MXPA06005885A - Tangential flow filtration devices and methods for stem cell enrichment - Google Patents

Abstract

The present invention provides methods for enriching a heterogenous mixture of bone marrow or blood constituents for stem cells by removal of non-stem cell constituents comprising separation of the non-stem cell constituents using a tangential flow filtration device.

Description

TANGENTIAL FLOW FILTRATION DEVICES AND METHODS FOR ENRICHMENT OF TRUNK CELLS Field of the Invention Cell populations enriched for stem cells are often desired for use in research or therapy. Typical stem cell sources include bone marrow, complete peripheral blood, leukapheresis products or apheresis, especially from "mobilized" donors, or other less common sources, such as umbilical cord blood and suspensions of tissues and organs. The enrichment of stem cells has been carried out in several ways. Typical methods include density stage gradients (eg, FICOLL-HYPAQUE®, colloidal silica and the like), elutriation, centrifugation, lysis of erythrocytes by hypotonic apoplexy, and various combinations of these methods. As an example, purification of stem cells from the bone marrow requires removal of erythrocytes and granulocytes, which is often achieved by FICOLL-HYPAQUE® density gradient centrifugation. There are disadvantages to each of these methods, one of which is the need for laborious washing steps after the enrichment step is performed, for example, to remove the density gradient centrifugation medium.

Background of the Invention After enrichment, the cells are typically washed by a repetitive process. The steps generally include placing the enriched cell suspension in a centrifuge tube and pelletizing the cells at the bottom of the tube by the use of a centrifuge. The tube is removed from the centrifuge, and the supernatant is decanted from the cells formed into pellets. A wash liquid was added to the tube, and the cell pellet was suspended again. These stages are typically repeated 2 to 4 times. A disadvantage of this washing process is that sequential suspension and centrifugation can decrease cell viability and increase cell lysis. Another disadvantage of washing by centrifugation is the opportunity to contaminate the cells by bacteria or other infectious agents. Even without all the material is kept sterile, the repeated opening of the centrifuge tubes, and the exposure of pipettes and bottles of washing solution to the air can result in contamination. The risk of contamination is sufficiently significant that some medical regulatory agencies have demanded that only "closed" systems be used for cellular management.

Filtration methods have also been used to remove cells from the blood, while retaining other blood constituents for later use. Such methods generally entrap the cells in a filter in a non-recoverable manner, while allowing other blood constituents to pass through the filter and into a collection vessel. For example, filters are available to remove leukocytes from the blood so that the incidence of alloimmune reactions is minimized after blood transfusions. The removal of leukocytes is typically carried out using a filter, which is made of matured plastic fiber mesh. The mesh is usually placed to trap the leukocytes in a cross-linked matrix that is deep enough for the cells to be trapped through the depth of the filter, thereby maintaining the blocking filter, as could happen if the leukocytes were trapped in a flat surface. In addition to the physical trapping of the cells, the materials and large surface area of the filter allow the leukocytes to attach irreversibly to the surface. Many of these adherent cells are very desirable for some medical procedures. The resulting combination of entrapment and adhesion to the filter creates highly efficient means for removing the leukocytes for disposition prior to blood infusion therapy. However, when leukocytes are the desired cells, this filtration method is not advantageous. One method that has been useful in the fractionation of several particles is the filtration by tangential flow (FFT) or filtration by "cross flow". The FFT has the movement of a fluid parallel to the surface of a porous membrane filter. The pores of the membrane allow the passage of fluid and particles within the fluid that are typically smaller than the pores. In addition, cross flow (or "tangential" flow) or fluid parallel to the filter prevents an accumulation of particles larger than the pores on the filter surface. The FFT has been used for the total separation of several materials. The use of tangential flow filtration in the pharmaceutical field has been reviewed by Genovesi (J. Parenter, Aci. Technol., 37:81, 1983), which includes filtration of sterile water by injection, clarification of a solvent system, and filtration of enzymes from broths and bacterial cultures. Marinaccio et al. (WO 85/03011), reports a process for the use in the removal of particulate blood components of the blood for plasmapheresis, and Robinson et al. (US Patent 5,423,738), describes the use of FFT for the removal of plasma from blood, allowing the reinfusion of blood cells and platelets in patients. In another use, FFT has been reported for beer filtration (EP 0 208 450), specifically for the removal of particulates such as yeast cells and other suspended solids. Kothe et al. (US Patent 4,644,056), describes the use of FFT in the purification of milk or colostrum immunoglobulins, and Castino (US Patent 4,420,398), describes its use in the separation of antiviral substances, such as interferons, from broths containing these substances as well as well as particles and viral cells. Similarly, FFT has been used in the separation of bacterial enzymes from cellular debris. (Quirk et al., Enzyme Microb. Technol., 6: 201, 1984). In addition, tangential flow filtration units have been employed in the concentration of cells suspended in the culture medium. (See, for example, Radlett, J-. R Appl. Chem. Biotechnol., 22: 495, 1972). FFT has also been reported to separate liposomes and lipid particles according to size. (Lenk et al., U.S. Patent 5,948,441). FFT allows the formation and isolation of liposomes and lipid particles that have a defined size range of heterogeneous populations of such particles. (See Lenk et al., Supra). However, while FFT has been used for total fractionation of biological fluids and separation of, for example, liposomes, the use of FFT for separation of living cell populations that differ in defined characteristics has not been appreciated in the art. In particular, the only problems associated with the selective separation of stem cells from other cells of the bone marrow or from blood cells and suspensions of tissues or organs, while maintaining sterility, have not been treated for cell viability and activity. regenerative In addition, the removal of other cell populations such as, for example, populations with overlapping size ranges, has not been resolved by current procedures. Therefore, a need remains in the art for additional devices and methods to selectively enrich stem cells from other blood constituents or bone marrow, including plasma, erythrocytes and / or platelets, while preserving sterility, cell viability and activity. cellular and regenerative. The present invention satisfies these and other needs.

