Aqueous Two-Phase System Strategies: In Route for the Potential Isolation of Stem Cells

Mirna González-González and Marco Rito-Palomares

Centro de Biotecnología-FEMSA, Tecnológico de Monterrey. Campus Monterrey,

Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, México.

Tel: (52) 81 8328-4132, Fax: (52) 81 8328-4136, e-mail: mrito@itesm.mx

            A fast, scalable and cost-effective cell isolation technique is required to promote the application of stem cells therapies. Aqueous two-phase system (ATPS) is a liquid-liquid extraction technique that offers high biocompatibility, scalability, short processing times, and a mild environment for the recovery of stem cells. In this context, we have implemented multiple bioengineering strategies based on ATPS for this purpose. In a first approach, the use of novel and traditional polymer-polymer ATPS composed of ficoll, polyethylene glycol (PEG), dextran (DEX) or UCON have been explored utilizing human umbilical cord blood as the experimental matrix to study the partition behavior and viability of CD133⁺ stem cells. Additionally, the comparison between these conventional systems and the ones complemented with antibodies, known as immunoaffinity ATPS, has also been carried out. From these experiments, it can be concluded that a different fractionation profile of the CD133⁺ cells and the contaminants can be observed within the three studied ATPS, highlighting that the CD133⁺ stem cells partition to the top phase of the ficoll 400,000-DEX 70,000 systems, meanwhile the contaminants showed a greater preference for the dextran rich bottom phase. On the other hand, an absolute preference for the bottom phase is obtained for the PEG 8,000-DEX 500,000 and Ucon-DEX 75,000 systems, for both the cell of interest and the contaminants. Furthermore, no significant differences were observed when compared the immunoaffinity and the conventional ATPS (without antibody addition). It is important to mention that in all experiments, cell viability was at least 98% after ATPS recovery, confirming the suitability of ATPS for stem cell recovery. In a further attempt to reach the objective, novel strategies including the chemical modification of the antibodies introduced into the immunoaffinity ATPS are being developed to enhance the partitioning of the antibody of interest into the desired phase. An example of this strategy is the site-specific PEGylation of the CD133 antibody via streptavidin-biotin conjugation. For this, two modified PEGs were developed: COOH-PEG-Biotin and NH₂-PEG-Biotin.

            The present investigation aims to contribute as an inspiration point for the development of novel, robust and scalable strategies for the purification of stem cells, which may find application in clinical settings in the near future.

 Acknowledgements

The authors wish to acknowledge the financial support of Tecnológico de Monterrey, Bioprocess research chair (Grant CAT161), Zambrano-Hellion Foundation and CONACyT for the fellowships of M. González-González No. 223963 and Grant 179775.