Name: stromal vascular fraction-enriched fat grafting for the treatment of symptomatic end-neuromata
Uploaded: 2017-11-23T13:00:00
Description: Watch this Scientific Journal Video about Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata at JoVE.com

The authors have no acknowledgements.

The protocol follows the guidelines of the University Hospital Zurich Human Research Ethics Committee.
1. Diagnoses of Neuromata and Patient Selection
<ol>
	<li>Perform preoperative nerve blocks proximal to the neuroma in order to confirm the diagnoses. Assess the blocks in the middle third of the forearm and exclude potential triggers of the overlapping innervation areas of the SBRN, such as the lateral antebrachial cutaneous nerve (LACN), posterior interosseous nerve (PIN), and a dorsal br...

<ol>
	<li>Mackinnon, S. E., Dellon, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+treatment+of+recurrent+dorsoradial+wrist+neuromas.">Results of treatment of recurrent dorsoradial wrist neuromas.</a> Ann Plast Surg. 19 (1), 54-61 (1987).</li><li>Hazari, A., Elliot, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+end-neuromas,+neuromas-in-continuity+and+scarred+nerves+of+the+digits+by+proximal+relocation.">Treatment of end-neuromas, neuromas-in-continuity and scarred nerves of the digits by proximal relocation.</a> J Hand Surg Br. 29 (4), 338-350 (2004).</li><li>Goldstein, S. A., Sturim, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraosseous+nerve+transposition+for+treatment+of+painful+neuromas.">Intraosseous nerve transposition for treatment of painful neuromas.</a> J Hand Surg Am. 10 (2), 270-274 (1985).</li><li>Koch, H., Haas, F., Hubmer, M., Rappl, T., Scharnagl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+painful+neuroma+by+resection+and+nerve+stump+transplantation+into+a+vein.">Treatment of painful neuroma by resection and nerve stump transplantation into a vein.</a> Ann Plast Surg. 51 (1), 45-50 (2003).</li><li>Vaienti, L., Merle, M., Battiston, B., Villani, F., Gazzola, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+fat+grafting+in+the+treatment+of+painful+end-neuromas+of+the+upper+limb:+a+pilot+study.">Perineural fat grafting in the treatment of painful end-neuromas of the upper limb: a pilot study.</a> J Hand Surg Eur. 38 (1), 36-42 (2013).</li><li>Zuk, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Fraser, J. K., 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+Celution+System:+Automated+Processing+of+Adipose-Derived+Regenerative+Cells+in+a+Functionally+Closed+System.">The Celution System: Automated Processing of Adipose-Derived Regenerative Cells in a Functionally Closed System.</a> Adv Wound Care (New Rochelle). 3 (1), 38-45 (2014).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+vascular+fraction:+A+regenerative+reality?+Part+2:+Mechanisms+of+regenerative+action.">Stromal vascular fraction: A regenerative reality? Part 2: Mechanisms of regenerative action.</a> J Plast Reconstr Aesthet Surg. 69 (2), 180-188 (2015).