FACS is more potent to fish IVD progenitor cells than magnetic and bead-based methods.

Low back pain related to intervertebral disc (IVD) degeneration has a major socioeconomical impact on our ageing society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role for IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (nucleus pulposus progenitor cells or NPPCs) was found positive for the angiopoietin-1 receptor (Tie2+) and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols, i.e., fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony forming units = CFUs) and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate efficiency of three wide-spread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in down-stream functional differentiation in adipogenesis, osteogenesis and chondrogenesis. The cell yield among the isolation methods differed widely, with FACS presenting the highest yield (5.0 ± 4.0%), followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2- NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2- populations in differentiation assays. To conclude, the isolation of NPPC was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells. .