This work was supported by the National Natural Science Foundation of China (82130012 and 81830010) and the Nurture projects for basic research of Shanghai Chest Hospital (Grant Number: 2022YNJCQ03).

The animal protocols were approved by the Institutional Animal Care and Use Committee at Shanghai Chest Hospital affiliated to Shanghai Jiao Tong University School of Medicine (approval number: KS23010) and were in compliance with relevant ethical regulations. Male and female C57BL/6 mice aged 4-8 weeks are to be preferred for this experiment.
1. Preparation of surgical tools, buffers, and culture media
<ol>
	<li>Autoclave surgical tools (e.g., surgical scissors and standard forceps) at 121 &#176;C for 30 min. Disinfect microsurgical instruments with 75% alcohol.</li>
	<li>Prepare sterile phosphate-buffered saline (PBS) supplemented with 1% v/v penicillin-streptomycin and another 10 mL of PBS supplemented with 10% v/v penicillin-streptomycin. 
	NOTE: In the following sections, if not specifically mentioned, PBS refers to regular sterile PBS.</li>
	<li>Prepare Krebs Ringer HEPES BSA buffer: 1% bovine serum albumin (BSA), 20 mM HEPES dissolved in Krebs Ringer solution.</li>
	<li>Prepare fresh digestion solution: type 1 collagenase (1 mg/mL) and dispase II (4 mg/mL) dissolved in Krebs Ringer HEPES BSA buffer. Sterilize the solution using a 0.2 &#181;m filter.</li>
	<li>Prepare a collagen-coated 12-well plate.
	<ol>
		<li>Dilute the collagen solution (1 mg/mL) with sterile deionized water to the concentration of 40 &#181;g/mL. Add 1 mL of the diluted collagen solution on the surface of each well to achieve a concentration of 6-10 &#181;g/cm2. Incubate the coating for 1 h at room temperature.</li>
		<li>Remove the remaining solution and rinse each well 2x with 1 mL of PBS. Use the plate immediately, or air dry the plate in a class II laminar flow hood and store the coated plate at 2-8 &#176;C. When storing, seal the coated plates with parafilm.</li>
	</ol>
	</li>
	<li>Prepare culture medium: high-glucose Dulbecco&#39;s-Modified Eagles Medium (DMEM), 10% fetal bovine serum (FBS), 1% v/v penicillin-streptomycin. Keep at 4 &#176;C for up to 2 weeks.</li>
	<li>Prepare lipogenic induction medium: high-glucose DMEM, 10% FBS, 1% v/v penicillin-streptomycin, 1 nM triiodothyronine, 1 &#181;M rosiglitazone, 1 &#181;M insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 1 &#181;M dexamethasone. Keep at 4 &#176;C for up to 2 weeks.</li>
	<li>Prepare maintenance medium: high-glucose DMEM, 10% FBS, 1% v/v penicillin-streptomycin, 1 nM triiodothyronine, 1 &#181;M rosiglitazone, and 1 &#181;M insulin. Keep at 4 &#176;C for up to 2 weeks.</li>
</ol>
2. Dissection and isolation of perivascular adipose tissue (PVAT)
<ol>
	<li>Euthanize the mouse by cervical dislocation under 2% isoflurane anesthesia or with carbon dioxide overdose. Spray with 75% alcohol for skin disinfection.</li>
	<li>Place the mouse in a supine position (facing upward).</li>
	<li>Lift the skin, make a small incision, and bluntly separate the skin and abdominal muscles with surgical scissors along the ventral midline from the pelvis to the neck. Expose the heart and lungs by cutting the diaphragm and ribs along both sides of the midline.</li>
	<li>Carefully remove the liver, spleen, bowels, and kidney. Avoid cutting the intestinal wall.</li>
	<li>Remove the lungs and esophagus.</li>
	<li>Gently lift the heart with forceps in one hand and separate the aorta from the spine with surgical scissors in the other hand. Then, place the heart and aorta in a 60 mm Petri dish with PBS containing 1% v/v penicillin-streptomycin.</li>
	<li>Remove microsurgical forceps and microsurgical scissors from alcohol and rinse in 25 mL of PBS to remove the excess alcohol. Under a stereo microscope, remove the thymus and other tissue from the heart and aorta, then remove the heart, leaving the aorta with the PVATs.</li>
	<li>Transfer the aorta with the PVATs to a clean 60 mm Petri dish with PBS containing 1% v/v penicillin-streptomycin.</li>
	<li>Use microsurgical forceps to strip PVATs, with one pair of forceps fixing the aorta and the other pulling off the adipose tissue. Remove the vasculature tissue as much as possible while minimizing damage to the perivascular adipose tissue.</li>
	<li>Collect the PVAT surrounding the thoracic aorta into the 2 mL microcentrifuge tube containing high-glucose DMEM supplemented with 1% v/v penicillin-streptomycin placed on ice.</li>
</ol>
3. Isolation of stromal vascular fraction (SVF)
<ol>
	<li>Rinse the collected tissues sequentially with PBS containing 10% v/v penicillin-streptomycin and PBS containing 1% v/v penicillin-streptomycin. 
	NOTE: From this step onwards, all experiments must be carried out under sterile conditions in a class II laminar flow hood.</li>
	<li>Transfer the tissues to a sterile 60 mm Petri dish, add 200 &#181;L of digestion solution, and mince them into 1 mm3 pieces using sterile scissors.</li>
	<li>Transfer the mix to a 15 mL centrifuge tube using a plastic pipette tip, the end broadened by a sterile scissor cut, and add 6 mL of digestion solution to start the digestion reaction. 
	NOTE: One digestion reaction (3 mL of digestion solution) is sufficient for PVAT depots from six mice.</li>
	<li>Incubate the tissues at 37 &#176;C in an incubator with an orbital shaker (150 rpm frequency for effective mixing) for 30-45 min. Tie the tubes flat on a rack to make the tubes shake horizontally. Shake up and down vigorously by hand for 10 s every 5-10 min. 
	NOTE: Digestion can be discontinued when a homogeneous, yellowish digestive fluid is seen with no tissue fragments left.</li>
	<li>Strain the digested tissues through a 70 &#181;m cell strainer into a 50 mL centrifuge tube. Rinse the cell strainer with an equal volume of culture medium to maximize cell yield and stop the digestion.</li>
	<li>Transfer the filtrate to a new 15 mL centrifuge tube and centrifuge at 1,800 &#215; g for 10 min. Invert the tube to discard the supernatant and resuspend the pellets in 5 mL of PBS. Centrifuge at 1,800 &#215; g for 5 min.</li>
	<li>Discard the supernatant by inverting the tube and resuspend the pellets in an appropriate volume of culture medium. 
	NOTE: The cell pellets collected from every six mice are resuspended in 1 mL of culture medium and seeded into one well of a 12-well plate.</li>
	<li>Seed the cells into a collagen-coated 12-well plate and incubate at 37 &#176;C in a humid atmosphere with 5% CO2 overnight.</li>
	<li>On the next day, aspirate the culture medium, wash the cells with prewarmed (37 &#176;C) PBS containing 1% v/v penicillin-streptomycin to remove cell debris and red blood cells, and add back 1 mL of fresh culture medium each well.</li>
</ol>
4. Adipogenic induction of SVF-derived preadipocytes from periaortic adipose tissue
<ol>
	<li>Change the culture medium every other day until the cells reach ~60-70% confluence. 
	NOTE: It usually takes 3-4 days for the cells to reach 60-70% confluence.</li>
	<li>When the cells reach 60-70% confluence, aspirate the culture medium and replace it with brown adipogenic induction medium. Consider the day of induction of adipogenic differentiation as day 0 of differentiation.</li>
	<li>After 72 h (day 3 of differentiation), refresh the medium with maintenance medium. Change the maintenance medium every 2 days until the cells are used for experiments.</li>
	<li>Analyze the adipogenic cells in an appropriate way, such as Oil Red O staining14 and western blotting15.</li>
</ol>

