Background
The lifetime prevalence of degenerative disc disease (DDD) is very high. Over 90% of over age 50 show radiographic signs of DDD in magnetic resonance imaging studies [
1]. Degeneration of the intervertebral disc (IVD) is a natural process occurring during ageing and is under continuous investigation [
2]. The IVD is a cartilage-like tissue and consists of two compartments, the inner nucleus pulposus (NP) and the surrounding annulus fibrosus (AF). Both contain specific cells that maintain the extracellular matrix (ECM) through synthesis and degradation of ECM proteins. Cellular activity and metabolism is dependent on nutrient supply. The IVD is an avascular tissue, and thus nutrients are only supplied by the blood system of the adjacent vertebral bodies via a physical barrier, the cartilage endplate (CEP). In mature discs, the distance between an AF or NP cell and CEP is up to 6–8 mm and can only be overcome by free diffusion of fluids through the IVD tissue [
3]. Although disc cells are adapted to the limited nutrient supply, an imbalance in the ECM maintenance occurs with age. The resulting impaired distribution of matrix degrading enzymes causes the DDD. To treat DDD, cell-based therapeutic approaches are of increasing clinical importance [
4]. In disc cell therapy, cells are isolated from the disc and are expanded ex vivo before they are implanted into the impaired disc. Cell expansion is necessary to obtain a sufficient cell number for implantation. These strategies aim to slow down the degeneration process and are predominantly tested in preclinical studies using cell culture experiments [
5].
Reviewing the literature of the two last decades, we found no common protocol available for disc cell expansion. Although cell growth and maintenance of disc cell viability is affected by nutrition [
6], the cell culture medium differs with respect to serum and glucose concentration, as well as in supplementation of ascorbic acid, growth factors and non-essential amino acids. Studies even utilise a variety of basal cell culture media: alpha-Minimal Essential Medium (alpha-MEM), Dulbecco’s Modified Eagle’s Medium (DMEM) or Ham’s F-12 medium (Ham’s F-12). These synthetic culture media were originally developed for different cell types, and nutrition is changed by the medium itself. Harry Eagle found the minimal nutritional requirements to support growth and multiplication of adherent mammalian cells in vitro [
7]. For more demanding cell types such as primary embryonic cells, Renato Dulbecco increased the concentration of amino acids and vitamins and added trace elements and bicarbonates resulting in DMEM [
8]. DMEM has been established as universal medium for various cell types of primary source and immortalised cell lines. Another modification of Eagle’s medium, alpha-MEM, is enriched with non-essential amino acids and further vitamins for cell-lines of mouse and hamster hybrid cells [
9]. Richard G. Ham, another pioneer in the field of nutritional biochemistry, defined different basal media for fibroblast cells, e.g. Ham’s F-12. Ham’s F-12 was the first medium that was used for chondrocyte expansion in cell culture [
10]. In addition, alpha-MEM supports cell growth and stimulation of collagen synthesis in primary osteogenic and chondrogenic cells [
11]. However, only few studies use Ham’s F-12 or alpha-MEM to culture the chondrocyte-like disc cells. For disc cell culture, DMEM is predominantly used as single medium or in a mixture with Ham’s F-12 (DMEM/F-12).
It is well known, that cell growth of primary disc cells is affected by the choice of serum and glucose concentration [
12]. Furthermore, cells are altered by supplementation of growth factors or change in oxygen concentration [
13,
14]. However, not much is known about the impact of the different basal media itself on human disc cells. Since nutrition is altered by the basal medium itself, we hypothesised that the choice of medium would influence the behaviour of cultured disc cells. Cells used for cell therapy could thus possibly show different characteristics, when cultured with different media. As cell expansion is the inevitable step in the manufacturing process of a cell therapeutic product, we put the focus on simple cell expansion rather than complex tissue engineering. Hence, the aim of our study was to set standards for 2D culture of human AF and NP cells with respect to the used basal medium. Therefore, we exposed human AF and NP cells to the different basal media and evaluated the cell’s response regarding cell morphology, cell growth, glycosaminoglycan (GAG) production and expression of cartilage and IVD-related genes. Although Ham’s F-12 and alpha-MEM are not commonly used as single medium for disc cell culture besides DMEM, we tested each medium separately and in a mixture of two media to see individual impacts. In order to prevent any bias by other culture conditions, we used consistent culture conditions (5% CO
2, 95% humidity, normoxia), and all samples from all donors were exactly treated the same way regarding seeding density, time point of media exchange and subcultivation, as well as media and serum batch.
