Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Summary

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Abstract

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Introduction

The BBB is a specialized interface between the peripheral blood circulation and the central nervous system, crucially responsible for the maintenance of brain hemostasis. It comprises distinct brain microvascular endothelial cells (BECs) which are functionally influenced by few cellular and acellular components (below) to form a tight and dynamic gateway into the brain. Under physiological conditions the BBB restricts the passage of blood cells, plasma components and harmful substances, all potentially neurotoxic, into the brain. In parallel, the BBB selectively exchanges key ions and nutrients (glucose and amino-acids) and metabolic waste products between the brain and the circulation to precisely maintain the brain environment^1,2. In recent years it is becoming evident that failure of the BBB occurs in a variety of chronic brain pathologies, such as neurodegenerative or inflammatory-related diseases (e.g. Alzheimer's disease and multiple sclerosis, respectively)³, as well as in acute conditions like ischemic stroke⁴.

The unique BBB properties of brain endothelial cells (BECs) are largely induced by their cerebral environment⁵, and in particular by astrocytes^6,7. There is a growing understanding that other cell-types, such as pericytes⁸, neurons and microglia^1,3, as well as the basement membrane⁹, support BECs and form together a functional unit termed the "neurovascular unit" (NVU) which simultaneously couples neuronal metabolic demands to their supplying capillaries¹⁰.

The involvement of the BBB in pathological situations underlies numerous attempts to develop in vitro BBB models to assist in BBB-related research^11,12. These models aim to mimic as close as possible in vivo BBB characteristics according to the NVU principle. In vitro BBB models generally rely on a monolayer of tight-junction-forming BECs (mainly from bovine¹³, human¹⁴, rat¹⁵, mouse¹⁶, and porcine^17,18 origins), cultured on a porous membrane together with supporting astrocytes (extensively reviewed by Deli et al. 2005¹¹).

Astrocytes can be grown in non-contact conditions on the bottom of a tissue culture well, separated from BECs (cultivated on the upper surface of the membrane) by the culture medium yet communicating with BECs via soluble factors¹⁶. In more advanced models which better resemble the anatomical structure of the BBB in vivo, astrocytes are maintained in contact conditions and cultured directly on the opposite side of the membrane in close proximity to BECs^13,15,17 (Figure 1). This configuration enables physical contact between BECs and astrocytes, established when astrocytes project their processes through the porous membrane. Importantly, for a true contact to occur the pores should be ≥1µm in diameter, since astrocytic end-feet cannot pass through smaller pore sizes (i.e. 0.4 µm)^14,15. Notably, contact BBB systems are demonstrated in some studies to be superior to their non-contact counterparts regarding their trans-endothelial electrical resistance (TEER) and endothelial permeability values of various tracers^13,17,18. An additional dimension of media flow was recently added in a number of in vitro BBB models to apply shear forces to the endothelium for closer simulation of the brain vasculature^12,19.

One technical obstacle to overcome when generating a contact BBB model is the seeding of astrocytes against gravity on the abluminal surface of the porous membrane. Previous protocols^13,20, where astrocytes were simply seeded in a drop of media on top of an inverted insert, allowed only short seeding times (i.e. 10 min¹³ or 2 hr²⁰) which were found in our hands to be insufficient for proper cell attachment. Using this basic method, a longer astrocyte attachment period requires constant monitoring of the inserts by frequent opening of the incubator (causing fluctuations in temperature, pH and humidity) and is also prone to uneven cell seeding due to leakage of media through the pores, especially if pores larger than 1 µm are employed.

Here, we describe a general protocol for preparation of a contact BBB model. Our procedure includes an alternative method for cell-seeding on inverted inserts, which addresses the above mentioned limitations. The method permits undisturbed adherence of astrocytes onto the abluminal membrane surface, in an equilibrated incubator, for an extended period of time. As a result, a uniform seeding of astrocytes is achieved which increases barrier quality and minimizes basal permeability variations between inserts.

As the use of human cells is important for human-relevant research²¹, we additionally demonstrate in this article the specific utilization of a novel combination of primary human BECs and immortalized human astrocytes for establishment of a contact human BBB model with a high throughput screening capacity. Since viewing of cells on porous membranes can be difficult, we also detail staining techniques which can assist in determination of confluence and cell morphology on the porous membranes. Finally, we exemplify how our human BBB model can be utilized to examine the effect of tissue-type plasminogen activator (t-PA) - a clot busting enzyme which serves as a sole treatment option for acute ischemic stroke - on the BBB.

