Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

This protocol will allow the researcher to dissect artery of interest and isolate vascular cells to perform a wide range of functional tests. Researcher can investigate different vascular beds and cell type to expand the knowledge of vascular cell biology and mechanism of vascular dysfunction. This method is most applicable to researchers investigating basic vascular cell biology, as well as vascular cell dysfunction in animal models of disease.

Special care should be taken to optimize the digestion protocol, especially where vascular beds of interest differ from those presented here. To begin, secure the mouse using dissection pins and spray the ventral surface with 70%ethanol. Use forceps to lift the skin right above the genitals, and make a small incision of approximately two millimeters in the skin.

Insert the tip of the scissors into the incision site, and make a four to five centimeter long midline incision beginning at the initial incision, and dissecting cranially to the base of the sternum. Make an incision of one centimeter extending laterally from the midline immediately below the fore limb. Make a diagonal one centimeter incision between the hind limb and genitals.

Make small cuts along the midline skin incisions to peel away the associated connective tissue, and expose the subcutaneous adipose depot. Identify the C-shaped subcutaneous adipose tissue and the artery or vein that runs perpendicular to the abdominal cavity. Start from the bottom of the C-shape near the genitals, and gently grasp the adipose tissue to expose the connective tissues.

Make small cuts in the connective tissue to separate the fat from the skin, and avoid disturbing the exposed vasculature. Continue dissecting away the connective tissue until the subcutaneous adipose tissue can be removed intact. Carefully separate the exposed artery from the surrounding connective tissue.

Store the isolated subcutaneous adipose in HEPES buffer on ice. Repeat the dissection for the remaining posterior subcutaneous adipose depot. To isolate the mesenteric adipose depot, use Graefe Forceps to lift the thin peritoneal cavity wall above the urinary bladder, and make a small incision using straight scissors.

Slowly lift the peritoneal cavity wall and insert the straight iris scissors into the incision site. Make an incision of four to five centimeters from the urinary bladder to the sternum. Then make two horizontal incisions of one centimeter with the straight iris scissors extending laterally from the midline to immediately above the hind limb and below the fore limb.

Use the Graefe forceps to peel back the abdominal musculature and expose the peritoneal viscera. Use a pair of number five forceps to lift the intestines out of the visceral cavity to reveal the mesenteric adipose. Use the curved scissors and number five forceps to separate the adipose along the colon, starting from the cecum to where the colon descends from view.

Then, separate the adipose along the small intestine to the pancreas. Remove a small part of the pancreas to completely isolate the mesenteric adipose. Store the isolated mesenteric adipose in HEPES buffer on ice.

To isolate the arteries, first transfer the adipose tissue to a dissecting dish containing 10 milliliters of cold HEPES buffer. Under a stereo microscope, position the subcutaneous adipose tissue to expose the artery vein pair. Position the visceral adipose tissue to expose the cauliflower shape and respective artery vein pair.

Use number 55 forceps to carefully remove the parenchymal adipose from the artery. Remove small pieces of adipose at a time without damaging the vasculature of interest. Continue until the arteries are completely void of parenchymal adipose tissue.

Use number five forceps to transfer the cleaned arteries to a new tube with fresh HEPES buffer, and keep on ice until ready for digestion. First, add one milligram each of dispase and elastase to a two milliliter micro centrifuge tube. Add two milliliters of dissociation solution, vortex, and incubate at 37 degrees Celsius until completely dissolved.

Use number five forceps to transfer the cleaned arteries to the respective sample tubes. Incubate the tubes at 37 degrees Celsius for one hour with gentle agitation by inversion every 10 to 15 minutes. In the meantime, add one milligram of collagenase type one per sample to new tubes.

After incubation, allow the arteries to settle at the bottom of the tube and transfer 500 microliters of the buffer from the top into the tubes containing collagenase. Vortex and return this solution to the respective sample tubes. Incubate the samples at 37 degrees Celsius for 15 minutes.

Ensure that the arteries separate into pieces upon shaking the sample tube. Using a glass pipette, vigorously triturate the digested tissue 10 to 15 times to mechanically dissociate the cells. Pass the digested samples through a 70 micron cell strainer or a flow cytometry tube strainer to obtain single cell suspensions.

Centrifuge the single cell suspension and remove the supernatant. Re-suspend the cell pellet in one milliliter of PBS with 5%BSA for 30 minutes. Add one microliter of cell viability stain and incubate in the dark for 30 minutes.

Centrifuge the cell suspension and re-suspend the cell pellet in 200 microliters of PBS with 1%BSA. Add primary antibodies conjugated to CD31 and CD45 and incubate on a rocker in the refrigerator for 15 minutes. Wash the cells by adding one milliliter of PBS with 1%BSA directly to the cell suspension.

Centrifuge the cell suspension and re-suspend the pellet in 200 microliters of 1%formaldehyde with 0.1%BSA. Cover the tubes with aluminum foil and store in the refrigerator until ready for flow cytometry. Cell preparations obtained from digested subcutaneous adipose arteries were used to identify the endothelial cell populations using flow cytometry.

CD31 positive CD45 negative cells were identified as endothelial cells. The cell viability stain allowed for the identification of only those viable cells that survived the isolation and digestion protocol prior to fixation. To identify the differences in membrane protein expression and endothelial cells from distinct adipose depot vasculature, the isolated cells were probed for fatty acid translocase CD36.

CD36 positive endothelial cells were identified in subcutaneous and mesenteric adipose. Quantification of CD36 expression and subcutaneous and mesenteric endothelial cells revealed that both the percentage of endothelial cells expressing CD36, and the intensity of CD36 expression were greater in subcutaneous endothelial cells. It is crucial to carefully dissect, identify, and clean the arteries.

Most importantly, the digestion protocol should produce a sufficient yield of viable cells for downstream applications. Following this procedure, the isolated cell population can be used for applications including but not limited to, electrophysiology, molecular profiling, and generation of primary cell line for drug screening in vitro.