JP5832591B2 - Composition of stem and progenitor cells recovered from bone marrow or umbilical cord blood, system and method for preparing them - Google Patents

Description

  The present invention generally relates to a method and apparatus for recovering specific cell populations from human bone marrow or umbilical cord blood. In particular, the present invention provides high efficiency of stem and progenitor cells present in bone marrow or umbilical cord blood without the use of xenobiotic additives or additives commonly used to improve the efficiency of stem cell recovery. Recovery and separation, and systems and methods for removing excess red blood cells, neutrophils, platelets, and plasma. The system separates sterile and sterilized functionally closed bag sets (a set of bags) and cell populations based on their size and density or concentration, and then separates these stem and progenitor cells It includes electrical, mechanical and optical devices designed to operate in a centrifugal field, controlled by a microprocessor, and transferred to a stem cell bag in a predetermined final volume. The invention also includes stem and progenitor cell compositions or compositions prepared or made from human bone marrow or umbilical cord blood, which are either ready for use or stored for later use. .

(Cross-reference of related applications)
35U. S. C. In accordance with the provisions of §120, this application claims priority and is a partially pending application of co-pending US patent application Ser. No. 10 / 118,291 filed Apr. 8, 2002. The entire disclosure of which is incorporated herein by reference. 35U. S. C. In accordance with the provisions of §120, this application also claims and is a co-pending application of co-pending US patent application No. 11 / 664,212 filed on March 28, 2007, The application is filed with International Application No. PCT / US2005 / 029288 published in English on April 13, 2006 as the International Publication No. WO 2006/038993 A2 based on Article 21 (2) PCT and designated as the US Priority, 35U. S. C. § 371, a national phase application, and this US patent application, on the other hand, was filed on September 30, 2004 and issued as US Patent No. 7,211,191 on May 1, 2007. No. 10 / 957,095, claimed in its entirety, the entire disclosures of which are incorporated herein by reference.

For the purposes of this specification and the following claims, the following definitions will be used.
“In-house use” means internal transplantation, transplantation, injection or transfer to return a person's cells or tissues to the individual from which they were collected.
“Crystalline” refers to isotonic salts and / or glucose solutions (isotonic salts) used to increase electrolyte replacement or intravascular volume, such as saline, lactated Ringer's solution, or 5% glucose solution. And / or a solution of glucose).
“Hematopoietic stem cells” are pluripotent stem cells that give rise to a variety of blood cells including red blood cells (RBC), white blood cells (Leukocytes), white blood cells (WBC), and platelets.
“Leukocytes” are cells of the hematopoietic system, mainly including monocytes, lymphocytes, and neutrophils, and express or exhibit the cell surface antigen CD45 (CD45 + cells).
“Lymphocytes” are cells of the lymphoid system (lymphoid cells), as measured by Sysmex XE-2100, such as mature lymphocytes (T cells, B cells, NK cells) and lymphoblasts. Contains immature or mature lymphocytes.
“Minimally treated bone marrow or umbilical cord blood” refers to bone marrow or umbilical cord blood for stem cells and progenitor cells (eg, anticoagulated or anticoagulated with heparin or citrate). , Meaning that there are no chemical additions (excluding water, crystals, or disinfectants (disinfectants), preservatives, preservatives) and the associated biological properties have not been altered.
“Monocytes” are cells of the myeloid lineage and include mature monocytes and mature monocytes such as monocytes, as measured by Sysmex XE-2100.
“Mononuclear cells (mononuclear cells)” (MNC) are hematopoietic cells and include monocytes and lymphocytes.
“Neutrophils” are myeloid cells, which are measured with Sysmex XE-2100 and include polymorphonuclear cells and mature neutrophils such as rodent nucleus, retromyelocytes, myelocytes, and myeloblasts. Including.
“Platelets” are megakaryocyte cells and include mature platelets as measured by Sysmex XE-2100.
“Progenitor cells” are direct progeny of stem cells and produce a direct cell lineage through a series of cell divisions.
“Red blood cells” are cells of the red blood cell lineage, measured with Sysmex XE-2100, which contain mature RBC but not nucleated red blood cells.
A “stem cell” is a multifunctional cell that has three general properties: (1) it can divide and replicate itself over a long period of time, (2) it is undifferentiated, and (3) various differentiation or special features. It is possible to produce a cell type that is The antigen marker on the cell surface of stem cells is CD34, which is a high concentration of the intracellular enzyme aldehyde dehydrogenase (aldehyde dehydrogenase).
“Stokes' law” is a mathematical expression (V _g = d ² (P1−P2) / 18 μ × G) representing the sedimentation or sedimentation velocity of particles in a viscous liquid (viscous liquid) during acceleration of gravity. Yes, V _g = precipitation rate, d = particle size (particle diameter), P1 = particle density (concentration), P2 = liquid concentration or density, G = gravity acceleration, and μ = liquid viscosity or viscosity. .
"Sysmex XE-2100" is an automated blood and cell analyzer manufactured by Sysmex Corporation in Kobe, Japan. It identifies and quantifies hematopoietic cells by flow cytometry. Radiate and detect three optical signals, forward scattered light, side scattered light, and side fluorescence intensity.
“Total nucleated cells” (TNC) are WBC and nucleated RBC.
“Xenobiotic additives” and “Xenobiotics” are substances that are intentionally added to collected bone marrow or umbilical cord blood for the purpose of causing changes in cell separation process performance characteristics that do not occur in the absence of addition. A chemical substance that is not a natural element.
Examples of xenobiotic additives include:
1. Precipitating agent (eg, hydroxyethyl starch (hydroxyethyl starch), dextran, or gelatin)
2. 2. density gradient carrier (eg Ficoll®, Percoll®), and Affinity molecules for cell surface macromolecular cells (eg, monoclonal antibodies, monoclonal antibodies that bind to paramagnetic particles)

  To date, despite the great clinical and medical potential of stem cells, FDA has not been approved for use in humans for stem cell therapy. The main obstacles to demonstrating the clinical efficacy and safety of stem cell therapy and obtaining regulatory approval are the following of existing devices and methods used to separate stem cells from bone marrow and umbilical cord blood Due to having one or more of the following serious disadvantages. In other words, these devices and methods are open systems (open systems) and there is a risk of microbial contamination, they are time and labor intensive (high rate of input of time and labor), The addition of unfavorable and expensive xenobiotic agents is necessary, the rate of stem cell recovery is very low, on average 40 to 75%, and these are cord blood used for tissue and cell regeneration Do not fit the amount of bone marrow.

Before using stem cells on a large scale to treat important and frequent human diseases such as myocardial infarction, ischemia, diabetes and skin wounds, the following three critical obstacles need to be overcome.
1. Demonstrate the efficiency and safety of stem cell therapy through clinical trials
2. 2. Must be approved by the national health authorities, especially the US Food and Drug Administration (FDA), Cell, Tissue and Gene Therapy Agency (OCTGT); Practical use for quick and easy recovery of viable stem and progenitor cells from minimally processed bone marrow or cord blood without the use of xenobiotic additives to remove excess plasma, red blood cells, and granulocytes Need to develop a practical way.

  In addition to hematopoietic stem cells, it is now known that bone marrow and umbilical cord blood contain other types of stem and progenitor cells with potential therapeutic evaluation for tissue regeneration and wound healing. Human mesenchymal stem cells, formerly called bone marrow stromal cells, can give rise to bone, cartilage, fat, muscle, and connective tissue and are ALDH Br +. Endothelial progenitor cells present in the bone marrow, which are primitive cells derived from hematopoietic stem cells, are those that can enter the bloodstream and go to the site of vascular injury to repair the damage and give rise to new blood vessels. is there.

  Stem cells are identified or identified by various cell surface markers including CD34 + and aldehyde dehydrogenase bright cells (ALDH Br +). CD34 + represents an antigen identified by CD34 and is a stem cell that can be detected using a luminophore-conjugated antibody specific for this particular antigen. Other researchers have identified adult stem cells based on the high level enzyme aldehyde dehydrogenase (ALDH) they represent. One company, Aldagen (Durham, NC), has developed a technology that uses an object or substrate to detect cells that exhibit high levels of ALDH by generating intense green fluorescence. These so-called ALDH bright cells (ALDH Br +) can be detected using flow cytometry. ALDH Br + cells contain different progenitor cells that can give rise to a variety of important cell lines, including neurons and endothelial cells.