Summary of the Invention The present invention relates to the separation of stem cells or progeny cells from bone marrow, blood and blood preparations, tissue and tissue and organ preparations. In particular, a cell population enriched in stem cells is prepared by the use of a tangential flow filtration device. Methods are provided for the use of the device for the preparation of enriched stem cell populations. Cell populations enriched in stem cells and the like, obtained by the use of devices and methods of the present invention, can be used to prepare compositions suitable for infusion in individuals for the purpose of e.g. reconstitution of the bone marrow, or for the repair of damaged tissue that includes heart muscle, and the like. A tangential flow filtration device of the present invention comprises removing units having a cross flow chamber, a filtering chamber and a filter disposed therebetween. The filter is in fluid communication on one side, the retained surface, with the cross flow chamber, and on the other side, the filtered surface, with the filtering chamber. The cross flow chamber has an inlet adapted to introduce a sample, such as bone marrow, or blood constituents, comprising stem cells, into the cross-flow chamber and parallel to the retained surface of the filter. An outlet is also provided in the cross flow chamber centrally disposed in a portion of the chamber opposite the retained surface of the filter. The filter suitable for use in the tangential flow filtration device typically has an average pore size ranging from about 1 to about 10 microns. In certain embodiments for use in stem cell enrichment, the filter has an average pore size of about 3 to about 7 microns, or about 3 to about 5.5 microns. Typically, stirring units are provided as a single-use disposable assembly. In addition, the device may comprise means for providing a predetermined input ratio of the sample at the inlet of the cross-flow chamber and means for controlling a filtration rate of the filtrate through the filter and in the filtration chamber. Filtration rate control means limiting the filtration rate to less than the filtering rate not opposite to the filter. The sample comprises stem cells which can be provided by a source device such as a leukapheresis device or a container comprising a sample collected from, for example, a leukapheresis device and the like. The tangential flow filtration device may further comprise a recovery unit. The recovery unit comprises an inlet and an outlet that can be interconnected in a loop format with the cross flow chamber of the stripping unit. In this embodiment of the device, the inlet of the cross flow chamber is in fluid communication with the outlet of the recovery unit, and the outlet of the cross flow chamber is in fluid communication with the inlet of the recovery unit. The recovery unit may further comprise a sample entry and a wash entry. In certain embodiments of the tangential flow filtration device, the sample inlet and the wash inlet are in a single common inlet. Typically, the wash inlet is in fluid communication with a source of replacement or flushing fluid. The replacement or washing fluid may be, for example, an isotonic buffer or tissue culture medium. The sample entry of the recovery unit is in fluid communication with a sample source such as bone marrow, or blood constituents comprising stem cells. In an embodiment of the present invention, the sample source comprising bone marrow or blood constituents is a syringe equipped with a needle, or a specialized device specifically designed for the removal of the bone marrow from a donor or a patient. The FFT device and operation of the device are described in greater detail in the US provisional patent application serial number 60 / 390,730 filed on June 19, 2002 and in WO 2004/000444, each incorporated herein by reference in its entirety One embodiment of the device of the present invention comprises a tangential flow filtration device for enriching a sample of bone marrow by stem cells. The device comprises a stirring unit comprising a cross flow chamber and a filtering chamber separated by a filter, wherein the cross flow chamber has an inlet and an outlet, the outlet centrally disposed in an upper portion of the chamber, and wherein the inlet is disposed above the filter and introduces the filter in the cross flow chamber substantially parallel to the filter; a means for providing a predetermined entry velocity of the sample through the inlet of the cross flow chamber; and a means for modulating a filtration rate through the filter; wherein the filter has a pore size of approximately 5 microns; and with this, the sample is enriched by stem cells in a retention in the cross-flow chamber. In another embodiment of the present invention, a tangential flow filtration device is provided for enriching a sample of blood constituents by stem cells, wherein the device comprises a stripping unit, wherein the stripping unit comprises a cross flow chamber below of a filtering chamber and separated by a filter, the cross-flow chamber has an inlet and an outlet, the inlet centrally disposed in a lower portion of the chamber, and wherein the inlet is disposed below the filter and introduces the fluid in the cross flow chamber substantially parallel to the filter; a means for providing a predetermined entry velocity of the sample through the inlet of the cross flow chamber; and a means for maintaining a filtration rate through the filter; wherein the filter has a pore size of approximately 5 microns; and thereby the sample is enriched for stem cells in a retention in the cross-flow chamber. The present invention also provides methods for separating stem cells from a sample of bone marrow constituents, blood constituents, tissue or tissue or organ preparations comprising stem cells. The steps of the method include: (1) entering the sample in a stripper unit through an inlet in the stripper unit; (2) subjecting the sample to cross flow substantially parallel to a filter having a pore size of about 1 to about 10 microns; (3) subjecting the fluid to filtration through the filter; and (4) removing selectively constituents of non-stem cells from the sample to form a cell population enriched for stem cells. The sample may be subjected to partial purification or enrichment by leukapheresis, density centrifugation, differential lysis, filtration or preparation of a mononuclear cell culture, before introduction into the stirring unit. In one embodiment, the sample is induced for cross flow on the filter surface with a swirling motion in the cross flow chamber. Additionally, the cell population enriched for stem cells can be washed with a wash solution. In a particular embodiment of the present invention, a cellular sample, such as a sample of bone marrow constituents comprising stem cells, is contacted with a pretreatment solution comprising an agent that causes contraction of the cells in the sample that They are of a nominal size similar to stem cells. The contracted cells are susceptible to pass through the filtration membrane providing a cell population more enriched for stem cells. In a specific embodiment, the cells induced to undergo contraction are granulocytes, such as neutrophils and the like. A particular solution useful in this embodiment comprises, for example, an effective amount of dimethyl sulfoxide (DMSO) in a physiologically acceptable solution. The physiologically acceptable solution can be, for example, a hypotonic saline solution such as diluted phosphate buffered saline (PBS). Alternatively, the pretreatment solution may comprise a hypertonic solution containing, for example, a sugar such as mannitol or glucose, or it may be a hypertonic saline solution. In yet another embodiment, the cells in which the contraction is induced, are prevented from re-distension by treating or pre-treating the cell sample with an agent that prevents distension of the contracted cells. In a modality, the anti-distension agent is an agent that prevents tyrosine phosphorylation, such as, for example, genistein and the like. In still another embodiment, the anti-distension agent inhibits the action of the sodium-hydrogen exchanger. In yet another embodiment, the solution in which the cellular sample, for example, comprising constituents of bone marrow, is suspended, is free of sodium salts which block the exchange of hydrogen and sodium by the sodium-hydrogen exchanger which prevents induction of re-distension of cells. In the methods of the present invention, the non-core cellular constituents removed from the cell sample include for example stroma, plasma, platelets, erythrocytes and the like. The enriched cell population may comprise at least about 10% of stem cells, but typically comprises at least 20% or more, of stem cells. In one embodiment of the method of the present invention, steps (1), (2) and (3) are repeated at least twice to form the cell population enriched for stem cells. The cell population enriched for stem cells can be used by infusion in patients in need of stem cell therapy. In additional embodiments, the cell population enriched for stem cells can be induced to form other cell types useful in therapy including, for example, endothelial cells, smooth muscle cells, heart muscle cells, neurons, dendritic cells and other cell types. Several methods of stem cell induction are well known to the person skilled in the art. Cell samples used in the methods of the present invention are typically collected from an individual donor. The donor can be the patient to receive stem cell therapy or another individual. Before the collection of the cellular sample from a donor, the donor can be subjected to treatment with an agent that mobilizes the stem cell, such as, for example, M-CSF, G-CSF, GM-CSF, or cyclophosphamide at high dose or low, and the like, to produce a cell population enriched for hematopoietic stem cells. The agent that mobilizes the stem cell induces the proliferation of CD34 + stem cells, which are released into the peripheral bloodstream. The bone marrow, blood, for example, a sample of leukapheresis, tissue, or tissue preparation or organ from the individual donor, are then introduced into a tangential flow filtration unit (FFT) of the present invention. The FFT unit comprises a cross-flow chamber, a filtering chamber and a filter in fluid communication with the cross-flow chamber and the filtering chamber. Typically, the filter used in the FFT device has a pore size of about 3 to about 5.5 microns. The cell sample enriched by hematopoietic cells is recirculated through the FFT unit at a predetermined feed rate and a predetermined filtration rate, the predetermined feed rate is typically at least five times the predetermined filtration rate; and the predetermined filtration rate is less than the filtration rate not opposed by the filter; providing an isolated cell population enriched by CD34 + leukocytes. The method can result in an enriched cell population that is substantially free of blood constituents without leukocytes including plasma, platelets and erythrocytes. The enriched cell population produced by this method can increase the percentage of CD34 + cells to comprise about 2% up to about 10%, up to about 5% up to about 40% or more of the cell population. A further understanding of the nature and disadvantages of the invention will become apparent by reference to the remaining portions of the specification.