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+adipose+tissue+in+vascular+function+and+disease:+a+review+of+current+research+and+animal+models.">Perivascular adipose tissue in vascular function and disease: a review of current research and animal models.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 34 (8), 1621-1630 (2014).</li><li>Ferrero, R., Rainer, P., Deplancke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+consensus+view+of+mammalian+adipocyte+stem+and+progenitor+cell+heterogeneity.">Toward a consensus view of mammalian adipocyte stem and progenitor cell heterogeneity.</a> Trends in Cell Biology. 30 (12), 937-950 (2020).</li><li>Angueira, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+lineage+of+thermogenic+perivascular+adipose+tissue.">Defining the lineage of thermogenic perivascular adipose tissue.</a> Nature Metabolism. 3 (4), 469-484 (2021).</li><li>Boucher, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab27a+regulates+human+perivascular+adipose+progenitor+cell+differentiation.">Rab27a regulates human perivascular adipose progenitor cell differentiation.</a> Cardiovascular Drugs and Therapy. 32 (5), 519-530 (2018).</li><li>Saxton, S. N., Withers, S. B., Heagerty, A. 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The authors have no conflicts of interest, financial or otherwise, to declare.

We propose a practical and feasible approach for the isolation and adipogenic induction of SVF-derived preadipocytes from mouse periaortic adipose tissue. The advantages of this protocol are that it is simple and economical. Adequate numbers of mice are critical for successful isolation, as insufficient tissue can result in low SVF density and poor growth state, ultimately affecting lipogenic efficiency. Additionally, mouse age is an important factor to consider as the adipogenic potential of SVF decreases with age. Rapi...