  Stem cell viability can be demonstrated by several methods, including the exclusion of vital dyes such as trypan blue or 7-amino-actinomycin D (7-AAD). Stem cell viability is usually assessed or determined by measuring the viability of leukocytes (CD45 + cells) as a surrogate for stem cells in bone marrow or umbilical cord blood using commercially available kits.

  Large amounts of bone marrow or umbilical cord blood are particularly cumbersome because of the presence of a large number of red blood cells (about 25,000 red blood cells for each CD34 + cell in the bone marrow and about each CD34 + cell in cord blood There are 180,000 red blood cells). These excess red blood cells make it difficult to utilize stem and progenitor cells. This is due to the extreme dilution of the stem and progenitor cell concentration at the site of the wound that requires tissue regeneration due to their presence, and these are in vitro cell growth cultures, gene therapy, or cell population affinity. By interfering with other treatments on stem cells, including sex purification. In addition, the presence of such large amounts of red blood cells causes other safety issues such as ABO incompatibility in allogeneic transplants. In some cases, minimizing neutrophils from reduced volume or volume stem cell concentrates can cause these cells to undergo an inflammatory response, and increase stem cell viability or flexibility. It is equally preferred in view of the presence of biologically active enzymes and cytokines in the cytoplasm that can adversely affect or alter their functionality.

  The cell migration rate and final placement after centrifugation for a specified period is a function of its size and density (concentration), and its movement is defined by Stokes' law. Stem cells are less dense than RBC, more dense than platelets, and have a concentration close to that of WBC. Thus, the conventional method of recovering stem cells by density and size involves stratifying the cell means by centrifugation, and then removing the stem cells from the spinal cord or cord blood residue after removal from the centrifuge An attempt has been made to win.

  One manual method of isolating WBC or stem cells from bone marrow or umbilical cord blood is to insert all bone marrow or umbilical cord blood into a centrifuge tube and spin (high speed rotation) at 1500-2000xg for 10-15 minutes at room temperature. It is. This separates bone marrow and umbilical cord blood into an upper plasma layer, a lower red blood cell (RBC), and a thin layer of WBC, stem cells and progenitor cells at the interface between RBC and plasma. After stratification (stratification) is completed, plasma (upper layer) from the RBC to about 1 mm below is aspirated using a disposable plastic transfer pipette (injector). When removing plasma, a layer of WBC / stem and progenitor cells removed by a similar pipette aspiration technique while monitoring for loss of stem and progenitor cells into the RBC or monitoring for RBC contamination in the stem cells It is necessary to pay close attention so as not to disturb. The disadvantages of this manual approach in vitro are the open system and the risk of microbial contamination of the bone marrow unit, the time and labor intensiveness, the amount of red blood cell contamination of the WBC / stem cells and progenitor cell layer. It is very easy to change and the loss (loss) of stem cells is very large and easy to change. It is important to avoid microbial contamination, which is a major concern in this method, because it negatively affects the culture of stem cells or there is a risk of transmission of infection to the body of the recipient of the stem cells (transplanter) It is.

  Other methods for recovering WBC / stem and progenitor cell populations from cord blood and spinal cord are the Compomat G4 device (Fresenius Kabi AG, Friedberg, Germany) and the COBE 2991 blood cell processor (COBE Laboratory, Lakewood, Colorado). ), A cell processing (cell conditioning, cell culture) apparatus is used. An example of this type of processing is presented by Tsubaki et al., “Concentration of Progenitor Cells Collected from Bone Marrow Fluid Using a Continuous Flow Cell Separator System” Apheresis and Dialysis 5 (1), 46-48, 2001. The amount of bone marrow or umbilical cord blood collected for stem cells depends on the intended clinical use. For cell-based therapy in tissue regeneration, typically 50-150 mL of bone marrow or umbilical cord blood is collected. These blood bank devices require larger amounts of bone marrow or umbilical cord blood (200 mL or more) to function properly, and are collected (less than 200 mL) for tissue regeneration medical applications. It becomes completely nonconforming. Furthermore, the final volume (50-100 mL) of these mechanized processing cell products is more than the preferred final volume of 3-20 mL, which provides a suitable concentration of stem and progenitor cells for medical applications. Is also substantially large. These devices require pumps to move blood and bone marrow from one container to another, and these pumps can cause physical (mechanical) damage to cells.

  Other methods for bone marrow and cord blood volume or volume reduction (volume reduction), purification or purification utilize gradient carriers such as Ficoll® and Percoll®, Combines or combines with bone marrow and umbilical cord blood, and itself intervenes or interrupts the cell population during centrifugation, reducing the mixing of the cell population while attempting to recover stem cells. Ficoll® is a neutral, highly branched, high-mass hydrophilic polysaccharide. Ficoll® (eg, density is 1.077) can be used to separate blood or bone marrow into its constituents (red blood cells, white blood cells, etc.). Ficoll® is usually placed at the bottom of the tube, and then bone marrow or umbilical cord blood diluted with saline is slowly layered on top of Ficoll®. After centrifuging, the Ficoll® at the bottom of the tube is replaced with heavier RBC (density 1.09-1.10) and each subsequent layer appears in tandem. From top to bottom, (1) plasma and platelets, (2) mononuclear cells (MNC) (density 1.06-1.07), (3) Ficoll®, and (4) erythrocytes and neutrophils (Density 1.08-1.09) appears in pellet form at the bottom. By this separation, the MNC can be collected in a state where the possibility that the MNC is mixed with the RBC is low. There is some red blood cell collection (red blood cells and neutrophils) in the MNC layer.

  A major disadvantage of processing bone marrow and umbilical cord blood with Ficoll (registered trademark) is that it is a release system in the process of adding Ficoll (registered trademark) to bone marrow and umbilical cord blood, and microorganisms (bacteria) are present in bone marrow and umbilical cord blood. It goes directly into it, which increases the risk of microbial contamination. In order to reduce this risk, this process must be carried out using a biological safety cabinet, which is not always available, is an expensive laboratory device (research equipment) and is acceptable Performance must be constantly maintained and monitored. In addition, Ficoll® is a xenobiotic and must be removed by washing before cells can be safely administered to the human body. This washing process is time consuming and labor intensive and increases the potential for microbial contamination and cell loss. Stem cell recovery is low and very variable, ranging from 20 to 70%. Percoll® is a colloidal silica coated with polyvinylpyrrolidone, another density gradient carrier similar to Ficoll®, with similar disadvantages.

  A technique for isolating bone marrow leukocytes using hydroxyethyl starch (HES) precipitating agent is described in “A technique for isolation of bone marrow cells using, hydroxyethyl starch (HES) sedimentation agent,” Haematologica 76 Suppl 1: 7-9, 1991. HES is clinically used as a plasma expander (plasma augmentation fluid) and is characterized by its molecular weight and its degree of substitution. Typically, the addition of HES to the bone marrow or cord blood volume at a final concentration of 0.5 to 2% by weight causes erythrocyte aggregation, which changes their sedimentation rate based on Stokes' law. To do. However, HES is a xenobiotic and is not associated with adverse events in some patients when administered intravenously. Furthermore, the use of HES adds cost and complexity to cell processing. Dextran polymer formulations and gelatin solutions are used for similar purposes, but have the same drawbacks as HES.

  Finally, stem and progenitor cells are selected using immunological methods including flow cytometric cell sorting (Beckton Dickinson or Coulter) and antibody-coated paramagnetic particle cell sorting such as Miltenyi CliniMacs or Baxter Isolex systems Can be separated from bone marrow or umbilical cord blood. Antibodies with specificity for stem cell antigens such as CD34 have been adopted as part of this separation technique. These methods have the disadvantage of requiring expensive xenobiotic agents, devices and disposable processing sets and are time consuming to implement. While the final product is a purer population of stem cells with little erythrocyte or leukocyte contamination, this level of purity is costly to generate and is not necessary to achieve the intended cell therapy effect. In addition, the addition of antibodies and beads to bone marrow and umbilical cord blood causes unintended harmful biological effects such as irreversible stem cell differentiation into the lineage committed pathway, The product is no longer “minimal processed” during processing.

  In a study by Minguell et al. "Preparative separation of nucleated cells from human bone marrow" Expertia 35: 548-549, 1978, dextran, dextran and Ficoll (registered trademark), and Ficoll (registered trademark) and the results of treatment with the buffy coat method Volume reduction, cell recovery and cell viability are being compared. In this paper, several steps of bone marrow cause loss of cell viability, and all have relatively low cell recovery of 68% or less of lymphoid cells (monocytes, lymphocytes or MNC) In this respect, it illustrates the lack of an ideal preparation or preparation process. In order to achieve an observed red blood cell (RBC) / nucleated cell ratio of 2.7: 1, the centrifugation of these cells initially present in centrifugation without the aid of precipitation, referred to as the buffy coat method The range of lineage cell recovery (low and variable) should be in the range of 36 to 68%.

