The authors would like to acknowledge the institutional support provided by LSU Health Sciences Center, which funded the project.

All tasks were performed in adherence to protocols #8759 and #9189, as approved by the IRB Office of LSUHSC-NO. All animal work was performed in adherence to protocol #3285 approved by the IACUC Office at LSUHSC-NO.
1. Seeding of Sandwiching Cell Sheets
NOTE: See Figure 1.
<ol>
	<li>Seed ADSCs at approximately 80% confluency in tissue culture plates (6 cm or 6-well plates). For each well of SWAT desired, seed 1 conventional tissue culture well and 1 well of corresponding size on poly(N-isopropylacrylamide (pNIPAAm)-coated tissue culture plastic plate. 
	NOTE: Accordingly, a 6-well plate of SWAT will require seeding cells on one 6-well standard tissue culture plate (base layer) and one 6-well pNIPAAm-coated tissue culture plate (upper layer). pNIPAAm-coated plates can be bought commercially or produced in-lab12,13,14.</li>
	<li>Maintain ADSCs at 37 &#176;C and 5% CO2 in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1x Penicillin/Streptomycin. Change media every 2 days.</li>
	<li>Allow cells to coalesce until they become 100% confluent and take on a striated pattern (approximately 6&#8211;8 days). 
	NOTE: These cells will need to function as a single intact cell sheet in order to stabilize the buoyant adipose tissue. Insufficiently confluent cells will fragment upon seeding with WAT.</li>
</ol>
2. Preparation of SWAT Supplies
<ol>
	<li>Prepare several 10 mL aliquots of 1x Hank&#39;s Balanced Salt Solution (HBSS); prepare enough for the desired number of plates with volume to spare.</li>
	<li>Prepare the plunger apparatus for SWAT seeding. Construct the plunger using simple acrylic plastics, comprised of a stem attached to a round disk. Ensure that it fits within the circumference of the tissue culture wells (diameter: &#60;6 cm for 6 cm dish, &#60;3.5 cm for 6-well plate; approximate mass: 6.7 g for a 6 cm dish, 5.1 g for a 6-well plate).
	<ol>
		<li>Prepare extra plungers to ensure that the procedure will run smoothly in case there is a problem with any individual plunger.</li>
		<li>Wrap lab tape around the rim of the plunger disk, at least 2x, to prevent leakage of gelatin solution.</li>
		<li>Spray a biosafety cabinet (BSC) with 70% EtOH. Spray down a 15 mL conical tube rack inside the BSC with empty, uncapped 15 mL conical tubes; tubes will help secure the placement of the gelatin plungers.</li>
		<li>Spray each tape-wrapped plunger thoroughly with 70% EtOH and place into a 15mL conical tube on the rack. Spray metal washers (approximate mass 6.3 g) and place them on the rack along with a pair of hooked forceps; the washers will add weight to the plungers during SWAT seeding.</li>
		<li>Close the BSC sash and turn on the UV light to facilitate drying and sterilization. 
		NOTE: Ideally, conduct this step 24 h prior to SWAT seeding. Alternatively, conduct the process on the day of tissue collection/SWAT seeding; however, in this case, allow 15&#8211;45 min for UV drying/sterilization.</li>
	</ol>
	</li>
</ol>
3. Preparation of Gelatin Plungers &#8212; Application to Upper Cell Sheets
<ol>
	<li>Heat a water bath to 75 &#176;C.</li>
	<li>Prepare the gelatin solution by adding 0.75 g of gelatin powder to 10 mL stock of 1x HBSS. Under a fume hood add 100 &#181;L of 1 M NaOH to balance the pH of the solution.
	<ol>
		<li>Add the 10 mL stocks to the water bath and shake vigorously every 5 min until the powder dissolves into solution. Aim to dissolve the powdered gelatin soon after adding to the heated water bath.</li>
		<li>Turn on the BSC blower, turn off the UV light, and raise the sash. Spray the BSC surface and prepare the filter supplies (5 mL luer-lock syringes and 0.2 &#181;m syringe filters). Prepare multiple filters to efficiently strain sufficient volumes of gelatin to apply to plungers.</li>
	</ol>
	</li>
	<li>When the gelatin solution reaches a homogenous consistency, filter the solution and apply it to the plungers. Load the syringe with gelatin. Apply the gelatin to the plastic plungers through the syringe filter (~2.5 mL for a 6-well plate, ~4.5 mL for a 6 cm dish) and allow to solidify (~20 min).</li>
	<li>Once the gelatin is solid, unwrap the tape from the edge of the plungers. With hooked forceps, remove the gelatin from the outer edge of the plunger (i.e.,&#160;the raised edges of the meniscus). Ensure that the remaining gelatin in the center of the plunger is completely level to maximize contact with ADSC sheet.</li>
	<li>Once excess gelatin is removed, gently apply the gelatin plungers to the pNIPAAm-coated ADSC plates. Use the metal washers to weigh down the plunger. Do not shear cell sheets while applying the plungers.</li>
	<li>Leave the plungers on the cell sheets for 1.5 h at room temperature. Incubate plates/plungers in an ice water bath for 1.5 h to complete dissociation of the cell sheet from the pNIPAAm-coated plate surface.
	<ol>
		<li>Exercise caution while incubating in the ice bath; do not allow the ice bath to contaminate the cell media during incubation.</li>
		<li>After completing incubation, clean the bottom of the pNIPAAm-coated plates to remove non-sterile water.</li>
	</ol>
	</li>
</ol>
4. White Adipose Tissue Processing
<ol>
	<li>When collecting human adipose tissue from the operating room, keep all samples in a sterile container that is on ice until SWAT is to be seeded. Add sterile maintenance media, such as phosphate-buffered saline (PBS), to adipose tissue container for tissue stability.</li>
