Name: clinical protocol of producing adipose tissue-derived stromal vascular fraction for potential cartilage regeneration
Uploaded: 2018-09-29T14:00:00.000+00:00
Duration: 14 min 49 s
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; (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 modification of the protocol is the incorporation of closed system syringes in obtaining and processing the lipoaspirates in order to minimize the air contact while maintaining the ease of performing the procedure. Using such closed system syringes, adipose tissue was obtained via liposuction. Then, the lipoaspirates were mixed with collagenase within the closed system syringes. Afterwards, the collagenase was removed from the mixture by diluting the collagenase concentration within the closed system syringes. The end result is a sterile adipose tissue-derived SVF obtained with ease of the process.&#160;
By using the similar method, Pak showed in 2011, for the first time, that autologous adipose tissue-derived SVF can be used to treat OA patients by potentially regenerating cartilage-like tissue. Later, Pak et al. also demonstrated that autologous adipose tissue-derived SVF can be used to treat meniscus tear and chondromalacia of patellae by regenerating fibrous cartilage-like tissue and hyaline cartilage-like tissue, respectively. In 2013, Pak et al. reported a safety study involving 91 patients treated with autologous adipose tissue-derived SVF. The mean efficacy of the study reported was 67%, paving the road to the possibility of applying autologous adipose tissue-derived SVF in general patient population with cartilage damage.
One of the most common side effects reported by Pak et al. in 2013 safety study was post-treatment knee joint swelling, which can be explained by 1) stem cell death, 2) inflammation by RBCs contained in the PRP, and/or 3) residual collagenase that has remained in the final volume of adipose tissue-derived SVF. Since MSCs exist within ECM of adipose tissue11,12, digesting the lipoaspirates with collagenase to break down the matrix to lease the stem cells is a mandatory process12.&#160;However, after breaking down the matrix to release the stem cells, the remaining residual collagenase should be fully removed from the final volume in order to prevent any potential inflammation that can be produced by the residual collagenase28. Although collagenase can cause inflammation, an insignificant amount of collagenase in the final volume of adipose tissue-derived SVF may not trigger a joint swelling. Such final volume of autologous adipose tissue-derived SVF with an insignificant amount of collagenase has been used safely for the last few years to treat OA of knees by regenerating cartilage-like tissue.
Recently two non-enzymatic methods of obtaining adipose tissue-derived SVF have been introduced29,30. These methods utilize either mechanical force or ultrasound (vibrational) force, instead of collagenase. In 2018, D&#39;Ambrosi et al. reported a result of treating four osteochondral lesions of the talus of the ankle with micro-fractured, purified autologous adipose tissue that was obtained by using mechanical force to breakdown the matrix29. No collagenase was used to break down the matrix. Four patients were treated with the micro-fractured adipose tissue (i.e., mechanically broken-down adipose tissue), for their talus29. All these four patients showed clinical improvement six months after the procedure. None had any complications. However, on this study29, there was no report of potential regeneration of cartilage. Another study published this year involving vibrational energy showed that vibrational energy in certain frequency was not sufficient to isolate stem cells30.
As shown above, different groups have different protocols for using an autologous adipose tissue to treat various degenerative joint disorders. The protocol can vary from the size of the cannula used for liposuction, the amount of adipose tissue used, type and amount of collagenase (if any), method of injections, and so on. Since MSCs exist within ECM of adipose tissue, the effect of collagenase, or purification method using mechanical forces, may play an important role. Especially, the efficacy of collagenase may depend on factors such as the size of the adipose tissue, amount of the adipose tissue, concentration of collagenase, time and temperature of incubation with collagenase, and the process of removing the leftover collagenase, which can cause cell damage28.
It should be noted that collagenase, as it is an important component of this procedure to produce stem cell containing adipose tissue-derived SVF, may have a negative effect on the final volume of adipose tissue-derived SVF. In this procedure, collagenase is mixed with condensed lipoaspirates, homogenized, incubated, and then washed off. Throughout this process, stem cells are exposed to collagenase, which can have a detrimental effect on the survival of the stem cells28. Too much concentration or prolonged exposure of collagenase may lower the stem cell viability in the final volume28. Further, too much of collagenase left in the final volume may cause inflammation of the joint since collagenase may break down the cartilage matrix of the joint injected28.
