The overall goal of this procedure is to obtain primary murine, brain microvascular endothelial cells or MBX. This is accomplished by first isolating and then homogenizing the meninges free forebrains of adult mice. In the second step, the tissue homogenate is further processed by enzymatic digestion.
Then in the final step, the endothelial cells are separated by density, gradient, centrifugation, and plated for cell culture. Ultimately, immunofluorescent staining of the isolated cells for CD 31 expression can be used to confirm the purity of the MB mech culture. The main advantage of this technique of existing methods, like the usage of immortalized brain, endothelial cell lines, is that the results obtained with primary cells provide a better transferability to the in vivo situation.
This method can help answer key questions in the field of blood brain barrier search, for example, which pathways are involved in immune cell thinking. The implications Of this technique extend towards the therapy of neurovascular, infectious, inflammatory, or degenerative diseases of the central nervous system. As blood-brain barrier dysfunction is critically involved in the pathogen of such diseases.
This procedure will be demonstrated by Lisa Eeping, a grad student from our lab. After isolating the brains from ten eight to 12 week old C 57 BL six mice store the tissues in five milliliters of sterile PBS. Next, use a forceps to remove the brain stems, cerebella and thalami, and then carefully roll each brain on sterile blotting paper.
To remove the meninges pool the brains in a 50 milliliter falcon tube and add 13.5 milliliters of DM em then mince the tissue first with a 25 milliliter pipette, and then with a 10 milliliter pipette until the media becomes milky. Digest the tissue in collagenase and DNA at 37 degrees Celsius on an orbital shaker at 180 RPM. After an hour, add 10 milliliters of DM em to the tissue suspension and centrifuge the cells for 10 minutes at 1000 Gs and four degrees Celsius.
Discard the supernatant taking care not to disturb the loose pellet, and then use a 25 milliliter pipette to tri the pellet approximately 25 times in 25 milliliters of B-S-A-D-M-E-M. After spinning down the cells again, use a broken glass pipette with a large diameter to remove the milky upper myelin layer. Then remove the BSA layer with a normal PEs or pipette.
Resuspend the pellet in nine milliliters of DM em, and then add one milliliter of collagenase disc space and 0.1 milliliter of DNAs to the cells. Digest the solution at 37 degrees Celsius on the orbital shaker at 180 RPM. During the digestion set up a perca density gradient in an ultracentrifuge.
After an hour, add 10 milliliters of DM E em to the digested cell suspension to stop the reaction and then spin down the cells and resuspend the pellet In two milliliters of DM E em, add the cell suspension carefully on top of the per call gradient, and then separate the cells by centrifugation. After the separation, the cells may be visible as a cloudy interface. Carefully dip along sterile needle into the interface at a 30 degree angle and collect approximately 12 milliliters of the solution.
The interface is often not visible to obtain as many cells with as little contamination as possible. A small amount of the interface should be aspirated. In addition to that, the tip of the sterile needle should be carefully and continuously move to every part of the interface, Transfer the cells into a new 50 milliliter Falcon containing five milliliters of DM em, and then after spinning down the cells again, reus, suspend the palate in 0.2 milliliters of endothelial cell medium per one square centimeter surface of a cell culture plate.
Next, remove the coating solution from coated cell culture plates and rinse every well with sterile PBS in two consecutive washing steps. Finally, maintain the cell cultures in endothelial cell medium at 37 degrees Celsius and 5%carbon dioxide in a sterile incubator. Changing the medium every two to three days immediately after isolation.
Murine BM e and contaminating cells form conglomerates that can be observed floating in the cell culture. Medium treatment with mycin leads to the selection of murine BMAX as contaminating cells are much more susceptible to pur mycin mediated cytotoxicity and the unaffected BM E begin to attach to the collagen four fibronectin coated cell culture plates until day five, the BME proliferate to a density of approximately 80 to 90%forming a monolayer of tightly packed, longitudinally aligned and non-overlapping contact inhibited cells characterized by their typical spindle shaped appearance by puram Mycin selection, A high purity of up to 99%murine BMAX is reached as confirmed by the endothelial cell marker CD 31. The BM E form tightly sealed monolayers interconnected by tight junction proteins.
After seven to eight days of culture, these cell layers build up stable values for electrical resistance between 20 to 30 ohms by square centimeter and a hearty capacitance at this time, functional assays such as cell migration experiments or permeability tests with Evans blue or dextran can be performed Once mastered. This technique can be done in four to Five hours following this procedure. Other methods like migration assays, permeability experiments, or electrophysiological measurements can be performed to address that visionary questions, for example, how is immune cell trafficking altered under certain experimental conditions?
Or how are iron channels involved in blood brain barrier regulation? After watching this video, you should have a good understanding of how to isolate urine, brain microvascular endothelial cells by mechanical homogenization, enzymatic, digestion, and density gradient centrifugation.
