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; (2) either male or female; (3) over 18 years of age; (4) sufficient (100-110 g) adipose tissue for liposuction; (5) unwillingness to proceed with surgical intervention; (6) failure of conservative management; and (7) ongoing disabling pain.</li>
	<li>Use the following exclusion criteria: (1) active inflammatory or connective tissue disease thought to impact pain condition (i.e.,, lupus, rheumatoid arthritis, fibromyalgia); (2) active endocrine disorder that might impact pain condition (i.e.,, hypothyroidism, diabetes); (3) active neurological disorder that might impact pain condition (i.e.,, peripheral neuropathy, multiple sclerosis); (4) active pulmonary disease requiring medication usage; and (5) no history of steroid joint injections past 3 months.</li>
	<li>Bring the patient into an operating room with a biohazard class A hood and place him (or her) in a supine position.</li>
	<li>Clean the abdominal area of the patient with 5% betadine (povidone-iodine) and drape the patient using the sterile technique, exposing the cleaned area of the abdomen for liposuction.</li>
	<li>Approximately 5 cm infero-laterally from the umbilicus, anesthetize two sites (the one on the left and the other on the right side of the umbilicus) of incision to-be-made using 2 mL of 2% lidocaine without epinephrine with a (25 gauge, 1&#189; inch) needle in a 5-mL syringe by injecting each site at the epidermal level.</li>
	<li>Anesthetize the site of incision to-be-made using 5 mL of tumescent solution (500 mL of normal saline, 40 mL of 2% lidocaine, 20 mL of 0.5% bupivacaine, 0.5 mL of 1:1000 epinephrine) in a 10-mL syringe with a needle (25 gauge, 1&#189; inch).</li>
	<li>Make 2 incisions of 0.5 cm approximately 5 cm below the umbilicus laterally by pinching the skin to raise the depth of the subcutaneous level.</li>
	<li>Using No. 11 blade, poke the raised skin to penetrate through to the subcutaneous level but not to penetrate through the abdominal wall.</li>
	<li>By using 20 cm 16-gauge cannula, anesthetize the subcutaneous level of the whole lower abdomen area, which is to-be-liposuctioned, with 700 to 800 mL of the tumescent solution.</li>
	<li>After finishing infiltration of the whole lower abdomen with the tumescent solution, prepare a liposuction apparatus by connecting a 3.0 mm cannula connected to a 60 mL (or a 30 mL) syringe for manual liposuction or a specially designed 3.0 mm cannula connected to a centrifuge kit, a closed-system syringe for the purpose of keeping the sterility, connected to a vacuum machine for vacuum-assisted liposuction.</li>
	<li>Perform liposuction to obtain 100-110 g of adipose tissue excluding tumescent solution. When performing liposuction, adipose tissue will be obtained along with the tumescent solution, which should be separated and removed.</li>
	<li>To separate the tumescent solution, first by gravity, transfer the adipose tissue in the centrifuge kit to a 60 mL syringe and place the syringe down (i.e., the portion of the syringe is at the bottom). By waiting 5-6 min, the adipose tissue and the tumescent fluid will be separated. Remove the fluid at the bottom of the syringe by pressing on the top part of the syringe plunger.</li>
	<li>Perform the above Steps 1.9-1.11 until a total of 100-110 g of adipose tissue (lipoaspirates) per knee has been accumulated.</li>
</ol>
2. Preparation of ASC/ECM Mixture with Sterile Closed System
<ol>
	<li>After separating the tumescent solution by gravity and accumulating 50-55 g of lipoaspirates per each 60 mL centrifuge kit, a sterile closed system, place the 2 centrifuge kits into a centrifuge container bucket and spin at 1600 x g for 5 min, condensing the lipoaspirates and separating fluid from the adipose tissue. In this process of further condenses, the lipoaspirates may produce fatty oil in certain cases.</li>
	<li>Being cautious not to shake, remove the safety cap and the plug at the bottom of the centrifuge kit.</li>
	<li>Remove the bottom fluid by slowly pressing down on the top of the plunger of the centrifuge kit.</li>
	<li>On separate 60 mL syringe, dissolve 10 mg of collagenase (5 mg of collagenase specific for connective tissue24 and 5 mg of collagenase specific for adipose tissue25) with 50 mL of normal saline.</li>
	<li>Mix approximately 25-30 mL of condensed lipoaspirate with dissolved collagenase (5 mg of collagenase specific for connective tissue&#160;and 5 mg of collagenase specific for adipose tissue) at a ratio of 1:1 (v:v) by connecting the 60 mL syringe to a centrifuge kit by using a specialized connector.</li>
	<li>Thoroughly mix the condensed lipoaspirate and the collagenase by pushing the content between the 60 mL syringe and the centrifuge kits by using a rod or a pusher.</li>
	<li>Transfer the mixture of the lipoaspirate and the collagenase back to 60 mL syringes.</li>
	<li>Connect each 60 mL syringe containing the mixture with a tissue homogenizer which contains blades.</li>
	<li>Connect an empty 60 mL syringe to the other end of the homogenizer.</li>
	<li>Push the mixture to the other 60 mL syringe through the homogenizer for 4 -6 times, resulting in cutting and mincing of the lipoaspirate.</li>
	<li>Transfer the homogenized lipoaspirate and the collagenase mixture back to 60 mL centrifuge kits through a specialized connector</li>
