We thank Xiao-Rong Peng and Stefan Hallen for providing resources and optimizing the adipocyte isolation, Martin Uhrbom for technical assistance, and Daniel Olausson and Malin L&#246;nn for coordinating and providing the human adipose.

Anonymous samples of adipose tissue were collected from the abdominal region of female patients undergoing elective surgery at Sahlgrenska University Hospital in Gothenburg, Sweden. All study subjects received written and oral information before giving written informed consent for the use of the tissue. The studies were approved by The Regional Ethical Review Board in Gothenburg, Sweden.
NOTE: An overview of the method is provided in Figure 1.
1. Preparation of Buffers, Tissue Culture Media, and Culture Plates
<ol>
	<li>Prepare a 5x Krebbs Ringer (KR) stock by dissolving 35.1 g of NaCl, 1.75 g of KCl, 0.82 g of KH2PO4, and 1.48 g of MgSO4&#183;7H2O in 900 mL of water. Add 1.84 g of CaCl2&#183;2H2O and adjust the volume to 1 L by adding water. Sterile filter through a 0.22 &#181;m filter and store at 4 &#176;C.</li>
	<li>From the 5x KR stock, prepare 1 L of buffer containing 1x KR, 25 mM HEPES, 2 mM glucose, and 2% bovine serum albumin (BSA) (referred to hereafter as wash buffer). Adjust the pH to 7.4.</li>
	<li>Prepare 500 mL of collagenase buffer containing 1x HBSS + CaCl2 + MgCl2, 2% BSA, and 450 mL of water (referred to hereafter as digestion buffer). 
	NOTE: The collagenase should not be added to the digestion buffer until after the adipose tissue has been weighed in step 2.2.</li>
	<li>Adjust the pH of Medium 199 to 7.4.</li>
	<li>Sterile filter both buffers and medium 199 through a 0.22 &#181;m filter flask and warm to 37 &#176;C. 
	NOTE: To save time, the tissue culture media can be prepared and added to plates before processing the adipose tissue. For a 24-well plate format (Figure 1A), use 0.5&#8722;1 mL/well of Dulbecco&#39;s modified Eagle&#39;s medium/Ham&#39;s nutrient mixture F12 (DMEM/F12), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (penn/strep) and 20 nM insulin. Small molecules and other stable pharmacological agents can also be added at this time to the media in the desired layout. Place the plates in a tissue culture incubator (37 &#176;C, 5% CO2).</li>
</ol>
2. Dissection of Human Subcutaneous Adipose Tissue
NOTE: Work within a biological safety cabinet and use sterile technique throughout the isolation process, as well as exclusive use of autoclaved and sterile equipment.
<ol>
	<li>Place the human adipose tissue in a 15 cm petri dish and add a small volume of medium 199 to keep it moisturized during the dissection.</li>
	<li>Working with pieces of adipose approximately the size of a golf ball, grasp large fibrotic vessels with tweezers and gently release the adipocytes by scraping the adipose with the back of closed scissors. Discard the large pieces of fibrotic tissue. Weigh the trimmed fat.</li>
</ol>
3. Collagenase Treatment
<ol>
	<li>Add collagenase to the digestion buffer (see step 1.3) at a concentration of 1 mg/mL. Sterile filter the solution using a 0.22 &#181;m sterile filter. 
	NOTE: Three mL of digestion buffer per gram of fat is recommended (i.e., for 100 g of fat, prepare 300 mL of digestion buffer with 300 mg of type 2 collagenase). 
	CAUTION: Type 2 Collagenase is hazardous to eyes, skin and may cause respiratory irritation. Wear gloves, eye protection, and work in a ventilated hood when handling the collagenase.</li>
	<li>Move approximately 10 g of adipose tissue to a 15 cm petri dish and mince the fat carefully using a pair of curved scissors until it becomes a smooth homogenous mixture and there are no large pieces of adipose left. Repeat the process until all of the fat has been processed. 
	NOTE: Each round should take approximately 2 min and the adipose pieces should be small enough so that they can be pipetted using a wide-bore pipette tip. This step is crucial to yielding high quality adipocytes. If the pieces are too large, digestion times will have to be extended, compromising cell viabilty.</li>
	<li>Transfer the minced fat into 50 mL conical tubes using a spoon. Add 10 mL of minced tissue and 30 mL of digestion buffer to each tube. Scale down to appropriate volumes if less than 10 mL of minced fat is available.</li>
	<li>Digest the tissue at 37 &#176;C in a shaking incubator with constant agitation at 150 rpm for 30&#8722;45 min. After 30 min, check on the process every 5 min to avoid over-digestion. 
	NOTE: The digestion is complete when the adipose solution is homogenous without any large pieces and has an apricot color.</li>
</ol>
4. Filtration of the Cell Suspension and Purification of the Mature Adipocytes
<ol>
