The overall goal of this procedure is to set up a highly reproducible in vitro model of rat blood-brain barrier. This is accomplished by removing the optic nerves, the cerebellum and the meninges under a stereo microscope, then separating the two cerebral hemispheres and dissecting them to obtain a clean piece of cortex. The second step is to isolate the microvessels from the cortex by enzymatic treatment and density dependent centrifugation.
Next, the microvessels are plated and incubated with pur mycin to purify the endothelial cells from contaminating cells such as parasites. Pure cultures of wrap brain endothelial cells proliferate until cells reach 90%confluence. The final step is to induce differentiation of the rat brain endothelial cells by the cells in the upper chamber of the insert filters and by co culturing them with rat astrocytes that have been previously prepared and plated.
Ultimately, the differentiated brain endothelial cell monolayer can be characterized by the expression of specific tight junction proteins and by the permeability index of a reference compound across the endothelial cell monolayer from the upper compartment. The in vitro BBB models, such as the one presented here are useful to explore the cellular distribution of key molecules and to unravel basic cellular and molecular mechanisms of BBB function. In particular, such models allow the exploration of BBB receptors their involvement in endocytosis trafficking and transcytosis in our hands.
The in vitro BBB models are mainly used to investigate the targeting of specific receptors with peptide vectors that we develop To facilitate CNS drug delivery across the BBB, we use a process that is called receptor mediated transcytosis, also known as the Trojan horse approach. Visual description is critical as individuals. New to this method will require dissection skill and there's a microscope to be fast and accurate enough to enter the tissue survival during cell production process.
Another critical point is a precocious ending of the fragile and the tele salmon area to avoid barrier opening during experimentations. Finally, this idea will show details along the protocol which will make the difference between red and visualized protocols demonstrating the procedure will done by myself and my colleague. Begin this procedure by coating two T 75 flasks with collagen type four and fibronectin at one microgram per square centimeter in sterile culture water, and allow them to adhere in the incubator at 37 degrees Celsius until micro vessels seeding.
Two days later, remove the brain from the skull of three five week old Vista rats and transfer the brains into a Petri dish filled with cold dissection buffer held on ice. Dissect out the brains without the cerebellum and optic nerves. Then cut each brain in half to separate the two cerebral hemispheres under the stereo microscope.
Make another cut to remove the midbrain and transfer all four brains into a clean Petri dish on ice with dissection buffer. Then transfer one forebrain into a new Petri dish filled with cold dissection buffer. Hold it with a clamp and carefully detach the meninges from the edges of the forebrain.
Afterward, turn the forebrain over and carefully pull off the meninges without tearing them. Make sure that the forebrain is cleared of meninges and cut the remaining part of the optic nerves. The aim of this step is to remove the duvet of white matter to obtain a shell of cortex.
Start by clamping the forebrain at the edge and then carefully drag the forceps along the surface, taking care not to tear the surface of the cortex. Then make sure that the brain is free of the meninges by rolling it over to check that there are no residual meninges or big surface vessels. Next, starting with the extraction of the three brains from their skull, the full time for the dissection should not take more than two hours for tissue preservation and cell survival.
Transfer the cortices of three brains to six milliliters of cold dissection buffer in a seven milliliter down homogenizer subsequently dissociate the cortices with 10 up and down strokes with the first pestle of larger clearance followed by 10 up and down strokes with the second pele of smaller clearance. Then transfer six milliliters of the suspension into a new 50 milliliter Falcon tube and wash the downs homogenizer with 24 milliliters of coal dissection buffer, divide the suspension into three falcon tubes to obtain the equivalent of one cortex per tube. Afterward, centrifuge at 1000 Gs for five minutes at room temperature.
Observation of the pellet shows a gradient of pink color, which represents the micro vessels part of the cortices. Homogenate now discard the snat and homogenize the pellet from one cortex with one milliliter of enzymatic solution containing a mix of collagenases dyse, DNA type one and Gentamycin. Place the tubes in a shaker for 30 minutes at 37 degrees Celsius.
Then mix one milliliter of digest with 10 milliliters of 25%B-S-A-H-B-S-S one X, and gently vortex the tubes subsequently separate them by density dependent centrifugation at 3, 600 Gs for 15 minutes at room temperature. After the density dependent centrifugation, the pellet contains the microvessels and the upper disc contains myelin brain parenchyma and residual microvessels. Carefully transfer the upper disc and the supernatant into clean 50 milliliter falcon tubes.
Take care not to aspirate the micro vessels pellet gently vortex the tube and repeat the density dependent centrifugation during the second centrifugation. Keep the resulting pellet containing the microvessels at four degrees celsius. After the second density dependent centrifugation carefully discard the upper disc and the snat mix and resuspend the resulting pellets containing the microvessels from both centrifugations with one milliliter of cold HBSS one x.
Next, wash the microvessels with 20 milliliters of cold hbss one x and centrifuge at 1000 Gs for five minutes. Then discard the supra natant, taking care not to lose the micro vessels. Pellet homogenize the micro vessels pellet from one cortex with one milliliter of the same enzymatic solution following a gentle vortex of the tubes.
