Brock Judith, Hörning Marcel
Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany.
Front Cell Dev Biol. 2024 Nov 22;12:1501540. doi: 10.3389/fcell.2024.1501540. eCollection 2024.
As a reliable alternative to animal testing in cardiovascular research, it is crucial to improve differentiation of immortalized cell lines. In this study, we focused on optimizing the differentiation efficiency of the H9c2 cell line into cardiomyocytes using a high-throughput, automated image processing approach. While previous studies used protocols involving retinoic acid to enhance cardiac differentiation, we applied a simplified medium composition that results in higher differentiation rates. Along that line, we differentiated H9c2 cells into cardiomyocytes, which not only showed sarcomere-characteristic striation but also periodic intracellular calcium signaling for the first time. As a second step, we examined the potential application of polyacrylamide hydrogels ( kPa) with defined fibronectin coating densities. The optimum fibronectin density of 2.6 μg/cm found for single cells was investigated to further improve the differentiation efficiency. However, the differentiation and proliferation dynamics dominate the adhesion forces between the cells and the hydrogel, and thus, result in premature clustering and detachment. In conclusion, we identified an optimized differentiation protocol and provided a basis for the further investigation necessary to potentially use hydrogels as natural cell environment, aiming to raise the differentiation efficiency even more.
作为心血管研究中动物实验的可靠替代方法，提高永生化细胞系的分化能力至关重要。在本研究中，我们专注于使用高通量自动化图像处理方法优化H9c2细胞系向心肌细胞的分化效率。虽然先前的研究使用涉及视黄酸的方案来增强心脏分化，但我们应用了一种简化的培养基成分，其可导致更高的分化率。据此，我们将H9c2细胞分化为心肌细胞，这些细胞不仅首次显示出肌节特征性条纹，还呈现出周期性的细胞内钙信号。第二步，我们研究了具有确定纤连蛋白包被密度的聚丙烯酰胺水凝胶（kPa）的潜在应用。研究了单细胞的最佳纤连蛋白密度为2.6 μg/cm²，以进一步提高分化效率。然而，分化和增殖动态主导着细胞与水凝胶之间的粘附力，从而导致过早聚集和脱离。总之，我们确定了一种优化的分化方案，并为进一步研究提供了基础，该研究可能将水凝胶用作天然细胞环境，旨在进一步提高分化效率。