  A system and method for preparing a composition of stem cells from bone marrow or umbilical cord blood that can selectively recover stem cells at a high rate and a very low rate of RBCs, which is quick, non-labor intensive and consistent Reduce the initial volume or volume of bone marrow or cord blood samples without the need for xenobiotic additives There is a need to provide one that can process various amounts or volumes of bone marrow and allows the user to select in advance the amount or volume of the final product. In addition, it contains a high percentage of stem cells from the original sample, and can keep these stem cells alive, contains a very low percentage of RBC from the original sample, does not contain any xenobiotic additives, and is selected by the user There is a need for a stem cell composition or composition having a final volume that can be made.

According to the present invention, a composition of stem cells derived from human bone marrow or umbilical cord blood and a system and method for preparing the composition are provided. The disclosed invention integrates the following ten characteristics or attributes into a single system.
1. Collect stem cells at a high rate.
2. By removing excess or excess plasma, RBC, platelets, neutrophils, the original volume or amount of bone marrow or cord blood sample is reduced.
3. While no xenobiotic additives are required to perform stem cell recovery, the product or product safety profile is enhanced, the need for cell washing is eliminated, and costs are minimized.
4). Stem cells are maintained in a viable state.
5. Can be completed quickly (less than 90 minutes).
6). Stem cell separation and acquisition steps are performed automatically and are not labor intensive.
7). It is done in a consistent manner and requires minimal operator training and expertise.
8). It can be used inside or adjacent to the operating room for self (self) use, or it can be used inside or adjacent to the cell processing laboratory for the same type of use.
9. Stem cells or progenitor cells can be isolated and sequestered in a sterile, functionally closed system to reduce the risk of microbial contamination of the product.
10. Any type of bone marrow and umbilical cord blood used for tissue regeneration can be processed.

  In one aspect, the present invention is a system including a processing bag set and a processing device. The bag set is sterile and preferably disposable and includes a processing bag, an RBC concentration bag, a stem cell bag, a throttle valve, and a line connected to each bag from the throttle valve. As bone marrow or umbilical cord blood is transferred into the processing bag, the bag set is placed in a processing device that fits into the bucket of the centrifuge. The processor interacts with the bag set and transfers the WBC / stem cell and progenitor cell layers from the bone marrow or umbilical cord blood into the stem cell bag during a single centrifugation run. The processing device includes an optical sensor, a microcontroller, a servo motor, one or more accelerometers, a load cell, and a battery. The microcontroller receives or receives and analyzes and analyzes inputs from the light sensors, accelerometers, and load cells, and commands the servo motor to open and close the throttle valve. With precise or precise valve movement, different cell layers are stratified by density (concentration) and size during centrifugation and move into different bags.

  From another aspect, the present invention also includes a method for isolating and concentrating stem cells from bone marrow or umbilical cord blood. This method includes the following steps. Bone marrow or umbilical cord blood is transferred into the processing bag. The bag set thus loaded is placed in the processing apparatus. The bag set is centrifuged in the processing apparatus at a g force and time sufficient for each cell of bone marrow or cord blood in the processing bag to be stratified based on their density (concentration) and size. The bag set is centrifuged in the processing bag at a lower g force than most of the RBC is separated from the processing bag into the RBC concentration bag. Optionally or optionally, the bag set is centrifuged in the processing apparatus for a g force and time sufficient for each cell in the processing bag to restratify based on cell density and size. The bag set is centrifuged in the processing unit, while the throttle valve is rapidly opened and closed continuously and repeatedly many times, without repeated movement of the WBC / stem cell and progenitor cell layers. , Separate the remaining portion of the RBC into the RBC concentration bag. While the bag set is being centrifuged, the processor tares the empty stem cell bag to zero weight. While the bag set is centrifuged in the processor, the throttling valve is rapidly opened and closed many times in succession (repeated opening and closing), a small amount of remaining RBC, a layer of WBC / stem cells and progenitor cells, and some Plasma is separated and transferred from the processing bag into the stem cell bag.

  In another aspect, the present invention includes a composition of stem and progenitor cells derived from bone marrow or umbilical cord blood prepared by an example of the method of the present invention. The composition of stem and progenitor cells from bone marrow has about 1 ratio of CD34 + cells to about 500 RBCs, and 1 ratio of CD34 + cells to 5,000 RBCs from cord blood. ing.

  The stem cell composition of the present invention provides stem cells and progenitors that are rapidly prepared in a functionally closed bag set without the need for xenobiotic additives to achieve a ratio of red blood cells to stem cells that cannot be achieved previously. It can address the currently unmet clinical need for cellular composition. Bone marrow composition is useful in that it reduces about 98% of RBC and can recover a high proportion of CD34 + cells (about 97%) and ALDH Br + cells (about 92%) without the inclusion of xenobiotic additives. The umbilical cord blood composition is useful in that it can recover a high percentage of CD34 + cells (about 95%) without reducing about 98% of RBC and containing xenobiotic additives.

Explanatory drawing which showed the two-dimensional arrangement | positioning of the Example of the bag set of this invention. The perspective view of the bag set of FIG. (A) is a schematic explanatory view showing one fluid path of the throttle valve of the bag set of FIG. 1, and (B) is a schematic showing a second fluid path of the throttle valve shown in FIG. 3 (A). (C) is a schematic explanatory view showing a third fluid path of the throttle valve shown in FIG. 3 (A). (A) is an illustrative diagram showing one fluid path of a throttle valve different from the embodiment of FIGS. 3 (A) to 3 (C), and (B) is the throttle shown in FIG. 4 (A). The schematic explanatory drawing which showed the 2nd fluid path of the valve. The perspective view of the Example of the processing apparatus of this invention. The perspective view of the processing apparatus of FIG. The perspective view of the processing apparatus of FIG. The perspective view of the processing apparatus of FIG. The side view of the processing apparatus of FIG. The perspective view of the Example of the baseplate of the processing apparatus of this invention by which the component was shown. FIG. 6 is an exploded view of the processing apparatus of FIG. 5. The perspective view of the bag set f of FIG. 1 arrange | positioned in the processing apparatus of FIG. The perspective view of the bag set and processing apparatus of FIG. The perspective view of the bag set and processing apparatus of FIG. The perspective view of the bag set and processing apparatus of FIG. FIG. 13 is a perspective view of the bag set and processing device of FIG. 12 in a centrifuge bucket. FIG. 17 is a partially exploded explanatory view showing a state where the centrifugal bucket and the centrifugal separator of FIG. 16 are mounted. The block diagram which showed the input and output of the microcontroller of the processing apparatus of FIG. The side view of the partial cross section which showed the processing bag of FIG. 1 which showed the cell layer after the hierarchy step. FIG. 6 is a graph showing the change in light transmission as a function of time during the centrifugation step of the method.

(System for recovering stem and progenitor cells from bone marrow or umbilical cord blood)
The system includes a bag set 100 and a processing device 200.
1 and 2 show an embodiment of the bag set 100. FIG. Bag set 100 is effectively closed and is preferably disposable. The bag set 100 includes three or more bags connected or connected to a throttle valve by a line, piping, or the like, and includes an input line, a clamp, a filter, and a sampling site (a place where sampling is performed). Bag set 100 preferably includes three bags: processing bag 102, red blood cell (RBC) concentration bag 104, and stem cell bag 106.

  The treatment bag 102 is made from ethylene vinyl acetate (EVA), but can also be made from polyvinyl chloride (PVC) or other plastics. RBC concentration bags are made of PVC or other plastics. The stem cell bag 106 is made from EVA, although other plastics can be used. These bags 102 and 106 can be made by blow molding. The RBC concentration bag is RF-welded (dielectric heating welding, electromagnetic wave welding), but may be blow-molded.

