Aqueous two-phase systems strategies to establish novel bioprocesses for stem cells recovery

July 8, 2017 | Autor: Marco Rito-palomares | Categoria: Technology, Stem Cells, Cell separation, Biological Sciences, Humans, Animals, Bioreactors, Animals, Bioreactors
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Descrição do Produto ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–10 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2013.794125


Aqueous two-phase systems strategies to establish novel bioprocesses for stem cells recovery Mirna Gonza´lez-Gonza´lez and Marco Rito-Palomares



During the past decade, stem cell transplantation has emerged as a novel therapeutic alternative for several diseases. Nevertheless, numerous challenges regarding the recovery and purification steps must be addressed to supply the number of cells required and in the degree of purity needed for clinical treatments. Currently, there is a wide range of methodologies available for stem cells isolation. Nevertheless, there is not a golden standard method that accomplishes all requirements. A desirable recovery method for stem cells has to guarantee high purity and should be sensitive, rapid, quantitative, scalable, non- or minimally invasive to preserve viability and differentiation capacity of the purified cells. In this context, aqueous twophase systems (ATPS) represent a promising alternative to fulfill the mentioned requirements, promoting the use of stem cell-based therapies for incurable diseases. This practical review focuses on presenting the bases for the development of a novel and scalable bioprocess for the purification of stem cells, with a case scenario of CD133þ cells. The bioengineering strategies include the application of immunoaffinity ATPS in its multiple variants, including antibodypolymer conjugation, antibody addition and antibody immobilization. Conclusions are drawn in the light of the potential generic implementation of these strategies as an initial step in the establishment of bioprocesses for the purification of stem cells.

Affinity partitioning, ATPS, CD133þ cells, scale-up, stem/progenitor cells isolation, purification

Introduction Stem cells are distinguished for their unique characteristics of self-renewal, proliferation and differentiation capacities. These properties have attracted the attention of researchers due to the potential results that can be achieved with stem cell transplantation. In this sense, purified stem cells have been used as a therapeutic alternative for several incurable, chronic and degenerative diseases, including critical limb ischemia (Burt et al., 2010), chronic ischemic heart disease (Stamm et al., 2007), amyotrophic lateral sclerosis (ALS) (Martinez et al., 2009) and chronic lymphocytic leukemia (Isidori et al., 2007). However, to apply these treatments, special attention must be given to the recovery and purification stages to guarantee the purity and number of stem cells required for a successful transplantation procedure. Isolation of highly purified stem cells is essential for the development of cellbased therapeutics to guarantee removal of undifferentiated and other unwanted cells that could be tumor forming. A desirable recovery method for stem cells has to assure high purity and should be sensitive, rapid, quantitative, scalable, non- or minimally invasive to preserve viability and biological functions (e.g. differentiation capacity) of the purified Address for correspondence: Marco Rito-Palomares, Centro de Biotecnologı´a-FEMSA, Tecnolo´gico de Monterrey. Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Me´xico. Tel: (52) 81 8328-4132. Fax: (52) 81 8328-4136. E-mail: [email protected]

History Received 17 July 2012 Revised 14 March 2013 Accepted 3 April 2013 Published online 16 May 2013

cells (Gonza´lez-Gonza´lez et al., 2012a; Pethig et al., 2010). Currently, there is a wide range of methodologies available for stem cells isolation. These techniques can be classified into three categories: (1) isopycnic centrifugation, including density gradient and cell culture; (2) immunochemical, employing immune labeling; and (3) novel, tagless procedures (Gonza´lez-Gonza´lez et al., 2012a). Table 1 highlights the advantages, limitations and performance parameters of some of the current methods employed for stem cell separation that require immuno-tags. In this context, the major constraints of employing immune-affinity separation methodologies are the availability of suitable antibodies and possible elimination of important primitive cell subsets that have not expressed the selection marker (Wognum et al., 2003). Another possible drawback is the need of removing the antibody from the isolated cells, particularly when the antibody alters the surface characteristic of the cells and affects its subsequent use (Tsukamoto et al., 2009). In this context, immunochemical affinity techniques including MACS (Magnetic Activated Cell Sorting) and FACS (Fluorescence Activated Cell Sorting) have become one of the most exploited methods for stem cells purification. This is due to the high specificity conferred by the cell surface marker (cluster of differentiation, CD) that they employ as molecular tagging. For example, one of the most recently used CD for identification of stem cells is the novel CD133. CD133, a five-transmembrane stem cell glycoprotein

