Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells

Abstract

BackgroundThe lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell’s behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture.MethodsHuman AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco’s Modified Eagle’s Medium (DMEM) or Ham’s F-12 medium (Ham’s F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation.ResultsAF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham’s F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham’s F-12.ConclusionThe impact of the different media itself on disc cell’s behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades

“…For RUNX2 and BGLAP, a gene expression in native tissue from mild degenerated AF could not be detected. These results confirm the microarray results and support the theory of a potential dedifferentiation in monolayer cultures [29].…”

“…Here, the signal intensity was highly reduced from 6901.30 (mild) and 2063.57 (severe) to 161.73 (mild) and 410.97 (severe) and the FC changed from 4.32 to −2.35. This indicates the possibility of dedifferentiation processes in monolayer cultures [29]. Another possibility of the reversed FC in monolayer culture compared to native AF tissue is due to the heterogeneity of the native tissue.…”

Adrenoceptor Expression during Intervertebral Disc Degeneration

“…The reason for this observation might be that monolayer culture conditions lead to dedifferentiation processes, which in turn could result in AR regulation [40]. Indeed, also degeneration results in dedifferentiation, therefore, we believe that our monolayer culture conditions used in the present study and as performed by others [41] optimally mimic the phenotype and behavior of IVD cells in vivo. Furthermore, isolated IVD cells were able to respond to NE stimulation by PKA and ERK1/2 phosphorylation.…”

Optimization of culture media to enhance the growth of tissue engineered cartilage

“…While the underlying mechanism is presently unknown, there may be a limit with respect to glucose, as concentrations lower than 5 mM can detrimentally affect chondrocyte function 37 . Although the effect of basal media formulation has not been widely studied, previous studies have also indicated that DMEM/F12 mixtures (1:1) can increase proteoglycan accumulation compared to F12 media alone 38,39 . This result may be due to the combined influence of the high amino acid content of DMEM and vitamin rich formulation of F12.…”

“…37 Although the effect of basal media formulation has not been widely studied, previous studies have also indicated that DMEM/F12 mixtures (1:1) can increase proteoglycan accumulation compared to F12 media alone. 38,39 This result may be due to the combined influence of the high amino acid content of DMEM and vitamin rich formulation of F12. Glutamine is a well-known essential nutrient for chondrocytes and is required for proteoglycan synthesis.…”