	<li>Place a funnel on top of a 1 L sterile flask and place a sterile 250 &#181;m mesh filter inside the funnel. Pour the digested fat solution into the filter to remove the undigested tissue.</li>
	<li>When all of the adipocyte suspension has passed through the filter, gently squeeze the mesh filter to increase the yield of adipocytes. Pour approximately 50&#8722;100 mL of wash buffer into the filter to rinse it and squeeze the filter again.</li>
	<li>Pour the isolated adipocyte suspension from the flask into a separation funnel and add wash buffer until the funnel is almost completely filled. Gently invert the funnel a few times to mix the adipocyte suspension with the buffer.</li>
	<li>Let the suspension stand for 2&#8722;3 min until there is a distinct separation of 2 layers (Figure 1B), with a top yellow layer containing the mature adipocytes and free lipid and a bottom layer containing the buffer and the adipose stroma vascular fraction (SVF).</li>
	<li>Open the nozzle on the separation funnel, and slowly elute the bottom solution into a sterile flask (cells from the SVF can be pelleted and collected after centrifugation at 200 x g for 7 min). Keep the top layer with the mature adipocytes in the separation funnel.</li>
	<li>Repeat the washing process in steps 4.3&#8722;4.5 three times to thoroughly wash the mature adipocytes and remove all of the collagenase.</li>
</ol>
5. Packing of the Mature Adipocytes
<ol>
	<li>Open the nozzle and collect the purified mature adipocyte suspension into 50 mL conical tubes.</li>
	<li>Lightly pack the mature adipocytes by spinning the tubes at 50 x g for 3 min.</li>
	<li>Use a 18 G needle and a syringe to remove the remaining wash buffer below the adipocyte suspension.</li>
	<li>Remove the free lipid layer (oil from the small portion of adipocytes that broke during the isolation procedure) floating on top of the mature adipocytes by using a pipette. 
	NOTE: To reduce the risk that the adipocytes will drip off the membranes in step 6.5, it is important that the lipid layer and all wash buffer have been removed when the mature adipocytes are seeded onto membranes. For this reason collect the adipocytes in 3 tubes. The samples that are collected last will have the most carryover lipid and will therefore be of the lowest quality. However, with careful pipetting these samples can be used.</li>
</ol>
6. Seeding of Mature Adipocytes
<ol>
	<li>Open the package containing the permeable membrane inserts (Table of Materials) and take out the membrane component. Flip it upside down and place on a sterile surface so the membranes face the ceiling.</li>
	<li>Pipette 30 &#181;L of packed mature adipocytes onto each of the membranes (Figure 1C). Avoid touching the membrane with the tip. Use wide-bore pipette tips to seed the cells, or use scissors to cut off a small piece of a pipette tip to make it wider.</li>
	<li>Gently invert the 50 mL tubes with packed adipocytes several times throughout the seeding process to ensure an even distribution of adipocytes with different sizes.</li>
	<li>Bring the prepared multiwell plates containing the media from the incubator to the biosafety cabinet and remove the lids. Pick up the membranes seeded with adipocytes and grasp it from the bottom so that it can be inverted in step 6.5.</li>
	<li>In one smooth movement invert the membranes with the adipocytes on top so that the seeded adipocytes are now facing down (Figure 1D). Lower the plate with adipocytes into the wells containing media (Figure 1E).</li>
	<li>Put the lid on the plate and carefully transfer the plate into a tissue culture incubator. Avoid rapid movement of the plate since adipocytes can easily be dislodged initially. 
	NOTE: It takes a few days for the cells to adhere more firmly to the membrane.</li>
</ol>
7. Maintenance of Adipocytes and Harvesting for Analysis
<ol>
	<li>Change the media at least every 7 days. Remove and add media via the cutout hole in each membrane insert. To remove the media, use a syringe and a needle, an aspirating wand, or a pipette with a p200 tip. To add media, use a pipette and slowly pipette new media into the wells along the side of the wall to avoid disturbing the adipocytes. 
	NOTE: Adipocytes have been cultured for 2 weeks with one media change after 7 days. However, different adipose origins and experimental questions may benefit from increased media changes.</li>
	<li>To harvest RNA, remove the media as described in step 7.1, and add 500 &#181;L of lysis buffer (Table of Materials) directly into the wells to lyse the cells. To fix the cells for imaging, add formaldehyde directly to the well at a final concentration of 1%. 
	NOTE: The cells can then be stored at 4 &#176;C.</li>
</ol>

