Focused Ion beam analysis of cell growth in 3D interconnected porous structure scaffold

Hydrogels are synthetic or natural polymer networks that have emerged as promising candidates for 3D tissue engineering scaffolds. In the past several years, research interest has shifted from hydrogel implants to injectable formulations, which have the advantage that cells and bioactive compounds can be mixed easily with precursor solutions prior to gelation to give homogeneously loaded gels. In addition, in situ gelation allows the formation of complex shapes and can be applied using minimally invasive surgery. However, electron imaging of cell growth in situ to understand cell behaviour and activity is still challenging, especially when cells are growing in porous extracellular matrix (ECM)-like structures. 3D porous hydrogels of high permeability and biocompatible structure can mimic the microenvironment of ECM, but for high resolution imaging, there are still obstacles to overcome. Porous microstructures, with or without residing cells, are not appropriate for microtomy, and thus transmission electron microcopy (TEM) imaging is extremely difficult to apply for study of cell-hydrogel interfaces. The other alternative, scanning electron microscopy (SEM), is limited to observation of the surface region only and is not suitable for probing 3D scaffolds. In this study, we obtained images of the cellhydrogel interface by exposing and probing target samples using focused ion beam (FIB) milling. Hydrogels were prepared and mixed with African green monkey kidney cells (COS-7 cells) followed by in situ polymerization catalyzed by horseradish peroxidase (HRP) and diluted H2O2. The samples were fixed using plunge freezing and then freeze-dried. We demonstrate that FIB milling and associated microanalysis techniques are effective for 3D characterization of cells grown inside hydrogel scaffolds. As thin nano-scale slices were removed continuously by FIB, cell growth inside the scaffold was captured in serial images acquired by SEM. 3D reconstruction was then used to elucidate the intricate porous microstructure of the scaffold and its relationship with the embedded cells.