Name: clinical protocol of producing adipose tissue-derived stromal vascular fraction for potential cartilage regeneration
Uploaded: 2018-09-29T14:00:00
Description: Watch this Scientific Journal Video about Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration at JoVE.com

The author acknowledges the support from the staff of Mipro Medical Clinic and the figure design by Jaepil/David Lee. This work was supported by research grants from the Bio &amp; Medical Technology Development Program of the NRF funded by the MSIT (number NRF-2017M3A9E4078014); and the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (numbers NRF-2017R1A2B4002315 and NRF-2016R1C1B2010308).

The approval and consent to report following case reports were waived by Myongji University Institutional Review Board committee (MJUIRB).&#160;Further, this clinical protocol was in compliance with the Declaration of Helsinki and regulation guidelines of the KFDA. For the procedures, informed consents were obtained from the patients.
1. Liposuction
NOTE: Perform with sterile technique.
<ol>
	<li>Use the following inclusion criteria: (1) MRI evidence of stage 3 OA...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Engineering. 7 (2), 211-228 (2001).</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. 2012, 812693 (2012).</li><li>Zhu, 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-derived+stem+cell:+a+better+stem+cell+than+BMSC.">Adipose-derived stem cell: a better stem cell than BMSC.</a> Cell Biochemistry and Function. 26 (6), 664-675 (2008).</li><li>Bellei, B., Migliano, E., Tedesco, M., Caputo, S., Picardo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+non-enzymatic+methods+for+harvesting+adipose-derived+stem+from+lipoaspirate:+technical+considerations+and+clinical+implications+for+regenerative+surgery.">Maximizing non-enzymatic methods for harvesting adipose-derived stem from lipoaspirate: technical considerations and clinical implications for regenerative surgery.</a> Scientific Reports. 7 (1), 10015 (2017).</li><li>Pak, J., Lee, J. H., Park, K. S., Jeong, B. C., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+Cartilage+in+Human+Knee+Osteoarthritis+with+Autologous+Adipose+Tissue-Derived+Stem+Cells+and+Autologous+Extracellular+Matrix.">Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix.</a> BioResearch Open Access. 5 (1), 192-200 (2016).</li><li>Alexander, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+Adipose-derived+Stromal+Vascular+Fraction+(AD-SVF)+Cell+Biology+and+Use+on+the+Basis+of+Cellular,+Chemical,+Structural+and+Paracrine+Components:+A+Concise+Review.">Understanding Adipose-derived Stromal Vascular Fraction (AD-SVF) Cell Biology and Use on the Basis of Cellular, Chemical, Structural and Paracrine Components: A Concise Review.</a> Journal of Prolotherapy. 4, e855-e869 (2012).</li><li>Benders, K. 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=Extracellular+matrix+scaffolds+for+cartilage+and+bone+regeneration.">Extracellular matrix scaffolds for cartilage and bone regeneration.</a> Trends in Biotechnology. 31 (3), 169-176 (2013).</li><li>Korean Food and Drug Administration (KFDA). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy:+Rules+and+Regulations.">Cell therapy: Rules and Regulations.</a> KFDA. , (2009).</li><li>Pak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+human+bones+in+hip+osteonecrosis+and+human+cartilage+in+knee+osteoarthritis+with+autologous+adipose-tissue-derived+stem+cells:+a+case+series.">Regeneration of human bones in hip osteonecrosis and human cartilage in knee osteoarthritis with autologous adipose-tissue-derived stem cells: a case series.</a> Journal of Medical Case Reports. 5, 296 (2011).</li><li>Pak, J., Chang, J. J., Lee, J. H., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+reporting+on+implantation+of+autologous+adipose+tissue-derived+stem+cells+with+platelet-rich+plasma+into+human+articular+joints.">Safety reporting on implantation of autologous adipose tissue-derived stem cells with platelet-rich plasma into human articular joints.