Methods
Tissue source and sample preparation
IVD tissue was obtained from five patients (female/male ratio 3/2) during anterior cervical fusion. All patients showed only mild radiographic degenerative changes of the IVD but failed conservative treatment (Miyazaki degeneration grade ≤ 3, Modic change ≤ 2) [
15]. The mean age was 49 years (range 42–52 years).
AF and NP were macroscopically resected from the IVD tissue using lamellae appearance and tissue colour as criteria. Other tissues such as CEP were discarded. Only tissue parts with clear assignment to either AF or NP in macroscopic evaluation were used for this study. In addition, no IVD tissue with macroscopic signs of degeneration, e.g. a greyish colour or sclerotic parts, were used for this study.
Histological analysis
To confirm correct separation of AF and NP in macroscopic preparation, we performed standard histological staining of each AF and NP sample. A representative small tissue part was embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek, Staufen, Germany), frozen in liquid nitrogen and stored at − 80 °C until sectioning. Samples were cryosectioned at a thickness of 6 μm. To show tissue morphology, sections were fixed with methanol/acetone (1:1 v/v %) and stained with Mayer’s haematoxylin (Dako, Germany) and eosin-G solution (Roth, Karlsruhe, Germany). To analyse sulphated GAGs, the sections were fixed with 4% formaldehyde (Herbeta, Germany) and stained with 1% alcian blue 8GS solution (Roth) and counterstained with nuclear fast red-aluminium sulphate solution (Roth). To demonstrate acidic GAGs, the sections were fixed with 92% ethanol and stained with 0.7% safranin O solution (Sigma-Aldrich) and subsequently counterstained with 0.2% fast green solution (Sigma-Aldrich).
Cell isolation and cell culture
AF and NP samples were washed with phosphate-buffered saline (PBS, Biochrom, Berlin, Germany), and wet weight was determined, separately. Samples were minced, and cells were released by enzymatically treating with 3333 U/mL collagenase CLS II (Biochrom), 1 U/mL collagenase P (Sigma-Aldrich, Taufkirchen, Germany) and 333 U/mL hyaluronidase (Sigma-Aldrich) in a spinner flask under gentle stirring for 4 h at 37 °C (5% CO
2, 95% humidity, normoxia) [
16]. After cellular release, the cell solution was put through a 100 μm nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA), centrifuged at 600×
g, and supernatant was discarded. The viable cell number was determined using trypan blue (Sigma-Aldrich) dye exclusion. From cellular release, three NP and five AF samples of all five donors had a sufficient cell yield to perform all experiments with all media and were thus included in this study.
Cells were cultured in 12-well plates with the different media in duplicates until passage P2 under consistent culture conditions (37 °C, 5% CO2, 95% humidity, normoxia). Medium was replaced every second day. For subcultivation, the cells were detached with trypsin/ethylenediaminetetraacetic acid (Biochrom) at day 6 of passage P0 and day 3 of passage P1 and P2, respectively. Seeding density was always 9000 cells/cm2.
AF and NP cells were cultured using six different media (all from Biochrom): (1) alpha-MEM, (2) DMEM, (3) Ham’s F-12, (4) 1:1 (v/v %) alpha-MEM and Ham’s F-12 (alpha/F-12), (5) 1:1 (v/v %) DMEM and alpha-MEM (DMEM/alpha) and (6) 1:1 (v/v %) DMEM and Ham’s F-12 (DMEM/F-12). The original formulation of alpha-MEM, DMEM and Ham’s F-12 is shown in Table
1. As the distributor only provided alpha-MEM without
l-glutamine, stable
l-glutamine (Biochrom) was added to alpha-MEM to achieve the glutamine concentration of the original alpha-MEM formulation (4 mM). All media were supplemented with 10% human serum (HS) (German Red Cross, Berlin, Germany) and 1% antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, Biochrom). To avoid donor variability, serum was pooled from eight donors (German Red Cross), and serum batch was not changed in this study.
