This work has been founded by Odense University Hospital (11/31936), The Danish Centre for Regenerative Medicine (14/50427) and the Danish Cancer Society.

All methods described in the protocol were approved by the Danish National Ethics Committee (No. 37054), The Danish Health and Medicines Authority (EUDRA-CT number 2013-004220-11) and the Danish Data Protection Agency (2008-58-0035). The study was registered at ClinicalTrials.gov (NCT02240823). The study was performed in accordance with the Declaration of Helsinki monitored by the Good Clinical Practice (GCP) unit at Odense University Hospital. ADRC preparation was carried out in an authorized tissue establishment for the handling of human tissues and cells at Odense University Hospital (Danish Health and Medicines Authority, Authorization no. 29035).
1. Recruitment of patient/participants
<ol>
	<li>To participate in the trial recruit patients who fulfil the following criteria of inclusion.
	<ol>
		<li>Recruit patients that are over 18 years of age, sexually active before RP and not infected with the sexually transmitted diseases e.g., human immunodeficiency virus infection, syphilis, or hepatitis.</li>
		<li>Ensure that they suffer from erectile dysfunction after a RP which was performed due to prostate cancer. 
		NOTE: In this study, the patients were enrolled into the study 5-18 months after the RP and were included regardless of the surgery method: open/robotic assisted or nervesparing/non-nervesparing.</li>
		<li>Ensure that the value of prostate-specific antigen (PSA) had to be undetectable at the clinical follow up after the RP.</li>
		<li>Ensure that patients were sexually active before the RP, and still expressing a wish to remain sexually active after the RP. Ensure that pharmacological intervention with a phosphodiesterase type 5 inhibitor (PDE5 inhibitor) or a synthetic analog of prostaglandin E1 (PGE1) have been tried before participating and deemed insufficient.</li>
		<li>Furthermore, ensure that the patients have enough subcutaneous fat on the abdomen or thigh.</li>
	</ol>
	</li>
	<li>Exclude patients from the trial, if there were severe events under the anesthesia during the RP or if they were treated with anticoagulants.</li>
</ol>
2. Liposuction
NOTE: Liposuction is an operation that removes fat or lipocytes from the subcutaneous area. This part of the protocol is performed as a standard liposuction and the procedure is performed under sterile conditions in an operating theater. All instruments used under the procedure must be sterile and the surgeon must wear scrubs, sterile surgical gown, sterile gloves, surgical mask and hat.
<ol>
	<li>Anesthetize the patient.
	<ol>
		<li>Ensure that the patient is under general anesthesia during the procedure, and this part is performed by an anesthesiologist.</li>
		<li>Disinfect the skin of the abdomen and scrotum with chlorhexidine 0.5% (clorhexidine spirit, 96% ethanol medicated chlorhexidine digluconate 77% W/W = 83% v/v). Use a surgical marker to draw up the areas on the abdomen were the liposuction is going to be performed. The area will usually be the area between the symphysis and the umbilicus.</li>
	</ol>
	</li>
	<li>Make two 6 mm wide incisions in the skin of the abdomen with a scalpel (number 11). Place the incisions symmetrically and laterally from the updrawn area of liposuction.
	<ol>
		<li>Put a bit of lubricant such petroleum jelly on the incisions, to make sure that the infiltration cannula slides in and out of the incisions easily during the liposuction.</li>
	</ol>
	</li>
	<li>Introduce a size 14 G infiltration cannula through the incisions in the skin. Inject modified Kleins solution subcutaneously in the marked areas parallel to the skin surface. Inject so much of the solution that the targeted tissue becomes swollen and firm, or tumescent. 
	NOTE: The modified Kleins solution consists of a solution of 1,000 mL Ringers lactate and 1 mg adrenalin (epinephrine (1:1,000,000)). Do not ass local anesthesia to the solution, as it may have a negative impact on the ADRCs.
	<ol>
		<li>Inject a volume of modified Kleins solution that corresponds to the ratio 1:1 to adipose tissue.</li>
		<li>Wait for 10 min to maximize the effect of adrenalin and reduce the amount of blood in harvested adipose tissue.</li>
	</ol>
	</li>
	<li>Perform a standard liposuction using a jet infusion liposuction to harvest 200-300 mL of adipose tissue. Use blunt cannulas that are hollow, to which an infusion tube and a nozzle are integrated. Attach the cannula to a suction device for liposuction. 
	NOTE: A continuous fan-shaped water infusion loosens the fat tissue into fragments that can be easily be suctioned out through the opening in the cannula.
	<ol>
		<li>Introduce a 3 mm syringe with a blunt tip through the incisions on the abdomen</li>
	</ol>
	</li>
	<li>Collect the adipose tissue in a lipo-collector to preserve the lipoaspirate in a sterile environment. The lipoaspirate will start to separate from the water phase.</li>
	<li>Use 50 mL sterile syringes to suck up the lipoaspirate from the lipo-collector.
	<ol>
		<li>Minimize the volume of water sucked up in the syringes and screw on a plug on the tip of the syringes to keep the adipose tissue sterile.</li>
	</ol>
	</li>
	<li>Place syringes with tip downwards in a sterile plastic bag to start the separation of lipoaspirate from the water phase. Place bag in sterile container still with the tips facing downwards to continue separation.
	<ol>
		<li>Cover the container with sterile drape to secure the sterile environment during transportation. 
		NOTE: According to the user manual of the machine used for the isolation, the lipoaspirate can be stored for up to a maximum of 4 h before isolation of the ADRCs however, we always process it immediately.</li>
	</ol>
	</li>
	<li>Close the skin incisions with the surgeons preferred suture material. Place a compression garment, an abdominal binder, around the abdomen to reduce the postoperative edema.</li>
	<li>Make a penile block by injecting 20 mL bupivacaine 5 mg/mL containing 5 &#181;g of adrenaline.
	<ol>
		<li>Use a 20 mL syringe and apply with a 23 G needle, 1 &#188; inches long.</li>
		<li>Apply 5 mL of bupivacaine in each quadrant in the subcutis.</li>
