This technology is available from Temarex Corporation.
ENDOTHELIAL CELL SEEDING
Title: APPARATUS AND METHOD FOR ELECTROSTATIC ENDOTHELIAL CELL SEEDING AND DNA TRANFECTION
Inventor(s): G. Bowlin, S. Rittgers
Disclosure 268-CIP U.S. Patent 5,723,324
An apparatus and method for electrostatic seeding ofendothelial cells (EC) and DNA transfection to the lumenwall of small diameter (<6 mm) synthetic vascular protheses has been developed. One objective is to raise the level ofclinical acceptance of small diameter vascular protheses by increasing the reliability and patency of the grafts. By concurrently transfecting DNA plasmids and seeding endothelial cells to a graft, the long term patency of the graft is extended.
As those skilled in the art will apreciate, specific DNA plasmids may be selected to incorporate anti-coagulant properties into the lumen wall of the graft to ensure non-adhesion of the platelets. Moreover, DNA plasmids may be transfected into a graft to facilitate the delivery of disease or cancer fighting agents to a particular area of the body.
The advantages of concurrent transfection/seeding are (a)facilitates the genetic alteration of seeded ECs to enhance the anti-thrombogenic properties as well as the smooth muscle anti-proliferative of the ECs, and (b) induced positive surface charge maintained at the graft luminal surface wall may allow passage of even more foreign DNA into the cells adhered on the luminal surface.
Clinical Research Findings (completed June 1997):
Simultaneous Electrostatic Endothelial Cell Seeding and Transfection (Electroporation) of Vascular Protheses
Electrostatic endothelial cell (EC) seeding/transfection(patent pending) of vascular prosthetics has potential application in gene therapy (direct delivery) using genetically altered Ecs. Simultaneous seeding/transfectionis conducted by a continuous, low voltage (+1.0 V D.C.) applied over 16 minutes while allowing high voltage pulses(100-2,000 V) at any time. The high voltage pulses (100 ms square waves) can either be a single pulse or four pulses at four second intervals to electroporate the Ecs.
The preliminary study used human umbilical vein endothelial cells (5x106 HUVEC/ml) to seed 4 mm I.D. GORE-TEX (R) (W.L.Gore & Associates) standard wall graft specimens. Electroporation was conducted with the pSV b-galactosidase Control Plasmid (25 mg/ml) (Promega Corp.) by applying 4 pulses (+700 V) midway (8 minutes) through the seeding procedure. After seeding/transfection, the ECs were removed(Trypsin) from the graft, washed, and placed in culture flasks (fibronectin coated) for three days (72 hours) in Endothelial Growth Media (Clonetic Corp.).The ECs were then rinsed with PBS, fixed for 15 minutes in 2% formaldehyde (4oC), rinsed with PBS, overlaid with an X-Gal solution (1 mg/ml), and incubated at 37oC overnight.
Preliminary results indicate a transfection efficiency of 54.8% +\- 4.6% (n=3) (73,540 +\- 8,514 HUVEC/cm2) with no transfection detected with the controls (0.0 V/16 minutes with no high voltage pulses). Thus, enhanced EC seeding and transfection can be accomplished in a single procedure over a clinically acceptable time period (16 minutes).
Title: APPARATUS AND METHOD FOR ELECTROSTATIC ENDOTHELIAL CELL SEEDING
Inventor(s): G. Bowlin, S. Rittgers
Disclosure 268 U.S. Patent - 5,714,359
An apparatus and method for electrostatic seeding of endothelial cells (EC) on the lumen wall of small diameter vascular protheses has been developed. One objective is to raise the level of clinical acceptance of small diameter vascular protheses by increasing the reliability and patency of the grafts.
All current techniques use a protein "glue" to enhance EC adhesion. This "glue" is permanent and thrombogenic. Thus,in the absence of 100% endothelialization and\or EC losses upon implantation, a failure quickly occurs in small diameter vascular protheses due to additional adherence of thrombogenic cells and material. This new procedure takes an available graft material which possesses a low thrombogenicity, a high negative surface charge, and temporarily makes it attractive to EC by changing its surface charge to a positive state. Once the seeding is completed, the graft luminal surface charge reverts back to its natural negative charge.Thus, any non-endothelialized surfaces remain non-thrombogenic and help maintain patency during the healing process. Advantages over prior art are: (1) increases implanted graft's condition of being open or unobstructed from clotting, (2) decreases seeding time approximately 80% from120 minutes to 20 minutes, and (3) the apparatus and process maintains flexibility to accommodate the different combinations of graft lengths and diameter required forimplantation.
Clinical Research Findings (completed June 1997):
In vivo Performance of Electrostatic Endothelial Cell Seeded 4 mm I.D. e-PTFE Vascular ProsthesesIn vivo (canine) evaluation of a recently introduced electrostatic seeding technique (patent pending) used autologous jugular vein (L=10 cm) endothelial cells (EC)harvested using a 0.2% collagenase solution. These Ecs were electrostatically seeded (+1.0 Volt/16 minutes) onto4.0 mm I.D. GORE-TEX (R) (W.L. Gore & Associates) graft (L=6 cm) and then implanted into a femoral artery along with a contralateral untreated control graft (n=5) for six weeks. Aspirin therapy (325 mg/day) was administered for the first two weeks post-operation and discontinued for the remaining four weeks.The external and internal qualitative visual examination of the explanted graft segments demonstrated a superiority of the electrostatically seeded grafts when compared to the controls.
The electrostatically seeded grafts resulted in a 100% patency rate while the control grafts had a patency rate of80%. The electrostatically seeded grafts experienced minimal graft encapsulation while the control grafts demonstrated a high degree of tissue in growth. Furthermore,the internal visual examination of the electrostatically EC seeded graft luminal surfaces revealed minimal to no thrombus formation with neointimal development while the control grafts demonstrated minimal pannus EC in growth and extensive thrombus formation on all luminal surfaces. Histological examination verified the qualitative visual examination showing the development of the neointima and media in the seeded grafts. Thus, the electrostatically seeded grafts demonstrated high thromboresistance despite discontinuation of the aspirin therapy.