Nanocellulose has emerged as a promising alternative to animal-derived hydrogels for 3D cell culture applications. In this study, a crosslinkable 3D granular hydrogel system was developed using cellulose nanofibril methacrylate (CNFMA), which possesses intrinsic dual-crosslinking capability. This system was designed with CNFMA alone or in combination with polyacrylamide (PAA) to form a copolymer network (CNFMA+PAA). The dual-crosslinking behavior stemmed from the distinct reactive groups present in CNFMA—carboxyl groups facilitating ionic crosslinking and methacrylate groups enabling photocrosslinking—allowing precise modulation of hydrogel mechanics.  
The dual-crosslinking strategy was implemented in two steps: first, photocrosslinking was employed to generate bulk CNFMA hydrogels, which were then fragmented into microgels through mechanical sieving. These microgels were subsequently reassembled via secondary ionic crosslinking, forming a macroporous granular hydrogel. The presence of interconnected macropores was evaluated using Calcofluor White staining. Rheological assessments indicated good injectability and handling properties, while nanoindentation measurements revealed localized stiffness variations introduced by PAA. The mechanical properties of the hydrogel were tunable by varying the CNFMA-only and PAA content, offering a customizable microenvironment.  
Long-term (14-day) 3D culture experiments demonstrated that MC3T3-E1 and PANC-1 cells exhibited distinct responses to different mechanical microenvironments. A homogeneous, low-stiffness CNFMA matrix promoted integrin-mediated adhesion of PANC-1 cells, leading to a spread, sheet-like morphology. Conversely, the CNFMA-PAA (CMPAA) system, with localized stiffness variations induced by PAA, enhanced cytoskeletal tension in MC3T3-E1 cells while encouraging PANC-1 cells to adopt a more compact spheroid structure.  
By integrating dual-crosslinking capability, tunable stiffness, and a xeno-free composition, this granular hydrogel system provides a versatile platform for 3D cell culture, with potential applications in tissue engineering and cancer research.