  The processing bag 102 is an asymmetric three-dimensional bag including an upper edge portion 108, a curved side portion 110, a straight side portion 112, a tapered bottom portion (tapered bottom portion) 113, and a bottom outlet portion 114. The upper edge 108 includes an inlet 115 and two holes 116. Alternatively, the processing bag 102 may have a symmetrical shape in which both side portions thereof are symmetrically directed to the bottom outlet portion 114 in a tapered manner. The overall volume of the processing bag 102 is about 240 mL, but in use it is usually filled with about 50-150 mL of bone marrow or umbilical cord blood. The processing bag 102 is supplied through an input line 118 connected to an inlet 115. The inlet line 118 includes a female luer 120 that connects the bag set 100 to a syringe containing bone marrow and umbilical cord blood to be moved into the processing bag 102. The female luer 120 is connected to a male luer 122 connected to a spike 124 covered by a cap 126 so that the bag set 100 can collect bone marrow and umbilical cord blood that is moved into the processing bag 102. Connected to the bag. Input line 118 includes a clot and bone fragment filter 128 (approximately 200-300 microns mesh). A line or plumbing clamp 130 is located between the clot and bone fragment filter 128 and the female luer 120. Optionally, the input line 118 may include a sampling site 132, a sampling pillow 134, and a sampling site 136, all located below the clot and bone fragment filter 128. Sampling sites 132 and 136 include a needleless (no needle) female luer and an apnea type luer cap, respectively. The bottom outlet 114 guides the output from the processing bag 102 to the throttle valve 138.

  The RBC concentrating bag 104 may be a flat bag, having a top 105, a bottom edge 107, and two side edges 109, and, if necessary, an RBC aliquot (overall) at the end of the process. A butterfly-type (foldable) spike port 140 that is used to remove a portion taken out of). The bottom edge 107 includes an inlet 111 at one corner. The volume or capacity of the RBC concentration bag 104 is 100 mL, but in use, it is usually filled with about 30-80 mL. The RBC concentrating bag 104 is connected at the inlet 111 to a supply line 142 that is connected to the throttle valve 138 by one of the connectors 139 of the throttle valve 138.

  The stem cell bag 106 is a square three-dimensional bag. The stem cell bag 106 includes an upper end portion 117, a bottom end portion 119, a large partition portion 144, and a small partition portion 146, and the partition portions 144 and 146 are connected by two channels (flow paths) 148. The upper end 117 includes an inlet 121 and two spike ports 150 that are used to remove stem cells at the end of the process. The volume or volume of the stem cell bag 106 is about 30 mL, but normally about 25 mL is filled in use, of which about 20 mL enters the large compartment 144 and about 5 mL enters the small compartment 146. The stem cell bag 106 is connected at an inlet 121 to a stem cell bag inlet line 152 connected to a supply line 156 via an F connector 154. The supply line 156 is connected to the throttle valve 138 by one of the connectors 139 of the throttle valve 138. The F connector 154 connects the supply line 156 and the stem cell bag input line 152 to the branch line 158. The branch line 158 is connected to the sampling line 162 and the cryoprotectant supply line 164 via a T connector 160. Sampling line 162 includes line clamp 164 and sampling site 166 and terminates at a sampling pillow 168. The cryoprotectant supply line 164 includes a sampling site 170 and a line clamp 172 and is terminated with a sterile (sterile) filter 174 (preferably about 0.2 μm mesh). Sampling sites 166 and 170 each include a needleless female luer and an apnea luer cap.

  Lines 118, 142, 156, and 162 are piping made of PVC, EVA or other materials. Lines 152 and 158 are piping made of EVA. The cryoprotectant supply line 164 is a coextruded pipe made of PVC on the outside and EVA on the inside. Plastic materials that come into contact with the cryoprotectant enter the stem cell bag 106 and contaminate the final product when leached into the cryoprotectant, so that in the line that comes into contact with the cryoprotectant, an anti-freeze such as EVA is used. It is preferred to use a material that does not contain a plasticizer that leaches into the freezing agent.

  The throttle valve 138 is also a stopper. In one embodiment, throttle valve 138 is a three-way plug and has three connectors 139 that can be connected to three bags, namely processing bag 102, RBC concentration bag 104, and stem cell bag 106. . Other types of throttle valves or plugs, such as a four-way plug with four connectors, can be used as well. The throttle valve 138 includes an outer portion 141 having three connectors 139 and an inner portion 143. The outer part 141 is made of polycarbonate. The inner portion 143 is integrally molded and includes a handle 145 and a barrel (cylindrical portion) 147 made of polyethylene. Barrel 147 is in a closed position (closed position) defined as a position where fluid flow through throttle valve 138 is not permitted, and two open positions (open position) defined as a position where fluid flow through throttle valve 138 is permitted. Move between several positions, including (position). The two open positions are one position where fluid flow is allowed from the processing bag 102 to the RBC concentrating bag 104 through the throttle valve 138 and fluid flow from the processing bag 102 to the stem cell bag 106 through the throttle valve 138. Includes one allowed position. In one embodiment, as shown in FIGS. 3A-3C, the barrel 147 of the throttle valve 138 is shown in three possible fluid paths, namely in FIG. 3A. The desired path from the processing bag 102 to the RBC enrichment bag 104, the desired path from the processing bag 102 to the stem cell bag 106 as shown in FIG. 3B, and the throttle valve 138 are two desired Three openings that allow fluid flow along an unintentional temporary path between the RBC concentrating bag 104 and the stem cell bag 106 as shown in FIG. (Opening position) is comprised. Such a temporary fluid flow is not preferred because some of the RBCs may additionally flow into the stem cell bag 106 and reduce the purity of the stem cell composition obtained. In order to address this problem, in another embodiment, as shown in FIGS. 4A to 4B, the barrel 147 of the throttle valve 138 includes only two openings (open positions) as an alternative. Configured as shown in FIG. 4 (B), only two possible fluid paths, ie, the desired path from the processing bag 102 to the RBC concentrating bag 104 as shown in FIG. 4 (A). Such a configuration is configured to allow fluid flow along a desired path from the processing bag 102 to the stem cell bag 106. This configuration can eliminate the possibility of temporary fluid flow between the RBC concentration bag 104 and the stem cell bag 106. The throttle valve 138 has a structure capable of withstanding the high pressure generated during the centrifugal separation, and satisfies, for example, a rating of up to about 500 psi. A supply line 164 leads from the throttle valve 138 to the RBC concentration bag 104. The supply line 164 leads from the throttle valve 138 to the F connector 154, and this connector leads to the stem cell bag 106 via the stem cell bag input line 152. Lines 142, 152, and 156 are each heat sealed and separated from bag set 100.

  If another component needs to be separated from the bone marrow or umbilical cord blood, another bag may be included in the bag set 100. If separate bags are included, the throttle valve 138 has an additional connector corresponding to each bag, and the bag set includes a separate supply line for connecting the throttle valve to each bag. For example, if it is desired to separate the last part of the RBC in the processing bag 102 from the RBC moved into the RBC concentration bag 104, four bag sets are used. The fourth bag is connected to the throttle valve 138 by a separate supply line. In that case, throttle valve 138 is a four-way plug with four connectors and includes a barrel that allows fluid flow from process bag 102 to each of the other four bags.

  5 to 15 show an embodiment of the processing apparatus 200. The processing apparatus 200 has a substantially cylindrical shape and includes an upper part 202, a bottom part 204, a front part 206, a rear part 208, a side part 210, and a side part 212. Front portion 206 includes a front door 214 and front walls 216 and 217. The processing apparatus 200 includes a main body 218, a stem cell bag chamber 220, a bottom plate 222, a support bracket 224, and a processing bag hanger 226. The body 218 and stem cell bag chamber 220 are preferably urethane molded, although other thermoplastic materials can be used. The bottom plate 222 is preferably made from stainless steel. Support bracket 224 is preferably made of aluminum. The treatment bag hanger 226 is preferably made from stainless steel. The processing device 200 is of a size that fits inside a centrifuge bucket (bucket) having a circular or elliptical cross section and a minimum volume of 1L. FIG. 16 shows the processing apparatus 200 including the bag set 100 inside the centrifugal bucket 228. FIG. 17 showed how to load the centrifuge bucket 228 inside a centrifuge with five other centrifuge buckets already in place.