Table 1
Original formulation of alpha-MEM, DMEM and Ham’s F-12
Nutrients | d-Glucose | 5.6 × 100 | 5.6 × 100 | 1.0 × 101 |
Sodium pyruvate | 1.0 × 100 | 1.0 × 100 | 1.0 × 100 |
Sodium bicarbonate | 2.4 × 101 | 4.4 × 101 | 1.4 × 101 |
Lipoic acid | 9.7 × 10−4 | – | 1.0 × 10−3 |
Linoleic acid | – | – | 3.0 × 10−4 |
NaCl | 1.2 × 102 | 1.1 × 102 | 1.3 × 102 |
KCl | 5.4 × 100 | 5.4 × 100 | 3.0 × 100 |
MgSO4·7H2O | 8.1 × 10−1 | 8.1 × 10−1 | – |
MgCl2·6H2O | – | – | 6.0 × 10−1 |
Na2HPO4·H2O | 1.1 × 100 | 8.7 × 10−1 | 1.0 × 100 |
CaCl2·2H2O | 1.8 × 100 | 1.8 × 100 | 3.0 × 10−1 |
Fe(NO3)3·9H2O | – | 2.5 × 10−4 | – |
FeSO4·7H2O | – | – | 3.0 × 10−3 |
CuSO4·5H2O | – | – | 1.0 × 10−5 |
ZnSO4·7H2O | – | – | 3.0 × 10−3 |
Hypoxanthine | – | – | 3.0 × 10−2 |
Thymidine | – | – | 3.0 × 10−3 |
Vitamins | Folic acid | 2.3 × 10−3 | 9.1 × 10−3 | 2.9 × 10−3 |
Vitamin B12 | 1.0 × 10−3 | – | 1.0 × 10−3 |
Pyridoxal·HCl | 4.9 × 10−3 | 2.0 × 10−2 | – |
Pyridoxine·HCl | – | – | 3.0 × 10−4 |
Niacinamid | 8.0 × 10−3 | 3.3 × 10−2 | 3.0 × 10−4 |
d-Ca-pantothenate | 2.1 × 10−3 | 8.4 × 10−3 | 1.0 × 10−3 |
Biotin | 4.1 × 10−4 | – | 3.0 × 10−5 |
Riboflavin | 2.7 × 10−4 | 1.1 × 10−3 | 1.0 × 10−4 |
Thiamine·HCl | 3.0 × 10−3 | 1.2 × 10−2 | 1.0 × 10−3 |
Ascorbic acid | 2.8 × 10−1 | – | – |
Amino acids essential | l-Isoleucine | 4.0 × 10−1 | 8.0 × 10−1 | 3.0 × 10−2 |
l-Leucine | 4.0 × 10−1 | 8.0 × 10−1 | 9.9 × 10−2 |
l-Lysine HCl | 4.0 × 10−1 | 8.0 × 10−1 | 2.0 × 10−1 |
l-Methionine | 1.0 × 10−1 | 2.0 × 10−1 | 3.0 × 10−2 |
l-Phenylalanine | 2.0 × 10−1 | 4.0 × 10−1 | 3.0 × 10−2 |
l-Threonine | 4.0 × 10−1 | 8.0 × 10−1 | 1.0 × 10−1 |
l-Tryptophan | 5.0 × 10−2 | 7.8 × 10−2 | 9.8 × 10−3 |
l-Valine | 4.0 × 10−1 | 8.0 × 10−1 | 1.0 × 10−1 |
Semi-essential | l-Cysteine·H2O | 2.2 × 10−1 | – | 2.3 × 10−1 |
l-Tyrosine | 2.0 × 10−1 | 4.0 × 10−1 | 3.0 × 10−2 |
l-Arginine·HCl | 6.0 × 10−1 | 4.0 × 10−1 | 1.0 × 100 |
l-Histidine·HCl·H2O | 2.0 × 10−1 | 2.0 × 10−1 | 1.0 × 10−1 |
Non-essential | l-Alanine | 2.8 × 10−1 | – | 1.0 × 10−1 |
l-Asparagine·H2O | 3.3 × 10−1 | – | 8.8 × 10−2 |
l-Aspartic acid | 2.3 × 10−1 | – | 1.0 × 10−1 |
l-Glutamine | 4.0 × 100* | 4.0 × 100 | 1.0 × 100 |
l-Glutamic acid | 5.1 × 10−1 | – | 1.0 × 10−1 |
Glycine | 6.7 × 10−1 | 4.0 × 10−1 | 1.0 × 10−1 |
l-Proline | 3.5 × 10−1 | – | 3.0 × 10−1 |
l-Serine | 2.4 × 10−1 | 4.0 × 10−1 | 1.0 × 10−1 |
l-Cystine | 4.2 × 10−1 | 2.0 × 10−1 | – |
Vitaminoids | Inositol | 1.1 × 10−2 | 4.0 × 10−2 | 1.0 × 10−1 |
Choline chloride | 7.2 × 10−3 | 2.9 × 10−2 | 1.0 × 10−1 |
Supplements | Putrescine·2HCl | – | – | 1.0 × 10−3 |
Phenol red | 2.8 × 10−2 | 4.2 × 10−2 | 2.8 × 10−2 |
Cell growth analysis
The cell growth of AF and NP cells was studied based on the population doubling (PD) in each passage P0, P1 and P2 and the cumulative population doubling level (cPDL) at the end of passage P2. The PD was calculated at each subcultivation with the equation PD = 3.32*(log(final cell number)−log(initial cell number)). Adding up PDs of all passages resulted in cPDL [
17]. The growth rate was studied using the population doubling rate (PDR) in each passage with the equation PDR = PD/culture time [days] [
18]. PD, cPDL and PDR are parameters to describe the proliferation capacity and growth rate of cell expansion, respectively.
