Optimizing Oxygenation and Serum Concentration in Ex Vivo Culture of Mouse Nucleus Pulposus Cells Derived from the Notochord

Abstract

Introduction It has been a long-time argument regarding the origin of NP cells in adult humans. Recent review on lineage studies suggests that all nucleus pulposus cells including chondrocyte-like cells are derived from notochordal precursors and those variations in morphology and size are representative of different stages of maturation, and or, function. To define fate of cells in the IVDs that originate from the notochord, we investigated the IVDs of double-transgenic (TG) mouse encoding Protein-0(P0)-Cre/Floxed-EGFP. Cells originating from the neural crest or notochord express positivity for GFP in this TG mouse. We analyzed the effect of hypoxia and serum concentration in notochordal NP cells. Materials and Methods The double transgenic P0-Cre/Floxed-EGFP mice ( n = 10, 5–7 Wks old) were used. The NP cells were isolated from the coccygeal IVDs of P0-Cre/Floxed-EGFP mouse under microscopy and cultured for 10 days without enzymatic processing. After passage these cells were divided into four groups according to the culture condition, Group A; Hypoxia (2% O2) and 20% FBS, Group B; Hypoxia and 10% FBS, Group C; Normoxia (21% O2) and 20% FBS, Group D; Normoxia and 10% FBS. These cells are cultured for over 7 days. We evaluated cell counts with trypan blue staining, cell cycle, and apoptosis by the flow cytometry on the third, fifth, and the seventh day of culture. Results The cell number in group A after the seventh day of culture showed the obviously largest increase. On the seventh day the cell number in group A showed a 17-fold increase, group B showed a 9-fold increase, group C showed a 2-fold increase, group D showed a 1.8-fold increase. The hypoxia (2% O2) groups showed increased cell proliferation rate than the normoxia (21% O2) groups, and the higher FBS concentration (20%) groups showed increased cell proliferation than the lower FBS (10%) groups. Though it seems that in GFP positive and negative ratio there is a difference between the hypoxia groups and the normoxia groups, it has not said clearly yet. We need to obtain some more number of culture. There were no significant changes in the cell cycle and apoptosis between these groups. Conclusion The condition of a low oxygen density and higher FBS concentration was the optimized condition for the mouse notochordal NP cells. Since notochordal NP cells may be important for NP tissue maintenance and phenotype, our result will be useful in ex vivo investigations and based therapy for disk disease. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared