Engineered hydrogels have been extensively used to direct cell function in 3D cell culture models, which are more representative of the native cellular microenvironment than conventional 2D cell culture. Previously, hyaluronan-furan and bis-maleimide polyethylene glycol hydrogels were synthesized via Diels–Alder chemistry at acidic pH, which did not allow encapsulation of viable cells. In order to enable gelation at physiological pH, the reaction kinetics were accelerated by replacing the hyaluronan-furan with the more electron-rich hyaluronan-methylfuran. These new click-cross-linked hydrogels gel faster and at physiological pH, enabling encapsulation of viable cells, as demonstrated with 3D culture of 5 different cancer cell lines. The methylfuran accelerates Diels–Alder cycloaddition yet also increases the retro Diels–Alder reaction. Using computational analysis, we gain insight into the mechanism of the increased Diels–Alder reactivity and uncover that transition state geometry and an unexpected hydrogen-bonding interaction are important contributors to the observed rate enhancement. This cross-linking strategy serves as a platform for bioconjugation and hydrogel synthesis for use in 3D cell culture and tissue engineering.

中文翻译：

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具有更高反应性的Diels–Alder点击交联水凝胶可实现3D细胞封装

工程水凝胶已广泛用于指导3D细胞培养模型中的细胞功能，该模型比常规2D细胞培养更能代表天然细胞微环境。以前，透明质酸呋喃和双马来酰亚胺聚乙二醇水凝胶是通过Diels-Alder化学方法在酸性pH值下合成的，无法封装活细胞。为了能够在生理pH下凝胶化，通过用电子含量更高的乙酰透明质酸-甲基呋喃代替乙酰透明质酸-呋喃来加速反应动力学。这些新的单击交联的水凝胶在生理pH值下更快地凝胶化，从而能够包裹活细胞，这是由5种不同癌细胞系的3D培养所证明的。甲基呋喃加速了Diels-Alder的环加成反应，但也增加了Diels-Alder的逆反应。通过计算分析，我们深入了解了Diels-Alder反应性增加的机理，并发现过渡态的几何形状和意外的氢键相互作用是观察到的速率提高的重要因素。这种交联策略可作为生物偶联和水凝胶合成的平台，用于3D细胞培养和组织工程。