Abstract:
The present invention relates to a method for separating particles. The invention has particular advantages in connection with separating and purifying progenitor cells or stem cells obtained from bone marrow. The method comprises removing a desired volume of stem cell staring product from a donor/patient and eluting off a first contaminating cell type in a fluid chamber to create an enriched stem cell product.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This Application claims priority from U.S. provisional patent application 60/521,552, filed May 21, 2004 and is a continuation-in-part of U.S. regular application Ser. No. 10/310,528, filed Dec. 4, 2002, which claims priority of U.S. provisional application 60/338,938, filed Dec. 5, 2001. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     1. Field of the Invention  
         [0003]     The present invention relates to a method for separating particles. The invention has particular advantages in connection with separating and purifying progenitor cells or stem cells obtained from bone marrow.  
         [0004]     2. Description of the Related Art  
         [0005]     Bone marrow transplants are used to treat diseases once thought incurable. Diseases such as leukemia, aplastic anemia, Hodgkin&#39;s lymphoma, multiple myeloma, immune deficiency disorders and some solid tumors such as breast and ovarian cancers have been successfully treated by bone marrow transplants.  
         [0006]     Bone marrow is a spongy tissue found inside bones. The majority of the bone marrow is found in the breast bone, skull, hips, ribs and spine, and contain stem cells or progenitor cells which produce the body&#39;s blood cells as well as other types of cells.  
         [0007]     A stem cell/progenitor cell is characterized by having the ability to both self-renew and differentiate into functionally distinct lineages. The differentiation pathway of a stem cell is unidirectional; that is, once committed to a particular cell lineage, the cell develops into a terminally differentiated cell. Stem cells are directed toward a particular lineage by exposure to growth factors and their receptors.  
         [0008]     Besides bone marrow, progenitor cells/stem cells are also found in some adult organs and tissues. These stem cells are known as adult stem cells (ASC). Stem cells are also found in embryos during early stages of development and in fetal tissue, as well as in the umbilical chord. These stem cells are known as embryonic stem cells (ESC).  
         [0009]     Until recently, it was believed that adult stem cell differentiation was restricted to the tissue in which the stem cell resides. Two examples are hematopoietic stem cells that generate blood cells and oval cells (liver progenator cells), which generate hepatocytes.  
         [0010]     Recently however, the concept of adult stem cells being only restricted to their own tissue has been challenged by numerous reports that adult stem cells can jump lineages barriers and differentiate into cells outside their own tissue, in a process called stem cell transdifferentiation. These reports have revealed that stromal cells obtained from adult bone marrow have many characteristics of mesenchymal stem cells. Pluripotent progenitor stromal cells may differentiate into various types of cells, including bone, muscle, fat, tendon or cartilage. Because of these recent findings, a process to obtain large amounts of stem cells or progenitor cells to differentiate into various cell types would be highly desirable.  
         [0011]     Adult stem cells are present in bone marrow, blood, skin, muscle, liver, adipose tissue and brain. However, the frequency of stem cells in these tissues is relatively low. For example, mesenchymal stem cell frequency in bone marrow is estimated at between 1 in 100,00 and 1 in 1,000,000 nucleated cells. Similarly, extraction of stem cells from tissue involves a complicated series of cell culture steps over several weeks. Any proposed clinical application using adult stem cells requires a high number of cells, high purity and external manipulation of cellular maturation by processes of cell purification and cell culture.  
         [0012]     Currently, purification of stem cells from bone marrow aspirate is done using Ficoll-Paque and Percol density gradients. Such methods of purification are problematic for several reasons. Firstly, such purifications are done manually by a technician. Although these separations are done under sterile conditions using laminar flow hoods and the like, this method of purification does not occur in a closed system, which increases the risk of contaminating the cells with microorganisms. Secondly, ficoll and percol are chemicals, which must be removed before the purified product may be given to a patient. Thirdly, in such separations, cells are lost during each step of the procedure. As discussed above, if the number of desired cells in a bone marrow aspirate is not high to begin with, every cell lost due to processing issues is critical to the end process.  
