Ionically crosslinked biohybrid gelatin-based hydrogels for 3D cell culture.

The transition from two-dimensional to three-dimensional cell cultures has transformed the understanding of cell physiology and cell-matrix interactions. Extracellular matrix (ECM) mimics tend to fall into either the natural or synthetic categories. Naturally occurring ECM mimics, such as collagen and gelatin, have superior bioactive properties but typically lack tuneability. Conversely, synthetic ECM mimics are highly defined but even with modifications, can lack the bioactivity of natural proteins. Therefore, to take advantage of the potential of both natural and synthetic ECM mimics, a biohybrid ionically crosslinked gelatin hydrogel was synthesised. This was achieved by utilising free amine groups along the gelatin backbone as the basis for a reversible addition - fragmentation chain-transfer (RAFT) reaction. The resulting polymers had tuneable stiffness and enhanced solubility compared to gelatin. The biohybrid gel also showed good biocompatibility, with MCF-7 cells forming larger spheroids when encapsulated within the biohybrid gel when compared to an unfunctionalized polyethylene-glycol (PEG) gel. Furthermore, due to the ionic crosslinking in the biohybrid gel, spheroids can be retrieved by digesting the matrix using 10 × phosphate-buffered saline (PBS). Retrieved cells were shown to be viable which allows for the potential of downstream analysis. Thus, this study highlights the potential of hybrid gelatin-PEG hydrogels for 3D cell culture. Graphical abstract: The biohybrid gelatin (Gelatin-SPMA) is crosslinked with a positively charged polymer (PEG-MAETMA) to form a gel within seconds. MCF-7 cells survived encapsulation and formed spheroids over 7 days. 10x phosphate buffered saline (PBS) was then used to digest the hydrogel, allowing for the recovery of encapsulated spheroids. Supplementary Information: The online version contains supplementary material available at 10.1007/s13233-025-00380-z.