		<li>Inject the bupivacaine at two sites, one placed ventrally the other dorsally. Introduce the needle in the whole of its length in the subcutaneous tissue pointing the needle laterally to the right and inject the anesthesia as the needle is retracted. Repeat the injection pointing the needle to the left. 
		NOTE: The patient can now be awakened from the general anesthesia by the anesthesiologist.</li>
	</ol>
	</li>
</ol>
3. Isolation of ADRCs
NOTE: The isolation process of ADRCs is performed as described in detail in the user manual following the device (see Table of Materials). It is important that the procedure is performed under sterile conditions to ensure that the lipoaspirate is not exposed to any contaminants during the purification of the ADRCs. The time taken for the isolation of ADRCs depends of the volume of lipoaspirate, but the whole procedure will approximately take 2.5 h using the semi-automatic device as described here.
<ol>
	<li>Place all consumables and enzymes aseptically on a table covered by a sterile disposable surgical towel. 
	NOTE: A procedure kit contains all necessary consumables and enzymes needed for the isolation of ADRCs from one patient. Besides this, three 37-39 &#730;C, one-liter infusion bags of Lactated Ringers must be available along with sterile clothing, gloves and towels and ethanol for disinfection. Note down all lot numbers of consumables and enzymes and also the temperature of the Lactated Ringers, when connected to the system.
	<ol>
		<li>Wipe down the device with ethanol and following the manufacturer&#8217;s instructions</li>
		<li>Load the consumable set onto the device and connect a bag of Lactated Ringers to the system.</li>
		<li>Perform the series of semi-automatic tests (system check and leak test) before adding the lipoaspirate.</li>
	</ol>
	</li>
	<li>Let the lipoaspirate stand in 50 mL tubes while the steps in 3.1 are performed. This will allow the fat to separate from the liquid phase. Note down the total amount (in mL) of fat tissue and use this to ensure that the amount of loaded fat will be in the range of the device capacity.</li>
	<li>Load the tissue when prompted to by the device. The machine will now drain off excess fluid and weigh the amount of fat tissue loaded before it is washed with Lactated Ringers.
	<ol>
		<li>When the tissue has been washed and drained again, connect a new infusion bag with 37-39 &#730;C Lactated Ringers when requested by the machine.</li>
		<li>Reconstitute the commercially available enzyme containing collagenase and protease blend in 5 mL Lactated Ringers (one vial of enzyme is enough for fat amounts up to 270 mL).</li>
		<li>Ensure that the device displays the amount of enzyme (based on the tissue weight) to inject into the canister with the fat tissue. Once injected, the enzymatic digestion that is carried out under agitation will last around 20 min.</li>
	</ol>
	</li>
	<li>After tissue digestion, let the agitator stop and check that the content is separated into two phases: an upper lipid-containing, yellow phase and a lower pink layer that contains the ADRCs. Let the latter layer drained into the cell processing chamber (that goes into the device&#160;built-in centrifuge) of the consumable set, while leaving the lipid layer behind.</li>
	<li>Let ADRCs concentrate in the cell processing chamber during multiple rounds of centrifugation. Now add 10 mL of Intravase (contains DNase, that helps to avoid clumping in the final ADRC suspension) reconstituted in Lactated Ringers. The enzymatic reaction lasts for 10 min after which the ADRCs are washed. 
	NOTE: This step is fully automatic, and the machine will inform when the process is done.</li>
	<li>When the isolation of ADRCs is completed, resuspend the cells in 5 mL of Ringer Lactate. Aspirate the solution into a 5 mL syringe.</li>
	<li>Mount a 3-way stopcock female Luer-lock on the 5 mL syringe containing the ADRCs, and aspirate 4 mL of ADRCs into another 5 mL sterile syringe.</li>
	<li>Transfer the last 1 mL into a 1 mL syringe. Use this 1 mL for ADRC characterization e.g., cell count, cell viability, analysis of surface markers by flow cytometry and ADRC differentiation ability.</li>
	<li>Put on a 25 G needle on the 5 mL syringe containing the 4 mL of ADRC&#8217;s before it is packed in a sterile drape. Use the ADRCs for injection into the recipient. In this case, the solution of ADRCs will on average contain 8.4-32.7 million cells.</li>
</ol>
4. Implantation of the ADRCs into corpora cavernosum
NOTE: This is a sterile procedure. All instruments must be sterile, and the person injecting the solution containing the stem cells into the corpora cavernosum must wear sterile gloves. The patient is awake during this procedure and will receive his own stem cells. The ADRCs are injected without counting the cells prior to injection.
<ol>
	<li>Keep the solution of ADRCs homogeneous by tilting the syringe gently until it is injected into the corpora cavernosum.</li>
	<li>Place an aperture drape over the penis. Place a tourniquet at the root of the penis by using a silicone vessel loop. Tighten the loop and secure it with forceps curved pean. Make the tourniquet tight enough to stop the blood flow from the penis.</li>
	<li>Use two antiseptic swaps to disinfect the penile skin at injection site (lateral site of corpora cavernosum). Inject 1 mL of the solution containing ADRCs, in a direct angle into the corpora cavernosum on the right side at two different places, repeat this step afterwards in the left corpora cavernosum. Total volume of injected ADRCs is 4 mL.</li>
	<li>Wait for 30 min and then remove the tourniquet.</li>
	<li>Discharge the patient after 2 h of observation after the injection of ADRCs, to secure that the patient is well recovered following the anesthesia.</li>
</ol>
5. Postoperative care
<ol>
	<li>Recommend the patient to not do any physical performance that can raise the blood pressure within the first week, to prevent development of hematomas.</li>
	<li>Recommend the patient to wear the abdominal binder for 24 h the first 14 days after the surgery and hereafter for 14 days during the daytime.</li>
	<li>Treat postsurgical pain with oral acetaminophen (e.g., paracetamol; 1,000 mg, 4 times a day) and oral anti-inflammatory (e.g., ibuprofen; 400 mg 3 times a day).</li>
</ol>