</a> BMC Musculoskeletal Disorders. 14, 337 (2013).</li><li>Pak, J., Lee, J. H., Kartolo, W. A., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+Regeneration+in+Human+with+Adipose+Tissue-Derived+Stem+Cells:+Current+Status+in+Clinical+Implications.">Cartilage Regeneration in Human with Adipose Tissue-Derived Stem Cells: Current Status in Clinical Implications.</a> BioMed Research International. 2016, 4702674 (2016).</li><li>Pak, J., Lee, J. H., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+biological+approach+to+treat+chondromalacia+patellae.">A novel biological approach to treat chondromalacia patellae.</a> PLoS One. 8 (5), e64569 (2013).</li><li>Pak, J., Lee, J. H., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+repair+of+damaged+meniscus+with+autologous+adipose+tissue-derived+stem+cells.">Regenerative repair of damaged meniscus with autologous adipose tissue-derived stem cells.</a> BioMed Research International. 2014, 436029 (2014).</li><li>Aust, 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=Yield+of+human+adipose-derived+adult+stem+cells+from+liposuction+aspirates.">Yield of human adipose-derived adult stem cells from liposuction aspirates.</a> Cytotherapy. 6 (1), 7-14 (2004).</li><li>De Ugarte, D. 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+multi-lineage+cells+from+human+adipose+tissue+and+bone+marrow.">Comparison of multi-lineage cells from human adipose tissue and bone marrow.</a> Cells Tissues Organs. 174 (3), 101-109 (2003).</li><li>Guilak, F., 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=Clonal+analysis+of+the+differentiation+potential+of+human+adipose-derived+adult+stem+cells.">Clonal analysis of the differentiation potential of human adipose-derived adult stem cells.</a> Journal of Cellular Physiology. 206 (1), 229-237 (2006).</li><li>Mitchell, J. 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=Immunophenotype+of+human+adipose-derived+cells:+temporal+changes+in+stromal-associated+and+stem+cell-associated+markers.">Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.</a> Stem Cells. 24 (2), 376-385 (2006).</li><li>Oedayrajsingh-Varma, M. 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=Adipose+tissue-derived+mesenchymal+stem+cell+yield+and+growth+characteristics+are+affected+by+the+tissue-harvesting+procedure.">Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.</a> Cytotherapy. 8 (2), 166-177 (2006).</li><li>. Liberase TL information available from Sigma Millipore online Available from: <a target="_blank" href="https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&amp;region=US">https://www.sigmaaldrich.com/catalog/product/roche/05401020001?lang=en&amp;region=US</a> (2018)</li><li>. Liberase TM information available from Sigma Millipore online Available from: <a target="_blank" href="https://www.sigmaaldrich.com/catalog/product/roche/Libtmro?lang=en&amp;region=US">https://www.sigmaaldrich.com/catalog/product/roche/Libtmro?lang=en&amp;region=US</a> (2018)</li><li>Childs, J. D., Piva, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychometric+properties+of+the+functional+rating+index+in+patients+with+low+back+pain.">Psychometric properties of the functional rating index in patients with low back pain.</a> European Spine Journal. 14 (10), 1008-1012 (2005).</li><li>Price, D. 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In 2001, Zuk et al. isolated stem cells from adipose tissue by breaking down the collagen matrix with collagenase6. Afterwards, the group showed that these stem cells isolated from the adipose tissue could transform into cartilage and other tissues of mesoderm in origin, proving that these stem cells were mesenchymal in origin.&#160;
Likewise, the procedure presented in this article is a modified protocol to apply the similar method to human patients. The main ...