</li><li>Lutz, B. S., Ma, S. F., Chuang, D. C., Chan, K. H., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interposition+of+a+pedicle+fat+flap+significantly+improves+specificity+of+reinnervation+and+motor+recovery+after+repair+of+transected+nerves+in+adjacency+in+rats.">Interposition of a pedicle fat flap significantly improves specificity of reinnervation and motor recovery after repair of transected nerves in adjacency in rats.</a> Plast Reconstr Surg. 107 (1), 116-123 (2001).</li><li>Calcagni, M., 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+novel+treatment+of+SVF-enriched+fat+grafting+for+painful+end-neuromas+of+superficial+radial+nerve.">The novel treatment of SVF-enriched fat grafting for painful end-neuromas of superficial radial nerve.</a> Microsurgery. , (2016).</li><li>Hawker, G. A., Mian, S., Kendzerska, T., French, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+adult+pain:+Visual+Analog+Scale+for+Pain+(VAS+Pain),+Numeric+Rating+Scale+for+Pain+(NRS+Pain),+McGill+Pain+Questionnaire+(MPQ),+Short-Form+McGill+Pain+Questionnaire+(SF-MPQ),+Chronic+Pain+Grade+Scale+(CPGS),+Short+Form-36+Bodily+Pain+Scale+(SF-36+BPS),+and+Measure+of+Intermittent+and+Constant+Osteoarthritis+Pain+(ICOAP).">Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP).</a> Arthritis Care Res (Hoboken). 63 Suppl. 63, S240-S252 (2011).</li><li>Stokvis, A., van der Avoort, D. J., van Neck, J. W., Hovius, S. E., Coert, 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=Surgical+management+of+neuroma+pain:+a+prospective+follow-up+study.">Surgical management of neuroma pain: a prospective follow-up study.</a> Pain. 151 (3), 862-869 (2010).</li><li>Kolle, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+autologous+fat+grafts+with+ex-vivo+expanded+adipose+tissue-derived+stem+cells+for+graft+survival:+a+randomised+placebo-controlled+trial.">Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial.</a> Lancet. 382 (9898), 1113-1120 (2013).</li><li>Zhu, M., 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=Adipocyte+regeneration+after+free+fat+transplantation:+promotion+by+stromal+vascular+fraction+cells.">Adipocyte regeneration after free fat transplantation: promotion by stromal vascular fraction cells.</a> Cell Transplant. 24 (1), 49-62 (2015).</li><li>Eto, H., 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=Characterization+of+human+adipose+tissue-resident+hematopoietic+cell+populations+reveals+a+novel+macrophage+subpopulation+with+CD34+expression+and+mesenchymal+multipotency.">Characterization of human adipose tissue-resident hematopoietic cell populations reveals a novel macrophage subpopulation with CD34 expression and mesenchymal multipotency.</a> Stem Cells Dev. 22 (6), 985-997 (2013).</li><li>Premaratne, G. U., 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=Stromal+vascular+fraction+transplantation+as+an+alternative+therapy+for+ischemic+heart+failure:+anti-inflammatory+role.">Stromal vascular fraction transplantation as an alternative therapy for ischemic heart failure: anti-inflammatory role.</a> J Cardiothorac Surg. 6, 43 (2011).</li><li>Paik, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+Fat+Grafting:+Part+V.+Cell-Assisted+Lipotransfer+to+Enhance+Fat+Graft+Retention+Is+Dose+Dependent.">Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent.</a> Plast Reconstr Surg. 136 (1), 67-75 (2015).</li></ol>

