Abstract：
The two-dimensional (2D) monolayer culture as a conventional method has been widely applied in molecular biology fields, but it has limited capability to recapitulate real cell environments, being prone to misinterpretation with poor prediction of in vivo behavior. Recently, the three-dimensional (3D) spheroid culture has been studied extensively. Spheroids are self-assembled cell aggregates that have biomimicry capabilities. The behavior of thyroid cancer under the 3D spheroid culture environment has been studied; however, there are no reports regarding differences in the degree of thyroid cancer cell differentiation under 2D and 3D culture conditions. This study investigated the expression of thyroid differentiation proteins related to iodide-metabolizing mechanisms in thyroid cancer cells under different culture conditions. Four thyroid cancer cell lines and one thyroid follicular epithelial cell line were grown in adherent 2D cell culture and 3D spheroid culture with agarose-coated plates. We observed changes in proliferation, hypoxia, extracellular matrix (ECM), cytoskeleton, thyroid-specific proteins, and thyroid transcription factors. All cell lines were successfully established in the spheroid following cell aggregation. Proliferation considerably decreased, while hypoxia-inducible factor 1-α(HIF1-α) was promoted in 3D spheroids; moreover, 3D spheroids with thyroid cancers showed diminished thyroid differentiation markers, but thyroid follicular epithelial cells revealed either a maintenance or weak decline of protein expression. We verified that the 3D spheroid culture environment can be similar to in vivo conditions because of its alterations in numerous cellular and functional activities, including morphology, cellular proliferation, viability, hypoxia, ECM, cytoskeleton, and thyroid differentiation, compared to the conventional 2D monolayer culture environment. An in vitro experimental study using 3D spheroid culture is ideal for the faster discovery of new drugs.
摘要：
二维(2D)单层培养作为一种常规方法已广泛应用于分子生物学领域,但是它概括真实细胞环境的能力有限，容易误解，对体内行为的预测较差。最近,三维（3D）球体培养已被广泛研究。球体是具有仿生能力的自组装细胞聚集体。已经研究了3D球体培养环境下甲状腺癌的行为；然而，没有关于2D和3D培养条件下甲状腺癌细胞分化程度差异的报道.本研究调查了不同培养条件下甲状腺癌细胞中与碘代谢机制相关的甲状腺分化蛋白的表达。将四种甲状腺癌细胞系和一种甲状腺滤泡上皮细胞系生长在粘附的2D细胞培养物中，并在琼脂糖包被的平板上进行3D球体培养。我们观察到增殖的变化，缺氧,细胞外基质（ECM），细胞骨架,甲状腺特异性蛋白质，和甲状腺转录因子。在细胞聚集后,在球状体中成功建立所有细胞系。增殖大大减少，而缺氧诱导因子1-α（HIF1-α）在3D球体中被促进；此外，甲状腺癌的3D球状体显示甲状腺分化标志物降低，但甲状腺滤泡上皮细胞显示蛋白质表达维持或弱下降。我们验证了3D球体培养环境可以类似于体内条件，因为它在许多细胞和功能活动中发生了变化。包括形态学，细胞增殖，生存能力，缺氧,ECM,细胞骨架,和甲状腺分化，与传统的2D单层培养环境相比。使用3D球体培养的体外实验研究对于更快地发现新药是理想的。