Glycosaminoglycan analysis
GAG deposition was analysed by alcian blue and nuclear fast red staining (see above) of the cell layer. Cells were seeded on 8-well chamber slides (BD Falcon) and cultured like the main culture. Before cells reached 100% confluency, slides were washed with PBS, fixed with acetone and stored at − 20 °C until staining.
GAG secretion was analysed with standard 1,9-dimethylmethylene blue assay using chondroitin sulphate sodium salt from shark (Sigma-Aldrich) to generate standard curves as described before [
19]. Standard and samples of each donor were measured in triplicates. Culture supernatants were collected from all media exchanges and pooled within same passage. Total sample volume was determined for calculation of total amount of secreted GAGs.
Gene expression analysis
Gene expression analysis was performed for standard cartilage marker aggrecan (ACAN), collagen type I (COL1A1), collagen type II (COL2A2) and IVD-related genes keratin 18 (KRT18) and forkhead box F1 (FOXF1). No common marker genes were available to analyse the human NP and AF phenotype, separately.
For isolation of total RNA, samples were treated with TriReagent (Sigma-Aldrich), 1-bromo-3-chloropropane extraction (Sigma-Aldrich) and purified using the RNeasy Mini Kit with on-column DNase I digestion (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA was synthesised using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The gene expression analysis was performed by quantitative PCR using the primer-UPL probe system of Roche and conducted on the LightCycler® 480 (Roche). Expression of housekeeping genes ATP5F1B and RPL13A was used to normalise the individual mRNA expression level. The data are expressed as relative gene expression levels and calculated using the E-method developed by Roche (
https://lifescience.roche.com). Primer and probes are shown in Table
2. The samples were measured in triplicates.
Table 2
Primer and probes
ACAN | NM_001135.3 NM_013227.3 | ctggaagtcgtggtgaaagg | tcgagggtgtagcgtgtaga | 21 |
COL1A1 | NM_000088.3 | gggattccctggacctaaag | ggaacacctcgctctcca | 67 |
COL2A1 | NM_001844.4 NM_033150.2 | ccctggtcttggtggaaa | cattggtccttgcattactcc | 19 |
FOXF1 | NM_001451.2 | cagcctctccacgcactc | cctttcggtcacacatgct | 5 |
KRT18 | NM_000224.2 NM_199187.1 | aagctggaggctgagatcg | tccaaggcatcaccaagatta | 70 |
ATP5FB1* | NM_001686.3 | agaggtcccatcaaaaccaa | tcctgctcaacactcatttcc | 50 |
RPL13A* | NM_012423.3 NM_00127049.1 | caagcggatgaacaccaac | tgtggggcagcatacctc | 28 |
Statistical analysis
Data are expressed as mean with standard deviation. Statistical analysis between groups (media 1–6) was performed with two-way ANOVA and Bonferroni compensation (GraphPad PRISM 6.0). A p value of less than 0.05 was considered statistically significant.
Discussion
Only few studies are available comparing the basal media regarding cell growth, cell morphology and proteoglycan synthesis of disc cells isolated from young rats and rabbits [
22,
23]. In this study, we analysed for the first time the impact of different culture media on the in vitro behaviour of primary cells isolated from AF and NP from human IVD.
The visual description of AF and NP tissue and the different AF and NP cellularity was consistent with other studies [
24,
25]. In addition, initial cell attachment was delayed for cells isolated from the NP [
26]. Furthermore, the histological examination of the AF and NP starting material suggested a successful separation of AF and NP during sample preparation. Hence, we exclude mixing-up of AF and NP cells, even though both cell types behaved very similar in cell culture.