Brief Description of the Figures Figures 1A to 1C depict modalities of the tangential flow filtration device for the separation of leukocytes and also monocytes from a blood product sample. Figure IA provides a device modality for leukocyte enrichment wherein the cross flow chamber is above the filtration chamber. Figure IB describes a front view of the device where the sample entry is below the filter and the filtrate passes up through the filter for onocyte enrichment. Figure 1C is a top view of the device shown in Figure IB.

Detailed Description of the Invention The present invention provides methods for processing a cellular sample comprising a heterogeneous mixture of bone marrow constituents, blood constituents, tissue, or suspensions of tissue or organ to provide an enriched population of stem cells. In one aspect of the invention, methods are provided for the enrichment of stem cells by the selective removal of non-stem cell constituents, for example, estrus, plasma, platelets and / or erythrocytes and the like. In another aspect, methods are provided for the enrichment of stem cells by the selective removal of other types of large cells, including polymorphonuclear cells, such as, for example, granulocytes of the mixture. In a particular method, a sample of the bone marrow can be treated with an agent that contracts non-stem cells of approximately the same size, such that the non-stem cells contracted pass through a filter of an FFT device and separate from the stem cells. A population enriched for stem cells is typically prepared from a sample, or fluid mixture, comprising constituents of bone marrow. The term "bone marrow constituents" as used herein, refers to any material typically present in the bone marrow, which includes such material typically present in disease as well as to disease-free states. Bone marrow constituents include stem cells and may include, for example, lymphocytes, monocytes, erythrocytes, neutrophils, eosinophils, natural killer (NK) cells and / or platelets, soluble and insoluble proteins or protein complexes (eg, enzymes, immunoglobulins or immunoglobulin-antigen complexes), other macromolecular components such as, for example, lipids or any other portion of whole blood that can be physically separated, independent of its precise molecular or cellular composition, which includes, example, stroma, plasma or serum. The sample, or fluid mixture, may be partially enriched by stem cells before carrying out the methods of the present invention. The term "stem cell" is used interchangeably with the term "precursor cells" "progenitor cells" or "CD34 + cells". These terms include hematopoietic stem cells, which include, for example, lymphoid, myeloid and erythroid progenitor cells, as well as progenitor cells that can give rise to endothelial cells; muscle cells, which include smooth muscle cells and heart muscle cells; neuronal cells and skeletal cells, which include those that form bone and cartilage. In certain aspects of the present invention, a cell population containing polynuclear cells (PMN) or granulocytes is separated from the stem cells. This solution typically contains neutrophils, eosinophils and basophils and their precursors, and is referred to as PMN in this application. As used herein, the term "stem cell population" refers to any group of cells that include stem cells. A population of stem cells may include, as previously, a broad range of sub-types of stem cells or of particular sub-types, such as, for example, endothelial cell precursor or muscle cell or progenitor cells. The terms "enrichment", "enrich" and "enriched", mean that the procedure of a mixture of constituents of the bone marrow using a device as briefly described herein and more fully described in the provisional US patent application number. serial 60 / 390,730, filed June 19, 2002, and WO 2004/000444, (each incorporated herein by reference in its entirety), and following the methods of the present invention, result in a cell population which has a high percentage of viable stem cells, in relation to other constituents, than in the initial cell sample (ie, before enrichment). As used herein, the term "viable" refers to a stem cell that is capable of differentiation under suitable culture conditions or in reinfusion in a patient or a suitable animal model. The devices according to the present invention use tangential flow filtration to enrich a population of stem cells. The terms "tangential flow filtration" and "cross flow filtration" are used interchangeably and refer to the separation of suspended particles (e.g., cells) from a fluid mixture, which includes the separation of particles from a characteristic. defined (eg, a desired size range) of a heterogeneous mixture of particles in the fluid mixture. The particles are separated by passing or circulating the fluid mixture (e.g., a sample fluid) in a sample chamber substantially parallel or tangential to a filter (e.g., the surface of the filter facing the sample fluid), typical under some positive pressure, with the fluid mixture comprising the concentrated particles, or stem cells, containing a tangential flow to the membrane surface. In general, the determination of which the particles are removed in the "filtered", that is, the portion of the fluid that passes through the filter, and those particles retained in the "retained", is dependent on a variety of factors. Such factors include, for example, filter pore size, inlet velocity, filtration rate, particle concentration in the fluid mixture, temperature and viscosity of the fluid mixture. As used herein, "pore size" refers to the average pore size in the filter. "Entrance speed" refers to the speed at which a sample (eg, a mixture of fluid) is introduced into the chamber that houses the filter. Where the sample is recirculated multiple times through a filter (eg, in a particular embodiment of the device according to the present invention), the input speed is also referred to as the "recirculation rate". "Cross flow" refers to the substantially parallel flow (i.e., parallel to the filter surface in any direction) of the fluid mixture through the filter. "Cross flow rate" refers to the flow rate of the sample, or fluid mixture, above and substantially parallel to the filter. The cross flow rate of the fluid mixture is generally dependent on a variety of parameters, including, for example, the rate of entry and the size and shape of the chamber housing the filter. "Filtration rate" refers to the flow velocity of the fluid mixture through the filter. The filtration rate for a device and the methods according to the present invention are typically less than the non-opposing filtration rate (ie, open tube). "Exit velocity" refers to the rate of removal of the fluid mixture from the cross flow chamber, different from the mixture of the fluid that passes through the filter (ie, the filtered). The exit velocity is generally equal to the minimum exit velocity of the filtration rate. As used herein, the term "filter" refers to any article made of any material or combination of materials having a plurality of pores that allow one or more components (e.g., blood and / or constituent of the bone marrow). ) of a sample or mixture of fluid, undergo cross-flow through the article that pass through it, thereby separating those components (eg, non-stem cells, proteins, plasma, serum, platelets and the like) from other components (for example, stem cells). The surface of a filter can have any suitable area, such as, for example, about 42 to about 145 mm in diameter, although filters of greater or lesser area can be used. In certain modalities, only one filter is used in an FFT device. In other modalities, additional filters may be used in an FFT device. The filter typically employed in the FFT device of the present invention may be selected from a wide range of organic polymeric filters. Such filters include, but are not limited to, microporous nylon membranes, polyvinylidene fluoride (PVDF), cellulose acetate / nitrate, polysulfone, polycarbonate, polyethylene, polyester, polypropylene and polyamide. Other filters, such as ceramic filters and metal filters, can also be used. Filters, both hydrophilic and hydrophobic, charged and discharged can be used. In certain applications, hydrophilic filters may be preferred. A filter of the present invention typically comprises a number of pores distributed through the filter area. In certain embodiments, the filter has a plurality of pores with a small variation in pore size. For example, the variability in the pore size may be about ± 20%, or within the range of about ± 0% to about ± 20%. In a typical embodiment, "nucleophore" or "etched channel" filters are used (eg, Poretics® polyethylene or polycarbonate etched channel filter membranes (Osmonics, Minnetonka, MN)). These filters typically have a smooth surface with tightly controlled pore sizes in the material. Such filters are typically prepared by exposing a flat sheet of non-porous plastic to a source of radioactive particles, which are energetic enough to penetrate the plastic sheet. The "channels" are then extended in diameter by exposure to chemical solvents or etching agents. The size of the pores can be controlled by the engraving conditions of the channel. The present invention takes advantage of differences between various cell types in the bone marrow, blood, tissue or suspensions of tissues or organs to enrich the stem cells. Such differences may include, for example, differences in size, shape and / or deformability. The size and deformability of cells in human bone marrow, blood, tissue, or suspensions of tissue or organ, typically vary by cell type. Erythrocytes (red blood cells) typically are enucleated, biconcave disc-shaped, measuring approximately 7 microns in the largest diameter and are relatively deformable. Polymorphonuclear leukocyte cells are typically spheroidal, also about 7 microns, but less deformable than erythrocytes. Of the mononuclear cells, the lymphocytes are typically 7 to 10 microns, and the monocytes are usually in the range of 10 to 15 microns. Stem cells are generally in the same size range as monocytes. In various modalities, the filter size is selected to enrich the stem cells, and / or to fractionate the bone marrow, blood constituents, tissue, or suspensions of tissue or organ, thereby enriching the cell population collected by stem cells. For example, in certain embodiments, stem cells having a nominal diameter of 10 to 15 microns, and erythrocytes having a nominal diameter of 7 microns, can be prepared by FFT using a filter having a pore size of 5 microns. In other embodiments, the pore size of the filter may be in the range of about 1 to about 10 microns, about 3 to 8 microns, or about 3 to about 5.5 microns. A pore size of the filter in the range of about 3 microns can retain more stem cells and leukocytes, and effect less efficient removal of erythrocytes from the stem cells. In contrast, a pore size of the filter in the approximately 8 micron range can effect more efficient removal of erythrocytes, but increase the loss of stem cells and leukocytes in the filtrate. Typically a filter size of about 3 to about 5.5 microns is used to enrich the stem cells. The enrichment of stem cells of other constituents of bone marrow, blood, tissue or suspensions of tissue or organ may also be affected by the rate of entry, the filtration rate and / or the concentration of cells in the sample or fluid mixture. . For example, erythrocytes are more deformable than other cell types and can therefore pass more easily through a filter with a smaller pore size than the larger diameter of erythrocytes (e.g., less than about 7 microns) . In a specific example, erythrocytes can be separated from leukocytes using filters having a pore size of about 5.5 microns. Enrichment of stem cells from other constituents of the cellular bone marrow, or constituents of tissue or organ suspension, may also be affected by maintaining a filtration rate that is lower than the non-opposite filtration rate (ie, open tube) under the same speed of entry or recirculation. In other embodiments, the loss of leukocytes to the filtrate can be reduced by maintaining an inlet or recirculation velocity that is greater than the filtration rate. In exemplary embodiments, the rate of entry or recirculation may be at least about five times, at least about 10 times, at least about 20 times, at least about 50 times, or at least about 100 times, the filtration rate. A sample, or mixture of fluid, comprising various constituents of bone marrow, blood constituents, tissue, or suspensions of tissue or organs for fractionation of the stem cell by FFT, may be obtained from a variety of sources and may include mixtures of the fluid of blood products in any of the various stages of the procedure. For example, the bone marrow and blood sources can be either human or non-human. In addition, mixtures of the fluid can be, for example, bone marrow, whole blood, various dilutions of whole blood, or whole blood or dilution of blood that has been subjected to processing of, for example, removal of plasma or other blood constituents. , or tissue or organ suspensions. Thus, the fluid mixture may include, for example, a population of blood cells that is already at least partially enriched for stem cells. Bone marrow or blood constituents, populations of bone marrow or blood cells, or suspensions of tissue or organ, may be prepared by methods known to those skilled in the art. Such methods typically include collecting heparinized bone marrow or blood, apheresis or leukapheresis, mononuclear cell culture preparation, rosettes, centrifugation, density gradient centrifugation (for example, density gradient materials including, FICOLL-HYPAQUE®, PERCOLL®, sucrose and the like), differential lysis of cells without leukocyte, filtration