Development of three-dimesional system for culturing notochordal cells

Abstract

Background: Notochordal cells (NCCs) appear to play an important role in the formation of the spine and loss of them is related to disc degeneration. Recently, NCCs have drawn increasing attentions from developmental biology, stem cells research and tissue engineering as they are important in development and maintenance of NP. In our group, NCCs have been isolated from Foxa2mNE– Cre/Z/EG heterozygous embryos by identifying EGFP signal using FACS. However, the yield of EGFP positive cells is low (1%). These cells can be cultured as monolayer but all EGFP signals are gone in one month’s time. As a result, development of a better culture system able to maintain their phenotype is warranted. Methods: FACS-sorted NCCs or notochord segments at the anterior, trunk and posterior regions were microencapsulated in type I collagen microspheres and cultured for up to 1 month before characterization for survival and phenotype maintenance. Results: We demonstrated that FACS-sorted NCCs maintained their survival within the collagen microspheres for at least 4 weeks. However, most GFP cells showed elongated morphology. When the notochord segments were cultured in collagen microspheres, clusters of round GFP positive cells co-expressing with major NCC markers were found after 1 month, suggesting that NCC phenotype was maintained. Longer period of culture and the role of niche cells present in the notochord in supporting NCC maintenance are warranted. Conclusion: Type I collagen microspheres present a potential 3D culture system for FACS-sorted NCCs or NC segments.