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A., 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=Integrative+genomic+analysis+implicates+limited+peripheral+adipose+storage+capacity+in+the+pathogenesis+of+human+insulin+resistance.">Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance.</a> Nature Genetics. 49 (1), 17-26 (2017).</li><li>Gustafson, B., Hedjazifar, S., Gogg, S., Hammarstedt, A., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+and+impaired+adipogenesis.">Insulin resistance and impaired adipogenesis.</a> Trends in Endocrinology and Metabolism. 26 (4), 193-200 (2015).</li><li>Gesta, 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=Culture+of+human+adipose+tissue+explants+leads+to+profound+alteration+of+adipocyte+gene+expression.">Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.</a> Hormone and Metabolic Research. 35 (3), 158-163 (2003).</li><li>Fain, J. 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The authors have nothing to disclose.

Membrane mature adipocyte aggregate culture (MAAC) is a new method for the long-term culture of freshly isolated mature adipocytes. In setting up MAAC there are a few critical steps in the protocol that greatly impact the yield, quality, and viability of the mature adipocytes. Much effort should be put into mincing the fat in step 3.2 since this step directly influences the amount of time the adipocytes are exposed to the collagenase. If the pieces of adipose are too large, the digestion time will have to be extended which negatively impacts the viability of the cells. Conversely, if the tissue is processed too finely with scissors, viability can be impacted as well. For successful culture of mature adipocytes as MAAC, one should give special attention to the following steps: for successful seeding of the adipocytes onto the membranes, it is crucial that free lipid and carryover wash buffer is removed from the mature adipocytes in steps 5.3 and 5.4. Remaining lipid or wash buffer will reduce the surface tension of the adipocytes and increase the risk of adipocytes dripping-off the membranes when they are flipped. When the adipocytes are seeded and in media, the cells remain in contact with the membrane primarily through buoyancy, thus a slow and gentle technique is recommended when changing media to not lose cells. Remove media from the bottom of the wells as described in step 7.1 and add media by slowly pipetting media down the sides of the walls. Lastly, a time saving suggestion is to prepare the plates with media and treatments before the adipocyte isolation process. Particularly for complex experimental designs, pre-preparing the plates can save much time and ensures that the adipocytes are placed in media with their treatments as soon as they are isolated.
One benefit of using MAAC compared to differentiating preadipocytes is that the MAAC media used is very simple and does not require an unphysiological hormone cocktail. Here we have cultured MAAC in glucose-rich media (DMEM/F12), 10% FBS, 1% penn/strep, and 20 nM insulin. Importantly, we have found that insulin is absolutely required for the rosiglitazone/pioglitazone driven induction of UCP110. Insulin, however, is not required to maintain the cells&#39; adipogenic phenotype. Thus, depending upon the experimental question, insulin can be included or omitted.
The procedure detailed above has been optimized for the isolation and culture of human adipocytes. However, mouse, and possibly other organism&#39;s adipocytes, can also be cultured as MAAC. If mouse adipocytes are to be cultured as MAAC there are additional considerations and precautions that should be kept in mind. We have found that mature adipocytes from mice are much more fragile than those from humans. As a result, the digestion time should be shortened to an absolute minimum to increase cell viability. We also found that adipocytes from young mice (8-week-old and younger) provided the most robust and reproducible results. Lastly, mouse MAAC can be cultured for up to one week, however given that their adipogenic phenotype appears less stable than humans (which can be cultured through at least two weeks) we recommend culturing mouse MAAC for the minimum required time to address experimental questions.
Since the MAAC model is based on using permeable membranes, one advantage of this technique is the possibility of co-culturing mature adipocytes with other cell types. We have previously demonstrated the ability of mature adipocytes and macrophages to crosstalk through the use of MAAC10. This opens up opportunities to further explore the linkage between obesity, insulin resistance, and immune responses15,16,17. Future experiments could incorporate other cell types such as hepatocytes, preadipocytes, endothelial cells, or pancreatic cells to further increase the complexity and the translational relevance of the MAAC model and investigate crosstalk between multiple cell types.
Even though MAAC has been shown to be superior at maintaining functionality and identity of the adipocytes relative to other adipocyte in vitro models, it also has limitations that need to be considered. In comparison to using adipocytes differentiated from precursor cells, MAAC is a more laborious and time-consuming model. Plates with membranes are also more expensive compared to regular cell culture plates. Importantly, mature adipocytes need to be freshly isolated each time upon seeding and cannot be expanded or frozen into stocks like precursor cells. Thus, this requires having access to fresh white adipose tissue samples, but also adds a level of complexity stemming from donor to donor variations.
Here we have presented a detailed protocol for isolating human mature adipocytes and setting up MAAC. We have demonstrated that adipocytes cultured as MAAC remains viable through two weeks, their adipogenic gene signature is preserved, and they respond to diverse pharmacological agonism. Using MAAC allows for the study of cross-talk betweeen adipocytes and other cell types, and the assessment of long term phenotypic changes of mature adipocytes in response to different stimuli.