and the like. The mixture of fluid comprising the bone marrow or blood constituents can optionally be diluted or concentrated, as desired. For example, in certain embodiments, the bone marrow or blood constituents are diluted 1: 2, 1: 5, 1:10, or any other suitable dilution. Bone marrow or blood constituents can be diluted in, for example, isotonic buffers (e.g., PBS or buffered saline with HEPES), tissue culture medium and the like. Typically, the sample of bone marrow or blood constituents subjected to FFT has a cell concentration of about 10 6 to about 10 8 cells per ml of which at least about 10 to 20% are stem cells. In addition, the number of PMN is reduced from about 60 to about 75% of the number of cells to about 50% or less. Blood cell or bone marrow populations, or suspensions of tissue or organ, can be obtained from a variety of subject types, in accordance with the intended use of the enriched population of stem cells. The subject, for example, can be a healthy subject. Alternatively, the cells can be obtained from a subject in need of bone marrow reconstitution, such as, for example, a cancer patient who has been found to have bone marrow damage due to chemotherapeutic treatments. A population of blood cells or bone marrow can also be collected from individuals that have been administered with a stem cell mobilizing agent, such as, for example, M-CSF, GM-CSF, G-CSF, or dose cyclophosphamide. high or low (Deliliers et al., Leuk, Lymphoma 43: 1957, 2002) and the like. The individual patient may be a patient who will receive the population of enriched cells, an HLA matched individual or relative. The devices in accordance with the present invention as represented in Figures IA to 1C, typically comprise a cross flow chamber (3) and a filtration chamber (4). A filter (5) is positioned between and with one surface in fluid communication with the cross flow chamber (the retention surface) and the other surface in fluid communication with the filtration chamber (the filtering surface). The cross-flow chamber, the filtering chamber and the filter comprise a stirring unit (1). The stripping unit may be provided as a disposable disposable array, sterilized and prepared for use in an isolation method of the present invention. A remover unit arrangement can be used for each sample to be enriched by the stem cells. In a particular embodiment of the present invention, the cross flow chamber typically has a volume of approximately 55 ml, and the filtration chamber has a volume of approximately 25 ml. The diameter of the filter is typically substantially the same as the diameter of the cross flow chamber. In certain embodiments used to demonstrate the utility of the present invention, the filter is from about 140 mm to about 143 mm in diameter. In the methods of the present invention, the fluid enters the transverse flow chamber (3) through a fluid inlet (6) which is typically located adjacent to the retention surface of the filter and in such a way that the mixture of fluid (eg, sample), enters the chamber substantially parallel to the filter. Typically, the fluid is removed from the cross flow chamber (3) through a fluid outlet (7), which is usually located in a portion of the cross flow chamber, perpendicular to the filter retaining surface. In certain exemplary modalities, the inlet diameter of the cross flow chamber (6) is about 7 mm to about 8 mm, and the outlet diameter of the cross flow chamber (7) is about 8 mm to about 10 mm. The filtrate is removed through an outlet (8) in the filtering chamber (4). Typically, the fluid mixture is introduced into the cross flow chamber at a sufficient inlet velocity, so that the cross flow of the fluid chamber that crosses the surface of the filter (retention surface) is at a sufficiently high velocity to interrupt gently and mix again the fluid and the cells on the contact surface of the filter, that is, the boundary layer. As used herein, "boundary layer" refers to such fluid layer adjacent to and on the filter retention side, typically left by the fluid passing through the filter. This interruption of the boundary layer facilitates efficient filtration, preventing the material on the contact surface of the filter, from bonding to the filter or becoming stagnant, which can hamper efficient filtration. The rate of entry of the fluid mixture is usually not sufficient, however, to cause lysis of a substantial number of leukocytes. In certain embodiments, the blood constituents or bone marrow are passed through the retention surface of the filter by pumping the fluid mixture in the crossflow chamber (3). The pump used to drive the cross flow of the fluid through the filter is referred to as "the cross flow pump" or "recirculation pump" (14). The cross flow pump can include any pumping device in fluid communication with the cross flow chamber (3) sufficient to introduce the fluid mixture into the chamber and through the filter at the specified inlet speed, without causing substantial damage to the cells (for example, cell lysis). A cross flow pump suitable for use in the present invention may include, for example, a peristaltic pump, piston pump, diaphragm pump, or roller pump. A peristaltic pump can be used for example, where it is desired to maintain the FFT device as part of a "closed" system. The fluid mixture is typically pumped into the crossflow chamber (3) at an inlet velocity that exceeds the filtration rate. In an exemplary embodiment, the rate of entry is about 1680 ml / minute, and the filtration rate is about 15 ml / minute. In other exemplary embodiments, the entry speed is about 1600 to about 1800 ml / minute, and the filtration rate is about 10 to about 20 ml / minute. Non-stem cell material (e.g., erythrocytes, immune complexes, proteins, PMN and the like), passes through the filter (5) in a filtration chamber (4). As discussed above, the filtration rate is typically less than the non-opposite velocity (ie, open tube). The filtration rate can be controlled, for example, by reducing or restricting the size of the filtration chamber outlet, by the use of a second pump means (eg, a "filtration pump), to restrict the flow and In another exemplary embodiment, the introduction of a mixture of fluid into the device creates a vortex movement within the fluid.This can be done, for example, by introducing the fluid mixture, substantially parallel to a circular filter into a chamber. cylindrical crossover flow, for example, an input speed of about 5 to about 10 to about 100 times the filtration rate.The passing flow is removed by means of an outlet (7) located in the cylindrical chamber perpendicular to the filter and Typically adjacent to the center of the filter surface, this arrangement causes the filter to flow spirally inward toward the center of the filter. turbulent, or at such high speed, to cause substantial lysis of the stem cells. As discussed above, the cross flow can also "sweep" the filter surface to prevent bonding - or stagnation in the boundary layer. By calibrating the input speed so that it is large (eg, at least about 5 times) relative to the filtration rate, the resulting enriched population of stem cells can be at least about 5, or at least about 20, or at least about 60 percent, or more, of stem cells, when compared to the percentage of stem cells with the number of total cells in the sample cell population. In another exemplary embodiment, the retentate is recirculated to increase the separation efficiency. For example, a mixture of fluid comprising blood constituents or bone marrow, or a tissue or organ preparation, can be introduced into the cross-flow chamber, and during filtration retention, it can be removed through the fluid outlet (7) in the cross-flow chamber to another chamber, such as, for example, a chamber from which the fluid was initially provided ("a recovery unit").; (2) ) . The fluid mixture in the recovery unit can then be re-introduced into the cross flow unit. By connecting the recovery unit (2) and the stirring unit (1) in a "loop format", recirculation and continuous filtration of the fluid mixture can be achieved. Alternatively, the retentate can be withdrawn through the fluid outlet (7) of the cross flow chamber (3) and directly reintroduced into the inlet of the cross chamber (i.e., without passing through a recovery unit or a another camera). The fluid mixture can be passed through the cross flow unit for any suitable period of time. In certain embodiments, the fluid mixture can be re-circulated for about 5 to about 60 minutes, or longer, to achieve the desired purity or enrichment of stem cells. In yet another embodiment, the volume of the fluid mixture can be adjusted by adding a buffer, a wash solution or another solution (collectively referred to as a "replacement fluid"). The washing solution can, for example, be combined with a mixture of fluid in the recovery unit (for example, through a solution inlet; (13)), in the stirring unit, in a pump (14), in casing that extends to or from the stripper unit, or in any other convenient location. The cells in the retention can thus be enriched and washed in the same operation. Typically, the wash solution is isotonic with the cells. Suitable washing solutions and buffers may include a variety of buffers (e.g., phosphate buffered saline (PBS) or buffered saline with HEPES), tissue culture medium and the like. In certain embodiments, a cell sample comprising a cell population of, for example, bone marrow, blood, tissue, or a tissue or organ preparation, is enriched by a population of stem cells in an aseptic, closed system. As used herein, the term "closed, aseptic system," or "closed system," refers to a system in which exposure to non-sterile environment, or circulating air or other non-sterile conditions, is minimized or eliminated. Closed systems for enriching cell populations generally exclude centrifugation in open top tubes, cell transfer by open air, cell culture in tissue culture plates or unsealed flasks and the like. The complete filtration system that includes, for example, any of the cell containers, incubators, tissue culture vessels, or other apparatus for cellular processing (posterior), can be maintained as a "closed" system. In a typical embodiment, the closed system allows aseptic enrichment of stem cells and optionally, transfer from an initial collection vessel to a sealed tissue culture vessel, without exposure to non-sterile air. Typically, a peristaltic pump means (Figure 1A and 1C; (15)), is used in a closed system. In another aspect of the invention, a heterogeneous mixture of blood constituents or bone marrow, or suspension of tissues or organs, is substantially enriched for stem cells by the selective removal of the mixture of blood constituents or bone marrow from non-stem cells, which include for example, stroma, plasma, platelets, erythrocytes and the like. As used herein, the term "substantially enriched" means that the population of cells recovered in retention, after many recirculation cycles as desired, is typically comprised of at least about 5%, more typically at least about 20%. %, or at least about 60% of the type of cells desired (e.g., stem cells). In other modalities, a heterogeneous mixture of blood constituents or bone marrow, and the like, is enriched for stem cells to form an enriched population of stem cells that are substantially free of non-stem cell constituents. As used herein, the term "substantially free" means that the enriched population of stem cells comprises at least about 10% up to about 50% of stem cells. It has been determined that certain variables affect the operation of the device. For example, filter pore size, total liquid volume, recirculation and filtration rates, as well as the speed between these two speeds and the running time, can affect the performance of the stem cells. To determine optimal separation conditions for stem cells, the separation runs can be performed using variations in the parameters, where the cellular concentrate (retained) and the filtered one, are sampled at time intervals to monitor the operation over time. From these results, the optional filter pore size, total system volume, recirculation and filtration rates can be determined. In an exemplary embodiment of this aspect of the present invention, the FFT device comprises a cross flow chamber (3) with a volume of approximately 55 ml and a filtering chamber (4) with a volume of approximately 25 ml. The device further comprises the following: a filter having a plurality of pores with a pore size of about 1 to about 10 microns, more typically about 2 to about 8 microns, or even more typically about 3 to about 5 microns; a filter diameter of approximately 142 minutes. In this embodiment, the entry speed is set to be about 1600 to about 1800 ml / min; and the filtration rate is from about 12 to about 17 ml / min. The initial fluid mixture typically has a cell concentration of at least about 10 7 cells per ml (eg, leukocytes and other cells). The population of enriched stem cells achieved with this embodiment of the invention, comprises approximately 11 to approximately 20 million cells, of stem cells representing from about 10% to about 20% of the total number of cells. In another aspect of the invention, a heterogeneous mixture of blood constituents or bone marrow is substantially enriched for stem cells by the selective removal of constituents of non-stem cells, including for example, the removal of stroma and lymphocytes from the mixture . As used herein, the terms "selective removal", "selectively removed" and "selectively stirring" refer to the preferential removal of one type of cell and enrichment for another type of cell. In an exemplary embodiment of this aspect, the FFT device comprises a cross flow chamber (3) with a volume of approximately 55 ml and a filtering chamber (4) with a volume of approximately 25 ml.

