An endothelial and astrocyte co-culture model of the blood–brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane

Abstract

The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.

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

“…A few studies made use of scanning electron microscopy to identify astrocytes processes which pass from the abluminal membrane surface (where astrocytes are seeded) to the luminal surface, as a proof of principle that contact between astrocytes and EC is plausible 14,15,27 . Following these studies we imaged by SEM the luminal membrane after seeding SVGs on the abluminal surface.…”

“…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).…”

“…Importantly, true contact between BECs and astrocytes occurs only on membranes with larger pore diameter (≥1 µm), which permit passage of astrocytic end-feet and subsequent induction of tighter (less permeable) BEC monolayers 14,15,27 . However, the seeding of astrocytes on inverted inserts with higher pore size -the core technical element in the preparation of a contact BBB model -presents a double technical challenge, since in addition to the limited medium volume which can be added to the inverted 6.5 mm inserts (~50 ul), the membranes are also prone to leakage of medium due to their large pore diameter.…”

Biomedical Technologies for In Vitro Screening and Controlled Delivery of Neuroactive Compounds

“…Some membranes also do not permit transmission of light for microscopic analysis. Attempts have been made to produce lower resistance membranes of thinner material containing larger pores [148]. However, these culture systems have not been well characterized in terms of reproducible fabrication, cell transport properties, and tight junction formation.…”

“…In vivo permeability is determined by intravenous administration of a compound, followed by attempts to determine the fraction that passes into the brain tissue. In vitro permeability screens can be accomplished by assay of the transport of molecules across endothelial cells grown as a monolayer on a membrane or other permeable surface [147,148]. Compounds commonly used in permeability studies include dyes such as Evans blue or Prussian blue.…”