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>...

Semi-Automated Isolation of Murine White Adipose Tissue

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+expression+of+tumor+necrosis+factor-alpha:+direct+role+in+obesity-linked+insulin+resistance.">Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance.</a> Science. 259 (5091), 87-91 (1993).</li><li>Zhang, Y., 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=Positional+cloning+of+the+mouse+obese+gene+and+its+human+homologue.">Positional cloning of the mouse obese gene and its human homologue.</a> Nature. 372 (6505), 425-432 (1994).</li><li>Kajimura, S., Saito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+era+in+brown+adipose+tissue+biology:+Molecular+control+of+brown+fat+development+and+energy+homeostasis.">A new era in brown adipose tissue biology: Molecular control of brown fat development and energy homeostasis.</a> Annual Review of Physiology. 76, 225-249 (2014).</li><li>Wu, J., 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=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and+human.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human.</a> Cell. 150 (2), 366-376 (2012).</li><li>Loos, R. 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J., 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=Adipose+subtype-selective+recruitment+of+TLE3+or+Prdm16+by+PPAR&#947;+specifies+lipid+storage+versus+thermogenic+gene+programs.">Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPAR&#947; specifies lipid storage versus thermogenic gene programs.</a> Cell Metabolism. 17 (3), 423-435 (2013).</li><li>Pearson, S., 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=Loss+of+TLE3+promotes+the+mitochondrial+program+in+beige+adipocytes+and+improves+glucose+metabolism.">Loss of TLE3 promotes the mitochondrial program in beige adipocytes and improves glucose metabolism.</a> Genes and Development. 33 (13-14), 747-762 (2019).</li><li>Shao, 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=Zfp423+maintains+white+adipocyte+identity+through+suppression+of+the+beige+cell+thermogenic+gene+program.">Zfp423 maintains white adipocyte identity through suppression of the beige cell thermogenic gene program.</a> Cell Metabolism. 23 (6), 1167-1184 (2016).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublines+of+mouse+3T3+cells+that+accumulate+lipid.">Sublines of mouse 3T3 cells that accumulate lipid.</a> Cell. 1 (3), 113-116 (1974).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+established+preadipose+cell+line+and+its+differentiation+in+culture+II.+Factors+affecting+the+adipose+conversion.">An established preadipose cell line and its differentiation in culture II. Factors affecting the adipose conversion.</a> Cell. 5 (1), 19-27 (1975).</li><li>Green, H., Kehinde, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+heritable+changes+leading+to+increased+adipose+conversion+in+3T3+cells.">Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells.</a> Cell. 7 (1), 105-113 (1976).</li><li>Aune, U. L., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+differentiation+of+stromal+vascular+cells+to+beige/brite+cells.">Isolation and differentiation of stromal vascular cells to beige/brite cells.</a> Journal of Visualized Experiments. (73), e50191 (2013).</li><li>Galmozzi, A., Kok, B. 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S., SundarRaj, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture:+a+simple,+reproducible,+efficient+and+economic+technique+for+isolation+of+mesenchymal+stromal+cells+from+human+adipose+tissue+and+lipoaspirate.">Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.</a> Journal of Tissue Engineering and Regenerative Medicine. 8 (9), 706-716 (2014).</li><li>Lockhart, R. A., Aronowitz, J. A., Dos-Anjos Vilaboa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+freshly+isolated+human+adipose+stromal+cells+for+clinical+applications.">Use of freshly isolated human adipose stromal cells for clinical applications.</a> Aesthetic Surgery Journal. 37, S4-S8 (2017).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene Therapy. 7 (12), 1063-1066 (2000).</li><li>Li, Y., Fromme, T., Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meaningful+respirometric+measurements+of+UCP1-mediated+thermogenesis.">Meaningful respirometric measurements of UCP1-mediated thermogenesis.</a> Biochimie. 134, 56-61 (2017).</li><li>Hiraike, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+with+mouse+adipocytes+using+hypotonic+buffer+to+enrich+nuclear+fraction+before+fixation.">Chromatin immunoprecipitation with mouse adipocytes using hypotonic buffer to enrich nuclear fraction before fixation.</a> STAR Protocols. 4 (1), 102093 (2023).</li><li>Rodeheffer, M. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Invitrogen&#160;</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.