In addition, the device comprises the following: a filter pore size of about 1 to about 10 microns, or about 2 to about 8 microns, or about 3 to about 5 microns; an input speed of about 1600 to about 1800 ml / min; a filtration rate of about 12 to about 17 ml / min; and a filter diameter of approximately 142 mm. The initial fluid mixture typically has a cell concentration of at least about 10 7 cells per ml (eg, stem cells and other cells). In this mode, the device was operated in an inverted manner. In yet another embodiment of the present invention, a heterogeneous mixture of blood constituents from bone marrow, blood, tissue or from a tissue or organ preparation, is pre-treated to facilitate the removal of certain types of cells of similar size. and deformability as stem cells. Such stem cells may include polymorphonuclear granulocytes, including neutrophils, eosinophils, basophils, and the like. In one embodiment, this pretreatment comprises the contact of the bone marrow or blood constituents with an agent that can effect an osmotic gradient across the cell membrane, thereby inducing contraction of the cell through the water jet. Such agents may include, and are not limited to, dimethyl sulfoxide, glycerol, sodium chloride and the like. When DMSO is used, the final effective DMSO concentration may be between about 5% and about 20%, or between about 10% and about 15%. In a particular embodiment, the effective concentration of DMSO is about 12.5% or about 15%. The DMSO can be dissolved in a buffer or physiologically acceptable solution without low ionic strength. Another agent that can be used is glycerol. The effective amount of glycerol is between 0.5 mol / L and approximately 2.5 mol / L. In a specific embodiment, the final concentration of glycerol is approximately 1 mol / L. Contacting the cells with these agents can lead to unwanted granulocyte lysis. Lysis can be done through sequential exposure to DMSO and glycerol. The solution used may comprise a solution medium with low osmotic force. The induced contraction of the unwanted cells makes these cells more imposed for removal through filtration and allow the selective removal of these cell populations by filtration by tangential flow. Effective amounts of an agent that prevents redistening of the cells during the separation process can also be included. Such agents may include, but are not limited to, an agent that prevents tyrosine phosphorylation, such as genistein, or an agent that inhibits the action of the sodium-hydrogen exchanger.

