This work was supported by the National Nature Science Foundation of China (81471881, 81601702, 81671931), the Natural Science Foundation of the Guangdong Province of China (2014A030310155), and the Administrator Foundation of Nanfang Hospital (2014B009, 2015Z002, 2016Z010, 2016B001).

This research was approved by the Ethical Review Board in Nanfang Hospital, Guangzhou, China. Adipose tissue was collected from healthy donors who gave written informed consent to take part in the study. All animal experiments were approved by the Nanfang Hospital Institutional Animal Care and Use Committee and performed according to the guidelines of the National Health and Medical Research Council (China).
1. ECM/SVF-gel Preparation
<ol>
	<li>Harvest fat.
	<ol>
		<li>Perform liposuction on...

<ol>
	<li>Bateman, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Fat+to+Fight+Disease:+A+Systematic+Review+of+Non-Homologous+Adipose-Derived+Stromal/Stem+Cell+Therapies.">Using Fat to Fight Disease: A Systematic Review of Non-Homologous Adipose-Derived Stromal/Stem Cell Therapies.</a> Stem Cells. 36 (9), 1311-1328 (2018).</li><li>Baer, P. C., Geiger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+mesenchymal+stromal/stem+cells:+tissue+localization,+characterization,+and+heterogeneity.">Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity.</a> Stem Cells International. , 812693 (2012).</li><li>Gimble, J. M., Katz, A. J., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+for+regenerative+medicine.">Adipose-derived stem cells for regenerative medicine.</a> Circulation Research. 100 (9), 1249-1260 (2017).</li><li>Halme, D. G., Kessler, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+regulation+of+stem-cell-based+therapies.">FDA regulation of stem-cell-based therapies.</a> New England Journal of Medicine. 355 (16), 1730-1735 (2006).</li><li>Cheng, N. C., Wang, S., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+spheroid+formation+of+human+adipose-derived+stem+cells+on+chitosan+films+on+stemness+and+differentiation+capabilities.">The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities.</a> Biomaterials. 33 (6), 1748-1758 (2012).</li><li>van Dongen, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+intraoperative+procedures+for+isolation+of+clinical+grade+stromal+vascular+fraction+for+regenerative+purposes:+a+systematic+review.">Comparison of intraoperative procedures for isolation of clinical grade stromal vascular fraction for regenerative purposes: a systematic review.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (1), 261-274 (2018).</li><li>van Dongen, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fractionation+of+adipose+tissue+procedure+to+obtain+stromal+vascular+fractions+for+regenerative+purposes.">The fractionation of adipose tissue procedure to obtain stromal vascular fractions for regenerative purposes.</a> Wound Repair and Regeneration. 24 (6), 994-1003 (2016).</li><li>Mashiko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Micronization+of+Lipoaspirates:+Squeeze+and+Emulsification+Techniques.">Mechanical Micronization of Lipoaspirates: Squeeze and Emulsification Techniques.</a> Plastic and Reconstructive Surgery. 139 (1), 79-90 (2017).</li><li>Feng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micronized+cellular+adipose+matrix+as+a+therapeutic+injectable+for+diabetic+ulcer.">Micronized cellular adipose matrix as a therapeutic injectable for diabetic ulcer.</a> Regenerative Medicine. 10 (6), 699-708 (2015).</li><li>Zhang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+flap+survival+improvement+by+composition-selective+fat+grafting+with+novel+adipose+tissue+derived+product+-+stromal+vascular+fraction+gel.">Ischemic flap survival improvement by composition-selective fat grafting with novel adipose tissue derived product - stromal vascular fraction gel.</a> Biochemistry and Biophysics Research Communication. 495 (3), 2249-2256 (2018).</li><li>Tonnard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofat+grafting:+basic+research+and+clinical+applications.">Nanofat grafting: basic research and clinical applications.</a> Plastic and Reconstructive Surgery. 132 (4), 1017-1026 (2013).</li><li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Extracellular+Matrix/Stromal+Vascular+Fraction+Gel:+A+Novel+Adipose+Tissue-Derived+Injectable+for+Stem+Cell+Therapy.">Adipose Extracellular Matrix/Stromal Vascular Fraction Gel: A Novel Adipose Tissue-Derived Injectable for Stem Cell Therapy.</a> Plastic and Reconstructive Surgery. 139 (4), 867-879 (2017).</li><li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Stromal+Vascular+Fraction+Gel+Grafting:+A+New+Method+for+Tissue+Volumization+and+Rejuvenation.">Adipose Stromal Vascular Fraction Gel Grafting: A New Method for Tissue Volumization and Rejuvenation.</a> Dermatologic Surgery. 44 (10), 1278-1286 (2018).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Long-Term+Volume+Retention+of+Stromal+Vascular+Fraction+Gel+Grafting+with+Enhanced+Angiogenesis+and+Adipogenesis.">Improved Long-Term Volume Retention of Stromal Vascular Fraction Gel Grafting with Enhanced Angiogenesis and Adipogenesis.</a> Plastic and Reconstructive Surgery. 141 (5), 676-686 (2018).</li><li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+stem+cell-derived+exosomes+in+tissue+engineering+and+neurological+diseases.">Applications of stem cell-derived exosomes in tissue engineering and neurological diseases.</a> Reviews in the Neurosciences. 29 (5), 531-546 (2018).</li><li>Allen, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grading+lipoaspirate:+is+there+an+optimal+density+for+fat+grafting.">Grading lipoaspirate: is there an optimal density for fat grafting.</a> Plastic and Reconstructive Surgery. 131 (1), 38-45 (2013).</li><li>Qiu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+Centrifuged+Lipoaspirate+Fractions+Suitable+for+Postgrafting+Survival.">Identification of the Centrifuged Lipoaspirate Fractions Suitable for Postgrafting Survival.</a> Plastic and Reconstructive Surgery. 137 (1), 67-76 (2016).</li></ol>

