Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

The overall aim of this procedure is to obtain a homogenized immortalized brain endothelial cell culture. This is accomplished by first isolating the brains from neonatal mice and obtaining a single cell suspension. The second step is to immortalize the cells with polyoma middle T antigen.

Next, the cells grow for about four weeks until a homogenous endothelial monolayer is formed. The final step is to characterize the new cell line using techniques such as immuno staining, Western blot and Q-Q-P-C-R. The implications of this technique extend to a therapy of neuroinflammatory or ischemic brain disorders because preclinical therapy evaluation and insights in pathophysiology can be obtained.

Demonstrating the procedure will be Dr.Elaine Salvador, a postdoc from my laboratory. After removing the brain from a three to five day old neonatal mouse transferred to a Petri dish containing buffer A, the brains of five to 10 mice are required for each preparation while working in a laminar flow hood. Remove the cerebellum, brainstem, meninges, and capillary fragments from each brain.

Then transfer the cerebrals to a second Petri dish containing buffer A.Use a sterile scalpel to cut the cerebrums into small pieces and then use a 10 milliliter pipette to aspirate and expel the tissue and buffer until no clumps appear. At this point, transfer the suspension to a 50 milliliter centrifuge tube after centrifuging the brain tissue at 250 times G for five minutes at room temperature. Discard the supernatant and dissolve the pellet in 4.5 milliliters of buffer A with 1.5 milliliters of 0.75%weight per volume.

Collagenase dis pace. Incubate the tissue and collagenase solution for 45 minutes in a 37 degree Celsius water bath with occasional agitation. Meanwhile, prepare the tissue culture plates by coating four wells of a 12 well plate with a sterile solution of 0.1 milligrams per milliliter.

Collagen four and 50 millimolar acetic acid allow the collagen to adhere to the wells at room temperature for one hour. Once the 45 minute incubation period has elapsed, stop digestion of the brain tissue by adding 15 milliliters of buffer A resuspend the pellet thoroughly in this solution. Then centrifuge at 250 times G for 10 minutes and discard the supernatant.

Next to remove myelin from the preparation, add 10 milliliters of sterile, 25%weight per volume B, s, A, and PBS to the tissue and centrifuge at 1000 times G for 20 minutes at four degrees Celsius. Following centrifugation, carefully discard the supernatant and then dissolve the pellet in 15 milliliters of buffer A and transfer to a fresh 50 milliliter centrifuge tube re centrifuge at 250 times G for five minutes at room temperature. Meanwhile, wash the collagen four coated plate twice with PBS.

After the second wash, leave two of the wells empty and add two milliliters of growth media to the other two wells. After discarding the supernatant resuspend the cell pellet, which is comprised primarily of endothelial cells in four milliliters of growth media containing four micrograms per milliliter, puram mycin, and plate two milliliters of the cell suspension into each of the two empty collagen coated wells incubate at 37 degrees Celsius for 45 minutes to kill rapidly adhering non endothelial cells. Next, aspirate the growth media from the two wells that do not contain cells and discard.

Then transfer the medium containing non-ad adhered cells, which includes the endothelial cell fraction into these two wells. Refill the previous wells with two milliliters of fresh medium. These wells contain other cell types such as fibroblasts and astrocytes, and can be grown in parallel and used for comparisons of the morphology.

Incubate the cells overnight in the tissue culture incubator the next day. Replace with fresh medium cultivate the GPE neo fibroblasts, which secrete a replication deficient virus with polyoma middle T oncogene in gelatin coated flasks containing GPE neomed Polyoma medial T oncogene transfection causes growth advantage of endothelial cells over non endothelial cells leading to a homogenous monolayer of cells with endothelial morphology. After four to six weeks of culture, GPE neo cells are not commercially available and can be obtained as shared public material.

After growing the GPE Neo Fiberblast for 24 hours, remove 10 milliliters of the media and add sterile poly brain solution to a final concentration of eight micrograms per milliliter. Pass the supernatant mixture through a 0.45 micron filter to remove any cellular fragments. Now aspirate the media from the endothelial cell cultures and add two milliliters of the supernatant poly brain mixture to the wells after incubating overnight.

Repeat this procedure the next day with a fresh aliquot of the supernatant poly brain mixture the following day. Remove the supernatant poly brain mixture. Wash the cells twice with PBS and then add fresh growth medium.

Continue to change the growth medium every three days when the cells become confluent passage, the cells one to two on collagen. Four coated plates. Stable cerebral endothelial cell lines are typically obtained after four to five weeks of subsequent culture.

When a homogenous endothelial mono layer fills the whole well. Once the stable cell lines have been obtained, transfer to collagen four coated tissue culture flasks passage the cells no more than once a week by trypsin with three milliliters of trypsin EDTA and incubation at 37 degrees Celsius for five to 15 minutes until the cell layer is dispersed. After the addition of five milliliters of medium seed, the new flasks avoid splitting higher than one to four.

Once there are enough cells to obtain a confluent T 75 flask, cryo quats of cells can be prepared for later use. To do this, harvest the cells with trypsin as before, then centrifuge the cell suspension at 250 times G for five minutes. At room temperature, resuspend the cell pellet in six milliliters of freezing medium, then pipette 1.5 milliliters of the cell suspension into four cryo vials.

Place the cryo vials into a Nalgene freezing container filled with isopropanol and keep them for one day at minus 80 degrees Celsius. Finally, store the cryo vials under liquid nitrogen. Vapor the cryo quad of cells in a water bath, and then transfer them to a 15 milliliter centrifuge tube containing 10 milliliters of growth.

Medium prewarm to 37 degrees Celsius and centrifuge the tube at 250 times G for five minutes at room temperature. After aspirating the medium resuspend the pellet in an appropriate volume of growth. Medium transfer the cell suspension to a collagen four coated T 25 cell culture flask with prewarm growth medium.

The cell density for plating should be at least one times 10 to the fourth cells per milliliter. Change the medium the next day and continue to passage the cells as before when the cells reach confluence around five days later when ready plate the cells at the desired density for your subsequent experiments. The C end cells were characterized by immuno staining of endothelial and blood brainin barrier markers.

This image shows the morphology of C and cells immuno stain for clain five. The C and cells have a morphology similar to primary cultures of brain ECS with monolayers of tightly packed elongated cells that exhibited growth inhibition. Net confluence, the cells expressed detectable levels of clain.

Here the C CN cells have been immuno stain with an antibody to occludin. Here we see the expression of VE cadherin proteins, which are localized at the cell cell junctions, again, as shown by immunofluorescence staining. Trans endothelial electrical resistance was measured using an assembly containing current passing and voltage measuring electrodes.

It can be seen from this figure that the trans endothelial electrical resistance of C end cells increases with time in culture After its development. This technique paved the way for researchers in the field of neuroscience to explore cerebrovascular disorders in a mouse model.