         [0013]     Recent studies examining the therapeutic effects of bone-marrow derived progenitor/stem cells have used essentially the whole bone marrow to avoid the problems of cell purification. However, this creates other problems. Firstly, if bone marrow is injected directly into a damaged organ, only a small percentage of stem cells are actually delivered to the organ. As discussed above, the majority of bone marrow aspirate contains other cells such as red blood cells and platelets. Secondly, there is a limited volume of cells which may be injected into an organ. It would be better therefore to maximize the amount of stem cells delivered to an organ without the problems associated with manual purification.  
         [0014]     In studies using animal models, it has been shown that unfractionated mixtures of hematopoetic mononuclear cells that include differentiated cells as well as progenitor stem cells, become incorporated into collateral vessels.  
         [0015]     The same principles used above in the animal studies are also being used to treat humans. In patients who have suffered myocardial infarctions, loss of cardiac myocytes may lead to regional contractile dysfunction, and necrotized cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissue. Recent studies have shown that transplanted fetal cardiomyocytes are able to survive in the damaged heart tissue and the transplanted cells limited scar expansion and prevented postinfarction heart failure. Such treatment is not currently available due to current ethical and legal considerations. However, based on the results from the studies described below, stem cells taken from adult bone marrow may potentially substitute for fetal cardiomyocytes in this type of treatment.  
         [0016]     In a clinical trial by Tateishi-Yuyama, autologous bone marrow mononuclear cells were injected into patients with ischemic peripheral vascular disease. Bone marrow cells were collected under general anesthesia and injected into the gastrocnemius muscle of the ischemic leg in multiple sites. After treatment, significant improvement was seen in the ankle-brachial index (ABI), transcutaneous oxygen pressure and pain-free walking.  
         [0017]     In another recent clinical trial, Hung-Fat Tse et al injected autologous bone marrow mononuclear cells into ischemic myocardium. The ischemic area was injected intramyocardially with a mixture of CD34 + , CD3 +  T cells and granulocytes. Following treatment, the number of anginal episodes and nitroglycerin tablet usage decreased. Postinjection cardiac MRI demonstrated improved wall motion and thickness.  
         [0018]     In one preliminary study done to date, one 50 mL aspiration of bone marrow from patients who suffered an acute myocardial infarction was aspirated from the iliac crest and immediately injected into the damaged area of the heart. Repair of the damaged cardiac muscle and improved cardiac function was seen.  
         [0019]     An approximate volume of around 20 mL of bone marrow cells appears to be the upper volume limit that can be injected into the heart. It may be surmised that cardiac repair and function may increase exponentially if a greater volume of stem cells were collected either through multiple sticks or a greater aspiration volume and then concentrated into a smaller volume.  
         [0020]     The present invention is directed towards avoiding the problems associated with manual purification of stem cells and towards the goal of purifying and concentrating large amounts of stem cells to be used in treating humans.  
       SUMMARY OF THE INVENTION  
       [0021]     This invention includes a method for enriching stem cells, which includes the steps of removing a desired volume of stem cell starting product from a donor/patient to obtain a stem cell starting product, loading the stem cell starting product into a fluid chamber, flowing a low density fluid to the loaded stem cell starting product in the fluid chamber, centrifuging the fluid chamber; and eluting off a first contaminating cell type from the stem cell starting product in the fluid chamber to create an enriched stem cell product.  
         [0022]     The method may further include a step of debulking the stem cell starting product to remove a first contaminating cell type.  
         [0023]     In a further aspect the invention relates to a method of concentrating the enriched stem cell product.  
         [0024]     It is another aspect of the present invention to treat a damaged organ with stem cells, which were collected from bone marrow and enriched and concentrated using the above method.  
         [0025]     Although the present invention is particularly directed to separating stem cells or progenitor cells from other cells contained within a bone marrow aspirate, it is understood that the techniques of the present invention can also apply to stem cells collected using other well known collection methods and from sources other than bone marrow aspirate, including, but not limited to, peripheral blood and umbilical cord blood. Therefore, both the foregoing general description and the following detailed description are exemplary, and are intended to provide further explanation of the invention as claimed. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0026]     The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification. The drawings illustrate an embodiment of the invention and, together with the description, serve to explain the principles of the invention. In the drawings,  
         [0027]      FIG. 1  is a perspective view of a disposable which could be used in the closed system.  
