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

Osteoarthritis is one of the most common debilitating disorders among the elderly population. At present, there is no definite cure for the underlying causes of osteoarthritis. This clinical protocol describes an innovative procedure for a promising, minimally invasive, and alternative treatment of human knee osteoarthritis by regenerating cartilage like tissue.

Osteoarthritis is one of the most common debilitating disorders among the elderly population. At present, there is no definite cure for the underlining causes of osteoarthritis. This clinical protocol is an innovative procedure for a promising, minimally invasive, and alternative treatment of human knee osteoarthritis by regenerating cartilage-like tissue.

Bring the patient into an operating room with a biohazard Class A hood, and place him or her in a supine position. Clean the abdominal area of the patient with 5%betadine-povidine iodine and drape the patient using the sterile technique, exposing the clean area of the abdomen for liposuction. Approximately five centimeters infero-laterally from the umbilicus, anesthetize two sites, one on the left and other on the right side of the umbilicus of incision to be made.

Using two milliliter of 2%lidocaine without epinephrine with a 25-gauge 1.5 inch needle in a five milliliter syringe by injecting each site at the epidermal level. Anesthetize the site of incision to be made using five milliliter of tumescent solution and 10 milliliter syringe and the needle 25-gauge 1.5 inch. Make two incisions of 0.5 centimeters approximately five centimeters below the umbilicus laterally by pinching the skin to raise depth of the subcutaneous level.

Using a number 11 blade, help the raised skin to penetrate through to the subcutaneous level but not to penetrate through the abdominal wall. By using 20 centimeter 60-gauge cannula, anesthetize the subcutaneous level of the whole lower abdomen area which is to be liposuctioned with 700 to 800 milliliter of the tumescent solution. After finishing infiltration of the whole lower abdomen with tumescent solution, prepare a liposuction apparatus by connecting a 3.0 millimeter cannula connected to a 60 milliliter or 30 milliliter syringe for manual liposuction or specially designed 3.0 millimeter cannula connected to a centrifuge kit.

A closed system syringe developed by ourselves and approved by FDA CDRH for the purpose of keeping the sterility connected to a vacuum machine for vacuum assisted liposuction. Perform liposuction to obtain 100 to 110 gram of adipose tissue excluding tumescent solution. When performing liposuction, adipose tissue will be obtained along with tumescent solution but should be separated and removed.

To separate the tumescent solution first by gravity, the adipose tissue in the centrifuge kit may be transferred to a 60 milliliter syringe and place the syringe down. In example, the opening portion of the syringe is at the bottom. By waiting five to six minutes, the adipose tissue and tumescent solution will be separated.

Remove the fluid at the bottom of the syringe by pressing on the top of the syringe plunger. Perform the above steps nine to 11 until total of the 100 gram to 110 gram of adipose tissue lipid aspirate per knee has been accumulated. After separating the tumescent solution by gravity and accumulating 50 gram to 55 gram of lipid aspirate per each 60 milliliter centrifuge kit sterile closed system, place the two centrifuge kits into a centrifuge container bucket and spin at 1600 G for five minutes, condensing the lipid aspirate and separating fluid from the adipose tissue.

This process further condenses the lipid aspirate and may produce fatty oil in certain cases. Being careful not to shake, remove the safety cap and the plug at the bottom of the centrifuge kit. Remove the bottom fluid by slowly pushing down on top of the plunger of the centrifuge kit.

On separate 60 milliliter syringe, dissolve 10 milligrams of collagenase, five milligrams collagenase specific for connective tissue, and five milligrams of collagenase specific for adipose tissue with 50 milliliter of normal saline. Mix approximately 25 to 30 milliliter of condensed lipid aspirate with dissolved collagenase, five milligrams of collagenase specific for connective tissue, and five milligrams of collagenase specific for adipose tissue at a ratio of one to one, volume to volume, by connecting the 60 milliliter syringe to a centrifuge kit by using a specialized connector. Thoroughly mix to condense the prep aspirate and collagenase by pushing the content between the 60 milliliter syringe and the centrifuge kit by using a rod or a pusher.

Transfer the mixture of the lipid aspirate and the collagenase back to the 60 milliliter syringe. Connect each 60 milliliter syringe containing the mixture with the tissue homogenizer which contains blades. Connect an empty 60 milliliter syringe to the other end of the homogenizer.

Push the mixture to the other 60 milliliter syringe through the homogenizer for four to six times, resulting in cutting and mincing of the lipid aspirate. Transfer the homogenized lipid aspirate and the collagenous mixture back to the 60 milliliter centrifuge kit through a specialized connector. Place the centrifuge kits in a container to be placed in an incubator that has been preheated at 37 degrees Celsius.

Incubate the two centrifuge kits with the homogenized mixture at 37 degrees Celsius for 40 minutes while rotating at 45 rpm. After 40 minutes of incubation, remove the container from the incubator in sterile fashion. Then remove the centrifuge kits and place them in a centrifuge machine.