The purpose of the present study is to methodically illustrate the technical aspects of SVF-enriched fat grafting as a novel treatment for symptomatic end-neuromata.
Neuromata of the SBRN are associated with a surgical success rate of only 33%12. Vaienti et al. presented a series of favorable results in early follow-up using perineural fat grafting (PFG) without adding the SVF in 8 patients with painful neuromata of the upper limb<sup c...

The management of symptomatic end-neuromata of the SBRN remains challenging with a multitude of existing surgical treatments. Principally, the neuroma is excised and the remaining nerve stump is relocated into neighboring structures such as muscles1,2, bone3, or veins4. This is assumed to prevent the out-sprouting of the regenerating neural fascicles by mimicking the ambience of an end-organ. However, none of these techniques proved superior, as high rates of pain relapse and insufficient pain reduction were often observed.
Recently, fat grafting around the remaining nerve stump yielded promising results and was advocated as a new treatment option for painful end-neuromata of the upper limb5. Besides acting as a mechanical barrier to prevent continuing motion and tap pain, the beneficial aspect of fat grafting is rather attributed to the cellular level. Human adipose tissue contains multiple cell types, including preadipocytes, mast cells, pericytes, endothelial cells, vascular smooth muscle cells, fibroblasts, and adipose derived stem cells (ADSCs) along with hematopoietic stem cells6. This heterogeneous cell population is referred to as Adipose-Derived Regeneration Cells (ADRCs) or SVF, and can be obtained from the lipoaspirate by an isolation system (see the Table Of Materials) and applied enzyme reagent (see Table of Materials)7. The exact mechanism of action is still not fully understood; however, SVF has been associated with improvements in angiogenesis, immunomodulation, differentiation, and secretion of extracellular matrix8.
The concentrated cell population of the SVF is added to the fat graft with the purpose to reduce the resorption rate and to increase the regenerative potential of the fat graft, as recent studies revealed pain relapse after 12 months with fat grafting without added SVF5. Moreover, the biological action also provides beneficial effects on the nerve itself by reducing disorganized axonal sprouting of the stump9 and subsequent scar adherences which might result in nerve entrapment.
The first clinical study to describe SVF-enriched fat grafting as a novel surgical option for the treatment of symptomatic end-neuromata of SBRN has been recently published10. In general, the technique can be applied to any neuroma occurring after accidental or iatrogenic nerve injury. The aim of the present subsequent study is to methodically illustrate the technical aspects of this procedure.

<table border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cytori Celution System</td>
			<td style="width:71px;">Cytori</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">isolation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Celase</td>
			<td style="width:71px;">Cytori</td>
			<td style="width:119px;"></td>
			<td>enyzme reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">liposuction system</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">tumescent solution (500 ml Lactated Ringers, 20 ml of 1% lidocaine and 1 mg epinephrine)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14-gauge infiltrator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mm-collecting cannula (15, 26, 32 cm)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mL Tommey syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mosquito clamp</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toomey syringe stand</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">4 cannulas (fat injecting cannulas 1.5 mm, syringe NO.2, 90 mm)</td>
			<td style="width:71px;">Chalybs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">10 mL syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">connecting device for syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">#11- and #15-blade scalpel</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">stevens scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pins pincette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">bipolar electrocautery</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">suture material</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">adhesive dressing</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">binocular loupes magnifier glasses (2.5-3.5x)</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">Statistical Package for Social Sciences, version 22</td>
			<td>IBM</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Data were analyzed using statistical software and presented as means &#177; standard deviations. The Kolmogorov-Smirnov test was applied to verify the nonparametric distribution of the study population and confirmed a negative empirical distribution. Based on the nonparametric nature of the study population, the Wilcoxon&#39;s test for related samples was employed to compare preoperative and postoperative outcomes. Statistical significance was regarded at p-values less than 0.05<s...

Watch this Scientific Journal Video about Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata at JoVE.com

