The authors wish to thank Claire Cryer and Fiona Rossi at the University of Edinburgh for their expert assistance with flow cytometry. We also wish to thank the personnel of the Murrayfield hospital who contributed by providing tissue specimens.
This work was supported by grants from the British Heart Foundation and Lipogems, which supplied adipose tissue processing kits. Human adult tissue samples were procured with full ethics permission of the South East Scotland Research Ethics Committee (reference: 16/SS/0103).

Ethical approval for the use of human tissues in this research was obtained from the South East Scotland Research Ethics Committee (reference: 16/SS/0103).
1. Subcutaneous Abdominal Adipose Tissue Collection
NOTE: All instruments used in the manual lipo-aspiration procedure are provided by the manufacturer of the micro-fragmentation device.
<ol>
	<li>Maintain sterility for all fluids, containers, instruments, and the operational area throughout the experiment.</li>
	<li>Place the abdominoplasty sample (procured by the surgical team in a sterile bag without any solution) on top of a surgical cloth with the skin facing upwards. No washing is required. For optimal use, keep the sample at 4 &#176;C and process the adipose sample within 16 h of harvesting.</li>
	<li>Inject 50 to 100 mL of 37 &#176;C 0.9% NaCl solution, depending on the size of the tissue sample(use 50 mL for maximum a 15 cm2 adipose tissue surface and increase the volume accordingly), using a disposable tissue infiltration cannula, into the subcutaneous adipose tissue. Handle the specimen by the cutaneous part.</li>
	<li>Connect a 10 mL Luer lock syringe to a disposable liposuction cannula.</li>
	<li>Carefully introduce the cannula inside the adipose tissue from the edges. Once inside, create the vacuum inside the syringe by pulling the plunger. Keep the plunger in position by hand to secure the vacuum suction.</li>
	<li>Make radial movements inside the adipose tissue sample until the syringe is full of lipoaspirate. Carefully remove the cannula from the tissue and disconnect the syringe. Connect a new empty syringe.</li>
	<li>Repeat the procedure until 60 mL of lipoaspirate have been collected. 
	NOTE: The maximum amount of lipoaspirate that can be processed changes according to the type of device used. Please refer to the manufacturer instructions (Table of Materials).</li>
</ol>
2. Micro-fragmentation of the lipoaspirate
NOTE: This protocol is meant for the research use only. Micro-fragmentation is performed with the help of a commercially available device (Table of Materials).
<ol>
	<li>Remove the device from the package and verify that the main unit is connected to the waste bag. Place the waste collection bag on the ground and make sure that the valves are secured to the process unit caps.</li>
	<li>Connect the terminal spike of the input line to the saline bag by piercing the bag connection port. Keep the saline bag higher than the processing unit. 
	NOTE: For the type of device used in this protocol, a 2,000 mL bag of sterile saline is recommended.</li>
	<li>Verify that all 5 clamps connected to the tubes of the circuits are open. Place the processing unit in a vertical position with the gray cap upwards.</li>
	<li>Allow the saline solution to fill the processing unit. Check that the flow reaches the waste bag. Remove all air bubbles from the processing unit by shaking it. 
	NOTE:&#160;All the following passages must be performed in the absence of air in the processing unit.</li>
	<li>Place the processing unit in a vertical position with the blue cap upwards and close the clamp next to the input line.</li>
	<li>Start injecting the lipoaspirate by connecting the syringe to the self-occluding valve of the blue input cap. Keep the processing unit vertical during this procedure and slowly pull the syringe plunger until all the lipoaspirate has been transferred to the unit. Repeat the process until all the lipoaspirate is inside the processing unit. 
	NOTE:&#160;For the processing unit used in the video, add maximum 30 mL of lipoaspirate at the time. The procedure can be performed maximum twice, until 60 mL of lipoaspirate have been processed.</li>
	<li>Open the input clamp to restore the saline flow.</li>