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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=A+12-month+Follow-up+after+a+Single+Intracavernous+Injection+of+Autologous+Adipose-derived+Regenerative+Cells+in+Patients+with+Erectile+Dysfunction+following+Radical+Prostatectomy:+An+Open-label+Phase+1+Clinical+Trial.">A 12-month Follow-up after a Single Intracavernous Injection of Autologous Adipose-derived Regenerative Cells in Patients with Erectile Dysfunction following Radical Prostatectomy: An Open-label Phase 1 Clinical Trial.</a> Urology. 121, 203 (2018).</li><li>Fedder, 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=In+vitro+exposure+of+human+fibroblasts+to+local+anesthetics+impairs+cell+growth.">In vitro exposure of human fibroblasts to local anesthetics impairs cell growth.</a> Clinical &amp; Experimental Immunology. 162 (2), 280-288 (2010).</li><li>D'Andrea, 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=Large-scale+production+of+human+adipose+tissue+from+stem+cells:+a+new+tool+for+regenerative+medicine+ad+tissue+banking.">Large-scale production of human adipose tissue from stem cells: a new tool for regenerative medicine ad tissue banking.</a> Tissue Engineering Part C: Methods. 14 (3), 233-242 (2008).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+therapeutic+effects+of+human+and+mouse+adipose-derived+stem+cells+in+a+murine+model+of+lipopolysaccharide-induced+acute+lung+injury.">Comparison of the therapeutic effects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury.</a> Stem Cell Research and Therapy. 4 (1), 13 (2013).</li><li>Yusuke, 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=Transplantation+of+freshly+isolated+adipose+tissue-derived+regenerative+cells+enhances+angiogenesis+in+a+murine+model+of+hind+limb+ischemia.">Transplantation of freshly isolated adipose tissue-derived regenerative cells enhances angiogenesis in a murine model of hind limb ischemia.</a> Biomedical Research. 34 (1), 23-29 (2013).</li></ol>