Culture, Expansion and Differentiation of Enriched Cell Populations In one embodiment of the present invention, the methods of the present invention are used to obtain an enriched population of stem cells which can be used to produce a composition useful in, for example, transplantation. allogeneic or autologous In particular embodiments, the enriched population of stem cells is further enriched by haematopoietic stem cells following the tangential flow separation procedure. Methods for enrichment of haematopoietic stem cells from a source of peripheral blood leukocytes are known in the art and can be adapted for use with an enriched population of isolated stem cells as described herein. For example, a population enriched for stem cells can also be enriched for CD34"r cells using for example, immunomagnetic separation techniques (see for example, Rowley et al., Bone Marrow Transplant, 21: 1253, 1998).; Denning-Kendall et al. , Br. J. Haematol. 105: 780, 1990). The donor of blood or bone marrow, can be isolated from the patient to receive the transplant, an HLA equalized individual, relative relative, or similar. In yet another embodiment, the methods of the present invention are also used to obtain a subset of non-stem cells, such as for example, a population of cells enriched in progenitor cells (e.g., hematopoietic or endothelial progenitor cells), or cells that secrete a factor of interest (eg, atopoietic or angiogenic growth factors). For example, circulating endothelial progenitor cells (CEP) can be identified as a subset of CD34 + cells by, for example, co-expressing VEGFR-2 and AC133 (as well as, for example, VE-cadherin and E- selectin). (See for example, Peichev et al., Blood 95: 952, 2000). A leukocyte enriched population can also be enriched by CEP, using for example, immunomagnetic separation techniques with antibodies directed to VEGFR-2 and AC133. Also, CEPs can be mobilized in an individual donor prior to the isolation of a cell population from the donor and enrichment using FFT. In this method, the donor can be treated with a cytosine such as, for example, VEGF. (See, for example, Gilí et al., Cir Res., 88: 167, 2001). In addition, in yet other embodiments, angiogenic circulation cells similar to endothelial cells (CAC) (which secrete, for example, VEGF, HGF, G-CSF and GM-CSF), are obtained by culturing an enriched population of leukocytes with, for Example, VEGF, bGFG, IGF-1, EGF and FBS, on a surface coated with fibronectin, and then discharging them into non-adherent cells (see for example, Rehman et al., Circulation 107: 1164, 2003). In addition, the enriched population of stem cells can be cultured to induce the expansion of pluripotent progenitors or stem cells. For example, CD34 + stem cells can be expanded in vitro by culture with hematopoietic growth factors such as, for example, a combination of IL-1, IL-3, IL-6, stem cell factor (SCF), stimulation factor. of the granulocyte-monocyte colony (GM-CSF) and G-CSF (see for example, Sun et al., Haematologica 88: 561, 2003). The stem or progenitor cells may subsequently be treated with any of several cytokines and growth factors to induce differentiation in cells of hematopoietic or non-hematopoietic lineages. In other embodiments, an enriched population of stem cells may also be cultured under conditions suitable for inducing differentiation (e.g., differentiation of progenitor cells or transdifferentiation of more differentiated cell types such as, e.g., monocytes or dendritic cells derived from monocytes. ). (As used herein, "transdifferentiation" refers to a process of phenotypic modulation of a differentiated cell, generally without the need for some cell division, thereby, the differentiated cells differentiate into a functional cell type between and / or morphologically different). For example, in addition to differentiation into dendritic cells, monocytes can be transformed into other types of hematopoietic or non-hematopoietic cells including, for example, acrophages, osteoclasts, and similar endothelial cells, depending on culture conditions (see for example, Becker, et al., J. Immunol., 139: 3703, 1987; Nicholson et al., Clin Sci. 99: 133, 2000; Havemann et al., In Novel Angiogenic Mechanisms: Role o_f Circulating Progenitor Endothelial Cells 47-57 (Nicanor I. Moldovan eds., 2003)). Also, a leukocyte enriched population can be cultured under conditions that induce differentiation of relatively undifferentiated cell subseries (e.g., pluripotent stem and progenitor cells) in hematopoietic or non-hematopoietic lineages using any of several cytokines or growth factors. Such differentiation can be induced prior to or after cell expansion by methods known to those skilled in the art. In certain embodiments of this invention, the population of cells enriched for stem cells can be used to affect the regeneration or repopulation of cells or tissues in a person in need of such regeneration or repopulation. Such a person may be suffering from any condition where the patient could benefit from the administration of stem cells, which include Parkinson's disease, diabetes, chronic heart disease, kidney disease, liver failure, cancer, spinal cord injury, multiple sclerosis , Alzheimer's disease, or you might be in need of gene therapy to prevent a genetic or epigénico defect. In certain embodiments of the present invention, the recipient of the stem cells is autologous or may be allogeneic to the donor. The container may be in need of regeneration of the bone marrow because the individual has undergone myeloablative therapy. In another example, the container may be in need of repair of cardiac tissue because the individual has undergone a cardiac infarction or is suffering from congestive heart failure or heart failure. In any case, the population of enriched stem cells, isolated by a method of the present invention, can be infused into the circulation. In the vessel that has had a myocardial infarction or is suffering from heart failure or heart failure, the population of enriched stem cells can be infused directly into the coronary artery or applied directly to the injured heart tissue. Methods and compositions for administration of stem cells and stem cell populations are known to the person skilled in the art and are not considered part of the novelty of the present invention. The following examples are merely provided as illustrative of various aspects of the invention, and should not be construed to limit the invention in any way.

