US20050250204A1

US20050250204A1 - Methods and apparatus for separation of particles

Info

Publication number: US20050250204A1
Application number: US11/131,063
Authority: US
Inventors: Glen Antwiler
Original assignee: Gambro Inc
Current assignee: Terumo BCT Inc
Priority date: 2001-12-05
Filing date: 2005-05-16
Publication date: 2005-11-10
Also published as: DE60230997D1; EP1454135B1; EP1454135A2; JP2005512089A; WO2003050536A2; US7201848B2; WO2003050536A3; ATE421348T1; US20030116512A1; AU2002353022A8; US7588692B2; CA2455964A1; JP4512367B2; US20070144978A1; AU2002353022A1

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

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

1. Field of the Invention
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.
2. Description of the Related Art
Bone marrow transplants are used to treat diseases once thought incurable. Diseases such as leukemia, aplastic anemia, Hodgkin'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.
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's blood cells as well as other types of cells.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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

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.
The method may further include a step of debulking the stem cell starting product to remove a first contaminating cell type.
In a further aspect the invention relates to a method of concentrating the enriched stem cell product.
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.
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

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,
FIG. 1 is a perspective view of a disposable which could be used in the closed system.
FIG. 2 is a perspective view of a closed system disposable containing a fluid chamber, concentrator and separation vessel mounted on a centrifuge rotor.
FIG. 3 is a table showing elutriation results from the stem cell enrichment protocol of the present invention.
FIGS. 4 a-f are graphs of the elutriation results from FIG. 3 above.

DESCRIPTION OF THE PREFERRED EMBODIMENT

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's pelvis via syringe draw. A typical bone marrow harvest for hematopoetic reconstitution yields around 2×10⁸nucleated 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.
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.
Stem cells are also found in umbilical cord blood. The procedure described below may also be used to enrich stem cells from cord blood.
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.
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.
Thus, centrifugal elutriation separates particles having different sedimentation velocities. Stoke's law describes sedimentation velocity (SV) of a spherical particle, as follows: $SV = \frac{2}{9} \frac{r^{2} (ρ_{p} - ρ_{m}) g}{η}$
where,

- r is the radius of the particle,
- ρ_pis the density of the particle,
- ρ_mis the density of the liquid medium,
- η is the viscosity of the medium, and
- g is the gravitational or centrifugal acceleration.

Because the radius of a particle is raised to the second power in the Stoke'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.
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.
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.
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.
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.
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.
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.
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'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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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

In the following experiment, stem cells were mobilized from bone marrow into the peripheral blood and an apheresis sample was collected using Spectra.

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

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.
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.
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.
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

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.
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.
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.
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.
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.

Claims

1. A method of enriching stem cells comprising 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 into 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; and

collecting the enriched stem cell product.

2. The method of claim 1 further comprising a debulking step to remove a first contaminating cell type from the stem cell starting product.

3. The method of claim 2 wherein the debulking step occurs before the eluting step.

4. The method of claim 1 further comprising concentrating the enriched stem cell product.

5. The method of claim 1 wherein the stem cell starting product further comprises bone marrow.

6. The method of claim 1 wherein the stem cell product further comprises peripheral blood.

7. The method of claim 1 wherein the stem cell product further comprises umbilical cord blood.

8. The method of claim 5 wherein the step of removing bone marrow further comprises filtering the bone marrow.

9. The method of claim 8 wherein the filtering step occurs before the loading step.

10. The method of claim 1 further comprising eluting off a second contaminating cell type from the starting product in the fluid chamber.

11. The method of claim 1 further comprising eluting off a third contaminating cell type from the starting product in the fluid chamber.

12. The method of claim 1 further comprising eluting off a fourth contaminating cell type from the starting product in the fluid chamber.

13. The method of claim 1 comprising repeating the loading, flowing, centrifuging, eluting and collecting steps as many times as necessary to obtain the desired number of enriched stem cells.

14. The method of claim 2 comprising repeating the loading, flowing, debulking, and centrifuging steps as many times as necessary to remove undesired cells before the eluting step.

15. The method of claim 4 further comprising repeating the concentrating step as many times as necessary to obtain the desired number of enriched, concentrated stem cells.

16. The method of claim 1 wherein the flowing step comprises flowing a low density fluid into the fluid chamber at a flow rate of around 37 mL/min at a volume of around 500 mL.

17. The method of claim 2 wherein the first contaminating cell type is red blood cells.

18. The method of claim 10 wherein the second contaminating cell type is platelets.

19. The method of claim 11 wherein the third contaminating cell type is T-cells.

20. The method of claim 12 wherein the fourth contaminating cell type is B-cells.

21. A method of treating a damaged organ with stem cells collected from bone marrow comprising the steps of:

removing a desired volume of bone marrow from a donor/patient to obtain a bone marrow starting product;

loading the bone marrow starting product into a fluid chamber;

flowing a low density fluid into the loaded bone marrow starting product in the fluid chamber;

centrifuging the fluid chamber;

debulking a first contaminating cell type from the bone marrow starting product in the fluid chamber;

eluting off a second contaminating cell type from the bone marrow starting product in the fluid chamber to create an enriched stem cell product;

concentrating the enriched stem cell product to obtain a concentrated, enriched stem cell product; and

injecting the concentrated enriched stem cell product into the damaged organ.

22. The method of claim 21 further comprising determining the amount of stem cells needed to treat the damaged organ.

23. The method of claim 21 wherein the step of injecting further comprises injecting the concentrated enriched sample into cardiac tissue.

24. The method of claim 21 wherein the step of injecting further comprises injecting the concentrated enriched stem cell product intravenously.

25. The method of claim 21 wherein the step of injecting further comprises injecting the concentrated enriched stem cell product intramuscularly.

26. The method of claim 21 wherein the step of injecting further comprises injecting the concentrated enriched stem cell product both intravenously and intramuscularly.

27. The method of claim 21 wherein the step of injecting further comprises injecting the concentrated enriched stem cell product in multiple injections.

28. The method of claim 21 wherein the step of injecting further comprises injecting the concentrated enriched stem cell product in a single injection.