20 13

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Centro de Biotecnologı´a-FEMSA, Tecnolo´gico de Monterrey, Monterrey, NL, Me´xico

KG1 bone marrow (CD34þ) CD34þ whole umbilical cord blood

Low specificity, instability of interphase, lab scale, recovery of separated cells, optimization requires repetitive extraction steps

Biocompatibility, label free, scalable, low energy input, continuous, short process time, recycling of costly affinity ligands, selective partition, single step, high cell viability, low viscosity

Hydrophobicity, size, net charge

Aqueous two phase systems (ATPS)

NR, not reported

CD34þ bone marrow CD34þ bone marrow

Requires washing step, low yield, unspecific adhesion, low resolution

Commercially available, short process time, scalable


80 245

70–90 94






CD34þ cryopreserved leukapheresis product CD34þ KG-1a and leukapheresis product

Nonlinear performance, cell loss due to magnetic deposition

Sterile disposable flow channel, continuous, highest throughput, scalable, high level of T-cell log depletion

Affinity, magnetism

Quadrupole Magnetic Cell Sorter (QMS)

75–80 95

NR 74

81 85

93 95

CD133þ leukapheresis product 50% SSEA-1 þ mESC cell mixture (CD34þ)

Yield (%)

Requires magnetic beads with antibody and magnetic field, long process time, sample preparation required, need label removal, alters cell viability, non-multiparametric, low cell recovery, might contribute to cell differentiation, optimized protocols are multisteps

Purity (%)

Stem cell source


High purity, lower cost vs. FACS, high yield, easy to use, commercially available in different capacities, allows positive and negative selection


Affinity, magnetism

Separation criteria

Magnetic Activated Cell Sorter (MACS)


Table 1. Advantages, limitations and performance parameters of current methods employed for stem cells separation. Adapted from Gonza´lez-Gonza´lez et al. (2012b).

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95 NR

85 NR



495 70–80

Viability (%)


Kumar et al. (2001) Sousa et al. (2011)

Lebkowski et al. (1992) Cardoso et al. (1995)

Jing et al. (2007b)

Jing et al. (2007a)

Lang et al. (2004) Schriebl et al. (2010)

2 M. Gonza´lez-Gonza´lez and M. Rito-Palomares Crit Rev Biotechnol, Early Online: 1–10

Aqueous two-phase systems strategies for stem cells recovery

DOI: 10.3109/07388551.2013.794125


Table 2. Advantages and limitations of the immunoaffinity ATPS strategies proposed for stem cells separation. Immunoaffinity ATPS strategy Antibody-polymer conjugation

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Antibody addition a) Free antibody

b) PEGylated antibody

Antibody immobilization



Most employed Possible modified phase recycling Smart polymer can be employed Different conjugation reactions available PEGs simple modification May apply to various ATPS Higher affinity interaction Positive selection strategy

Conjugation step Detaching step Long reaction times More reagents Laborious

Faster Simple cell recovery No extra steps Less reagents Less time-consuming No polymer forming phase activation Positive selection strategy Benefits of PEGylation Biotin-streptavidin fast conjugation Commercially available modified PEGs No polymer forming phase activation May apply to various ATPS Highly effective reaction Site-specific reaction conserves antibody’s affinity Positive selection strategy

Antibody recycling challenging

Possible phase recycling Nontoxic, biodegradable matrix Different immobilization approaches No polymer activation Greater surface area for selective binding Positive selection strategy

(Miraglia et al., 1997), that appears to be a reliable marker for the isolation of neural stem cells (Wu and Wu, 2009) and has the ability to promote neural growth (Martinez et al., 2009). Particularly, researchers from Hospital San Jose´ Tec de Monterrey (Mexico) have isolated CD133þ stem cells and transplanted them into the frontal motor cortex in ALS patients (Martinez et al., 2009). ALS is a neurodegenerative disease characterized by the rapid weakening and selective death of neurons. Unfortunately, current purification techniques employed for stem cells treatments are limited by their potential scale-up feasibility, high costs and complex infrastructure (specialized instrument, reagents, facilities, maintenance and expertise personnel), resulting in a non-generic process application. Aqueous two-phase systems (ATPS) represent an attractive alternative for the recovery of stem cells. ATPS are a liquidliquid extraction technique (polymer-polymer, polymer-salt or novel components) that exhibits several advantages including biocompatibility, economically attractive, scalable and low processing time (Benavides & Rito-Palomares, 2008; Benavides et al., 2011; Hatti-Kaul, 2001; Sinha et al., 2000). Moreover, if this methodology is complemented with the use of antibodies (known as immunoaffinity ATPS), a novel strategy with improved selectivity for the purification of stem cells that satisfies the requirements previously mentioned could be achieved. Even though the method reported by Martinez and collaborators (2009) obtained successful clinical results, a latent niche exists for the development of a faster, scalable and cost-effective procedure that guarantees purity, yield and

