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Ideal culture condition for notochordal cells yet to be identified
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Although researchers determined it was best to culture notochordal cells in 3-D to most closely maintain their phenotype — preferably in hypoxia and with supplementation of fetal calf serum in the culture media — the ideal culture condition has not yet been identified, according to study findings.
The researchers kept porcine notochordal cells (NC) in a 2-D monolayer and in 3-D alginate bead culture to identify a suitable culture system. Co-cultures of NC and bovine-derived coccygeal intervertebral disc (IVD) cells were conducted in a 1:1 ratio to test the stimulating effects of NC. For 7 to 14 days, no direct cell contact was permitted between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3-D alginate beads under normoxia and hypoxia (2%). NPC and AFC were stimulated with NC-derived conditioned medium (NCCM) as a control for the study.
After 34 days, the researchers found much more NC could be recovered from 3-D alginate bead culture (around 50%) compared with 2-D monolayer, indicating the 3-D alginate was likely superior for NC culture.
NC was shown to activate NPC in 3-D alginate culture, either through indirect co-culture or through the addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2, according to the researchers. The researchers found these effects to be strongest with 10% fetal calf serum and under hypoxia. However, AFC seemed to be unresponsive to co-culture with bigger notochordal cells or to the NCCM.
Although hypoxia resulted in the deceleration of metabolic activity, it did not lead to any significant changes in the glycosaminoglycan / DNA ratio, according to the researchers. – by Robert Linnehan
Disclosures: The authors report no relevant financial disclosures.
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