Name: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells (Video) | JoVE Journal
Uploaded: 2013-03-28T12:00:00.000+00:00
Duration: 7 min 22 s
Description: Learn to isolate primary adipocytes from mouse adipose tissue and induce differentiation into beige/brite cells using collagenase digestion. This method enables study of molecular mechanisms regulating adipocyte browning for metabolic research.

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5 minutes

We thank Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda, and Lauren Ruiz for discussion, technical help and editorial assistance on the manuscript. This work was supported by grants from the NIH (DK087853), from the Program for Breakthrough Biomedical Research and from Asubio Pharm Inc. to S.K. U.L.A. was supported by a SHARE PhD fellowships from The University of Copenhagen and the EU FP7 project DIABAT (HEALTH-F2-2011-278373) to Lise Madsen and Karsten Kristiansen. We also acknowledge the DERC center grant (NIH P30 DK063720).

1. Prepare Digestion Medium
Make 5 ml per 5 mice per tissue (approximately 1 ml/1 g adipose tissue).
<ol>
 <li>Weigh in digestion enzymes:
 - Collaginase D: 1.5 u/ml (1108874103, 1 g, Roche, 70334223)
 - Dispase II: 2.4 u/ml (04942078001, 0,980 mg/lyo, Roche, 11466200)
 </li>
 <li>Add 25 ml PBS and mix well to dissolve</li>
 <li>Add CaCl2 just prior to digestion of the tissue at a final concentration of 10 mM</li>
</ol>
2. Dissect Adipose Tissue from Mice
<ol>
 <li>Dissection should be performed carefully, and immediately after the mice (6-8 weeks old) are sacrificed. Work sterile with ethanol sprayed on the mouse fur before opening it. Place the tissue directly in clean PBS, separate interscapular BAT and subcutaneous inguinal WAT.</li>
 <li>For interscapular BAT: have the mouse placed with the back faced up, cut open along the back and all the way to the neck. The brown adipose tissue can be found right under the skin between the shoulders (interscapular). BAT can be seen as two lobes, butterfly shaped. A thin layer of white fat covering the BAT should to be carefully removed.</li>
 <li>For subcutaneous WAT (inguinal WAT): remove skin. Inguinal WAT is found directly under the skin on both sides, starting almost on the back and going alongside, inside of the thighs and down toward the testicle.</li>
</ol>
3. Cut and Digest Adipose Tissue
<ol>
 <li>Quickly remove all contamination, like hair, skeletal muscle and connective tissue from the tissue sections.</li>
 <li>Dry tissue quickly on paper to not dilute dissection medium with PBS and place it on a dry plate.</li>
 <li>Add digestion medium (added CaCl2 prior to digestion) and mince the tissue into small pieces. Mix very well by pipetting up and down with a 5 ml pipette. Transfer the minced tissues in 50 ml tubes with the rest of the digestion medium, mix even more by pipetting up and down.</li>
 <li>Digest in 37 &deg;C with constant agitation at 150 rpm for 40-50 min. Check every 10-15 min to make sure the digestion is going well and to prevent over-digestion.</li>
</ol>
Note: Correct digestion medium and time are important. The tissue needs to be well digested, but excess digestion can damage the cells.