	<li>Vigorously shake the processing unit for 2 min at least, regularly checking the saline solution flow into the waste bag.</li>
	<li>When the saline solution in the processing unit turns transparent, after approximatively 2 min of shaking, place the unit with the gray cap upwards, and close the clamp located on the drain near the gray cap. The processed tissue will float at the top.</li>
	<li>Fill a 10 mL Luer lock syringe with saline solution and connect it to the loading valve of the blue cap. Close the clamp located near the blue cap. Connect an empty 10 mL Luer lock syringe, with the plunger completely inserted, to the valve of the gray cap. 
	NOTE: Use Luer lock syringes only.</li>
	<li>Firmly inject the saline into the processing unit from the blue cap. Ensure that the syringe connected to the gray valve is consequently being filled with the processed tissue. Once having injected all the saline, carefully remove both syringes.</li>
	<li>Repeat steps 2.10 and 2.11 until all the processed tissue is collected. 
	NOTE: The average yield of MAT is 30 mL from 60 mL of manual lipoaspirate. Some tissue will remain inside the processing unit and some clumps might be present attached to the blue edge inside the unit.</li>
	<li>At the end of the processing, remove all syringes, close all clamps, detach the saline bag and dispose the device according to local protocols. 
	NOTE: The processing device cannot be re-used.</li>
</ol>
3. Fluorescence Immunohistochemistry
<ol>
	<li>Transfer 300-500 &#181;L of MAT into a 1.5 mL tube. Add 1 mL of 4% buffered paraformaldehyde (PFA), mix gently by hand (no vortexing) and leave at 4 &#176;C overnight (from 16 to 24 h).</li>
	<li>Collect the specimen and transfer to a clean 1.5 mL tube containing 1 mL of PBS. Repeat the step twice to remove PFA excess.</li>
	<li>Transfer the specimen in 15% (w/v) sucrose in PBS solution and incubate at 4 &#176;C overnight (from 16 to 24 h)</li>
	<li>Transfer the specimen in a well of a 24 well plate and add an embedding solution made of 15% sucrose and 7% gelatin (w/v) in PBS. Fill the well halfway. Incubate for 4 h at 37 &#176;C.</li>
	<li>Transfer the samples to 4 &#176;C. After 2-4 h for the gelatin to solidify, fill up the well with the embedding solution and leave at 4 &#176;C overnight.</li>
	<li>Remove the samples from the wells. Remove excess embedding compound and freeze on dry ice. Store the samples at -80 &#176;C.</li>
	<li>Cut 8-10 &#181;m thick sections using a cryostat. For optimal sectioning, use the cryostat at -30 &#176;C. Slides can be stored at -80 &#176;C.</li>
	<li>Prior to proceeding with the immunofluorescence, fix the sections in 4% PFA for 7 min. Remove the PFA and wash twice with lukewarm PBS (37 &#176;C) in order to remove gelatin from the sections.</li>
	<li>Block non-specific antibody binding by incubating the slides with 10% goat serum in PBS (v/v) for 1 h at room temperature.</li>
	<li>Dilute the primary antibodies in antibody diluent or 0.2% (w/v) bovine serum albumin (BSA) in PBS (w/v): mouse anti-human-NG2 (1:100, stock 0.5 mg/mL), rabbit anti-human-PDGFR&#946; (1:100, stock 0.15 mg/mL).</li>
	<li>Remove the blocking solution and add the diluted primary antibodies. Incubate at 4 &#176;C overnight. 
	NOTE: Perform all the incubation steps from now on in the dark.</li>
	<li>Remove the primary antibodies and wash 3 times with PBS for 10 min each time.</li>
	<li>Dilute the secondary antibodies in antibody diluent or 0.2% (w/v) BSA in PBS: goat anti-mouse- 555 (1:300), goat anti-rabbit- 647 (1:300). Incubate 1 h at room temperature.</li>
	<li>Remove the secondary antibodies and wash three times with PBS for 10 min each time.</li>
	<li>If a biotin conjugated lectin is used, use the avidin/biotin blocking kit. Incubate for 10 min with each reagent provided with two washes in between with PBS.</li>
	<li>Repeat the blocking step by incubating the slides for 1 h at room temperature with 10% goat serum in PBS.</li>
	<li>Add the biotinylated lectin, biotinylated Ulex europaeus lectin (1:200, stock 2 mg/mL), diluted either in 0.2% BSA (w/v) in PBS or antibody diluent. Incubate 1 h and 30 min at room temperature.</li>
	<li>Remove excess biotinylated lectin and wash three times with PBS for 10 min each time.</li>
