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

This isolation technique can be used to explore whether adipose stem cells can help patients regenerate erectile function following radical prostatectomy. This method is not limited for a specific disease. It could be used within all areas of adipose stem cell research.

The isolated stem cells are used to treat erectile dysfunction, but the isolation technique is a general procedure and the stem cells can be useful in other areas within stem cell research. Begin by disinfecting the skin of the abdomen and scrotum with 0.5%chlorhexidine. Use a surgical marker to draw up the areas on the abdomen where the liposuction is going to be performed, usually the area between the synthesis and the umbilicus.

Make two six millimeter wide incisions in the skin of the abdomen with a number 11 scalpel. Place the incisions symmetrically and laterally from the up drawn area of liposuction. Place a bit of lubricant on the incisions to make sure that the infiltration cannula slides in and out of the incisions easily during the liposuction.

Then introduce a size 14 gauge infiltration cannula through the incisions in the skin. Inject modified Klein solution subcutaneously in the marked areas parallel to the skin surface until the targeted tissue becomes swollen and firm or tumescent. Inject a volume of modified Klein solution at a one-to-one ratio to the adipose tissue.

Wait for 10 minutes to maximize the effects of adrenaline, and reduce the amount of blood in the harvested adipose tissue. Perform a standard jet infusion liposuction to harvest 200 to 300 millimeters of adipose tissue. Use blunt cannulas that are hollow with an integrated infusion tube and nozzle.

Attach the cannula to a suction device for liposuction. Introduce a three millimeter syringe with a blunt tip through the incisions on the abdomen and collect the adipose tissue in the Lipo Collector to preserve the lipoaspirate in a sterile environment. The lipoaspirate will start to separate from the water phase.

Use 50 milliliter sterile syringes to suck up the lipoaspirate from the LipoCollector, minimizing the volume of water sucked up in the syringes. Screw a plug on the tip of the syringes to keep the adipose tissue sterile. Place syringes with tip downward in a sterile plastic bag to start the separation of lipoaspirate from the water phase.

Then place the bag in a sterile container. Cover the container with a sterile drape to secure the sterile environment during transportation. Close the skin incisions with the surgeon's preferred suture material.

Place a compression garment around the abdomen to reduce postoperative edema. To make a penile block, inject the bupivacaine at two sites, one placed ventrally and the other dorsally. Introduce the whole needle in the subcutaneous tissue, pointing it laterally to the right, and inject the anesthesia as the needle is retracted.

Repeat the injection, pointing the needle to the left. Place all consumables and enzymes aseptically on the table covered by a sterile disposable surgical towel. Wipe down the device with ethanol and load the consumable set.

Then connect a bag of lactated ringers to the system. Perform the series of semiautomatic tests before adding the lipoaspirate. While the device is being prepared, let the lipoaspirate stand in a 50 milliliter tube to allow the fat to separate from the liquid phase.

Note the total amount of fat tissue and use this to ensure that the total amount of loaded fat will be in the range of the device capacity. Load the tissue when prompted to by the device. The machine will drain off excess fluid.

Weigh the amount of fat tissue and wash it with lactated ringers. When the tissue has been washed and drained, connect a new infusion bag with 37 to 39 degrees Celsius lactated ringers. Reconstitute the commercially available enzyme containing collagenase and protease blend in five milliliters lactated ringers.

Ensure the device displays the amount of enzyme to inject into the canister with the fat tissue. Once injected the enzymatic digestion will last around 20 minutes. After tissue digestion, check that the content is separated into an upper lipid containing yellow phase and a lower pink layer that contains the adipose derived regenerative cells, or ADRCs.

Let the pink layer drain into the cell processing chamber of the consumable set, leaving the lipid layer behind. Allow the ADRCs to concentrate in the cell processing chamber during multiple rounds of centrifugation. Then add 10 milliliters of intrabase reconstituted in the lactated ringers.

The enzymatic reaction lasts for 10 minutes and the ADRCs are washed afterwards. When the isolation of ADRCs has completed, re-suspend the cells in five milliliters of Ringer's lactate and aspirate the solution into a five milliliter syringe. Mount a three-way stopcock female Luer lock on the syringe and aspirate four milliliters of ADRCs into another five milliliter sterile syringe.

Transfer the last one milliliter of ADRCs into a one milliliter syringe. Use this aliquot for ADRC characterization analyses, such as cell count, cell viability, analysis of surface markers by flow cytometry, and ADRC differentiation ability. Put a 25 gauge needle on the five milliliter syringe containing the four milliliters of ADRCs.

Then pack it in a sterile drape. This solution of ADRCs will be injected into the recipient and should on average contain 8.4 to 32.7 million cells. Make sure that the solution of ADRCs remains homogenous by tilting the syringe gently until it is injected into the corpora cavernosum.

Place an aperture drape over the penis. And a tourniquet at the root of the penis with a silicone vessel loop. Tighten the loop and secure it with forceps, making the tourniquet tight enough to stop the blood flow from the penis.

Use two antiseptic swabs to disinfect the penile skin at the injection site. Then inject one milliliter of the ADRC solution in a direct angle into the corpora cavernosum, on the right side at two different places. Repeat this step in the left corpora cavernosum, injecting a total volume of four milliliters.

This method was tested in an open label phase one clinical trial on 21 men with a mean age of 60 years and a normal erection and active sex life before a radical prostatectomy. Six of the men also suffered from incontinence. All men received a single inter cavernous injection of isolated ADRCs and were followed with four visits in the outpatient clinic at 1, 3, 6 and 12 months after the injection.

No serious events occurred during the time of observation. Eight men reported transient redness and swelling at the injection sites, and three reported reaction in the penile area. Eight men experienced reversible minor events related to the liposuction, such as light abdominal discomfort and minor abdominal hematomas.

No patients reported discomfort at the one month follow up. Their sexual function was evaluated with the International Index of Erectile Function Five and the Erection Hardness Score. Eight out of 15 men in the continent group reported that their erectile function was sufficient for intercourse at 12 months.

The group of six incontinent men did not show any improvement of erectile function When attempting this protocol, keep in mind that the enzymes that are used during the procedure of isolation must be taken out of the freezer or fridge two hours before the procedure is started. The results from this technique have paved the way for an RCT where the effects of adipose stem cells are explored on a bigger scale.