MX2013005204A - Process for recovering and purifying cd133+ mononuclear cells using aqueous two-phase systems. - Google Patents

Abstract

The invention relates to a process for the fractionation, recovery and purification of CD133+ mononucleated cells from a biological matrix rich in stem cells (umbilical cord, mobilized peripheral blood, bone marrow) using an aqueous two-phase system having at least one step. The invention is suitable for use in the medical field for the recovery of stem cells. The process consists of: construction of the polymer-polymer aqueous two-phase system (in its different modes), addition of the sample, agitation, centrifugation and separation of the phases for the subsequent recovery of same and quantification of CD133+ cells by means of flow cytometry. The phase rich in CD133+ is washed and resuspended in a medium suitable for its subsequent use.

Description

PROCESS FOR RECOVERY AND PURIFICATION OF CD133 + MONONUCLEAR CELLS BY SYSTEMS OF TWO AQUEOUS PHASES DESCRIPTION OBJECT OF THE INVENTION The present patent application provides a process of fractionation, recovery and purification of CD133 + mononuclear cells from a biological matrix rich in stem cells using a two-phase aqueous system of at least one stage.

BACKGROUND In the last decades, the stem cell transplant has aroused a growing interest in the area of clinical research due to the promising results that are expected derived from the particular characteristics of these cells. Stem cells are distinguished by their capacity for self-renewal and differentiation (Tárnok, A., Ulrich, H., &Bocsi, J. (2010) Phenotypes of stem cells from diverse origin Cytometry. Journal of the International Society for Analytical Cytology, 77 (1), 6-10), as well as the presence of unique cell surface proteins for this particular type of cell, called a cluster of differentiation (CD). The stem cells express the CD34 antigen presented by the hematopoietic progenitor cells.

This type of cells can be obtained from various sources such as umbilical cord, bone marrow or mobilized peripheral blood (Namiri, M., Baharvand, H., &Aghdami, N. (201 1) .Methods for Isolation of Bone Marrow Stem Cells : Comparative Analysis, Cell Journal, 12 (4), 439-446). The self-renewal implies that the cell can be divided for long periods of time by mitosis in order to replicate and proliferate without losing the capacity for differentiation in almost all existing cell types. It is precisely these properties which turn stem cells into excellent tools in cell transplantation, where the use of these cells is proposed with the aim of regenerating or replacing the damaged cells of some tissue to restore the required tissue function (Miwa, J., Suzuki, Y., &Kasagi, N. (2008) Adhesion-Based Cell Sorter With Antibody-Coated Amino-Functionalized-Parylene Surface, Journal of Microelectromechanical Systems, 17 (3), 61 1-622).

Cellular regeneration is usually presented as a viable treatment for chronic degenerative diseases and genetic defects that until now have no cure (Yu, RK, Suzuki, Yusuke, &Yanagisawa, M. (2010) .Membrane glycolipids in stem cells. Letters, 584 (9), 1694-1699, Federation of European Biochemical Societies, Peters, A., Burridge, PW, Pryzhkova, MV, Levine, M., Park, T.-S., Roxbury, C, et al. (2010) Challenges and strategies for the therapeutic patient-specific hemangioblasts and hematopoietic stem cells from human pluripotent stem cells, The International Journal of Developmental Biology, 54 (6-1), 965-990) as they are musculoskeletal disorders, neurological, cardiovascular and hematopoietic (Wu, X., Wang, S., Chen, B., & An, X. (2010).

Muscle-derived stem cells: isolation, characterization, differentiation, and application in cell and gene therapy. Cell and Tissue Research, 340 (3), 549-567; Miwa, J., Suzuki, Y., & Kasagi, N. (2008). Adhesion-Based Cell Sorter With Antibody-Coated Amino-Functionalized-Parylene Surface. Journal of Microelectromechanical Systems, 77 (3), 61 1-622).

A particular case is the disease of Amyotrophic Lateral Sclerosis (ALS), which is a neurodegenerative disease characterized by the rapid deterioration and selective death of neurons in the cerebral cortex. Currently, autologous transplantation of CD133 + stem cells in the cerebral cortex is being implemented as a treatment for this disease. (Martínez, H., Gonzalez-Garza, M., Moreno-Cuevas, J., Caro, E., Gutiérrez-Jiménez, E., &Segura, J. (2009) Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients.Cytotherapy, 11 (1), 26-34). CD133 + cells are considered pluripotent (Ruzicka, K., Grskovic, B., Pavlovic, V., Qujeq, D., Karimi, A., &; Mueller, M. M. (2004). Differentiation of human umbilical cord blood CD133 + stem cells towards myelo-monocytic lineage. Clínica Chimica Acta, 343 (1-2), 85-92) and have the ability to differentiate into neurons, which is why they are implanted in the patient with ALS with the aim of improving neuronal functions (Martínez, H., Gonzalez- Garza, M., Moreno-Cuevas, J., Caro, E., Gutierrez-Jimenez, E., &Segura, J. (2009) Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients. , 11 (1), 26-34).

The CD133 antigen is a 5-transmembrane glycoprotein conserved among mammals, fish and insects (Handgretinger, R., Gordon, P., Leimig, T., Chen, X., Buhring, H.-J., Niethammer, D., et al. (2003). Biology and plasticity of CD133 + hematopoietic stem cells. Annals of the New York Academy of Sciences, 996, 141-151). Although the specific function of the CD133 antigen or its potential ligands is unknown, it has been proposed that CD133 + cells can also be differentiated to CD34 + cells and other hematopoietic precursors with endothelial capacity (Handgretinger, R., Gordon, P., Leimig, T. , Chen, X., Buhring, H.-J., Niethammer, D., et al. (2003), Biology and plasticity of CD133 + hematopoietic stem cells, Annals of the New York Academy of Sciences, 996, 141-151) .

