Impact of 3D co-culture on epigenome and cell phenotype

体外细胞培养已经发展到允许单株细胞系连续繁殖。它被广泛应用于从基础研究实验室到生物技术公司的各个领域。然而，经典的二维细胞培养方法缺乏不同细胞类型之间密集的相互作用和通信。细胞对经典体外条件的适应诱导表型、转录组和表观遗传漂移，从而改变细胞行为、转录组和表观基因组。这些影响不利于我们对细胞生物学原理和分子机制的理解。事实上，一些研究表明，在从体内培养到体外培养的几个小时内，细胞转录组和表观基因组（DNA甲基化和染色质修饰）在体内的原始状态发生了深刻的改变。因此，更复杂的培养条件，如共培养（混合不同类型的细胞）和3D细胞培养（球体或类器官），已经开发出来，以更好地概括体内环境。三维共培养模型被认为可以更好地模拟微环境。然而，支持这一结论的数据缺乏，3D细胞培养和共培养对表型、转录组和表观基因组的影响也没有很好的表征。为了回答这些关于细胞培养方法影响的基本问题，我们开发了荧光上皮细胞和成纤维细胞的三维细胞培养模型。我们的研究计划有三个不同的目标：目标1：确定从2D到3D细胞培养条件下发生的表型，转录组和表观遗传变化。目的2：表征二维和三维共培养对转录组和表观基因组对单个细胞类型的影响。目的3：确定在体外和体内共培养中，直接和间接细胞相互作用对基因表达和表观遗传修饰的影响。本研究项目将揭示三维细胞培养和共培养模型中细胞相互作用对细胞表型、转录组和表观基因组的一般影响。该计划的结果将适用于几乎所有从事涉及细胞系、iPS细胞的基础研究的实验室；以及参与细胞治疗的生物技术。从我们的研究项目中得出的结论可能会为在实验室中使用细胞培养整合常规3D共培养提供理论依据。

In vitro cell culture has been developed to allow continuous propagation of single cell lines. It is being used in a wide range of applications from basic research laboratories to biotech companies. However, classical cell culture methods in 2 dimensions (2D) lack cell-cell dense interactions and communication between different cell types. Cell adaptation to classical in vitro conditions induces phenotypic, transcriptomic and epigenetic drifts, which alter cell behavior, transcriptome and epigenome. These effects are detrimental to our understanding of cell biology principles and molecular mechanisms. Indeed, several studies have revealed that within several hours after the transition from in vivo to in vitro culture, cell transcriptome and epigenome (DNA methylation and chromatin modifications) are modified deeply from the original status in vivo. Therefore, more complex culture conditions, such as co-culture (mixing different cell types together) and 3D cell culture (with spheroids or organoids), have been developed to better recapitulate the in vivo context. 3D co-culture models are considered to better mimic the microenvironment. However, data supporting this conclusion are lacking and the effects of 3D cell culture and co-culture on phenotype, transcriptome and epigenome are not well-characterized. To answer these basic questions on the effects of cell culture methods, we developed a 3D cell culture model of fluorescent epithelial cells and fibroblasts. Our research program has 3 different aims: AIM 1: Determine phenotypic, transcriptomic, and epigenetic changes occurring from 2D to 3D cell culture conditions. AIM 2: Characterize the effects of 2D and 3D co-culture on transcriptome and epigenome on individual cell type. AIM 3: Determine the impact of direct and indirect cell interactions on gene expression and epigenetic modifications in co-culture in vitro and in vivo. This research program will reveal general effects of cell interactions in 3D cell culture and co-culture models on cell phenotype, transcriptome and epigenome. The results of this program will be applicable to nearly all laboratories doing basic research involving cell lines, iPS cells; and to biotech involved in cell therapy. The conclusions emerging from our research program may bring the rationale to integrate routinely 3D co-culture in labs using cell culture.