The overall goal of this procedure is to isolate Sca1 positive mesenchymal stem cells from various mouse fat pads. The main advantage of this technique is that by using the immunomagnetic beads-based cell sorting adipose-derived stem cells from different fat depots can be effectibely enriched for subsequent experimentations. Generally, investigators new to this method will struggle because the strength and duration of the collagenase-mediated tissue digestion should be optimized to procure an adequate number of cells for sorting.
To isolate the subcutaneous fat pads, begin by placing the mouse on a foam dissecting board in the supine position and spraying the animal with 70%ethanol. After securing the paws to the board, make a small incision in the skin of the lower abdomen. Then, grasping the top of the cut with forceps, use scissors to separate the skin and the peritoneum from the abdomen to the thorax.
Making lateral cuts in the skin, along the side of the body, reflect the skin away from the peritoneum towards the head. Then, pull back the remaining skin, holding the inguinal fat away from the body, and use 22 gauge needles to pin the skin to the board. Next, use the forceps to grab one inguinal fat pad at its origin near the peritoneum, and use fine scissors to snip between the skin and inguinal fat, progressing toward the groin, and avoiding contaminating the subcutaneous fat pad with skin.
Then, place the isolated inguinal fat pad in an SQ-labeled 60 millimeter dish containing 37 degrees Celsius culture medium, and harvest the inguinal fat pad from the other side of the animal in the same way. To isolate the visceral fat pads, cut the peritoneum to expose the visceral fat pads and gut. Then move the gut aside, towards the thorax.
Grasping the visceral fat pad at the distal end, gently pull up on the fat tissue, and dissect it out, taking care to exclude the epididymal tissue. Then place the fat pad in a VIS-labeled 60 millimeter dish containing 37 degrees Celsius culture medium. When all of the fat pads have been collected, transfer the dishes to a tissue culture hood, and aspirate the medium.
Use curved scissors to mince the pads. Then, add the fat pieces to individual 50 milliliter tubes containing freshly prepared collagenase. Next, shake the tubes at 300 RPM and 37 degrees Celsius for 10 to 20 minutes, until the tissues are digested.
Some pieces of subcutaneous fat pad tissue may still be visible. Add 20 milliliters of culture medium to the collagenase solution to stop the digestion, and use a 10 milliliter serological pipette to aspirate and dispense the tissue slurry 10 times to fully dissociate the digested tissues. Filter the cells through 100 micron cell strainers, and centrifuge the tissues.
Then carefully decant the medium, taking care not to disturb the pellets, and gently resuspend both groups of cells in five milliliters of sterile water. Wait two minutes for the erythrocytes to be lysed. Then arrest the reaction in each tube with 25 milliliters of medium supplemented with FBS.
Filter both adipocyte populations through new 100 micron cell strainers, spin them down again, and then resuspend the pellets in one to three milliliters of medium. After counting by trypan blue exclusion, separately plate one times ten to the sixth subcutaneous fat pad cells and visceral cells into individual wells of a six-well cell culture plate. After trypsinizing the cells, dilute the trypsin with one milliliter of separation buffer, and then centrifuge the culture plates.
We suspend the cells in 500 microliters per tube of separation buffer, and centrifuge the cells again. Now, resuspend the cells in 90 microliters per tube of separation buffer, and mix 10 microliters of anti-Sca1 FITC primary antibody into the cell suspension. After 10 minutes of incubation at four degrees Celsius in the dark, wash the cells with one milliliter of separation buffer per tube.
After spinning, cells are resuspended in 80 microliters of separation buffer per cell suspension. Then mix 20 microliters of Anti-FITC Microbeads into each well, and place the plates at four degrees Celsius, protected from light for 15 minutes. At the end of the incubation, wash the cells in one milliliter of separation buffer, and resuspend the cells in 500 microliters of fresh buffer per tube.
Now, place a column in the magnetic holder, and rinse the column with 500 microliters of separaton buffer. Transfer the cells from the subcutaneous fat pad tissue culture onto the column, taking care to avoid bubbles, and collect the unlabeled Sca1 negative cells in a 15 milliliter conical tube. When all of the cells have run through, wash the column three times with 500 microliters of separation buffer.
Then transfer the column from the magnet into a new 15 milliliter conical tube, and plunge one milliliter of fresh separation buffer through the column to elute the Sca1 positive cells. Finally, spin down both Sca1 positive and negative cell fractions, and resuspend the pellets in one milliliter of culture medium. Then count the number of cells in each fraction, and plate the fractions in individuals wells of a new six-well cell culture plate.
Vascular stromal cells isolated from subcutaneous fat display a fibroblast-like, stretched cell shape regardless of their level of Sca1 expression. Like subcutaneous inguinal fat-derived Sca1 high cells, visceral epididymal fat-derived Sca1 high cells display a stretched fibroblast-like cell shape. Whereas VIS Sca1 low cells exhibit an epithelioid shape.
Sca1 high cells isolated from isolated Sca1-GFP mice are easily identified as GFP positive cells in tissue culture. Indeed, when these cells are assessed by flow cytometry, most of the GFP positive cells are confirmed to express Sca1 proteins on their cell surfaces, as detected by anti-Sca1 antibody. Coupled with real-time PCR analyses, the differential expression of the collagenolytic matrix metalloproteinases between inguinal and epididymal fat-derived Sca1 high adipose-derived stem cells can be demonstrated.
Further, when fluorescein labeled type I collagen gels are used to assess pericellular degradation activity, a markedly increased collagen remodeling activity mediated by visceral Sca1 high adipose-derived stem cells is observed. Once mastered, the fat pad cell isolation can be completed in two hours if it is performed properly. While attempting this procedure, it's important to remember to maintain a clean and organized surgical field to ensure efficient fat pad isolation.
Following this procedure, other methods, like a collagen degradation assay, can be performed to answer additional questions about adipose-derived stem cell-mediated tissue remodeling. After its development, this technique paved the way for researchers in the field of obesity and matrix biology to explore the role of adipose-derived stem cells in adipose tissue remodeling and function in mice and humans. After watching this video, you should have a good understanding of how to procure and enrich primary Sca1 positive cells from adipose tissue depots.
