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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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><tbody><tr><td>0.2 &amp;mu;m syringe filters</td><td>PALL</td><td>4612</td><td></td></tr><tr><td>12-well plate&amp;nbsp;</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&#039;,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&amp;alpha;</td><td>Abcam</td><td>ab191838</td><td>1:1,000 working concentration</td></tr><tr><td>anti-PPAR&amp;gamma;</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-&amp;alpha;-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 &amp;micro;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 &amp;mu;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&amp;nbsp;</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&amp;nbsp;</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)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>11070-73-8</td><td>1 &amp;mu;M</td></tr><tr><td>Isoflurane</td><td>RWD</td><td>R510-22-10</td><td></td></tr><tr><td>Krebs-Ringer&#039;s Solution</td><td>Pricella&amp;nbsp;</td><td>PB180347</td><td>protect from light&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 &amp;mu;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.

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

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

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.

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 ...

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Research

JoVE Journal

Biology

2.1K Views.  Shanghai Jiao Tong University School of Medicine. We streven naar een gestandaardiseerde aanpak voor de isolatiecultuur en adipogene inductie van stromale vasculaire fractie-afgeleide preadipocyten uit massa PVAT. Dit protocol stelt ons in staat om verder onderzoek te doen naar de PVAT-functie en naar de communicatie tussen periaorta-adipocyten en vasculaire cellen in vitro. De belangrijkste uitdagingen waarmee we momenteel in onze experimenten worden geconfronteerd, zijn onder meer de beperkte beschikbaarheid van PVAT en de vereisten voor e...

Video: Auteur in de schijnwerpers: het belang van isolatie, cultuur en adipogene inductie van SVF-afgeleide preadipocyten uit perivasculair vetweefsel van muizen 

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Developmental Biology

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.

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 ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunology and Infection

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

Source: Qi, Y., et al. <a href="https://app.jove.com/v/60691">Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for isolating and culturing fluorescently labeled neural crest-derived adipose-derived stem cells (ADSCs) from murine periaortic arch adipose tissue. Following isolation, the NCADSCs are detected, sorted using fluorescence-activated cell sorting, and subsequently cultured on a plate, promoting cell proliferation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of the stromal vascular ...

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

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