The worldwide increase in obesity and obesity-related co-morbidities necessitates the development of new therapeutics. White adipose tissue (WAT) is an important regulator of whole-body metabolism, energy homeostasis, and is a central player in the development of insulin resistance and type 2 diabetes (T2D)1,2. During chronic excess calorie consumption, adipocytes enlarge to handle the surplus of energy. However, adipocyte lipid storage capacity can become exceeded, resulting in an elevation of circulating levels of fatty acids and increased storage in peripheral non-adipose tissues and leading to lipotoxicity3,4.
The lack of adipocyte in vitro models with high translational relevance is a key challenge in the development of new treatments for obesity and T2D. The ex vivo explant model, where small pieces of adipose tissue are cultured, is associated with rapid alterations in adipogenic gene expression driven by hypoxia and inflammation5,6. Ceiling cultures (CCs) where mature adipocytes float and adhere to the top of media-filled flasks, rapidly dedifferentiate into fibroblast-like cells lacking lipid7,8,9,10. The most commonly used model is adipocytes differentiated in vitro from committed precursors. The differentiated cells are, however, morphologically distinct from mature adipocytes in vivo since they are much smaller in size and lack a unilocular lipid droplet. Other limitations with this model include the unphysiological need of a chemical cocktail to drive differentiation, as well as variability in differentiation efficiency which can be affected by a number of factors11,12,13,14.
We have recently developed membrane mature adipocyte aggregate culture (MAAC), a method for long term culture of freshly isolated mature adipocytes, where adipocytes are cultured under permeable membranes10. Unbiased analysis of RNA sequencing data has shown that relative to adipose tissue explants and in vitro-differentiated adipocytes, MAAC is most similar to freshly isolated adipocytes. MAAC can be used to culture mature adipocytes isolated from both subcutaneous and visceral adipose tissue, as well as adipocytes from both obese and lean subjects. This methodology permits the study of long-term adipocyte phenotypic changes and facilitates co-culture of adipocytes with other cell types. Here we provide a detailed protocol for the isolation of mature adipocytes from human adipose tissue and how to set up the MAAC system.

&#160; &#160;

<table><tbody><tr><td>Autoclaved scissors</td><td></td><td></td><td></td></tr><tr><td>Autoclaved spoons</td><td></td><td></td><td></td></tr><tr><td>Autoclaved tweezers</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A6003</td><td></td></tr><tr><td>Buffer RLT</td><td>QIAGEN</td><td>79216</td><td>Lysis buffer</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>Conical tubes, 50 mL</td><td></td><td></td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>31331-028</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Filter flask 0.2 &amp;micro;m pore size, 75 mm</td><td>Thermo Scientific</td><td>156-4020</td><td>500 mL</td></tr><tr><td>Filter flask 0.2 &amp;micro;m pore size, 75 mm</td><td>Thermo Scientific</td><td>158-0020</td><td>1000 mL</td></tr><tr><td>HBSS+CaCl&lt;sub&gt;2&lt;/sub&gt;+MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Gibco</td><td>14065-49</td><td></td></tr><tr><td>HEPES buffer solution (1M)</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>Insulin (Actrapid Penfill)</td><td>Novo Nordisk A/S</td><td></td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>104873</td><td></td></tr><tr><td>Medium 199</td><td>Gibco</td><td>10012-011</td><td></td></tr><tr><td>Mesh filter (250 &amp;micro;M)</td><td>Sintab AB</td><td>6111-025043</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M1880</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Needles, 18 G, 1.20 mm x 40 mm</td><td>Sterican</td><td>613-2948</td><td></td></tr><tr><td>Pencillin-Streptomycin (Penn/Strep)</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>Petri dishes, 150 mm x 21 mm</td><td>Thermo Scientific</td><td>168381</td><td></td></tr><tr><td>Power SYBR Green PCR master mix</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>Quantstudio 7 Flex Real-Time PCR machine</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>RNeasy Mini kits</td><td>QIAGEN</td><td>74106</td><td></td></tr><tr><td>Separation funnel</td><td>VWR</td><td>527-0008</td><td>For large scale preparation</td></tr><tr><td>Separation funnel</td><td>VWR</td><td>527-0005</td><td>For small scale preparation</td></tr><tr><td>Shaking incubator (37 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Syringes, 5 mL</td><td>Omnifix</td><td>612-2892</td><td>100 st</td></tr><tr><td>Tissue culture incubator (37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td></td><td></td><td></td></tr><tr><td>Transwells, 24-well (6.5 mm)</td><td>Costar</td><td>3397</td><td>Permeable membrane inserts</td></tr><tr><td>TRIzol reagent</td><td>Invitrogen</td><td>10296010</td><td>Lysis buffer</td></tr><tr><td>Type 2 Collagenase</td><td>Worthington</td><td>LS004177</td><td></td></tr></tbody></table>

