This innovative protocol is designed for LLEC cultivation by other researchers, only extravasated. We established the multi procedure to harvest and culture primary LLECs in vitro. Our protocol ultimately led to the establishment of a primary culture of LLECs with a purity level exceeding 95%There is currently no existing protocol for harvesting and culturing LLEC in vitro.
Our results pave the way for further investigating the similar function of LLECs in vitro. LLEC are a reason to discover the intracranial cellular population, the biological significance of LLEC secure annum. We will conduct further research on the cellular function and reveal its clinical implication.
To begin, carefully incise the skin of a sacrificed mouse, starting from the skull's opening and extending towards the frontal area. Using scissors, delicately lift and remove the skull without damaging the leptomeninges, collecting the entire brain. Submerge the whole brain in the washing buffer and gently flush it to remove surface blood.
Transfer the brain into a sterile Petri dish without chopping. Then, under a microscope, use fine point tweezers to extract the leptomeninges from the brain's surface. Using sterile microscissors, cut the leptomeninges tissue into fragments.
Prepare the digestive enzyme mix using the given components. Add 10 milliliters of mix to the fragments and incubate at 37 degrees Celsius for 15 minutes. Gently agitate to detach the fragments from the bottom of the tube.
Then add 10 milliliters of stopping buffer. Centrifuge the suspension at 300g for five minutes at four degrees Celsius. And using a pipette, carefully remove the supernatant.
Add 10 milliliters of cold PBS and filter any clumps through a 70 micron strainer into a sterile 50 milliliter tube. Plate 10 to the fifth cells per square centimeter into a fibronectin coated T25 flask with five milliliters of culture medium. And incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
After incubation, replace the medium with a fresh medium to eliminate non-attached cells. Begin by attaching a magnetic separator to its stand. Connect a selection column to the magnetic separator and place a 70 micron cell strainer on top of the selection column.
Place a 50 milliliter tube beneath the selection column to collect the flow. Once the mouse brain cells reach 80%confluency, aspirate the medium and rinse the cells with PBS. Add 0.25%trypsin to detach the adherent cells and incubate at 37 degrees Celsius for five minutes.
Then, add a stopping buffer. Centrifuge the cell suspension at 300g for five minutes and remove the supernatant. For antibody staining, resuspend 10 to the 7 cells in 100 microliters of PBS.
Then add 10 microliters of LYVE-1 antibody solution and mix thoroughly. Incubate the mixture for 30 minutes in the dark at four degrees Celsius. After incubation, centrifuge the cells and discard the supernatant.
Then rinse the cells by adding one milliliter of PBS and centrifuging at 300g for five minutes. For microbead labeling, resuspend the pellet in 100 microliters of PBS and 20 microliters of microbeads. Mix well and incubate for 30 minutes in the dark at four degrees Celsius.
Then, centrifuge the suspension and wash the pellet with one milliliter of PBS. Again, centrifuge and resuspend the pellet in four milliliters of PBS for magnetic negative exclusion. Pass the cell suspension through a 70 micron strainer to eliminate clumps.
Prepare the selection column by rinsing it with three milliliters of PBS. Then, add cell suspension into the selection column. Wash the column with three milliliters of PBS and collect LYVE-1 negative cells into a 50 milliliter tube.
For magnetic positive selection, pipette six milliliters of PBS into the selection column. Then, flush the magnetically labeled cells by firmly pushing the plunger into the selection column to obtain LYVE-1 positive LLECs. Next, centrifuge the positive cell suspension and remove the supernatant.
Plate 10 to the fifth cells per square centimeter into a T-25 flask with five milliliters of culture medium. Maintain the culture by replacing 50%of the medium every alternate day. When the cells reach 80%confluency, detach them as demonstrated previously before performing cell passage.
Flow cytometry analysis revealed that the percentage of LYVE-1 positive cells in passage two was not significantly different from passage three after max, indicating a purity greater than 95%Immunofluorescent staining confirmed co staining of LYVE-1 with PDPN, VEGFR-3, and PROX1, establishing the identity of LLECs. LYVE-1 positive cells did not express F4/80 and platelet derived growth factor beta. Effectively distinguishing LLECs from macrophages and fibroblasts.
Leptomeningeal cells before max displayed heterogeneous morphology, ranging from round spheres to fused fiber shapes. However, post max, LLECs exhibited typical endothelial-like features, such as spindle and cobblestone shapes.