Currently the recovery of CD133 + cells is through Percoll gradients and immunomagnetic separation using the CD133 antigen conjugated with magnetic microparticles and lasts approximately eight hours. The methodology used is as follows: patients with ALS are induced daily for three days by a subcutaneous injection containing filgastrim or better known as human granulocyte colony stimulating factor (G-CSF) at a concentration of 300μg. This with the aim of mobilizing and releasing the cells of interest to the bloodstream. On the day after the last dose, patients undergo leukapheresis, a process in which peripheral blood is removed from a vein to separate leukocytes (white blood cells) and the rest of the blood is reinfused into the patient. Subsequently, the recovered cells are washed three times with phosphate buffered saline (PBS) and then incubated with the CD133 antigen conjugated with magnetic microparticles and separated using a magnetic field in the MiniMACS column.

Finally, the enriched CD133 + cells are validated by flow cytometry and are resuspended in sterile tubes at a concentration of 2.5-7.5 X 105 in 300 μl, of cerebrospinal fluid previously extracted from the patient, for transplantation (Martínez, H., Gonzalez- Garza, M., Moreno-Cuevas, J., Caro, E., Gutierrez-Jimenez, E., &Segura, J. (2009) Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients. , 11 (1), 26-34).

It should be noted that commercially there are other equipment that allows obtaining these cells also based on the use of antibodies conjugated to supermagnéticas microperlas. An example of this is the Isolex equipment (Nexell Therapeutics Inc., Irvine, CA, USA) which differ from the columns described above in allowing the use of the leukapheresis product as obtained without the need to make a Percoll gradient to obtain * White cells and use for cell separation two stages of immunological labeling. In the first, the specific antibody is added to the cells to be separated and in the second, the antibodies are added to the supermagnetic microbeads that recognize the first antibody. Subsequently the cells are mobilized through a column where a magnetic field is applied that separates the marked cells from the rest of the suspension. Likewise, the cells have to be washed and suitably prepared for transplantation to the patient (Rowley, S., Loken, M., Radich, J., Kunkle, L., Mills, B., Gooley, T., et. (1998) Isolation of CD34 + cells from blood stem cell components using the Baxter Isolex system, Bone Marrow Transplantation, 21 (12), 1253-62).

The purification of CD133 cells by the techniques described above has achieved successful results in various clinical tests when obtaining high yields, however, the procedures tend to be long (approximately 5 hours) and costly. In this sense, there is a latent need to develop a faster and more scalable process that guarantees the purity and performance of CD133 + mononuclear cells. Purity is an essential requirement to ensure the success of separation, since the incidence of graft-versus-host disease caused by an inefficient depletion of T cells after an allogeneic transplant should be reduced (Bitan, M., Shapira, MY, Resnick, IB, Zilberman, I., Mirón, S., Samuel, S., et al. (2005), Successful transplantation of haploidentically mismatched blood stem cells using CD133 + -purified stem cells, Experimental Hematology, 33 (6), 713-718) and eliminating the risk of contamination by viral pathogens in syngeneic transplants.

In this context, the use of aqueous two-phase systems (SDFA) for the primary recovery and partial purification of CD133 + cells presents multiple benefits. Since its discovery for the separation of biological compounds, SDFA have been used to recover a large number of products such as proteins, genetic material, low molecular weight compounds, etc. (Benavides, J., &Rito-Palomares, M. (2008).) Practical experiences from the development of two-phase processes for the recovery of high biological production, Journal of Chemical Technology And Biotechnology, 83 (2), 133-142). Additionally they have been used for the fractionation and recovery of cells and organelles, including blood components (Delgado, C, Anderson, R., Francis, G., &Fisher, D. (1991).

Separation of cell mixtures by immunoaffmity cell partitioning: strategies for low abundance cells. Analytical Biochemistry, 192 (2), 322-8; Walter, H., Krob, E., Garza, R., & Ascher, G. (1969). Partition and countercurrent distribution of erythrocytes and leukocytes from different species. Experimental Cell Research, 55 (1), 57-64). These systems are a liquid-liquid extraction technique with advantages such as high biocompatibility, low degradation of biomolecules and ease of scaling (Benavides, J., &Rito-Palomares, M. (2008). Two-phase processes for the recovery of high valué biological production, Journal of Chemical Technology and Biotechnology, 83 (2), 133-142; Rito-Palomares, M. (2004) Practical application of aqueous two-phase partition to process development for the recovery of biological production, Journal of Chromatography B, 807 (1), 3-1 1; Rito-Palomares, M., &Lyddiatt, A. (2002) Process integration using aqueous two-phase partition for the recovery of intracellular proteins, Chemical Engineering Journal, 87 (3), 313-319).

The SDFA are obtained by mixing two hydrophilic components (two polymers, a polymer and a salt, a low molecular weight alcohol and a salt, or an ionic liquid and a salt) that, over certain critical concentrations, become insoluble, generating two phases whose main component is water (Carrea, G. (1984), Biocatalysis in water-organic solvent two-phase systems, TRENOS in Biotechnology, 2 (4), 102-106; Culi, S., Holbrey, I, Vargas-Mora , V., Seddon,., &Lye, G. (2000), Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations, Biotechnology &Bioengineering, 69 (2), 227-233; Palomares, M. (2004). Practical application of aqueous two-phase partition to process development for the recovery of biological products. Journal of Chromatography B, 807 (1), 3-1 1) There are also SDFA formed by mixtures of detergents which, under certain conditions, form phases rich in micelles (reverse micelles and micelles (Tani, H., Kamidate, T., & Watanabe, H. (1998) Aqueous micellar two-phase systems for protein separation, Analytical Sciences, 14 (5), 875-888).

The partition of solutes such as biomolecules and cells in this type of systems is directly related to several physicochemical properties intrinsic to the surface of said solutes as well as to their hydrodynamic radius (size) (Albertsson, P.-Á. (1961). of particles and macromolecules in aqueous two-phase systems, Biochemical Pharmacology, 5 (4), 351-358, Hartounian, H., Kaler, E., &Sandler, S. (1994) Aqueous Two-Phase Systems. Protein Partitioning, Industrial &Engineering Chemistry Research, 33 (10), 2294-2300). In the case of the fractionation of cells and organelles, characteristics such as surface charge and hydrophobicity, together with particle size, have great relevance for determining the partition behavior (Olivera-Nappa, A., Lagomarsino, G., Andrews, B. , &Asenjo, J. (2004). Effect of electrostatic energy on partitioning of proteins in aqueous two-phase systems, Journal of Chromatography B, 807 (1), 81-86; Salgado, J., Andrews, B., Ortuzar, M., &Asenjo, J. (2008) Prediction of the partitioning behavior of proteins in aqueous two-phase systems using only their amino acid composition, Journal of Chromatography A, 1178 (1-2), 134-144 Schmidt, A., Andrews, B., &Asenjo, J. (1996).

