Abstract
3D hydrogels better replicate in vivo conditions, and yield different results from 2D substrates. However, imaging interactions between cells and the hydrogel microenvironment is challenging because of light diffraction and poor focal depth. Here, cryosectioning and vibrating microtomy methods and fixation protocols are compared. Collagen I/III hydrogel sections (20-100 µm) are fixed with paraformaldehyde (2%-4%) and structurally evaluated. Cryosectioning damaged hydrogels, and vibrating microtomy (100 µm, 2%) yielded the best preservation of microstructure and cell integrity. These results demonstrate a potential processing method that preserves hydrogel and cell integrity, permitting imaging of cell interactions with the microenvironment.