4. Filter Cell Suspension
<ol>
 <li>Stop digestion by adding 5 ml complete medium (DMEM/F12 containing 10% FPS and P/S). The cells should almost be completely homogenous. Mix well by pipetting.</li>
 <li>Centrifuge at 700 x g for 10 min.</li>
 <li>SVF can now be seen as a brownish pellet on the bottom of the tube. Aspirate the oily mature adipocyte layer on top and most of the liquid layer - keep the pellet and dissolve it in 10 ml complete medium. Mix well.</li>
 <li>Place a cell strainer (50-70 &mu;m diameter) over a new 50 ml tube and filter the cell suspension.</li>
</ol>
5. Plate Cells 
<ol>
 <li>Transfer cell suspension to a 15 ml tube and centrifuge at 700 x g for 10 min.</li>
 <li>Aspirate medium and re-suspend pellet in 10 ml complete medium. Pipette and mix well.</li>
 <li>Estimate how many plates are needed. This may vary and depends on the pellet size. A general rule is 2 of 10 cm plates from 5 mice for inguinal WAT and one 10 cm plate for BAT.</li>
 <li>Plate cells on collagen coated dishes (R&amp;D) in complete medium</li>
 <li>1-2 hr after plating the cells, aspirate medium, wash with PBS twice and add fresh medium. This step is important as this can remove red blood cells, immune cells and other contaminants.</li>
</ol>
6. Differentiate Cells
<ol>
 <li>Make maintenance and induction mediums.</li>
</ol>
Maintenance medium
Complete medium with added: 
Insulin, final conc. 5 &mu;g/ml (5 mg/ml stock, ***100 &mu;l of acetic acid in 10 ml H2O to prepare acidified water pH 2.5. dissolve insulin in the acidified water. Store stock at -20 &deg;C)
3,3',5-Triiodo-L-thyronine (T3), final conc. 1 nM (10 &mu;M stock, ***dissolve T3 in 1N NaOH and add medium to make 10 &mu;M stock. Sigma cat# T-2877)
Store at 4 &deg;C, good for one week
Induction medium
Maintenance medium containing following compounds:
Indomethacin, final conc. 125 &mu;M (0.125 M stock in ethanol, Sigma cat#I-7378). Indomethacin must be heated to 60 &deg;C to be dissolved.
Dexamethasone, final conc. 2 &mu;g/ml (2 mg/ml sock in ethanol, Sigma cat# D-1756) 3-Isobutyl-1-methylxanthine (IBMX), final conc. 0.5 mM (0.25 M stock in DMSO, Sigma cat# I-5879) Rosiglitazone, final conc. 0.5 &mu;M (10 mM stock in DMSO, Sigma cat# R-2408)
Note: Make fresh induction medium each time.
<ol start="2">
 <li>Grow primary adipocytes to 95-97% confluence in complete medium (confluent but not too packed)</li>
 <li>Change regular complete medium with induction medium (day 0)</li>
 <li>After 48 hr (day 2), change medium to maintenance medium with rosiglitazone (0.5 &mu;M)</li>
 <li>After additional 48 hr (day 4), change to fresh maintenance medium with rosiglitazone (1 &mu;M) for additional 2-3 days. 6-7 days after adding the induction medium, cells are fully differentiated to mature fat cells and are filled with oil droplets. Droplets will appear around 3-4 days after the induction.</li>
</ol>
Note: Change medium every 2-3 days until the cells are fully differentiated.
Cells can now be harvested and mRNA expression of Ucp1 and other brown adipocyte-specific genes can be measured by qRT-PCR. Western blot will be used to detect proteins.