Conjugation step More reagents Laborious

Immobilization step Detaching step with glass bead matrix More processing time prior ATPS step More reagents Applied to selective types of ATPS Laborious

the biological activity required for the final application of the process. This article focuses on presenting a strategic review, based on our working experience, that provides general rules and pre-establish the bases for the development of a novel, faster and scalable procedure with lower downstream costs for the selective recovery and purification of stem cells employing immunoaffinity aqueous two-phase systems. The proposed bioengineering strategies include the potential implementation of immunoaffinity ATPS in three major variants: (i) antibody-polymer conjugation, (ii) antibody addition and (iii) antibody immobilization. In this sense, immunoaffinity ATPS represent an alternative technique to establish a potential bioprocess viable for clinical use, thus promoting the widespread application of stem cells therapy.

Application of ATPS for stem cells recovery and purification Stem cells are mostly present in a limited amount in adult tissues and organs. Moreover, if a rare population of stem cells is the target object (e.g. CD133þ cells), an efficient purification method is required. This procedure is hindered by the considerations of employing a simplified, mild, fast, reproducible, cost-effective and scalable procedure to obtain the purity and amount of cells required for clinical settings. An aqueous two-phase system is a liquid-liquid fractionation technique first employed in the 1950s by Albertsson that has demonstrated to be a gentle procedure for the recovery and primary purification of viable and fully functional high-value biological products, including proteins

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M. Gonza´lez-Gonza´lez and M. Rito-Palomares

(Albertsson, 1958; Albertsson et al., 1987; Johansson 1985), cells (Walter et al., 1968; Walter et al., 1969a,b) and organelles (Albertsson, 1974; Albertsson, 1988; Morre´ & Morre, 2000; Morre´ et al., 1998). The biphasic system contains more than 80% water as it is composed of two hydrophilic aqueous solutions. When mixing these two liquids above certain critical concentrations, immiscible phase formation is induced. ATPS can be classified depending on their composition in (i) polymer-polymer, (ii) polymer-salt and (iii) novel systems including ionic liquids, tree gum, starch, copolymers and alcohol, among others. The two structurally distinct hydrophilic and high molecular weight polymer forming phases could be polyethylene glycol (PEG), dextran and ficoll, while the salts could be phosphates, sulfates or citrates. The reader is referred to the volumes by Walter et al. (1985), Albertsson (1986), Walter & Johansson (1994), Zaslavsky (1995), and Hatti Kaul (2000) for a broader explanation of ATPS. For cell separation, ATPS exploit the affinity of the cells for the components of either the top, bottom phase or the interface between phases in one or multiple steps (Kumar & Bhardwaj, 2008) positioning the cell in the most energetically favorable location within the system (SooHoo & Walker, 2009). The separation is based on the physicochemical properties of the cell such as hydrophobicity, size, net surface charge and membrane properties (Gossett et al., 2010; Kamihira & Kumar, 2007). As well as the polymeric and ionic composition of the phases (Malmstrom et al., 1978) and the selected systems parameters of volume ratio (VR), tie line length (TLL), pH and temperature (Benavides & RitoPalomares, 2008). Moreover, ATPS are advantageous for cell separation as they are safe, suitable for large-scale separation, noninvasive, nondestructive, inexpensive, technologically simple and biocompatible to preserve cell viability and biological functions. Other advantages of the ATPS separation method is that the cell fractions are not exposed to differences in pH, osmolarity or ion concentration during the separation procedure (Malmstrom et al., 1978). Furthermore, if the technology of ATPS is combined with affinity ligands (e.g. dyes, metal ions, enzyme inhibitors or antibodies) a powerful and versatile separation method known as affinity ATPS is developed (Delgado et al., 1991, 1992; Johansson, 1984; Karr et al., 1988; Kopperschla¨ger & Birkenmeier, 1990) to achieve specific partitioning through cell surface receptors. This technology has the advantage of exploiting the highly specific interaction between an antigen and an antibody raised against it, known as immunoaffinity ATPS. Thus, is capable of separating the product of interest from the contaminants even though only small differences in physical properties such as charge, size and hydrophobicity exist. Immunoaffinity ATPS can be constructed mainly in three different ways: (i) antibody-polymer conjugation, (ii) antibody addition or (iii) antibody immobilization. In most cases, the upper phase (frequently PEG) is the polymer that suffers the chemical modification or where the added antibody must partition, because the target cell and contaminants have preference to the bottom phase. In this way, the antibody will bind the specific target antigen on the cell surface and will promote the cell’s partition to the phase to which the affinity ligand is partitioned, enabling them to be easily isolated.