	<li>Add the secondary streptavidin conjugated antibody diluted in either 0.2% (w/v) BSA in PBS or antibody diluent. Incubate for 1 h.</li>
	<li>Remove the secondary antibody and wash three times with PBS for 10 min each time.</li>
	<li>Counterstain nuclei with 4&#8242;,6-diamidino-2-phenylindole (DAPI, 1:500, stock 5 mg/mL), diluted in 0.2% BSA (w/v) in PBS, for 10 min at room temperature.</li>
	<li>Remove the DAPI solution and wash twice with PBS, 10 min each time.</li>
	<li>Mount the slides with an aqueous mounting agent. Let it dry completely, either overnight on the bench in the dark or 1 h at 37 &#176;C.</li>
	<li>Keep the slides at 4 &#176;C in the dark and acquire images on an epifluorescence microscope.</li>
</ol>
4. Digestion of the micro-fragmented adipose tissue and cell isolation
<ol>
	<li>Transfer the micro-fragmented adipose tissue into a sterile container.</li>
	<li>Freshly make up the digestion medium by dissolving type-II collagenase in DMEM at the concentration of 1 mg/mL.</li>
	<li>Add the same volume of digestion medium to the micro-fragmented adipose tissue (i.e., for 30 mL of sample add 30 mL of digestion medium).</li>
	<li>Place the sealed container in a 37 &#176;C shaking water bath set to 120 rpm for 45 min. Please note that the container should allow a proper shaking of the sample. If a large container is not available, use two 50 mL tubes (30 mL of sample/digestion medium mixture in each) placed horizontally.</li>
	<li>After the incubation, block the digestion by adding an equal volume of blocking solution (2% (v/v) FCS/PBS) and filter sequentially through 100 &#181;m and 70 &#181;m cell strainers.</li>
	<li>Centrifuge the filtered suspension for 5 min at 200 x g. Discard the supernatant.</li>
	<li>Resuspend the pellet in approximately 5 mL of erythrocyte lysis buffer (155 mM NH4Cl, 170 mM Tris, pH 7.65). The volume of buffer may vary according to the erythrocyte contamination observed in the cellular pellet. Incubate at room temperature for 15 min.</li>
	<li>Add an equal volume of blocking solution and filter through a 40 &#181;m cell strainer.</li>
	<li>Centrifuge the cell suspension for 5 min at 200 x g and discard the supernatant.</li>
	<li>Resuspend the pellet in blocking solution. Mix well by pipetting. Count viable cells with Trypan blue exclusion on a hemocytometer.
	<ol>
		<li>Mix an equal volume of the cell suspension with Trypan blue stain. Aspirate 10 &#181;L of the solution and place it on a hemocytometer.</li>
		<li>Count the number of live cells (bright and round) present in at least 5 squares and get the mean cell number. In order to include the stain dilution factor, multiply the mean cell number by 2. To get the total number of live cells per 1 mL of sample multiply the obtained number by 104. 
		NOTE: The obtained single cell suspension, namely the stromal vascular fraction (SVF), can be either seeded at 20,000 cells/cm2 in perivascular cell growth medium (DMEM supplemented with 20% heat inactivated FCS, 1% penicillin/streptomycin and 1% L-glutamine) or used for flow cytometry analysis and sorting. The average yield of nucleated cells in the SVF is approximately 3 x 104 cells per mL of MAT. Sorting experiments require at least 8 x 105 cells to obtain enough perivascular cells for culturing.</li>
	</ol>
	</li>
</ol>
5. Cell labelling and sorting
<ol>
	<li>Centrifuge the cell suspension at room temperature for 5 min at 200 x g, and re-suspend the pellet in blocking medium at a concentration of 1 x 106 cells per 100 &#181;L.</li>
	<li>Aliquot at least 50,000 cells for the unstained control and the fluorescence minus one (FMO) controls into polystyrene round bottom flow cytometry tubes. Use the rest of the cells for multicolor staining by placing in another tube. 
	NOTE: Once the settings of the experiment have been established (i.e., laser intensity, gating strategy), the number of cells used for the unstained control and the FMOs can be reduced, if, the antibodies used are the same.</li>
	<li>Add CD31-V450 (1:400), CD34-PE (1:100), CD45-V450 (1:400) and CD146-BV711 (1:100) antibodies to the single cell suspension for multi-color staining. For the FMO controls, add: for the V450 FMO equivalent volumes of CD34-PE and CD146-BV711 plus V450 isotype control; for the PE FMO equivalent volumes of CD31-V450, CD45-V450 and CD146-BV711 plus PE isotype control; for the BV711 FMO equivalent volumes of CD31-V450, CD45-V450 and CD34-PE plus BV711 isotype control.</li>