In addition to the potential negative effects of collagenase, liposuction can carry other potential complications. In order to obtain adipose tissue-derived SVF, liposuction must be performed first. Performing liposuctions, as other medical procedures, carries few potential complications such as irregularities in the skin contour, seromas, infection, perforation of the abdominal wall, necrotizing fasciitis, fat embolism, and pulmonary embolism. Among these complications, skin contour irregularities are a very common side effect that can be easily prevented by using small cannulas and avoiding superficial liposuctions31,32. Seromas, which is a collection of serous fluid in the suctioned area, can also be a result of aggressive liposuction33. Other potential complications such as infection, necrotizing fasciitis, perforation of abdominal walls, fat embolism, or pulmonary embolism are all possible. However, these complications can also be prevented by using strict sterile techniques and by improving patient compliance34.
Further, all three patients included in the study initially presented with a mild joint effusion, a clear sign of synovitis. After the treatment with adipose tissue-derived SVF, all these patients joint swelling improved. For this reason, it is also reasonable to assume that the improvement of clinical symptoms is not due to the possible cartilage regeneration, but instead, due to the modulatory and anti-inflammatory effect of stem cells and/or PRP on synovial membrane35,36. Alternatively, the symptom improvement can be contributed to both modulatory and anti-inflammatory effect of stem cells and/or PRP on synovial membrane along with possible regeneration of cartilage-like tissue. Although more research is necessary to delineate the true mechanism of symptom improvement, this percutaneous intra-articular injection of cell therapy in the form of autologous, homogenized adipose tissue-derived SVF with autologous PRP activated with calcium chloride and /or HA may offer an alternative treatment to the current strategy of treating knee OA.
As for the addition of autologous PRP is concerned, it is widely accepted that PRP contains various growth factors and differentiating factors for injected MSCs to grow and differentiate37,38,39. PRP also has shown to have a collagenase neutralizing effect40. Regarding the addition of hyaluronic acid (HA), HA has been shown to have a potential role as a scaffolding material, due to its high affinity to cartilage tissue, and a potential role to assist stem cells in penetrating the cartilage matrix41.
As more new processes of obtaining adipose tissue-derived SVF are available, an optimization of a method of processing lipoaspirates is necessary to standardize the process. However, this may be a difficult task due to the number of potential variations in the process of obtaining and processing autologous adipose tissue-derived SVF. For example, differences in the texture of subcutaneous adipose tissue in each individual patient, due to aging or the degree of obesity, may have different responses to collagenase activities, resulting in a different number of ASCs in the autologous adipose tissue-derived SVF42. In addition, the number of stem cells in each gram of adipose tissue may differ from one patient to another as shown by the great variation of reported stem cell numbers in numerous publications7,19,20,21,22,23.
This protocol presented in this article is a novel, innovative procedure attempting to improve the current strategy of treating human knee OA by potentially regenerating cartilage-like tissue with percutaneous injections of autologous adipose tissue-derived SVF. The correct use of collagenase is an important and critical step in this protocol. Using closed system syringes to keep the sterility while maintaining the convenience of performing the procedure is the major modification of the protocol developed by Zuk et al.5 In addition, for human application, the residual amount of collagenase contained in the final adipose tissue-derived SVF should be insignificant to prevent any joint inflammation. There are limitations of this protocol: the collagenase residues in the final volume (although this may be negligible), no control groups that singly injected PRP and HA, no biopsies, no evidence excluding the improvement of clinical symptoms due to PRP, the small number of patients (pilot study), no defying components of the adipose tissue-derived SVF, and no specification of the number of stem cells in each of the 3 patients. Applying adipose tissue-derived SVF for disorders such as a meniscus tear, chondromalacia patellae, spinal disc disorders, a delayed healing (or non-healing) bone fracture, a non-healing skin ulcer, breast reconstruction, and other medical disorders may improve the clinical outcome. Although these are potential clinical applications of adipose tissue-derived SVF, more thorough and vigorous clinical researches are necessary for incorporating adipose tissue-derived SVF in actual clinical settings.

