The overall goal of this procedure is to prepare pure cultures of endothelial cells from embryonic four brains to study cerebral angiogenesis and neurovascular interactions. This is accomplished by first peeling away the peel membrane and isolating the cephalon from embryonic brains. The second step of the procedure is to produce a single cell suspension by using dissociation, filtration and digestion of the harvested tissue.
The third step is to magnetically label the cells using CD 31 microbeads. The final steps are to purify the CD 31 labeled cells by magnetic separation and to then culture them. Ultimately, pure populations of paraventricular endothelial cells are generated for further study through immunofluorescence microscopy and angiogenesis assays.
This method will benefit studies focusing on cellular and molecular mechanisms. For brain anis, it can serve as a tool for elucidating, pre ventricular endothelial cell interactions and crosstalk with neuronal cell types. It also holds tremendous potential for therapeutic ansis Mission.
Demonstration of this method is critical to eliminate some of the technical difficulties associated with isolating and culturing pure populations of endothelial cells from embryonic for brain. For this protocol, have heads of E 15 pups from CD one dams in ice cold PBS under a stereoscope. Remove the brains with fine microtip, scissors and fine forceps.
A good dissection is critical and will come with practice and with having the tools in excellent condition. Next, use the forceps to get a firm grip on the brain and the microtip scissors to peel away the peel membrane. This step is the most challenging of the procedure, but the skills necessary will come with practice.
Then remove the mesencephalon and met Cephalon. Isolate the cephalon. Next, transfer it free of peel membrane to a 35 millimeter culture dish with two milliliters of PBS on ice.
Collect all the talons in a single dish. Begin by mincing the talons into one or two millimeter fragments with a scalpel blade. Collect the tissue in a 15 milliliter tube containing two milliliters of complete DMEM.
Dissociate the tissue gently but thoroughly by tri with a one milliliter pipette until the clumps disappear and a milky suspension is obtained. Centrifuge the suspension at 800 to 1000 G for five minutes. At room temperature, carefully remove the supernatant by aspiration and resus.
Suspend the palate in DMEM with DS one. Now dissociate the cells again using gentle tation and repeat the centrifugation step. Then resuspend the pellet in warmed complete DMEM.
Filter the suspended cells through a 70 micron nylon mesh and collect the cell portions retained on the mesh and digest further in two milliliters of DMEM with collagenase and dis bays. Wait a couple of minutes and then wash the tissue. Three to using centrifugation and resus of the pellet in DMEM with DS one pool, the washed collection with the filtrate kept on ice.
Then wash the combination with complete DMEM using centrifugation and resus. Now determine the cell number with a hemo cytometer and proceed with magnetic labeling. The volumes in this protocol are good for up to 10 million cells For greater cell numbers just scale upwards.
Begin by centrifuging the cell suspension at 300 G for 10 minutes at room temperature, completely aspirate the supernatant and add 90 microliters of chilled purification buffer to the cell pellet, followed by 10 microliters of CD 31 conjugated micro beads. Mix the cells well using a 200 microliter pipette and incubate the suspension for 15 minutes in a refrigerator but not longer so that labeling remains specific. Wash the cells by adding one to two milliliters of chilled purification buffer and centrifuging at 300 G for 10 minutes.
Remove the supernatant and resuspend the cells in 500 microliters of chilled purification buffer. Now load an MS column into the magnetic field of the max separator. Next, rinse the column with 500 microliters of purification buffer at room temperature.
Then apply the cell suspension to the column. It is good for up to 20 million cells. Collect and discard all the flow through which contains the unlabeled cells.
Wash the column with 500 microliters of purification buffer three times. Always make sure that the column reservoir is empty before adding subsequent purification buffer aliquots. Now remove the column from the max separator and place it on a 15 milliliter tube.
Add one milliliter of purification buffer to the column and immediately flush out the magnetically labeled cells by firmly pushing the supplied plunger into the column plate. The flushed out cells on a 35 millimeter culture dish pre-coded with collagen. Type one.
Now grow the cells with ECCM in an incubator set to 37 degrees Celsius with 95%oxygen and 5%carbon dioxide. Change the medium every four days. Observe the cells growing in the culture dish through a microscope every day.
The phenotypic characterization of periventricular endothelial cells or PVE CS changes from day one to day 12 as seen by phase contrast light microscopy. The cells attached to the dish on day one, show morphology characteristic of cell division between five to eight days. The PVE CS transition from a cobblestone to a spindle shaped morphology typical for endothelial cells and more akin to their in vivo state.
By day 12. The PVEC culture achieves full confluence. The p vs can be subculture easily to expand the colony after subculture.
The purity of the endothelial cell cultures was determined to be 100%using the following endothelial cell markers. ISO selectin before von Willbrand factor CD 31 PE cam one and VE cadherin high magnification images of PV Cs prepared from CD one embryos as well as tie two GFP embryos whose endothelial cells expressed GFP were labeled with ISO lectin before they showed the common cobblestone and spindle shaped morphologies as well as polygonal morphologies with slender processes in some collagen coated culture dishes. In the absence of matrigel pve, CS formed lattice patterns resembling the unique characteristic of the in vivo periventricular vascular network.
This reflects the high angiogenic potential of pve. Cs.An angiogenesis assay was performed in which 20, 000 PVE CS were added to culture dishes coated with matrix gel matrix. These PV Cs showed robust tube formation within 18 hours.
Long-term study of pve CS including migration, motility morphology, cell function assays, as well as in vivo experiments is possible by labeling PV vs. Isolated from CD one embryos with Q dot nano crystals. These nano crystals deliver an intense stable fluorescence into the cytoplasm of living cells.
Pve EECS can be transfected with cell tracers like cell light plasma membrane. RFP back MAM 2.0 for clear visualization of cell morphology in live cell imaging experiments. Pve CS were cultured in several medias and the cell morphology was compared by phase contrast microscopy and ISO selectin before staining.
While PV CS grown in ECCM showed spindle shaped morphology and were confluent by day 12, pve CS grown in rat brain endothelial cell growth medium showed very elongated morphology and were confluent by day 20. On the other hand, PV CS cultured in D-M-E-M-F 12. Media showed only flat and polygonal morphology with no spindle formation, had very rapid growth and were conent by day four.
Pve eecs have been grown in ECCM for up to three passages without loss of phenotype and function. Therefore, ECCM is the medium preferred for PVEC culture. Once mastered, this stick can be done in three hours if it is perform properly.
Co culture of periventricular endothelial cells with neuronal precursors is likely to stimulate neurogenesis and neuronal migration much better than endothelial cells from adult brain or other sources. They may thus be valuable for aiding recovery of neurological function in many different scenarios.