Stem cell-based regenerative therapy has shown a great potential benefit in different diseases. ASCs are outstanding therapeutic candidates because they are easy to obtain and have the capacity for tissue repair and the regeneration of novel tissues15. However, there are limitations to expanding its clinical application, since it requires complicated procedures to isolate cells and collagenase for processing6. Thus, it is essential to develop a simple technique to obtain st...

Stem cell therapies provide a paradigm shift for tissue repair and regeneration so that they may offer an alternative therapeutic regimen for various diseases1. Stem cells (e.g., induced pluripotent stem cells and embryonic stem cells) have great therapeutic potential but are limited due to cell regulation and ethical considerations. Adipose-derived mesenchymal stromal/stem cells (ASCs) are easy to obtain from lipoaspirates and not subject to the same restrictions; thus, it has become an ideal cell type for practical regenerative medicine2. In addition, they are nonimmunogenic and have abundant resources from autologous fat3.
Currently, ASCs are obtained mainly by collagenase-mediated digestion of the adipose tissue. The stromal vascular fraction (SVF) of adipose tissue contains ASCs, endothelial progenitor cell, pericytes, and immune cells. Although obtaining a high density of SVF/ASCs enzymatically was shown to have beneficial effects, the legislation in several countries strictly regulates the clinical application of cell-based products using collagenase4. Digesting the adipose tissue with collagenase for 30 min to 1 h to obtain SVF cells increases the risk of both exogenous material in the preparation and biological contamination. The adherent culture and the purification of ASCs, which takes days to weeks, require specific laboratory equipment. Moreover, in most studies, SVF cells and ASCs are used in suspension. Without the protection of extracellular matrix (ECM) or another carrier, free cells are vulnerable, cause a poor cell retention after injection, and compromise the therapeutic result5. All of these reasons limit the further application of stem cell therapy.
To obtain ASCs from adipose tissue without collagenase-mediated digestion, several mechanical processing procedures, including centrifugation, mechanical chopping, shredding, pureeing, and mincing, have been developed6,7,8,9. These methods are thought to condense tissue and ASCs by mechanically disrupting mature adipocytes and their oil-containing vesicles. Moreover, these preparations, containing high concentrations of ASCs, showed considerable therapeutic potential as regenerative medicine in animal models8,9,10.
In 2013, Tonnard et al. introduced the nanofat grafting technique, which involves producing emulsified lipoaspirates by intersyringe processing11. The shearing force created by intersyringe shifting can selectively break mature adipocytes. Based on their findings, we developed a purely mechanical processing method that removes most of the lipid and fluid in the lipoaspirates, leaving only SVF cells and fractionated ECM, which is ECM/SVF-gel12. Herein, we describe the details of the mechanical process of human-derived adipose tissue to produce the ECM/SVF-gel.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488-conjugated isolectin GS-IB4</td>
			<td style="width:232px;">Molecular Probes</td>
			<td style="width:119px;">I21411</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">guinea pig anti-mouse perilipin</td>
			<td>Progen</td>
			<td>GP29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Thermofisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">wide tip pipet</td>
			<td>Celltreat</td>
			<td>229211B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>TCS SP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nude nice&#160;</td>
			<td>Southern Mdical University</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">light microscope&#160;</td>
			<td>Olympus</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL tube</td>
			<td>Cornig</td>
			<td>430828</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">sterile bag</td>
			<td>Laishi</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microtome</td>
			<td>Leica&#160;</td>
			<td>CM1900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td>Heraus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanical micronization of lipoaspirates for regenerative therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

After processing the Coleman fat to ECM/SVF-gel, the volume of discarded oil takes up 80% of the final volume, and only 20% of adipose tissue preserved under the oil layer is regarded as ECM/SVF-gel (Figure 1A). ECM/SVF-gel has a smooth liquid-like texture that enables it to go through a 27 G fine needle; however, Coleman fat is comprised of an integral adipose structure with large fibers and can only go through an 18 G cannula (<strong class...