The author S&#248;ren P. Sheikh is CEO at Blue Cell Therapeutics, Copenhagen, Denmark.

The presented procedure for isolating ADRCs is not limited to be used only for ED therapy, but can be used in multiple other forms of treatment and experiments. Our trial showed that autologous, freshly isolated ADRCs are safe to use, and the treatment is well tolerable in a 12 month follow up.
Before the procedure is used there are some considerations to be made. A disadvantage of this procedure is that the patient must be under general anesthesia during the liposuction. Liposuction is possible to perform under local anesthesia, but a previous trial has shown that a combination of local anesthetics and adrenaline may have a negative impact on cell growth of fibroblasts20. The risk of being under general anesthesia is generally low, but a negative outcome is still seen. This risk must be taken in mind when patients are selected for the treatment. Liposuction is a surgical procedure and will, therefore, always carry at risk of complications. As with all other surgical procedures, there is a risk of post-operative bleeding resulting in formation of hematomas and a risk of infection. Some of our patients did also report a transient reduced sensitivity of the abdominal skin. An immediate complication to liposuction is bleeding. It is known that patients risk getting systemic complications when large amounts of tissue is removed. The liposuction in this procedure was relatively small and therefore not considered a risk factor.
Complications to the injection of cells is reported as transient redness, tenderness, and hematomas.
The device used for the isolation of the ADRCs, was chosen because the system is CE approved. In Denmark, it is mandatory to use CE approved equipment because the authorities categorize treatment with stem cells as a drug testing trial, when the ADRC is used for injection in humans.
The device does have advantages such as the whole procedure being standardized and performed in a closed sterile environment without requiring a highly classified laboratory. The risk of contamination is, therefore, reduced. This ensures that the procedure is uniform and reproducible, and that the quality of the end-product is always the same each time (however, it also depends on the quality of the input tissue). Performing the isolation on the device is easy and does not require specially educated operators.
One limitation of the device is that the minimum input into the machine is 100 g drained lipoaspirate. As the liposuction is completely finished before the cell processing starts (and in our case, at a different location, not in the OR) in our experience, this will require the amount of lipoaspirate to be at least 125 mL using the rough measure of the 50 mL syringes. Otherwise there is a risk that there will not be enough material for the machine to proceed with. Also, using the upper end limit (max input is 425 mL) would result in a very long isolation procedure.
Adult stem cells from both adipose tissue and bone marrow seem to have capacity for self-renewal and differentiation like embryonic stem cells. One of the advantages of ADRCs over haemopoietic stem cells is a 100-500 times higher yield per tissue volume as compared to bone marrow21,22, and, therefore, ADRCs do not need to be cultured. Furthermore, haemopoietic stem cells are more difficult and more painful to harvest from the patient than adipose tissue. In many situations, adipose tissue is just a waste product after surgery. It is possible to culture stem cells to give a higher yield suitable for angiogenesis, however freshly isolated ADRC&#8217;s may have higher angiogenic potential than cultured23.