  The main body 218 of the processing apparatus 200 includes a main chamber 230 that is elongated and elliptical in size to receive the processing bag 102. The main chamber 230 is open at the top, and can be accessed (utilized) by opening the front door 214 attached to the front portion 206 of the processing apparatus 200 by the hinge 232. The main chamber 230 has side walls 234 and 236 and a rear wall 238 for fitting to and supporting the processing bag 102 and a channel (groove) 240 sized to receive the tapered bottom 113 of the processing bag 102. It becomes a taper shape, that is, a tapered shape. The side walls 234 and 236 place the processing bag 102 gently at the center and more firmly at the tapered bottom 113. Less tolerance near the tapered bottom 113 of the processing bag 102 to support the processing bag 102 and its contents during the high pressure of the centrifuge and to minimize disturbance of the bag contents. Is preferred. The front door 214 includes a recessed inner recess 242 that provides a continuous surface corresponding to the side walls 234 and 236 of the main chamber 230 and the channel 240 on the inner surface thereof, so that the processing bag 102 is in the closed state when the front door 214 is closed. Fit and support this.

  The recess 244 is disposed on the front portion 206 of the processing apparatus 200 below the front walls 216 and 217 and has a shape that supports the connector 139 of the throttle valve 138. A valve actuator cuff (valve actuator fold) 246 is provided inside the recess 244 and is sized and shaped to receive the handle 145 of the throttle valve 138. The valve actuator cuff 246 is attached to the shaft of the servo motor 248 by a screw 250. Corresponding recesses 252 are provided on the inside of the front door 214 with dimensions and shapes to receive the protrusions and connectors of the throttle valve 138.

  One or more photosensors 254 are gathered through the front wall 217 of the main chamber 230, and their openings are provided in the side wall 236, and the same number of LEDs 256 are arranged on the opposite side wall 234. LED 256 includes red LED light, but other types of LEDs may be used. One light sensor 254 and one LED 256 are preferably located approximately 2 cm above channel 240 and the volume or amount of processing bag 102 between the level of light sensor 254 and LED 256 and the level of throttle valve 138 is approximately Although it becomes 2 mL, you may use another distance and corresponding volume thru | or quantity. If another (additional) light sensor and LED need to be included, these are placed above or below the light sensor 254 and LED 256 depending on the desired volume or amount of RBC to be separated.

  The side 212 of the processing apparatus 200 includes a notch 258, a storage cavity 260, a channel (groove) 262, an RBC concentration bag recess 264, a support shelf 266, and a hook 268. Notch 258 is sized and shaped to receive spike port 140 of RBC concentration bag 104. RBC concentrating bag recess 264 descends from notch 258 to support shelf 266 and is sized and shaped to receive RBC concentrating bag 104. The storage cavity 260 is located below the notch 258 and is connected to it behind the RBC concentrating bag recess 264. The channel 262 extends along the inner edge of the RBC concentrating bag recess 264 and is adjacent to the front door 214, the upper portion 202 and the rear portion 208. The support shelf 266 is a horizontal shelf and forms the floor surface of the recess 264 for the RBC concentration bag.

  Stem cell bag chamber 220 is a horizontal, hollow, square section and has a large side 270 located below hinge 232 of front door 214. The stem cell bag chamber 268 is located below the bottom plate 232 and is attached to the load cell 272. The stem cell bag chamber 220 is accessed through a hinged door 274 that opens downward, and this door is fitted in place via a latch 276 at the upper end located inside the stem cell bag chamber 220. Hinged door 274 includes a recess 278 sized and shaped to accommodate spike port 150 and inlet 121 of stem cell bag 106 therein. The hinged door 274 includes a slot 280 and a channel 282 sized to receive the stem cell bag inlet line 152 on the outside thereof.

  The bottom channel 284 extends in parallel at the top of the bottom plate 222 and crosses the side 210 and rear 208 from the lower front 206 of the hinge 232 back to the side 212. The bottom channel 284 is sized and shaped to accommodate the line 142.

  The processing apparatus 200 includes a processing bag hanger 226 that extends to the top of the top 202 at the side 210. The processing bag hanger 226 engages with the hole 116 on the processing bag 102 and has a tab 227 for holding the processing bag 102 in a predetermined position inside the processing apparatus 200. The LED window 229 is disposed on the upper portion 202 at the side portion 210.

  The support bracket 224 is a U-shaped bracket that is attached to the bottom plate 222 with screws 225 and oriented parallel to the side portions 210 and 212 of the processing apparatus 200.

  As shown in FIG. 10, the following components are preferably attached to the bottom plate 222: a printed circuit board 286, a servo motor 248, a load cell 272, and an interface printed circuit board 288. The printed circuit board 286 includes a microcontroller 290 with programmable read-only memory. Microcontroller 290 requires temperature compensation due to heat generation during centrifugation. The load cell 272 is a temperature compensated strain gauge load cell. The servo motor 248 is a reduction gear type motor. One or more accelerometers 292 are mounted on the printed circuit board 286, and when two or more accelerometers 292 are used, one is mounted on the other. Preferably, the accelerometer 292 includes a low g accelerometer 291 that measures approximately 0-200 × g and a high g accelerometer 293 that measures approximately 1000-1700 × g. The printed circuit board 286 includes a status LED 294 that is visible from the LED window 229. One or more status LEDs 294 display the state of charge of the battery 296, the steps currently being performed during processing, and other information in the sequence. The interface printed board 288 is provided for connection to an external charger and for communication connection with a personal computer. The bottom plate 222 is attached to the main body 218 with screws 298.

  FIG. 18 is a block diagram showing the inputs and outputs of the microcontroller 290. Photosensor 254, battery voltage 302, load cell 272, and accelerometers 291 and 293 generate analog signals that are converted to digital signals and received by microcontroller 290. Microcontroller 290 records, stores, and analyzes these inputs at predetermined time intervals. The microcontroller controls servo motor 248, LED 256 for optical center 254, and status LED 294. When the bag set 100 including the throttle valve 138 is correctly installed in the processing apparatus 200, the servo motor 248 is connected to the valve actuator cuff 246 via its drive shaft, and in response to a signal from the microcontroller 290, the servo motor 248 Motor 248 moves throttle valve 138 to a different position to open and close the fluid flow to each bag of bag set 100.

  The battery 296 is disposed in the battery cavity 300 on the top 202 at the side 210 of the processing apparatus 200. Battery 296 is a rechargeable nickel metal hydride battery that includes three cells that are approximately 3.8-4 volts in total, and this voltage is adjusted to provide a constant voltage of 5 volts. Battery 296 powers all electrical circuits within processing unit 200 including microcontroller 290, optical sensor 254, accelerometers 291 and 293, load cell 272, optical sensor LED 256, status LED 294, servo motor 248, and communication with a personal computer. Supply.

  The processing unit 200 is disposed in another docking station (connection rack) including a charger and is connected to a personal computer. The data from the microcontroller 290 is transferred to the personal computer through the docking station via the interface printed circuit board 288, and the command (command) from the personal computer is received.

  As shown in FIGS. 12 to 15, the bag set 100 is inserted into the processing apparatus 200 as follows. The stem cell bag 106 is disposed in the stem cell bag chamber 220 so that the bottom end portion 119 is first fitted and the small partition 146 is folded and disposed in the large side portion 270. The inlet 121 and spike port 150 are disposed inside the recess 278 of the hinged door 274. Stem cell bag input line 152 is disposed in slot 280 of hinged door 274. The hinged door 274 is then closed and fastened by a latch 276. A stem cell bag input line 152 is disposed in the channel 282. A handle 145 of the throttle valve 138 is disposed within the valve actuator cuff 246. The processing unit 102 is directed into the main chamber 230 such that the front door 214 closes the straight side 112 of the processing bag 102 from above. The F connector 154 is disposed inside the recess 264 for the RBC concentration bag. The line clamp 165 is closed and placed inside the channel 262 along the sampling line 162. A cryoprotectant supply line 165, a line clamp 172 (closed), and a sampling site 170 are disposed in the storage cavity 260 and a sterile filter 174 is disposed in the right side wall container of the storage cavity 260. . Sampling site 166 is secured at hook 268. The sampling pillow 168 is housed in a recess immediately below the filter container on the right side of the recess 264 for the RBC concentration bag. The supply line 142 is then placed in the bottom channel 284 and the RBC concentration bag 104 is placed in the RBC concentration bag recess 264. Input line 118 and sampling site 136 are folded down and directed to processing bag hanger 226 on side 210. The hole 116 of the processing bag 102 is attached to the tab 227 of the hanger 226. The front door 214 is closed. With the bottom edge 107 on the support shelf 266, the spike port 140 is fitted into the notch 258 and the RBC concentration bag 104 is fitted over the storage cavity 260. The branch line 158 is arranged vertically along the left side of the storage cavity 260.

