Efficacy of CD14 + blood monocytes/macrophages isolation: Positive versus negative MACS™ protocol

Abstract

Cardiovascular implants such as stents or artificial vascular grafts established in clinical application to date are generally designed as long-term implants. An important requirement for the successful application of these devices is the haemocompatibility of their interface to the blood stream, which often is approached by polymeric coatings. Modern therapies are aiming at curative treatments (Regenerative Medicine) by inducing the in vivo formation of a new endothelial layer on the implant surface ensuring an optimal haemocompatibility based on the generated glycocalyx. A promising approach for providing the required biological stimulus is the secretion of pro-angiogenic factors (such as VEGF-A) by autologous CD14⁺ monocytes/macrophages (mo/mΦ), which can be induced by previous angiogenic stimulation. While protocols for negative and positive immune-isolation are already established, this study was aimed to evaluate the efficacy of a positive and a negative MACS™ based immune-isolation protocol by analysing the yield, the purity and the activation status of the isolated CD14⁺ mo/mΦ. Both protocols enabled the isolation of more than 80% (positive isolation) and 86% (negative isolation) CD14⁺ mo/mΦ representing approximately 1.3 × 10⁷ CD14⁺ mo/mΦ from 1 ml buffy coat. Less than 10% of these cells were also positive for CD69. Latter is an important selection criterion as CD69 is a marker characteristic for classically activated and catabolic active mo/mΦ which can negatively affect the endothelialisation process. As the negative immune-isolation protocol resulted in a cell fraction meeting the requirements on a pure cell fraction (percentage of contaminating cells < 15%) this isolation method is advantageous although it was more time consuming than the positive isolation protocol.