1 Immunoaffinity aqueous two-phase system bioengineering strategies to establish novel bioprocess for stem cells recovery Mirna González-González and Marco Rito-Palomares Centro de Biotecnología-FEMSA, Tecnológico de Monterrey. Campus Monterrey, Monterrey, México. mrito@itesm.mx Recovery and purification of stem cells are crucial steps in order to guarantee the purity and viability required for successful transplantations. Even though a wide variety of isolation techniques exist, there is not a golden method that accomplishes the goals of high purity and yield with a fast, scalable process that does not affect viability and differentiation capacity of recovered cells (1). Researchers from Hospital San José Tec de Monterrey have isolated and transplanted CD133 + In this context, aqueous-two phase systems (ATPS) represent an attractive alternative for the recovery of stem cells. ATPS is a liquid-liquid extraction technique that exhibits several advantages including: biocompatibility, economically attractive, scalable, and low processing time (3). Moreover, if this methodology is complemented with the use of antibodies (known as immunoaffinity ATPS (4)), a novel strategy for the purification of CD133 into amyotrophic lateral sclerosis patients (2). However, the current isolation protocol is limited by its potential scale up feasibility, resulting in a non-generic process application. + The objective of this research is to establish the bases for the development of a novel and scalable purification bioprocess for the selective recovery of CD133 stem cells satisfying the requirements previously mentioned, could be achieved, thus promoting the use of stem cell based therapies for incurable diseases. + The first strategy consist in the implementation of novel ATPS composed of UCON and density gradients (Lymphoprep, Percoll or Ficoll) in order to prove the viability of CD133 stem cells employing immunoaffinity aqueous two-phase systems. The proposed bioengineering strategies include 1) implementation of novel ATPS and 2) immunoaffinity ATPS in its multiple variants. + stem cells and to characterize the partitioning behavior of the target product and contaminants in these types of systems. The second approach includes the implementation of immunoaffinity ATPS, in order to concentrate the contaminants and the stem cells of interest in opposite phases. The first immunoaffinity ATPS strategy implies the addition of free antiCD133, the second approach consists in the addition of PEGylated CD133 antibody to the system. The PEGylated antibody is obtained through a site-specific PEGylation of CD133 via streptavidin-biotin conjugation (5). The third possibility is to construct ATPS composed of Ficoll 400,000 and Dextran 70,000 to which immobilized microbeads are introduced. In this type of system, the product of interest and the contaminants partition into the Ficoll rich top phase and after the addition of the immobilized micro- glass beads, the CD133 + stem cells would ideally get attached to the beads and could be recovered in the bottom phase. References 1. González-González M, Vázquez-Villegas P, García-Salinas C, Rito-Palomares M. Current strategies and challenges for the purification of stem cells. Journal of Chemical Technology & Biotechnology 2012; 87 (1), 2-10. 2. Martinez HR, Gonzalez-Garza MT, Moreno-Cuevas JE, Caro E, Gutierrez-Jimenez E, Segura JJ. Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients. Cytotherapy 2009; 11 (1), 26-34. 3. Benavides J, Aguilar O, Lapizco-Encinas BH, Rito-Palomares M. Extraction and purification of bioproducts and nanoparticles using Aqueous Two-Phase Systems strategies. Chemical Engineering & Technology 2008; 31 (6), 838-845. 4. Ruiz-Ruiz F, Benavides J, Aguilar O, Rito-Palomares M. Aqueous two-phase affinity partitioning systems: Current applications and trends. Journal of Chromatography A 2012; 1244, 1-13. 5. González-González M, Mayolo-Deloisa K, Rito-Palomares M. PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation. Journal of Chromatography B 2012; 893–894 (0), 182-186.