Optimization of H9c2 differentiation leads to calcium-active and striated cardiac cells without addition of retinoic acid

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 ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>E</mml:mi> <mml:mo>=</mml:mo> <mml:mn>12</mml:mn></mml:mrow> </mml:math> kPa) with defined fibronectin coating densities. The optimum fibronectin density of 2.6 μg/cm² 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.