The authors would like to thank Takahito Wada and Saiko Yoshida (The University of Tokyo, Tokyo, Japan) for their experimental assistance. This work was funded by the following grants to Y.H.: research grant from the University of Tokyo Excellent Young Researcher Program; Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists, grant number 19K17976; grant for the Front Runner of Future Diabetes Research (FFDR) from the Japan Foundation for Applied Enzymology, grant number 17F005; grant from the Pharmacological Research Foundation; grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research; grant from the MSD Life Science Foundation; grant from the Daiwa Securities Health Foundation; grant from the Tokyo Biochemical Research Foundation; Life Science Research grant from the Takeda Science Foundation; and grant from the SENSHIN Medical Research Foundation.

All animal experiments described in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tokyo and performed according to the institutional guidelines of the University of Tokyo.
1. Preparation of enzyme solution and medium
<ol>
	<li>Put both sides of inguinal white adipose tissue (right and left side, approximately 150 mg) from a 7-8-week-old mouse and 2.5 mL of enzyme solution into tube C of the dissociator.</li>
	<li>Reconstitute Enzyme D with 3 mL of Dulbecco&#39;s modified eagle medium (DMEM)/F12 without fetal bovine serum (FBS) or antibiotics, Enzyme R with 2.7 mL of DMEM/F12 without FBS or antibiotics, and Enzyme A with 1 mL of buffer A.</li>
	<li>Prepare 2.5 mL of enzyme solution by adding 100 &#181;L of Enzyme D, 50 &#181;L of Enzyme R, 12.5 &#181;L of Enzyme A, and 2.35 mL of DMEM/F12 without FBS or antibiotics into tube C. Set tube C with enzyme solution on ice.</li>
</ol>
2. Isolation of adipose tissue
<ol>
	<li>Euthanize 7-8-week-old male C57BL/6J mice (approximately 20 g) by cervical dislocation.</li>
	<li>At a clean bench, to isolate inguinal white adipose tissue, cut the skin with blunt/sharp scissors at the abdomen and toward the lower extremities. Isolate the adipose tissue from the inside of the thighs using sharp/sharp scissors. One side of inguinal adipose tissue wieghs ~75 mg.</li>
	<li>Put the isolated adipose tissue into tube C with enzyme solution on ice, and keep the tube within the clean bench to maintain sterility.</li>
</ol>
3. Mincing and digestion of adipose tissue
<ol>
	<li>At the clean bench, add 2.5 mL of enzyme solution to tube C.</li>
	<li>Mince the adipose tissue into small pieces by cutting with sharp/sharp scissors 50 times. Cut the adipose tissue into ~2 mm2 pieces.</li>
	<li>Tightly close the cap of tube C, turn the tube upside down, and attach it to the sleeve of the tissue dissociatorwith the cap on. Then, digest the sample for ~40 min at 37 &#176;C. 
	&#8203;NOTE: This study used gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec 130-096-427), and the preinstalled program &#34;37C_mr_ATDK_1&#34; was followed.</li>
</ol>
4. Filtration of cell suspension
<ol>
	<li>Pre-warm DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin to 37 &#176;C.</li>
	<li>Detach tube C from the tissue dissociator and stop digestion by adding 5 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and gently pipetting four times.</li>
	<li>Centrifuge at 700 x g for 10 min at 20 &#176;C.</li>
	<li>Carefully aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend the pellet by adding 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and gently pipetting five times.</li>
	<li>Filter the cell suspension through a 70 &#181;m diameter cell strainer placed on a new 50 mL tube.</li>
	<li>Centrifuge at 250 x g for 5 min and resuspend the pellet in 10 mL of phosphate-buffered saline (PBS).</li>
</ol>
5. Cell plating
<ol>
	<li>Centrifuge at 500 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet by adding 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and pipetting ten times.</li>