Centrifuge the mixtures at 800 G for five minutes to separate the adipose tissue-derived SVF. After the centrifuge, the supernatant, which includes collagenase and digested adipose tissue is removed from each centrifuge kit by removing the syringe cap on top of the plunger and placing a 30 milliliter syringe on the plunger opening through a syringe-lock connection. Slowly press down on the barrel part of the 30 milliliter syringe for the supernatant to fill into the 30 milliliter syringe.

Press down the 30 milliliter syringe barrel all the way to the last three to four milliliters of the bottom of centrifuge kit, leaving only the last three to four milliliters of adipose tissue-derived SVF. The supernatant is discard. Remove the 30 milliliter syringe from the top of the plunger and fill the syringe with 5%dextrose lactated ringer solution, also known as D5LR.

By attaching the 30 milliliter syringe filled with D5LR on the top of the plunger opening, fill the centrifuge kits containing three to four milliliter of adipose tissue-derived SVF with D5LR up to 55 milliliters. Then remove the 30 milliliter syringe, cap the plunger opening, and centrifuge the centrifuge kits again at 300 G for about four minutes. Repeat steps number 17 to number 21 for a total of four washings.

After the fourth centrifuge, in order to obtain the final SVF for injection, remove the safety cap and the plug at the bottom opening of the centrifuge kit without shaking or turning the centrifuge kit. Attach a 20 milliliter syringe to the centrifuge kit bottom opening by using a specially designed connector. Pull the plunger of the 20 milliliter syringe several times back and forth to shake up the cells that have settled on the bottom of the centrifuge kit.

Remove desired total volume of the SVF containing both ASTs and ECM along with other cells and tissue. Usually about three to four milliliter from each centrifuge kit for knee joint injections. We did 30 milliliters of autologous blood along with 2.5 milliliters of anticoagulant citrate dextrose solution.

Then the drawn blood was transferred to the 60 milliliter centrifuge barrel kits. It was then centrifuged at 730 gravity for five minutes. And the supernatant was removed and put into a new 60 milliliter centrifuge barrel kit.

The supernatant was centrifuged at 1350 gravity for four minutes, obtaining 4.4 milliliters of PRP. Clean the knee of the patient with 5%betadine and drape it in a sterile manner. Palpate the anterior of the knee for the joint space between tibial and femoral bones.

Anesthetize the injection site superficially with diluted lidocaine, one milliliter of 1%lidocaine diluted with four milliliters of normal saline, from skin to just outside the joint capsule. Then anesthetize the inside of the joint capsule with diluted ropivacaine, one milliliter 0.75%ropivacaine diluted with three milliliter of normal saline. Mix two milliliter of HA to the six to eight milliliter of SVF contained in a 20 milliliter syringe through a syringe to syringe connector.

By using a syringe to syringe connector, add 0.4 milliliters of calcium chloride to the three to four milliliters of autologous PRP that has been already prepared and being ready in a five milliliter syringe. Combine 3.5 to 4.5 milliliters PRP calcium chloride in a five milliliter syringe with eight to 10 milliliters of HA-SVF mixture in a 20 milliliter syringe through a syringe to syringe connector. Immediately inject the mixture, about 12 to 15 milliliter, slowly into the anterior tibial-femoral joint of the knee, using 38 millimeter 18-gauge needle with or without the ultrasound guidance.

After the injection, bandage the injection site with pressure by folding 4x4 cotton gauze four times and placing tapes over the folded 4x4 gauze. Instruct patient to remain still for 60 minutes to allow for cell attachment. Instruct patient to limit activities for minimal of one week after discharge from the clinic.

Return to clinic for three additional injections of PRP activated by calcium chloride over three weeks. Patient with stage three OA received the treatment as described in the protocol section. After the second week of the ASV-ECM mixture injection, the patient's pain and ROM improved.

By the 16th week, the patient's pain and ROM significantly improved more than 70%Post treatment MRI, taken after the 16th week, showed the increased thickness of cartilage-like tissue on the medial side of the knee. Another patient with stage three OA received the treatment as described in the protocol section. After the second week of the ASV-ECM mixture injection, the patient's pain and ROM improved.

By the 18th week, the pain and ROM significantly improved to 80%Repeated MRI taken after the 18th week showed an increase in the height of cartilage-like tissue on the anterior-medial side of the knee. The third patient with stage three OA received the treatment as described in the protocol section. After second week of ASV-ECM mixture injection, the patient's pain and ROM improved by approximately 50%By the 22nd week, the pain and ROM significantly improved over 80%Repeated MRI taken after the 22nd week showed an increase in the height of the cartilage-like tissue on the medial side of the knee.

Currently no curative therapy is available for the treatment of OA.However, percutaneous injection of stem cell therapy with ASCs and homogenized ECM along with HA and analogous PRP activated with calcium chloride may provide an alternative to current treatment therapy of treating knee OA.