Protocol

1. Cell Culture (3-7 Days Prior to BBB Assembly)

1.1. Primary Human Brain Microvascular Endothelial Cells (BECs)

BECs were commercially obtained. The cells were produced by dispase dissociation of normal human brain cortex tissue and provided frozen at passage 3 (<12 population doublings).

1. Substratum: Coat tissue-culture vessels with "Attachment Factor" as per the manufacturer instructions.
2. Cell maintenance: Maintain BECs in serum-containing complete medium. For experimentation, culture BECs in complete serum-free medium. Maintain the cells in a humidified 5% CO₂, 21% O₂ incubator at 37 °C
   Note: As an alternative to specialized reagents, 0.1% (w/v) gelatine solution can serve as a coating reagent while DMEM/F-12 supplemented with 15 mM HEPES, 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 50 µg/ml gentamycin, 20 µg/ml heparin and 20 µg/ml endothelial cells growth supplement can be used for BEC maintenance.
3. Sub-culturing for experimentation: Split confluent BECs at a ratio of 1:3 to 1:6, depending on the intended time for next use (3-7 days). Change the maintenance medium every 3 days. Use BECs up to 15 passages.

1.2. SVG Human Fetal Astroglial Cell-line

SVG human fetal astroglial cells²² were originally derived from primary cultures of human fetal brain transformed with replication-deficient simian virus 40.

1. Maintenance: Culture SVG cells in minimum essential medium with Earle's balanced salt solution supplemented with 20% (v/v) FCS (same serum percentage used to maintain their parental primary cells ²²), 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. Maintain the cells in a humidified 5% CO₂, 21% O₂ incubator at 37 °C.
2. Sub-culturing for experimentation: Split confluent SVGs at a ratio of 1:5 to 1:10, depending on the intended time for next use (3-7 days). Removal of trypsin is not required due to the high content of serum in the medium. Change the medium every 3 days. Use SVGs up to 20 passages.

2. Assembly and Coculturing of the Human In vitro BBB Model (Day 0)

Comment: The human in vitro contact BBB model is prepared according to published protocols ^13,23, with modifications.

2.1. Coating of Inserts

1. Place tissue-culture inserts (6.5 mm in diameter with polyester porous membrane, 3 µm pore-size) in the wells of the supplied 24-well plate. Coat the luminal surface of the inserts overnight in a humidified 37 °C incubator with rat collagen I (20 µg/cm²; 50 µl of 132 µg/ml collagen I in 0.02% acetic acid in Milli-Q water (MQH₂O)). Wash the inserts once (both from the abluminal and luminal side) with MQH₂O to remove the residual acid.
   Note: Basement membrane of brain capillaries contains no collagen I but mainly laminins, collagen type IV isoforms, nidogens, and heparan sulfate proteoglycans⁹. For this reason, some researchers utilize coating agents such as collagen IV, fibronectin¹³, or Matrigel (BD biosciences)¹⁶ for a more authentic BBB modeling.