Further digest the microvessels for one hour at 37 degrees Celsius in a shaker. After that, mix the digested micro vessels from the three cortices into one tube. Again, separate the mixture into two 50 milliliter falcon tubes to obtain the microvessels extracted from the equivalent of one and a half cortices per tube.
Then centrifuge at 1000 Gs for five minutes. At room temperature just before plating the microvessels, take two coated T 75 flasks from the incubator and aspirate the excess of coating. Discard the snat and resuspend the micro vessels pellet in one milliliter of ECM, bring each tube to 10 milliliters with ECM supplemented with four micrograms per milliliter.
Pur mycin to start the purification process and plate the microvessels in the flask. Then put the flasks in the incubator at 37 degrees Celsius and 5%CO2 to allow micro vessels adhesion to the collagen fibronectin matrix to follow the purification process. 24 hours after micro vessels plating, wash the cell debris with fresh ECM and replace the culture media with ECM supplemented with four micrograms per milliliter pur mycin until day five in the incubator at 37 degrees Celsius and 5%CO 2 24 hours after micro vessels plating the endothelial cells start to migrate and spread on the collagen fibronectin matrix on day five to finish the purification process, wash the cell debris with fresh ECM and replace the culture media with ECM supplemented with two micrograms per milliliter.
Puram mycin for three more days for proliferation. On day eight, wash the cells and add insulin transferrin and sodium selenite. Supplement to the culture media until the cultures reach 90%confluence on day 10.
On day eight, prepare the filters of three 12 well plates by coating them with a mix of collagen. Type four and fibronectin both at 0.5 micrograms per square centimeters in sterile culture water. Start by adding 1.5 milliliters of sterile culture water in the lower compartment.
Then add 0.5 milliliters of mix in the upper compartment. Allow the matrix to adhere at 37 degrees Celsius until seeding of the endothelial cells on the same day, five days before the establishment of the co-culture. Thaw the astrocytes from cryo vials at 37 degrees Celsius and transfer the cells to a 15 milliliter falcon tube with 10 milliliters of GCM centrifuge.
The suspension at 120 Gs for eight minutes at room temperature. Then resuspend the astrocyte pellet in GCM and plate at a density of 30, 000 cells per square centimeter. In the 12 well plates, put the 12 well plates in the incubator at 37 degrees Celsius, 5%CO2 on day 10 just before the dissociation of RBEC with trypsin.
Wash the pre-coded filter with DMM F 12 medium pre-fill the chambers with ECM and put them back in the incubator until RBEC plating on the same day. Wash the RBEC twice with DPBS without calcium and magnesium. Add four milliliters of warm trypsin EDTA solution per T 75 flask at 37 degrees Celsius for exactly 30 seconds and remove 3.5 milliliters of trypsin EDTA solution.
Taking care to always have a layer of trypsin EDTA covering the endothelial cells. Then follow the detachment of cells by observation under the microscope. When the cell layer starts to detach from the matrix, gently tap the edge of the T 75 flask until all the cells are floating.
Add three milliliters of ECM to each T 75 flask and transfer into a 50 milliliter falcon tube. Repeat this step twice. By flushing the medium to retrieve all the cells.
Very gently dissociate the cell suspension by pipetting up and down four times with a 10 milliliter pipette equipped with a yellow tip. Count the cells in the suspension and immediately plate them on the pre-coded and pre-filled filters at a high density on the following day. Change the medium of the upper compartment twice to remove cell debris in order to avoid cell monolayer disruption during the medium replacement.
Slightly angle the plate and do not approach the aspiration pipette always leaving some media above the cell monolayer similarly, to avoid drying of the cell monolayer during medium replacement. Wash only six inserts at a time. 24 hours after plating the cell monolayer has to be at confluence.
Then on the same day, one day before the establishment of the co-culture, replace the astrocyte culture media with 1.5 milliliters of supplemented differentiation media on day 12, which is defined as the first day of differentiation. Replace the culture media from the filters containing the RBEC with differentiation media. Afterwards transfer the RBEC filters into the wells containing the astrocytes under these conditions.
In vitro models differentiate and express junction related proteins within three days and the optimal differentiation lasts for three more days. Here the cell monolayer was immunostain with menton to reveal a confluent brain endothelial cell monolayer with non-overlapping morphology and the typical spindle shaped cells with endothelial morphology, as well as the expression of tight junction proteins clawed in five ZO one and occludin monolayer tightness in the 12. Well, BBB model was assessed by measuring the transport of Lucifer yellow.
The results are expressed in permeability or PE in 10 to the minus three centimeters per minute, and the barrier is considered permeable or open when the PE of LY is above 0.6 times 10 to the negative three centimeters per minute. In the last decades, many publication and reviews provided information on static and dynamic in vitro BBB model from different species. However, such models remain difficult to handle and the reproducibility of the barrier quality remains a major goal ago.
After watching this video, scientists should have a good understanding of how to produce and characterize an in vitro model of the PBB.
