Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan; Department of Materials Science and Engineering, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan.
Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.
Acta Biomater. 2021 Apr 15;125:100-111. doi: 10.1016/j.actbio.2021.01.042. Epub 2021 Jan 30.
Transmembrane transport of exogenous genes is widely investigated because of high demand for gene therapy. Both gene carriers and cellular conditions can affect gene transfection efficiency. Although cell morphology has been reported to affect cell functions, the influence of cell adhesion area and cell spreading area on the transfection of exogenous genes remains unclear because it is difficult to separate the individual influence of these areas during normal cell culture. In this study, micropatterns were prepared to separately control the adhesion and spreading areas of human bone marrow-derived mesenchymal stem cells (hMSCs). Transfection efficiency of the green fluorescent protein gene to hMSCs cultured on the micropatterns was compared. Cells with a larger adhesion area showed higher transfection efficiency, while cell spreading area hardly affected gene transfection efficiency. Cell adhesion area had dominant influence on gene transfection. Microparticle uptake and BrdU staining showed that the cellular uptake capacity and DNA synthesis activity increased with the increase in cell adhesion area, but were not affected by cell spreading area. The different influence of cell adhesion area and cell spreading area on gene transfection was correlated with their influence on cellular uptake capacity, DNA synthesis activity, focal adhesion formation, cytoskeletal mechanics, and mechanotransduction signal activation. The results suggest that cell adhesion area and cell spreading area had different influence on gene transfection; this finding should provide useful information for the manipulation of cell functions in gene therapy, protein modification, and cell reprogramming. STATEMENT OF SIGNIFICANCE: Cell adhesion and spreading are important morphological factors during the interaction of cells with biomaterial surfaces or interfaces. However, the predominant morphological factor that affects cellular functions such as gene transfection remains unclear. In the present study, special micropatterns were used to precisely control cell adhesion and spreading areas independently. Mesenchymal stem cells cultured on the micropatterns were transfected with the green fluorescent protein gene to compare the different influence of cell adhesion and spreading areas on gene transfection efficiency. Cell adhesion area showed dominant influence on gene transfection, while cell spreading area did not affect gene transfection. The dominant influence of cell adhesion area could be explained by cellular uptake capacity and DNA synthesis activity through the formation of FAs, cytoskeletal mechanics, and YAP/TAZ nuclear localization. The results provide new insights of correlation between cell morphology and cellular functions for designing functional biomaterials.
外源性基因的跨膜转运因其对基因治疗的高需求而受到广泛研究。基因载体和细胞状态都可以影响基因转染效率。尽管细胞形态已被报道会影响细胞功能，但由于在正常细胞培养过程中难以分离这些区域的单独影响，细胞黏附面积和细胞铺展面积对外源性基因转染的影响仍不清楚。在本研究中，制备了微图案以分别控制人骨髓间充质干细胞（hMSC）的黏附面积和铺展面积。比较了在微图案上培养的 hMSC 的绿色荧光蛋白基因的转染效率。具有较大黏附面积的细胞显示出更高的转染效率，而细胞铺展面积几乎不影响基因转染效率。细胞黏附面积对基因转染具有主导影响。微颗粒摄取和 BrdU 染色表明，细胞摄取能力和 DNA 合成活性随细胞黏附面积的增加而增加，但不受细胞铺展面积的影响。细胞黏附面积和细胞铺展面积对基因转染的不同影响与它们对细胞摄取能力、DNA 合成活性、焦点黏附形成、细胞骨架力学和机械转导信号激活的影响有关。结果表明，细胞黏附面积和细胞铺展面积对基因转染有不同的影响；这一发现应该为基因治疗、蛋白质修饰和细胞重编程中细胞功能的操作提供有用的信息。
细胞黏附和铺展是细胞与生物材料表面或界面相互作用过程中的重要形态学因素。然而，影响细胞功能（如基因转染）的主要形态学因素仍不清楚。在本研究中，使用特殊的微图案精确地独立控制细胞的黏附面积和铺展面积。将绿色荧光蛋白基因转染培养在微图案上的间充质干细胞，以比较细胞黏附面积和铺展面积对基因转染效率的不同影响。细胞黏附面积对基因转染表现出主导影响，而细胞铺展面积不影响基因转染。细胞黏附面积的主导影响可以通过 FA 的形成、细胞骨架力学和 YAP/TAZ 核定位来解释，这与细胞摄取能力和 DNA 合成活性有关。结果为设计功能性生物材料提供了细胞形态与细胞功能之间相关性的新见解。