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<li>Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+identification+of+bipotential+adipocyte+progenitors+recruited+by+beta3-adrenoceptor+activation+and+high-fat+feeding%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding.</a> Cell Metabolism. 15, 480-491 (2012).</li>
<li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transdifferentiation+properties+of+adipocytes+in+the+adipose+organ%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Endocrinology+and+Metabolism%22%5BJournal%5D)">Transdifferentiation properties of adipocytes in the adipose organ.</a> American Journal of Physiology. Endocrinology and Metabolism. 297, E977-E986 (2009).</li>
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</ol>

The authors declare that they have no competing financial interests.

Here we present a reliable cellular system to study the development of beige/brite cells in primary cultured adipocytes in mice. As compared to several available immortalized cell lines, this system is likely to offer enhanced relevance to the browning of white fat in vivo.
Even though the study of these primary adipocytes offers some advantages, there also exist some limitations and concerns that are important to consider. First, this system is highly reliant on differentiation poten...

Obesity is dramatically increasing worldwide and is now considered one of the most serious concerns to public health 1. This condition is related to a misbalance in energy intake relative to expenditure and results in excess energy stored as lipid in white adipose tissue (WAT). Enlarged WAT is associated with increased body mass and weight, while brown adipose tissue has the ability to dissipate excess energy to produce heat. Hence BAT can function as protection against both cold and obesity 2,3 . This is achieved by uncoupling of the electron transport in mitochondria by uncoupling protein 1 (UCP1). This protein is considered a hallmark for nonshivering thermogenesis in BAT3. Several studies in recent years revealed that adult humans have functional BAT 4-8 and, consequently, manipulation of BAT in humans can be a potential therapeutic intervention in the battle against obesity and its related diseases.
Current evidence indicates that two types of brown adipocytes exist in rodents; "classical" or "pre-existing" brown fat develops during the prenatal stage and forms dedicated brown adipose depots in interscapular region and other peripheral tissues. On the other hand, an "inducible" form of brown fat (so called brite or beige cells) develops during post-natal stage and appears interspersed in white adipose tissues. The two types of brown adipocytes are also separated by different developmental origins. While the pre-existing brown adipocytes arise from myoblastsic-like Myf5 precursors, the inducible brite/beige cells interspersed in WAT arise from a non-Myf5 lineage9,10 . In addition, regulatory pathways of this cell type is likely to be different from the Myf5-derived brown adipocytes 11. The development of the beige cells (i.e. "browning" of white fat) can be activated in response to chronic cold exposure and to &beta;3-adrenoceptor-agonists or PPAR&gamma; agonists in adults12-14. The beige/brite cells are likely to be a promising therapeutic target for manipulation of overall energy balance and can potentially become part of obesity treatment; hence, it is important to understand precise molecular mechanisms and signaling pathways by which environmental cues control the development of beige cells.
To understand the molecular control of the browning of white fat, in vitro experiments are best suited as differentiation of preadipocytes takes place rather asynchronously and it is difficult to detect the cells in situ 15. Although studies on adipocyte development have thus far been performed primarily on cell lines such as 3T3-L1, 3T3-F442A or HIB1, these cell lines appear to lack the molecular signature of beige cells. On the other hand, primary adipocytes isolated from subcutaneous WAT are most likely to recapitulate the process of browning of white fat in a cell autonomous fashion. Here we provide a protocol for effective isolation of the stromal-vascular fraction from adipose tissues and for inducing the browning of white fat in response to PPAR&gamma; agonists. Rosiglitazone has been shown to be an especially effective mediator of browning in these cells. As previously suggested 16, this cellular system can be used to serve a reliable cellular system to study the development of beige/brite cells.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Collaginase D</td><td>Roche</td><td>11088874103</td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>04942078001</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM medium</td><td>Fisher</td><td>10017-CV</td><td>With 2,5 g/l glucose &amp;amp; L-glutamine without sodium pyruvate </td></tr><tr><td>Insulin</td><td></td><td></td><td></td></tr><tr><td>T3 (3,3&#039;,5-Triiodo-L-thyronine) </td><td>Sigma</td><td>T-2877</td><td></td></tr><tr><td>Indomethacin </td><td>Sigma</td><td>I-7378</td><td></td></tr><tr><td>Dexamethasone </td><td>Sigma</td><td>D-1756</td><td></td></tr><tr><td>IBMX </td><td>Sigma</td><td>I-5879</td><td></td></tr><tr><td>Rosiglitazone </td><td>Sigma</td><td>R-2408</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Collagen coated dishes</td><td>BD</td><td>354450</td><td>10 cm plates</td></tr><tr><td> 70 &amp;mu;m filter </td><td>BD Falcon</td><td>352350</td><td>Cell strainer,70 &amp;mu;m nylon 1/ea</td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

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.

Browning of primary adipocytes can be accessed by measuring mRNA expression of Ucp1 and other brown fat-specific or selective genes by qRT-PCR. Presented in Figure 1 is gene expression data in inguinal WAT-derived primary adipocytes. The cells were induced to differentiate in the presence of two different doses of rosiglitazone at 50 nM and 500 nM, respectively. A subset of cells was treated with forskolin at 10 μM for 4 hr prior to harvest. This will induce cyclic-AMP (cAMP) in the cells and ac...

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 ...

