2013

http://informahealthcare.com/btyISSN: 0738-8551 (print), 1549-7801 (electronic)

Crit Rev Biotechnol, Early Online: 1–10! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2013.794125

REVIEW ARTICLE

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

Mirna Gonzalez-Gonzalez and Marco Rito-Palomares

Centro de Biotecnologıa-FEMSA, Tecnologico de Monterrey, Monterrey, NL, Mexico

Abstract

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

Keywords

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

History

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

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 cell-

based 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 bio-

logical functions (e.g. differentiation capacity) of the purified

cells (Gonzalez-Gonzalez 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

(Gonzalez-Gonzalez 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

Address for correspondence: Marco Rito-Palomares, Centro de Biotec-nologıa-FEMSA, Tecnologico de Monterrey. Campus Monterrey, Ave.Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico. Tel: (52)81 8328-4132. Fax: (52) 81 8328-4136. E-mail: mrito@itesm.mx

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(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 tech-

niques employed for stem cells treatments are limited by their

potential scale-up feasibility, high costs and complex infra-

structure (specialized instrument, reagents, facilities, main-

tenance 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 liquid-

liquid 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

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 andpurification

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 fraction-

ation 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

Table 2. Advantages and limitations of the immunoaffinity ATPS strategies proposed for stem cells separation.

Immunoaffinity ATPS strategy Advantages Limitations

Antibody-polymer conjugation Most employed Conjugation stepPossible modified phase recycling Detaching stepSmart polymer can be employed Long reaction timesDifferent conjugation reactions available More reagentsPEGs simple modification LaboriousMay apply to various ATPSHigher affinity interactionPositive selection strategy

Antibody additiona) Free antibody Faster Antibody recycling challenging

Simple cell recoveryNo extra stepsLess reagentsLess time-consumingNo polymer forming phase activationPositive selection strategy

b) PEGylated antibody Benefits of PEGylation Conjugation stepBiotin-streptavidin fast conjugation More reagentsCommercially available modified PEGs LaboriousNo polymer forming phase activationMay apply to various ATPSHighly effective reactionSite-specific reaction conserves antibody’s affinityPositive selection strategy

Antibody immobilization Possible phase recycling Immobilization stepNontoxic, biodegradable matrix Detaching step with glass bead matrixDifferent immobilization approaches More processing time prior ATPS stepNo polymer activation More reagentsGreater surface area for selective binding Applied to selective types of ATPSPositive selection strategy Laborious

DOI: 10.3109/07388551.2013.794125 Aqueous two-phase systems strategies for stem cells recovery 3

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(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 & Rito-

Palomares, 2008). Moreover, ATPS are advantageous for

cell separation as they are safe, suitable for large-scale

separation, noninvasive, nondestructive, inexpensive, techno-

logically 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; Kopperschlager & 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) anti-

body 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.

Potential immunoaffinity ATPS bioengineeringstrategies for stem cells recovery and purification

Before discussing the proposed bioengineering ATPS strate-

gies, 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 charac-

terization 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 empha-

sized, as careful handling is important to allow the preser-

vation 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 polymer-

salts, 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 invest-

ment 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 antibody-

antigen 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 bio-

availability; 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.

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The final objective of immunoaffinity ATPS is to concen-

trate 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 repre-

sentation of each immunoaffinity ATPS strategy, derived

from our work experience, for the particular case scenario of

CD133þ cells purification. Before entering to the immunoaf-

finity strategies, description of a brief explanation concerning

the experimental sample matrix and sample preparation is

addressed.

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 contam-

inants. 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

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 advan-

tages 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.

DOI: 10.3109/07388551.2013.794125 Aqueous two-phase systems strategies for stem cells recovery 5

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not the exception. The conjugation of the CD34 antibody

with the temperature-sensitive polymer polyNIPAM (poly-N-

isopropylacrylamide) 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).

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 intro-

duced 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 CD133-

Biotin antibody has been recently reported through a site-

specific PEGylation reaction via streptavidin-biotin conjuga-

tion (Gonzalez-Gonzalez 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%

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 immo-

bilized 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 purifi-

cation. 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) PEGylatedantibody approach.

6 M. Gonzalez-Gonzalez and M. Rito-Palomares Crit Rev Biotechnol, Early Online: 1–10

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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 comple-

ment 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.

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

DOI: 10.3109/07388551.2013.794125 Aqueous two-phase systems strategies for stem cells recovery 7

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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

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

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

8 M. Gonzalez-Gonzalez and M. Rito-Palomares Crit Rev Biotechnol, Early Online: 1–10

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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 immunoaffi-

nity 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 Tecnologico

de Monterrey, Bioprocess research chair (Grant CAT161),

of the Zambrano-Hellion Foundation and of the CONACyT

for the fellowship of M. Gonzalez-Gonzalez No. 223963.

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