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Potential immunoaffinity ATPS bioengineering strategies for stem cells recovery and purification Before discussing the proposed bioengineering ATPS strategies, special attention must be placed when working on the purification of stem cells. Considerations derived from our experience are presented with the aim of providing a complete scenario that could facilitate the understanding and characterization of the partitioning of stem cells in ATPS. First, the most recommended types of ATPS in the case of stem cell separation are the polymer-polymer systems. This is emphasized, as careful handling is important to allow the preservation of the integrity of the cells. In this respect, the most adequate solvent is phosphate buffered saline (PBS, pH 7.4, 150 mM NaCl), providing suitable media for the separation of viable cells. It is not advised to use PEG/salt ATPS due to the fact that biospecific interactions are usually obstructed by high salt concentration (Cabral, 2007) and because of the hypertonicity of the salt component. Even though traditional polymer-polymer systems are more expensive than polymersalts, it is anticipated that the investment and operational costs of immunoaffinity ATPS represent a lower budget compared to the MACS and FACS technologies. Moreover, the investment in polymer-polymer ATPS for the purification of specific stem cells with potential medical applications appears highly justified. The speed of the operation is another important logistical factor, but thanks to the simplicity of ATPS technology this does not represent an obstacle. The process requires a few minutes for the mixing step, after the sample and antibodies (in the case of immunoaffinity ATPS) have been added. Afterwards, phase separation is achieved and this could usually be performed by low-speed centrifugation. Affinity ATPS is exploited with the introduction of ligands, for which receptors exist on the material of interest. The most selective type of affinity ATPS is the antibodyantigen interaction or so-called immunoaffinity ATPS. Ideally, the product of interest would be recovered in the upper phase, leaving the contaminants in the bottom phase. In such scenario, the recommended ATPS for affinity approaches are the ones that conserve the top phase clean, meaning that the target cells and contaminants partition naturally to the bottom phase (e.g. PEG 10 000-dextran 10 000 and PEG 8000-dextran 500 000). The affinity ligand must partition into the upper phase and this can be achieved with two of the previously mentioned immunoaffinity strategies: (i) antibody-polymer conjugation and (ii) antibody addition. The first strategy implies that the chemical modification of one of the phase forming polymers (i.e. PEGylation). PEGylation is the process of attaching PEG to a molecule and when performed to an antibody it confers the advantages of increase circulating half-lives; reduce antigenicity, immunogenicity and toxicity; improve solubility and bioavailability; and enhance proteolytic resistance (Chapman, 2002). On the other hand, the antibody addition method consists of loading free ligands or modified antibodies (e.g. PEGylated antibody) to the ATPS. The three proposed strategies imply positive selection, in which the antibody would be used against a specific surface marker to label the desired cells.

DOI: 10.3109/07388551.2013.794125

The final objective of immunoaffinity ATPS is to concentrate the contaminants and the stem cells of interest in opposite phases. In this context, the next subsection will focus on providing a deeper description and the schematic representation of each immunoaffinity ATPS strategy, derived from our work experience, for the particular case scenario of CD133þ cells purification. Before entering to the immunoaffinity strategies, description of a brief explanation concerning the experimental sample matrix and sample preparation is addressed.

