Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

The overall goal of this experiment is to seed vascular implants with autologous blood derived endothelial Progenitor cells then observe the effect of this treatment on thrombosis in a large animal model. This is achieved by obtaining a peripheral blood sample from swine to isolate late outgrowth porcine endothelial progenitor cells. Next, an implantable device.

The titanium tube in our proof of principle study is seated with the autologous PCs. The device is then implanted into the pig's inferior vena cava. Results show there's protection against thrombosis based on patency of the EPC seated titanium tube as opposed to the control and occluded bare metal tube.

Duke surgery is committed to fostering groundbreaking, innovative research. The following presentation is one example of how we work across departments and disciplines to advance medicine. Dr.EK has been leading a research effort to develop a cell seating technology that will prevent thrombosis by lining the blood contacting surface of vascular devices with patient's own progenitor cells.

Since these cells have been derived from the same patients that will receive the implant, we avoid rejection by the immune system. Our success and the methods described herein are the results of a large team effort. The surgical implantation will be demonstrated by Sean Gage from the section of vascular surgery, Whitney Lane, a Duke medical student, and myself.

I would now like to introduce Alexander Janssen, a PhD student from the Department of Biomedical Engineering, who will take us through the cell seeding process In detail, I will demonstrate how to seed titanium tubes with blood derived endothelial progenitor cells just prior to implantation into a pig model. This method serves as proof of concept and the same basic principles can be applied to many other implantable devices such as nitinol stents and mechanical circulatory assist devices. EPCs are isolated by plating the mononuclear cell fraction of porcine blood and usually appear after about seven days.

They can be recognized by their typical cobblestone morphology. Expand EPCs to a total number of 9 million cells in culture. Rinse the prepared EPCs twice in serum free medium, taking care to limit their light exposure during and after labeling.

Then cover the cells with a fluorescent label, PKH 26 and record how much solution is applied. Incubate the cells at room temperature for four minutes. Stop the labeling reaction by adding an equal volume of porcine serum to the dye solution one minute later.

Dilute the combined solutions one-to-one with full growth medium. Then aspirate the liquid and rinse the cells three times with full growth medium followed by two washes and DPBS without calcium or magnesium chloride. Next, cover the cells in trypsin taking note of the volume used and incubate them at 37 degrees Celsius.

After three minutes of incubation, use microscopy to check that the cells have detached, and if so, add twice as much trypsin neutralization solution as was added. Trypsin, combine the cell solutions into a single tube and measure the cell density with a hemo cytometer. Lastly, pellet the cell suspension and resuspend the cells in at least 4.5 milliliters of serum free medium.

After preparing the pig for surgery, incise the midline with a number 15 scalpel blade from the second set of memory glands. Cran lead to the second to last set coddly using electrocautery. Carry the dissection down to the abdominal fascia.

Lift the peritoneum with mosquito forceps and carefully enter it with mets andal scissors, externalize the urinary bladder and place a three oh Vicryl purse string suture into the bladder wall. Make a stab incision at its center and insert a 16 French Foley catheter into the incision. Then administer intravenous fluids to titrate the urine output to at least one milliliter per kilogram per hour during the surgery.

Next, externalize the small enlarged bowel and place two bour surgical retractors to expose the posterior aspect of the peritoneal cavity and identify the IVC using sharp and blunt dissection. Carefully free the IVC from surrounding tissue. Then skeletonize the vessel from the right renal artery proximal to the bifurcation of the IVC distal exercise.

Extreme care during the IVC dissection as even a very small defect in the IVC may lead to rapid hemorrhage and exsanguination of the animal ligate. All of the side branches of the IVC segment take special care dissecting and ligating the two large posterior lumbar veins immediately proximal to the bifurcation of the IVC. There's often a large lumbar vein on the posterior side.

Dissect this vein free and control it with vessel loops. Now in parallel to the surgery, begin seeding the implant with EPCs prior to beginning this procedure. The titanium tube tissue seating scaffolds must be fully assembled and ready for use in a sterile field.

Open the gas sterilized titanium tube assembly in a five cc syringe. Then remove the plunger from the five cc syringe, but do not dispose of it A fixed allure cap to the syringe. Use a pipette to fill the syringe with the EPC suspension.

Then replace the plunger. Remove the cap and insert the lure end of the syringe into the open silicone tubing of the assembly until it is just snug. But no further remove any bubbles by slowly advancing the cell solution until it reaches the top of the headpiece.

Then close the assembly with a lure cap and tape it shut in a sterile glove. Insert this entire assembly into a machined syringe holder and place it in a 37 degree Celsius incubator. Use a gauge to ensure that the seating chambers level and allow the assembly to incubate with gentle rotation.

Continue the surgery just after ligating the IVC by administering 100 units of heparin per kilogram pig. Then immediately clamp the IVC at a 45 degree angle distally on the IVC and lumbar vein, and then proximally. Next, use a number 11 scalpel blade and pot scissors to make a four centimeter longitudinal vein otomy between the proximal and distal clamps.

Evacuate any blood from the IVC and flush its inner lumen with sterile DPBS. Now insert the EPC seated implant or a bare metal control into the IVC to prevent the cells from drying and to evacuate air. Fill the IVC with DPBS with calcium chloride and magnesium chloride.

Then close the vein otomy with a four oh proline running suture and remove one proximal clamp to de air the IVC through a small amount of back bleeding. After the bleed, remove the distal clamp to keep the implant in place. Apply a stay suture into the PVC tubing going through the vein wall.

Close the fascia with OPDS on a CT needle. Close the subcutaneous space with two o Vicryl running. Then staple the skin closed.

Administer up to 20 milliliters of 0.25%Marcaine subcutaneously along the incision site and cover the wound with gauze and tegaderm for pain. Give flu Nixon and oxymorphone subcutaneously as needed. Discontinue the anesthesia and when the pig awakens, return it to its cage over the next seven days.

Administer atri every 24 hours as an antibiotic prophylactic and be sure that the fentanyl patch applied prior to surgery stays adhered for a total of three days. After three days, the seated titanium tube implants were removed and the inside surfaces were examined. Under 550 nanometer excitation wavelength, a confluent layer of fluorescently labeled cells was found in another tube.

Section cells were additionally stained with a platelet endothelial cell adhesion marker to visualize cell borders. A wide variety of stains can be used in this procedure. By contrast, a bare titanium tube implant completely occludes in the prothrombotic low share environment of the inferior vena cava.

As illustrated in images A and C, the cell seated titanium tube is shown in image B and remained free of clot Following our say isolation procedure with peak blood. EPC appear after a average seven days in culture While performing the surgical procedure, it is very important to both carefully and gently skeletonize the IVC cause even the smallest tear in the IVC or its side branches can lead to exsanguination of the pig. Following this procedure, other blood contacting materials can be rapidly endothelial lies to prevent thrombosis.

Endothelial vascular stents, for example, with our EPCs mimics the native lining of the blood vessel wall and may prevent stent re-stenosis and thrombosis After its development. Our quick see technology will allow for lining intravascular devices with patients own blood derived endothelial progenitor cells within minutes in the operating room or catheterization laboratory. Therefore, prolonged exvivo culture.

Time on the device itself is avoided and the technology becomes practical for translation into clinical practice.