Characterization of ECM produced by MC3T3-E1 cellular spheroids encapsulated in alginate hydrogel

Abstract

The application of cellular spheroids in bone tissue engineering research has gained significant interest in the last decade. Compared to monolayer cell cultures, the 3D architecture allows for more physiological cell-cell and cell-extracellular matrix (ECM) interactions that make cellular spheroids a suitable model system to investigate bone ECM in vitro. The use of 3D model systems requires fine-tuning of experimental methods used to study cell morphology, ECM deposition and mineralization, and cell-ECM interactions. In this study, we use MC3T3-E1 cellular spheroids encapsulated in an alginate hydrogel to study and characterize the deposited ECM. Spheroid shape and structure were evaluated using confocal microscopy. The deposited collagenous ECM was characterized using quantitative assay and microscopy, in particular Second Harmonic Imaging Microscopy (SHIM), hydroxyproline (HYP) assay and Transmission Electron Microscopy (TEM). The use of hydrogel constructs allows easy handling and imaging of the samples and helps to preserve the spheroids stability by preventing cells from adhering to the culture dish surface. We use a non-modified alginate hydrogel that does not facilitate cell attachment and therefore functions as an inert encapsulating scaffold. Constructs were cultured for up to 4 weeks. SHIM, HYP assay and TEM confirmed the deposition of collagenous matrix by the spheroid, with most of it taking place between week 2 and 4. We demonstrated that alginate encapsulated bone spheroids are a promising model for studying bone ECM in vitro.