. The usefulness of the model is illustrated by measuring the transport of polymeric nanogels.
Treatment of brain diseases remains a challenge considering the difficulty of the drugs to hurdle over the BBB to reach their cellular and molecular targets in the brain parenchyma.
Drug development for brain diseases currently exhibits a low success rate since most drugs displaying promising results in preclinical models failed to show any benefit when used in the clinic. Following the "3R rule," which aims at reducing the number of animals used for experimentation, in vitro models of the BBB are developed to study brain pathologies and to predict brain penetration of drugs29. In vitro models of BBB have mainly been developed using animal cells and have become more sophisticated to improve the relevance of the results obtained16. One of the significant advances in the use of human cells, which brings undeniable new insight and more specificity, at the cellular and molecular levels, to study human disease mechanisms16. However, the development of relevant models requires considering the improvement of the BBB in vitro model settings and the knowledge arising, thanks to animal models. Hence, it needs to consider the complexity of the BBB architecture and the importance of the cell-cell communications to study the BBB under physiological and pathological conditions30.
The protocol presented here describes a method to set up a full human BBB in vitro model comprising the three main cell types of the BBB, without limitation of access to brain tissue. As a multiple cell system, the induction and the maintenance of BBB properties, without the artificial use of tightening compounds, but instead induced by cell-cell communications is more physiologically relevant and in line with the in vivo induction of the BBB properties31. Hence, the respect of the chronology of the protocol is prime of importance for the success of the protocol. Moreover, the incubation times during the setting of the triple culture and once the three cell types are assembled represent the main critical steps of the protocol.
The BBB properties in ECs are induced by the co-culture with pericytes, as described for the co-culture model24. Hence, the culture of pericytes at the reverse side of the insert filter is the most critical point and requires strictly following the protocol at the risk of not having enough pericytes for the induction of the BBB properties. First of all, during the coating procedure and also cell seeding, attention has to be put not to have the cover of the Petri dish in contact with the coating and also the medium once the cells are seeded to ensure a good coating of the filter and not to lose cells (steps 2.2.1 and 2.2.4). Moreover, once the pericytes are seeded, it is essential to wait the indicated time for the attachment of the pericytes (step 2.2.4) before reverting the insert filter for the coating and seeding of ECs on the other side (steps 2.2.5 and 2.3). Once seeded, six days are required to induce the BBB properties through cell-cell communications (step 2.4).
The model is validated in terms of restricted permeability (associated with the setting of the tight junctions) since the ECs of the triple culture model display permeability values to BBB integrity markers similar to the validated co-culture model and also measured in validated animal or human models16,27,32. Moreover, the validation of an in vitro BBB model requires, in addition to the restricted permeability, the responsiveness to other cell types of the NVU and the expression of functional receptors and transporters16. In addition, the model is reproducible and produces multiple insert filters and wells to perform numerous analyses (gene and protein expression, fluorescent staining, toxicity tests) on each cell type separately without requiring a cell sorting method.
The model was developed using a 0.4 µm pore size filter to have one cell type on each side of the insert filter. The insert filter system allowed the study of cell-cell communications in physiological conditions by transferring it upon well-containing astrocytes. The presence of astrocytes in the system represents a plus value compared to the initial co-culture in vitro model24. Indeed, considering the importance of astrocytes in the physiology of the BBB, this third cell type allows further understanding of the cell-cell communications within the BBB. Moreover, the triple cell culture system can also be studied in pathological conditions such as stroke, in which the astrocytes play an essential role33,34,35. In addition, the design of BLECs/ pericytes on both sides of the insert filter can easily be placed upon other cell types to mimic pathological conditions such as brain cancer23.
The pore size of the insert filter can bring limitations with some experiments, such as cell transmigration across the BBB. However, the development of the model with a larger pore size requires the adaptation of the protocol to ensure the formation of a physiological monolayer of ECs and not multiple layers, which is not physiologically relevant to mimic the BBB36.
