Hydrogel Scaffolds with Immobilized Bacteria for 3D Cultures Marı ´a C. Gutie ´rrez, ² Zaira Y. Garcı ´a-Carvajal, ² Matı ´as Jobba ´gy, ² Luis Yuste, ‡ Fernando Rojo, ‡ Concepcio ´n Abrusci, § Fernando Catalina, ⊥ Francisco del Monte,* ,² and Marı ´a L. Ferrer ² Institute of Materials Science at Madrid (ICMM) and Biotechnology National Center (CNB), Spanish Research Council (CSIC), Cantoblanco, 28049 Madrid, Spain, Institute of Polymers Science and Technology (ICTP), Spanish Research Council (CSIC), Juan de la CierVa, 7. 28006 Madrid, Spain, and Department of Microbiology III, Faculty of Biology, UniVersity Complutense of Madrid, Jose ´ Antonio NoVais, 2. 28040 Madrid, Spain ReceiVed December 5, 2006. ReVised Manuscript ReceiVed January 25, 2007 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. Introduction The pressures of an ever-increasing population and industrial development have led to the addition of an array of man-made chemicals in the environment, leading to tremendous deterioration in environmental quality. Contami- nation of soil, air, water, and food is one of the major problems facing the industrialized world today. Significant regulatory steps have been taken to eliminate or reduce production and/or release of these chemicals into the environment. Microbial cells and bacteria are being widely used for bioremediation and biocatalysis, offering the pos- sibility to decontaminate polluted environmental media and implement chemo-enzymatically catalyzed, environmentally friendly synthetic methods. 1 Immobilization of microbial cells in membranes and bioreactors provides enhanced catalytic activity and stability, protecting microorganisms from mechanical degradation and deactivation and allowing for an overall intensification of biochemical reactions. 2 Such membranes and bioreactors must indeed be suitable supports for cells growth, which implies they must be composed of biocompatible materials and processed into a porous matrix of suitable morphology (e.g., scaffolds). 3 Cell immobilization typically occurs after scaffold preparation (e.g., by soaking the scaffold into a cell suspension), which eventually makes cells proliferate within the whole scaffold structure difficult. Note that the presence of the support itself introduces mass transfer restrictions for the diffusion of any substance (nutrients and oxygen, among others), which impedes cell proliferation deep inside the scaffold. 4 This event (e.g., cell proliferation within the scaffold limited to just a few layers of cells) has also been corroborated for animal cells growing in inverted colloidal crystals, for which rational design is ideal for the study of cell-cell and cell-matrix interactions, cell growth, and cell motility. 5 This problem can be overcome if cells grow from the inner to the outer side of the scaffold, in search of nutrients and oxygen. For this purpose, one should design chemical processes suitable for preparation of scaffolds with in situ immobilized cells. Unfortunately, the vast majority of preparation processes devised so far to prepare scaffolds (phase emulsion, air bubbling, or use of templates, among * Corresponding author. E-mail: delmonte@icmm.csic.es. Phone: 34 91 3349033. Fax: 34 91 3720623. ² Institute of Materials Science at Madrid, Spanish Research Council. ‡ Biotechnology National Center, Spanish Research Council. § University Complutense of Madrid. ⊥ Institute of Polymers Science and Technology, Spanish Research Council. (1) (a) Ishige, T.; Honda, K.; Shimizu, S. Curr. Opin. Chem. Biol. 2005, 9, 174. (b) White, C.; Sharman, A. K.; Gadd, G. M. Nat. Biotechnol. 1998, 16, 572. (c) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Nature 2001, 409, 258. (2) (a) Hecht, V.; Langer, O.; Deckwer, W. D. Biotechnol. Bioeng. 2000, 70, 391. (b) Pekdemir, T.; Keskinler, B.; Yildiz, E.; Akay, G. J. Chem. Technol. Biotechnol. 2003, 78, 773. (c) Giorno, L.; Drioli, E. TIBTECH 2000, 18, 339. (d) Erhan, E.; Keskinler, B.; Akay, G.; Algur, O. F. J. Membr. Sci. 2002, 206, 361. (e) Kwak, M. Y.; Rhee, J. S. Biotechnol. Bioeng. 1992, 39, 903. (3) (a) Yang, J.; Webb, A. R.; Ameer, G. A. AdV. Mater. 2004, 16, 511. (b) Shea, L. D.; Smiley, E.; Bonadio, J.; Mooney, D. J. Nat. Biotechnol. 1999, 17, 551. (c) Stachowiak, A. N.; Bershteyn, A.; Tzatzalos, E.; Irvine, D. J. AdV. Mater. 2005, 17, 399. (d) Zhang, Y.; Wang, S.; Eghtedari, M.; Motamedi, M.; Kotov, N. A. AdV. Funct. Mater. 2005, 15, 725. (e) Dankars, P. Y. W.; Harmsen, M. C.; Brouwer, L. A.; Van Luyn, M. J. A.; Meijer, E. W. Nat. Mater. 2005, 4, 568-574. (4) (a) Wolffberg, A.; Sheintuch, M. Chem. Eng. Sci. 1993, 48, 3937. (b) Akay, G.; Erhan, E.; Keskinler, B. Biotechnol. Bioeng. 2005, 90, 180. (5) (a) Kotov, N. A.; Liu, Y.; Wang, S.; Cumming, C.; Eghtedari, M.; Vargas, G.; Motamedi, M.; Nichols, J.; Cortiella, J. Langmuir 2004, 20, 7887. (b) Shanbhag, S.; Wang, S.; Kotov, N. A. Small 2005, 1, 1208. 1968 Chem. Mater. 2007, 19, 1968-1973 10.1021/cm062882s CCC: $37.00 © 2007 American Chemical Society Published on Web 03/17/2007