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Sample preparation Hematopoietic stem cells CD133þ, our product of interest, is present at very low concentrations in bone marrow, mobilized peripheral blood or human umbilical cord blood (HUCB), thus the isolation for further analysis is a complex challenge. HUCB is selected as the experimental matrix based upon abundance, simplicity of collection and as the recovery of suitable samples is a noninvasive and painless procedure. A pre-enrichment step employing Lymphoprep (Axis-Shield, Norway) is performed to eliminate water and other contaminants. Hence, the mononuclears are separated from platelets and red blood cells. Additionally, the volume of the sample is drastically reduced (from 100 mL obtained during a typical HUCB collection to a concentrated pellet). Antibody-polymer conjugation In this approach (Figure 1), the antibody that recognizes the stem cells of interest would be conjugated to one of the phase forming polymers through a covalent or noncovalent reaction. The PEG is commonly selected as the modified phase as most

Aqueous two-phase systems strategies for stem cells recovery


products partition preferentially to the dextran-rich phase (Azevedo et al., 2009), leaving the PEG-rich top phase clean and available to capture the stem cell of interest. Furthermore, PEG is easily derivatized due to its terminal hydroxyl groups. For this various reactions have been reported (Azevedo et al., 2009; Ruiz-Ruiz et al., 2012). Another possibility is to employ commercially available derivatized PEGs. Likewise, dextran or other phase-forming polymers could be chemically modified, in cases where the samples added concentrate in the opposite polymer phase. The fundamentals behind this strategy are the positive selection performed by the antibody coupled to one of the phase-forming polymers during the mixing step. After phase formation, the stem cells of interest would be isolated in the modified phase, leaving contaminants in the opposite phase. Even though antibody-polymer conjugation has the advantages of exploiting a higher affinity interaction by allowing the homogenous distribution of the antibody in one of the phases, it requires additional time and costs for the derivatization. One alternative to overcome these limitations is to recycle the modified polymer after detaching the stem cells of interest. Another variant of the antibody-polymer conjugation methodology is to bind the antibody of interest into a third ligand carrier polymer, which concentrates mainly in one of the ATPS phases. The advantage of the ligand carriers is that smart polymers (SP) (sensitive to temperature, pressure, pH or light) can be employed to facilitate the detaching step. Examples of this type of SP are presented in various reports (Kumar et al., 2007; Liu, 2011). This strategy is the most common way of purifying products within the affinity strategies, and for stem cells, it is

Figure 1. Schematic representation of the first proposed immunoaffinity ATPS: antibody-polymer conjugation.


M. Gonza´lez-Gonza´lez and M. Rito-Palomares

not the exception. The conjugation of the CD34 antibody with the temperature-sensitive polymer polyNIPAM (poly-Nisopropylacrylamide) in an ATPS composed of 4% PEG 8000–5% dextran T500 to isolate CD34þ human acute myeloid leukemia cells (KG-1) from human T lymphoma cells (Jurkat) has been reported (Kumar et al., 2001).

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Antibody addition This immunoaffinity ATPS strategy implies the addition of CD133 antibodies into traditional polymer-polymer systems. Its advantages are the elimination of the pre-treatment step required for the chemical activation of the polymer, which increases the time and cost of the process. The main difference with the previous strategy is the addition of antibodies after ATPS construction, instead of being introduced within one of the polymers. The incorporation of antibodies into ATPS can be achieved by adding them freely into the solution (Figure 2A). Alternatively, the antibodies could first be modified to increase their partition to the desired phase. An easy and fast approach to perform this improvement is through PEGylation (Figure 2B). The PEGylation of the CD133Biotin antibody has been recently reported through a sitespecific PEGylation reaction via streptavidin-biotin conjugation (Gonza´lez-Gonza´lez et al., 2012b). The molecular weight and charge of the PEG used in the reaction are factors that could help in achieving a better partition of the antibodies to the desired phase. Sousa and coworkers implemented an immunoaffinity ATPS strategy to recover CD34þ from whole umbilical cord blood (Sousa et al., 2011). Traditional 5.6% PEG 8000–7.5%