(Method of recovering stem cell and progenitor cell composition from bone marrow or umbilical cord blood)
This method involves centrifugation, where bone marrow or umbilical cord blood is stratified and separated by cell density and size (dimensions). The bone marrow or umbilical cord blood to be treated contains plasma, RBC, WBC, and stem cells as indicated by the presence of CD34 + and ALDH Br + stem cell markers. The resulting stem cell product is a composition of stem cells and progenitor cells and contains a small amount of WBC and significantly reduced RBC and plasma. This method uses the system described above to process various functionally closed volumes or amounts of bone marrow or umbilical cord blood and produce a volume or amount of end product preselected by the user. Can be implemented in the system. This method significantly reduces bone marrow volume by removing excess or excess RBC and plasma, and neutrophils and platelets if clinically preferred, thereby concentrating the stem cells within the final stem cell composition. This method does not require a xenobiotic additive and recovers stem cells and progenitor cells from bone marrow or umbilical cord blood at a high rate.

  As used herein, the term “g force” refers to relative centrifugal force, and all references to a particular g force are determined at the location of the optical sensor 254 within the processing device 200. Here, the specific or specific g force and time period referred to in the present specification are those exemplified in one embodiment of the present invention, and the g force and time period other than those mentioned are the same. And is encompassed in other embodiments of the method.

1. Bone marrow or umbilical cord blood is transferred into the processing bag.
Bone marrow or umbilical cord blood is collected in a collection container such as a bag, syringe, or other container, stored with an anticoagulant or anticoagulant such as heparin or CPD, and processing bag 102. Moved into. This movement is accomplished by inserting the spike 124 into the collection bag and attaching a female luer to the syringe or aseptically coupling the collection bag tubing to the processing bag using an aseptic connection device. The line clamp 130 is opened. The bone marrow or umbilical cord blood then moves from the collection container to the processing bag 102 by gravity flow via the input line 118. The bone marrow or umbilical cord blood passes through the clot and bone fragment filter 128, which removes the clot (thrombus, fat globule, bone fragment, etc.) from the bone marrow or umbilical cord blood before reaching the processing bag 102. After the bone marrow or umbilical cord blood has moved, the input line 118 is heat sealed at the sampling site 132 described above, and the collection container, clot and bone fragment filter 128 and the remainder of the line 118 are removed.

  A volume of bone marrow or cord blood of about 25-200 mL, preferably about 50-170 mL, is placed in the processing bag 102. If the initial volume of bone marrow or cord blood collected is about 100 mL and the hematocrit value of bone marrow or cord blood is about 30%, the volume of RBC is about 30 mL, and the volume of WBC and stem and progenitor cells is About 1 mL, the rest is plasma.

  Surprisingly, as described above, no precipitant or other xenobiotic additive is required to perform this method to recover stem and progenitor cells from bone marrow or umbilical cord blood. If a precipitating agent such as HES is required, it is added to the bone marrow in the processing bag 102 as appropriate.

  If necessary, a sample of bone marrow or cord blood to be processed is taken from the processing bag 102 after heat sealing at the sampling site 132 described above. Sampling pillow 134 is pressed and released to take the sample into the pillow. The input line 118 is then heat sealed at the sampling site 136 and the sampling pillow 134 is removed along with the sampling site 132. The bone marrow or umbilical cord blood in the sampling pillow 134 is then accessed via the sampling site 132 for further analysis such as cell count, cell activity, microbial contamination, and HLA analysis.

  If no sample is removed from the processing bag 102, the input line 118 is heat sealed at the sampling site 136 described above and all components at the top of the sampling site 136 are removed.

2. The loaded bag set is placed in the processing apparatus.
A bag set 100 including each bag, each line, and a throttle valve is placed in the processing apparatus 200 as described above.
As shown in FIGS. 16 and 17, the processing device 200, which may be configured to cool, is placed with the bag set 100 in a centrifuge bucket 228 of the centrifuge. The centrifuge is balanced by another processing device 200 or a suitable counterweight.

3. The bag set is centrifuged in the processing unit with sufficient g force and sufficient time to allow each cell of bone marrow or umbilical cord blood to be stratified into layers in the processing bag based on their density and size. The

  The bag set 100 in the processing apparatus 200 is set in advance enough for the g force set in advance and the bone marrow or umbilical cord blood cell population in the processing bag 102 to be stratified into layers with cell density and size. It is centrifuged for a certain time. For example, centrifugation is performed at about 1400 × g for only about 20 to 40 minutes. During this stratification step, the throttle valve 138 is in its closed position so that there is no fluid flow through the throttle valve 138. The accelerometer 293 measures or measures g force during centrifugation.

  Centrifugation is preferably continued until each cell is stratified into three layers. Referring to FIG. 19, this figure shows the processing bag 102 after this tiering step has been completed. The lower most dense layer 400 is mainly RBC, the middle density middle layer 402 is WBC / stem and progenitor cells, and a few platelets, and the lowest density layer 404 is mainly plasma. There are a few platelets. The proportion of platelets present with stem and progenitor cells increases with increasing centrifugation time. With 1400 g force and 20-40 centrifugation time, more than 70% of the stem cells are sufficient to migrate to the WBC / stem and progenitor layer, but within this layer at least about 80% to 90% of the stem cells Is preferably transferred.

4). The bag set 100 is centrifuged in the processing apparatus with a small g force to separate and move most of the RBC from the processing bag into the RBC concentration bag.
After each cell is stratified in the processing bag 102 in the previous step, with a predetermined small g force for a time sufficient to separate and move most of the RBC from the processing bag 102 into the RBC concentration bag 104. The bag set 100 is centrifuged in the processing apparatus 200. This g force flows from the processing bag 102 through the throttle valve 138 through the supply line 142 and into the RBC concentrating bag 104 with the RBC layer under control and with minimal risk of cell damage. Less than the g force used in step 3. For example, centrifugation is performed at about 80 × g for about 10 minutes. This centrifugation step is performed continuously with the previous stratification step centrifugation by automatically reducing the previous step g force to a lower preset g force, or the previous centrifugation step It is done after the completion and stop.

  The accelerometer 291 measures or measures g force during centrifugation. As soon as the microcontroller 290 receives the input from the accelerometer 291 with the preset g force stabilized, for example, for about 30 seconds, the microcontroller 290 commands the servo motor 248 to cause the valve actuator cuff 246 to The throttle valve 138 is opened to a position where a fluid path from the processing bag 102 to the RBC concentration bag 104 through the supply line 142 is formed. While the throttle valve 138 is open, much of the lower layer in the processing bag 102 consisting of concentrated (crushed) RBC flows into the RBC concentration bag.

  At the same time as the throttle valve 138 opens, the microcontroller 290 commands the optical sensor 254 to read the light transmission from the LED 256 through the bone marrow or umbilical cord blood in the processing bag 102 as a fluid flow through the LED 256. Microcontroller 290 continuously records and analyzes the light transmission received from optical sensor 254 as an input as a function of time. FIG. 20 shows the relative light transmission as a function of time during centrifugation as each cell layer passes through the LED 256 and the light sensor 254, after the throttle valve 138 is opened. Starting from step 4 and continuing through step 8, the optional step 5 is omitted. This line is a substantially S-shaped curve with a light transmittance of little or zero and the line is a substantially horizontal region A, a region B where the amount of light transmittance is significantly increased and the slope of the line is steep, And a region C in which the light transmittance is high and the line is substantially horizontal.

  During this step, the RBC layer flows past the optical sensor 254. Since the RBC layer has a high cell concentration, it prevents light from being transmitted to the optical sensor 254. Therefore, during this step, as shown in region A of FIG. 20, the optical sensor 254 detects little or no light from the LED 256.

  After the RBC layer has flowed over the light sensor 254, the interface between the RBC layer and the WBC / stem cell and progenitor cell layer flows by the light sensor 254. Since this layer has a lower cell concentration than the RBC layer, the optical sensor 254 detects more light from the LED 256. When the microcontroller 290 communicates from the optical sensor 254 that a predetermined amount of light transmission has been reached, meaning the onset of passage through the WBC / stem cell and progenitor cell composition layer, the microcontroller 290 The servo actuator 248 is commanded to close the throttle valve 138 by the valve actuator cuff 246, thereby stopping fluid flow through the throttle valve 138. This is indicated by point D in FIG. Thus, the throttle valve 138 is closed as soon as the bottom layer of the WBC / stem cell and progenitor cell layer begins to reach the photosensor 256.

