Perivascular adipose tissue surrounds blood vessels and regulates cardiovascular physiology. This protocol is used to answer key questions related to the function of human adipose progenitors in the vascular microenvironment. Adipose progenitor cells vary according to their anatomical location.
We show that a population of multipotent progenitors can be derived successfully from perivascular adipose tissue from patients with cardiovascular disease. Demonstrating the procedure will be Spencer Scott, a medical student from my laboratory. Obtain a 500 milligram piece of Human Perivascular Adipose Tissue or PVAT from the operating room as described in the text protocol.
Transfer fresh human PVAT from DMEM to a 50 milliliter conical tube containing 25 milliliters of antibiotic solution. Incubate with rocking for 20 minutes at four degrees Celsius. While the PVAT is in antibiotic solution, thaw an aliquot of dissociation buffer at 37 degrees Celsius.
Add 50 microliters of 100x antibiotic/antimycotic solution to five milliliters of dissociation buffer and sterilize using a 0.22 micron syringe filter. Add one milliliter of gelatin solution to one well of a 24-well plate. In a laminar flow hood, use sterile forceps and scissors to transfer PVAT from the antibiotic solution to a sterile Petri dish.
Add one milliliter of pre-warmed dissociation buffer to the tissue. Finely mince the entire tissue into a slurry using sterile forceps and dissection scissors. Transfer the one milliliter slurry to four milliliters of dissociation buffer.
Incubate the tube on its side in a pre-warmed 37 degrees Celsius orbital shaker at 200 rpm for one hour. After one hour, no visible tissue pieces should be present and the solution will appear as a cloudy cell suspension. Filter the solution through a 70 micron cell strainer set atop a 50 milliliter conical tube.
Rinse the strainer with an additional 10 milliliters of antibiotic solution to capture as many cells as possible. Do not squeeze the strainer. Now pellet the cells for 12 minutes at 300 times g in a swinging bucket centrifuge.
After centrifugation, the tube will be separated into a fatty top layer of adipocytes, an interphase and a pellet. The pellet is the stromal vascular fraction containing endothelial cells, immune cells, blood cells and progenitor cells. Resuspend the pellet in 10 milliliters of HBSS and centrifuge for five minutes at 300 times g.
Repeat this step for a total of two washes in HBSS. Now aspirate the gelatin from a 24-well plate. Gently wash the well once with HBSS to remove unbound gelatin.
Resuspend the stromal vascular fraction pellet with intact red blood cells in one milliliter of growth medium and seed onto the gelatin-coated well. Then add human FGF2 to a final concentration of 25 nanograms per milliliter in culture medium. Incubate for 24 hours at 37 degrees Celsius with 5%CO2.
After growing the cells for 24 hours, remove the growth media and wash the cells five times with HBSS. This washing step removes red blood cells and dead cells. Then add one milliliter of fresh growth media supplemented with 25 nanograms per milliliter FGF2 to each well.
Change the media every 48 hours making sure to supplement with 25 nanograms per milliliter fresh FGF2 each time. Passage cells once they reach 100%confluence seven to 10 days after explant. To do so, aspirate growth media and wash the monolayer twice with one milliliter HBSS.
Aspirate all HBSS from the wells and add a few drops of cell dissociation solution. Tap and swirl the plate several times and incubate at 37 degrees Celsius with 5%CO2 for five to seven minutes to lift the cells. Then add approximately one milliliter of fresh culture medium to the detached cells.
Distribute 500 microliters of the detached cells to two wells of a 24-well plate each containing 500 microliters of growth media and 25 nanograms FGF2. Also, culture human bone marrow Mesenchymal Stem Cells or MSC colonies as described in the text protocol. Plate the appropriate numbers of bone marrow and PVAT-derived cells per well of a 12-well plate.
For adipogenic and osteogenic conditions, plate approximately 200, 000 to 225, 000 cells per well. Whereas for chondrogenic conditions, plate 150, 000 to 175, 000 cells per well. Then dissociate cells from both the human PVAT progenitor cell population and the human bone marrow MSC population by adding cell detachment solution.
Incubate the cells in the detachment solution at 37 degrees Celsius and 5%CO2 for five minutes. Pull the populations into separate 15 milliliter conical vials. Spin the vials down at 500 times g for seven minutes to pellet the cells.
Then resuspend the cells in one milliliter of PBS and use a hemocytometer to estimate the cell number. Plate the cells in 12-well dishes as before. Provide separate dishes for the induced and non-induced adipogenic and osteogenic conditions.
And add 1.5 milliliters of adipogenic and osteogenic conduction media to each well of the induced condition. Then add 1.5 milliliters of adipogenic and osteogenic non-induction media to each well of the non-induced condition. Begin incubation of the adipogenic and osteogenic induced and non-induced cell populations at 37 degrees Celsius and 5%CO2.
Spin down the remaining volume of human PVAT progenitor cells and human bone marrow MSCs for seven minutes at 500 times g. Determine the volume needed to resuspend the remaining bone marrow and PVAT-derived cell pellets to achieve a density of 100, 000 cells per 10 microliters. Then resuspend the pellets in the calculated volume of MSC growth media for chondrogenic lineage induction.
Gently move the volume of cells up and down using a pipette to ensure a homogenous distribution. Now pipette a 10 microliter droplet of the concentrated cell solution into the center of each well to form a micro mass of 100, 000 cells. Place one milliliter of sterile water in the adjacent well to prevent evaporation.
Incubate the micro mass cultures for two hours at 37 degrees Celsius and 5%CO2 to allow the micro mass to aggregate. After two hours, carefully add chondrogenic differentiation media spiked with 10 nanograms per milliliter human TGF-beta-one to each of the induced condition wells. Now carefully add 1.5 milliliters of the non-induction media to the non-induced condition wells.
Scrape or pour the micro mass in the induced chondrogenic condition into a cassette in order to dehydrate, embed, and stain the sample as described in the text protocol. Adipogenic differentiation studies were performed in parallel with human bone marrow-derived MSC and PVAT-derived progenitor cells. In the non-induced condition, no lipid accumulation is evident.
This is in contrast to the induced condition shown following staining of neutral lipids with Oil Red O.While the degree of differentiation in the human aortic PVAT-derived cells is more robust, both human cell sources exhibited the ability to differentiate for the adipogenic lineage. The osteogenic differentiation protocol was used for human bone marrow-derived MSCs and PVAT-derived cells. Non-induced cells did not stain with Alizarin Red.
After the osteogenic differentiation protocol, the human MSCs developed calcified nodules that stained with Alizarin Red while human aortic PVAT cells did not. Cells derived from both human bone marrow MSCs and human PVAT displayed features characteristic of chondrogenic differentiation with abundant collagen accumulation in the micro mass. Micro mass is formed from human bone marrow MSCs and aortic PVAT-derived cells also exhibited abundant accumulation of glycosaminoglycans as indicated by Alcian Blue staining.
Morphologically, structures similar to lacunae were detected with cells sitting in cavities surrounded by collagen deposition. It is critical to finely mince perivascular adipose tissue prior to enzymatic disassociation and to ensure proper cell densities are plated for each lineage differentiation assay. Following this procedure, quantitative PCR combined with western blot and flow cytometry should be used to characterize lineage-specific markers of differentiated progenitors from perivascular adipose and bone marrow sources.
With this technique, we can begin to understand the mechanisms regulating perivascular adipose tissue expansion and dysfunction during obesity and the implications that progenitor cells have on vascular function and cardiovascular disease.