Correlations for the partition of proteins in aqueous two-phase systems: effect of overall protein concentration. Biotechnolog & Bioenginee ng, 50 (6), 617-626; Walter, H., & Krob, E. J. (1976). Partition in two-polymer aqueous phases reveals differences between membrane surface properties of erythrocytes, ghosts and membrane vesicles. Biochimica et Biophysica Acta, 455 (1), 8-23). The partition of the biological products of interest in the SDFA is characterized by the determination of the partition coefficient (Kp), defined as the ratio of the concentrations of the partitioned products in the upper and lower phase of the system (Albertsson, P.-Á (1961), Fractionation of particles and macromolecules in aqueous two-phase systems, Biochemical Pharmacology, 5 (4), 351-358). The value of P and therefore the percentages of recovery is usually dependent on system parameters such as the chemical nature of the constituents, the length of the cut line (LLC), which is a function of the concentration difference of the constituents of the system. in upper and lower phase), the volume ratio between the upper and lower phase (V), the pH of the system and the percentage of sample fed to the system, among others (Benavides, J., &Rito-Palomares, M. (2008) .Practical experiences from the development of aqueous two-phase processes for the recovery of high-value biological products, Journal of Chemical Technology and Biotechnology, 83 (2), 133-142).

The present patent application is intended to provide a process for carrying out the fractionation, recovery and purification of blood cells, particularly CD133 + mononuclear cells, from a product rich in stem cells by implementing a system of two aqueous phases of at least one stage. Although works by other authors report the fractionation of blood cells in SDFA from whole blood, no studies have been found that show the partition behavior in SDFA of cells from a product rich in stem cells. Additionally, said works do not focus on obtaining a fraction enriched in CD133 + cells.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Block diagram of the process for recovery and purification of CD133 + mononuclear cells from a biological matrix rich in stem cells using two-phase aqueous systems of at least one stage as the primary purification method. The optional stages of the process are shown with dotted lines.

Figure 2: Block diagram of the sub-stages of stage A) of the process for this patent application.

Figure 3: Block diagram of the sub-stages of stage B) of the process for this patent application. The optional stages of the process are shown with dotted lines.

Figure 4: Percentages of recovery of each of the cell families (white blood cells, red blood cells and platelets) in a PEG-dextran aqueous phase system. Upper Phase), Interphase), Lower Phase).

Figure 5: Percentages of recovery of each of the cell families (white blood cells, red blood cells and platelets) in an aqueous phase system PEG-dextran added with NaCl. Upper Phase), Lower Phase (^), Precipitate (H).

Figure 6: Percentages of recovery of CD133 + cells in each of the two-phase systems analyzed. Upper Phase (|), Interface (0), Lower Phase (?).

Figure 7: Percentages of recovery of CD133 + cells in each of the two-phase systems with free antibody analyzed. Upper Phase (|), Interface (0), Lower Phase < H.H ).

DETAILED DESCRIPTION OF THE INVENTION The present patent application provides a process for carrying out the fractionation, recovery and purification of stem cells, particularly CD133 + mononuclear cells, from a product rich in stem cells, which comprises any of the following: umbilical cord blood, mobilized peripheral blood, leukapheresis product, cell culture, bone marrow, adipose tissue, to name a few, by implementing a two-phase aqueous system of at least one stage . This involves liquid-liquid fractionation strategies with a two-phase aqueous system (SDFA) that can consist of the implementation of a single-stage system to achieve partition, recovery and partial purification until the implementation of at least two systems of two consecutive aqueous phases called multi-stages in order to increase the final purity of the desired product. Furthermore, it should be noted that the two aqueous phase systems can include free affinity ligands or these can be conjugated to one or both of the polymers used for the construction of the system, in order to increase the selectivity of the SDFA by the cells of interest . The multi-stage SDFA can in turn be operated continuously, for example centrifugal countercurrent partition and countercurrent distribution chromatography, among others. After the implementation of the SDFA it is possible to couple other purification techniques, such as chromatography, to achieve a greater purification of the product of interest.

Figure 1 shows the general block diagram of the process proposed in this patent application developed for the fractionation, recovery and purification of CD133 + cells by SDFA, which generally comprises the steps of: A. Select the SDFA (100), B. Submit a product rich in stem cells to at least one SDFA (200) to obtain CD133 + stem cells (200).

According to the intended use of the CD133 + stem cells obtained with the proposed process, they must be quantified (300) to know their percentage of purity (400), and determine whether they are again submitted to an SDFA or taken to a polishing stage. , so that after step B, optionally at least one quantization step (300) and / or polish (500) is implemented.

It is the interest of the inventors to describe the obtaining of the product rich in stem cells on which the present process is applied, so that the obtaining of said product from biological matrices is described below: a) Leukapheresis product from mobilized peripheral blood The mobilization of hematopoietic stem cells and their subsequent release into the bloodstream of the patients is achieved by subcutaneous administration of between 5 and 10 μg / kg of the patient's weight of filgastrim® or better known as human granulocyte colony stimulating factor (G). -CSF). This dose is administered every 12 or 24 hours for 3 to 6 days (Beelen, D., Peceny, R., Elmaagacli, A., Ottinger, H., Kummer, G., Opalka, B., et al. (2000 ).

Transplantation of highly purified HLA-identical sibling donor blood CD34 + cells without prophylactic post-transplant immunosuppression in adult patients with first chronic phase chronic myeloid leukemia: results of a phase II study. Bone Marrow Transplantation, 26 (8), 823-829; Lang, P., Handgretinger, R., Niethammer, D., Schlegel, P., Schumm, M., Greil, J., et al. (2003). Transplantation of highly purified CD34 + progenitor cells from unrelated donors in pediatric leukemia. Blood, 01 (4), 1630-6; Martínez, H., Gonzalez-Garza, M, Moreno-Cuevas, J., Caro, E., Gutiérrez-Jiménez, E., & Segura, J. (2009). Stem-cell transplantation into the frontal motor cortex in amyotrophic lateral sclerosis patients. Cytotherapy, 11 (1), 26-34).). Once the last dose is administered, the patient is subjected to the leukapheresis procedure in order to recover the mobilized mononuclear cells.