	<li>Gently pipette, or vortex slowly the solution in the tube to mix and incubate the tubes at 4 &#176;C for 20 min in the dark.</li>
	<li>Prepare compensation control beads. Add 15 &#181;L of positive beads and 15 &#181;L of negative beads to 70 &#181;L of blocking medium in 3 polystyrene round-bottom flow-cytometry tubes. Add CD34-PE, CD31-V450 or CD45-V450, and CD146-BV711 antibodies, one per tube.</li>
	<li>Gently pipette, or vortex slowly, to mix the antibodies with the beads and incubate all tubes at 4 &#176;C for 20 min in the dark. 
	NOTE: The procedure described can be used either for flow cytometry analysis or cell sorting.</li>
	<li>Prepare collection tubes by pre-wetting the inner surface of the tubes with endothelial growth medium (EGM), leaving approximately 50 &#181;L of medium on the bottom of each tube.</li>
	<li>After antibody incubation, remove the excess of antibody by washing the cells and the beads with 2 mL of blocking medium.</li>
	<li>Remove the solution by centrifuging the tubes at 200 x g for 5 min and carefully aspirating the supernatant. Replicate the washing step.</li>
	<li>Resuspend the cells in blocking medium at a final concentration of 1 x 106 cells per 250 &#181;L. Resuspend the beads in 100 &#181;L of blocking medium.</li>
	<li>Transfer the cells into new polystyrene round-bottom flow cytometry tubes with cell strainer cap. This will allow the disruption of cell clumps.</li>
	<li>Transport all cell and bead suspensions to the cell sorter on ice in the dark.</li>
	<li>Run unstained control cells to establish the background fluorescence and set the voltages.</li>
	<li>Run the compensation control tubes to set the fluorescence compensation.</li>
	<li>Use forward scatter area (FSC-A) vs side scatter area (SSC-A) to identify cells, and then use forward scatter area (FSC-A) vs forward scatter height (FSC-H) to select single cells.</li>
	<li>Add DAPI, 1 &#181;L of 5 &#181;g/mL solution for each mL of sample, to the unstained control to set the gating for dead cells (Figure 2). No washing is required after DAPI staining.</li>
	<li>Run isotype controls to establish background fluorescence thresholds related to non-specific binding and set the gating.</li>
	<li>Run the multi-color stained sample. Exclude hematopoietic and endothelial cells by gating on CD31 and CD45 negative cells. Collect the pericyte (CD146+ CD34-) and adventitial cell (CD146- CD34+) populations into collection tubes (Figure 2). 
	NOTE: Optimal viable cell yields are approximately 2% of the total live cell dissociation for pericytes and 1.5% for adventitial cells.</li>
</ol>
6. Cell culture
<ol>
	<li>Seed freshly sorted pericytes and adventitial cells at a density of 30,000 to 40,000 cells/cm2 on gelatin coated culture plates</li>
	<li>To coat the culture plates, cover the plate area with sterile 0.2% (w/v) gelatin in PBS solution (approximately 100 &#181;L of solution per cm2). Incubate at room temperature for 10 min and remove the solution. Keep freshly sorted cells on ice during culture plate coating.</li>
	<li>Centrifuge freshly collected cells at 200 x g for 5 min and gently re-suspend the cell pellet in an appropriate amount of endothelial growth medium (EGM). If the number of cells collected is very low, avoid the centrifugation and plate directly the collected cells.</li>
	<li>Seed the cells on gelatin-coated plates. Incubate at 37 &#176;C in 5% CO2.</li>
	<li>Once cells have settled and adhered to the plate (after at least 72 h), exchange EGM with perivascular cell growth medium (DMEM supplemented with 20% heat inactivated FCS, 1% penicillin/streptomycin and 1% L-glutamine) for both pericytes and adventitial cells.</li>
	<li>Once pericytes and adventitial cells have reached 80%-90% confluence, dissociate cells using 0.05% trypsin-EDTA. Collect with 5% FCS/PBS, centrifuge at 200 x g for 5 min, re-suspend in perivascular cell growth medium, and then passage the cells from 1:3 to 1:5 ratio (or at approximately 7,000 cells/cm2) onto uncoated polystyrene culture plates or flasks.</li>
</ol>