  After this separation step, approximately 2 mL of concentrated RBC remains in the processing bag 102 and most of the RBC volume has moved into the RBC concentration bag 104. For example, the original 100 mL volume of harvested bone marrow with a hematocrit value of 30% is about 28% RBC (about 93%) in the RBC concentration bag 104 and about 2 mL (7%) volume. The RBC remains in the processing bag 102.

5. The bag set is optionally centrifuged in the processing apparatus with sufficient g force and elapsed time to stratify the cells in the processing bag based on cell density and size.
This is an optional or optional step. If this step is not performed, the method proceeds to the next step.

  As a result of the previous separation step and the Coriolis force generated when the cell solution is removed through the throttle valve 138, the rest of the RBC layer, all of the WBC / stem and progenitor cell layers, and all of the plasma layer are removed. Each cell layer in the processing bag 102 to be included becomes a state in which the boundary is not clear and becomes more mixed at the interface. In this optional step, the bag set 100 in the processing apparatus 200 is pre-determined with a predetermined g force that allows the remaining cells to stratify into more distinct layers depending on cell density and size. Centrifuge over time. For example, this centrifugation is performed at about 1400 × g for only about 5 to 15 minutes. This centrifuge step may be continuous with the previous centrifuge step by programming the centrifuge to automatically increase the g force, or it may begin after the previous centrifuge step is complete and stopped. May be performed.

  During this centrifugation step, the throttle valve 138 is placed in a closed position, thereby preventing fluid flow through the throttle valve 138. Centrifugation is preferably where each cell is re-stratified into three layers: the lower remaining RBC layer, the middle WBC / stem and progenitor layer, and the upper plasma phase and at the interface between each layer. Continue until mixing is reduced. The centrifugation force and time are sufficient if it results in more than 70% of the stem cells being placed in the WBC / stem and progenitor layer, but at least about 80% to 90% of the stem cells are in this layer. It is more preferable if the result arranged inside can be obtained.

6). While the bag set is centrifuged in the processor, the throttling valve repeats a number of successive open and closes quickly, allowing most of the remaining RBCs to be transferred without moving the WBC / stem and progenitor cell layers. Is more accurately moved from the processing bag into the RBC concentration bag.
After the previous optional re-layering step, or after the previous separation step if this re-layering step is not performed, the bag set 100 in the processor 200 completes this step and steps 7 and 8 Centrifugation at a predetermined g force sufficient to do and a predetermined time interval. For example, the centrifugation is performed for about 5 to 15 minutes at about 80 × g, the same g force used in step 4.

  In this step, most of the remaining RBC in the processing bag 102 is correctly moved into the RBC concentration bag 104. The purpose of the low g force is to allow the RBC to flow from the processing bag 102 into the RBC concentration bag 104 in a controlled manner that minimizes the risk of cell damage. This centrifugation step can be made continuous with the previous step by programming the centrifugation to automatically reduce the g force if the previous step is a re-stratification, or the previous step Can be continued with the same g force, or can be started and executed after the previous centrifugation step is completed and stopped.

  The accelerometer 291 measures g force during centrifugation. As soon as the microcontroller 290 receives input from the accelerometer 291 that the predetermined g force has stabilized, for example, for about 30 seconds, the microcontroller 290 issues a command to the servo motor 248 and the valve actuator cuff 246 is throttled. The valve 138 is in the open position to create a fluid path from the processing bag 102 to the RBC concentrating bag 104 via the supply line 142 and then closed, the opening and closing for a predetermined period of time with a predetermined number of cycles. The amount of discontinuous time sufficient for the plasma layer to reach the level of the photosensor 254, repeated from the processing bag 102 to the RBC concentrating bag 104 by repeating a number of times continuously at predetermined intervals. Is repeated. For example, the throttle valve 138 is set to open for a period of about 0.1 to 0.2 seconds and closed for about 10 seconds, and this cycle is repeated about 10 to about 30 times, but other periods, intervals, and Repeated combinations may be used. The remaining portion of the RBC in the processing bag 102 flows into the RBC concentrating bag 104 as the throttle valve 138 opens and closes. By this accurate red blood cell removal step, about 1 to 1.5 mL of 2 mL of RBC amount remaining in the processing bag 102 after the previous step flows from the processing bag 102 to the RBC concentration bag 104, The amount of RBC remaining in is further reduced. This is because a small amount of RBC moves each time the throttle valve 138 is quickly opened and closed. The RBC is not mixed with the stem cells and progenitor cells, and the stem cells and progenitor cells are caused by Coriolis force. Is not sucked into the transferred red blood cells. This approach allows a large portion of the remaining RBC to be moved into the RBC concentration bag 104 as needed.

  This last fine metering action is directed by the microcontroller 290 that has received a measurement at the light sensor 254 of the light transmission from the LED 256 as each cell layer in the processing bag 102 flows by the LED 256. It is. The microcontroller 290 continuously records and analyzes the light transmission as a function of time and compares the previous value with the current value. During this step, the WBC / stem cell and progenitor cell layers flow past the optical sensor 254. This layer has a higher cell density at the interface with the RBC layer and a lower cell density at the interface with the plasma phase, so that the WBC / stem cell and progenitor cell layers pass through the photosensor 254 to the plasma. The light transmittance gradually increases as the interface with the phase approaches. Thus, during this step, the light sensor 254 detects a uniform increase in the light transmission from the LED 256. This is indicated by the slope of the line in region B of FIG.

  After the WBC / stem cell and progenitor cell layer passes through the photosensor 254, the interface between the WBC / stem cell and progenitor cell layer and the plasma phase flows by the photosensor 254. Since the plasma phase has fewer cells than the WBC / stem cell and progenitor cell layer, more light is detected by the light sensor 254 from the LED 256. The microcontroller 290 indicates from the optical sensor 254 that the rate of change (change rate) in the light transmittance is a predetermined decrease based on the fact that the optical sensor 254 has detected a predetermined change rate value. Is received, the servo actuator 248 instructs the valve actuator cuff 246 to close the throttle valve 138, thereby terminating the movement of the RBC through the throttle valve 138. This is indicated by point E in FIG. 20 and is a transition from the steep slope of the line in region B to a substantially horizontal line in region C, where the composition layer of WBC / stem cells and progenitor cells is of photosensor 254. This is the time when the plasma phase begins to pass through the light sensor 254 after passing through the lower side. Therefore, as soon as the lowest part of the plasma phase starts to reach the optical sensor 254, the throttle valve 138 is closed.

  After this separation step, an approximately 0.5 to 1.0 mL amount of RBC remains in the processing bag 102, and another 1 to 1.5 mL amount of RBC moves into the RBC concentration bag 104.

7). While the bag set is being centrifuged, the processor tares empty stem cell bags to zero (zero dead weight of empty stem cell bags).
After the throttle valve 138 is closed in the previous step, centrifugation continues with the g force and time set in step 6. When the accelerometer 291 confirms the set g force, the load cell 272 and the microcontroller 290 cooperate at this point to tare the empty stem cell bag 106 to zero.

8). While the bag set is centrifuged in the processor, the throttling valve repeats a number of successive opening and closings rapidly, resulting in a small amount of remaining RBC, a layer of WBC / stem and progenitor cells, and a small amount Plasma is separated and transferred from the treatment bag into the stem cell bag.
Centrifugation of the bag set 100 in the processing bag 102 is continued with the g force and time set in step 6. If the microcontroller 290 receives as an input from the load cell 272 that the weight of the empty stem cell bag 106 has been tared to zero, it instructs the servo motor 248 to perform the next instruction. That is, the valve actuator cuff 246 opens the throttle valve 138 to a position where a fluid path from the processing bag 102 through the supply line 156 and the stem cell bag input line 152 to the stem cell bag 106 is generated and then closed. Opening and closing is repeated continuously for a predetermined period of time with a predetermined number of cycles, sufficient to fill the stem cell bag until it reaches its predetermined weight The access from the processing bag 102 to the stem cell bag 106 is repeated for an individual amount of time. For example, throttle valve 138 is set to open for a period of about 0.1 to 0.2 seconds, close for about 10 seconds, and repeat this cycle many times. When the throttle valve 138 opens or closes, some of the plasma in the processing bag 102 flows into the stem cell bag 106. The purpose of the low g force in this step is to direct these cells from the treatment bag 102 into the stem cell bag 106 in a controlled manner that minimizes the risk of cell damage that occurs at high g forces. .