Leukapheresis is carried out in a continuous flow separator of blood cells equipped with a computer program that allows the collection of mononuclear cells. On average, the procedure lasts between 3 and 10 hours in one, two or three consecutive daily sessions, respectively (Lang, P., Handgretinger, R., Niethammer, D., Schlegel, P., Schumm, M., Greil, J., et al. (2003) .Transplantation of highly purifi ed CD34 + progenitor cells from unrelated donors in pediatric leukemia, Blood, 101 (4), 1630-6). According to published studies, leukapheresis products from people induced with granulocyte colony stimulating factor have a median concentration of 1.3x1010 mononuclear cells (range 0.7-3.8x1010) with a median percentage of 0.75% (range 0.39 - 2.03). %) of CD133 + cells. b) Umbilical cord blood With the prior consent of the mother to donate the umbilical cord of her baby, obtaining the signature of the corresponding persons for the donation of the umbilical cord and having previously informed the doctor in turn; The latter will be asked to remove cord blood after delivery and place it directly in the bag to collect blood. Preferably, this procedure should be carried out in a laminar flow hood by puncturing the umbilical vein, thus obtaining 40 to 160 mL of blood and stored in the bag with anticoagulant to be transported to the laboratory in a cooler at 4 o'clock. ° C and will remain at this temperature for up to 24 hours before processing. Try to process the blood immediately to obtain mononuclear cells.

Once in the laboratory, using a blood count, determine the total blood volume and the number of initial nucleated cells with which the processing begins. The cord blood collected in the bag will be diluted in PBS in a 1: 1 ratio and then placed slowly on the density gradient layer to be used (Lymphoprep, Percoll, Ficoll) in several 50 mL tubes, in a proportion 2: 1 (diluted blood: density gradient medium). Said mixture will be separated into aliquots that will be treated with a means for separation by density gradient (Lymphoprep, Percoll, Ficoll). It should be centrifuged between 500 to 1,000 X g for 10 to 40 min at a temperature between 4 - 25 ° C. If the blood was stored for more than 2 hours, increase the centrifugation time.

Subsequently, remove the mononuclear cell band with Pasteur pipettes. Dilute the fraction collected in PBS to decrease the density of the solution (at least 2 washes). Concentrate the cells in a pellet by centrifugation for 5 - 15 min between 100 - 300 X g. If it is not necessary to use the sample immediately, store in medium with 10% (v / v, max) of DMSO in a stepwise process (30 min at 4 ° C, 2 hours at -20 ° C and move to -80 ° C ). Finally, thaw, transfer to medium or PBS without DMSO by washing as quickly as possible.

Next, each of the stages and sub-steps of the process that is the motive of this patent application is described in detail.

A. Select the SDFA (100) This stage has the purpose of selecting a system of two aqueous phases based on the different design parameters, in order to find the optimal conditions for the separation of the cells of interest according to the specific needs of the process. The sub-steps that make up this stage are the following and are represented in Figure 2: a) Select polymers (110) For the development of the invention SDFA polymer-polymer was selected due to the biological nature of the product of interest. Other types of SDFA can be used in the same way for the fractionation of cells. However the viability of cells is generally better conserved in polymer-polymer systems, without ruling out the potential use of other types of systems. The polymers selected for the elaboration of the SDFA were polyethylene glycol (PEG), dextran, fícoll and UCON which have been used previously for the partition of blood cells (Backman, L. (1990). by aqueous two-phase partition FEBS Letters, 262 (1), 107-1 10; Soohoo, J., &Walker, G. (2009) .Microfluidic aqueous two phase system for leukocyte concentration from whole blood. 11 (2), 323-9; Walter, H., &Kiob, EJ (1976) .Partition in two-polymer aqueous phases reveals differences between membrane surface properties of erythrocytes, ghosts and membrane vesicles Biochimica et Biophysica Acta, 455 (1), 8-23).

The polymer-polymer SDFAs are prepared using a fixed mass base by making a mixture of solutions with the appropriate amounts of each of the polymers. These polymer solutions are conveniently maintained at concentrations between 20 and 80% w / w and their pH is adjusted by the addition of acid (orthophosphoric acid or hydrochloric acid 1-6 N) or base (sodium hydroxide or potassium hydroxide 1 - 6 N) as necessary. For the preparation of the PEG-dextran systems, PEG with molecular weight between 3,000 and 35,000 g / mol and dextran with molecular weight between 10,000 and 500,000 g / mol can be used. For the ficoll-dextran and ficoll-PEG systems, ficoll of molecular weight of 70,000 or 400,000 g / mol, dextran of molecular weight between 10,000 and 300,000 g / mol and PEG of molecular weight between 3,000 and 35,000 g / mol can be used. He process for the selection of a two-phase aqueous system for the fractionation, recovery and primary purification of CD133 + cells is shown in Figure 2. b) Determine SDFA parameters such as: LLC, VR, pH, percentage of sample, concentration of sodium chloride (NaCl) and concentration of antibody (120).

As for the LLC systems can be managed in the range of 15 to 50% p / p. With regard to VR can be handled at convenience (depending on the proportion of volumes required between upper and lower phase) from values of 0.05 to 20. For its part the pH of the system should be managed at an approximate value at pH physiological (7.0 ± 1.0) to which the cells of interest are exposed in order to avoid damage due to extreme conditions. The amount of sample that is fed into the system (any cell suspension, in this particular case the biological product rich in stem cells) can represent 1 to 50% w / w of the total weight of the system. Optionally it is possible to add neutral salt to the SDFA to manipulate the ionic strength in the phases and generate a more selective fractionation towards some of them. The most commonly used salt for these purposes is sodium chloride (NaCl), which can reach a concentration of 0.0 - 0.25 M (± 0.01) in the system, since salt concentrations outside this range can affect cell viability. c) Build the SOFA (130) Once defined the characteristics of the polymers to be used in the SDFA (type and molecular weight) and to select all the variables of the system, we proceed to build the systems of two aqueous phases. For this, stock solutions (at a certain concentration) of each of the polymers are prepared and the necessary amount of each is weighed (stock solution of polymer 1, stock solution of polymer 2 and solvent) into a tube.

