title: Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia.
abstract: BACKGROUND
  Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen).
  
  
  METHODS
  Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted.
  
  
  RESULTS
  We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM.
  
  
  CONCLUSIONS
  Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.
authors.affiliation: Tissue & Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland. Benjamin.Gantenbein@istb.unibe.ch.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gantenbein
authors.forename: Elena
authors.initials: E
authors.lastname: Calandriello
authors.forename: Karin
authors.initials: K
authors.lastname: Wuertz-Kozak
authors.forename: Lorin M
authors.initials: LM
authors.lastname: Benneker
authors.forename: Marius J B
authors.initials: MJ
authors.lastname: Keel
authors.forename: Samantha C W
authors.initials: SC
authors.lastname: Chan
mesh_terms: D000818:Animals; D002417:Cattle; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D018920:Coculture Techniques / Q000379:methods; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009672:Notochord / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
chemical_list: D017077:Culture Media, Conditioned