         [0028]      FIG. 2  is a perspective view of a closed system disposable containing a fluid chamber, concentrator and separation vessel mounted on a centrifuge rotor.  
         [0029]      FIG. 3  is a table showing elutriation results from the stem cell enrichment protocol of the present invention.  
         [0030]      FIGS. 4   a - f  are graphs of the elutriation results from  FIG. 3  above. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0031]     In the present invention, bone marrow is taken from a donor or patient using any means known in the art. Typically, bone marrow is removed from the iliac crest of the donor/patient&#39;s pelvis via syringe draw. A typical bone marrow harvest for hematopoetic reconstitution yields around 2×10 8  nucleated cells/kg body weight of the recipient. To obtain the necessary amount of cells, it is usually required to remove around 1 L of bone marrow. Anywhere between 0.1-25 mL of bone marrow may be aspirated from the bone with any one draw. Multiple aspirations are typically necessary to obtain the desired amount of cells. If multiple aspirations are collected, they may be combined into a single source bag to provide a single source of stem cell starting product collected from multiple syringe draws, or may be collected into multiple source bags, each containing stem cell starting product collected from a single syringe draw. The single source bags may be processed individually, or may be combined either before or after processing.  
         [0032]     Stem cells may also be separated from peripheral blood. A COBE® SPECTRA™ blood component centrifuge manufactured by Gambro BCT, Inc. of Colorado may be used to initially separate blood into components. Stem cells are typically found in the white blood cell fraction. The separated cell fraction containing white blood cells and stem cells may then be used as the stem cell starting product in the enrichment procedure described below.  
         [0033]     Stem cells are also found in umbilical cord blood. The procedure described below may also be used to enrich stem cells from cord blood.  
         [0034]     One way to enrich a specific subset of cells from a fluid containing many cell types is to use elutriation technology. Elutriation could be used to separate progenitor/stem cells from other cells contained in bone marrow, peripheral blood or umbilical cord blood. The enriched product may then be concentrated to a final volume appropriate for the desired application.  
         [0035]     In one common form of elutriation, a cell batch such as the stem cell starting product collected in the source bag/bags is introduced into a funnel-shaped chamber located in a spinning centrifuge. A flow of liquid elutriation buffer is then introduced into the chamber containing the cell batch. As the flow rate of the liquid buffer solution is increased through the chamber (usually in a stepwise manner), the liquid sweeps smaller sized, slower-sedimenting cells toward an elutriation boundary within the chamber, while larger, faster-sedimenting cells migrate to an area of the chamber where the centrifugal force and the sedimentation (drag) forces are balanced.  
         [0036]     Thus, centrifugal elutriation separates particles having different sedimentation velocities. Stoke&#39;s law describes sedimentation velocity (SV) of a spherical particle, as follows:  
       SV   =       2   9     ⁢           r   2     ⁡     (       ρ   p     -     ρ   m       )       ⁢   g     η           
 
 where, 
        r is the radius of the particle,     ρ p  is the density of the particle,     ρ m  is the density of the liquid medium,     η is the viscosity of the medium, and     g is the gravitational or centrifugal acceleration.        
 
         [0042]     Because the radius of a particle is raised to the second power in the Stoke&#39;s equation and the density of the particle is not directly related to the size of a cell, its density greatly influences its sedimentation rate. This explains why larger particles/cells generally remain in a chamber during centrifugal elutriation, while smaller particles/cells are released, if the particles have similar densities.  
         [0043]     Specific cell subsets to date have initially been separated from, or debulked of, red blood cells by density gradient centrifugation, using various separation media. In density gradient centrifugation, a sample is layered on top of a media support and centrifuged. Under centrifugal force, the particles in the sample will sediment through the media in separate zones according to their density. As discussed above, manual density gradient separation is not done in a closed system and requires both a contamination free environment and chemical gradients, both of which are undesirable.  
         [0044]     It is known that red blood cells under proper conditions have the tendency to adhere to each other forming red blood cell rouleaux. Rouleaux formation and size, and therefore red cell sedimentation velocity, is influenced by the hematocrit of the cell suspension, exposure to shear, protein concentration, and presence of sedimentation agents.  
         [0045]     Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.  
