Designing Cross-Linked Nanogel Microfibers for the Fabrication of Hybrid Cell Spheroids with Potential Biomedical Applications

Cell spheroids are three-dimensional spherical aggregates of cells that show advantages for mimicking living tissues, owing to their structure. However, issues such as cell necrosis often result in limited cell survival and functional expression. The introduction of biocompatible functional material spacers into cell spheroids can not only improve cell survival by facilitating free diffusion of oxygen, nutrients, and waste products but also enables the incorporation of novel functions into the cell spheroids. In this study, we focused on cholesterol-modified pullulan (CHP) nanogels, which exhibit protein complexation and controlled release properties. The microfiber spheroid spacers were produced by cross-linking the CHP nanogels with poly(ethylene glycol) (PEG). Using freeze-thaw cycles and ultrasonic irradiation, we successfully developed a simple and high-yielding method for microfiber fabrication. The microfibers retained the protein complexation and controlled the release properties of the CHP nanogels. Furthermore, fluorescence-labeled microfibers were integrated uniformly with mouse myoblast cells (C2C12) to give hybrid cell spheroids. The hybrid cell spheroids were scaled up to millimeter size, demonstrating that the cells maintained a high viability. The hybrid cell spheroids are expected to support not only the construction of organoids but also advances in cell therapy and the development of new treatment modalities.