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Journal of Orthopaedic Surgery and Research
Published in: Journal of Orthopaedic Surgery and Research 1/2018

Open Access 01-12-2018 | Research article

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

Authors: Ann-Kathrin Schubert, Jeske J. Smink, Matthias Pumberger, Michael Putzier, Michael Sittinger, Jochen Ringe

Published in: Journal of Orthopaedic Surgery and Research | Issue 1/2018

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Abstract

Background

The 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.

Methods

Human 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.

Results

AF 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.

Conclusion

The 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.
Literature
1.
Cheung KM, Karppinen J, Chan D, Ho DW, Song YQ, Sham P, Cheah KS, Leong JC, Luk KD. Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine (Phila Pa 1976). 2009;34(9):934–40.CrossRef
2.
Wang F, Cai F, Shi R, Wang XH, Wu XT. Aging and age related stresses: a senescence mechanism of intervertebral disc degeneration. Osteoarthr Cartil. 2016;24(3):398–408.CrossRefPubMed
3.
Urban JP. The role of the physicochemical environment in determining disc cell behaviour. Biochem Soc Trans. 2002;30(Pt 6):858–64.CrossRefPubMed
4.
5.
Mern DS, Beierfuss A, Thome C, Hegewald AA. Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases: a systematic review. J Tissue Eng Regen Med. 2014;8(12):925–36.CrossRefPubMed
6.
Bibby SR, Urban JP. Effect of nutrient deprivation on the viability of intervertebral disc cells. Eur. Spine J. 2004;13(8):695–701.
7.
8.
9.
Stanners CP, Eliceiri GL, Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971;230(10):52–4.CrossRefPubMed
10.
Ham RG, Sattler GL. Clonal growth of differentiated rabbit cartilage cells. J Cell Physiol. 1968;72(2):109–14.CrossRefPubMed
11.
12.
13.
Wang SZ, Chang Q, Lu J, Wang C. Growth factors and platelet-rich plasma: promising biological strategies for early intervertebral disc degeneration. Int Orthop. 2015;39(5):927–34.CrossRefPubMed
14.
Gorth DJ, Lothstein KE, Chiaro JA, Farrell MJ, Dodge GR, Elliott DM, Malhotra NR, Mauck RL, Smith LJ. Hypoxic regulation of functional extracellular matrix elaboration by nucleus pulposus cells in long-term agarose culture. J Orthop Res. 2015;33(5):747–54.CrossRefPubMedPubMedCentral
15.
Miyazaki M, Hong SW, Yoon SH, Morishita Y, Wang JC. Reliability of a magnetic resonance imaging-based grading system for cervical intervertebral disc degeneration. J Spinal Disord Tech. 2008;21(4):288–92.CrossRefPubMed
16.
Stich S, Stolk M, Girod PP, Thome C, Sittinger M, Ringe J, Seifert M, Hegewald AA. Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs. PLoS One. 2015;10(5):e0126954.CrossRefPubMedPubMedCentral
17.
Cristofalo VJ, Allen RG, Pignolo RJ, Martin BG, Beck JC. Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc Natl Acad Sci U S A. 1998;95(18):10614–9.CrossRefPubMedPubMedCentral
18.
Kaji K, Matsuo M. Aging of chick embryo fibroblasts in vitro--I. Saturation density and population doubling rate. Exp Gerontol. 1978;13(6):439–45.CrossRefPubMed
19.
Bartz C, Meixner M, Giesemann P, Roel G, Bulwin GC, Smink JJ. An ex vivo human cartilage repair model to evaluate the potency of a cartilage cell transplant. J Transl Med. 2016;14(1):317.CrossRefPubMedPubMedCentral
20.
Risbud MV, Schoepflin ZR, Mwale F, Kandel RA, Grad S, Iatridis JC, Sakai D, Hoyland JA. Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the spine research interest group at the 2014 annual ORS meeting. J Orthop Res. 2015;33(3):283–93.CrossRefPubMedPubMedCentral
21.
van den Akker GG, Surtel DA, Cremers A, Rodrigues-Pinto R, Richardson SM, Hoyland JA, van Rhijn LW, Welting TJ, Voncken JW. Novel immortal human cell lines reveal subpopulations in the nucleus pulposus. Arthritis Res Ther. 2014;16(3):R135.CrossRefPubMedPubMedCentral
22.
Ichimura K, Tsuji H, Matsui H, Makiyama N. Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis. J Spinal Disord. 1991;4(4):428–36.CrossRefPubMed