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

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

We are interested in brown and beige subtypes of adipocyte which dissipate energy in the form of heat, while white adipocyte generally store energy as Our goal is to develop a new treatment for obesity and diabetes based on promotion of energy expenditure, rather than suppression of energy intake. We have identified a transcription factor, NFI, as a critical regulator of brown adipocyte differentiation by a genome-wide open chromatin analysis. NFI and the master transcriptional regulator of adipogenesis, PPAR-gamma, co-localize as the brown-fat-specific enhancers.In the process of isolating stromal vascular fraction from mouse adipose tissue, manual mincing and collagenase digestion of adipose tissue can lead to experimental variability and contamination. Our method uses a tissue dissociator for collagenase digestion, which facilitates SVF isolation, reduce experimental variability and contamination, and improves reproducibility. Experiment using individually-differentiated adipocyte are indispensable for research in our field.Our protocol would offer easy and reproducible SVF isolation for subsequent adipocyte differentiation and would accelerate research in our community. Currently, we are investigating the role of NFI of all-body metabolism, such as body weight and glucose, using gain and loss of function mouse models. We will also explore other pharmacological activation of NFI for each downstream pathway in near future.

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...

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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 ...

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JoVE Journal

Biology

Isolation, Digestion, Filtration and Cell Plating of Murine Adipose Tissue

Begin by placing a seven to eight week old euthanized male mouse on a clean bench surface. Cut the abdominal skin of the mouse with a pair of scissors towards the lower abdomen. Excise the adipose tissue from the inner thighs.Place the excised tissue into tube C on ice containing 2.5 milliliters of an enzyme mixture of enzymes D, R, A and 2.35 milliliters of DMEM/F12 without FBS or antibiotics. Using scissors, cut the adipose tissue into approximately two square millimeter sized pieces. Tightly close the cap of tube C and invert the tube.Attach the tube to the sleeve of a tissue dissociator and digest the samples at 37 degrees Celsius for 40 minutes. Next, prewarm DMEM/F12 media containing FBS and antibiotics to 37 degrees Celsius. After incubation, remove tube C from the tissue dissociator and stop digestion by adding five milliliters of DMEM/F12 with FBS and antibiotics.Gently pipette four times. Centrifuge the suspension at 700 g for 10 minutes at 20 degrees Celsius. Without disturbing the cell palette, carefully aspirate the supernatant.Resuspend the pellet in 10 milliliters of DMEM/F12 containing FBS and antibiotics, and gently pipette five times. Filter the cell suspension through a 70 micron diameter cell strainer placed on a 50 milliliter tube. Centrifuge the filtrate at 250 g for five minutes.Resuspend the pellet in 10 milliliters of PBS. Before plating, centrifuge the cell suspension at 500 g for five minutes. Discard the supernatant.Resuspend the pellet in 10 milliliters of DMEM/F12 containing FBS and antibiotics. Mix by pipetting the suspension gently, 10 times. Plate 10 milliliters of the suspension on a 10 centimeter collagen coated dish.Place the dish in a cell culture incubator. Aspirate the medium and wash the cells three times with three milliliters of PBS per wash. Add 10 milliliters of DMEM/F12 containing FBS and antibiotics and incubate.Oil Red O staining showed lipid laden adipocytes seven days after inducing adipocyte differentiation. The degree of full differentiation was confirmed by mRNA expression analysis of adipogenesis regulators. Rosiglitazone induced a dose dependent effect on the expression levels of brown fat specific genes such as Ucp1 and Ppargc1a.However, for Fabp4, the effect saturated at 0.1 micromole rosiglitazone concentration. Differentiated adipocytes can be used for various functional and mechanistic analyses such as oxygen consumption rate analysis and chromatin immunoprecipitation.

1.3K 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.

Semi-Automated Isolation of Murine White Adipose Tissue

概要