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><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5% Betadine (povidone-iodine)&amp;nbsp;</td><td>Firson Co., Ltd.</td><td>657400260</td><td></td></tr><tr><td>2% Lidocaine&amp;nbsp;</td><td>Daehan Pharmaceutical Co.</td><td>670603480</td><td></td></tr><tr><td>Tumescent solution&amp;nbsp;</td><td>Myungmoon Pharm. Co. Ltd.</td><td>N01BB01</td><td>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><td>Liberase TL and TM research grade&amp;nbsp;</td><td>Roche Applied Science</td><td>5401020001</td><td></td></tr><tr><td>D5LR</td><td>Dahan Pharm. Co., Ltd.</td><td>645101072</td><td>Dextrose 5% in lactated Ringer&#039;s solution&amp;nbsp;</td></tr><tr><td>Anticoagulant citrate dextrose solution&amp;nbsp;</td><td>Fenwal, Inc.</td><td>NDC:0942-0641</td><td>The solution was composed of 0.8% citric acid,&lt;br/&gt; 0.22% sodium citrate, and 0.223% dextrose.</td></tr><tr><td>3% (w/v) Calcium chloride&amp;nbsp;</td><td>Choongwae Pharmaceutical Co.</td><td>644902101</td><td></td></tr><tr><td>0.5% (w/v) HA (Hyaluronic acid )</td><td>Dongkwang pharm. Co., Ltd.</td><td>645902030</td><td></td></tr><tr><td>0.25% Ropivacaine</td><td>Huons Co., Ltd.</td><td>670600150</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3.0 mm Cannula&amp;nbsp;</td><td>WOOJU Medical Instruments Co.</td><td>ML30200</td><td></td></tr><tr><td>60-mL Luer-Lock syringe</td><td>BD (Becton Dickinson)&amp;nbsp;</td><td>309653</td><td></td></tr><tr><td>Centrifuge Barrel Kit&amp;nbsp;</td><td>CPL Co., Ltd.</td><td>30-0827044</td><td></td></tr><tr><td>Tissue homogenizer that contains blades</td><td>CPL Co., Ltd.</td><td>30-0827045</td><td></td></tr><tr><td>Rotating incubator mixer</td><td>Medikan Co., Ltd</td><td>MS02060092</td><td></td></tr><tr><td>Centrifuge</td><td>Hanil Scientific Inc.</td><td>CE1133</td><td></td></tr><tr><td>Magnetic Resonance Imaging</td><td>Philips Medical Systems Inc.</td><td>18068</td><td></td></tr><tr><td>Ultrasound Imaging System</td><td>Samsung Medison co., Ltd</td><td>CT-LK-V10-ICM-09.05.2007</td><td></td></tr></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, all three patients had received multiple injections of steroids and HA without any prolonged improvement.
The 87-year-old Korean female patient, at the time of examination, complained of severe pain (VAS score of 8; Figure 2A) while on rest. She described the pain to be increasing ([FRI: 37; Figure 2A) when walking up and down stairs. On physical exam, mild joint swelling with decreased ROM and tenderness with flexion (Figure 2B) were noted. However, no ligament laxity was appreciated. McMurray&#39;s and Apley&#39;s tests were negative. A before-treatment MRI demonstrated a decreased size medial meniscus along with deformation and maceration. (Figure 2C, 2E, 2G, and 2I).
The second patient, a 68-year-old Korean male, complained of severe left knee pain (VAS score: 7; Figure 3A) while on rest. The pain (FRI: 33; Figure 3A) was described as increasing when walking up and down stairs. On physical exam, there was mild deformity with mild joint swelling. ROM (Figure 3B) was decreased. Further, no ligament laxity was appreciated. McMurray&#39;s and Apley&#39;s tests were negative. A before-treatment MRI showed cartilage thinning along with a decreased size and deformed medial meniscus secondary to the previous meniscectomy (Figure 3C, 3E, 3G, and 3I). The patient was diagnosed to have stage 3 OA.
The third patient, a 60-year-old Korean female, also reported severe pain (VAS score: 8; Figure 4A) on rest. The pain (FRI: 36; Figure 4A) was described as increasing when walking. The patient also had mild knee swelling and decreased ROM (Figure 4B). No ligament laxity was appreciated. McMurray&#39;s and Apley&#39;s tests were negative. A before-treatment MRI showed decreased size medial meniscus with deformation and maceration, and there was cartilage thinning (Figure 4C, 4E, 4G, and 4I).
Treatment Plan. All three representative patients were restricted from taking steroids, aspirin, non-steroidal anti-inflammatory drugs (NSAIDs), and Asian herbal medications at least for 1 week prior to the procedure. After taking MRI imaging studies, lipoaspirates were obtained and processed as referenced above. Afterwards, the autologous adipose tissue-derived SVF containing ASCs, ECM, HA, and calcium-chloride-activated autologous PRP were injected to the knees at day 0. There was no complication due to liposuctions and joint injections. Subsequently, the patients returned to the clinic in one week, then 2 weeks, and then 3 weeks for additional injections of HA and autologous PRP activated with calcium chloride.
Outcome. After the second week of the ASC/ECM mixture injection, the 87-year-old female patient&#39;s pain and ROM improved (Figure 2A and 2B). By the 16th week, the patient&#39;s pain and ROM significantly improved by more than 70% (Figure 2A and 2B). Post-treatment MRI taken after the 16th week showed the increased thickness of hyaline cartilage-like tissue on the medial side of the knee (Figure 2D, 2F, 2H, and 2J). As a comparison, the mean efficacy rate was 67% in data using this clinical protocol, involving 91 patients, and reported in 201315.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58363/58363fig2.jpg" /> 
