Techniques for isolating and purifying porcine aortic valve endothelial cells.

摘要

BACKGROUND AND AIM OF THE STUDY: Existing methods to isolate aortic valve endothelial cells (ECs) are unreliable and often yield populations that are inadequate for long-term studies in vitro because of valve interstitial cell (IC) contamination. The study aim was to test various isolation protocols to improve the yield and purity of isolated ECs, and to assess two purification techniques to further deplete contaminating ICs and improve the quality of long-term EC cultures. METHODS: Porcine aortic valve leaflets were digested in different concentrations of collagenase and dispase over various incubation times. Isolated cells were counted, and the purities of the populations determined by immunocytochemical staining and image analysis. Improvements in purity after magnetic cell sorting (MACS) or clonal expansion were assessed. RESULTS: Enzymatic digestion using 60 U/ml collagenase yielded the largest number of cells. Digestion with 2.0 U/ml dispase and 60 U/ml collagenase produced significantly more pure populations of ECs than solutions containing 0.5 or 1.0 U/ml dispase (p <0.05). A 2-h digestion produced similar yields compared to longer digestion times. MACS improved purity (p <0.01) and was efficient and economical, but purified populations were contaminated with ICs post-confluence. Clonal expansion produced the highest quality EC cultures, with no IC contamination after weeks of post-confluent culturing. CONCLUSION: The results of these studies have provided recommendations for the improved isolation of aortic valve ECs, and guidelines for the further purification of isolated EC populations based on quality, time and economical considerations.