         [0046]     The COBE® SPECTRA™ centrifuge incorporates a one-omega/two-omega sealless tubing connection as disclosed in U.S. Pat. No. 4,425,112 to Ito, the entire disclosure of which is incorporated herein by reference. Although the embodiments of the invention are described in combination with the COBE® SPECTRA™ centrifuge, this reference is made for exemplary purposes only and is not intended to limit the invention in any sense.  
         [0047]     As will be apparent to one having skill in the art, the present invention may be advantageously used in a variety of centrifuge devices commonly used to separate cell subsets into desired cell types. In particular, the present invention may be used with any centrifugal apparatus regardless of whether or not the apparatus employs a one-omega/two-omega sealless tubing connection.  
         [0048]     As embodied herein and illustrated in  FIG. 1 , the present invention includes a particle separation disposable system  10  for use with a centrifuge rotor  12 . Preferably, the centrifuge rotor  12  is coupled to a motor (not shown) via an arm  14 , shown in  FIG. 2 , so that the centrifuge rotor  12  rotates about its axis of rotation A-A.  
         [0049]     As shown in  FIG. 2 , a holder  16  is provided on a top surface of the rotor  12 . The holder  16  releasably holds a fluid chamber  18  on the rotor  12  such that an outlet  20  for cells other than red blood cells, hereinafter called the outlet of the fluid chamber  18 , is positioned closer to the axis of rotation A-A than the inlet  22  of the fluid chamber  18 . The holder  16  preferably orients the fluid chamber  18  on the rotor  12  with a longitudinal axis of the fluid chamber  18  in a plane transverse to the rotor&#39;s axis of rotation A-A. In addition, the holder  16  is preferably arranged to hold the fluid chamber  18  on the rotor  12  with the fluid chamber outlet  20  for cells other than red blood cells facing the axis of rotation A-A. Although the holder  16  retains the fluid chamber  18  on a top surface of the rotor  12 , the fluid chamber  18  may also be secured to the rotor  12  at alternate locations, such as beneath the top surface of the rotor  12 . It is also understood that the fluid chamber  18  could be secured by other well known fixative devices or by other methods other than the holder as shown.  
         [0050]     The fluid chamber  18  has smooth sides as shown in  FIGS. 1 and 2  as described below. As shown in  FIGS. 1 and 2 , the inlet  22  and outlet  20  of the fluid chamber  18  are arranged along a longitudinal axis of the fluid chamber  18 . A wall  21  of the fluid chamber  18  extends between the inlet  22  and outlet  20  thereby defining inlet  22 , the outlet  20 , the side and an interior of the fluid chamber  18 .  
         [0051]     The fluid chamber  18  includes two frustoconical shaped sections  25 ,  27  joined together at a maximum cross-sectional area  23  of the fluid chamber  18 . The interior of the fluid chamber  18  tapers (decreases in cross-section) from the maximum cross-sectional area  23  in opposite directions toward the inlet  22  and the outlet  20 . Although the fluid chamber  18  is depicted with two sections ( 25 ,  27 ) having frustoconical interior shapes, the interior of each section may be paraboloidal, or of any other shape having a major cross-sectional area greater than the inlet or outlet area.  
         [0052]     The fluid chamber  18  may be constructed from a unitary piece of plastic or from separate pieces joined together using known fixative or sealing methods to form separate sections of the fluid chamber  18 . The fluid chamber  18  may be formed of a transparent or translucent copolyester plastic, such as PETG, to allow viewing of the contents within the chamber interior with the aid of an optional strobe (not shown) during a separation or debulking procedure.  
         [0053]     As shown in  FIG. 1 , the system  10  which depicts a closed system disposable further includes a first conduit or line  28 , second or debulk conduit or line  30 , an inlet conduit or line  32  in fluid communication with the inlet  22  of the fluid chamber  18 , and a three-way or Y connector  34  having three legs for flow or fluidly connecting the first conduit  28 , second or debulk conduit  30 , and inlet line  32 . The first conduit  28  includes a coupling  36  for flow-connecting the first conduit  28  with conduit or line  27 , coupling  39  and the single (or multiple) source bag/s  38  containing stem cell starting product to be separated into stem cells and other cells. Likewise, the first conduit  28  is connected by coupling  36  to conduit or line  37  which includes couplings  40  for flow-connecting the first conduit  28  with a second source  42  containing a low density diluting, sedimentation or elutriation fluid. An in-line filter  3  may or may not be placed within conduit  37  to filter fluid from source  42 . The couplings  36 ,  39  and  40  are preferably any type of common medical coupling devices, such as spikes or sterile tubing connectors.  