As expected, both AF and NP cells showed proliferation in all tested media that are commonly used for disc cell expansion. Both AF and NP cells underwent a morphological change from an isodiametric to a spindle-shaped appearance through passaging. Furthermore, cells lost the ability to produce GAGs, as well as to express ACAN and COL2A1 after passaging, whereas COL1A1 was still present. These are typical characteristics of dedifferentiation, a process that is known for 2D culture of high specialised cells such as disc cells or cartilage cells [
25,
27,
28]. The primary cells switch from their native state into a proliferative state, triggered by their attachment to the cell culture surface. Thereby, cells stop synthesising ECM molecules, e.g. GAGs and aggrecan, and switch their collagen expression from type II to type I. This was seen in all media and confirmed previous results [
22]. Hence, the presence of different media did not affect dedifferentiation of cultured disc cells.
Nevertheless, our results showed that the choice of medium affected cell growth and gene expression of the IVD-related marker KRT18. The proliferation capacity of both AF and NP cells was impaired in Ham’s F-12 compared to alpha-MEM and DMEM. A lower cell growth in Ham’s F-12 compared to DMEM and a similar cell growth between alpha-MEM and DMEM was reported before for rat AF and rabbit NP cells, respectively [
22,
23]. Although cell growth was highest in alpha-MEM and lowest in Ham’s F-12, both media increased the KRT18 expression in NP cells. This is a major change in the cell’s behaviour in vitro, because disc cells should typically show a stable expression of the IVD-related marker KRT18 through 2D expansion [
20,
21]. As the differences between the media were only seen after proceeding culture in passage P2 but not in P0 or P1, the change in cell’s behaviour cannot be simply explained by dedifferentiation processes, but was indeed driven by the culture media and its different nutrient supply. In addition, all differences seen between the single media were compensated when the media were used in a mixture.
Comparing the media formulations, the glucose concentration is two times higher in Ham’s F-12 than in alpha-MEM and DMEM (Table
1). As glycolysis is the main energy source in disc cells [
29] and glucose consumption increases with higher glucose concentration [
30], we would have expected a higher performance of the cells in Ham’s F-12 compared to alpha-MEM or DMEM. However, cell growth and GAG production was lower in Ham’s F-12, and expression of cartilage-related genes was unaffected by the different media. Disc cells are used to glucose concentrations in vivo comparable to alpha-MEM, DMEM (0.56 mM) and Hams’ F-12 (1 mM). Furthermore, cell growth of human NP cells is not variant with glucose levels of either 1.8 or 2.5 mM [
12]. Hence, the difference in glucose supplied by the different media was too low to have an impact that was previously described for human NP cells when cultured with either 0.5 mM or 5 mM glucose [
6,
31]. This indicates that glucose was not the most important factor triggering the different performance of AF and NP cells observed in the different media.
The glutamine concentration is apparently four times higher in alpha-MEM and DMEM (4 mM) than in Ham’s F-12 (1 mM). Cell growth was higher in alpha-MEM and DMEM compared to Ham’s F-12. Other studies showed that proliferation capacity is more dependent on glutamine rather than glucose [
32]. Glutamine is an alternative energy source for rapidly dividing cells that have a high demand on energy, especially when glucose level is low. So, if both glutamine and glucose are present in vitro, cells might prefer glutamine as it is faster metabolised. Nevertheless, glucose is mandatory in cell culture as the stimulatory effect of glutamine is only seen in presence of glucose [
33]. The higher concentration of glucose in Ham’s F-12, however, appeared to be insufficient to compensate its lower glutamine concentration. Therefore, the continuous low glutamine level could be one reason for the lower cell growth of AF and NP cells in Ham’s F-12.
In passage P2, when decrease in cell growth was observed for NP cells in Ham’s F’12, NP cells detained in a honeycombed orientation and did not spread into free spaces between cells. A similar stratification pattern is described for immortalised AF cells when cultured in Ham’s F-12 [
34]. Another study associates the cell’s behaviour with a low calcium concentration [
35], comparable to the level existing in Ham’s F-12 (0.3 mM). Furthermore, calcium mediates cell attachment and cell–cell interaction through adhesion molecules, and is a major regulator in cell proliferation [
36]. At a calcium concentration lower than 0.5 mM, cell proliferation is retarded [
37]. Hence, AF and NP cells showed higher cell growth in alpha-MEM and DMEM compared to Ham’s F-12, because alpha-MEM and DMEM contain more calcium (1.8 mM) that is comparable to the physiological range in the blood [
38]. In addition, a calcium concentration like in Ham’s F-12 was found to be more favourable for the expansion of other cell types like keratinocytes [
39].
