Various three-dimensional (3D) culture methods have been introduced to overcome the limitations of in vitro culture and mimic in vivo conditions. This study aimed to evaluate two microsphere-forming culture methods and a monolayer culture method. We evaluated cell morphology, viability, osteo-, adipo-, and chondrogenic differentiation potential of dental pulp stem cells (DPSCs) cultured in 3D culture plates: ultra-low attachment (ULA) and U-bottomed StemFit 3D (SF) plates, and a two-dimensional (2D) monolayer plate. RNA sequencing (RNA-seq) revealed differentially expressed gene (DEG) profiles of the DPSCs. In contrast to an increasing pattern in the 2D group, cell viability in 3D groups (ULA and SF) showed a decreasing pattern; however, high multilineage differentiation was observed in both the 3D groups. RNA-seq showed significantly overexpressed gene ontology categories including angiogenesis, cell migration, differentiation, and proliferation in the 3D groups. Hierarchical clustering analysis revealed a similar DEG regulation pattern between the 3D groups; however, a comparatively different DEG was observed between the 2D and 3D groups. Taken together, this study shows that DPSCs cultured in microsphere-forming plates present superior multilineage differentiation capacities and demonstrate higher DEG expression in regeneration-related gene categories compared to that in DPSCs cultured in a conventional monolayer plate.

译文

已经引入了各种三维 (3D) 培养方法,以克服体外培养和模拟体内条件的局限性。本研究旨在评估两种微球形成培养方法和单层培养方法。我们评估了在3D培养板中培养的牙髓干细胞 (DPSCs) 的细胞形态,活力,骨,脂肪和软骨分化潜力: 超低附着 (ULA) 和U底StemFit 3D (SF) 板,以及二维 (2D) 单层板。RNA测序 (RNA-seq) 揭示了dpsc的差异表达基因 (DEG) 谱。与2D组的增加模式相反,3D组 (ULA和SF) 的细胞活力显示出降低的模式; 然而,在两个3D组中均观察到高多向分化。RNA-seq在3D组中显示出显着过表达的基因本体类别,包括血管生成,细胞迁移,分化和增殖。层次聚类分析揭示了3D组之间相似的DEG调节模式; 但是,在2D和3D组之间观察到相对不同的DEG。总之,这项研究表明,与在常规单层平板中培养的DPSCs相比,在微球形成平板中培养的DPSCs具有优异的多向分化能力,并在再生相关基因类别中显示出更高的DEG表达。

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