	<li>Plate the cell suspension on a 10 cm collagen-coated dish and place the dishes in a cell culture incubator (37 &#176;C, 5% CO2) for 1-2 h. 
	NOTE: Collagen-coated dishes are not be absolutely required. However, based on experience, collagen-coated dishes do help the adherence of preadipocytes and thus increase the survival of the cells.</li>
	<li>Aspirate the medium and wash the cells twice with 3 mL of PBS per wash.</li>
	<li>Add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and return the dishes to the cell-culture incubator (37 &#176;C, 5% CO2).</li>
</ol>
6. Passaging of preadipocytes
<ol>
	<li>The next day, aspirate the medium (the cells will be adherent, and thus the medium can be aspirated), wash the cells twice with 3 mL of PBS per wash, and add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin.</li>
	<li>When the cells reach 80% confluence (usually 4 days after SVF isolation), split the cells into four 10 cm collagen-coated dishes.
	<ol>
		<li>Specifically, aspirate the medium, wash the cells with PBS (room temperature [RT]), add 1 mL of pre-warmed 0.05% trypsin, and incubate for 5 min in the cell-culture incubator.</li>
		<li>Add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin to quench the trypsin activity. After pipetting, centrifuge at 440 x g for 5 min.</li>
		<li>Aspirate the supernatant, resuspend the cells by adding 40 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin, and plate the cells in four 10 cm collagen-coated dishes.</li>
	</ol>
	</li>
	<li>Passage the cells again when they reach 80% confluence.</li>
</ol>
7. Preparation of retrovirus expressing SV40 large T antigen for immortalization of preadipocytes (optional)
<ol>
	<li>At least 3 days before immortalization, plate Platinum-E (Plat-E) packaging cells29 at a density of 3.0 x 105 cells/mL in 2 mL of DMEM with 10% FBS without antibiotics. Use a hemacytometer to count the cells.</li>
	<li>The next day, transfect 4 &#181;g of pBABE-neo largeTcDNA (add gene plasmid #1780) using Lipofectamine 2000, according to the manufacturer&#39;s instructions.</li>
	<li>After 24 h, aspirate the medium and add 2 mL of DMEM with 10% FBS and 1% penicillin-streptomycin.</li>
	<li>The next day, harvest the retroviral supernatant. The retrovirus can be used for immortalization immediately or stored at -80 &#176;C.</li>
</ol>
8. Immortalization of preadipocytes with SV40 large T antigen (optional)
<ol>
	<li>Passage and expand the preadipocytes twice after SVF isolation.</li>
	<li>Plate the cells in 6-well plates at a density of 0.5-1.0 x 104 cells/mL in 2 mL of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin. Use a hemacytometer to count the cells.</li>
	<li>On the next day, prepare 250 &#181;L of fresh or thawed retrovirus expressing SV40 large T antigen per well of the 6-well plates. Centrifuge at 440 x g for 5 min and place the supernatant in a new tube.</li>
	<li>Add 750 &#181;L of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and 1 &#181;L of polybrene per 250 &#181;L of retroviral supernatant to make the working virus.</li>
	<li>Aspirate the medium and add 1,000 &#181;L of the working retrovirus to each well containing preadipocytes.</li>
	<li>Add 1 mL of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin 4 h later.</li>
	<li>The next day, separate the infected preadipocytes into a 10 cm dish and select the infected cells with DMEM/F12 containing 10% FBS, 1% penicillin-streptomycin, and 500 &#181;g/mL neomycin. To monitor the antibiotics selection, prepare a dish of uninfected cells with neomycin.</li>
	<li>Continue to passage the infected cells with neomycin until all uninfected cells in the monitor dish are dead.</li>
</ol>
9. Adipocyte differentiation
<ol>
	<li>Plate the cells. For 12-well plates, plate the cells at a density of 4.0 x 104 cells/mL in 1 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin.</li>
	<li>When the preadipocytes become confluent (48-72 h after plating), aspirate the medium and replace it with differentiation medium (see Table 1 for composition).</li>