23.
Rastogi A, Thakore P, Leung A, Benavides M, Machado M, Morschauser MA, Hsieh AH. Environmental regulation of notochordal gene expression in nucleus pulposus cells. J Cell Physiol. 2009;220(3):698–705.CrossRefPubMed
24.
Chou AI, Bansal A, Miller GJ, Nicoll SB. The effect of serial monolayer passaging on the collagen expression profile of outer and inner anulus fibrosus cells. Spine (Phila Pa 1976). 2006;31(17):1875–81.CrossRef
25.
Kluba T, Niemeyer T, Gaissmaier C, Grunder T. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976). 2005;30(24):2743–8.CrossRef
26.
Horner HA, Roberts S, Bielby RC, Menage J, Evans H, Urban JP. Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype. Spine (Phila Pa 1976). 2002;27(10):1018–28.CrossRef
27.
von der Mark K, Gauss V, von der Mark H, Muller P. Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature. 1977;267(5611):531–2.CrossRefPubMed
28.
Gorensek M, Jaksimovic C, Kregar-Velikonja N, Gorensek M, Knezevic M, Jeras M, Pavlovcic V, Cor A. Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes. Cell Mol Biol Lett. 2004;9(2):363–73.PubMed
29.
Urban JP, Winlove CP. Pathophysiology of the intervertebral disc and the challenges for MRI. J Magn Reson Imaging. 2007;25(2):419–32.CrossRefPubMed
30.
Bibby SR, Jones DA, Ripley RM, Urban JP. Metabolism of the intervertebral disc: effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells. Spine (Phila Pa 1976). 2005;30(5):487–96.CrossRef
31.
Rinkler C, Heuer F, Pedro MT, Mauer UM, Ignatius A, Neidlinger-Wilke C. Influence of low glucose supply on the regulation of gene expression by nucleus pulposus cells and their responsiveness to mechanical loading. J Neurosurg Spine. 2010;13(4):535–42.CrossRefPubMed
32.
Slivac I, Gaurina Srcek V, Radosevic K, Porobic I, Bilic K, Fumic K, Kniewald Z. Growth characteristics of channel catfish ovary cells-influence of glucose and glutamine. Cytotechnology. 2008;57(3):273–8.CrossRefPubMedPubMedCentral
33.
Slivac I, Blajic V, Radosevic K, Kniewald Z, Gaurina Srcek V. Influence of different ammonium, lactate and glutamine concentrations on CCO cell growth. Cytotechnology. 2010;62(6):585–94.CrossRefPubMedPubMedCentral
34.
van den Akker GG, Surtel DA, Cremers A, Richardson SM, Hoyland JA, van Rhijn LW, Voncken JW, Welting TJ. Novel immortal cell lines support cellular heterogeneity in the human annulus fibrosus. PLoS One. 2016;11(1):e0144497.CrossRefPubMedPubMedCentral
35.
Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell. 1980;19(1):245–54.CrossRefPubMed
36.
Ko KS, Arora PD, Bhide V, Chen A, McCulloch CA. Cell-cell adhesion in human fibroblasts requires calcium signaling. J Cell Sci. 2001;114(Pt 6):1155–67.PubMed
37.
Boynton AL, Whitfield JF, Isaacs RJ, Morton HJ. Control of 3T3 cell proliferation by calcium. In Vitro. 1974;10:12–7.CrossRefPubMed
38.
39.
Dahm AM, de Bruin A, Linat A, von Tscharner C, Wyder M, Suter MM. Cultivation and characterisation of primary and subcultured equine keratinocytes. Equine Vet J. 2002;34(2):114–20.CrossRefPubMed
40.
Lee YJ, Kong MH, Song KY, Lee KH, Heo SH. The relation between Sox9, TGF-beta1, and proteoglycan in human intervertebral disc cells. J Korean Neurosurg Soc. 2008;43(3):149–54.CrossRefPubMedPubMedCentral
41.
Flagler DJ, Huang CY, Yuan TY, Lu Z, Cheung HS, Intracellular Flow GWY. Cytometric measurement of extracellular matrix components in porcine intervertebral disc cells. Cell Mol Bioeng. 2009;2(2):264–73.CrossRefPubMedPubMedCentral
42.
Kim MH. Effect of L-ascorbic acid on collagen synthesis in 3T6 fibroblasts and primary cultured cells of chondrocytes. J Korean Soc Food Sci Nutr. 2006;35:42–7.CrossRef
43.
Tallheden T, van der Lee J, Brantsing C, Mansson JE, Sjogren-Jansson E, Lindahl A. Human serum for culture of articular chondrocytes. Cell Transplant. 2005;14(7):469–79.CrossRefPubMed
44.
Gruber HE, Fisher EC Jr, Desai B, Stasky AA, Hoelscher G, Hanley EN Jr. Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. Exp Cell Res. 1997;235(1):13–21.CrossRefPubMed
45.