	<li>Place the centrifuge kits in a container to be placed in an incubator that has been pre-heated at 37 &#176;C.</li>
	<li>Incubate the two centrifuge kits with the homogenized mixture at 37 &#176;C for 40 min while rotating at 45 rpm.</li>
	<li>After the 40 min of incubation, remove the container from the incubator in a sterile fashion. Then, remove the centrifuge kits and place them in a centrifuge machine.</li>
	<li>Centrifuge the mixtures at 800 x g for 5 min to separate the adipose tissue-derived SVF.</li>
	<li>After the centrifuge, remove the supernatant (which includes collagenase and digested adipose tissue) from each centrifuge kits by removing the syringe cap on the top of the plunger and placing a 30 mL syringe on the plunger opening via syringe lock connection.</li>
	<li>Slowly press down the barrel part of the 30 mL syringe for the supernatant to fill into the 30 mL syringe.</li>
	<li>Press down the 30 mL syringe barrel all the way down to the last 3-4 mL of the bottom of the centrifuge kit, leaving only the last 3-4 mL of adipose tissue-derived SVF. The supernatant is discarded.</li>
	<li>Remove the 30 mL syringe from the top of the plunger and fill the syringe with 5% dextrose in lactated Ringer&#39;s solution (D5LR).</li>
	<li>By attaching the 30 mL syringe filled with D5LR on the top of the plunger opening, fill the centrifuge kits, containing 3-4 mL of adipose tissue-derived SVF, with D5LR up to 55 mL.</li>
	<li>Remove the 30 mL syringe, cap the plunger, and centrifuge the centrifuge kits again at 300 x g for 4 min.</li>
	<li>Repeat Steps 2.17-2.21 for a total of 4 washings. The used collagenase is xenogenic. Therefore, most collagenase is removed by 4 washings. However, for FDA approval, fine-tuning of the protocol may be necessary to completely remove the collagenase residues in the final volume, although the current amount of collagenase residues may be negligible enough for the patients that do not have any clinical side effects.</li>
	<li>After the 4th centrifugation, 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.</li>
	<li>Attach a 20 mL syringe to the centrifuge kit bottom opening by using a specially designed connector.</li>
	<li>Pull the plunger of the syringe several times back and forth to shake up the cells that have settled on the bottom of the centrifuge kit.</li>
	<li>Remove the desired total volume of the SVF containing both ASCs and ECM along with other cells and tissue (usually about 3-4 mL from each centrifuge kit for knee joint injections).</li>
</ol>
3. PRP Preparation with Sterile Technique
<ol>
	<li>While preparing the ASCs and ECM, draw 30 mL of autologous blood with 2.5 mL of anticoagulant citrate dextrose solution.</li>
	<li>Transfer the drawn blood to the 60 mL centrifuge kits.</li>
	<li>Centrifuge the drawn blood at 730 x g for 5 min and remove the supernatant into a new 60 mL centrifuge kit. Centrifuge the supernatant at 1300 x g for 4 min, resulting in 3-4 mL of PRP.</li>
	<li>Right before the injection, add 3% (w/v) calcium chloride at a ratio of 10:2 (PRP: calcium chloride, v:v) to the PRP to activate it.</li>
	<li>Add 0.5% (w/v) HA, as a scaffold, to the PRP activated with calcium chloride. These ASCs with ECM, along with PRP, activated by calcium chloride, and HA stand for the ASC/ECM mixture.</li>
</ol>
4. ASC/ECM Mixture-Based Treatment
<ol>
	<li>Clean the knee of the patient with 5% betadine and drape it in a sterile manner.</li>
	<li>Palpate the anterior of the knee for the joint space between the tibial and femoral bones.</li>
	<li>Anesthetize the injection site superficially with diluted lidocaine (1 mL of 1% lidocaine diluted with 4 mL of normal saline) from the skin to just outside of the joint capsule.</li>
	<li>Anesthetize the inside of the joint capsule with diluted ropivacaine (1 mL 0.75% ropivacaine diluted with 3 mL of normal saline).</li>
	<li>Mix 2 mL of HA to the 6-8 mL of SVF contained in a 20 mL syringe through the syringe-to-syringe connector.</li>
	<li>By using a syringe-to-syringe connector, add 0.4 mL of calcium chloride to the 3-4 mL of autologous PRP that has been already prepared and being ready in a 5-mL syringe.</li>
	<li>Combine 3.5 - 4.5 mL of PRP/calcium chloride in a 5 mL syringe with 8-10 mL of HA/SVF mixture in a 20 mL syringe via syringe-to-syringe connector.</li>
	<li>Immediately inject the mixture (about 12-15 mL) slowly into the anterior tibio-femoral joint of the knees using 38-mm 18-gauge needle with or without the ultrasound guidance.</li>
	<li>After the injection, bandage the injection site with pressure by folding 4x4 cotton gauze 4 times and placing tapes over the folded 4x4 gauze.</li>
	<li>Instruct the patient to remain still for 60 min to allow for cell attachment.</li>
	<li>Instruct the patient to limit activities for minimal of 1 week after discharge from the clinic.</li>
	<li>Return to the clinic for three additional injections of PRP activated by calcium chloride over 3 weeks.</li>
</ol>
5. Post-Treatment Follow Up
<ol>
	<li>Assess patient at the week of 2, 4, and 16 (18 or 22) for pain improvement in terms of visual analog scale (VAS) and function improvement in terms of physical therapy parameters. Determine functional rating index (FRI), VAS, and range of motion (ROM) as previously described26,27.</li>
	<li>Follow the patient by post-treatment MRI 3 months after the treatment.</li>
</ol>