	<li>At day 2 of differentiation (i.e., 48 h after inducing differentiation), replace the medium with maintenance medium (see Table 1 for composition). Afterward, change the maintenance medium every 2 days. Terminal differentiation is achieved at day 7 of differentiation.</li>
	<li>Oil red o staining
	<ol>
		<li>For oil red o staining, aspirate the medium and rinse the cells with 1 mL of PBS per well. Fix the cells by adding 1 mL of 4% formaldehyde per well, and incubate the cells for 15 min at RT.</li>
		<li>During the incubation, dilute the 0.5% oil red o stock solution (in isopropyl alcohol) to 0.3% using ddH2O and filter the working solution using a filter paper.</li>
		<li>After the 15 min of incubation, remove the formaldehyde, rinse the cells with 60% isopropyl alcohol, add 1 mL of filtered oil red o working solution, and incubate for 1 h at RT.</li>
		<li>After the incubation, wash the cells with running water. Then, observe lipid-laden adipocytes under an inverted microscope (magnification: 20x objective and 10x eyepiece).</li>
	</ol>
	</li>
	<li>mRNA expression analysis 
	NOTE: Refer to Table 2 for the list of primers used in this study.
	<ol>
		<li>Aspirate the medium and add 1 mL/well of trizol to the well.</li>
		<li>Incubate for 15 min at RT, detach the cells by pipetting, and collect the samples in 1.5 mL tubes. The samples can be stored at -80 &#176;C until RNA extraction.</li>
		<li>Thaw the frozen sample to RT, add 200 &#181;L of chloroform to the sample, and shake vigorously for 15 s.</li>
		<li>Incubate the sample at RT for 3 min and then spin at 20,000 x g for 15 min at 4 &#176;C.</li>
		<li>Carefully remove the aqueous phase (top, colorless) and transfer to new tubes. Never transfer the protein phase (middle, white) or the phenol/chloroform phase (bottom, pink).</li>
		<li>Slowly add an equal volume of 70% EtOH and mix gently. Do not vortex.</li>
		<li>Load the sample (up to 700 &#181;L) into an RNA spin column seated in a collection tube. Be sure to include any precipitate that may have formed.</li>
		<li>Spin at 9,000 x g for 30 s at 4 &#176;C , and then discard the flow-through.</li>
		<li>Add 700 &#181;L of buffer RW1, spin at 9,000 x g for 30 s at 4 &#176;C , and then discard the flow-through.</li>
		<li>Transfer the column into a new collection tube and add 500 &#181;L of buffer RPE.</li>
		<li>Spin at 9,000 x g for 30 s at 4 &#176;C and then discard the flow-through.</li>
		<li>Add 500 &#181;L of buffer RPE, spin at 9,000 x g for 2 min at 4 &#176;C, and then discard the flow-through.</li>
		<li>Transfer the column to a new collection tube, and spin (columns without buffer) at 9,000 x g for 1-2 min at 4 &#176;C.</li>
		<li>Transfer the column into a new 1.5 mL collection tube and add 30 &#181;L of RNase-free water directly onto the column membrane.</li>
		<li>Incubate the sample at RT for 1-2 min. Then, spin at 9,000 x g for 1 min at 4 &#176;C to elute the RNA.</li>
		<li>Check the RNA concentration using a fluorospectrometer. 
		NOTE: It is recommended to align the concentration here.</li>
		<li>Perform reverse transcription using ~200 ng of RNA template. Use a commercial quantitative real-time polymerase chain reaction (qPCR-RT) kit and follow the manufacturer&#39;s protocol. After the reaction, dilute the cDNA 20 times to 200 &#181;L.</li>
		<li>Add 2.0 &#181;L of cDNA, 3.0 &#181;L of Power Sybr Green Master Mix, 0.018 &#181;L of 100 &#181;M qPCR forward primer, 0.018 &#181;L of 100 &#181;M qPCR reverse primer, and 0.96 &#181;L of ddH2O to each well of a 384-well plate.</li>
		<li>Run a qPCR (hold stage: 50&#176;C for 2 min and 95 &#176;C for 2 min; PCR stage: 40 cycles of 95 &#176;C for 15 s and 60 &#176;C for 1 min; melt curve stage: 95 &#176;C for 15 s, 60 &#176;C for 1 min, and 95 &#176;C for 15 s). Use the standard curve method for quantification.</li>
	</ol>
	</li>
</ol>

