Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

All procedures described in this paper follow guidelines established by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University. All buffers and medias should be protected from light.

1. Preparation of Buffers, Media, and Instruments

1. Prepare Krebs Ringer Bicarbonate-Buffered solution (KRBB): 135 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.4 mM potassium phosphate dibasic, 5.5 mM glucose, 1% antibiotic/antimycotic (10,000 units/mL penicillin, 10,000 µg/mL streptomycin, 25 µg/mL amphotericin B), and 10 mM HEPES (pH = 7.4). This solution is stable for 3 weeks when kept sterile and at 4 °C.
2. Prepare Collagenase Type 1 Solution: 1 mg/mL in KRBB with 4% Bovine Serum Albumin (BSA). This solution should be kept at 37 °C and is stable for 4 h.
3. Prepare Erythrocyte Lysis Buffer Solution: 154 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA. Keep at 4 °C for up to one month.
4. Prepare MCS Blocking Buffer: DMEM/F12 Media base, 10% Fetal Bovine Serum (FBS), 5% normal donkey serum, 40 µL/mL F(ab) Fragment Donkey Anti-Rat IgG. Keep at 4 °C for up to one month.
5. Prepare MCS Buffer Solution: Phosphate-Buffered Saline (PBS, pH = 7.5), 0.5% BSA, and 2 mM EDTA. Keep at 4 °C for up to one month. De-gas solution by heating to 37 °C in a glass container and then applying a vacuum for15 s. Leave unused buffered sealed.
6. Prepare Stromal Vascular Fraction (SVF) Basal Media: Dulbecco's-Modified Eagles Medium (DMEM):F12, 15% Fetal Calf Serum (FCS), 1% antibiotic/antimycotic (10,000 units/mL penicillin, 10,000 µg/mL streptomycin, 25 µg/mL amphotericin B), 44.05 mM sodium bicarbonate, 100 µM ascorbic acid, 33 µM biotin, 17 µM pantothenate, 2 mM L-glutamine, and 20 mM HEPES. Keep sterile and at 4 °C for up to 2 weeks.
7. Prepare APC Media: Basal Media with additional growth factors including epidermal growth factor 10 ng/mL), leukemia inhibitory factor (10 ng/mL), platelet-derived growth factor BB (10 ng/mL), and basic fibroblast growth factor (5 ng/mL). Keep sterile and at 4 °C for up to 2 weeks.
8. Prepare APC Induction Media: APC Media with 10% FBS, 2.5 µg/mL insulin, 0.5 mM 2-isobutyl-1-methylaxanthine (IBMX), 1 µM dexamethasone, and 200 pM T3 (triiodothyronine thyroid hormone). Keep sterile and at 4 °C for up to 2 weeks.

2. Adipocyte Progenitors Isolation

1. Anesthetize rat according to institutional guidelines. Place the rat in dorsal recumbency. Confirm depth of anesthesia via a toe-pinch and the loss of reflex response to this painful stimulus.
   NOTE: This protocol uses 10-week-old Sprague Dawley rats and 70 mg/kg of pentobarbital delivered via an intraperitoneal injection.
2. Make a vertical midline incision with scissors along the sternum in the thoracic area and to the perineal area. Access the abdominal cavity and expose the superior mesenteric artery, the small mesenteric resistance vessels (mPVAT) and the thoracic aorta (aPVAT).
   1. Sever all connections to the mesentery and aorta and remove vessels from animal. Isolate PVAT by using a dissecting microscope and Petri dish filled with KRBB to view vessels and isolate PVAT.
   2. In this experiment, collect gonadal (GON) adipose to represent a non-PVAT adipose depot. Place isolated fat pads on ice in KRBB with 10 mM HEPES (pH = 7.4).
   3. In a biosafety hood, transfer about 50 mg of tissue to a 1.7 mL tube with 1 mL of collagenase type I solution and mince with tissue scissors (1 – 3 mm pieces).
3. Digest samples by incubating at 37 °C in a rotisserie incubator (or incubator with an orbital shaker) for 1 h. In a biosafety hood, sequentially filter digested material through 100 and 40 µm cell strainers into a 50-mL tube. Centrifuge resulting filtrate at 4 °C for 10 min at 300 × g.
   NOTE: All steps in the protocol from here forward are to be performed in a biosafety hood to keep cells sterile.
4. Pour off supernatant and resuspend pellets containing the SVF cells in 1 mL of 1X Erythrocyte Lysis Buffer Solution and transfer to a 1.7 mL microfuge tube. Incubate cells for 5 min at RT protected from light and centrifuge at 4 °C for 5 min at 300 x g.
5. Pour off supernatant and resuspend remaining cell pellet in SVF Basal Media. Collect a 20 µL sub-sample to count live cells with Trypan Blue Solution.
   NOTE: The number of SVF cells that can be isolated will vary by site. Average numbers of SVF harvested per mg of tissue are: aPVAT = 5.0 ±2.0×10³, mPVAT = 1.04 ±0.62×10⁴, GON = 2.4 ±1.2×10⁵.