The model's applicability has been demonstrated using NGs transport experiment exhibiting the possibility to do transport experiment using a multicellular system. Nevertheless, one should be aware of the difficulties in having a control compound or molecule for the transport experiment, sharing comparable properties with NGs since each nanostructure exhibits a unique set of properties (molecular weight, charge, shape, physical properties, protein corona formation).
One limitation of the model is the absence of shear stress, which was demonstrated to influence the differentiation of ECs and the expression of TJ proteins37. However, developing a fluidic system mimicking the brain capillary is challenging considering the complexity of adding a fluidic part, requiring a specific device, in a multiple cell system. Moreover, the particular device is usually not commercially available and does not allow many replicates, thus making fluidic systems less adapted for high-throughput use.
In summary, this triple culture system consisting of human cells reproduces in vitro the architecture of the BBB. It allows the generation of many inserts that can be used for extensive screening of compounds.
The authors have nothing to disclose.
This work is granted by European Union's Horizon 2020 research and innovation program under grant agreement No 764958, as part of the NANOSTEM project, a Marie Skłodowska-Curie Innovative Training Network (ITN) (Fellowship Eleonora Rizzi). This study is granted by the 'Conseil régional du Nord-Pas-de-Calais' (Fellowship to Clémence Deligne), the "Société Française de lutte contre les Cancers et les leucémies de l'Enfant et de l'adolescent"(SFCE), the Association "l'étoile de Martin" and the Association"Cassandra contre la leucémie".
Cell Culture | |||
Astocyte Medium (AM) | ScienCell | 1801 | |
Astrocyte Growth Supplement | ScienCell | 1852 | Astrocyte Growth Supplement is provided in the AM set. |
Cell culture dish 100 mm | Corning | 430293 | 100 mm x 20 mm; dish used for the thawing of ECs and PCs before the triculture setting |
Cell culture dish 150 mm | Corning | 430599 | The height of these dishes (25 mm) allows the seeding of PCs in the reverted insert for the setting of the triculture model. |
Collagen I | Corning | 354236 | Rat tail |
Dulbecco's Modified Eagle Medium | Gibco | 31600-083 | Powder |
Endothelial Cell Growth Supplement | ScienCell | 1051 | Endothelial Cell Growth Supplement is provided in the ECM set. |
Endothelial Cell Medium (ECM) | ScienCell | 1001 | |
Fetal Calf Serum | Sigma | F7524 | |
Gelatin | Sigma | G2500 | 2% gelatin from porcine skin in PBS-CMF |
Gentamicin | BiochromA6 | A-2712 | |
Glucose | Sigma | G6152 | Powder |
Glutamine | Merck | 1002891000 | |
Human Brain Cortex Astrocytes | ScienCell | 1800-SC | |
Malassez cell counting chamber | vWR | HECH40453702 | The count was performed manually. |
Matrigel | Corning | 354230 | Extracellular matrix-based hydrogel |
Penicillin/Streptomycin | ScienCell | 0503 | Penicillin/Streptomycin solution is provided in the ECM and AM sets. |
Poly-L-lysine | ScienCell | 0413 | |
Steritop | Millipore System | SCGPT0SRE | 0.22 µm pore size |
Transwell insert | Corning | 3401 | 0.4 µm pore polycarbonate filter |
Trypsin/EDTA neutralization solution | ScienCell | 0113 | |
Trypsin/EDTA solution | ScienCell | 0103 | |
Immunocytochemistry | |||
SEA BLOCK blocking buffer | ThermoScientific | 37527 | |