B. Submit the product rich in CD133 + stem cells to the SDFA selected in A.) This step of submitting the product rich in stem cells is detailed in Figure 3, and consists of the following sub-steps: a) Add sample (210) Once the adequate quantities of the stock solutions of polymer 1 and 2 are added and the corresponding quantity of the solvent added, the sample is added in the determined volume. b) Mix (220) Once all the components of the system are added, they are shaken very gently by inversion of 5 to 30 minutes, in such a way that the mixture of all the compounds is carried out and cell damage is avoided to the maximum. c) Stabilize (230) The stabilization of the phases of the system is achieved by natural sedimentation or by using centrifugation with moderate gravity forces (between 100 and 5,000 X g) low to moderate temperatures (4 - 25 ° C) between 10 and 30 min. The centrifugation streamlines the process of phase separation and helps the compaction of the different cell groups in some of the phases of the system.

Under optimal conditions a selective separation of white cells (including CD133 +) is obtained towards one of the phases of SDFA (upper, lower, interface or surface), while erythrocytes or red blood cells (main pollutant) are concentrated in a different phase. In this way the phase in which the red blood cells are concentrated can be removed by pipetting or any other form of suction. d) Recover (240) The phase that includes the set of white blood cells (where the CD133 + cells are located) is recovered in the same way for further processing. This phase of interest can be centrifuged (between 100 and 5,000 X g at 4 - 25 ° C for 10 and 30 min.) To promote the precipitation of white blood cells towards the bottom of the container. The concentrated fraction of white blood cells can be recovered by dripping, pipetting or other suctioning. The remaining cells are finally washed with PBS of physiological pH.

Several strategies can be followed to select a system of two aqueous phases to carry. Here are some of them: a) Implementation of a single-stage SDFA, in the present patent application it is understood that a single-stage SDFA consists of once submitting the rich product in stem cells to a system of two aqueous phases. b) Implementation of two or more stages of SDFA consecutively (multistage SDFA), in the present patent application it is understood that a SDFA of two or more stages consists of subjecting the product rich in stem cells obtained more than once of the separation of the phases of a first SDFA to a new system of two aqueous phases. c) Implementation of one or more SDFAs modified by the addition of free form affinity ligands (see figure 3-250): to. Antigens: CD45, CD34, CD133 b. Antibodies: anti-CD45, anti-CD34, anti-CD 133 c. Particles with conjugated antibodies d. Magnetic particles conjugated with antibodies and. Mix of the above d) Implementation of one or more SDFA constructed with constituents chemically derivatized with affinity ligands e) Implementation of SDFA operated continuously by centrifugal countercurrent partition and / or countercurrent distribution chromatography f) A combination of the aforementioned.

After stage B, the following stages can be implemented: C. Quantify the CD133 + stem cells obtained in B.) In this stage of quantifying the CD133 + stem cells (300) once all the phases of the system are separated, it consists in taking a sample of 250 μ ?, of each one of the phases to quantify CD133 +. This sample is divided into three samples of 70 μL ·. Two of the samples will be used for flow cytometry staining (viability, detection and quantification of cell families) and the other will be resuspended in 1 mL of FACS FLOW® (Becton-Dickinson, California, USA), for granulometry observation and size. The above, with a BD FACSCanto II equipment (Becton-Dickinson, California, USA). The surface markers that will be used for the counting of mononuclear cells will be: CD45-FITC, CD34-APC and CD133-PE (Miltenyi Biotech GmBH, Germany). To obtain the viability of the cells, the samples will be stained with 7-AAD (7-Amino-actinomycin D from BD Biosciences).

D. Polishing and final preparation of CD133 * cells There is the possibility of coupling one or more unit operations upon completion of CD133 + cell recovery by SDFA. This allows the final purity of the cells of interest to be increased by a polishing step (500). An alternative is to use the fluorescence activated cell separator (FACS), which consists of incubating the cells with a specific antibody to the CD133 marker and linked to a fluorochrome (Gordon, P., Leimig, T., Babarin-Dorner, A., Houston, J., Holladay, M, Mueller, I., et al., (2003) Large-scale isolation of CD133 + progenitor cells from G-CSF mobilized blood stem cells Bone Marrow Transplantation, 31 (1), 17-22). They can also be incubated first with a specific antibody to CD133 and then with a secondary antibody linked to a fluorochrome akin to the first antibody. The FACS collects fluorescence signals and specific characteristics of light scattering (lateral and frontal scattering) and based on this data selects the cells of interest and cuts the flow separating them into small droplets (Kamihira, M., &Kumar, A. ( 2007), Development of Separation Technique for Stem Cells, Advances in Biochemical Engineering / Biotechnology, 106, 173-193).

Another option is affinity chromatography, which can be coupled to the methodology described here to achieve a higher final purity of CD133 + cells. In this procedure the cells recovered and resuspended from the SDFA were passed through a column packed with agarose, polymethyl methacrylate or dextran beads to which specific antibodies were bound to the CD133 + cells. This allows the elution of cells that do not possess this antigen. Once these cells leave the column, the cells attached to the stationary phase can elute from the column and thus recover them (Matsumoto, U., &; Shibusawa, Y. (1980). Surface affmity chromatographic separation of blood cells. I. Separation of human and rabbit peripheral granulocytes, lymphocytes and erythrocytes using polyethylene glycol-bonded column packings. Journal of Chromatography A, 187 (2), 351-362). In case of using any additional technique, the recovered cells have to be counted, washed and resuspended in a suitable medium for later use.

Example 1: Recovery of mononuclear cells from a leukapheresis product with the process motif of this patent application.

A. Select the SDFA To build a two-phase aqueous system of 1.0 g total weight (5.4% w / w PEG 4,000 g / mol, 10% w / w dextran 110,000 g / mol, 30% w / w leukapheresis product and line length of cut 30% w / w) in a conical plastic tube with a capacity of 2 cm3, 0.15 g of PEG solution 4,000 g / mol (50% w / w), 0.52 g of dextran solution 1 10,000 g / mol were mixed. (30% w / w), 0.30 g of the leukapheresis product and 0.03 g of distilled water.