3. Magnetic-activated Cell Sorting

NOTE: Isolate APC from SVF based on CD34 and PDGFRα cell surface markers by performing all steps at 4 °C.

1. Spin cells for 5 min at 300 x g. Pour off supernatant and resuspend the cell pellet in MCS Blocking Buffer at 1 x 10⁶ cells/mL and incubate for 20 min.
   NOTE: Cell suspensions of 1 x 10⁶ – 2 x 10⁸ cells/mL can be separated effectively.
2. Incubate cells with 5 µL of FITC-conjugated Mouse anti-CD34 (1 µg/1 x 10⁶ cells) for 30 minutes at 4 °C. Spin cells for 5 min at 300 x g and 4 °C.
   1. Incubate with 4 µL of anti-FITC microbeads and 96 µL of MCS buffer (total volume of 100 µL) for 5 min at 4 °C in the dark to separate CD34⁺ and CD34^– cells.
3. Attach magnetic separator to the stand and place the MultiSort (MS) Column, with the column wings to the front, into the separator. Place a 5 mL collection tube under the MS Column in the upper tube holder.
4. Prepare MS Column by rinsing with MCS Buffer Solution. Apply 500 µL of MCS Buffer on top of the column and let the buffer run through. Discard effluent and change collection tube.
5. Load antibody labeled cell suspension onto the prepared MS Column. Collect flow-through containing unlabeled cells.
6. Wash MS Column with 500 µL of degassed MCS Buffer 3x. Collect unlabeled cells that pass through and combine with the flow through from previous step.
7. Remove MS Column from the magnetic separator and place it on a new collection tube. Pipette 1 mL of MCS Buffer onto the MS Column. Immediately flush out fraction with the magnetically labeled cells by firmly, but slowly, applying the plunger supplied with the column as to not allow excess gas into the column.
   NOTE: Isolated cell numbers will vary by site. Average numbers of CD34⁺ APC isolated from the SVF population per mg of tissue are: aPVAT = 2.6 ±0.43×10², mPVAT = 9.6 ±1.4×10², GON = 1.3 ±0.22×10³.
8. Spin cells for 5 min at 300 × g and 4 °C. Incubate the CD34⁺ fraction collected in 10 µL of a 1:200 solution/1 x 10⁶ cells Rabbit anti-PDGFRα for 30 min at 4 °C.
   1. Centrifuge cells again for 5 min at 300 x g and 4 °C. Incubate labeled cells with 4 µL of anti-rabbit IgG microbeads and 96 µL of MCS buffer by repeating steps 3.3 through 3.7 to isolate.
      NOTE: Isolated cell numbers will vary by site. Average numbers of PDGFRα⁺ APC isolated from the CD34⁺ population per mg of tissue are: aPVAT = 2.4 ±0.64, mPVAT = 8.4 ±2.4, GON = 10.4 ±1.9, which is 0.5 – 10% of the previously isolated population.

4. Cell Culture and Adipogenesis Induction

1. Culture the SVF and APC in 6-Well tissue culture plates in basal media with replacement every 2 days. After 3 serial passages, plate in black 96-Well tissue culture plates at 1×10² cells/well for proliferation assays, which are evaluated at 8, 24, 48, and 96 h, and at 50,000 cells/well in 24-well plates or 10,000 cells/well in 48-Well tissue culture plates for adipogenesis assays, which are qualitative and quantitative.
2. Supplement APC basal media for 48 h post-confluency and prior to induction with bone morphogenic protein 4 (3.3 nmol/L) as indicated¹⁰ for differentiation. Induce cells after 48 h of 100% confluency (day 0) using the APC Induction Media to incubate the cells.
3. After 48 h, change media to maintain cells in APC Induction media without IBMX and dexamethasone, for 14 days with media changes every 48 h.
4. MCS isolation validated by FACS.
   1. Take a 50 µL (50,000 cell) sub sample of magnetically separated cells and wash with FACS solution.
   2. Centrifuge cells and resuspend in 100 µL of a 1:1,000 solution of donkey anti-rabbit IgG Dylight 405 to label the PDGFRα⁺ cells. Incubate for 30 min protected from light and at 4 °C.
   3. Wash cells and resuspend in 200 µL of 2% formaldehyde solution until time for analysis using 488 nm (FITC) and 405 nm (Dylight 405) filters on a flow cytometer.
      NOTE: Lipid accumulation by cultured cells is assessed quantitatively using a lipophilic adipogenesis fluorescence assay in a microplate reader measuring fluorescence and using preadipocytes as calibrators for lipid accumulation. Lipid accumulation is also measured qualitatively by lipid dye staining and imaging performed on an inverted microscope equipped with a camera, making the percentage of total cells that do or do not contain lipid observable. Any plate reader that is able to measure fluorescence with excitation at 485 nm and emission at 572 nm is suitable for analysis as well as any microscope with a camera capable of capturing digital images.