         [0054]     As shown in  FIG. 1 , the first conduit  28  includes a first tubing loop  44 . During use, the first tubing loop  44  is mounted in a peristaltic pump (not shown) for respectively pumping the stem cell starting product to be separated and the diluting, sedimentation or elutriation fluid from the first and second sources  38  and  42 , respectively.  
         [0055]     The stem cell starting product from the first source bag  38  and the diluting, sedimentation or elutriation fluid from the second source  42  flow through the respective first conduit  28  to the three-way connector  34 . These substances then flow through the inlet line  32  into the inlet  22  of the fluid chamber  18 . In the fluid chamber  18 , turning with rotor  12 , the cells in the bone marrow in the centrifugal field separate according to differences in sedimentation velocity leaving faster sedimenting cells in the fluid chamber  18  and allowing some slower sedimenting cells to flow from the fluid chamber  18  as will be described below.  
         [0056]     As the fluid chamber  18  is loaded with stem cell starting product as is more fully described below, the fluid and cells having a relatively slower sedimentation velocity, which generally includes white blood cells and stem cells, will flow through the fluid chamber outlet  20  into conduit tubing or line  48 . As shown in  FIG. 2 , the tubing  48  may optionally be connected to an inlet  50  of a separation vessel  52  or optional cellular concentrator mounted to the centrifuge rotor  12 .  
         [0057]     If an optional concentrator is used, it will be placed adjacent to an outer portion of the centrifuge rotor  12 . The concentrator  52  has a collection well  54  for collecting particles flowing into the concentrator  52 . Rotation of centrifuge rotor  12  sediments particles into the collection well  54 , while slower sedimenting fluid and possibly some slower sedimenting particles remain above a top boundary of the collection well  54 . The collected particles in the collection well  54  can include any cells or particles that have exited the fluid chamber  18 , or separated subsets of white blood cells and stem cells, as noted above.  
         [0058]     In the embodiment shown in  FIG. 2 , the optional concentrator  52  is placed in a groove  64  formed in the rotor  12 . Preferably, the concentrator  52  is a channel formed of a semi-rigid material so that a valley  66  in an outer wall of the groove  64  forms the collection well  54  when the concentrator  52  expands in response to fluid and particles in the concentrator  52  encountering centrifugal forces. As shown in  FIG. 2 , the top surface of the rotor  12  preferably includes retainer grooves for receiving the first and second conduits  28  and  30 , three-way connector  34 , inlet line  32 , tubing  48 , particle concentrate line  58 , and fluid outlet line  62 . If a tubing set without a concentrator is used, such as shown in  FIG. 1 , the rotor will not have groove  64  or valley  66 .  
         [0059]     As shown in  FIG. 1 , the fluid outlet line  62  is fluidly coupled at one end to outlet  20  and at the other end to a fluid collection container  61  for collecting fluid removed from the fluid chamber  18 , and the particle concentrate line  58  is fluidly coupled to one or more particle collection containers  70  for collecting particles removed from the fluid chamber  18 . Although only one particle collection container  70  is shown, it should be appreciated that as many particle containers as needed to collect elutriation fractions may be used. For example, if twelve fractions (such as shown in  FIG. 3 ) are collected, each fraction may be collected in a separate collection container. Therefore, twelve collection containers  70  would be attached to particle concentrate line  58 .  
         [0060]     Preferably, the particle concentrate line  58  includes a tubing loop  72  capable of being mounted in a peristaltic pump for pumping particles through the particle concentrate line  58 . The pump for tubing loop  72  regulates the flow rate and concentration of particles in particle concentrate line  58 . The stem cells will be collected into bag  70 . It is understood that any number of bags  70  can be used to collect the desired subsets of stem cells. Platelets, which are considered to be contaminating cells in a stem cell enrichment procedure such as described here, can also be collected in a separate bag if desired.  