Example 1: This example briefly describes the enrichment of a population of stem cells from a sample of the bone marrow collected from a normal donor. The bone marrow sample was treated with an agent that induces contraction, prior to enrichment by filtration by tangential flow. Briefly, 50 ml of bone marrow are removed from the hip bone of a normal volunteer, and 15 ml of phosphate buffered saline (PBS) supplemented with histamine was added to prevent coagulation.

After storage overnight, one third (approximately 33 ml) of this preparation was mixed with 33 ml of 30% dimethylsulfoxide (DMSO) in water. After a 10 minute incubation at room temperature, 3 ml of a 25% solution of human serum albumin (HSA) is added and the mixture is loaded into a recirculation chamber of the tangential flow filtration device briefly described above, and more fully in the US Provisional Patent Application Series No. 60 / 390,730, filed June 19, 2002 and WO 2004/000444 (each incorporated herein by reference). After two volume adjustments, the cells in the mixture were subjected to tangential flow filtration, with continuous filling of PBS with 0.625% HSA. At the end of the run, the cells were collected and analyzed. The preparation was found to be substantially clear of red blood cells and platelets. The percentage of CD34 + cells increased from 4.78% to 18.2% of the total cell number, while the percentage of cells in the neutrophil output as measured by fluorescent flow analysis decreased from 53.9% to 51.7%. The mean direct dispersion of the cells at the neutrophil output decreased from about 400 to about 250, indicating cell damage. The optimal method to date consists of treating the bone marrow aspirated for 10-20 minutes with a mixture of DMSO and PBS, followed by filtration in the FFT device for 60 minutes. The base solution of DMSO results in the contraction of PMN without collateral damage to the other populations of cells present. The result is the population concentration of CD34 + / CD45 + and CD133 + cells, while providing a reduced population of PMN cells and lymphocytes. In one protocol, the aspirated bone marrow is treated with DMSO in PBS diluted for 20 minutes, followed by addition of human serum albumin (for cell stabilization) and subsequent loading on the FFT device. The results may vary depending on the specific cell concentration of the input material. The runs of two examples are illustrated in Table I.

Table I. Characterization of cellular preparation derived from aspirated bone marrow of two volunteers after the concentration of stem cells in the FFT system.

Table I shows a partial reduction in PMN from as high as 74% initially up to 53%, due to the reduction in cell size and removal by the FFT system. Enrichment of the population of CD34 + / 45 + cells was from 5% to 18% in the CORRIDA 1 and from 4.2% to 13% in the CORRIDA 2. In addition, there was a reduction in the lymphocyte population from 16% up to 7% in the CORRIDA 1 and from 18% to 10% in the CORRIDA 2. The number of progenitor cells recovered was 11 million in the CORRIDA 1 and 16 million in the CORRIDA 2, far in excess, of that recovered in clinical trials referenced in this document. These stem cells derived from bone marrow, are capable of the function of normal stem cells, in which colony formation tests have been carried out, with the formation of a vibrant colony in a short period of time. It should be noted that CD133 + cells were also present in the population of concentrated and isolated stem cells as illustrated in CORRIDA 2, 0.1% is presented as the starting material and 1.3% in the final preparation.

