Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Essen 45117, Germany.
Imaging Center Campus Essen (ICCE), University of Duisburg-Essen, Universitaetsstr. 5-7, 45117 Essen, Germany.
Acta Biomater. 2019 Jan 15;84:391-401. doi: 10.1016/j.actbio.2018.11.051. Epub 2018 Nov 29.
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.
将核酸转入活细胞，即转染，是当前分子生物学和医学的主要技术。由于核酸本身不能穿透细胞膜，因此需要一种有效的载体。由于具有生物相容性、可生物降解性和与 DNA 或 RNA 等核酸的高亲和力，磷酸钙纳米颗粒可用作载体。在这里，它们的应用从二维 (2D) 扩展到了包括共培养物在内的三维 (3D) 细胞培养模型。与 2D 单层细胞培养相比，3D 培养系统更能代表真实的空间、生化和细胞环境。通过共焦激光扫描显微镜研究了荧光磷酸钙纳米颗粒（直径 40-70nm；阳离子）在 2D 和 3D 细胞培养模型中的摄取情况。通过 2D 和 3D 细胞培养比较了磷酸钙纳米颗粒对 eGFP 的转染，包括在 2D 和 3D 模型中对绿色荧光 HeLa-eGFP 细胞和红色荧光蛋白 mCherry 的 MG-63 细胞的共培养。这允许对局部转染效率进行细胞特异性评估。一般来说，纳米颗粒进入球体的穿透率明显高于由 Lipofectamine 携带的模型寡核苷酸。3D 细胞培养中的转染效率与 2D 细胞培养相当，但它主要发生在球体的表面，遵循纳米颗粒的摄取途径。
三维细胞培养模型可以作为体外细胞培养与体内情况之间的桥梁，特别是在需要考虑传质效应的情况下。对于纳米颗粒来说就是这种情况，因为在二维细胞培养中的孵育效果与三维细胞培养或活体组织有很大的不同。我们比较了纳米颗粒的摄取以及随后在二维和三维细胞培养模型中荧光蛋白的转染。使用共培养的 HeLa-eGFP 细胞（绿色荧光）与用红色荧光蛋白 mCherry 转染的非荧光 MG-63 细胞一起开发了一种研究转染的优雅模型。由此，很容易区分共培养物中两种细胞类型的转染。
