We aim to establish a standardized approach for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mass PVAT. This protocol enables us to conduct further investigations into PVAT function and into the communication between periaortic adipocytes and vascular cells in vitro. The main challenges we currently face in our experiments include the restricted of availability of PVAT and the requirements for additional human and the material resources when compared to using immortalized preadipocytes.
Our protocol allows for the study of perivascular adipose tissue function and it's relationship with vascular cells. We also offers a basis for studying PVAT ring and genes, and heterogeneity, and provides a platform for exploring the important regulator of PVAT differentiation and the adipogenesis in vitro. Begin by spraying the properly euthanized mouse with 75%alcohol to disinfect the skin.
Then, place the mouse in a supine position. Lift the skin of the animal's abdominal region, and using surgical scissors, make a small incision in it. Then, using the scissors, bluntly separate the skin and abdominal muscles from the pelvis to the neck along the ventral midline.
Expose the heart and lungs by cutting the diaphragm and ribs along both sides of the midline. Next, carefully remove the liver, spleen, bowels, and kidney, avoiding damage to the intestinal wall. Following that, remove the lungs and esophagus.
Now gently lift the heart using forceps held in one hand and separate the aorta from the spine with surgical scissors held in the other hand. Then, place the heart and aorta in a 60 millimeter Petri dish containing antibiotic supplemented PBS. Retrieve the microsurgical forceps and microsurgical scissors from the alcohol and rinse them in 25 milliliters of PBS to remove excess alcohol.
Working under a stereo microscope, remove the thymus and other tissues from the heart and aorta. Then remove the heart, leaving the aorta with the perivascular adipose tissues or PVATs. Transfer the aorta along with the PVATs to a clean 60 millimeter Petri dish containing antibiotic supplemented PBS.
Using one pair of microsurgical forceps, hold the aorta and using another pair, pull off the attached pose tissues to separate the PVATS from the aorta. Remove as much vascular tissue as possible while causing minimal harm to the PVATs. Finally, transfer the collected PVATs into a two-milliliter micro centrifuge tube containing antibiotic supplemented high glucose DMEM placed on ice.
While working under a class two laminar flow hood, begin by sequentially washing the perivascular adipose tissues or PVATs collected from mice two times. First, using PBS containing 10%penicillin-streptomycin. And next, using PBS supplemented with 1%penicillin-streptomycin.
Transfer the rinsed tissues into a sterile 60-millimeter Petri dish and add 200 microliters of digestion solution to it. Using sterile scissors, mince the tissues into pieces, having a dimension of one cubic millimeter. With the help of sterile scissors, cut a plastic pipette tip to broaden its end.
Then, transfer the minced tissue mix to a 15-milliliter centrifuge tube using the broad ended pipette tip. Add six milliliters of the digestion solution to the tissues to initiate digestion. After tying the tube horizontally on a rack, incubated at 37 degrees Celsius in an orbital shaker for 30 to 45 minutes.
Every five to 10 minutes, remove the tube from the shaker in the incubator and manually shake it up and down vigorously. Pass the digested tissue mix through a 70 micron cell strainer into a 50-milliliter centrifuge tube. Rinse the strainer with an equal volume of culture medium to maximize cell yield and quench digestion.
Transfer the filtrate to a new 15-milliliter centrifuge tube. Centrifuge the tube to isolate the stromal vascular fraction or SVF. Invert the tube to discard the supernatant and resuspend the pellet in five milliliters of PBS.
Centrifuge the tube again at 1800 g for five minutes before discarding the supernatant. Resuspend the pellet in an appropriate volume of culture medium. Seed the cells into a collagen coated 12 well plate and incubate overnight at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide.
On the next day, remove cell debris and red blood cells by aspirating the culture medium and washing the cells with antibiotic supplemented PBS, prewarm to 37 degrees Celsius. Finally, add one milliliter of fresh culture medium to each well, and continue culturing the cells until they reach the desired stage of confluency.