2.2. Seeding SVGs on the Underside (Abluminal) Membrane Surface

1. Invert the insert and gently fit around the rim of the porous membrane a short piece of elastic silicone tubing ("external tubing"; ~10 mm long, 8 mm internal diameter, 1.6 mm wall), creating essentially a new well above the abluminal surface (Figure 2A).
2. The underside of the insert needs to be sealed to avoid media leakage. First, prepare a plug (Figure 2A and 2B) made of a silicone tubing (termed "internal tubing"; ~8 mm long, 3.2 mm internal diameter, 1.6 mm wall; Figure 2B), sealed at one end with a plastic sealing cone (~3mm long; prepared by cutting the bottom of a 0.2 ml polymerase chain reaction (PCR) tube; Figure 2B). This cone not only seals the internal tubing lumen, but also widens its edge for tighter fitting into the insert. Next, using a sterile forceps, insert the silicone plug into the luminal cavity and advance it until it reaches up-to ~1-2 mm from the membrane (Figure 2A).
3. Next, seed 4 x 10⁴ SVG cells in 200 µl SVG maintenance medium (section 1.2.2 above) directly into the silicone well above the abluminal membrane surface (Figure 2A). Allow SVGs to adhere for at least 4 hr in the incubator.
   Note: To maintain sterility transport the assembled inserts in between two 6-well plates (one plate inverted over the other; Figure 2C) to minimize exposure to unfiltered air during the procedure. Silicone tubes and plugs are washed in ethanol and autoclaved for later use.
   Note: Use plugs (section 2.2.2) only for inserts with membrane pore sizes larger than 1 µm. Membranes with pore diameter ≤1 µm rarely leak and require only the fitting of the external silicone tubing (section 2.2.1). In addition, some coating agents other than collagen I (see note to section 2.1) may prevent leakiness even through 3 µm pores. Determine this empirically.
4. In order to monitor the adherence state of your astrocytes, seed in parallel into a standard 96-well an identical volume of the astrocyte cell-suspension. This well has the same surface area as the 6.5 mm insert (0.33 cm²) and permits easier visualization of cells by phase-contrast microscopy than the insert membrane. When you observe sufficient adherence of the astrocyte in the control 96-well, the astrocytes have adequately adhered also to the insert membrane.
5. When astrocytes have sufficiently adhered in the control 96-well, transfer the insert assemblies to the tissue culture hood (see note above) and gently remove the external silicone tubing and plugs. Return inserts to the normal (i.e. upright) orientation into the supplied wells containing 800 µl/well of BEC maintenance medium (section 1.1.2 above).

2.3. Seeding of BECs into the Luminal Chamber above the Astrocytes

Seed 2x10⁴ BECs in 200 μl onto the collagen-coated luminal surface and return the inserts into the incubator.

2.4. Coculturing

Coculture the SVG/BEC-seeded inserts for 3 days in BEC medium without media change before experimentation.

3. Experimentation with the Human BBB Model (Day 3)

1. To stimulate the in vitro BBB first wash cells by replacing the abluminal and luminal media with 600 µl and 100 µl of serum-free BECs medium, respectively.
2. Aspirate the luminal medium again and replace with 100 µl of serum-free medium with stimulating agents.
   Note: If stimulation of the abluminal chamber is required (to activate the in vitro BBB from the astrocytic compartment), aspirate the abluminal medium and replace with 600 ul of serum free medium with stimulating agents.
3. Test each experimental group in triplicate (hence, if luminal stimulation is performed, which requires 3 x 100 µl, we recommend diluting the reagents to their final concentrations in 350 µl of serum-free medium per group to minimize pipetting errors between wells).
4. Perform standard paracellular and/or transcellular permeability assays of labeled tracers ^16,24 (for the latter we use fluorescein isothiocyanate (FITC)-conjugated albumin) and/or measurement of TEER ^13,16,25 as previously described.

To account for fluctuations in baseline permeability between experiments, analyze changes in permeability relative to a coated insert without cells (which serves as a reference for maximal permeability) and to the vehicle-treated group using the formula: permeability (% of max) = (permeability value of experimental insert - average permeability value of the vehicle group) / (permeability value of the blank insert - average permeability value of the vehicle group) X 100.

4. Visualization of Cells on Porous Membranes

Comment: Unstained cells on insert membranes are difficult to observe by phase contrast or differential interference contrast (DIC) microscopy since the membrane is quite opaque and interferes with light transmission. To deal with this issue we developed various staining procedures of cells on inserts, which greatly enhance cell appearance on the membrane. Generally, stain the insert membranes while still attached to the insert, in the wells. The membranes should be removed only at the end of the staining procedure for mounting purposes.

4.1. Hematoxylin Staining

1. Fix inserts with ice-cold 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 130 mM NaCl, 10 mM Na₂HPO₄, 10 mM NaH₂PO₄, pH 7.2) for 20 min. Wash once in PBS.
2. Submerge the insert in 0.1% Mayer's hematoxylin solution for 2 min.
3. Gently wash the insert with tap water (by submerging the insert in a beaker containing tap water and gently pipetting the excess water out). Transfer the insert to Scott's tap water solution (2 g/L NaHCO₃, 20 g/L MgSO₄) for ~20 sec until the desired blue color develops. Wash the insert again with tap water (section 4.1.3). An optional eosin stain can be introduced here by dipping the insert in 1% eosin solution for 10 sec followed by a tap water wash.
4. Air-dry the insert membrane. Using a scalpel (cutting around the membrane edge) remove the membrane and mount it on a glass slide for bright-field imaging
   Note: If di-N-butylphthalateinxylene (DPX) mounting medium is utilized wash the insert with 100% ethanol followed by xylene before removing and mounting the membrane.