Perivascular adipose tissue (PVAT), a perivascular structure composed of a mixture of mature adipocytes and a stromal vascular fraction (SVF), is believed to interact with the adjacent vessel wall via its secretome paracrineally1. As a critical regulator of vascular homeostasis, PVAT dysfunction is implicated in the pathogenesis of cardiovascular diseases2,3,4. The SVF of adipocyte tissue consists of several expected cell populations, including endothelial cells, immune cells, mesothelium cells, neuronal cells, and adipose stem and progenitor cells (ASPCs)5,6. It is well known that ASPCs residing in the SVF of adipose tissue can give rise to mature adipocytes5. SVF is inferred to be a critical source of mature adipocytes in PVAT. Several studies have shown that PVAT-SVF can differentiate into mature adipocytes under specific induction conditions6,7,8.
Currently, there are two isolation systems for isolating SVF from adipose tissue, one is enzymatic digestion and the other is non-enzymatic9. Enzymatic methods typically result in a higher yield of nucleated progenitor cells10. To date, the benefits of SVF in promoting vascular regeneration and neovascularization in wound healing, urogenital, and cardiovascular diseases have been widely demonstrated11, especially in dermatology and plastic surgery12,13. However, the clinical application prospects of PVAT-derived SVF have not been well explored, which may be attributed to the lack of a standardized method for the isolation of SVF from PVAT. The objective of this protocol is to establish a standardized approach for the isolation, culture, and adipogenic induction of SVF-derived preadipocytes from mouse PVAT surrounding the thoracic aorta, enabling further investigation of PVAT function. This protocol optimizes tissue processing and cell differentiation techniques for culturing periaortic adipocytes obtained from young mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m syringe filters</td>
			<td>PALL</td>
			<td>4612</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate&#160;</td>
			<td>Labselect</td>
			<td>11210</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Labserv</td>
			<td>310109003</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;,5-triiodo-L-thyronine (T3)</td>
			<td>Sigma-Aldrich</td>
			<td>T-2877</td>
			<td>1 nM</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Labselect</td>
			<td>CT-002-50A</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-adiponectin</td>
			<td>Abcam</td>
			<td>ab22554</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>anti-COX IV</td>
			<td>CST</td>
			<td>4850</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>anti-FABP4</td>
			<td>CST</td>
			<td>2120</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>anti-PGC1&#945;</td>
			<td>Abcam</td>
			<td>ab191838</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>anti-PPAR&#947;</td>
			<td>Invitrogen</td>
			<td>MA5-14889</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>anti-UCP1</td>
			<td>Abcam</td>
			<td>ab10983</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>anti-&#945;-Actinin</td>
			<td>CST</td>
			<td>6487</td>
			<td>1:1,000 working concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beyotime</td>
			<td>ST023-200g</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>C57BL/6 mice aged 4-8 weeks of both sexes</td>
			<td>Shanghai Model Organisms Center, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#181;m, nylon</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from calf skin</td>
			<td>Sigma-Aldrich</td>
			<td>C8919</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>D4693-1G</td>
			<td>4 mg/mL</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-25G</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>High glucose DMEM</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Incubator with orbital shaker</td>
			<td>Shanghai longyue Instrument Eruipment Co.,Ltd.</td>
			<td>LYZ-103B</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin (cattle)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>11070-73-8</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Ringer&#39;s Solution</td>
			<td>Pricella&#160;</td>
			<td>PB180347</td>
			<td>protect from light&#160;</td>
		</tr>
		<tr>
			<td>Microsurgical forceps</td>
			<td>Beyotime</td>
			<td>FS233</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissor</td>
			<td>Beyotime</td>
			<td>FS217</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd</td>
			<td>A600395-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd</td>
			<td>B548117-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>115-035-146</td>
			<td>1:5,000 working concentration</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-035-144</td>
			<td>1:5,000 working concentration</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>Standard forceps</td>
			<td>Beyotime</td>
			<td>FS225</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Beyotime</td>
			<td>FS001</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

We aim to establish a standardized approach for the isolation culture and adipogenic induction of stromal vascular fraction-derived preadipocytes from mass PVAT. This protocol enables us to conduct further investigations into PVAT function and into the communication between periaortic adipocytes and vascular cells in vitro. The main challenges we currently face in our experiments include the restricted of availability of PVAT and the requirements for additional human and the material resources when compared to using immortalized preadipocytes.Our protocol allows for the study of perivascular adipose tissue function and it's relationship with vascular cells. We also offers a basis for studying PVAT ring and genes, and heterogeneity, and provides a platform for exploring the important regulator of PVAT differentiation and the adipogenesis in vitro.