Stem cells have the ability to differentiate into different cell types, and they secrete paracrine factors that are thought to promote the healing process in damaged tissue1,2,3,4. They are, therefore, attractive within the field of regenerative medicine, because they can represent a possible curative treatment.
Radical prostatectomy (RP) is the golden curative treatment for patients with low/intermediate risk localized prostate cancer and a life expectancy &#62; 10 years. The aim of the surgery is eradication of the cancer, but it has several side effects. The prevalence of post-prostatectomy incontinence ranges from 2-60% and erectile dysfunction (ED) is experienced by 20-90% of the patients5. Nerve-sparing technique is an option in some patients (Gleason score &#60; 7, low risk of extracapsular disease)5. This technique spares the nerves that are responsible for erection, but even though this is possible, many patients still report ED postoperatively.
Treatment options for penile rehabilitation after RP consist primarily of treatment with PDE-5 inhibitors, injection or instillation therapy and vacuum pumps. Medicaments used for penile rehabilitation differ pharmacologically, but their mechanism of action includes relaxation of the smooth muscle cells in the corpus cavernosum. However, many patients experience treatment failure and never achieve an effect of the medicine that enables intercourse6.
The ED that occurs after RP is thought to be due to structurally irreversible changes7. These changes occur in the cavernous tissue and include apoptosis of the smooth muscle- and endothelial cells and fibrosis. The veno-occlusive mechanism that is responsible for a central part of the erection, is impaired by the changes, resulting in poorer filling and hardness of the penis7.
Many of the patients report that the ED they experience has a negative impact on their quality of life8. They are not ready to give up their sexual activity after surgery and, therefore, a curative therapy for ED is attractive, when the other available treatments for penile rehabilitation fail.
In previous trials, including animal and small phase 1 trials on humans, stem cells have shown promising results as an alternative treatment for ED2,9,10,11,12. Results show that it is safe to use ADRCs, and that the erectile function is significantly improved after a single injection into the corpora cavernosum2,9,10,11,12. Adipose-derived regenerative cells (ADRCs) are thought to support the tissue regeneration by paracrine mechanisms through liberation of multiple hormones, neurotrophic- and other growth factors, cytokines and possibly, micro-RNAs13. In addition, ADRCs are able to differentiate into several mature cell-types including endothelial and vascular muscle cells, cartilage cells, osteocytes and neurons14,15. These properties make stem cells interesting for developing a permanent new treatment for ED.
Stem cells are divided into several groups, basically those derived from the early embryo (embryonic stem cells) and those from adult tissue (adult stem cells). Adult stem cells include mesenchymal stem cells (MSC) that are multipotent and can be found in the bone marrow, adipose tissue, umbilical cord blood, placenta and dental pulp17.
Stem cells from the adipose tissue are easy to get access to, unlike stem cells derived from bone marrow. Harvesting stem cells from bone marrow is a risky and painful procedure compared to liposuction. The number of cells possible to harvest from bone marrow will be limited, while only the patient&#8217;s depots of adipose tissue sets the limit for the number of cells that can be harvested. It is, therefore, possible to isolate a large amount of stem cells from the adipose tissue without a subsequent need to culture the cells to obtain a satisfactory amount. The adipose-derived regenerative cells, also often referred to as the stromal vascular fraction (SFV), is composed of many cell types including MSCs, endothelial cells, pericytes, immune cells and progenitors hereof18. These may all play a role in the regenerative process.
The aim of the present study is to investigate the effect of stem cells on ED after RP by using 4 mL of autologous ADRCs isolated from freshly harvested adipose tissue after injection into the corpora cavernosum.

