This procedure aims for an in vitro simulation of the blood-brain barrier by co culturing of astrocytes and endothelial cells on opposite sides of a porous membrane. First coat the luminal surface of the porous membrane with an extracellular matrix. Protein like collagen fit the inverted insert with an external silicone tubing and an internal silicone plug to create a removable well above the abluminal surface of the membrane.
Now seed the astrocyte cells on the inverted insert so that they adhere to the abluminal surface. Then remove the tubing and place the insert in its normal orientation inside a medium filled well. Next seed the endothelial cells into the luminal compartment of the insert and co-culture with the opposing astrocytes.
Ultimately, the passage of fluorescent tracers or recording of trans endothelial electrical resistance is used to measure permeability changes in the in vitro BBB in response to various stimulating agents. Demonstrating this procedure will be Mr.Barry Nigo, a postdoc in my laboratory, who during the course of his PhD, developed this modified seeding protocol that we have used to simulate the blood brain barrier. We first had an idea of this procedure after testing more crude seating methods and having a much frustration with leakage of medium through the pause, as well as small medium volumes.
This dictated a short and insufficient astrocyte seating periods, which we are trying to tackle in our procedure. Place the tissue culture inserts in the wells of a 24 well plate. Then add 50 microliters of 132 microgram per milliliter rat collagen, one to coat the luminal surface of the inserts, incubate overnight in a humidified 37 degrees Celsius incubator in order to remove any residual acid, wash both the abluminal and luminal sides of the inserts with double distilled water.
Then invert the inserts and gently fit a short piece of elastic silicone tubing around the rim of the porous membrane. Essentially creating a new well above the abluminal surface. To seal the underside.
Prepare a plug made of silicone tubing sealed at one end with the cut tip of a 0.2 milliliter PCR tube. Now using a sterile forcep, insert the silicone plug into the luminal cavity and advance it until it reaches up to approximately one to two millimeters from the membrane. Next seed, 40, 000 astrocytes in 200 microliters of their maintenance medium directly into the silicone well above the abluminal membrane surface.
To monitor the adherence state of the astrocytes, use a standard 96 well plate as it has the same surface area as the 6.5 millimeter insert and permits. Easier visualization of cells by phase contrast microscopy seed, an identical volume of the astrocyte cell suspension into the 96 well plate eight. This will be monitored over time to determine when the astrocytes in the 96 well and by extension in the insert membrane have sufficiently adhered to maintain sterility.
Transport the assembled inserts in between two six well plates to minimize exposure to unfiltered air. Place the cells in the incubator to allow the astrocytes to adhere. Also place the control 96 Well plate into the incubator at this time.
After about four hours, check the control 96 well to see if the astrocytes have adhered. If so, then transfer the insert assembly back to the tissue culture hood. Gently remove the external silicone tubing and plugs.
Then return the inserts to the normal upright orientation into wells containing 800 microliters per well of endothelial cell maintenance Medium. See 20, 000 brain endothelial cells in 200 microliters onto the collagen coated luminal surface co-culture for three days in BEC medium without any media change. Before experimentation.
Wash the silicone tubes and plugs in ethanol before autoclaving for later use. To stimulate the in vitro blood brainin barrier, first, replace the abluminal and luminal media with 600 microliters and 100 microliters of serum free becs medium respectively. Aspirate the luminal wash and replace with 100 microliters of serum free medium containing stimulating agents.
If inducing the in vitro BBB from the astrocytic compartment, then aspirate the abluminal medium and replace with 600 microliters of serum free. Medium containing stimulating agents continue with the standard paracellular or transcellular permeability assays of the label tracers in order to establish a human contact blood-brain barrier model. Human immortalized, astrocytes and brain endothelial cells are cultivated on three micron porous membranes that permit passage of astrocyte and feet for contact with endothelial cells.
The method allowed an optimal uninterrupted seeding period of astrocytes and becs, which in turn attach strongly to the porous surface with minimal cell loss and grow to co fluency by three days. Post seeding. Both cell types maintain their cell specific markers on porous membranes as indicated by the expression patterns of glial fibrillary acidic protein GFAP in SVGs and von Vand factor VWF NECs.
Consistent with the most fundamental feature of a contact BBB model, the astrocytic NFE can pass through the three micron pores into the luminal compartment. Thus, contact can potentially exist between SVGs and BES seated on pores of this dimension. Furthermore, the SEM imaging indicates that SVGs and BBCs were capable of growing directly over the membrane pores a phenomenon which artificially contributes to the physical barrier function of in vitro BBB contact models.
In this study, the human contact BBB model is used to explore the effect of TPAA fibrinolytic agent on the BBB. These in vitro studies confirm that TPA indeed increased the permeability of the intact BBB in a concentration and time-dependent manner, which were both within a pharmacological relevant range. Dramatic morphological changes of SVG astrocytes are evident on the porous membranes post exposure to TPA suggesting that astrocytic response to TPA underlie TPA induced BBB opening.
This method played a key role in our research, which was aiming to investigate how the blood brain barrier responds to TPAA thrombolytic drug used in stroke patient to dissolve blood clots. It is the first demonstration of this method in the literature, and we hope that other researchers could adopt it for their own purposes.
