Generation of multicellular tumor spheroids via 3D cell culture utilizing a hydrogel comprising chitosan and allylthiourea

Abstract

Hydrogels made with chitosan (CS) biopolymer can undergo mechanical and chemical modifications that can lead to new properties and functions in the biomedical field, which can be used for 3D cell culture as well as the formation of tumor spheroids that mimic the microenvironment of a tissue or organ. Chitosan-poly(allylthiourea) (CSATU)-based hydrogels were synthesized, which exhibit self-healing properties, biocompatibility, nontoxicity, and reasonable mechanical strength. The ratio of chitosan was varied to synthesize a series of CSATU hydrogels viz. CSATU-1 containing (CS : ATU) in a (2 : 1) ratio, CSATU-2 containing (CS : ATU) in a (1 : 1) ratio, and CSATU-3 containing (CS : ATU) in a (1 : 2) ratio. The swelling of all CSATU hydrogels was found to be maximum in a neutral medium. The maximum mechanical strength was demonstrated by the CSATU-1 hydrogel among all hydrogels. The surface morphology of the hydrogels was analyzed using AFM and SEM, while the thermal stability was determined using TGA, revealing that the hydrogels were stable up to 200 °C, and the T_g values of CS and ATU were observed at approximately 97 °C and 74 °C, respectively. Although there is minimal variation among the T_g values of each hydrogel, a significant endothermic peak was observed at around 60–70 °C for all hydrogels, and the semi-crystalline nature was analyzed using powder XRD. However, the CSATU hydrogels were characterized using FT-IR spectroscopy. Furthermore, the CSATU hydrogels have been explored to investigate the biocompatibility of the cells. Our results revealed that the CSATU hydrogels were nontoxic to the T-cell lymphoma and MCF-7 cells. Additionally, all three hydrogels have successfully established the formation of multicellular tumor spheroids. Overall, our present study demonstrated that the synthesized CSATU hydrogels can be used as a promising platform for spheroid development, which has potential applications in tissue engineering and drug development.