Construction of Hybrid Cell Spheroids Using Cell-Sized Cross-Linked Nanogel Microspheres as an Artificial Extracellular Matrix

The introduction of functional material supports or spacers into cell spheroids increases the free volume, allowing oxygen, nutrients, and waste products to diffuse in and out more freely. Here, a biocompatible polysaccharide spacer material was investigated. Microspheres were prepared by cross-linking cholesterol-modified pullulan (CHP) nanogels with poly(ethylene glycol) (PEG). The ratio of modified CHP nanogel to PEG cross-linker was optimized to give uniform microspheres with an average diameter of approximately 14 μm. Rhodamine B-labeled microspheres showed a homogeneous assembly with bone marrow-derived mesenchymal stem cells (1:1 ratio) to create hybrid cell spheroids. The addition of the cross-linked nanogel spacers did not affect the cell viability, indicating that the microspheres provided a biocompatible scaffold that supported cell proliferation. In addition, the microspheres were stable under culture conditions over 14 days. The hybrid cell spheroids were scaled up to millimeter size to demonstrate their potential as a transplantable treatment, and the cells were found to maintain their high viability. The hybrid cell spheroids are expected to support the production of organoids.