Figure 2: Outcome of pain measurements (A) and range of motion (B); and MRI sagittal (C-F) and coronal (G-J) sequential T2 views of the knee from the patient case 1.* indicates a statistically significant finding (p &#60; 0.05). Pre-treatment MRI scans (C: sequential image, 5/20; E: 6/20; G: 10/20; and I: 11/20) show cartilage lesions (arrows). Post-treatment MRI scans at 16 weeks (D: 6/20; F: 7/20; H: 10/20; and J: 11/20) indicate cartilage-like tissue regeneration (arrowhead) that has been repaired by ASC/ECM mixture-based treatment. This figure has been modified from the previous report of Pak et al.10. <a href="https://www.jove.com/files/ftp_upload/58363/58363fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The 68-year-old male patient&#39;s pain and ROM improved after the second week of the ASC/ECM mixture injection (Figure 3A and 3B). By the 18th week, his pain and ROM significantly further improved to 80% (Figure 3A and 3B). Repeated MRI taken after 18th week showed an increase in the height of hyaline cartilage-like tissue on the anterior medial side of the knee (Figure 3D, 3F, 3H, and 3J).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58363/58363fig3.jpg" /> 
Figure 3: Outcome of pain measurements (A) and range of motion (B); and MRI sagittal (C-F) and coronal (G-J) sequential T2 views of the knee from the patient case 2.* indicates a statistically significant finding (p &#60; 0.05). Pre-treatment MRI scans (C: sequential image, 6/20; E: 7/20; G: 13/20; and I: 14/20) show cartilage lesions (arrows). Post-treatment MRI scans at 16 weeks (D: 6/20; F: 7/20; H: 13/20; and J: 14/20) indicate cartilage-like tissue regeneration (arrowhead) that has been repaired by ASC/ECM mixture-based treatment. This figure has been modified from the previous report of Pak et al.10. <a href="https://www.jove.com/files/ftp_upload/58363/58363fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The 60-year-old female patient&#39;s pain and ROM improved approximately 50% after the second week of the ASC/ECM mixture injection, (Figure 4A and 4B). By the 22nd week, the pain and ROM significantly improved over 80% (Figure 4A and 4B). Repeated MRI taken after the 22nd week showed an increase in the height of the hyaline cartilage-like tissue on the medial side of the knee (Figure 4D, 4F, 4H, and 4J).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58363/58363fig4.jpg" /> 
Figure 4: Outcome of pain measurements (A) and range of motion (B); and MRI sagittal (C-F) and coronal (G-J) sequential T2 views of the knee from the patient case 3.* indicates a statistically significant finding (p &#60; 0.05). Pre-treatment MRI scans (C: sequential image, 4/20; E: 5/20; G: 10/20; and I: 11/20) show cartilage lesions (arrows). Post-treatment MRI scans at 16 weeks (D: 4/20; F: 5/20; H: 10/20; and J: 11/20) indicate cartilage-like tissue regeneration (arrowhead) that has been repaired by ASC/ECM mixture-based treatment. This figure has been modified from the previous report of Pak et al.10.&#160;<a href="https://www.jove.com/files/ftp_upload/58363/58363fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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41.8K 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

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Developmental Biology

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

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. ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioengineering

Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several studies have focused their attention on mesenchymal stem cells derived from adipose tissue. Adipose-derived stem cells (ADSCs) exhibit morphological characteristics and properties similar to other mesenchymal cells, and are able to differentiate into several cellular lines. Moreover, these cells are also widely available in the subcutaneous tissue, representing 10 - 30% of the normal body weight, with a concentration of 5,000 cells per gram of tissue.
In the presented technique, the first step involves harvesting ADSCs from the abdomen and a process of microfracture and purification; next, the surgical procedure is performed entirely arthroscopically, with less soft tissue dissection, better joint visualization, and a faster recovery compared with standard open procedures. Arthroscopy is characterized by a first phase in which the lesion is identified, isolated, and prepared with microperforations; the second step, performed dry, involves injection of adipose tissue at the level of the lesion.
Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery with no reported complications. Functional scores at the latest follow-up are encouraging and confirm that the technique provides reliable pain relief and improvements in patients with osteochondral lesion of the talus.

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells.

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

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

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

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

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

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

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

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

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...