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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			<td></td>
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		<tr height="21">
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			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:261px;">Equipment</td>
			<td style="width:221px;"></td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3.0 mm Cannula&#160;</td>
			<td style="width:221px;">WOOJU Medical Instruments Co.</td>
			<td style="width:156px;">ML30200</td>
			<td></td>
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			<td style="width:221px;">BD (Becton Dickinson)&#160;</td>
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			<td>CPL Co., Ltd.</td>
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			<td height="21" style="height:21px;">Tissue homogenizer that contains blades</td>
			<td>CPL Co., Ltd.</td>
			<td style="width:156px;">30-0827045</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Rotating incubator mixer</td>
			<td style="width:221px;">Medikan Co., Ltd</td>
			<td style="width:156px;">MS02060092</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:261px;">Centrifuge</td>
			<td style="width:221px;">Hanil Scientific Inc.</td>
			<td style="width:156px;">CE1133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Magnetic Resonance Imaging</td>
			<td style="width:221px;">Philips Medical Systems Inc.</td>
			<td style="width:156px;">18068</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>
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</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 ...

Osteoarthritis is one of the most common debilitating disorders among the elderly population. At present, there is no definite cure for the underlying causes of osteoarthritis. This clinical protocol describes an innovative procedure for a promising, minimally invasive, and alternative treatment of human knee osteoarthritis by regenerating cartilage like tissue.Osteoarthritis is one of the most common debilitating disorders among the elderly population. At present, there is no definite cure for the underlining causes of osteoarthritis. This clinical protocol is an innovative procedure for a promising, minimally invasive, and alternative treatment of human knee osteoarthritis by regenerating cartilage-like tissue.Bring the patient into an operating room with a biohazard Class A hood, and place him or her in a supine position. Clean the abdominal area of the patient with 5%betadine-povidine iodine and drape the patient using the sterile technique, exposing the clean area of the abdomen for liposuction. Approximately five centimeters infero-laterally from the umbilicus, anesthetize two sites, one on the left and other on the right side of the umbilicus of incision to be made.Using two milliliter of 2%lidocaine without epinephrine with a 25-gauge 1.5 inch needle in a five milliliter syringe by injecting each site at the epidermal level. Anesthetize the site of incision to be made using five milliliter of tumescent solution and 10 milliliter syringe and the needle 25-gauge 1.5 inch. Make two incisions of 0.5 centimeters approximately five centimeters below the umbilicus laterally by pinching the skin to raise depth of the subcutaneous level.Using a number 11 blade, help the raised skin to penetrate through to the subcutaneous level but not to penetrate through the abdominal wall. By using 20 centimeter 60-gauge cannula, anesthetize the subcutaneous level of the whole lower abdomen area which is to be liposuctioned with 700 to 800 milliliter of the tumescent solution. After finishing infiltration of the whole lower abdomen with tumescent solution, prepare a liposuction apparatus by connecting a 3.0 millimeter cannula connected to a 60 milliliter or 30 milliliter syringe for manual liposuction or specially designed 3.0 millimeter cannula connected to a centrifuge kit.A closed system syringe developed by ourselves and approved by FDA CDRH for the purpose of keeping the sterility connected to a vacuum machine for vacuum assisted liposuction. Perform liposuction to obtain 100 to 110 gram of adipose tissue excluding tumescent solution. When performing liposuction, adipose tissue will be obtained along with tumescent solution but should be separated and removed.To separate the tumescent solution first by gravity, the adipose tissue in the centrifuge kit may be transferred to a 60 milliliter syringe and place the syringe down. In example, the opening portion of the syringe is at the bottom. By waiting five to six minutes, the adipose tissue and tumescent solution will be separated.Remove the fluid at the bottom of the syringe by pressing on the top of the syringe plunger. Perform the above steps nine to 11 until total of the 100 gram to 110 gram of adipose tissue lipid aspirate per knee has been accumulated. After separating the tumescent solution by gravity and accumulating 50 gram to 55 gram of lipid aspirate per each 60 milliliter centrifuge kit sterile closed system, place the two centrifuge kits into a centrifuge container bucket and spin at 1600 G for five minutes, condensing the lipid aspirate and separating fluid from the adipose tissue.This process further condenses the lipid aspirate and may produce fatty oil in certain cases. Being careful not to shake, remove the safety cap and the plug at the bottom of the centrifuge kit. Remove the bottom fluid by slowly pushing down on top of the plunger of the centrifuge kit.On separate 60 milliliter syringe, dissolve 10 milligrams of collagenase, five milligrams collagenase specific for connective tissue, and five milligrams of collagenase specific for adipose tissue with 50 milliliter of normal saline. Mix approximately 25 to 30 milliliter of condensed lipid aspirate with dissolved collagenase, five milligrams of collagenase specific for connective tissue, and five milligrams of collagenase specific for adipose tissue at a ratio of one to one, volume to volume, by connecting the 60 milliliter syringe to a centrifuge kit by using a specialized connector. Thoroughly mix to condense the prep aspirate and collagenase by pushing the content between the 60 milliliter syringe and the centrifuge kit by using a rod or a pusher.Transfer the mixture of the lipid aspirate and the collagenase back to the 60 milliliter syringe. Connect each 60 milliliter syringe containing