Hegewald AA, Cluzel J, Kruger JP, Endres M, Kaps C, Thome C. Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells. J Orthop Surg Res. 2014;9:73.CrossRefPubMedPubMedCentral
46.
Wallenborn M, Petters O, Rudolf D, Hantmann H, Richter M, Ahnert P, Rohani L, Smink JJ, Bulwin GC, Krupp W, et al. Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array. Eur Cell Mater. 2018;35:225–41.CrossRefPubMed
47.
Chen YF, Zhang YZ, Zhang WL, Luan GN, Liu ZH, Gao Y, Wan ZY, Sun Z, Zhu S, Samartzis D, et al. Insights into the hallmarks of human nucleus pulposus cells with particular reference to cell viability, phagocytic potential and long process formation. Int J Med Sci. 2013;10(13):1805–16.CrossRefPubMedPubMedCentral
48.
van den Akker GGH, Koenders MI, van de Loo FAJ, van Lent P, Blaney Davidson E, van der Kraan PM. Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc. Eur Spine J. 2017;26(8):2053–62.CrossRefPubMed
49.
Rutges J, Creemers LB, Dhert W, Milz S, Sakai D, Mochida J, Alini M, Grad S. Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration. Osteoarthr Cartil. 2010;18(3):416–23.CrossRefPubMed
50.
Lv FJ, Peng Y, Lim FL, Sun Y, Lv M, Zhou L, Wang H, Zheng Z, Cheung KM, Leung VY. Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers. Osteoarthr Cartil. 2016;24(10):1826–36.CrossRefPubMed
51.
Sun Y, Lv M, Zhou L, Tam V, Lv F, Chan D, Wang H, Zheng Z, Cheung KM, Leung VY. Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation. Osteoarthr Cartil. 2015;23(7):1194–203.CrossRefPubMed
52.
Wan S, Borland S, Richardson SM, Merry CL, Saiani A, Gough JE. Self-assembling peptide hydrogel for intervertebral disc tissue engineering. Acta Biomater. 2016;46:29–40.CrossRefPubMed
53.
Park JY, Kuh SU, Park HS, Kim KS. Comparative expression of matrix-associated genes and inflammatory cytokines-associated genes according to disc degeneration: analysis of living human nucleus pulposus. J Spinal Disord Tech. 2011;24(6):352–7.CrossRefPubMed
54.
Choi EH, Park H, Park KS, Park KS, Kim BS, Han IB, Shin DA, Lee SH. Effect of nucleus pulposus cells having different phenotypes on chondrogenic differentiation of adipose-derived stromal cells in a coculture system using porous membranes. Tissue Eng Part A. 2011;17(19–20):2445–51.CrossRefPubMed
55.
Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, Freemont AJ. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol. 2002;55(2):91–7.CrossRefPubMedPubMedCentral
56.
Meisel HJ, Siodla V, Ganey T, Minkus Y, Hutton WC, Alasevic OJ. Clinical experience in cell-based therapeutics: disc chondrocyte transplantation a treatment for degenerated or damaged intervertebral disc. Biomol Eng. 2007;24(1):5–21.CrossRefPubMed
57.
Tschugg A, Diepers M, Simone S, Michnacs F, Quirbach S, Strowitzki M, Meisel HJ, Thome C. A prospective randomized multicenter phase I/II clinical trial to evaluate safety and efficacy of NOVOCART disk plus autologous disk chondrocyte transplantation in the treatment of nucleotomized and degenerative lumbar disks to avoid secondary disease: safety results of phase I-a short report. Neurosurg Rev. 2017;40(1):155–62.CrossRefPubMed
58.
Moore RJ, Vernon-Roberts B, Fraser RD, Osti OL, Schembri M. The origin and fate of herniated lumbar intervertebral disc tissue. Spine (Phila Pa 1976). 1996;21(18):2149–55.CrossRef
59.
Rajasekaran S, Babu JN, Arun R, Armstrong BR, Shetty AP, Murugan S. ISSLS prize winner: a study of diffusion in human lumbar discs: a serial magnetic resonance imaging study documenting the influence of the endplate on diffusion in normal and degenerate discs. Spine (Phila Pa 1976). 2004;29(23):2654–67.CrossRef
Metadata
Title
Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells
Authors
Ann-Kathrin Schubert
Jeske J. Smink
Matthias Pumberger
Michael Putzier
Michael Sittinger
Jochen Ringe
Publication date
01-12-2018
Publisher
BioMed Central
Published in
Journal of Orthopaedic Surgery and Research / Issue 1/2018
Electronic ISSN: 1749-799X
DOI
https://doi.org/10.1186/s13018-018-0914-y

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