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dextran 500 000 ATPS was added with a pretreated sample with the monoclonal antibody produced against the CD34 antigen. It was reported an enrichment of CD34þ cells at the interface, reaching purification factors up to 245 with a recovery yield of 95%. The addition of PEGylated antibodies to the polymer-polymer ATPS has not been extensively addressed. Hence, more investigation should be conducted to exploit this immunoaffinity ATPS for the purification of stem cells. The potential of phase recycling to reduce the operational costs, especially for a scale-up process, is also interesting. Antibody immobilization The last proposed strategy involved the immobilization of antibodies on a solid matrix. It is anticipated that the cells of interest will be coupled to the immobilized matrix. This strategy considers the use of ficoll-dextran ATPS added with microbeads containing anti-CD133 (Figure 3A). The immobilized micro-beads have the advantage of possessing a greater surface area for selective cell binding. In this type of ATPS, both the product of interest and contaminants partition to the ficoll rich top phase. However, it is expected that the CD133þ stem cells would bind the immobilized antibody on the microbeads. As a result, the product of interest would be recovered from the bottom phase. Further removing of the cell from the separating agent via trypsinization, can be implemented, to obtain a product suitable for further purification. Alternatively, a nontoxic and biodegradable matrix (as in the case of MACS technology) can be used with the final aim of eliminating the need of removing the cells after the separation process. In comparison to the MACS technology,

Figure 2. Schematic representation of the second proposed immunoaffinity ATPS: antibody addition. (A) Free antibody strategy and (B) PEGylated antibody approach.

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DOI: 10.3109/07388551.2013.794125

Aqueous two-phase systems strategies for stem cells recovery


Figure 3. Schematic representation of the third proposed immunoaffinity ATPS: antibody immobilization. (A) Immobilized micro-beads and (B) immobilized bottom phase walls.

this proposed protocol does not require the usage of magnetic particles. The main limitation of this strategy is the need of an immobilization step, which consumes time and reagents. Another approach for this strategy is to immobilize the antibody of interest on the wall of the tubes that will be in touch with the clean bottom phase (Figure 3B). In this way, the cells of interest will be in contact with the immobilized antibodies during the mixing step and will be retained on the tube. A mild detaching step will be necessary to recover the product of interest. Other creative ways could be developed to introduce the solid phase into the two polymer phases and exploit the already mentioned advantages of ATPS. This proposed technology fuses the benefits of several existing purification technologies, including ATPS, MACS and panning, but should be further investigated to fully develop its potential. In this context, the herein proposed strategies have the objective to serve as an inspirational strategy to unlock other possible isolation mechanisms that may gather the advantages of existing methods and complement them with novel approaches. In an attempt to increase the recovery and purity of the target cells, counter current distribution (CCD, a multiple-step extraction procedure) could be implemented. This technology enhances the high selectivity of the affinity step and the aforementioned advantages of ATPS. Briefly, immunoaffinity ATPS-CCD implies the use of the immunoaffinity rich top phase of the selected system and transferring it to a fresh bottom phase. Likewise, the bottom phase of the original ATPS is mixed with fresh immunoaffinity-rich top phase (Figure 4). This approach can be repeated consecutively. Hence, a number of immunoaffinity-rich top phases are

sequentially moved over a set of fresh bottom phases, and vice versa. The required time and effort involved in this strategy needs to be analyzed. As general considerations for all the proposed strategies (Figure 5), special attention must be given to the operational conditions to preserve cell viability and function. Thus, after the estimation of the recovery yield and purification factor obtained from each of the isolation procedure proposed, the purified stem cells must be cultured to monitor their viability, differentiation and propagation capacities.

Conclusions Today, stem cell researchers are focused on the discovery of interesting functional phenotypes or are directing their efforts toward the application of stem cells to try to cure several diseases. In this sense, stem cells have the potential to revolutionize tissue regeneration and cell-based treatments by providing a therapy for incurable diseases in the near future. However, it is important to realize that there will be a need to develop novel isolation protocols. In the coming years, stem cell purification, to some degree now neglected, will play a crucial role once effective cell-based clinical protocols have been tested and approved. Hence, it is important for stem cells to be efficiently and accurately isolated from their original matrix. Currently, there are numerous challenges regarding the purification and isolation of stem cells that must be addressed before therapeutic stem cell transplantations can be widely applied. Moreover, it is well known that a key problem for the recovery of stem cells is the high cost and scale-up limitations of the existing methods.

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Crit Rev Biotechnol, Early Online: 1–10

Figure 4. Scheme of the immunoaffinity ATPS counter current distribution (CCD) process.

Figure 5. Summary of the proposed immunoaffinity ATPS bioengineering strategies.