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S., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+effects+of+mesenchymal+stem+cells:+novel+concept+for+future+therapies.">Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies.</a> Expert Opinion on Biological Therapy. 8 (5), 569-581 (2008).</li><li>Zhang, J., Middleton, K. K., Fu, F. H., Im, H. J., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HGF+mediates+the+anti-inflammatory+effects+of+PRP+on+injured+tendons.">HGF mediates the anti-inflammatory effects of PRP on injured tendons.</a> PLoS One. 8 (6), e67303 (2013).</li><li>Li, N. Y., Yuan, R. T., Chen, T., Chen, L. Q., Jin, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+platelet-rich+plasma+and+latissimus+dorsi+muscle+flap+on+osteogenesis+and+vascularization+of+tissue-engineered+bone+in+dogs.">Effect of platelet-rich plasma and latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered bone in dogs.</a> Journal of Oral and Maxillofacial Surgery. 67 (9), 1850-1858 (2009).</li><li>Parsons, 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=The+biological+effect+of+platelet+rich-plasma+on+the+fracture+healing+process.">The biological effect of platelet rich-plasma on the fracture healing process.</a> The Journal of bone and joint surgery. British volume. 91-B, 293 (2009).</li><li>Wu, W., Chen, F., Liu, Y., Ma, Q., Mao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+injectable+tissue-engineered+cartilage+by+using+platelet-rich+plasma:+experimental+study+in+a+rabbit+model.">Autologous injectable tissue-engineered cartilage by using platelet-rich plasma: experimental study in a rabbit model.</a> Journal of Oral and Maxillofacial Surgery. 65 (10), 1951-1957 (2007).</li><li>Cooper, T. W., Eisen, A. Z., Stricklin, G. P., Welgus, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+collagenase+inhibitor:+characterization+and+subcellular+localization.">Platelet-derived collagenase inhibitor: characterization and subcellular localization.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (9), 2779-2783 (1985).</li><li>Uzuki, M., Sawai, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+affinity+of+sodium+hyaluronate+of+various+molecular+weights+for+degenerated+cartilage:+a+histochemical+study+using+hyaluronic+acid+binding+protein.">A comparison of the affinity of sodium hyaluronate of various molecular weights for degenerated cartilage: a histochemical study using hyaluronic acid binding protein.</a> International Congress Series. 1223, 279-284 (2001).</li><li>Pagano, C., 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=Molecular+and+morphometric+description+of+adipose+tissue+during+weight+changes:+a+quantitative+tool+for+assessment+of+tissue+texture.">Molecular and morphometric description of adipose tissue during weight changes: a quantitative tool for assessment of tissue texture.</a> International Journal of Molecular Medicine. 14 (5), 897-902 (2004).</li></ol>

The authors have nothing to disclose.

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>

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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;">3% (w/v) Calcium chloride&#160;</td>
			<td>Choongwae Pharmaceutical Co.</td>
			<td style="width:156px;">644902101</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.5% (w/v) HA (Hyaluronic acid )</td>
			<td>Dongkwang pharm. Co., Ltd.</td>
			<td style="width:156px;">645902030</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.25% Ropivacaine</td>
			<td>Huons Co., Ltd.</td>
			<td style="width:156px;">670600150</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>
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mL Luer-Lock syringe</td>
			<td style="width:221px;">BD (Becton Dickinson)&#160;</td>
			<td style="width:156px;">309653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Barrel Kit&#160;</td>
			<td>CPL Co., Ltd.</td>
			<td style="width:156px;">30-0827044</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
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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 ...

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41.7K Views.  Mipro Medical Clinic. 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. 

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

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

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

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

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...