The transfer of nucleic acids into living cells, i.e. transfection, is a major technique in current molecular biology and medicine. As nucleic acids alone are not able to penetrate the cell membrane, an efficient carrier is needed. Calcium phosphate nanoparticles can serve as carrier due to their biocompatibility, biodegradability and high affinity to nucleic acids like DNA or RNA. Their application was extended here from two-dimensional (2D) to three-dimensional (3D) cell culture models, including co-cultures. Compared to 2D monolayer cell cultures, a 3D culture system represents a more realistic spatial, biochemical and cellular environment. The uptake of fluorescent calcium phosphate nanoparticles (diameter 40-70 nm; cationic) was studied in 2D and 3D cell culture models by confocal laser scanning microscopy. The transfection of eGFP by calcium phosphate nanoparticles was compared in 2D and 3D cell culture, including co-cultures of green fluorescing HeLa-eGFP cells and MG-63 cells in 2D and in 3D models with the red fluorescent protein mCherry. This permitted a cell-specific assessment of the local transfection efficiency. In general, the penetration of nanoparticles into the spheroids was significantly higher than that of a model oligonucleotide carried by Lipofectamine. The transfection efficiency was comparable in 3D cell cultures with 2D cell cultures, but it occurred preferentially at the surface of the spheroids, following the uptake pathway of the nanoparticles. STATEMENT OF SIGNIFICANCE: Three-dimensional cell culture models can serve as a bridge between the in-vitro cell cultures and the in-vivo situation, especially when mass transfer effects have to be considered. This is the case for nanoparticles where the incubation effect in a two-dimensional cell culture strongly differs from a three-dimensional cell culture or a living tissue. We have compared the uptake of nanoparticles and a subsequent transfection of fluorescent proteins in two-dimensional and three-dimensional cell culture models. An elegant model to investigate the transfection in co-cultures was developed using HeLa-eGFP cells (green fluorescent) together with MG-63 cells (non-fluorescent) that were transfected with the red-fluorescing protein mCherry. Thereby, the transfection of both cell types in the co-culture was easily distinguished.

中文翻译：

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磷酸钙纳米颗粒介导的2D和3D单培养和共培养细胞模型中的转染。

核酸转移到活细胞中，即转染，是当前分子生物学和医学中的一项主要技术。由于仅核酸不能穿透细胞膜，因此需要有效的载体。磷酸钙纳米颗粒由于其生物相容性，生物可降解性以及对核酸（如DNA或RNA）的高度亲和力而可以用作载体。他们的应用在这里从二维（2D）扩展到了三维（3D）细胞培养模型，包括共培养。与2D单层细胞培养相比，3D培养系统代表了更现实的空间，生化和细胞环境。通过共聚焦激光扫描显微镜研究了2D和3D细胞培养模型中荧光磷酸钙纳米颗粒（直径40-70 nm；阳离子）的摄取。在2D和3D细胞培养中比较了磷酸钙纳米颗粒对eGFP的转染，包括在2D和3D模型中将绿色荧光HeLa-eGFP细胞和MG-63细胞与红色荧光蛋白mCherry共培养。这允许对局部转染效率进行细胞特异性评估。通常，纳米颗粒对球体的渗透明显高于脂转染胺所携带的模型寡核苷酸的渗透。转染效率在3D细胞培养物中与2D细胞培养物中可比，但是它优先发生在纳米颗粒的摄取途径之后的球体表面。意义说明：三维细胞培养模型可以充当体外细胞培养与体内情况之间的桥梁，特别是在必须考虑传质效应的情况下。对于纳米颗粒而言，在二维细胞培养物中的孵育效果与三维细胞培养物或活组织有很大不同。我们在二维和三维细胞培养模型中比较了纳米颗粒的摄取和随后荧光蛋白的转染。使用HeLa-eGFP细胞（绿色荧光）和用红色荧光蛋白mCherry转染的MG-63细胞（非荧光），开发了一种研究共培养中转染的优雅模型。由此，容易区分共培养中两种细胞类型的转染。对于纳米颗粒而言，在二维细胞培养物中的孵育效果与三维细胞培养物或活组织有很大不同。我们在二维和三维细胞培养模型中比较了纳米颗粒的摄取和随后荧光蛋白的转染。使用HeLa-eGFP细胞（绿色荧光）和用红色荧光蛋白mCherry转染的MG-63细胞（非荧光），开发了一种研究共培养中转染的优雅模型。因此，容易区分共培养中两种细胞类型的转染。对于纳米颗粒而言，在二维细胞培养物中的孵育效果与三维细胞培养物或活组织有很大不同。我们在二维和三维细胞培养模型中比较了纳米颗粒的摄取和随后荧光蛋白的转染。使用HeLa-eGFP细胞（绿色荧光）和用红色荧光蛋白mCherry转染的MG-63细胞（非荧光），开发了一种研究共培养中转染的优雅模型。因此，容易区分共培养中两种细胞类型的转染。使用HeLa-eGFP细胞（绿色荧光）和用红色荧光蛋白mCherry转染的MG-63细胞（非荧光），开发了一种研究共培养中转染的优雅模型。由此，容易区分共培养中两种细胞类型的转染。使用HeLa-eGFP细胞（绿色荧光）和用红色荧光蛋白mCherry转染的MG-63细胞（非荧光），开发了一种研究共培养中转染的优雅模型。因此，容易区分共培养中两种细胞类型的转染。