Neuroscience
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A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain
Chapters
Summary April 9th, 2019
Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.
Transcript
This human blood-brain interface model is useful for studying the transfer of molecules into the neuro system. As well as for studying of microbes reach an infected brain pain tumor. The addition of the mini brain to this blood brain barrier or BBB model allows the study of our pathogens and molecules that cause the BBB behave in the brain.
Demostrating the procedure will be Florian Bakao, the PhD students for my laboratory. To set up a BBB mini brain polyester membrane culture insert device, dilute the cells to a five times 10 to the fourth cells per insert concentration incomplete endothelial cell medium and add the appropriate volume of cells to each polyester membrane culture insert in a 12 well plate. Then, place the inserts and the cell culture incubator until the cells reach 100%confluency.
Hold a 12 well plate with one milliliter of poly-D-lysine her well for four hours at room temperature. Followed by one milliliter of laminin per well overnight. Replace the laminin with one milliliter of endothelial cell medium supplemented with 5%fetal bovine serum, or FBS.
Place the place in an incubator for one hour. To prepare the mini grain gently trypsinize the human neurons astrocytes in CHME clone five cells. Associate the cell pellets with complete endothelial cell medium.
After counting at 3.6 times 10 to the fifth neurons and astrocytes to 0.4 times 10 to the fifth CHME clone five cells per well have a 12 well plate. And seed the 12 well plate. To construct the BBB mini brain 24 hours after seeding replace the used medium with fresh complete endothelial cell medium and transfer the cerebral micro vascular endothelial cell coated polyester membrane culture inserts over the mini brain cells.
Then, place the BBB many brain device into the incubator. To validate the BBB mini brain endothelial permeability add 1.5 milliliters of transport buffer per well to a new 12 well plate. And flip each filter upside down to carefully remove the medium without affecting the endothelial cell barrier.
Place each filter in one well of the transport buffer filled plate, and add 0.5 milliliters of Lucifer yellow into each well. Then place the plate into the incubator for 10 minutes before transferring the filters into a new transport buffer filled 12 well plate. After setting up a BBB many brain device as demonstrated, add 3500 platforming units of the French neurotrophic virus strain of yellow fever virus diluted and 50 microliters of 2%FPS endothelial cell medium, very carefully, on top of luminal compartment.
Inoculate the control BBB mini brain with 50 microliters of 2%FBS endothelial cell medium without virus. Then place the virus expose BBB many brain device in the incubator for 24 hours. The next day, remove the inserts to determine the endothelial cell permeability and a companion well as just as demonstrated.
Save one milliliter of sample from each well to determine the virus titer in the middle compartment. Under the insert gently to avoid leakage of the luminal compartment into the abluminal compartments. To study the transport of a neuron targeting biomolecular across the blood brain barrier, add the bio molecule of interest to the inserts and return the plate to the cell culture incubator.
After 24 hours remotely inserts and determine endothelial permeability. Then stain the many brain cells with the appropriate antibody for detecting the presence of the bio molecule of interest within the mini brain. Human cerebral micro vascular endothelial cells express all the subsets of rececptors, Efflux transporters or transporters that are key relevant proteins for their biological functions.
Some French neuro tropic virus strain of yellow fever virus can cross the blood brain barrier at 24 hours. The viruses then amplify for the next two days by the multiplication of the virus inside the brain cells. Further, the expression of specific viral biomarkers is stimulated when this neuro invasive viral population is serially passaged on the mini brain cells and strictly correlate to the viral load.
After being added to the limit of compartment cell penetrating molecule Neuro-Tag Neurovita crosses the blood brain barrier and is able to target human neurons. Cell penetrating molecule neuro tag Delta neurovita crosses the endothelial cell barrier, but targets less efficiently the human neurons. After being added to the little middle compartment, cell penetrating molecule Neuro-Tag Neurovita crosses the blood brain barrier and is able to regenerate the axons of human neurons, after wounding.
In contrast to cell penetrating molecule Neuro-Tag Neurovita Delta, the inactive form of Neurovita. When applied after the axon scratching cell penetrating molecule Neuro-Tag Neurovita triggers the neuro protection of the wounded neurons and the x zonal regeneration. Strictly for the good sake of practices.
do not over use I-cell passages. Try to maintain some semblance of medium and regions and check for the absence of mycoplasma. Interactions between the mini brain and the drugs are microbes of interest can be further studied by transcript that chemicalize this or classical biology called techniques.
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