Even though ATPS have been mainly used for the recovery and purification of proteins, immunoaffinity ATPS represent a promising and suitable option to develop a selective purification system capable of processing large quantities of cell mixtures. ATPS are able to isolate a specific target stem

cell population without the requirement of specialized and expensive instruments or of highly trained personnel. This article proposes potential ATPS bioengineering strategies that can be effectively followed in order to obtain the desired purity and recovery required for further studies. Thus, these

DOI: 10.3109/07388551.2013.794125

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strategies may be used as a starting point for the development of novel and more ambitious stem cell purification processes. Additionally, the aim of this work is to present immunoaffinity ATPS as a relatively inexpensive approach compared to currently existing affinity-based purification technologies. In this sense, immunoaffinity ATPS represent a viable technique that can meet the future necessities, thus promoting the acceleration of the widespread application of stem cells therapy.

Declaration of interest The authors report no declarations of interest and wish to acknowledge the financial support of Tecnolo´gico de Monterrey, Bioprocess research chair (Grant CAT161), of the Zambrano-Hellion Foundation and of the CONACyT for the fellowship of M. Gonza´lez-Gonza´lez No. 223963. References ˚ . (1958). Partition of proteins in liquid polymer-polymer Albertsson PA two-phase systems. Nature, 182, 709–11. ˚ . (1974). Countercurrent distribution of cells and cell Albertsson PA organelles. In: Fleisher S, Packer L, eds. Methods in Enzymology. Vow. 31. San Diego: Academic Press, 761–9. ˚ . (1986). Partition of cell particles and macromolecules. Albertsson PA New York: Wiley. ˚ . (1988). Separation of cell organelles and membrane Albertsson PA vesicles by phase partition. Prog Clin Biol Res, 270, 227–35. ˚ , Cajarville A, Brooks DE, Tjerneld F. (1987). Partition Albertsson PA of proteins in aqueous polymer two-phase systems and the effect of molecular weight of the polymer. BBA-Gen Subjects, 926, 87–93. Azevedo AM, Rosa PAJ, Ferreira IF, et al. (2009). Affinity-enhanced purification of human antibodies by aqueous two-phase extraction. Sep Purif Technol, 65, 31–9. Benavides J, Rito-Palomares M. (2008). Practical experiences from the development of aqueous two-phase processes for the recovery of high value biological products. J Chem Technol Biotechnol, 83, 133–42. Benavides J, Rito-Palomares M, Asenjo JA. (2011). Downstream processing and product recovery j Aqueous two-phase systems. In: Murray MY, ed. Comprehensive Biotechnology. 2nd ed., Vol. 2. Burlington: Elsevier, 697–713. Burt RK, Testori A, Oyama Y, et al. (2010). Autologous peripheral blood CD133þ cell implantation for limb salvage in patients with critical limb ischemia. Bone Marrow Transpl, 45, 111–6. Cabral JMS. (2007). Cell partitioning in aqueous two-phase polymer systems. Adv Biochem Eng Biotechnol, 106, 151–71. Cardoso AA, Watt SM, Batard P, et al. (1995). An improved panning technique for the selection of CD34(þ) human bone-marrow hematopoietic-cells with high recovery of early progenitors. Exp Hematol, 23, 407–12. Chapman AP. (2002). PEGylated antibodies and antibody fragments for improved therapy: a review. Adv Drug Deliver Rev, 54, 531–45. Delgado C, Anderson RJ, Francis GE, Fisher D. (1991). Separation of cell mixtures by immunoaffinity cell partitioning: strategies for low abundance cells. Anal Biochem, 192, 322–8. Delgado C, Sancho P, Medieta J, Luque J. (1992). Ligand-receptor interactions in affinity cell partitioning: studies with transferrin covalently linked to monomethoxypoly(ethylene glycol) and rat reticulocytes. J Chromatogr A, 594, 97–103. Gonza´lez-Gonza´lez M, Va´zquez-Villegas P, Garcı´a-Salinas C, RitoPalomares M. (2012a). Current strategies and challenges for the purification of stem cells. J Chem Technol Biot, 87, 2–10. Gonza´lez-Gonza´lez M, Mayolo-Deloisa K, Rito-Palomares M. (2012b). PEGylation, detection and chromatographic purification of sitespecific PEGylated CD133-Biotin antibody in route to stem cell separation. J Chromatogr B, 893–4, 182–6. Gossett DR, Weaver WM, Mach AJ, et al. (2010). Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem, 397, 3249–67. Hatti-Kaul R. (2000). Aqueous two-phase systems: methods and protocols. New Jersey: Humana Press.

Aqueous two-phase systems strategies for stem cells recovery


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