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CT is a founder of Lipogems.

This paper describes the physical fractionation, using a closed system device, of human adipose tissue into small clusters displaying normal adipose tissue microanatomy.
Manually aspirated human subcutaneous adipose tissue and saline solution are loaded into a transparent plastic cylinder containing large pinball-style metallic spheres that, upon vigorous manual shaking of the device, rupture the fat into sub-millimeter fragments. Attached filters and outlet allow eliminating debris, blood and...

Adipose tissue, long used as a filler in reconstructive and cosmetic surgery, has recently become more popular in regenerative medicine once recognized as a source for mesenchymal stem cells (MSCs)1. Lipoaspirates dissociated enzymatically into single-cell suspensions yield an adipocyte-free stromal vascular fraction (SVF) that is used unaltered in the patient or, more commonly, is cultured for several weeks into MSCs2.
However, enzyme dissociation ruptures the tissue microenvironments, secluding neighboring regulatory cells from presumptive regenerative cells that become considerably modified by in vitro culture. To avoid such experimental artifacts and consequent functional alterations, attempts have been made to process adipose tissue for therapeutic use while maintaining its native configuration as intact as possible3,4. Notably, mechanical tissue disruption has started to replace enzymatic dissociation. To this end, the full immersion closed system micro-fragments lipoaspirates into sub-millimeter, blood- and oil-free tissue clusters (e.g., Lipogems) via a sequence of sieve filtration and steel marble induced disruption3. Autologous transplantation of micro-fragmented adipose tissue, using this closed system technology, has been successful in multiple indications, spanning cosmetics, orthopedics, proctology and gynaecology4,5,6,7,8,9,10,11,12,13.
Comparison between human micro-fragmented adipose tissue (MAT) obtained with the closed system device and isogenic SVF revealed that with respect to vascular/stromal cell distribution and secretory activity in culture, MAT contains more pericytes, which are presumptive MSCs14, and secretes higher amounts of growth factors and cytokines15.
The present article illustrates the enzyme-free micro-fragmentation of human subcutaneous adipose tissue using a closed system device, and the further processing of such micronized adipose tissue for in vitro culture, immunohistochemistry and FACS analysis, in order to identify the cell types present and the soluble factors secreted (Figure 1). The described method safely generates adipose derived sub-millimeter organoids containing viable adipose tissue cell populations in an intact niche, suitable for further applications and studies.