Alexa Fluor 568 anti-Mouse secondary antibody | Thermofisher | A11031 | Dilution 1:500 |
Alexa Fluor 568 anti-Rabbit secondary antibody | Thermofisher | A11036 | Dilution 1:500 |
Anti-Claudin-5 primary antibody | InVitrogen | 34-1600 | Dilution 1:100 |
Anti-Desmin primary antibody | Abcam | ab6322 | Dilution 1:200 |
Anti-Glial Fibrillary Acidic Protein primary antibody | Dako | Z0334 | Dilution 1:500 |
Anti-Platelet-Derived Growth Factor-β primary antibody | Abcam | ab51090 | Dilution 1:200 |
Anti-VE-cadherin primary antibody | Abcam | ab207732 | Dilution 1:200 |
Anti-Zona Occludens-1 primary antibody | InVitrogen | 61-7300 | Dilution 1:200 |
Normal Goat Serum | Sigma | G6767 | |
ProLong Diamond Antifade Mountant with DAPI | Invitrogen | P36962 | |
Gene expression | |||
NucleoSpin Rna/Protein Macherey Nagel Kit | Macherey-Nagel | 740,933,250 | |
96 multiplate well | Biorad | HSP9601 | |
iSCRIPT | Biorad | 1708841 | |
Sealer sheet | Biorad | MSB1001 | |
SsoFast EvaGreen Supermix | Biorad | 172-5201 | |
Protein expression | |||
2x Laemli Sample Buffer | Biorad | 161-0737 | Add 50 µL of bMercaptoetanol to 950 µL of Laemmli Buffer and store at -20°C. Dilute 1:1 with the protein sample for the assay. |
Anti-Breast Cancer Resistance Protein primary antibody | Abcam | ab207732 | Pre-treatment 15 minutes at RT under agitation; dilution 1:1000, O.N. at 4°C |
Anti-Claudin-5 primary antibody | Abcam | ab15106 | Pre-treatment 5 minutes at 95°C; dilution 1:1000, O.N. at 4°C |
Anti-Glucose Transporter 1 primary antibody | Millipore | 07-1401 | Pre-treatment 5 minutes at 95°C; dilution 1:1000, O.N. at 4°C |
Anti-Mouse secondary antibody | Dako | P0447 | Dilution 1:5000 in TBS-Tween |
Anti-P-glycoprotein primary antibody | Genetex | GTX23364 | Pre-treatment 15 minutes at RT under agitation; dilution 1:400, 3 hours at RT |
Anti-Rabbit secondary antibody | Dako | P0448 | Dilution 1:8000 in TBS-Tween |
Anti-Transferrin Receptor primary antibody | Abcam | ab84036 | Pre-treatment 5 minutes at 95°C; dilution 1:1000, O.N. at 4°C |
Anti-Zona occludens-1 primary antibody | Abcam | ab216880 | Pre-treatment 5 minutes at 95°C; dilution 1:1000, O.N. at 4°C |
Criterion TGX Gel | Biorad | 5678083 | |
ECL Prime Solution | Amersham | RPN2236 | Revelation solution to keep in the dark |
Phospatase inhibitor cocktail 2 | Sigma | P5726 | |
Phospatase inhibitor cocktail 3 | Sigma | P0044 | |
Protease Inhibitor | Sigma | P8340 | |
Protein Standards | Biorad | 161-0373 | Molecular weight markers |
RIPA 10x | Millipore | 20-188 | |
TBS 10x | Biorad | 1706435 | |
TRIS-Glycine | Biorad | 1610771 | |
Tween | Biorad | 1706531 | |
BBB integrity assay | |||
Sodium Fluorescein | Ampresco | 0681 | λex= 490 nm; λem= 525 nm |
Elacridar | Sigma | SML0486 | GF120918 |
FITC-Dextran 20 kDa | Sigma | FD-20S | λex= 490 nm; λem= 525 nm |
Rhodamine 123 | Sigma | R8004 | λex= 501 nm; λem= 538 nm |
SynergyTM H1 | BioTek Instruments | Fluorescent multiplate reader | |
Nanogel Transport | |||
Syringe | Terumo | SS+01T1 | 1 mL syringe |
Filter | FisherScientific | 15161499 | 0.2 µm PTFE membrane filter, 15 mm diameter |
N-Isopropylacrylamide (NIPAM)-based hydrogels | The nanogels (NGs) used in the study are provided by our collaborator in Queen Mary University London, Department of Chemistry. The NGs are covalently tagged with a fluorescent molecule (λex= 477 nm; λem= 540 nm). NGs are freeze dried and shipped as powder, in this state they are stable at room temperature for long period of time. |