<table>
	<tbody>
		<tr>
			<td>Liposuction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Abdominal Binder</td>
			<td>Dale</td>
			<td></td>
			<td>Size depent of the patient</td>
		</tr>
		<tr>
			<td>Adhessive OP-towel</td>
			<td>M&#246;lnlycke Health Care</td>
			<td>906677</td>
			<td>90x75 cm</td>
		</tr>
		<tr>
			<td>Adrenalin 1 mg/ml</td>
			<td>Amgros I/s</td>
			<td>74 44 23</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Basic OP supplies</td>
			<td>M&#246;lnlycke Health Care</td>
			<td>97010873-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel blade 11</td>
			<td>Swann-Morton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhixidine Ethanol</td>
			<td>Faarborg Pharma</td>
			<td>5% colored</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable set Lipocollector 3</td>
			<td>Human Med</td>
			<td>670200</td>
			<td></td>
		</tr>
		<tr>
			<td>Extension hoses</td>
			<td>Extrudan</td>
			<td></td>
			<td>5.8/8.3 mm, Ch 25, 3,5 M long</td>
		</tr>
		<tr>
			<td>Gaze Rags</td>
			<td>Barrier</td>
			<td>175201</td>
			<td>30x45 cm</td>
		</tr>
		<tr>
			<td>Jelonet</td>
			<td>Smith and Nephew Medical Ltd</td>
			<td>90509225</td>
			<td>10 x 10 cm</td>
		</tr>
		<tr>
			<td>Marcaine 5 mg/ml + Adrenaline 5 micrigram</td>
			<td>Astra Zeneca</td>
			<td></td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Mesorb Bandage</td>
			<td>M&#246;lnlycke Health Care</td>
			<td>677001</td>
			<td>10x13 cm</td>
		</tr>
		<tr>
			<td>Microlance</td>
			<td>Becton Dickinson</td>
			<td>304622</td>
			<td>18 G + 23 G</td>
		</tr>
		<tr>
			<td>Monocryl suture</td>
			<td>Ethicon</td>
			<td></td>
			<td>04:00</td>
		</tr>
		<tr>
			<td>Ringer-lactat</td>
			<td>Fresinus Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Gammex</td>
			<td>330052065</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Gown</td>
			<td>M&#246;lnlycke Health Care</td>
			<td>690103-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Marker</td>
			<td>Richard-Allan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Mask</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Codan Medical</td>
			<td>6,28,402</td>
			<td>50/60 ml cath tip</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Becton Dickinson</td>
			<td>700016181</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>WAL- Applikator for cannulae 25/30 cm</td>
			<td>Human Med</td>
			<td>REF 500001</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation of ADRC&#39;s</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>Becton Dickinson</td>
			<td>REF 303172</td>
			<td></td>
		</tr>
		<tr>
			<td>3 way stopcock</td>
			<td>One Med</td>
			<td>REF 10554-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive OP-towel</td>
			<td>M&#246;lnlycke Health Care AB</td>
			<td>REF 906677</td>
			<td>90x75 cm</td>
		</tr>
		<tr>
			<td>Cytori Celution 800IV device</td>
			<td>Cytori</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desinfection swaps</td>
			<td>Mediq Danmark</td>
			<td>3340010</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>25G (0,5*25 mm)</td>
		</tr>
		<tr>
			<td>Nitril Gloves</td>
			<td>Abena</td>
			<td></td>
			<td>Powder free</td>
		</tr>
		<tr>
			<td>OR drape Sheet, 2 layers</td>
			<td>Lohmann &#38; Rauscher</td>
			<td>REF 33005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringers Lactate</td>
			<td>Fresinus Kabi</td>
			<td>06756 (DK)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplemental kit for Cytori Celution 800IV devise</td>
			<td>Cytori</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Termometer</td>
			<td>Thomas Scientific</td>
			<td></td>
			<td>traceable</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection of ADRC&#39;s</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aperture drape</td>
			<td>One Med</td>
			<td>REF1565-01</td>
			<td>75*90 cm</td>
		</tr>
		<tr>
			<td>Desinfection swap</td>
			<td>Mediq Danmark</td>
			<td>3340010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pean</td>
			<td>Leibinger</td>
			<td>32-01257</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Vessel Loop</td>
			<td>Purple Surgical</td>
			<td>REF PS3203</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Sempermed</td>
			<td></td>
			<td>Or Use your favorite</td>
		</tr>
	</tbody>
</table>

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.

