Name: human adipose tissue micro-fragmentation for cell phenotyping and secretome characterization
Uploaded: 2019-10-20T14:00:00
Description: Watch this Scientific Journal Video about Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization at JoVE.com

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 th...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+fat+grafting:+more+than+a+permanent+filler.">Structural fat grafting: more than a permanent filler.</a> Plastic and Reconstructive Surgery. 118 (3), 108S-120S (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editorial:+An+update+on+organoid+research.">Editorial: An update on organoid research.</a> Nature Cell Biology. 20 (6), 633 (2018).</li></ol>

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.

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			<td>4% Buffered paraformaldehyde (PFA)</td>
			<td>VWR chemicals</td>
			<td>9317.901</td>
			<td></td>
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			<td>0.9% NaCl Solution</td>
			<td>Baxter</td>
			<td>3KB7127</td>
			<td></td>
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			<td>AlexaFluor 555 goat anti-mouse IgG&#160;</td>
			<td>Life Technologies</td>
			<td>A21422</td>
			<td></td>
		</tr>
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			<td>AlexaFluor 647 goat anti-Rabbit IgG</td>
			<td>Life Technologies</td>
			<td>&#160;A21245</td>
			<td></td>
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			<td>Ammonium chloride</td>
			<td>fisher chemicals</td>
			<td>1158868</td>
			<td></td>
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			<td>Antigent Diluent</td>
			<td>Life Technologies</td>
			<td>3218</td>
			<td></td>
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			<td>Anti-Mouse Ig, &#954;/Negative Control (BSA) Compensation Plus</td>
			<td>BD Biosciences</td>
			<td>560497</td>
			<td></td>
		</tr>
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			<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&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Laser 405nm (violet)/375nm (UV) &#8211; filter V450/50 for DAPI and V450 antibodies; Laser 561nm (Yellow-green) &#8211; filter YG582/15 for PE antibodies; Laser 405nm (violet)/375nm (UV) &#8211; filter V710/50 for BV711 antibodies&#160;</td>
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			<td>Biotinylated Ulex europaeus lectin</td>
			<td>Vector Laboratories</td>
			<td>Vector-B1065</td>
			<td></td>
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		<tr>
			<td>BV711 Mouse IgG1, k Isotype Control</td>
			<td>BD Biosciences</td>
			<td>563044</td>
			<td></td>
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			<td>CD146-BV711</td>
			<td>BD Biosciences</td>
			<td>563186</td>
			<td></td>
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			<td>CD31-V450</td>
			<td>BD Biosciences</td>
			<td>561653</td>
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			<td>CD34-PE</td>
			<td>BD Biosciences</td>
			<td>555822</td>
			<td></td>
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			<td>CD45-V450</td>
			<td>BD Biosciences</td>
			<td>560367</td>
			<td></td>
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			<td>DAPI</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td>stock concentration: 5mg/mL</td>
		</tr>
		<tr>
			<td>Disposable liposuction cannula (LGI 13Gx185 mm &#8211; AR 13/18) &#160;</td>
			<td>Lipogems&#160;</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>
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			<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&#160;</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>
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			<td>Fluoromount G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
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			<td>Gelatin</td>
			<td>Acros Organics</td>
			<td>410870025</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipogems Surgical Kit</td>
			<td>Lipogems&#160;</td>
			<td>LG SK 60</td>
			<td></td>
		</tr>
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			<td>Mouse anti human- NG2</td>
			<td>BD Biosciences</td>
			<td>554275</td>
			<td>stock concentration: 0.5 mg/mL</td>
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		<tr>
			<td>PE Mouse IgG1, &#954; 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&#160;</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>
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			<td>Rabbit anti human - PDGFRb</td>
			<td>Abcam</td>
			<td>32570</td>
			<td>stock concentration: 0.15 mg/mL</td>
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			<td>Streptavidin conjugated-488</td>
			<td>Life Technologies</td>
			<td>&#160;S32354</td>
			<td></td>
		</tr>
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			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84100-5kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue infiltration cannula (17GX185 mm-VG 17/18)&#160;</td>
			<td>Lipogems&#160;</td>
			<td></td>
			<td>provided in the Lipogems surgical kit</td>
		</tr>
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			<td>Tris base</td>
			<td>fisher chemicals</td>
			<td>BP152-500</td>
			<td></td>
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			<td>Type- II Collagenase</td>
			<td>Gibco</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>V450 Mouse IgG1, &#954; 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 385nm; 488: Blue, excitation 475nm;&#160; 555: Green, excitation 555nm;&#160; 647:Red, excitation 630nm&#160;</td>
		</tr>
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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 (<...

Watch this Scientific Journal Video about Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization at JoVE.com

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. ...