Mesenchymal stem cells (MSCs) are known to have the capability to regenerate cartilage1,2,3,4,5,6. They can be easily obtained from various sources: bone marrow, cord blood, and adipose tissue among many others. Among these sources, adipose tissue is the only source where a sufficient number of MSCs can be obtained without any culture expansion to regenerate cartilage in clinical settings7,8. Autologous bone marrow stromal vascular fraction (SVF) can be easily obtained as well. However, the number of stem cells contained in the non-culture expanded marrow is very low7,8. Cord-blood may contain a sufficient number of MSCs. However, cord blood is not a readily available source of autologous SVF.
Numerous methods of processing adipose tissue to obtain SVF are available for clinical applications. Among these, the method of obtaining MSCs from adipose tissue using collagenase, developed and confirmed by Zuk et al.5,6, is very well accepted. This method of using collagenase has been modified for clinical applications in orthopedics. In order to be applied to clinical settings, the system must be a closed system to maintain the sterility while keeping the convenience. One particular modification presented in this article involves homogenization of the lipoaspirates. Small sized lipoaspirates are digested relatively faster than the larger ones that are resulting in the uneven breakdown of adipose tissue. Furthermore, these larger sized lipoaspirates may produce fibrous tissues that can clog up the syringes and needles while performing joint injections9,10. In order to prevent these issues, the lipoaspirates may be homogenized by cutting and mincing the lipoaspirates before the incubation with collagenase. The resulting adipose tissue-derived SVF may contain more uniform extracellular matrix (ECM)&#160;compared to lipoaspirates that are not homogenized11.&#160;The broken-down ECM contained in the SVF may work as a scaffold12.
In 2009, autologous adipose tissue-derived SVF has been allowed by the Korean Food and Drug Administration (KFDA) when processed within a medical facility with minimal processing by a physician13. Afterwards, autologous adipose tissue-derived SVF has been utilized as a potential agent to improve knee functions in osteoarthritis (OA) patients by potentially regenerating cartilage-like tissue10,14,15,16,17,18. 
In 2011, Pak showed for the first time that adipose tissue-derived stem cells (ASCs) contained in the adipose tissue-derived SVF can improve knee functions potentially regenerating cartilage-like tissue in human OA patients when injected with platelet-rich plasma (PRP)&#160;14. In addition, Pak et al. have reported safety data in 2013 involving 91 patients. The mean efficacy rate reported in this safety data was 67%15. Subsequently, additional studies by Pak et al. showed improved knee functions potentially due to cartilage-like tissue regeneration in patients with a meniscus tear and chondromalacia patellae10,16,17,18. Based on articles reported, it is known that the number of stem cells contained in 100 g of adipose tissue processed by the protocol presented in this article may range from 1,000,000 - 40,000,000 depending on patients&#39; characteristics8,19,20,21,22,23.
Here, we present a clinical protocol of human knee OA by using autologous adipose tissue-derived SVF with HA and PRP activated with calcium chloride. The first version of this clinical protocol, involving a closed, manual system to maintain the sterility, was reported in 201114. The identical protocol was optimized, maintaining sterility, and was reported in 2013 and 201610,15. Here, the optimized protocol is presented. The schematic overview of the protocol is presented in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58363/58363fig1.jpg" /> 
Figure 1: The schematic overview of the protocol. <a href="https://www.jove.com/files/ftp_upload/58363/58363fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:941px;" width="941">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Material</td>
			<td style="width:221px;"></td>
			<td style="width:156px;"></td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5% Betadine (povidone-iodine)&#160;</td>
			<td>Firson Co., Ltd.</td>
			<td style="width:156px;">657400260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% Lidocaine&#160;</td>
			<td>Daehan Pharmaceutical Co.</td>
			<td style="width:156px;">670603480</td>
			<td></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;">Tumescent solution&#160;</td>
			<td style="width:221px;">Myungmoon Pharm. Co. Ltd.</td>
			<td style="width:156px;">N01BB01</td>
			<td style="width:303px;">The solution was composed of 500 mL normal saline, 40 mL 2% lidocaine, 20 mL 0.5% marcaine, and 0.5 mL epinephrine 1:1000.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Liberase TL and TM research grade&#160;</td>
			<td style="width:221px;">Roche Applied Science</td>
			<td style="width:156px;">5401020001</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D5LR</td>
			<td>Dahan Pharm. Co., Ltd.</td>
			<td style="width:156px;">645101072</td>
			<td>Dextrose 5% in lactated Ringer&#39;s solution&#160;</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;">Anticoagulant citrate dextrose solution&#160;</td>
			<td>Fenwal, Inc.</td>
			<td style="width:156px;">NDC:0942-0641</td>
			<td style="width:303px;">The solution was composed of 0.8% citric acid, 
			0.22% sodium citrate, and 0.223% dextrose.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3% (w/v) Calcium chloride&#160;</td>
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		<tr height="21">
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			<td>Dongkwang pharm. Co., Ltd.</td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.25% Ropivacaine</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Equipment</td>
			<td style="width:221px;"></td>
			<td style="width:156px;"></td>
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		<tr height="42">
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			<td style="width:156px;">30-0827045</td>
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			<td style="width:221px;">Hanil Scientific Inc.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Magnetic Resonance Imaging</td>
			<td style="width:221px;">Philips Medical Systems Inc.</td>
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			<td height="42" style="height:42px;width:261px;">Ultrasound Imaging System</td>
			<td style="width:221px;">Samsung Medison co., Ltd</td>
			<td style="width:156px;">CT-LK-V10-ICM-09.05.2007</td>
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	</tbody>
</table>

clinical protocol of producing adipose tissue-derived stromal vascular fraction for potential cartilage regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Three patients (one 87-year-old female with stage 3 OA, one 68-year-old male with stage 3 OA, and one 60-year-old female with stage 3 OA) without any significant past medical history presented to the clinic with persistent knee pain and desired for potential autologous adipose tissue-derived SVF treatment. All three patients had their knee examined by an orthopedic surgeon and were offered to have total knee replacement (TKR) and were reluctant to have the surgery. Prior to the procedure,...

Watch this Scientific Journal Video about Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration at JoVE.com

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

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