isolation and culture of human mature adipocytes using membrane mature adipocyte aggregate cultures (maac)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Mature adipocytes cultured as MAAC preserves their function, phenotype, and can be used to study adipocyte responses to various pharmacological treatments. After 1 week of culture MAAC isolated from subcutaneous adipose tissue maintain the characteristic unilocular lipid droplet found only in mature adipocytes (Figure 2A). MAAC was cultured for 1 week while treated with either the PPAR&#947; agonists rosiglitazone (Rosi) and pioglitazone (Pio), or the glucocorticoid receptor (GR) agonist dexamethasone (Dex) to determine if different nuclear hormone receptor (NHR) agonists drive predicted changes in downstream target genes in MAAC. Rosi and Pio increased the expression of the PPAR&#947; responsive genes FABP4 and LPL by 4 and 2&#8722;3 fold, respectively, whereas dexamethasone had no effect (Figure 2B). Similarly, dexamethasone robustly drove the gene expression of the GR target genes APOD and FKBP5 by 13- and 55-fold respectively, while the PPAR&#947; agonists had no significant effects. We have previously demonstrated that freshly isolated human mature white adipocytes can transdifferentiate into a brown-like phenotype in MAAC when treated with Rosi10. A 7 day treatment with Rosi or Pio robustly induced the gene expression of the brown fat-specific gene UCP1 by 44-65 fold, as well as increased the expression of the brown fat marker PDK4 12-18 fold (Figure 2C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60485/60485fig1a.jpg" /> 
Figure 1: Visual diagram of MAAC setup. (A) Preparation of medium. (B) Isolation and packing of mature adipocytes. (C) Seeding mature adipocytes onto membranes. (D) Inverting membranes while keeping adipocytes attached. (E) Lowering the membranes into the medium and changing the medium. This figure has been modified from Harms et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60485/60485fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60485/60485fig2a.jpg" /> 
Figure 2: MAAC maintains unilocular appearance through one week and respond to diverse pharmacological agonism. (A) Representative 4x and 10x bright field images of MAAC after one week of culture. Adipocytes that have an average diameter of 100 &#181;m with large unilocular lipid droplets are easily discernable. (B) mRNA levels of PPAR&#947; target genes and glucocorticoid receptor (GR) target genes in MAAC after 7 days of treatment with vehicle (Vehc), rosiglitazone (Rosi), pioglitazone (Pio), or dexamethasone (Dex). Rosi, Pio and Dex were all used at a final concentration of 10 &#181;M. (C) mRNA levels of brown fat-enriched genes in MAAC after 7 days of treatment with vehicle (Vehc), rosiglitazone (Rosi), pioglitazone (Pio), or dexamethasone (Dex). Rosi, Pio and Dex were all used at a final concentration of 10 &#181;M. For all gene expression data, TATA-binding protein (TBP) was used as an internal normalization control. Statistics were calculated using one-way ANOVA with Tukey&#39;s multiple comparisons test. (mean &#177; SD, n = 3, *p &#60; 0.05; **p &#60; 0.01; ***p &#60; 0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60485/60485fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

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Nanyang Technological University

Research

JoVE Journal

Biology

18.9K Views.  AstraZeneca. Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Video: Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

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 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

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Bioengineering

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 ...

Immunology and Infection

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

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 ...