Different isolation methods for nucleus pulposus progenitor cells

Frauchiger, Daniela A; Tekari, Adel; May, Rahel; Dzafo, Emina; Chan, Samantha C.W.; Stoyanov, Jivko; Bertolo, Alessandro; Zhang, Xingshuo; Sakai, Daisuke; Schol, Jordy; Grad, Sibylle; Tryfonidou, Marianna; Benneker, Lorin M.; Gantenbein, Benjamin (2019). Different isolation methods for nucleus pulposus progenitor cells. In: 7th International Congress on Biotechnologies for Spinal Surgery (BioSpine7). Rom, Italy. 3-5 April 2019.

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Introduction: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared.

Methods: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays.

Results: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS resulted in more colonies than pluriSelect (p < 0.05). Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted NPPCs tended to produce more proteoglycan vs Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found.

Conclusion: Isolation of Tie2+ NPPC is possible with all three methods tested. However, FACS resulted in the highest cell yield and a clearer separation after differentiation making it the method of choice for Tie2+ NPPC isolation.

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