METHOD FOR ISOLATING THE ENDOTHELIUM OF RAT BRAIN MICROVESSELS Russian patent published in 2022 - IPC G01N33/50 

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, in particular to neurology, and can be used for isolating and cultivating the endothelium of brain microvessels. The method includes: extraction of the rat brain, removal of the meninges and large vessels, dissection of the cerebral cortex in cold Hank's solution and its grinding, tissue dissociation by pipetting 25-30 times, sedimentation by centrifugation to separate microvessels from neuronal tissue, digestion of 0.1 % collagenase II solution at 37°C, precipitation and removal of the enzyme, followed by inoculation on cultural plastic in a growth medium, characterized in that the brain of a rat aged from 14 to 20 days is used, while the cerebral cortex is crushed into pieces of 5-7 mm in size, 3 milliliters of Hank's solution is added, and tissue dissociation first, pipetting is carried out 25-30 times with a Pasteur pipette with a wide opening - 1.5-2 mm, after which sedimentation is carried out by centrifugation, the supernatant is removed, another 3 milliliters of Hanks' solution are added and again pipetted 25-30 times with a Pasteur pipette with a thin lumen - 0, 5-0.7 mm, then centrifuged for 5 minutes at 1000 rpm, the supernatant is removed and a cold 4-8°C 25% solution of bovine serum albumin (BSA) is added in sodium phosphate buffer solution (PBS), centrifuge again for 10 minutes at 2000 rpm, then the supernatant is pipetted another 25-30 times and centrifuged for 10 minutes at 2000 rpm, a part of the dissociated tissue over BSA is taken along with an albumin solution, 3-4 milliliters of Hank's solution is added to the sediment and the sediment is centrifuged for 5 minutes at 1000 rpm, remove the supernatant, add 2 ml of 0.1% collagenase II solution in PBS to the sediment and pipette again, then, the tube with the tissue is placed in a roller installation at 37°C for 40 minutes, then after incubation, 5 milliliters of Hank's solution is poured into the tube to stop the digestion process, centrifuged for 5 minutes at 1000 rpm, the supernatant is taken, the cell mass is resuspended in growth medium and seeded on culture plastic coated with Matrigel, while the growth medium has the following composition per 100 ml: DMEM/F-12 Gibco - 76 ml; fetal calf serum 20% -20 ml; GlutaMAX Gibco x100 - 1 ml; glucose 40% - 750 mcl; hydrocortisone 1.4 mm - 100 mcl; sodium pyruvate 11 mg/ml - 1 ml; antibiotic (penicillin-5000 units/ml: streptomycin-5000 mcg/ml) - 1 ml.

EFFECT: application of the invention provides an expansion of the arsenal of technical means for isolating the endothelium of rat brain microvessels with a better separation of microvessels from neuronal and glial cells and a higher yield of endothelial cells.

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