Semi-Automated Isolation of Murine White Adipose Tissue

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None of the authors have a competing financial interest related to this work.

Here, we described a protocol for isolation of the SVF from murine adipose tissue to obtain preadipocytes and perform adipocyte differentiation in vitro. The use of a tissue dissociator for collagenase digestion decreased experimental variation, decreased the risk of contamination, and increased reproducibility. While this procedure is a critical step within the presented protocol, the process is highly automated and optimization is not needed. However, depending on the mouse age and adipose tissue depot, optimi...

Adipose tissue biology has been attracting ever-increasing attention because of the growing prevalence of obesity and type 2 diabetes globally1. Adipocytes store excess energy in the form of lipid droplets, which are released upon starvation. Moreover, adipose tissue maintains systemic energy homeostasis by serving as an endocrine organ and communicating with other tissues2,3. Intriguingly, both excess adipose tissue (obesity) and adipose loss (lipodystrophy) are linked to insulin resistance and diabetes1. Adipocytes are divided into three types: white, brown, and beige1. White adipocytes mainly store excess energy as lipids, whereas brown and beige adipocytes dissipate energy in the form of heat via mitochondrial uncoupling protein-1 (Ucp1)1,4. Notably, beige adipocytes (also called &#34;inducible&#34; brown adipocytes) appear in white adipose tissue in response to cold or sympathetic stimulation and exhibit gene expression patterns that overlap with but are distinct from those of &#34;classical&#34; brown adipocytes5. Recently, brown and beige adipocytes have been anticipated as potential targets of anti-obesity and anti-diabetes treatments aimed at &#34;enhancing energy dissipation&#34; rather than &#34;suppressing energy intake&#34;4. Supportively, the risk allele of the FTO obesity variant rs1421085 in humans, which exhibits the strongest association with higher body mass index (BMI) among common variants6,7 and exhibits various gene-environment interactions8,9, is reported to negatively regulate beige adipocyte differentiation and function10. Peroxisome proliferator-activated receptor &#947; (PPAR&#947;) is known as a master transcriptional regulator of adipogenesis and is necessary and sufficient for adipocyte differentiation11. Transcriptional regulators, such as PRD1-BF1-RIZ1 homologous domain containing 16 (PRDM16), early b cell factor 2 (EBF2), and nuclear factor I-A (NFIA), are crucial for brown and beige adipocyte differentiation and function12,13,14,15,16,17,18. On the other hand, white adipocyte gene programming requires transcriptional regulators, such as transducin-like enhancer protein 3 (TLE3) and zinc finger protein 423 (ZFP423)19,20,21.
In vitro model systems enable molecular studies to be performed that aim to improve the understanding of the mechanism(s) underlying the functions and dysfunctions of adipocytes. Although publicly available and immortalized preadipocyte cell lines such as 3T3-L1 and 3T3-F442A exist22,23,24, the culture of primary preadipocytes and differentiation into adipocytes would be a more suitable model for studying in vivo adipogenesis. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is a well-known method for obtaining primary preadipocytes25,26. However, collagenase digestion of adipose tissue, which is commonly performed using a bacterial shaker with a tube rack, can result in experimental variation and is prone to contamination27,28. Here, we describe an alternative protocol that uses a gentle magnetic-activated cell sorting (MACS) tissue dissociator for collagenase digestion to achieve easier isolation of the SVF.

