The overall goals of these isolation and differentiation protocols are to obtain adipocyte progenitor cells from perivascular adipose tissues, and to induce their differentiation into mature adipocytes. This method allows the analysis of specific and defined adipocyte progenitor cell populations from perivascular adipose tissue, with minimal impact on their morphology, viability, and potential to proliferate and differentiate. The main advantage of this technique is that it allows an increased isolation of antigen-presenting cells per gram of adipose tissue, compared to fluorescence-activated cell sorting.
After confirming a lack of response to toe pinch, make a vertical midline incision along the sternum to the perineal area of the rat, to access the abdominal cavity. Expose the superior mesenteric artery, the small mesenteric resistance vessels, and the thoracic aorta, and sever all of the connections to the mesentery and aorta. Collect the gonadal adipose tissue and transfer the isolated fat pads into a new container of Krebs-Ringer bicarbonate buffer, or KRBB solution, supplemented with 10 millimolar heaps.
Transfer the vessels to a petri dish containing KRBB solution, and place the dish under a dissecting microscope. Remove perivascular adipose tissue from the small mesenteric resistance vessels and aorta. In a biosafety hood, transfer about 50 milligrams of tissue into a 1.7 milliliter tube containing 1 milliliter of collagenase type I solution, and mince the tissue into 1 to 3 millimeter pieces.
It is important to adequately mince the adipose tissue to allow the collagenase to thoroughly infiltrate the connective tissue. Incubate the fragments at 37 degrees Celsius with shaking for 1 hour. Then sequentially filter the digested material through individual 100-and 40-micron cell strainers into a 50-milliliter tube.
Collect the resulting filtrate by centrifugation, and resuspend the pellet in 1 milliliter of erythrocyte lysis buffer. Transfer the cell suspension into a new 1.7 milliliter Microfuge tube for a 5-minute incubation at room temperature, protected from light. Then collect the stromal vascular fraction cells with another centrifugation and resuspend the pellet in stromal vascular fraction basal medium for counting.
To isolate the adipocyte progenitor cells by magnetic-activated cell sorting, collect the cells by centrifugation, and resuspend the pellet in magnetic-activated cell sorting blocking buffer at a 1 times 10 to the 6th cells per milliliter concentration for a 20-minute incubation at 4 degrees Celsius. It is essential that all steps of the isolation are done at 4 degrees Celsius, and not over ice. Next, incubate the cells with 5 microliters of FITC-conjugated mouse anti-CD34 for 30 minutes.
Collect the cells by centrifugation. Then incubate the cells with 4 microliters of anti-FITC MicroBeads in 96 microliters of magnetic-activated cell sorting buffer for 5 minutes in the dark. While the cells are incubating, place a multi-sort column into the magnetic separator with a 5-milliliter waste tube under the column.
Rinse the column with 500 microliters of magnetic-activated cell sorting buffer, and discard the effluent and waste tube. Then place a new tube under the column, and load the cells onto the top of the column, collecting the unlabeled CD34 negative cells in the new collection tube. When all of the cells have passed through the top of the column, wash the column 3 times with 500 microliters of degassed magnetic-activated cell sorting buffer, continuing to pool the eluate in the collection tube.
After the last wash, transfer the column into a new collection tube, and use the column plunger to flush 1 milliliter of magnetic-activated cell sorting buffer through the column into the tube, to collect the CD34 positive cells. Collect the adipocyte progenitor cells by centrifugation. Then incubate the CD34 positive fraction in 10 microliters of rabbit anti-platelet-derived growth factor receptor alpha for 30 minutes.
At the end of the incubation, centrifuge the cells again, and incubate the pellet in 4 microliters of anti-rabbit IgG MicroBeads in 96 microliters of magnetic-activated cell sorting buffer for magnetic bead isolation of the platelet-derived growth factor receptor alpha positive cells, as just demonstrated. To culture the CD34 positive platelet-derived growth factor receptor alpha positive adipocyte progenitor cells, seed the cells in individual 6-well tissue culture plates in basal medium, and place the plates in a 37 degree Celsius and 5%carbon dioxide cell culture incubator. After 3 serial passages, seed the cells into black 96-well tissue culture plates at 1 times 10 to the 2 cells per well, and evaluate their proliferation at 8, 24, 48, and 96 hours at 5 times 10 to the 4th cells per well, in 24-well assay plates, according to standard proliferation assay protocols.
To induce adipogenesis, when the adipocyte progenitor cell culture reaches confluency, feed the cells with adipocyte progenitor basal medium for 48 hours, then treat the cultures with bone morphogenic protein 4 for another 48 hours. On the third day, replace the medium with adipocyte progenitor cell induction medium, without IBMX and examethasone for 14 days. The degree of adipogenesis in the cultures can then be assessed at 1 times 10 to the 4th cells per well in 48-well tissue culture plates, according to standard adipogenesis assay protocols.
Although both methods demonstrate a similar cell population distribution and viability, magnetic-activated cell sorting isolation produces a greater number of cells for culture, compared to fluorescence-activated cell sorting. In this representative experiment, the in vitro proliferation of magnetic-activated cell sorted stromal vascular fraction and adipocyte progenitor cells, isolated from the thoracic aorta, small mesenteric resistance vessels, and gonadal adipose tissue of male rats, was evaluated at 8, 24, 48, and 96 hours after plating, using a quantitative DNA assay. No site differences in stromal vascular fraction expansion rates were observed at any time point, except for the adipocyte progenitor cells from the thoracic aorta, which demonstrated less proliferation by 96 hours, compared to stromal vascular cells from the same site.
Stimulation of confluent adipocyte progenitor cells with bone morphogenic protein 4 for 48 hours induces adipocyte differentiation, evidenced by a greater lipid accumulation in droplets, as evaluated by both fluorescent lipid uptake assay, and Oil Red O staining. Once mastered, this technique can be completed in 8 hours, if it is performed correctly. While attempting this procedure, it is important to remember to maintain 4 degree Celsius temperatures during the isolation steps.
After watching this video you should have a good understanding of how to isolate and differentiate adipocyte progenitor cells.
