Novel Bioengineering Strategies for Stem Cells Recovery 

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.

Researchers from Hospital San José Tec de Monterrey have isolated and transplanted CD133⁺ into amyotrophic lateral sclerosis patients. However, the current isolation protocol is limited by its potential scale up feasibility, resulting in a non-generic process application.

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. Moreover, if this methodology is complemented with the use of antibodies (known as immunoaffinity ATPS), a novel strategy for the purification 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 objective of this study is to establish the bases for the development of a novel, fast, scalable and cost-effective purification bioprocess for the selective recovery of CD133⁺ stem cells employing traditional and immunoaffinity aqueous two-phase systems in its multiple variants. The proposed bioengineering strategies include the implementation of traditional ATPS composed of polyethylene-glycol, dextran and/or ficoll, as well as novel systems with Ucon in order to prove the viability of CD133⁺ stem cells and to concentrate contaminants and the stem cells of interest in opposite phases. Human umbilical cord blood (HUCB) is selected as the experimental matrix based upon abundance and simplicity of collection as it is a non-invasive and painless procedure to obtain suitable samples.

The versatility of ATPS can be visualized on the obtained results, as the product of interest partitions to the top or bottom phase of the systems depending on the polymers employed. For example, the ficoll 400,000-DEX 70,000 concentrates CD133+ stem cells in the top phase, while most of the contaminants are contained in the bottom phase. On the other hand, the PEG 8,000-DEX 500,000 and Ucon-DEX 75,000 ATPS are suitable for the concentration of these cells of interest in the bottom phase. The results achieved with this investigation represent a firm platform for the development of new strategies.

The present work demonstrates the feasibility of polymer-polymer ATPS for the selective separation of CD133+ stem cells from human umbilical cord blood. The aqueous environment of the systems preserves the viability of the stem cells of interest, as do the most exploited stem cell separation technologies. Moreover, the ATPS are easily and rapidly implemented; they do not require the use of highly specialized equipment with expensive maintenance programs that must be run by well-trained personnel, as other purification technologies do. Additionally, ATPS can be easily scaled-up and are performed with common lab equipment.