the mixture with the tissue homogenizer which contains blades. Connect an empty 60 milliliter syringe to the other end of the homogenizer.Push the mixture to the other 60 milliliter syringe through the homogenizer for four to six times, resulting in cutting and mincing of the lipid aspirate. Transfer the homogenized lipid aspirate and the collagenous mixture back to the 60 milliliter centrifuge kit through a specialized connector. Place the centrifuge kits in a container to be placed in an incubator that has been preheated at 37 degrees Celsius.Incubate the two centrifuge kits with the homogenized mixture at 37 degrees Celsius for 40 minutes while rotating at 45 rpm. After 40 minutes of incubation, remove the container from the incubator in sterile fashion. Then remove the centrifuge kits and place them in a centrifuge machine.Centrifuge the mixtures at 800 G for five minutes to separate the adipose tissue-derived SVF. After the centrifuge, the supernatant, which includes collagenase and digested adipose tissue is removed from each centrifuge kit by removing the syringe cap on top of the plunger and placing a 30 milliliter syringe on the plunger opening through a syringe-lock connection. Slowly press down on the barrel part of the 30 milliliter syringe for the supernatant to fill into the 30 milliliter syringe.Press down the 30 milliliter syringe barrel all the way to the last three to four milliliters of the bottom of centrifuge kit, leaving only the last three to four milliliters of adipose tissue-derived SVF. The supernatant is discard. Remove the 30 milliliter syringe from the top of the plunger and fill the syringe with 5%dextrose lactated ringer solution, also known as D5LR.By attaching the 30 milliliter syringe filled with D5LR on the top of the plunger opening, fill the centrifuge kits containing three to four milliliter of adipose tissue-derived SVF with D5LR up to 55 milliliters. Then remove the 30 milliliter syringe, cap the plunger opening, and centrifuge the centrifuge kits again at 300 G for about four minutes. Repeat steps number 17 to number 21 for a total of four washings.After the fourth centrifuge, in order to obtain the final SVF for injection, remove the safety cap and the plug at the bottom opening of the centrifuge kit without shaking or turning the centrifuge kit. Attach a 20 milliliter syringe to the centrifuge kit bottom opening by using a specially designed connector. Pull the plunger of the 20 milliliter syringe several times back and forth to shake up the cells that have settled on the bottom of the centrifuge kit.Remove desired total volume of the SVF containing both ASTs and ECM along with other cells and tissue. Usually about three to four milliliter from each centrifuge kit for knee joint injections. We did 30 milliliters of autologous blood along with 2.5 milliliters of anticoagulant citrate dextrose solution.Then the drawn blood was transferred to the 60 milliliter centrifuge barrel kits. It was then centrifuged at 730 gravity for five minutes. And the supernatant was removed and put into a new 60 milliliter centrifuge barrel kit.The supernatant was centrifuged at 1350 gravity for four minutes, obtaining 4.4 milliliters of PRP. Clean the knee of the patient with 5%betadine and drape it in a sterile manner. Palpate the anterior of the knee for the joint space between tibial and femoral bones.Anesthetize the injection site superficially with diluted lidocaine, one milliliter of 1%lidocaine diluted with four milliliters of normal saline, from skin to just outside the joint capsule. Then anesthetize the inside of the joint capsule with diluted ropivacaine, one milliliter 0.75%ropivacaine diluted with three milliliter of normal saline. Mix two milliliter of HA to the six to eight milliliter of SVF contained in a 20 milliliter syringe through a syringe to syringe connector.By using a syringe to syringe connector, add 0.4 milliliters of calcium chloride to the three to four milliliters of autologous PRP that has been already prepared and being ready in a five milliliter syringe. Combine 3.5 to 4.5 milliliters PRP calcium chloride in a five milliliter syringe with eight to 10 milliliters of HA-SVF mixture in a 20 milliliter syringe through a syringe to syringe connector. Immediately inject the mixture, about 12 to 15 milliliter, slowly into the anterior tibial-femoral joint of the knee, using 38 millimeter 18-gauge needle with or without the ultrasound guidance.After the injection, bandage the injection site with pressure by folding 4x4 cotton gauze four times and placing tapes over the folded 4x4 gauze. Instruct patient to remain still for 60 minutes to allow for cell attachment. Instruct patient to limit activities for minimal of one week after discharge from the clinic.Return to clinic for three additional injections of PRP activated by calcium chloride over three weeks. Patient with stage three OA received the treatment as described in the protocol section. After the second week of the ASV-ECM mixture injection, the patient's pain and ROM improved.By the 16th week, the patient's pain and ROM significantly improved more than 70%Post treatment MRI, taken after the 16th week, showed the increased thickness of cartilage-like tissue on the medial side of the knee. Another patient with stage three OA received the treatment as described in the protocol section. After the second week of the ASV-ECM mixture injection, the patient's pain and ROM improved.By the 18th week, the pain and ROM significantly improved to 80%Repeated MRI taken after the 18th week showed an increase in the height of cartilage-like tissue on the anterior-medial side of the knee. The third patient with stage three OA received the treatment as described in the protocol section. After second week of ASV-ECM mixture injection, the patient's pain and ROM improved by approximately 50%By the 22nd week, the pain and ROM significantly improved over 80%Repeated MRI taken after the 22nd week showed an increase in the height of the cartilage-like tissue on the medial side of the knee.Currently no curative therapy is available for the treatment of OA.However, percutaneous injection of stem cell therapy with ASCs and homogenized ECM along with HA and analogous PRP activated with calcium chloride may provide an alternative to current treatment therapy of treating knee OA.

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