Using this protocol described above, we carefully isolated PVATs surrounding mouse thoracic aortas (Figure 1A-D). After washing and mincing the PVATs into small pieces using sterile scissors (Figure 1E,F), tissue fragments were digested in a digestion solution containing type 1 collagenase (1 mg/mL) and dispase II (4 mg/mL) and incubated at 37 &#176;C on a shaker for 30-45 min (Figure 1G</st...

Watch this Scientific Journal Video about The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue at JoVE.com

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

isolation-culture-adipogenic-induction-stromal-vascular-fraction

Research

JoVE Journal

Biology

1.9K vues.  Shanghai Jiao Tong University School of Medicine. Notre objectif est d’établir une approche standardisée pour la culture d’isolement et l’induction adipogénique de préadipocytes dérivés de la fraction vasculaire stromale à partir de PVAT de masse. Ce protocole nous permet d’approfondir nos recherches sur la fonction PVAT et sur la communication in vitro entre les adipocytes périaortiques et les cellules vasculaires. Les principaux défis auxquels nous sommes actuellement confrontés dans nos expériences comprennent la dispon...

Vidéo: Pleins feux sur l’auteur : L’importance de l’isolement, de la culture et de l’induction adipogénique de préadipocytes dérivés de la SVF à partir du tissu adipeux périvasculaire de souris 

Dissection and Isolation of Murine Perivascular Adipose Tissue (PVAT)

Begin by spraying the properly euthanized mouse with 75%alcohol to disinfect the skin. Then place the mouse in a supine position. Lift the skin of the animal's abdominal region, and using surgical scissors, make a small incision in it.Then using the scissors, bluntly separate the skin and abdominal muscles from the pelvis to the neck, along the ventral midline. Expose the heart and lungs by cutting the diaphragm and ribs along both sides of the midline. Next, carefully remove the liver, spleen, bowels, and kidney, avoiding damage to the intestinal wall.Following that, remove the lungs and esophagus. Now gently lift the heart using forceps held in one hand and separate the aorta from the spine with surgical scissors held in the other hand. Then place the heart and aorta in a 60 millimeter petri dish, containing antibiotic supplemented PBS.Retrieve the microsurgical forceps and microsurgical scissors from the alcohol, and rinse them in 25 milliliters of PBS to remove excess alcohol. Working under a stereo microscope, remove the thymus and other tissues from the heart and aorta. Then remove the heart, leaving the aorta with the perivascular adipose tissues or PVATs.Transfer the aorta along with the PVATs to a clean 60 millimeter petri dish containing antibiotic supplemented PBS. Using one pair of microsurgical forceps, hold the aorta, and using another pair, pull off the attached adipose tissues to separate the PVATs from the aorta. Remove as much vascular tissue as possible while causing minimal harm to the PVATs.Finally, transfer the collected PVATs into a two milliliter microcentrifuge tube, containing antibiotic supplemented, high glucose, DMEM, placed on ice.

Isolation of Stromal Vascular Fraction (SVF) from Perivascular Adipose Tissue (PVAT)

While working under a class two laminar flow hood, begin by sequentially washing the perivascular adipose tissues or PVATs collected from mice two times. First, using PBS containing 10%penicillin-streptomycin, and, next, using PBS supplemented with 1%penicillin-streptomycin. Transfer the rinsed tissues into a sterile 60-millimeter Petri dish and add 200 microliters of digestion solution to it.Using sterile scissors, mince the tissues into pieces having a dimension of one cubic millimeter. With the help of sterile scissors, cut a plastic pipette tip to broaden its end. Then transfer the minced tissue mix to a 15-milliliter centrifuge tube using the broad-ended pipette tip.Add six milliliters of the digestion solution to the tissues to initiate digestion. After tying the tube horizontally on a rack, incubate it at 37 degrees Celsius in an orbital shaker for 30 to 45 minutes. Every 5 to 10 minutes, remove the tube from the shaker in the incubator and manually shake it up and down vigorously.Pass the digested tissue mix through a 70-micron cell strainer into a 50-milliliter centrifuge tube. Rinse the strainer with an equal volume of culture medium to maximize cell yield and quench digestion. Transfer the filtrate to a new 15-milliliter centrifuge tube.Centrifuge the tube to isolate the stromal vascular fraction or SVF. Invert the tube to discard the supernatant and re-suspend the pellet in five milliliters of PBS. Centrifuge the tube again at 1, 800 G for five minutes before discarding the supernatant.Re-suspend the pellet in an appropriate volume of culture medium. Seed the cells into a collagen-coated 12-well plate and incubate overnight at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide. On the next day, remove cell debris and red blood cells by aspirating the culture medium and washing the cells with antibiotic-supplemented PBS pre-warmed to 37 degrees Celsius.Finally, add one milliliter of fresh culture medium to each well, and continue culturing the cells until they reach the desired stage of confluency.