The presented procedure has been used for an open-label Phase 1 clinical trial including 21 patients19. The primary endpoint of the trial was safety of the use of ADRC&#8217;s in humans and secondary endpoint was the effect of ADRC&#8217;s on the erectile function.
Twenty-one men were included in the trial with a mean age of 60 years (range 46-69) and a normal erection and active sex life before the RP due to prostate cancer. They all suffered from ED after the RP, with no signs of recovery from medicine available for penile rehabilitation. Six men suffered from incontinence as a side effect to the RP. All men received a single intracavernous injection of ADRC&#8217;s isolated with the presented method.
All 21 men were followed up with 4 visits in the outpatient clinic at 1,3,6 and 12 months after the injection. The sexual function was evaluated with validated questionnaires &#8211; the International Index of Erectile Function-5 (IIEF-5) and Erection Hardness Score (EHS) (Attached as appendices).
No serious events occurred during the in the time of observation. Eight men reported transient redness and swelling at the injection sites, three reported reaction in the penile area. Eight experienced reversible minor events related to the liposuction were reported such as light abdominal discomfort and minor abdominal hematomas. No patients reported discomfort at the 1 month follow up. Eight out of fifteen (53%) in the continent group reported the erectile function were sufficient for intercourse at 12 months. The group of 6 incontinent men did not show any improvement of erectile function.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Continent</td>
			<td>Incontinent</td>
		</tr>
		<tr>
			<td>Included patients</td>
			<td>15</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Significant effect</td>
			<td>8</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: The table shows the differences in significant effect of the treatment between continent and incontinent patients.

Watch this Scientific Journal Video about Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy at JoVE.com

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

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 ...

isolation-adipose-derived-regenerative-cells-for-treatment-erectile

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3.2K Views.  Odense University Hospital. 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).

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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 ...

Intense Pulsed Light for the Treatment of Dry Eye Owing to Meibomian Gland Dysfunction

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the so-called &#34;evaporative&#34; subtype, that is mainly caused by meibomian gland dysfunction (MGD). Intense pulsed light (IPL) devices employ high intensity pulses of polychromatic lights with a broad range of wavelength (515-1200 nm). IPL treatment has been utilized for years in the field of dermatology, and then its use was applied to ophthalmology for the treatment of MGD. Recently, a new device employing IPL was specifically designed for the periocular application. This procedure determines the thermal selective coagulation and ablation of superficial blood vessels and telangiectasias of the eyelids skin, reducing the release of inflammatory mediators and tear cytokines levels, and improving meibomian glands outflow. IPL treatment is noninvasive and easy to perform, lasts for only a few minutes and can be conducted in an office setting. In the present study, 19 patients underwent 3 sessions of IPL treatment. After treatment, both mean noninvasive break-up time and lipid layer thickness grade significantly increased, as a result of an improvement of tear film stability and quality, respectively. Conversely, no statistically significant changes were found for meibomian gland loss and tear osmolarity. Furthermore, the vast majority of the treated patients (17/19; 89.5% of the total) perceived an improvement of their ocular discomfort symptoms after IPL treatment. Although IPL treatment provides an improvement of both ocular surface parameters and ocular discomfort symptoms after one cycle of three sessions, regular repeated treatments are usually required to maintain the persistence over the time of its beneficial effects.

Dry eye disease is an increasingly common condition, which strongly impair patients&#39; life quality. Recently, a new device employing intense pulsed light, specifically designed for the periocular area, has been shown to improve tear film stability and ocular discomfort symptoms in dry eye disease owing to meibomian gland dysfunction.

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

Retzius-Sparing Robot-Assisted Radical Prostatectomy

The technique of Retzius-sparing robot-assisted radical prostatectomy (RS-RARP) and initial experience with it at a single center are provided. The technique is described step-by-step and further illustrated by a video to enhance reproducibility. Early oncological and functional results were evaluated. In total, 77 patients were included with a median follow-up of 11 months (range: 3-21 months). Fifty-one percent of patients had local high-risk or locally advanced prostate cancer. There were no intra-operative complications, and all high-grade complications (2.6%) were related to pelvic lymph node dissection performed concomitant with RS-RARP. Median operation time was 160 min (range: 122-265 min) and median hospital stay was 3 (range: 3-8) days. A positive surgical margin was reported in 42.9%. One-year biochemical recurrence-free survival was 90.1%. After 6 months, all patients were socially continent and after 1 year, 94.3% were fully continent. Of sexually active patients who underwent at least unilateral nerve-sparing, 43.3% were able to have sexual intercourse. This series underlines the surgical safety of performing RS-RARP by a standardized technique and confirms the beneficial effect on the early return of continence. The patient needs to be informed about the risk of a positive surgical margin.

Robot-assisted Retzius-sparing radical prostatectomy is a technique that enables to preserve urinary continence or facilitates recovery of urinary continence in the majority of patients. Patients must be informed about the risk of a positive surgical margin.

The technique of Retzius-sparing robot-assisted radical prostatectomy (RS-RARP) and initial experience with it at a single center are provided. The ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...