Three-dimensional (3D) cell culture systems have promising applications compared with conventional two-dimensional cell culture systems. Herein, we report a facile method for the formation of 3D spheroids using novel thermo-reversible polysaccharide-based hydrogels. A series of thermo-reversible hydrogels consisting of N-acyl glycol chitosans (NAGCs) are synthesized through a simple N-acylation reaction, and the degree of acylation is finely tuned to obtain adequate thermo-reversible properties and gel stability. Among the NAGCs, N-hexanoyl glycol chitosan is the most thermo-sensitive and is highly effective for forming multi-cellular spheroids when used to coat the surfaces of cell culture dishes. Cell spheroids are effectively formed at various cell concentrations, and their spheroid shape and cellular functions are well maintained for longer times. The hydrogel culture system is also useful for co-cultures that mimic a biological microenvironment. Our thermo-reversible hydrogels may offer a convenient method for the development of in vitro 3D cell culture systems to provide enhanced performance in tissue regeneration, organ-on-chips, drug screening research and other biomedical applications.

与传统的二维细胞培养系统相比，三维（3D）细胞培养系统具有广阔的应用前景。在此，我们报告了一种使用新型热可逆多糖基水凝胶形成3D球体的简便方法。通过简单的N-酰化反应合成了一系列由N-酰基乙二醇壳聚糖（NAGC）组成的热可逆性水凝胶，并对酰化度进行了微调以获得足够的热可逆性和凝胶稳定性。在NAGC中，N-己酸二醇壳聚糖是最热敏感的，当用于覆盖细胞培养皿的表面时，对形成多细胞球体非常有效。细胞球体可以在各种细胞浓度下有效形成，并且它们的球体形状和细胞功能可以长期保持良好状态。水凝胶培养系统还可用于模拟生物微环境的共培养。我们的热可逆水凝胶可为体外3D细胞培养系统的开发提供便利的方法，从而在组织再生，芯片上的器官，药物筛选研究和其他生物医学应用中提供增强的性能。