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We have studied the suitability of a cryogenic process (e.g., ice-segregation-induced self-assembly, ISISA) for preparation of polyvinyl alcohol (PVA) scaffolds with in situ immobilized bacteria (e.g., Escherichia coli). Confocal fluorescence microscopy and impedance measurements were used to evaluate the extension of bacteria proliferation within the scaffold macrostructure. The bacteria efficiency for colonization of the scaffold macrostructure is compared for bacteria immobilized with and without the use of additional cryoprotectans. Cryoprotection by bacteria entrapment in alginate beads containing glucose results in a significant improvement (more than 2-fold as compared to non-cryoprotected) of bacteria proliferation within the PVA scaffold. Results are also compared with the most widely used method for cells colonization of scaffolds; i.e., soaking of a preformed PVA scaffold in bacteria culture medium.