	<li>Add cold cell culture medium to 1.5 mL microcentrifuge tubes for each well/dish to be seeded (100 &#181;L for a 6-well plate, 200 &#181;L for a 6 cm dish).</li>
	<li>Mince the adipose tissue.
	<ol>
		<li>For solid adipose tissue segments, do as follows.
		<ol>
			<li>Wash large segments of adipose tissue 3x in sterile PBS and remove as much PBS as possible.</li>
			<li>Coarsely mince tissue with forceps and sterile razor and remove as much vasculature and fascia as possible (see Discussion for more detail).</li>
			<li>Finely mince fat with razor until minced tissue takes on thick, liquid consistency. 
			NOTE: Ideally tissue will appear homogenous with no visible individual segments of WAT although this is not always possible.</li>
		</ol>
		</li>
		<li>Process lipoaspirate as follows.
		<ol>
			<li>Under the BSC, tape sterile gauze over the top of a beaker, and place this beaker in a larger beaker to collect any excess liquid.</li>
			<li>Using a 25 mL serological pipette, draw as much lipoaspirate as needed and apply it to the surface of the sterile gauze. Apply PBS directly to this surface to wash lipoaspirated fat and to remove excess blood and lipid residue.</li>
			<li>Use forceps to recover drained tissue, transfer it to a sterile mincing surface, and mince the lipoaspirate.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use a sterile razor to cut off the distal end of p1000 pipette tips to transfer minced tissue; this will minimize shear stress that can lead to adipocyte lysis. Once a proper tissue consistency is reached transfer the desired volume of minced tissue to each 1.5 mL tube (300&#8211;400 &#181;L for 6-well plate, 500&#8211;600 &#181;L for 6 cm dish). Mix minced WAT and media briefly in the tubes.</li>
	<li>Take the base ADSC plates and decant/aspirate the media. Replace the media with the WAT/media mixture from each 1.5 mL tube.
	<ol>
		<li>Gently remove the gelatin plungers from the pNIPAAm-coated plates and apply them to the WAT mixture on the base ADSC plates. Examine the monolayer of the pNIPAAm-coated plates under a microscope to confirm cellular detachment.</li>
	</ol>
	</li>
	<li>Set a heat block to approximately 37&#8211;40 &#176;C under the BSC. With the plungers still in place, move the plates to the heat block&#39;s surface. Add 2&#8211;3 mL of warmed culture media to incubate the cells and facilitate gelatin melting.</li>
	<li>After ~30 min, gently remove the plungers from the plate surface. Replace the lids of the base tissue culture plates and move to a cell culture incubator. Once the gelatin has completely liquefied at 37 &#176;C, aspirate and replace cell culture media.</li>
	<li>Maintain SWAT at 37 &#176;C and 5% CO2 in phenol red-free M199 medium with 7 &#181;M insulin, 30 &#181;M dexamethasone, 1x Penicillin/Streptomycin. Maintain in approximately 2 mL media for 6-well plates and 3 mL for 6 cm dishes. Change media every 2 days.</li>
</ol>
5. SWAT Harvest
<ol>
	<li>Prepare collagenase (0.5 mg/mL collagenase, 500 nM adenosine, in PBS) aliquots in 15 mL conical tubes (approximate volume of 10 mL). Freeze the tubes and store them at -20 &#176;C.</li>
	<li>Aspirate any culture medium from cells, wash 1x with PBS, and then aspirate PBS. Prep the collagenase aliquots by thawing them in a 37 &#176;C water bath. 
	NOTE: Ideally, the collagenase solution will reach 37 &#176;C; immediately thereafter, add tissue.</li>
	<li>Add all tissue from the SWAT plate to the individual aliquots. Harvest SWAT using a sterile cell scrapper and transfer it to the collagenase aliquots with a cut-off p1000 pipette tip. Add tissue directly to the 15 mL conical tubes containing collagenase solution.
	<ol>
		<li>Alternatively, incubate the tissue/collagenase mixture in a 50 mL conical tube; the increased surface area will further facilitate enzymatic digestion.</li>
	</ol>
	</li>
	<li>Place sample tubes in an incubated orbital shaker at a 45&#176; angle. Incubate at 200 rpm, at 37 &#176;C for 30&#8211;60 min.</li>
	<li>Place a 250 &#181;m mesh filter into a new 15 mL conical tube for collection. Pour the digested adipocyte solution through the filter. 
	NOTE: This will allow all cells to pass through while filtering out fibrous tissue.</li>
	<li>Allow flow-through to sit for 5 min at room temperature to allow for phase separation. 
	NOTE: The adipocytes will float to the top of the solution while the adipose stromal cells (ASCs) will settle into the lower phases. Centrifugation for 5 min at 500 x g can also maximize cell separation or harvest surrounding ADSCs in the pellet.</li>
	<li>Using a cut-off p1000 pipette tip, transfer the adipocytes (the floating layer at top of collagenase solution) to a 1.5 mL microcentrifuge collection tube. Taking ~250 &#181;L at a time, pipette slowly along the edge of the tube to collect the adipocytes.
	<ol>
		<li>Rotate the tube slowly while collecting the adipocytes to maximize the recovery of cells adhering to the inside. Keep drawing more cells until the 1.5 mL microcentrifuge tube is full.</li>
	</ol>
	</li>
	<li>Remove excess liquid from the isolated adipocytes using a syringe attached to a needle (~21 G). Submerge the needle under the floating adipocyte layer. Agitate the needle briefly to dislodge any cells adhering to the needle shaft, and then wait for the dislodged adipocytes to float to the top.</li>
	<li>Slowly draw the excess liquid, being careful to avoid unintended removal of adipocytes. Use microcentrifuge tube graduations to consistently isolate sample volumes (e.g., .0.1 mL for each sample).</li>
	<li>Use the isolated cells for DNA/RNA extraction, glucose uptake assay, lipolysis assay, etc.</li>
</ol>

