Yuan Hongbo, Liu Kaizheng, van Velthoven Melissa J J, Kumari Jyoti, Bao Yuying, Rocha Susana, Kouwer Paul H J
Innovation and Research Institute of Hebei University of Technology in Shijiazhuang, Shijiazhuang, China.
Molecular Imaging and Photonics, Chemistry Department, KU Leuven, Leuven, Belgium.
Nat Protoc. 2025 May 30. doi: 10.1038/s41596-025-01159-3.
Three-dimensional (3D) cell culture models based on hydrogels are rapidly evolving into a prominent tool for tissue engineering, mechanobiology, disease modeling and drug screening. While a vast variety of synthetic gels have emerged in recent years, they fail to penetrate the market substantially for two major reasons: they poorly mimic the extracellular matrix or they are difficult to use in gel formation and cell extraction. Mimicking the complexity of nature is challenging: the extracellular matrix plays a crucial role in cell development and function, which goes well beyond simple mechanical support. Recently, we introduced polyisocyanide (PIC) hydrogels for 3D cell culture applications. The fibrous architecture and associated (non)linear mechanical behavior closely mimic the physical properties of biogels such as collagen and fibrin. As fully synthetic materials, PIC gels benefit from high tailorability and reproducibility. Moreover, the thermoresponsive properties of PIC gels make them easy to handle in the lab; the gels form instantly at 37 °C and cells are easily extracted after cooling to 5 °C. The potential of PIC gels has been demonstrated in a quickly expanding library of papers discussing different cell lines, primary cells and organoids, as well as in vivo experiments. This manuscript provides protocols on how to handle PIC gels in the chemistry and cell biology laboratories. Material preparation requires 72 h. Cell encapsulation takes 1 h and the time for downstream analysis depends on the (commercial) methods used. The protocols described are suitable for researchers with expertise in cell culture and molecular biology.
基于水凝胶的三维（3D）细胞培养模型正迅速发展成为组织工程、力学生物学、疾病建模和药物筛选的重要工具。近年来，虽然出现了各种各样的合成凝胶，但它们未能在市场上取得重大突破，主要有两个原因：它们对细胞外基质的模拟效果不佳，或者在凝胶形成和细胞提取方面难以操作。模仿自然的复杂性具有挑战性：细胞外基质在细胞发育和功能中起着至关重要的作用，这远远超出了简单的机械支撑。最近，我们引入了聚异氰化物（PIC）水凝胶用于3D细胞培养应用。其纤维结构和相关的（非）线性力学行为与胶原蛋白和纤维蛋白等生物凝胶的物理特性非常相似。作为完全合成的材料，PIC凝胶具有高度的可定制性和可重复性。此外，PIC凝胶的热响应特性使其在实验室中易于操作；凝胶在37°C时立即形成，冷却至5°C后细胞很容易被提取出来。PIC凝胶的潜力已在越来越多讨论不同细胞系、原代细胞和类器官的论文库以及体内实验中得到证明。本手稿提供了在化学和细胞生物学实验室中处理PIC凝胶的方案。材料制备需要72小时。细胞封装需要1小时，下游分析的时间取决于所使用的（商业）方法。所描述的方案适用于具有细胞培养和分子生物学专业知识的研究人员。