         [0061]     After sedimentation in chamber  18 , as is more fully described below, red blood cells, which are considered to be contaminating cells in a stem cell enrichment procedure, are removed through inlet  22  to inlet conduit  32 . The debulked red blood cells then pass through Y connector  34  to debulking conduit  30 . As shown in  FIG. 1 , conduit  30  is fluidly coupled to a red blood cell collection container or debulked cell collection container  31  for collecting red blood cells collected during the debulking procedure. Preferably the red blood cell collection or debulk line or conduit  30  includes a tubing loop  46  capable of being mounted in a peristaltic pump for pumping red blood cells through conduit  30 .  
         [0062]     To control flow rates of substances and rotational speed of the rotor  12  during operation of the system  10 , a controller (not shown) controls pumps (not shown) for pumping substances through the tubing loops  44 ,  46  and  72  and controls a motor (not shown) for rotating the centrifuge rotor  12 .  
         [0063]     A preferred method of separating components of blood and, in particular, separating stem cells and white blood cells from red blood cells is discussed below with reference to  FIGS. 1-4 . Although the invention is described in connection with a blood component separation process and specifically a stem cell separation or fractionation process, it should be understood that the invention in its broadest sense is not so limited.  
         [0064]     Initially, bone marrow aspirate is drawn from a patient using a syringe  2  and needle  4 . Depending upon the number of stem cells desired, bone marrow may be collected from a donor/patient in very small volumes of around 0.1 mL, up to larger volumes of around 25 mL, using one or more needle sticks. This bone marrow aspirate will henceforth be known as the stem cell starting product regardless of the way it was collected from a donor/patient. It should be noted that the larger the amount of bone marrow removed from a donor/patient in a single draw, the more contaminated the sample may be with other components of bone marrow such as red blood cells and platelets, and the more separation and enrichment will be required. Small aspirates (around 0.2 mL) will be less contaminated with platelets and red blood cells than larger volumes. The aspirates may be injected into a storage bag (not shown) or may be injected directly into source bag  38  as shown in  FIG. 1 .  
         [0065]     Filtration of contaminating bone fragments and other solid material may be necessary before the elutriation procedure. Any filters  6  known in the art may be used. The filter may be placed anywhere within the closed system so long as it is placed before the elutriation chamber  18 . As examples, not meant to be limiting, the filter may be connected anywhere within the tubing line leading to source bag  38  (as shown in  FIG. 1 ). The bone marrow aspirate may gravity drain through tubing  8 , through filter  6  and into source bag  38 . The filter may also be placed directly on the end of the syringe used to aspirate the bone marrow. The bone marrow is forced through the filter into source bag  38  by application of downward pressure to the syringe. The filter may also be placed within the tubing line  10  leading out of source bag  38 .  
         [0066]     The stem cell starting product is placed in the first source  38  shown in  FIG. 1 , and the first source  38  is coupled to the first conduit  28  through conduit  27 . In addition, the second source  42  containing the diluting, sedimentation or elutriation fluid is coupled to the conduit  28  through the conduit  37 . The centrifuge rotor  12  is rotated about the axis of rotation A-A (see  FIG. 2 ), at approximately 2400 rpm. The stem cell starting product is pumped from source  38  at a low flow rate and loaded into the fluid chamber  18 . The flow of stem cell starting product from source  38  is then stopped by a valve or other well-known mechanism. Flow of diluting, sedimentation or elutriation fluid is then started to rinse conduit  28  and/or wash the loaded stem cell starting product. Small particles (such as platelets) may be removed from conduit  28  simply by the flow of the fluid during this flowing step. The diluting, sedimentation fluid or elutriation fluid passes through conduit  28  and Y connector  34 , and inlet conduit  32  into the inlet  22  of chamber  18 .  
         [0067]     The inlet pump  44  associated with the tubing loop is stopped to stop the flow of low density diluting, sedimentation or elutriation fluid into the chamber  18 . As the centrifuge continues to rotate, the stem cell starting product loaded in the chamber sediment under the resulting centrifugal force.  
         [0068]     After sedimentation of the particle constituents of the stem cell starting product, the pump associated with tubing loop  46  is activated to remove or debulk at a low flow rate the sedimented red blood cells through the inlet  22  of the chamber  18  and then through inlet conduit  32  and debulking conduit  30  to container  31 .  
