Cell seeding in a biomaterial is an important process for tissue engineering applications. It helps to modulate tissue formation or to control initial conditions for mechanobiological studies. The compression release-induced suction (CRIS) seeding technique leads to active infiltration of the cell suspension toward the central region of the scaffold. Its effectiveness, however, may significantly vary depending on several controlling factors such as the rate of loading and unloading or scaffold architecture. We utilized a 2D axisymmetric finite element model to numerically evaluate the influence of a seeding loading regime on suction pressure and infiltration velocity of the cell suspension. The in vitro application of optimized CRIS seeding obtained from simulation showed significant effectiveness over a static seeding method. As simulation results predicted, the permeability of the scaffold directly influenced CRIS seeding effectiveness in vitro. By supplementing an optimized CRIS loading regime with slow rotation after seeding treatment, cell distribution through thickness was improved especially for scaffolds showing low permeability. Finally, we systematically analyzed the relative importance of permeability, thickness, or coating on cell seeding efficiency and uniformity using a full factorial design of experiments. We observed that permeability has a higher impact on the CRIS seeding than scaffold coating and thickness.