B. Submit the product rich in CD133 + stem cells to the SDFA selected in A.) Once the determined amount of sample was added, the contents of the tube were gently stirred for 5 minutes by inversion and then centrifuged at 5 ° C for 10 minutes at 2,000 X g. The upper and lower phases were carefully removed in their entirety leaving the cells concentrated in the interface in the tube. 0.2 cm3 of each of the samples of the phases were taken and resuspended in 1.2 cm3 of Isoton® II solution (Beckman Coulter, Brea, CA, USA). Cells recovered at the interface, whose approximate volume is 0.2 cm3 were similarly resuspended in 1.2 cm3 of Isoton® II solution (Beckman Coulter, Brea, CA, USA).

C. Quantify the cells obtained in B) Once the recovered cells were resuspended in each of the phases of the SDFA, the concentration of red blood cells, white blood cells and platelets was analyzed using the Beckman Coulter HMX Blood Cell Counter (Beckman Coulter, Brea, CA, USA). In the lower phase, 29.36xl06 white blood cells, 0.20xl09 red blood cells and 277.60xl06 platelets were recovered, representing respectively 47.54%, 53.68% and 44.14% of the total of each of the cells in the system. In the interface, 32.40x106 white blood cells, 0.18xl09 red blood cells and 1440.00xl06 platelets were recovered, representing respectively 52.46%, 46.32% and 55.86% of the total of each of the cells in the system. In the upper phase, no cells were recovered. Finally, the viability of the cells was analyzed by the trypan blue assay for each of the phases, obtaining an average viability of 91.1% (living cells / total cells). Figure 4 shows the partition behavior of each of the cell families in the selected SDFA.

Example 2: Effect of the addition of sodium chloride (NaCl) on the recovery of mononuclear cells from a leukapheresis product with the process motif of this patent application.

A. Select the SDFA To build a system of two aqueous phases of 1.0 g of total weight (5.6% p / p PEG 10,000 g / mol, 15% w / w dextran 10,000 g / mol, 30% w / w leukapheresis product, 0.05 M NaCl and cutting line length 25% w / w) in a conical plastic tube with a capacity of 2 cm, 0.1 1 g of PEG solution 10,000 g / mol (50% w / w), 0.38 g of dextran solution 10,000 g / mol (40% w / w), 0.30 g of the leukapheresis product and 2.92 were mixed. mg of NaCl dissolved in 0.21 g of distilled water.

B. Submit the product rich in CD133 * stem cells to the SDFA selected in A.) Once the amount of sample was added, the contents of the tube were gently stirred by inversion for 5 minutes and then centrifuged for 10 minutes at 2,000 X g and 5 ° C. The upper and lower phases were carefully removed in their entirety leaving in the tube the cells concentrated in the precipitate located at the bottom of the tube and separated from the rest of the phases. 0.2 cm of each of the samples of the phases were taken and resuspended in 1.2 cm of Isoton® II solution (Beckman Coulter, Brea, CA, USA). The cells recovered in the precipitate, whose approximate volume is 0.2 cm3 were similarly resuspended in 1.2 cm3 of Isoton® II solution (Beckman Coulter, Brea, CA, USA).

C. Quantify the cells obtained in B) Once the cells recovered in each part of the SDFA were resuspended, the concentration of red blood cells, white blood cells and platelets was analyzed using the Beckman Coulter HMX Blood Cell Counter (Beckman Coulter, Brea, CA, USA). In the precipitate recovered 36.76xl06 white blood cells, 0.19xl09 red blood cells and 494.40x106 platelets, representing respectively 93.42%, 74.60% and 88.68% of the total of each of the cells in the system. In the lower phase 2.44x106 white blood cells, 0.06xl09 red blood cells and 61.60xl06 platelets were recovered, representing respectively 6.20%, 25.40% and 1.05% of the total of each of the cells in the system. In the upper phase 0.15x106 white blood cells, 1.5x106 platelets were recovered and no red blood cells were detected, which represents 0.38%) of the total white blood cells and 0.27% of the total platelets added to the system. Finally, the viability of the cells once the separation process was finished was analyzed by the trypan blue assay for each of the phases, obtaining an average viability of 87.20% (live cells / total cells). Figure 5 graphically shows the partition behavior of each of the cell families in this two-phase aqueous system. As can be seen, the addition of sodium chloride to the system favored the fractionation of the different cellular families to the lower phase.

Example 3: Effect of the type of polymers used in the construction of aqueous two-phase systems on the recovery of CD133 + cells from umbilical cord blood with the process motif of this patent application.

After obtaining the written consent of the mother, umbilical cord blood is collected. The cord blood collected in the bag is diluted in a 1: 1 ratio in phosphate buffered saline (PBS) and then slowly placed on the Lymphoprep density gradient layer in 50 mL tubes. in a 2: 1 ratio (diluted blood: density gradient medium). The samples are centrifuged at 800 X g for 30 min at 20 ° C. Subsequently, the band of mononuclear cells is collected with a Pasteur pipette, smoothed and the fraction harvested in PBS is washed to decrease the density of the solution (at least 2 used and 2 washes). The cells are concentrated in a pellet by centrifugation for 10 min at 250 X g and resuspended in the amount of PBS suitable to be introduced into the two-phase systems.

A. Select the SDFA The polymer-polymer two-phase systems are constructed with a total weight of 1.0 g (5.6% PEG 8,000-7.5% dextran 500,000, 13.6% ficoll 400,000- 1 1.6% dextran 70,000 and 8.23% dextran 75,000-6.83% UCON) with 10 % w / w of sample and a length of cutting line 20% w / w) in a conical plastic tube with a capacity of 2 cm3. In all cases, the solvent used was PBS (pH 7.4, 154 mM NaCl) filtered with a syringe filter (0.20 μp?), Which was mixed with the predetermined amounts of stock solution of each of the polymers used in the construction. of the systems (50% p / p PEG 8,000, 30% p / p dextran 500,000, 40% p / p fícoll 400,000, 40% p / p dextran 70,000, 42% p / p dextran 75,000, and 70% p / p UCON).