         [0069]     After removal of red blood cells, the stem cells and white blood cells remaining in chamber  18  can be further separated as described below, or the inlet pump associated with tubing loop  44  can be restarted to reintroduce a second batch of blood product from source  38  into chamber  18 . This would be desirable if multiple bone marrow aspirations were done.  
         [0070]     The elutriating step for separating stem cells and white blood cells into the desired subsets can be done after each debulking procedure or after the source  38  is empty of stem cell starting product. The only requirement is that there be a sufficient number of stem cells and white blood cells in chamber  18  to achieve effective separation or fractionation. Therefore, the white blood cell and stem cell content of the stem cell starting product should be considered in determining the sequence order of the elutriation step.  
         [0071]     For collection of fractionated or separated white blood cells or stem cells, an operator, after debulking or after the first source  38  is empty, slowly increases the inlet pump speed associated with tubing loop  44 , decreases the centrifuge speed, or increases the density or viscosity of the diluting, sedimentation or elutriation fluid to separate the cells in chamber  18  into subsets by elutriation, as is well known in the art. Such separated subsets may then be concentrated in the optional concentrator  52  (if used), or simply be removed to bag/s  70 .  
         [0072]     Although the preferred embodiment discloses separating the white blood cells and stem cells in subsets using elutriation in chamber  18 , it is also understood that a second separate chamber (not shown, but similar to chamber  18 ) could be fluidly connected between chamber  18  and optional concentrator  52  (if one is used) wherein the white blood cells and stem cells can be further separated into subsets or concentrated using the elutriation separation process in the second chamber. Also, the elutriative separation can occur after the white blood cells and stem cells are collected into a bag  70  as a separate processing step.  
         [0073]     The loading, flowing of low density fluid, sedimenting, debulking and elutriating steps, (if done after debulking), described above may be repeated until the entire stem cell starting product from one or more aspirations has been separated or enriched into desired components or desired subsets and debulked of red blood cells. Alternatively, as mentioned above, the loading, flowing of low density fluid, sedimenting and debulking steps may be repeated multiple times until the entire stem cell starting product has been debulked of red blood cells. The entire debulked stem cell product may than be elutriated in one elutriation step.  
         [0074]     It is understood that the protein and sedimentation agents used to form the diluting, sedimentation fluid could be any fluid known in the art. It is also understood that the low density fluid could be media or plasma.  
         [0075]     Although the diluting, sedimentation or elutriation fluid is added only at certain parts of the process, it is understood that other configurations are possible. For example, the fluid chamber  18  could be modified to include separate inlets for blood components and diluting or sedimentation fluid. The diluting or sedimentation fluid could also be added to the blood components in the first source  38  before, or at the beginning of, a batch separation process. It is further understood that the selection of elutriation fluid may depend on whether the subsets will be separated by an elutriation technique after debulking.  
         [0076]     As the stem cell starting product is being loaded into the separation chamber  18  and during the elutriating step, the diluting, sedimentation or elutriation fluid, plasma, platelets, and the white blood cells and stem cells flow from the fluid chamber outlet  20  through the particle collect line  58  to the collect bag/s  70 , while the diluting fluid and plasma flow through the fluid outlet  60  and fluid outlet line  62  to container  61 . This separates the platelets and other particles from the diluting fluid and plasma.  
         [0077]     The instant debulking procedure could achieve effective removal of RBCs without a significant loss of stem cells, and can achieve such in a closed system. The capacity of the system of the instant invention can be increased by placing several small chambers in parallel or in series, or by using one large chamber. Ideally, either the combined chambers or a single chamber should be capable of debulking and/or elutriating between approximately 10 to 150 ml of stem cell starting product. The current disposable could easily be adapted to accommodate multiple chambers or one large chamber, provided the chamber could be recessed in the rotor  12 .  
         [0078]     The disposable particle separation system may also optimally include sensors at various output locations such as in the particle concentrate line for monitoring the types of cells and concentration being collected. Any known type of a sensor could be used.  
       EXAMPLES  
     Example 1  
       [0079]     In the following experiment, stem cells were mobilized from bone marrow into the peripheral blood and an apheresis sample was collected using Spectra.  
