3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions.. 3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions.

Adipose tissues are closely related to physiological functions and pathological conditions in most organs. Although differentiated 3T3-L1 preadipocytes have been used for in vitro adipose studies, the difference in cellular characteristics of adipogenic differentiation in two-dimensional (2D) culture and three-dimensional (3D) culture remain unclear. In this study, we evaluated gene expression patterns using RNA sequencing and metabolic functions using an extracellular flux analyzer in 3T3-L1 preadipocytes with and without adipogenic induction in 2D culture and 3D culture. In 2D culture, 565 up-regulated genes and 391 down-regulated genes were identified as differentially expressed genes (DEGs) by adipogenic induction of 3T3-L1 preadipocytes, whereas only 69 up-regulated genes and 59 down-regulated genes were identified as DEGs in 3D culture. Ingenuity Pathway Analysis (IPA) revealed that genes associated with lipid metabolism were identified as 2 out of the top 3 causal networks related to diseases and function in 3D spheroids, whereas only one network related to lipid metabolism was identified within the top 9 of these causal networks in the 2D planar cells, suggesting that adipogenic induction in the 3D culture condition exhibits a more adipocyte-specific gene expression pattern in 3T3-L1 preadipocytes. Real-time metabolic analysis revealed that the metabolic capacity shifted from glycolysis to mitochondrial respiration in differentiated 3T3-L1 cells in the 3D culture condition but not in those in the 2D cultured condition, suggesting that adipogenic differentiation in 3D culture induces a metabolic phenotype of well-differentiated adipocytes. Consistently, expression levels of mitochondria-encoded genes including mt-Nd6, mt-Cytb, and mt-Co1 were significantly increased by adipogenic induction of 3T3-L1 preadipocytes in 3D culture compared with those in 2D culture. Taken together, the findings suggest that induction of adipogenesis in 3D culture provides a more adipocyte-specific gene expression pattern and enhances mitochondrial respiration, resulting in more adipocyte-like cellular properties. Overall design: Two independently prepared samples of 2D cultured 3T3-L1 cells or 3D cultured 3T3-L1 spheroids with or without adipogenic induction were subjected to RNA isolation, and total RNA was initially extracted from each sample. After checking the RNA content and quality, cDNA libraries were prepared, and their quality and quantity were validated. Thereafter, sequential steps including cluster generation, sequencing the multiplexed samples, image analysis, base calling, and quality filtering were carried out. The differentially expressed genes (DEGs, fold-change 2.0 and FDR adjusted P < 0.05 and q < 0.2) were statistically determined by using mapping of sequence data (Qiagen, Redwood City, CA, USA). To elucidate possible gene functions, gene ontology (GO) enrichment analysis as well as Ingenuity Pathway Analysis (IPA, Qiagen, USA) were performed.

脂肪组织与绝大多数器官的生理功能及病理状态均密切相关。尽管已分化的3T3-L1前脂肪细胞已被广泛应用于体外脂肪组织相关研究，但二维（two-dimensional, 2D）与三维（three-dimensional, 3D）培养体系中，成脂分化的细胞特征差异仍未明确。
本研究通过RNA测序（RNA sequencing）分析基因表达谱，并利用细胞外流量分析仪（extracellular flux analyzer）检测3T3-L1前脂肪细胞在2D及3D培养环境下，经成脂诱导与否的代谢功能。在2D培养体系中，3T3-L1前脂肪细胞经成脂诱导后，共鉴定出565个上调基因与391个下调差异表达基因（differentially expressed genes, DEGs）；而在3D培养体系中仅鉴定出69个上调基因与59个下调DEGs。Ingenuity路径分析（Ingenuity Pathway Analysis, IPA）结果显示，在3D细胞球中，与脂代谢相关的基因位列疾病与功能关联的前3个核心因果网络之中；而在2D单层培养细胞中，脂代谢相关基因仅出现在前9个核心因果网络中的1个，这表明3D培养环境下的成脂诱导可使3T3-L1前脂肪细胞呈现更具脂肪细胞特异性的基因表达谱。
实时代谢分析显示，3D培养环境下的分化3T3-L1细胞的代谢能力从糖酵解转向线粒体呼吸，而2D培养细胞未出现该变化，提示3D培养中的成脂分化可诱导出成熟脂肪细胞的代谢表型。与之一致的是，相较于2D培养体系，3D培养体系中3T3-L1前脂肪细胞经成脂诱导后，包括mt-Nd6、mt-Cytb及mt-Co1在内的线粒体编码基因的表达水平显著升高。
综上，本研究结果表明，3D培养环境下的成脂诱导可使3T3-L1前脂肪细胞呈现更具脂肪细胞特异性的基因表达谱，并增强线粒体呼吸功能，最终形成更接近成熟脂肪细胞的细胞特性。
总体实验设计：分别制备成脂诱导与未诱导的2D培养3T3-L1细胞、3D培养3T3-L1细胞球样品，各独立制备2份，随后进行RNA分离，每份样品均先提取总RNA。在检测RNA含量与质量后，构建cDNA文库并验证其质量与数量。后续依次完成簇生成、多重样本测序、图像分析、碱基识别及质量过滤等步骤。通过序列数据比对（凯杰公司，美国加利福尼亚州红木城），统计学鉴定差异表达基因（DEGs，倍数变化≥2.0，错误发现率（FDR）校正P值<0.05且q值<0.2）。为阐明潜在基因功能，本研究开展了基因本体（Gene Ontology, GO）富集分析及Ingenuity路径分析（IPA, 凯杰公司，美国）。