B. Submit the product rich in CD133 + stem cells to the SDFA selected in A.) Once the determined amount of sample has been added, the contents of the tube are stirred gently for 15 minutes by inversion to later reach the equilibrium of the phases by means of natural sedimentation at 20 ° C. Once the SDFA is stabilized, each of the phases of the system (upper, lower and interphase phases) are carefully separated by pipetting.

C. Quantify the CD133 + stem cells obtained in B) Once these have been separated, a sample of 250 μL · is taken from each of the phases to quantify CD133 +. This sample is divided into three samples of 70 μ ?. Two of the samples are used for flow cytometry staining (viability, detection and quantification of cell families) and the other is resuspended in 1 mL of FACS FLOW® (Becton-Dickinson, California, USA), for granulometry and size observation. The above, with a BD FACSCanto II equipment (Becton-Dickinson, California, USA). The surface markers used for the mononuclear cell count are: CD45-FITC, CD34-APC and CD133-PE (Miltenyi Biotech GmBH, Germany). To obtain the viability of the cells, the samples are stained with 7-AAD (7-Amino-actinomycin D from BD Biosciences). In the PEG 8,000-dextran 500,000 systems, 45% of the CD133 + cells divide into the lower phase and the rest are located in the interface, leaving the upper phase clean. In the case of dextran 75,000- systems UCON 56% of CD133 + cells are partitioned to the lower phase and the rest are located at the interface, leaving the upper phase clean. In the SDFA ficoll 400,000-dextran 70,000 100% of the CD133 + cells are partitioned towards the upper phase. Figure 6 shows graphically the partition behavior of CD133 + cells in each of the two-phase systems analyzed. Where it is observed that in the ficoll system 400,000-dextran 70,000 the CD133 + cells are completely partitioned to the upper phase. While in the PEG systems 8,000-dextran 500,000 and dextran 75,000-UCON a similar partition is obtained between the lower phase and the interphase.

Example 4: Effect of the addition of free antibody in aqueous two-phase systems on recovering the fractionation of CD133 + cells from umbilical cord blood with the process motif of this patent application.

After obtaining the written consent of the mother, umbilical cord blood is collected. The cord blood collected in the bag is diluted in a 1: 1 ratio in phosphate buffered saline (PBS) and then slowly placed on the Lymphoprep density gradient layer in 50 mL tubes. in a 2: 1 ratio (diluted blood: density gradient medium). The samples are centrifuged at 800 X g for 30 min at 20 ° C. Subsequently, the band of mononuclear cells is collected with a Pasteur pipette, smoothed and the fraction harvested in PBS is washed to decrease the density of the solution (at least 2 lysates and 2 washes).

The cells are concentrated in a pellet by centrifugation for 10 min at 250 X g and resuspended in the amount of PBS suitable to be introduced into the two-phase systems.

A. Select the SDFA The polymer-polymer two-phase systems are constructed with a total weight of 1. 0 g (5.6% PEG 8,000-7.5% dextran 500,000, 13.6% ficoll 400,000- 1 1.6% dextran 70,000 and 8.23% dextran 75,000-6.83% UCON) with 10% w / w of the sample and length of •to cutting line 20% p / p) in a conical plastic tube with a capacity of 2 cm. In all cases, the solvent used was PBS (pH 7.4, 154 mM NaCl) filtered with a syringe filter (0.20 μp?), Which was mixed with the predetermined amounts of the stock solution of each of the polymers used in the construction of each of the systems tested (50% p / p PEG 8,000, 30% p / p dextran 500,000, 40% p / p fícoll 400,000, 40% p / p dextran 70,000, 42% p / p dextran 75,000, and 70% w / w UCON) and with 2.5 μg of pure CD133 antibody (CD133 / 2, clone 293C3, Miltenyi Biotech GmBH, Germany).

B. Submit the product rich in CD133 + stem cells to the SDFA selected in A.) Once the determined amount of sample has been added, the contents of the tube are stirred gently for 15 minutes by inversion to later reach the equilibrium of the phases by means of natural sedimentation at 20 ° C. Once the SDFA is stabilized, each of the phases of the system (upper, lower and interphase phases) are carefully separated by pipetting.

C. Quantify the CD133 + stem cells obtained in B) Once all the phases of each system have been separated, a 250 μ ?, sample is taken from each of the phases to quantify CD133 +. This sample is divided into three samples of 70 μ?,. Two of the samples are used for flow cytometry staining (viability, detection and quantification of cell families) and the other is resuspended in 1 mL of FACS FLOW® (Becton-Dickinson, California, USA), for granulometry observation and size. The above, with a BD FACSCanto II equipment (Becton-Dickinson, California, USA). The surface markers used for the counting of mononuclear cells are: CD45-FITC, CD34-APC and CD133 / 1-PE (Miltenyi Biotech GmBH, Germany). To obtain the viability of the cells, the samples are stained with 7-AAD (7-Amino-actinomycin D from BD Biosciences). In PEG 8 systems, 000-dextran 500,000 50% of the CD133 + cells are partitioned to the lower phase and the rest are located at the interface, leaving the upper phase clean. In the case of dextran 75,000-UCON systems, 52% of CD133 + cells are partitioned to the lower phase and the rest are located at the interface, leaving the upper phase clean. In the SDFA ficoll 400,000-dextran 70,000 100% of the CD133 + cells are partitioned towards the upper phase. Figure 7 shows graphically the partition behavior of CD133 + cells in each of the two-phase systems with free antibody. Where it is observed that in the ficoll system 400,000-dextran 70,000 the CD133 + cells are completely partitioned to the upper phase. While in the PEG systems 8,000-dextran 500,000 and dextran 75,000-UCON a similar partition is obtained between the lower phase and the interphase. Comparing the behavior of two aqueous phase systems without antibody (Figure 6) and with free antibody (Figure 7) it is observed that the addition of the antibody under the conditions tested does not represent a benefit in the recovery of CD133 + stem cells, as has been reported for the recovery of other cell types .

Example 5: Quantification of CD133 + cells and their percentage of viability in the process motif of this patent application.