         [0080]     The elutriation protocol used to enrich stem cells from the starting stem cell product is set out in the table below. The columns set out the flow rate (mL/min), rotor speed and volume of elution fluid used to flow through the fluid chamber to enrich stem cells and white blood cells from peripheral blood. This procedure may also be used to debulk and enrich stem cell aspirated from bone marrow. The volume of elution fluid used was 500 mL. The rotor speed was maintained at 2400 rpm, except for the last fraction, which was collected with the rotor off. Twelve fractions were eluted and collected as well as a pre-fraction, which was collected before the elutriation procedure was begun.  
                                                           Fraction   ml/min   rotor   volume                           Pre   na   na   500 ml           1   37   2400   500 ml           2   77   2400   500 ml           3   81   2400   500 ml           4   85   2400   500 ml           5   90   2400   500 ml           6   95   2400   500 ml           7   100   2400   500 ml           8   105   2400   500 ml           9   110   2400   500 ml           10   115   2400   500 ml           11   120   2400   500 ml           12   120   off   250 ml                      
 
         [0081]     In the experiments, the eluted fractions were analyzed using flow cytometry to count and classify blood cell types. Fluorescent antibodies which are specific to receptors on the surface of the cells were used as markers to measure the different cell types. CD45 is a marker for white blood cells, CD34 is a marker for stem cells, CD3 is a marker for T-cells, CD 14 is a marker for monocytes, and CD19 is a marker for B-cells. Traditional flow cytometry gating/counting methods were used.  
         [0082]     The results are shown in the table of  FIG. 3  below. The table shows the total number of each cell type which elutes off in each fraction.  
         [0083]     The cell count data is also depicted graphically  FIGS. 4   a - 4   f .  FIG. 4   a  shows the elutriation profile of red blood cells.  FIG. 4   b  shows the elutriation profile of the general category of white blood cells, which will include all subsets of white blood cells as well as stem cells and other similarly sized cells.  FIG. 4   c  shows the elutriation profile of stem cells.  FIG. 4   d  shows the elutriation profile of T-cells.  FIG. 4   e  shows the elutriation profile of monocytes, and  FIG. 4   f  shows the elutriation profile of B-cells.  
         [0084]     The graphs show three white blood cell peaks: an early CD19 (B-cells) peak ( FIG. 4   f ) which overlaps with the RBC peak ( FIG. 4   a ); a mid-peak containing CD3 (T-cells) ( FIG. 4   d ); and a late peak containing CD34 (stem cells) ( FIG. 4   c ), and CD14 (monocytes) ( FIG. 4   e ). Using these results, elutriation fractions which contain enriched fractions of different cell types may be selectively collected in bag/s  70 . For example, if primarily stem cells were desired, fractions  9 - 12  should be collected. However, as can be seen from  FIG. 3   e , monocytes will also be collected in this enriched stem cell fraction. A further processing step, such as antibody specific adsorption as discussed above may be desired. Alternatively, other contaminating cell types such as B cells and T cells may be eluted off before the desired enriched fraction is collected.  
       Example 2  
       [0085]     The above-described method may be incorporated into a method for enriching progenitor cells from bone marrow aspirate. The enriched progenitor cells obtained by the described method may be further concentrated into a smaller volume. Progenitor cells obtained by this method may be injected directly into damaged tissue to heal and re-grow the injured tissue.  
         [0086]     Depending on the type of injured tissue to be treated, variable amounts of bone marrow may be collected. The bone marrow could be collected from the patient to be treated, or could be collected from a suitable donor.  
         [0087]     The final volume and number of cells that will be concentrated will depend on the type of organ to be treated. As one example, if it is desired to treat cardiac muscle, the starting volume of the stem cell starting product may be concentrated down to a volume of approximately 20 mL, which may be the approximate maximum volume practical to inject (in one or more injections) into the heart. The injection/s may be given either intramuscularly or intravenously, or both.  
         [0088]     An additional step may be to select for specific progenitor cell types from the final enriched product. Such selection may be done by any means known in the art, but may include cell selection using antibodies specific to subtypes of progenitor cells such as mesenchymal stem cells, which could differentiate into different tissue types upon injection/transplantation into the damaged organ, as but one example, not meant to be limiting.  
         [0089]     It will be apparent to those skilled in the art that various modifications and variations can be made to the structure and methodology of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims and their equivalents.