The presence of other components, for example ascorbic acid, could further have affected the cell’s behaviour in the different media. Ascorbic acid functions as a cofactor in collagen assembling and therefore facilitates ECM development. Despite the other media, alpha-MEM contains ascorbic acid (280 μM). However, both AF and NP cells were not stimulated in collagen expression on mRNA level or in GAG protein expression when cultured in alpha-MEM. By comparing with the literature, 280 μM ascorbic acid has a visible effect on GAG synthesis by disc cells [
40], whereas supplementation of 1000 μM is necessary to increase collagen synthesis [
41,
42]. Hence, ascorbic acid at a low concentration as used here or commonly supplemented in literature (100–280 μM) might not be relevant for disc cell culture.
We assume that low levels of other media components that are additionally present in Ham’s F-12 or rather missing in either alpha-MEM or DMEM were introduced to the cell culture as a component of the added serum. Therefore, trace elements, fatty acids, vitamins or other amino acids with slightly different concentration in the basal medium are not discussed here. In this study, we used the same serum batch constantly for all experiments. However, serum obtained from different donors contains variant concentrations of growth factors [
43]. Furthermore, growth factor concentration is different in serum obtained from different origins (autologous, allogeneic, xenogeneic) and serum-replacements (like platelet rich plasma) [
13,
43]. This is of importance, as the disc cell’s behaviour is altered when growth factors are supplemented to the cell culture medium [
44,
45]. Hence, both the serum batch and serum origin could possibly have a greater impact on disc cells in vitro than has been seen here between the different basal media.
The overall observed difference between the tested media in passage P0 to P2 was lower than expected. It would be interesting to see the cell’s response in higher passages. However, we decided to stop cell culture in passage P2, when AF and NP cells reached a cPDL of 9.4 ± 0.8 and 8.5 ± 0.8 on average, respectively, to avoid a bias by general cell culture effects. It is known from other cartilage cells that the genetic stability is altered, when cells are cultured higher than a cPDL of 10 [
46].
In agreement with other studies, the cultured AF cells were indistinguishable from NP cells regarding cell morphology and cell growth [
2,
25,
47]. Furthermore and in line with literature, AF and NP cells showed similar mRNA expression level of the IVD-related marker FOXF1 and KRT18 [
48,
49]. The higher KRT18 expression level in NP cells compared to AF cells was triggered by the proceeding culture in Ham’s F-12, as this was not seen for the other media. Hence, the KRT18 expression was influenced by the choice of medium and therefore nutrient supply. A previous study showed that KRT18 is down-regulated in cultured NP cells when cells are obtained from degenerated human IVD tissue [
50]. An increase in KRT18 expression is only reported for NP cells, when the cell culture system is changed from 2D to 3D or throughout prolonged culture in a 3D environment [
51,
52]. This further suggests that KRT18 expression in NP cells is sensitive to environmental conditions. A high ratio of aggrecan and collagen type II is another characteristic for human NP tissue [
20]. Apparently, this is only true for native tissue at protein level, because we and others could not recover that ratio in primary NP cells on mRNA level [
53,
54]. In addition, there was no difference between AF and NP cells in overall gene expression of ACAN, COL2A1 and COL1A1 [
55]. Therefore, the common cartilage and IVD-related markers used in this study were not suitable to discriminate AF and NP cells in vitro.
Nevertheless, our results indicate that the choice of medium has an impact on the behaviour of disc cells in vitro. Furthermore, AF and NP cells were influenced differently. Ham’s F-12 impaired cell growth in NP but not in AF cells. In addition, the proceeding culture in Ham’s F-12 changed the KRT18 expression only in NP cells. Hence, it is possible that different culture media are required for AF and NP cells in order to preserve their individual cell characteristics. This is of relevance as cell-based therapeutic approaches often utilise herniated IVD tissue as starting tissue material for cell isolation [
56,
57]. The herniated tissue contains a heterogeneous cell population as it originates either in the NP or AF compartment of the disc depending on the hernia location [
58,
59]. Therefore, as long as no marker is available to separate human AF and NP cells, the choice of medium is important for the development of cell-therapeutic products targeted for either AF or NP repair.