Abstract
BackgroundNotochordal 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).MethodsPorcine 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.ResultsWe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-422) contains supplementary material, which is available to authorized users.
Highlights
Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status
We investigated the hypothesis that notochordal cells (NC) are sensitive to culture conditions [28] and that their activation capacity on disc cells in indirect co-culture experiments can be stimulated by exposure to fetal calf serum (FCS)
Porcine and bovine nucleus pulposus (NP) tissues were digested by 0.19% pronase (Roche, Basel, Switzerland) for 1 hour and subsequent collagenase type 2 at a concentration of 32 U/mL in 25 mL of HG-DMEM supplemented with 10% FCS (Worthington, London, UK) overnight (~14 hours) at 37°C and standard culture conditions (5% CO2 and 100% humidity) [14,23]
Summary
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). NC or their cultured media — the so-called notochord-derived conditioned medium (NCCM) — demonstrated regenerative effects by inducing the glycosaminoglycan per DNA (GAG/DNA) ratio [13,18,21] These cells, or, possibly, merely their released factors, are of high interest for regenerative medicine to re-populate degenerated IVDs or to stimulate native disc cells. We replicated the experiments in normoxic (~21%) and hypoxic (~2%) conditions to study the effect of the physiological oxygen environment as it was reported in the literature that NC should be preferentially cultured in hypoxia for a better aligned extracellular matrix [20,26] and a more authentic phenotype as defined by the production of aggrecan with respect to collagen type 2, which was found to be around 27:1 in human tissue [31] or around 1:1 as found in rabbit tissue [32]
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