Example 2 This example describes how a population of cells enriched in stem cells as described above, could be used to treat a patient who has an acute myocardial infarction. Briefly, stem cells purified by FFT from bone marrow, are infused into the coronary artery of a patient who has undergone an acute myocardial infarction, after a spiral implant. Subsequent remodeling of the cardiac muscle results in improvement of the volume of the left ventricular end and reduces the change of death from subsequent cardiac failure. The examples provided in this document are proposed to illustrate, but not limit the scope of the claimed invention. Other variants of the invention will be readily apparent to those of ordinary skill in the art and encompassed by the appended claims. All publications, patents, patent applications and other references cited in this document are also incorporated by reference in this document.

Claims (43)

NOVELTY OF THE INVENTION Having described the present is considered as a novelty, and therefore, it is claimed as property contained in the following: CLAIMS

1. A method for separating stem cells from a sample of a subject, wherein the sample comprises stem cells and constituents of non-stem cells, characterized in that the method comprises separation in a tangential flow filtration device by: (i) introducing the shows in a stirring unit (1) comprising a cross flow chamber (3) through an inlet (6) in the stirring unit; (ii) subjecting the sample to substantially parallel transverse flow to a filter (5) having a pore size of about 1 to about 10 microns; (iii) subjecting the fluid to filtration through the filter; and (iv) selectively removing constituents of non-stem cells from the sample to form a population of cells enriched for stem cells.

2. The method according to claim 1, characterized in that it further comprises: preparing the sample of the subject by leukapheresis, density centrifugation, differential lysis, filtration or preparation of a mononuclear cell culture, for introduction in the stirring unit.

3. The method according to claim 1, characterized in that the sample is bone marrow, a suspension of tissue or a suspension of blood organ or constituents.

4. The method according to claim 1, characterized in that the constituents of non-stem cells are stroma, erythrocytes, plasma and platelets.

5. The method of compliance with the claim 1, characterized in that it further comprises repeating steps (i), (ii) and (iii) at least twice to form the population of cells enriched for stem cells.

6. The method according to claim 1, characterized in that the stem cells are hematopoietic stem cells, mesenchymal stem cells or pluripotent stem cells.

7. The method according to claim 5, characterized in that the haematopoietic stem cells are CD34 + cells.

8. The method according to claim 3, characterized in that the stem cells are hematopoietic stem cells, mesenchymal stem cells or pluripotent stem cells.

9. The method of compliance with the claim 8, characterized in that the hematopoietic stem cells are CD34 + cells.

The method according to claim 1, characterized in that the subject has been subjected to stem cell mobilization by administering a stem cell mobilizing agent.

The method according to claim 10, characterized in that the stem cell mobilizing agent is M-CSF, G-CSF, GM-CSF or cyclophosphamide.

The method according to claim 1, characterized in that the tangential flow filtration device has a means for providing a predetermined inlet velocity of the sample at the inlet of the cross flow chamber; means for controlling a filtration rate through the filter and in the filtering chamber; and wherein the means controlling the filtration rate limit the filtration rate to less than the filtering rate not opposite to the filter.

13. The method according to claim 1, characterized in that the sample is pretreated to induce the contraction of cells from cell populations with essentially the same size as the stem cells.

14. The method according to claim 13, characterized in that the population of cells with essentially the same size are granulocytes.

The method according to claim 13, characterized in that the pretreatment comprises contacting the cells with a physiologically acceptable solution comprising dimethyl sulfoxide (DMSO).

16. The method according to claim 15, characterized in that the final concentration of DMSO is between 5% and 20%.

17. The method according to claim 16, characterized in that the final concentration of DMSO is between 10% and 15%.

18. The method according to claim 17, characterized in that the final concentration of DMSO is 12.5%.

19. The method according to claim 17, characterized in that the final concentration of DMSO is 15%.

20. The method according to claim 15, characterized in that the physiologically acceptable solution is of low ionic strength.

The method according to claim 13, characterized in that the pretreatment comprises contacting the cells with a physiologically acceptable solution comprising glycerol.

22. The method according to claim 21, characterized in that the final concentration of glycerol is between 0.5 mol / L and 2.5 mol / L.

23. The method according to claim 21, characterized in that the final concentration of glycerol is 1 mol / L.

24. The method according to claim 14, characterized in that the granulocytes are preferentially removed from the cell mixture by lysis.

25. The method according to claim 24, characterized in that the lysis is effected through sequential contact of the sample with an effective amount of DMSO and glycerol.

26. The method according to claim 25, characterized in that the contact of the sample is with a solution with low osmotic force.

27. A method for enriching a sample of bone marrow or blood constituents by stem cells, characterized in that it comprises: (i) introducing the sample into a tangential flow filtration unit (FFT), the FFT unit comprising a cross-flow chamber, a filtering chamber, and a filter in fluid communication with the cross flow chamber and the filtering chamber, the filter has a pore size of about 1 to about 10 microns; (ii) recirculating the sample through the FFT unit at a predetermined feed rate and a predetermined filtration rate, the predetermined feed rate is at least five times the predetermined filtration rate; wherein the predetermined filtration rate is less than the filtering rate not opposite for the filter; and (iii) isolating the population of cells enriched for stem cells.

28. The method according to claim 27, characterized in that the population of enriched cells is substantially free of blood constituents without leukocytes.

29. The method according to claim 1, characterized in that it further comprises the step of administering the population of cells enriched by stem cells to an individual.

30. The method according to claim 29, characterized in that the sample is from a subject that has been treated with at least one agent that mobilizes stem cells.

31. The method according to claim 30, characterized in that the agent that mobilizes the stem cell is M-CSF, G-CSF, GM-CSF or cyclophosphamide.

32. The method according to claim 29, characterized in that the stem cells are reinfused in a container.

33. The method according to claim 32, characterized in that the container is autologous to the subject.

34. The method according to claim 32, characterized in that the container is allogeneic to the subject.

35. The method according to claim 32, characterized in that the container is in need of regeneration of bone marrow.

36. The method according to claim 32, characterized in that the container has been subjected to myeloablative therapy.

37. The method according to claim 32, characterized in that the container is in need of cardiac tissue repair.

38. The method according to claim 32, characterized in that the container has been subjected to a cardiac infarction.

39. The method according to claim 32, characterized in that the container is suffering from congestive heart failure.

40. The method according to claim 32, characterized in that the container is suffering from heart failure.

41. The method according to claim 32, characterized in that the population of enriched stem cells is infused in the circulation.

42. The method according to claim 32, characterized in that the population of enriched stem cells is infused into the coronary artery.

43. The method according to claim 32, characterized in that the population of enriched stem cells is applied directly to the damaged heart tissue.