4.2. Scanning Electron Microscope (SEM)

1. Fix inserts with ice-cold 4% (w/v) PFA in PBS for 20min. Wash three times in PBS.
2. Post-fix the membranes for 30 min in 1% (w/v) osmium tetroxide in MQH₂O.
3. Wash inserts in water (section 4.1.3) and dehydrate in an escalating ethanol gradient (50%, 70%, 90% for 5 min each, then 3x with 100% ethanol for 10 min per interval).
4. Treat dehydrated inserts with a mixture of hexamethyldisilazane (HMDS):ethanol (1:2 then 2:1 for 5 min).
5. Treat inserts with 100% HMDS for 5 min, air-dry and store under desiccated conditions.
6. Remove the membrane using a scalpel (cutting around the membrane edge). Examine membranes by scanning electron microscopy.

4.3. Immunofluorescence

1. Fix inserts with ice-cold 4% (w/v) PFA in PBS for 20 min, wash once in Tris-buffered saline (TBS; 50 mM Tris-HCl pH 7.6, 154 mM NaCl, 0.22 µm filtered ).
2. Perform immunostaining of the cells on the insert membrane as previously described²⁶.
3. Remove the membrane using a scalpel (cutting around the membrane edge) and mount it on a glass slide with fluorescence mounting medium. Examine membrane by fluorescent microscopy.

Results

In order to establish a human, contact BBB model we had to cultivate SVGs and BECs on porous membranes with a 3 µm pore-size, shown to permit passage of astrocyte end-feet for contact with endothelial cells^14,15,27,28. A schematic representation of the complete contact model is illustrated in Figure 1 (left illustration). The main technical challenge presented by a contact system is the need to seed astrocytes against gravity on the abluminal surface of the membrane. We have succeeded in this ...

Discussion

Medical research into brain pathologies suffers much translational difficulty. In the area of acute ischemic stroke, for example, many drugs which showed great promise in animal models failed at the clinic^38,39. The reasons for these disappointing results are diverse and include infidelity of the preclinical test systems to the human stroke scenario and overstatement of results obtained from animal studies²¹. One strategy to improve the predictive value of preclinical findings to the clinical phase ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Attachment Factor	Cell-Systems Corporation	4Z0-210
Brain microvascular endothelial cells (BECs), primary, human	Cell-Systems Corporation	ACBRI 376
Complete medium	Cell-Systems Corporation	4Z0-500	Supplemented with CSC JetFuel
Complete serum-free medium	Cell-Systems Corporation	SF-4Z0-500	Supplemented with CSC RocketFue
DMEM/F-12 with 15 mM HEPES	Life Technologies	11330-032
Endothelial cells growth supplement (from bovine origin)	Sigma-Aldrich	E2759-15MG
External silicone tubing	Watson-Marlow	913.A080.016	Pumpsil brand, 8 mm internal diameter, 1.6 mm wall
Foetal calf serum	Lonza	14-501F
Gentamycin sulfate	Life Technologies	15750-060
Heparin sodium	Pfizer		1,000 U/ml
Hexamethyldisilazane (HMDS)	Sigma-Aldrich	H4875
Human brain microvascular endothelial cells	Cell-Systems Corporation	ACBRI 376
Internal silicone tubing	Watson-Marlow	913.A032.016	Pumpsil brand, 3.2 mm internal diameter, 1.6 mm wall
L-Glutamine	Life Technologies	25030
Mayer’s hematoxylin solution	Amber Scientific	MH
Minimum essential medium with Earle’s balanced salt solution	HyClone Laboratories	SH30244.01
Penicillin/Streptomycin	Life Technologies	15140
Rat collagen I	Trevigen	3440-100-01	Cultrex brand
Tissue culture inserts	Corning Life Sciences	3472	Transwell brand, 6.5 mm in diameter, with polyester porous membrane, 3 µm pore-size