Our group's research focuses on delineating the mechanisms regulating endothelial cell physiology. This protocol describes a method for magnetic bead-based isolation of renal endothelial cells, specifically from thermal lymphatic capillaries in facilities where fluorescence-activated cell sorting is not available. The protocol allows the isolation of lymphatic endothelial cells from transgenic mice to study their physiology in vitro.
It is particularly suitable where the purity of the lymphatic endothelial cells can be compromised for downstream applications. This process provides a high yield of lymphatic endothelial cells obtained from the lymphatic capillaries and not other collecting vessels. It's also a nice alternative in the case of the absence of cell sorting facilities.
Perform the protocol inside a biosafety cabinet on the euthanized mouse, appropriately shaved and disinfected with 70%ethanol. Create a 0.5 inch superficial incision in the animal's abdomen using scissors. Cut longitudinally along the midline towards the neck and lower abdomen.
Additionally, make lateral cuts to flap open the skin. Using blunt forceps, gently separate the skin from surrounding tissues, while going around the animal's torso, cutting the skin laterally around the neck and the lower abdomen. Carefully place the skin with the dermis side facing down in a 100 millimeter tissue culture plate, and ensure the skin is as stretched as possible.
At six milliliters of dispase solution to the plate, ensuring the skin is completely covered, then cover the plate. Wrap the plate with Parafilm. Incubate the plate at room temperature for 10 minutes to flatten the explant.
The next day, using a pipette, remove the liquid from the plate containing the isolated skin. Then with the aid of forceps, separate the dermis from the epidermis while removing any visible adipose tissue from it, and place the isolated dermis into a clean tissue culture plate. Add one milliliter of DMEM containing 1x antibiotic antimycotic solution to the dermis in the culture plate.
Tilt the plate to accumulate the liquid on one side of the plate. Then mince the dermis into pieces of one to two square millimeters and transfer them to a new 50 milliliter conical tube. Add 10 milliliters of Collagenase Type II solution to the tube.
Incubate it for two hours at 37 degrees Celsius with continuous agitation at 30 to 50 RPM to digest the dermis. Filter the cell suspension through a 70 micron cell strainer and centrifuge it at 250 G for five minutes. After discarding the supernatant, resuspend the pellet in 10 milliliters of DMEM containing antibiotic antimycotic solution.
Filter the cell suspension through a 40 micron cell strainer before centrifuging, as demonstrated previously. Resuspend the resulting cell pellet in one milliliter of complete LEC media. Then plate the cells in complete LEC media in a collagen-coated 60 millimeter tissue culture plate.
Replace the media every two days by washing cells with PBS twice and adding fresh LEC media. Low magnification images of cells isolated from the murine dermis showed a mixed cell population. To purify murine lymphatic endothelial cells, use cells isolated from the murine dermis after they reach about 80 to 90%confluency.
Begin by washing pre-coated magnetic beads in a magnetic separator using one milliliter of magnetic bead coating solution. Resuspend the beads in 150 microliters of DMEM, containing 0.1%BSA. Next, wash the cells in the 60 millimeter tissue culture plate twice with PBS.
Mix 50 microliters of the antibody conjugated beads with three milliliters of DMEM containing 0.1%BSA, and add it to the cells. Seal the dish with Parafilm. Then incubate the dish on a shaker, agitating at 10 RPM at room temperature for exactly 10 minutes.
Discard the medium before washing the cells once with PBS. Quickly inspect the cells to verify the presence of LEC colonies on the plate. Add one milliliter of trypsin EDTA to the cells and incubate for three minutes at 37 degrees Celsius to trypsinize the cells.
Inspect the cells for successful cell detachment. After neutralizing the solution with two milliliters of complete LEC media, transfer the cell solution to a five milliliter sterile polypropylene tube. Using a magnetic separator, wash the cells with three milliliters of DMEM containing 0.1%BSA to remove unbound cells.
Resuspend the remaining bead-bound cells in complete LEC media and plate them onto collagen-coated 60 millimeter tissue culture plates. Bright field and fluorescence imaging easily identified the LYVE1 positive lymphatic endothelial cells after purification, and showed several beads attached to them. The cells displayed low confluency 24 hours after purification, while they were highly confluent seven days after purification.