Using this system, we can collect the sub-millimeter clusters of human adipose tissue, in which the whole microenvironment of regenerative cells is intact. The whole microvascular system is also intact, and so we can use these sort of micro organs for in-vivo transplantation for in-vitro culture and for cell isolation for further characterization. The main rationale for the use of the system in the research laboratory is that the process is totally enzyme-free, and so it allows the rapid and efficient processing of human adipose tissue while maintaining a micro-architecture and it will be the microvasculature of the tissue totally intact.And so we can directly study a cell therapy product which has proven already very useful in the clinic. This closed system technology is an easy to use technique for harvesting, processing and re-injection refined fat tissue intra-operatively, and can be successfully applied in cosmetics orthopedics, proctology and also gynecology. This method allows further study of the impact of fat in tissue regeneration.The procedure will be demonstrated by Bianca Vezzani, a postdoctoral fellow and by Mario Gomez-Salazar, who is a PhD student. Working under aseptic conditions within 16 hours of harvesting, place the abdominoplasty sample on top of a surgical cloth with the skin facing upwards. Use a disposable tissue infiltration cannula to inject 50 to 100 milliliters of 37 degrees Celsius 0.9%sodium chloride solution into the subcutaneous adipose tissue.Connect a 10 milliliter Luer lock syringe to a disposable liposuction cannula and grasping the sample at the cutaneous region near an edge of the sample, carefully introduce the cannula into the adipose tissue. Once the cannula is inside the tissue, extract the plunger and move the cannula radially within the adipose tissue sample. It is important to handle the adipose tissue firmly during the lipo-aspiration.If not enough fat is collected, consider performing an additional saline injection. When the syringe is full of lipo-aspirate, carefully remove the cannula from the tissue and replace the full syringe with a new empty syringe until a total of 60 milliliters of aspirate has been harvested. For micro-fragmentation of the lipo-aspirate, 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. Pierce the bag connection port to connect the terminal spike of the input line into the saline bag and position the saline bag higher than the processing unit. Verify that all five of the clamps connected to the tubes of the circuits are open, place the processing unit in a vertical position, allow the saline solution to fill the processing unit, and confirm that the flow reaches the waste bag.Shake the processing unit to remove any air bubbles and place the unit in a vertical position with the blue cap facing upwards. Close the clamp next to the input line and connect a syringe of lipo-aspirate to the self-occluding valve of the blue input cap to begin injecting the lipo-aspirate. Keeping the processing unit vertical, slowly push down on the syringe plunger until all of the lipo-aspirate has been transferred to the unit.When all of the lipo-aspirate has been injected, open the input clamp to restore the saline flow and vigorously shake the processing unit for at least two minutes, regularly checking the saline solution flow into the waste bag. When the saline solution in the processing unit turns transparent, place the unit with the gray cap upwards and close the clamps located on the drain near both the blue and the gray caps. The processed tissue will float at the top.Next, fill a 10 milliliter Luer lock syringe with saline solution, and connect the syringe to the loading valve of the blue cap. Connect an empty 10 milliliter Luer lock syringe with the plunger completely inserted to the valve of the gray cap. Firmly inject the saline into the processing unit from the blue cap, checking that the syringe connected to the gray valve is consequently being filled with the processed tissue.When all of the saline has been injected, carefully remove both syringes and repeat the saline infusion until all of the processed tissue has been collected. At the end of the processing, remove all of the syringes, close all of the clamps, detach the saline bag, and dispose of the device according to local protocols. When all of the processed tissue has been collected, transfer the micro-fragmented adipose tissue into a sterile container, and add an equal volume of digestion medium to the container.Place the sealed container in a 37 degrees Celsius shaking water bath, set to 120 rotations per minute for 45 minutes, stopping the digestion with an equal volume of blocking solution at the end of the incubation. Sequentially filter the sample, first through a 100 micrometer cell strainer followed by filtration through a 70 micrometer cell strainer and centrifuge the filtered suspension for five minutes at 200 times g. We suspend the pellet in approximately 5 milliliters of erythrocyte lysis buffer for 15 minutes at room temperature.Stop the lysis with an equal volume of blocking solution and filter the solution through a 40 micrometer cell strainer. Then collect the micro-fragmented adipose tissue cells by centrifugation and we suspend the pellet in fresh blocking solution with floure-mixing for counting. After dissociation and antibody staining, micro-fragmented fat cells can be taken to the flow cytometer for cell analysis and/or sorting.Mechanical dissociation of manual lipo-aspirates results in the production of micro-fragmented adipose tissue which consists of an aggregate of adipocytes enveloping a microvascular network. Immunofluorescence analysis of gelatin embedded in cryofixed micro-fragmented adipose tissue highlights the structure, showing the vascular network marked by the endothelial cell marker Ulex Europaeus Agglutinin 1 receptor and mainly consisting of small capillary-like vessels. Notably, pericytes expressing neuron-glial antigen 2 or platelet-derived growth factor receptor beta are normally distributed, ensheathing endothelial cells.By excluding CD31 positive endothelial and CD45 positive hematopoietic cells, the presence of CD146 positive CD34 negative pericytes and CD146 negative and CD34 positive adventitial cells can be identified within the MAT. Manual lipo-aspiration is a critical part of the procedure and must be performed under sterile conditions no more than 16 hours after the abdominoplasty. Micro-fragmented adipose tissue can be further processed by immunohistochemical staining, in vitro culture or secretome analysis to obtain a deeper insight into adipose tissue biology.This technique allows the study of diverse tissues in their natural, native, three-dimensional arrangement as a sort of small organoids, which are small enough to be transplanted in mice or to be maintained in long-term culture. And we use this system principally to approach experimentally the different organ-specific microenvironments. Our adipose tissue donors are not known as carriers of transmissible infections.However in the absence of any further screening, all human tissues should be considered as potentially contaminated and handled accordingly.

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

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

Author Spotlight: 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 ...

Semi-Automated Isolation of Murine White Adipose Tissue

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

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

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...

Morphological and Molecular Characterization of the scBAT in Mice

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue

Author Spotlight: Optimizing Supraclavicular Brown Adipose Tissue Extraction for Genetic Analysis

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly ...

Single Cell Nuclei Isolation from IMAT for Studying Gene Expression Profiles 

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Author Spotlight: Deciphering the Cellular Mysteries of Intermuscular Adipose Tissue in Humans

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and ...