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

The overall goal of this surgical intervention is to reduce the pain of end-neuromas by neuroma excision and fat grafting around the remaining nerve stump. SVF-enriched fat grafting can prevent the pain recurrence in peripheral neuromas. Both the mechanical and biologic features of the graft may contribute to this effect.Specifically, painful neuromas of the superficial branch of radial nerve can be treated, but the technique can be extended to any neuroma throughout the body. This method can also provide insight into the biologic effects of the stromal vascular fraction. Furthermore, it can also be applied to scleroderma, osteoarthritis, and thermal burn injuries.The text protocol goes over diagnosis and surgical preparations. Here, we begin with the surgery performed on a human patient. To start, make several small stab incisions in the area of liposuction, using a number 11 scalpel.Each incision should be approximately three to five millimeters in diameter and be incised down to the subcutaneous layer. Next, inject the tumescent solution containing a dilute lidocaine and epinephrine via a 14 gauge infiltrator, three millimeters into the subcutaneous fat layer where the fat will be removed. Allow about 20 minutes for the tumescent solution to take effect before proceeding.To harvest adipose tissue, attach three millimeter collecting cannula to a 60 milliliter Toomey syringe. Operate the cannula with the dominant hand and control placement and course of the cannula with the non-dominant hand. To prepare the syringe, pull back the plunger to the 60 milliliter mark and secure it using a mosquito clamp to maintain a vacuum therein.Then, harvest at minimum 220 milliliters of adipose tissue. Use additional syringes, as needed, and collect from different locations to avoid taking blood. Keep the tissue-loaded syringes vertical in a dedicated stand to allow the fat to separate from the fluid.Once enough lipoaspirate has been harvested, suture the stab incisions with simple stitches and apply pressure, as required. Then, set aside one syringe of lipoaspirate and transfer the remaining bulk of it into a single-use, disposable receptacle for the isolation system. To operate the isolation system, follow the instructions on the display.The process is mostly software controlled and only requires user affirmation at the display prompts. The processing system moves the lipoaspirate into a collecting canister, weighs the sample, and automatically cleans it with ringers lactate solution to remove residual blood cells and wetting solution. If requested, add more fat, then, add the required volume of enzyme reagent for the digestion.Once digested, the lipids and SVF will be separated. Next, the SVF is automatically separated by centrifugation and washed with ringers lactate solution to reduce residual enzyme levels. After 90 minutes, when the processing is completed, five milliliters of clear SVF fluid is produced.To approach the neuroma, make a skin incision over the diagnosed site, which in this case, is three centimeters distal to the wrist. To identify the involved nerve, proximal to the neuroma, wear surgical binocular loupes and dissect the subcutaneous tissue. Once the full extent of the neuroma is exposed, use a straight, transneural scissor cut to excise the neuroma.Proper identification of the neuroma is critical. Dissect until the nerve is visible and to identify the neuroma, simply follow the nerve distally until the neuroma formation is found. Next, perform neurolysis on the remaining nerve stump until the end of the nerve stump is located in the middle of the principle incision.In this case, as no neuroma was present, neurolysis only was performed. Now, through epidermal and dermal cutis around the principle approach, make two to four small, one millimeter puncture incisions using a scalpel. Then, through the incisions, place four blunt cannulas paraneural to the nerve stump, with the cannula tips directed at the stump.To ensure that the cannulas remain in situ, secure the cannulas with adhesive dressing. Special care should be taken to direct the cannulas precisely at the nerve stump and secure them in place, thus guaranteeing that the nerve stump can be totally covered by the stromal vascular fraction-enriched fat graft. Next, suture the main incision tightly so the graft solution that is to be delivered into the tissue via the cannulas will remain in situ.Now, aspirate the five milliliters of processed SVF into one barrel of a ten milliliter communicating syringe. Then, transfer two milliliters of the sedimented lipid from the set aside Toomey syringe into the opposing barrel of the communicating syringe. Now, using the plungers, mix the SVF and lipids until the sedimented lipid is evenly distributed in the SVF.Next, divide the seven milliliters of SVF-enriched fat graft equally into two to four 10 milliliter syringes. Then, carefully secure the two to four syringes to the cannulas and distribute the graft mixture around the nerve stump via the blunt cannulas. Once the graft has been delivered, remove the cannulas carefully.Then, tape the small puncture incisions with Steri-Strips. Finally, the arm and wrist are bandaged for 10 days. The described procedure was implemented on the superficial branch of the radial nerve of five patients.Their mean age was 50 years. Given the small sample size, the measured pain reductions were not statistically significant, however, there were reductions in all pain modalities at two months and at 36 months after the operation, with no reports of pain relapse. After watching this video, you should have a good understanding of the novel technique of SVF-enriched fat grafting for the treatment of painful neuromas.Once mastered, this technique can be done in two hours, if it's performed properly. While attempting this procedure, it's important to remember that the neurolysis and/or neuroma removal needs to be done thorough in order to guarantee the maximum effect of SVF-enriched fat graft. Going forward, random trials with larger study population will be needed to demonstrate a statistically significant pain reduction.Given the early positive results, the technique has been put to use to treat other body regions affected by painful neuromas.

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Medicine

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 ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

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 ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

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.

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 ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

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 ...