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NIH-CASIS Coordinated Microphysiological Systems Program for Translational Research in Space (#RFA-TR-18-001) Available from: <a target="_blank" href="https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html">https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html</a> (2017)</li><li>Lau, F. H., Vogel, K., Luckett, J. P., Hunt, M., Meyer, A., Rogers, C. L., Tessler, O., Dupin, C. L., St Hilaire, H., Islam, K. N., Frazier, T., Gimble, J. M., Scahill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sandwiched+White+Adipose+Tissue:+A+Microphysiological+System+of+Primary+Human+Adipose+Tissue.">Sandwiched White Adipose Tissue: A Microphysiological System of Primary Human Adipose Tissue.</a> Tissue Engineering Part C: Methods. 24 (3), (2018).</li><li>Ebara, M., Hoffman, J. M., Stayton, P. S., Hoffman, A. 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The authors have nothing to disclose.

This protocol details the use of ADSCs to sandwich human white adipose tissue; human ADSC cell lines can be isolated via well-established protocols15. However, the system can be adapted for individualized research requirements (such as using 3T3L-1 cells to sandwich mouse WAT). This process involves handling primary human tissue. Standard safety precautions should be employed; handle human tissues as BSL-2 pathogens (e.g., HIV, HepC). Only handle tissue directly under a BSC. Wear all appr...