The overall goal of this procedure is to isolate primary adipocytes and induce their differentiation in culture to beige or bright cells. The first step is to dissect adipose tissue from mice. Next, the tissue is digested with collagenase.Once the stroma vascular fraction is isolated, the cells are plated. The final step is to induce differentiation of the cells to beige or bright cells. Ultimately, analytical methods are used to show increased expression of brown adipocyte specific genes.This method can help address the key questions in brown adipose tissues, which is what molecular mechanism regulates the development of bright or be cells. The implications of this technique extend toward treatment of obesity and other lifestyle diseases. Since the manipulation of brown adipose tissue in humans can be used as a potential therapeutic intervention, The first step is to prepare the digestion medium.Then after euthanasia, the adipose tissue is removed. First, isolate the interscapular brown adipose tissue or bat by cutting along the back of the animal all the way to the neck. Two lobes of brown adipose tissue can be found right under the skin between the shoulders.Next, carefully remove the thin layer of white fat covering the bat. Once isolated, place the tissue directly into clean PBS. The inguinal white adipose tissue or wat is found directly under the skin on both sides.It extends to the inside of the thighs and down toward the testicles. Remove the tissue and place in clean PBS. The following steps are applied to both the bat and wet samples.Quickly remove all contamination such as hair, skeletal muscle, and connective tissue from the tissue sections. Next, dry the tissue quickly on a paper towel to prevent the dilution of dissection medium with PBS and place it on a dry plate. Add the digestion medium and mince the tissue into small pieces.Next, transfer the tissue into a 50 milliliter tube containing the rest of the digestion medium. Place the tube in a 37 degree Celsius incubator with constant agitation for 40 to 50 minutes to digest. Check every 10 to 15 minutes to make sure the digestion is going well and to prevent over digestion.After the tissue is well digested, stop the digestion. By adding five milliliters of complete medium and mix well, the cells should be almost completely homogenous. Next, centrifuge the sample at 700 times G for 10 minutes.The stromal vascular fraction should now be visible as a brownish pellet on the bottom of the tube aspirate the oily mature adipocyte layer on top and most of the liquid layer. Next, dissolve the pellet in 10 milliliters, complete medium and mix well. Once mixed, place a cell strainer over a new 50 milliliter tube and filter the cell suspension.Transfer the filtered cell suspension to a 15 milliliter tube and centrifuge at 700 times G for 10 minutes. Next, aspirate the medium and resuspend the pellet. In 10 milliliters of complete medium pipette and mix well plate the cells as needed.Generally, five mice will yield two 10 centimeter plates of inguinal wat and one plate of bat. One or two hours after plating. Aspirate the medium wash with PBS twice and add fresh medium.This step is important as this can remove red blood cells, immune cells, and other contaminants. The maintenance and induction mediums are prepared ahead of time. Once the primary adipocytes have grown to 95 to 97%confluence, change the regular complete medium with induction medium.After 48 hours, change the medium to maintenance medium containing 0.5 micromolar RO glitazone. After an additional 48 hours, change to fresh maintenance medium containing one micromolar RO glitazone for additional two to three days. Droplets will appear around three to four days after the induction as seen here six to seven days after first adding the induction medium.The cells should be fully differentiated to mature fat cells and should be filled with oil droplets. Cells can now be harvested and used for further analysis. Browning of primary adipocytes can be assessed by measuring mRNA of brown fat specific or selective genes by Q-R-T-P-C-R as seen here.RO glitazone, robustly induced UCP one mRNA expression FORSKOLIN treatment designated here as cyclic A MP.Further augmented the RO glitazone induced UCP one expression.Cdia. Another brown fat selective gene was also robustly increased in a dose-dependent manner. RO glitazone treatment also increased the expression of FABP four and adipogenic marker for both brown and white fat.This browning effect was not simply due to an enhancement of adipogenesis per se. Since the induction of UCP one and CDIA expression was still significant, even if the mRNA levels were normalized to those of FABP four, this Western blot confirms the increased protein level of UCP one. After watching this video, you should have a good understanding of how to isolate preadipocytes and how to differentiate these cells into beige or bright cells in culture.

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Research

JoVE Journal

Biology

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Learn to isolate primary adipocytes from mouse adipose tissue and induce differentiation into beige/brite cells using collagenase digestion. This method enables study of molecular mechanisms regulating adipocyte browning for metabolic research.

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