Watch this Scientific Journal Video about Mechanical Micronization of Lipoaspirates for Regenerative Therapy at JoVE.com

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...

Obtaining stromal vascular fractions, or SVFs, from lipoaspirates requires digestion with collagenase, which increases the risk of biological contamination. A better way to obtain SVF without collagenase should be developed. These techniques enable us to obtain the SVF in its native extracellular matrix through a simple mechanical process.Demonstrating the procedure will be Xihang Chen, a grad student from our lab. After obtaining the fat samples, transfer the lipoaspirates into one 50 milliliter tube, and allow the harvested fat to equilibrate for 10 minutes. Next, use a wide tip pipette to pull the fat from the top layer of the tube into a new 50 milliliter tube and spin down the fat by centrifugation.The upper layer in the tube is the Coleman fat fraction. Transfer the upper 2/3 of the Coleman fat into a new 15 milliliter tube. This portion is considered the low density fat.Transfer the lower third of the Coleman fat into a second 15 milliliter tube. This portion is considered the high density fat. To produce an extracellular matrix stromal vascular fraction gel from the low density fat, use two 20 milliliter syringes connected by a female to female luer lock connector to intershift 10 milliliters of the low density fat.This is the key step to destroy adipocytes. The speed and the time that the intersyringe shifting is performed contributes to the quality of the final product. After the last intershift session, collect the fat by centrifugation and transfer the top oil layer to a 15 milliliter tube.Then transfer the sticky middle extracellular matrix stromal vascular fraction gel layer into a new 15 milliliter conical tube. To produce an extracellular matrix stromal vascular fraction gel from the high density fat, add two milliliters of the low density oil fraction to five milliliters of the high density fat sample. Intershift the mixed fat as demonstrated until a flocculate is observed within the emulsion.Collect the flocculate by centrifugation and discard the top oil layer. Then collect the sticky middle layer and mix this fraction with the low density extracellular matrix stromal vascular fraction gel sample. After processing the Coleman fat to extracellular matrix stromal vascular fraction gel, the volume of discarded oil takes up 80%of the final volume, and only 20%of the adipose tissue preserved under the oil layer is regarded as extracellular matrix stromal vascular fraction gel.Extracellular matrix stromal vascular fraction gel has a smooth liquid-like texture that enables it to pass through a 27 gauge fine needle. However, Coleman fat is comprised of an integral adipose structure with large fibers that can only pass through an 18 gauge cannula. On day three after the transplantation, large numbers of small sized pre-adipocytes with multiple intracellular lipid droplets, extensive well-vascularized connective tissue, and infiltrated inflammatory cells appear.Beginning on day 15, the number of inflammatory cells starts to reduce gradually, and the adipocytes begin to mature. By day 90, most of the vascularized connective tissue in the grafts has been replaced by mature adipocytes. Extracellular matrix stromal vascular fraction gel grafts also contain a few perilipin positive adipocytes three days after the transplantation, with small sized pre-adipocytes, with multiple intracellular lipid droplets beginning to appear on day 15.Each field of the day 90 extracellular matrix stromal vascular fraction gel graft sections exhibit numerous perilipin positive adipocytes and newly formed blood vessels. Flow cytometry can be performed to determine the cellular population in the product. And scanning electron microscopy can be performed to visualize the microstructure of the product.This technique provides a simple method for surgeons and researchers to obtain a product with high concentration of SVF cells with native extracellular matrix fibers from adipose tissue.

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7.4K visualizações.  Southern Medical University. A obtenção de frações vasculares estromes, ou SVFs, de lipoaspiratas requer digestão com colagemnase, o que aumenta o risco de contaminação biológica. Uma melhor maneira de obter SVF sem colagemnase deve ser desenvolvida. Essas técnicas nos permitem obter o SVF em sua matriz extracelular nativa através de um simples processo mecânico.Demonstrando o procedimento será Xihang Chen, um estudante de pós-graduação do nosso laboratório. Após a obtenção das amostras de gordu...