Adipose tissue is the primary organ of obesity, which carries direct annual medical costs between $147 billion and $210 billion in the U.S.1. The accumulation of adipose tissue also contributes to other leading causes of death such as heart disease, type II diabetes, and certain types of cancer2. In vitro culture models are essential for metabolic studies and drug development, but current research models of adipose tissue have major deficiencies. Adipocytes are fragile, buoyant, and terminally differentiated cells that will not adhere to cell culture plastics, and therefore cannot be cultured using conventional cell culture methods. Since the 1970s, several methods have been used in attempts to overcome these barriers, including the use of glass coverslips, ceiling culture, suspension culture, and extracellular matrices3,4,5,6,7. However, these methods have been marked by cell death and dedifferentiation, and they are typically used for no more than a two-week study period. Moreover, these models do not attempt recapitulate the native adipose microenvironment as they do not maintain the intact ECM, the interactions between adipocytes and stromal support cells, nor the contractile forces cells exert on each other in in vivo WAT.
In the absence of a gold-standard primary adipose culture method, adipose research has relied primarily on differentiated pre-adipocytes (diffAds). DiffAds are multilocular, adherent, and metabolically active. By contrast, primary white adipocytes are unilocular, nonadherent, and demonstrate relatively low metabolism. The failure of current adipose culture models to recapitulate the physiology of healthy mature adipose tissue is likely a major factor in the absence of FDA-approved medications that directly target adipocytes. In fact, the lack of physiologic in vitro organ models is a major problem across most organs and disease.
In its position paper announcing the creation of its Microphysiological Systems (MPS) program, the National Institutes of Health (NIH) reported that the 2013 success rate across all human pharmaceutical clinical trials was only 18% for phase II and 50% for phase III clinical trials8.The MPS program is designed to directly address the inability of in vitro monoculture to model human physiology. The NIH defines MPSs as culture systems comprised of human primary or stem cells in multicellular 3D constructs that recapitulate organ functioning. Unlike reductionist models of homogeneous, immortalized cell cultures, MPSs should accurately model cell-cell, drug-cell, drug-drug, and organ-drug interactions9. Unlike short-term primary culture methods, NIH standards dictate MPS sustainability over 4 weeks in culture8. Further details of the MPS program can be found at the NIH&#39;s RFAs (#RFA-TR-18-001)10.
We have developed a simple, novel, adaptable, and inexpensive adipose MPS termed &#34;sandwiched white adipose tissue&#34; (SWAT)11. We overcome the natural buoyancy of adipocytes by &#34;sandwiching&#34; minced primary adipose tissue between sheets of adipose-derived stromal cells (ADSCs) (Figure 1). The resulting 3D construct recapitulates the cell-cell contact and the native adipose microenvironment by surrounding mature adipocytes with a natural adipocyte support cell population. SWAT has been validated by demonstrating 8-week viability, response to exogenous signaling, adipokine secretion, and engraftment into an animal model.

<table><tbody><tr><td>10x HBSS</td><td>Thermofisher</td><td>14185052</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-aldrich</td><td>G9391</td><td></td></tr><tr><td>Collagenase</td><td>Sigma-aldrich</td><td>C5138</td><td></td></tr><tr><td>Adenosine</td><td>Sigma-aldrich</td><td>A9251</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher</td><td>11995065</td><td></td></tr><tr><td>M199 Media</td><td>Thermofisher</td><td>11043023</td><td>Phenol red-free&amp;nbsp;</td></tr><tr><td>250 &amp;micro;m Mesh Filter</td><td>Pierce</td><td>87791</td><td></td></tr><tr><td>0.2 &amp;micro;m Syringe Filter</td><td>Celltreat</td><td>229747</td><td></td></tr><tr><td>5 mL Luer-Lok syringes&amp;nbsp;</td><td>BD</td><td>309646</td><td></td></tr><tr><td>Metal Washers</td><td></td><td></td><td>These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3 g&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Heated Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubated Orbital Shaker</td><td>VWR</td><td>10020-988</td><td>Samples should be pitched at 45&amp;deg; angle to facilitate collagenase digestion</td></tr><tr><td>Heat Block</td><td></td><td></td><td>Set to 37-40 &amp;deg;C and placed under Biosafety Cabinet</td></tr><tr><td>Water Bath</td><td></td><td></td><td>Set to ~75 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Specialized Plastics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Upcell Dishes 6cm of 6-multiwell</td><td>Nunc</td><td>174902&amp;nbsp; or 174901</td><td>These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &amp;lt;6 cm for 6 cm dish, &amp;lt;3.5 cm for 6-well plate; approximate mass: 6.7g for 6 cm dish, 5.1 g for 6-well plate</td></tr><tr><td>Plastic Plunger Apparatus</td><td></td><td></td><td>These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td></tr></tbody></table>

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.

Viability of SWAT was initially assessed by serial brightfield imaging of individual WAT clusters (n = 12) over approximately 7.6 weeks. Clusters remained secured in place on the monolayer throughout this time. Slight morphological changes were observed with individual adipocytes warping slightly or shifting positions. However, adipocytes neither become multilocular over time, indicating a lack of dedifferentiation, nor did they exhibit any visible signs of cell death such as cellular ble...

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A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

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 ...

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Research

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6.2K Views.  Louisiana State University Health Sciences Center. 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.

Video: A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

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

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Bioengineering

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

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

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

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

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