The sample to be analyzed is homogenized and an aliquot of 70 is deposited in three 5 mL round bottom polystyrene tubes for cytometry (BD Falcon, California, USA). The following reagents are added to tube No. 1: 20 μ ?. of FcR Blockig (Miltenyi Biotech GmBH, Germany) and 20 μ ?, of 7AAD (7-Amino-actinomycin D from BD Biosciences). In tube No. 2 and No. 3 is added: 20 μ ?, of FcR Blockig (Miltenyi Biotech GmBH, Germany), 20 μ? of 7AAD (7-Amino-actinomycin D from BD Biosciences) and 10 μ ?, of each of the following antibodies CD45-FITC, CD34-APC and CD133-PE (Miltenyi Biotech GmBH, Germany). The tubes are incubated for 10 minutes at 4 ° C in the dark. After the time, 1 mL of red blood cell lysis buffer IX (Miltenyi Biotech GmBH, Germany) is added and incubated for 10 minutes at room temperature in the dark. At the end of the time, they are centrifuged for 5 minutes at 300 X g at room temperature, the supernatant is removed and the pellet is resuspended in 1 mL of FACS FLOW® (Becton-Dickinson, California, USA).

Continue reading the samples in the BD FACSCanto II cytometer (Becton-Dickinson, California, USA) calibrated with the BD FACS 7 Color Setup beads kit (Becton- Dickinson, California, USA). The template used for the quantification of CD133 + is constructed following the guidelines of the ISHAGE protocol and the data obtained are complemented with the results of the blood count (30.7% of mononuclear cells), obtaining the following: 104 CD133 + / CD34 + cells per μ? with a 98.5% viability.

Claims (20)

1. A process for recovering and purifying CD133 + mononuclear cells from a product rich in stem cells such as umbilical cord blood, mobilized peripheral blood, leukapheresis product, cell culture, bone marrow or adipose tissue by means of a two aqueous phase system, characterized in that it comprises The following stages: A. Select a system of two aqueous phases (SDFA) polymer-polymer, consisting of: a) Select polymers, b) Determine system parameters of two aqueous phases and Build the system of two aqueous phases; B. Submit a product rich in stem cells to the two aqueous phase system selected in A), from at least one stage to fractionate the product rich in stem cells in their different compounds according to the affinity for each phase, which depends on factors of hydrophobicity, surface charge, size of the blood cells contained in the product rich in stem cells, having the CD133 + stem cells are fractionated in one of the phases, which consists of: to. Add a certain volume of sample, b. Mix the SDFA, c. Stabilize the SDFA, d. Separate the phases of the SDFA and and. Recover CD133 + cells
2. The process according to claim 1 characterized in that prior to step A), the product rich in stem cells is pre-fractionated by density, optionally by Lymphoprep, Percoll or ficoll.
3. The process according to claim 1, characterized in that after step B) at least one of the following steps is included: quantifying the CD133 + stem cells recovered in B); evaluate the purity required, or polish the CD133 + cells.
4. The process according to claim 1, characterized in that the polymer selection is made up of any of the following combinations: polyethylene glycol (PEG) dextran, PEG-ficoll, PEG-UCON, dextran-ficoll, dextran-UCON or ficoll- UCON.
5. The process according to claim 1, characterized in that in step A) the polymers used for the construction of the sequential stages of fractionation or polishing by two-phase aqueous systems can be the same or different from each other.
6. 6. The process according to claim 1, characterized in that the parameters of the SDFA to be determined are; cutting line lengths between 15 and 50% w / w, with volume ratios between 0.05 and 20, pH between 6.0-8.0, sodium chloride (NaCl) concentration between 0 and 0.25 M and a sample percentage of 1 at 50% p / p.
7. 7. The process according to claim 1, characterized in that the parameters of the SDFA preferably have cut-off line lengths of 20% w / w, pH of 7.4, sodium chloride (NaCl) concentration of 0.15 M and a percentage of the 10% p / p.
8. 8. The process according to claim 1, characterized in that between the step of adding a certain volume of sample and the step of mixing the SDFA, optionally at least one free antibody is added.
9. 9. The process according to claim 1, characterized in that between the step of adding a certain volume of sample and the step of mixing the SDFA, optionally at least one conjugated antibody is added.
10. 10. The process according to claim 7, characterized in that the antibody is preferably free anti-CD133.
11. 1. The process according to claim 8, characterized in that the antibody is preferably anti-CD133 conjugated.
12. 12. The process according to claim 1, characterized in that in the stage of mixing the SDFA is carried out by mixing by inversion of the two-phase systems with a duration of between 5 and 30 minutes and a frequency of between 15 and 60 investments per minute.
13. 13. The process according to claim 1, characterized in that in the step of mixing the SDFA is preferably carried out by reversing the two-phase systems with a duration of 15 minutes and a frequency of 20 inversions per minute.
14. 14. The process according to claim 1, characterized in that the stabilization of the SDFA is carried out by natural sedimentation or centrifugation.
15. 15. The process according to claim 1, characterized in that the stabilization of the SDFA or is preferably carried out by natural sedimentation leaving the system at rest for 5 minutes and 3 hours.
16. 16. The process according to claim 1, characterized in that the stabilization of the SDFA or is preferably carried out by centrifugation under gravity forces of between 100 and 5,000 X g, with temperature between 4 and 25 ° C and centrifugation time between 10 and 30 minutes.
17. 17. The process according to claim 1, characterized in that the separation of the phases of the SDFA is carried out by means of any of the following: suction, dripping or pipetting.
18. 18. The process according to claim 1, characterized in that the recovery of cells from the phase rich in CD 133 + cells is carried out by centrifugation.
19. 19. The process according to claim 1, characterized in that recovery of cells from the phase rich in CD133 + cells is carried out by centrifugation between 100 and 5,000 X g, 4 at 25 ° C and 10 to 30 minutes.
20. 20. The process according to claim 1, characterized in that after the recovery of the CD133 + cells a polishing step can be implemented to increase the purity or concentration of the CD133 + stem cells by at least one recovery and purification technique such as, but not limited to, chromatography, microfiltration, precipitation, adsorption, cytometry, capillary electrophoresis and dielectrophoresis.