<table><tbody><tr><td>100 mm dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>12 well plate</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>60 mm dish</td><td>IWAKI</td><td>3010-060</td><td></td></tr><tr><td>Adipose Tissue Dissociation Kit, mouse and rat</td><td>Miltenyi Biotec</td><td>130-105-808</td><td>contents: Enzyme D, Enzyme R, Enzyme A and Buffer A</td></tr><tr><td>Cell strainer 70 &amp;micro;m</td><td>BD falcon</td><td>#352350</td><td></td></tr><tr><td>Collagen coated dishes, 100 mm</td><td>BD</td><td>#356450</td><td></td></tr><tr><td>Collagen coated dishes, 60 mm</td><td>BD</td><td>#354401</td><td></td></tr><tr><td>Collagen I Coat Microplate 6 well</td><td>IWAKI</td><td>4810-010</td><td></td></tr><tr><td>Dexamethasone</td><td>Wako</td><td>041-18861</td><td></td></tr><tr><td>Dissecting Forceps</td><td>N/A</td><td>N/A</td><td>autoclave before use</td></tr><tr><td>Dissecting Scissors, blunt/sharp</td><td>N/A</td><td>N/A</td><td>autoclave before use</td></tr><tr><td>Dissecting Scissors, sharp/sharp</td><td>N/A</td><td>N/A</td><td>autoclave before use</td></tr><tr><td>DMEM/F-12, GlutaMAX supplement</td><td>Gibco</td><td>10565-042</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>gentleMACS C Tubes</td><td>Milteny Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentleMACSOcto Dissociator with Heaters</td><td>Miltenyi Biotec</td><td>130-096-427</td><td></td></tr><tr><td>Humulin R Injection U-100</td><td>Eli Lilly</td><td>872492</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma</td><td>I7378-5G</td><td></td></tr><tr><td>Isobutylmethylxanthine (IBMX)</td><td>Sigma</td><td>17018-1G</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668-019</td><td></td></tr><tr><td>Neomycin Sulfate</td><td>Fujifilm</td><td>146-08871&amp;nbsp;</td><td></td></tr><tr><td>Opti-MEM</td><td>Invitrogen&amp;nbsp;</td><td>31985-062</td><td></td></tr><tr><td>pBABE-neo largeTcDNA (SV40)</td><td>Add gene</td><td>#1780</td><td></td></tr><tr><td>PBS tablets</td><td>Takara</td><td>T900</td><td></td></tr><tr><td>Platinum-E (Plat-E) Retroviral Packaging Cell Line</td><td>cell biolab</td><td>RV-101</td><td></td></tr><tr><td>Polybrene</td><td>Nacalai Tesque</td><td>12996-81</td><td></td></tr><tr><td>Power Sybr Green Master Mix</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>ReverTra Ace qPCR RT Master Mix</td><td>TOYOBO</td><td>#FSQ-201</td><td></td></tr><tr><td>RNeasy Mini Kit (250)</td><td>QIAGEN</td><td>74106</td><td></td></tr><tr><td>Rosiglitazone</td><td>Wako</td><td>180-02653</td><td></td></tr><tr><td>T3</td><td>Sigma</td><td>T2877-100mg</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Gibco</td><td>25200-056</td><td></td></tr></tbody></table>

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.

This protocol yields fully differentiated, lipid-laden adipocytes 7 days after inducing adipocyte differentiation. The degree of adipocyte differentiation can be evaluated by the oil red o staining of triglycerides and lipids (Figure 1A), or mRNA expression analysis using qPCR-RT of adipocyte genes, such as the master regulator of adipogenesis Pparg and its target Fabp4 (Figure 1B). To induce beige adipocyte differentiation in vitro, a...

Watch this Scientific Journal Video about Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator at JoVE.com

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.

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 ...

semi-automated-isolation-stromal-vascular-fraction-from-murine-white

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.0K Views.  The University of Tokyo. 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.

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

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Genetics

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioengineering

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 ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Medicine

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

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 

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.