The Contreras and Watts Laboratories and Dr. William Raphael. These experiments were supported by NHLBI F31 HL128035-01 (tissue digestion protocol standardization), NHLBI 5R01HL117847-02 and 2P01HL070687-11A1 (animals), and NHLBI 5R01HL117847-02 (cell isolation and culture).

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
<ol>
	<li>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...

<ol>
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The central focus of the present experiment is the isolation, expansion, and adipogenic induction of APC from PVAT depots. Here we present a protocol for the isolation of APC based on the identification of cells expressing the surface markers CD34 and PDGFR&#945;. These surface proteins were previously identified on APC with high proliferation rates and the potential to differentiate into white or brown adipocytes in various adipose depots14,15. By selecting cell...

Perivascular Adipose Tissue (PVAT), due to its close proximity to blood vessels, is a major paracrine signaling component in vasculature function1. Expansion of this adipose tissue is dependent on the phenotype of the Adipocyte Progenitor Cells (APC) present2,3. Isolation of cells from adipose tissues is difficult as primary adipocytes are fragile, buoyant, and range in size. Certain isolation techniques can also alter cell phenotype and morphology by increasing inflammatory protein synthesis and reducing adipogenic gene expression4, emphasizing the importance of a protocol that maintains the integrity of the cells.
Culture of primary cells and specific preadipocyte subpopulations gives a reductionist approach to in vivo growth and maintains equivalent cellular genetic makeup5, although working time with these cells is limited due to deterioration with aging, or senescence6. Preadipocytes from various adipose depots, including subcutaneous and omental depots, also demonstrate differences in proliferation7, which emphasizes the importance of collecting cells from specific anatomical sites. Precursor cells from non-PVAT white adipose depots have been characterized in previous studies7,8,9, but less is known about PVAT APC phenotypes.
The techniques described here allow for the analysis of specific and defined APC populations with minimal impact on their morphology, viability, and potential to proliferate and differentiate. Magnetic-activated Cell Sorting (MCS) is amenable to downstream applications, such as culture, as the beads dissolve without altering the cell. MCS is also economical, and once the antibody concentrations have been standardized, the need for flow cytometry assays is minimal. In vitro studies with PVAT precursors can also give a glimpse of the potential that these primary cells may have.

<table border="0" cellpadding="0" cellspacing="0" width="757">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:325px;">Tissue Dissection</td>
			<td style="width:168px;"></td>
			<td style="width:108px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Dishes</td>
			<td>Handmade with Silicone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture Petri Dish</td>
			<td>Pyrex</td>
			<td></td>
			<td>7740 Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 170</td>
			<td>Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Braided Silk Suture</td>
			<td>Harvard Apparatus</td>
			<td>51-7615</td>
			<td>SP104</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope MZ6</td>
			<td>Leica</td>
			<td></td>
			<td>10447254</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomaster Microscope Fiber-Optic Light Source</td>
			<td>Fisher Scientific</td>
			<td>12562-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splinter Forceps</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>105-400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KRBB Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Phosphate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>7758-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic/Antimicotic</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type 1</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Thermo-Fisher Scientific</td>
			<td>2876</td>
			<td>Reciprocal Shaking Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Cabinet</td>
			<td>Thermo-Fisher Scientific</td>
			<td>1385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotisserie Incubator</td>
			<td>Daigger</td>
			<td>EF4894C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-549</td>
			<td>Yellow</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Blue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OctoMACS Kit</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(DMEM):F12 Medium</td>
			<td>Corning</td>
			<td>90-090</td>
			<td>Medium Base</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35016CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Donkey Serum</td>
			<td>AbCam</td>
			<td>AB7475</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD34</td>
			<td>Santa Cruz</td>
			<td>SC-7324</td>
			<td>FITC conjugated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PDGFR&#945;</td>
			<td>Thermo-Fisher Scientific</td>
			<td>PA5-17623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-007-003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS 10X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 Cell Incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>51030285</td>
			<td>Heracell 160i&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48- Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Plates, Black Wall</td>
			<td>Corning</td>
			<td>353376</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>144-55-8</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Calf Serum</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>58-85-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>137-08-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Corning</td>
			<td>61-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone Morphogenic Protein 4</td>
			<td>Prospec Bio</td>
			<td>CYT-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal Growth Factor</td>
			<td>PeproTech</td>
			<td>400-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia Inhibitory Factor</td>
			<td>PeproTech</td>
			<td>250-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived Growth Factor BB</td>
			<td>Prospec Bio</td>
			<td>CYT-740</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic Fibroblast Growth Factor</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Corning</td>
			<td>25-800-CR</td>
			<td>ITS Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>28822-58-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>50-02-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T3 (Triiodothyronine)</td>
			<td>Sigma-Aldrich</td>
			<td>6893-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyQUANT Proliferation Assay</td>
			<td>Thermo-Fisher Scientific</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AdipoRed Fluorescence Assay Reagent</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O Lipid Dye Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>O1391</td>
			<td>In Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M1000 Microplate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse Inverted Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Sight DS-Qil Camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Proliferative capacity of preadipocytes and adipogenic potential of adipocyte precursors are characteristics that are maintained in vitro11. In vitro proliferation of isolated SVF and APC from aPVAT, mPVAT, and GON of male rats was evaluated at 8, 24, 48, and 96 h after plating using a quantitative DNA assay. No site differences in SVF expansion rate were observed at any time point except for the APC from aPVAT, which had less proliferation by 96 ...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

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

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

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.

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9.3K Views.  Michigan State University. 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 increase...