- Abstract The pore architecture in 3-D polymeric scaffoldings plays a critical role in tissue engineering as it provides the framework for the seeded cells to organize into a functioning tissue. In the present paper, we investigate the effect of freezing regime on the pore microstructure in 3-D alginate scaffolds, fabricated by the freeze-dry method. The scaffolds have shown isotropic pore structure, when the calcium crosslinked alginate solutions were slowly frozen at −20°C, in a nearly homogenous cold atmosphere; the pores were spherical and interconnected. In contrast, when the cooling process was performed in liquid nitrogen or oil bath, where a temperature gradient was formed along the freezing solution, two main regions of pore structure were noted; at the interface with the cooling medium, small spherical pores were seen and above them a region with elongated pores. The different pore shape affected the compressibility of the scaffolds, while it had no effect on albumin diffusion. Rat hepatocytes seeded within the scaffolds were arranged according to the their pore shape. In scaffolds with elongated pores, the cells were lining along the pores, thus forming lines of interacting cells. In the scaffolds with the isotropic spherical pores, the hepatocytes clustered into spheroid-like aggregates. Thus, it appears that pore shape can modulate hepatocyte morphogenesis.