Human mesenchymal stem cells (hMSC) currently represent a major cell resource in the research laboratory, to study differentiated-cell behavior in 3D scaffolds during the regeneration processes. Adhesion and differentiation of stem cells to a specific phenotype are achieved by culturing them in apposite culture media under precise conditions. Meanwhile, hydrolytic degradation of polymeric scaffolds allows implanted cells to synthesize their own extracellular matrix in situ after implantation so that the degeneration of the foreign scaffold is temporally matched by creation of the new innate one. In this context, structural properties and biochemical signals may concur to influence the cell response to the environmental stimuli during the culture. So, it becomes mandatory to introduce robust protocols to treat hMSC alone-before the culture-and in combination with the scaffolds for the next investigation by scanning electron microscopy. Here, we describe the protocols used to manage hMSC before and during the culture in order to obtain more detailed information on cell mechanisms mediated by polymeric scaffolds.