Unicellular and multicellular spheroids are innovative culture platforms to recapitulate the in vivo microenvironment for various pathological conditions, such as tumor models and therapeutic research. Despite their advantages in biomedical fields, the lack of integrity and compactness is the main challenge in a prolonged culture system. This study aimed to develop and scale up high-throughput, long-term, and stable cancer spheroids with certain hydrogel composites using the hanging drop approach.

To better mimic tumor spheroid formation and enhance HT-29 cell cohesion, a novel hydrogel-based matrix composed of methylcellulose (MC) was fabricated. To this end, MC was modified using 3-aminopropyltriethoxysilane (3-APTES) and cross-linked with polyethylene glycol (PEG) and gelatin (GEL). HT-29 cells were encapsulated inside MC, modified methyl cellulose (MMC), MMC/PEG, MMC/GEL, and MMC/PEG/GEL hydrogels. General features of hydrogels were characterized using FTIR, NMR, Raman spectroscopy, XRD, DLS, FE-SEM, EDS, and rheology. The survival rate, angiogenesis capacity, compactness, circularity, and Wnt signaling pathway activity were also monitored over time.

Data indicated appropriate physicochemical properties of MC-based spheroids, especially in the MMC/PEG/GEL group. The incorporation of different substrates into the final composite was also confirmed. MTT assay indicated enhanced HT-29 cell viability inside spheroids composed of MMC/PEG/GEL compared to the other groups (p < 0.05). These features coincided with proper integrity, circularity, compactness, and up-regulation of VEGF and HIF-1α in the MMC/PEG/GEL group (p < 0.05). PCR array analysis showed the up-regulation of several effectors related to the Wnt signaling pathway in MMC/PEG/GEL spheroids as compared to the other groups (p < 0.05).

Taken together, the MMC/PEG/GEL hydrogel composite provides a suitable niche for proper cell-to-cell and cell-to-matrix interaction, leading to the generation of comparable 3D cultured spheroids to a biomimetic tumor mass.