<table><tbody><tr><td>4% Buffered paraformaldehyde (PFA)</td><td>VWR chemicals</td><td>9317.901</td><td></td></tr><tr><td>0.9% NaCl Solution</td><td>Baxter</td><td>3KB7127</td><td></td></tr><tr><td>AlexaFluor 555 goat anti-mouse IgG&amp;nbsp;</td><td>Life Technologies</td><td>A21422</td><td></td></tr><tr><td>AlexaFluor 647 goat anti-Rabbit IgG</td><td>Life Technologies</td><td>&amp;nbsp;A21245</td><td></td></tr><tr><td>Ammonium chloride</td><td>fisher chemicals</td><td>1158868</td><td></td></tr><tr><td>Antigent Diluent</td><td>Life Technologies</td><td>3218</td><td></td></tr><tr><td>Anti-Mouse Ig, &amp;kappa;/Negative Control (BSA) Compensation Plus</td><td>BD Biosciences</td><td>560497</td><td></td></tr><tr><td>Avidin/Biotin Blocking Kit</td><td>Life Technologies</td><td>4303</td><td></td></tr><tr><td>BD LSR Fortessa 5-laser flow cytometer&amp;nbsp;</td><td>BD Biosciences</td><td></td><td>Laser 405 nm (violet)/375 nm (UV) &amp;ndash; filter V450/50 for DAPI and V450 antibodies; Laser 561nm (Yellow-green) &amp;ndash; filter YG582/15 for PE antibodies; Laser 405 nm (violet)/375 nm (UV) &amp;ndash; filter V710/50 for BV711 antibodies&amp;nbsp;</td></tr><tr><td>Biotinylated Ulex europaeus lectin</td><td>Vector Laboratories</td><td>Vector-B1065</td><td></td></tr><tr><td>BV711 Mouse IgG1, k Isotype Control</td><td>BD Biosciences</td><td>563044</td><td></td></tr><tr><td>CD146-BV711</td><td>BD Biosciences</td><td>563186</td><td></td></tr><tr><td>CD31-V450</td><td>BD Biosciences</td><td>561653</td><td></td></tr><tr><td>CD34-PE</td><td>BD Biosciences</td><td>555822</td><td></td></tr><tr><td>CD45-V450</td><td>BD Biosciences</td><td>560367</td><td></td></tr><tr><td>DAPI</td><td>Life Technologies</td><td>D1306</td><td>stock concentration: 5mg/mL</td></tr><tr><td>Disposable liposuction cannula (LGI 13 G x185 mm &amp;ndash; AR 13/18) &amp;nbsp;</td><td>Lipogems&amp;nbsp;</td><td></td><td>provided in the Lipogems surgical kit</td></tr><tr><td>Diva software 306 (v.6.0)</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td><td>Life Technologies</td><td>61965026</td><td></td></tr><tr><td>EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM</td><td>Lonza&amp;nbsp;</td><td>CC-3156</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>FlowJo (v.10.0)</td><td>FlowJo</td><td></td><td></td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>Gelatin</td><td>Acros Organics</td><td>410870025</td><td></td></tr><tr><td>Lipogems Surgical Kit</td><td>Lipogems&amp;nbsp;</td><td>LG SK 60</td><td></td></tr><tr><td>Mouse anti human- NG2</td><td>BD Biosciences</td><td>554275</td><td>stock concentration: 0.5 mg/mL</td></tr><tr><td>PE Mouse IgG1, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>555749</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Polystirene round bottom 5 mL tube with cell strainer snap cap&amp;nbsp;</td><td>BD Biosciences</td><td>352235, 25/Pack</td><td></td></tr><tr><td>Polystyrene round bottom 5 mL tubes</td><td>BD Biosciences</td><td>352003</td><td></td></tr><tr><td>Rabbit anti human - PDGFRb</td><td>Abcam</td><td>32570</td><td>stock concentration: 0.15 mg/mL</td></tr><tr><td>Streptavidin conjugated-488</td><td>Life Technologies</td><td>&amp;nbsp;S32354</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84100-5kg</td><td></td></tr><tr><td>Tissue infiltration cannula (17 G x 185 mm-VG 17/18)&amp;nbsp;</td><td>Lipogems&amp;nbsp;</td><td></td><td>provided in the Lipogems surgical kit</td></tr><tr><td>Tris base</td><td>fisher chemicals</td><td>BP152-500</td><td></td></tr><tr><td>Type- II Collagenase</td><td>Gibco</td><td>17101-015</td><td></td></tr><tr><td>V450 Mouse IgG1, &amp;kappa; Isotype Control</td><td>BD Biosciences</td><td>560373</td><td></td></tr><tr><td>Widefield Zeiss observer</td><td>Zeiss</td><td></td><td>Objective used: Plan-Apo 20x/0.8</td></tr><tr><td>Zeiss Colibri7 LED light source ( LEDs: 385, 475, 555, 590, 630 nm)</td><td>Zeiss</td><td></td><td>DAPI: UV, excitation 385 nm; 488: Blue, excitation 475 nm;&amp;nbsp; 555: Green, excitation 555 nm;&amp;nbsp; 647:Red, excitation 630 nm&amp;nbsp;</td></tr></tbody></table>

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.

Mechanical dissociation of manual lipoaspirates resulted in the production of micro-fragmented adipose tissue (MAT), consisting of an aggregate of adipocytes enveloping a microvascular network (Figure 3). Immunofluorescence analysis of gelatin-embedded and cryofixed MAT highlights this structure, showing the vascular network marked by the endothelial cell marker Ulex europaeus agglutinin 1 (UEA-1) receptor mainly consisting of small, capillary-like vessels (<...

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

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. ...

human-adipose-tissue-micro-fragmentation-for-cell-phenotyping

Research

JoVE Journal

Biology

9.7K Views.  University of Edinburgh. Met behulp van dit systeem kunnen we de sub-millimeter clusters van menselijk vetweefsel verzamelen, waarin de hele micro-omgeving van regeneratieve cellen intact is. Het hele microvasculaire systeem is ook intact, en dus kunnen we dit soort microorganen gebruiken voor in-vivo transplantatie voor in-vitro cultuur en voor celisolatie voor verdere karakterisering. De belangrijkste reden voor het gebruik van het systeem in het onderzoekslaboratorium is dat het proces volledig enzymvrij is, en du...

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

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

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

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

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

Behavior

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Immunology and Infection

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

Genetics

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 ...

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin ...

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

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 ...

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

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

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

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

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

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

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