Towards clinical application of microvascular endothelial cell seeding

Abstract

The central question in this thesis is whether microvascular endothelial cells (MVEC) from subcutaneous fat tissue are suitable for the seeding of prosthetic vascular grafts and deendothelialized surfaces. The aim of the application of endothelial cells (EC) is the inhibition of thrombogenicity and intimal hyperplasia. * Seeding of MVEC from fat tissue onto prosthetic vascular grafts results in a confluent EC layer on top of a multiplayer of (myo)fibroblasts. Contaminating cells from the transplant contribute to this intimal hyperplasia. * The seeding of blood vessels after percutaneous transluminal angioplasty (PTA) with MVEC from fat tissue results in an increased intimal hyperplasia of the vessel wall. Again contaminants from the transplant seem to contribute to this intimal hyperplasia. * Cells isolated from fat tissue consist of 40% EC, and 60% contaminating cells ((myo)fibroblasts and macrophages). A combination of magnetic spheres coated with cell-specific antibodies can be used to remove the contaminants. The purity is increased from 40 to more than 90%. * Laboratory experiments show that thrombogenicity and intimal hyperplasia decrease significantly after seeding of purified cells compared to nonpurified ones. * In a laboratory model blood vessels were purfused continuously with cell culture medium. The seeding of purified cells resulted in a reduced intimal hyperplasia compared to the seeding of nonpurified cells. * One of the two antibodies necessary to remove contaminating cells is not suitable for clinical application. The CD34-selection method from Nexell selects the EC instead of the contaminating cells. The necessary products are approved for clinical grade application. The purity obtained is the same, however the yield is lower. * Definitive evidence for the suitability of purified MVEC cell seeding purposes is a clinical study. The ideal scenario is to utilize a graft for which thrombus formation and intimal hyperplasia are frequently events. In addition, the risk to the patient in case the procedure fails must be as low as possible. For this reason, we chose prosthetic arteriovenous (AV) grafts instead of lower extremity bypass grafts, for which the cell seeding procedure was originally developed. In ten patients, an AV graft will be implanted that is seeded with purified autologous fat derived MVEC. This is the ECLIPSE-studie (endothelial cell lining on prosthetic shunts evaluation). In case the pilot-study shows that the seeding of purified MVEC from fat tissue is safe, feasible, and effective, a randomised study will be performed.