  During this separation step, the load cell 272 weighs and weighs the stem cell bag 106 and the microcontroller 290 receives this input from the load cell 272. The desired weight of the WBC / stem cell and progenitor cell layer is preset, which is set from about 3 grams to about 30 grams. For example, if the desired amount of final stem cell product in the stem cell bag 106 is 10 mL, a weight of 10 grams is used. If the microcontroller 290 receives an input from the load cell 272 that the stem cell bag 106 has reached a preset weight, the microcontroller 290 causes the valve actuator cuff 246 to close the throttle valve 138 and the throttle valve 138 to the servo motor 248. Instruct the fluid flow through to stop.

  After the throttle valve 138 is closed, centrifugation continues with the same g force until the predetermined time set in step 6 is reached.

9. When centrifugation is complete, the bag set is removed from the processing apparatus.
When the predetermined centrifugation period ends, the centrifugation stops and the processing device 200 is removed from the centrifuge. The bag set 100, including all bags, throttle valves 138, and each line, is removed from the processing unit 200.

  Stem cell bag 106 contains the remaining RBC, WBC / stem cells and progenitor cells, and some plasma. The contents of the stem cell bag 106 are referred to herein as “stem cell composition (composition)” or “stem cell composition (composition)”. The RBC concentration bag 104 contains RBC. The processing bag 102 contains the remainder of the plasma that is not included in the stem cell bag 106.

  If necessary, samples from the RBC enrichment bag 104 and the stem cell bag 106 are removed. Sampling line 162, sampling pillow 168, sampling site 166, and line clamp 164 are used to sample stem cell bag 106.

  If the stem cell composition is used immediately, eg, for self (self) use, the stem cell bag 106 is then separated from the rest of the bag set 100 using a Sebra sealer (Sebra Corp., Tucson, Arizona). Is done.

  When the stem cell composition is frozen, an cryoprotectant solution, such as DMSO solution, is added to the stem cell bag 106 via the sterile filter 174 and the cryoprotectant supply line 165.

Example of using bone marrow and umbilical cord blood as raw material for stem cells Collected 5 units of human plasma and 6 units of umbilical cord blood within 1 to 3 days from an authorized or certified vendor (seller, vendor) Or collected. The following table shows data obtained from bone marrow and umbilical cord blood processed by the method of the present invention. Bone marrow was anticoagulated with heparin and umbilical cord blood with CPD. A desktop centrifuge of dimensions including the processing unit provides a centrifugal force field. In this experiment, no extraneous foreign matter additive for precipitation assistance was used. Table 1 below provides a summary of the steps of the present invention utilized for bone marrow and cord blood experiments.

  Bone marrow and umbilical cord blood samples were removed before treatment, and a final product (stem cell composition) sample was removed after treatment. Cell counts for RBC, WBC, platelets, neutrophils, lymphocytes, monocytes were performed using Sysmex XE-2100. For samples before and after treatment, CD34 + cells and CD45 + cells (all using a commercially available Stem-Count kit (Beckman Coulter, Inc., Miami, Florida) according to the manufacturer's instructions) Of white blood cells and most expressed CD34 + cells). The kit can perform simultaneous identification and counting of CD34 + and binary CD45 +, cell density rate of CD34 +, and absolute cell number in a biological sample by flow cytometry. Viability of CD45 + cells is determined based on dye exclusion of vital stain 7-AAD. Cell population measurements are obtained by flow cytometry with appropriate control and analysis on an appropriately configured Beckman Coulter FC-500 flow cytometer (Beckman Coulter, Inc., Miami, Florida). ALDH Br + cells are identified and counted using a commercially available Aldecount kit (Aldagen, Durham, NC) and a Beckman Coulter FC-500 flow cytometer before and after processing for samples from bone marrow units 1, 2 and 3. And performed according to the instruction manual. The reagent provided in the kit stained stem cells in bright fluorescent green. These cells were shown to be Aldagen clear cells (ALDH Br +).

A. Bone marrow data Table 2 shows bone marrow volume and cell counts in the five trials prior to treatment. The total number of RBCs, platelets, TNCs, neutrophils, lymphocytes, monocytes, MNCs, CD34 + cells, and ALDH Br + cells in each unit are shown. The ND item means that the data is “undecided”.

  Table 3 shows the volume and cell counts for the stem cell composition obtained in 5 tests from the 5 units referenced in Table 2. The total number of RBCs, platelets, TNCs, neutrophils, lymphocytes, monocytes, MNCs, CD34 + cells, and ALDH Br + cells in each unit are shown.

Table 4 shows the recovery of each cell type based on the results of the five experiments shown in Tables 2 and 3. The recovery rate is calculated by dividing the cell count within the stem cell composition (Table 3) by the corresponding value in the bone marrow treated sample (Table 2) and multiplying this quotient by 100. For example, in Experiment 1, the recovery rate of RBC is calculated by dividing “6,700 × 10 ⁶ ” by “329,000 × 10 ⁶ ” and multiplying the quotient by 100, resulting in a recovery rate of 2.0% ( Table 4) is obtained. The reduction rate is easily calculated by subtracting the recovery rate from 100%. For example, in Experiment 1, the red blood cell reduction rate is 100% −2.0% = 98%.

  The data in Table 4 surprisingly achieved a reduction in RBC (2.1% recovery) at an average of 97.9%, while CD34 + cells and ALDH Br + cells (average recoveries of 98.9%, 92 respectively) 0.0%) demonstrates that more than 90% of stem cells can be recovered. Even more surprising, Table 4 shows that over 90% of the stem cells are recovered, while neutrophils are recovered only 59% on average. This level of selective separation of bone marrow is expected to require the use of a xenobiotic precipitant.

  Tables 5 and 6 show the ratio of RBCs to nucleated cell types in bone marrow and stem cell composition before treatment based on the data in Tables 2 and 3. The ratio of these cells to each other is a unique and unusual stem cell composition that cannot be found in nature or cannot be made by any other cell processing system. Creating a stem cell composition with very few RBCs is an important advance for stem cell therapy, without the negative effects associated with excess red blood cells in stem cell transfusion, and further purification with immunoaffinity or flow cytometry methods Safe cell products can be made for cell products that contribute to the process. In particular, the data in Tables 5 and 6 show that the average ratio of RBC to CD34 + in the bone marrow before treatment is initially 25,642: 1 and after treatment this ratio in stem cell composition is 539: 1. This dramatic reduction in the ratio of RBC to stem cell composition reflects the success of the method for selectively removing RBC without losing CD34 + cells. A similar reduction is observed in the ratio of red blood cells to ALDH Br + cells.

  The unity of these cell populations is unique to the viable cells contained in them. Table 7 contains an analysis of the CD45 + cell viability (viability) results in bone marrow as measured using a commercially available kit (Stem-Kit, Beckman Coulter, Fullerton, Calif.). There was no significant change in the viability of CD45 + cells after treatment, which illustrates the biocompatibility of the process.

B. Umbilical cord blood data Table 8 shows the volume and cell count of six cord blood units before treatment. The total number of RBC, platelets, TNC, neutrophils, lymphocytes, monocytes, MNC, and CD34 + cells in each unit are shown.

  Table 9 shows volume and cell counts for stem cell composition obtained in 6 experiments from the corresponding units of cord blood referenced in Table 8. The total number of RBC, platelets, TNC, neutrophils, lymphocytes, monocytes, MNC, and CD34 + cells in each unit after treatment is shown.

  Table 10 shows the recovery of each cell type based on the results of the six experiments presented in Tables 8 and 9. The recovery rate is obtained by dividing the cell count in the stem cell composition (Table 9) by the corresponding value in the sample before treatment with cord blood (Table 8) and multiplying this quotient by 100.

  The data in Table 10 illustrate that while it is possible to achieve 98.0% for an average reduction in RBC (2.0% recovery), stem cells as measured by the CD34 + marker can be recovered at an average of 95%. Yes. In addition, Table 10 illustrates that neutrophils recovered on average only 55%, whereas stem cells recovered on average 95%. For the bone marrow, it is anticipated that it will be necessary to use xenobiotic precipitation to achieve this level of selective cell separation.

  Tables 11 and 12 below show the ratios of RBCs to various other cell types in cord blood before treatment and stem cell composition of cord blood based on the data in Tables 8 and 9. The ratio of these cells to each other defines a unique stem cell composition that cannot be found in nature without the presence of xenobiotic additives or cannot be generated by any other cell processing system. The unity of these stem cell compositions requires that the stem cells be alive, along with a significant reduction in red blood cells and the absence of xenobiotic additives. The availability of a stem cell composition that contains most of the stem and progenitor cells that survive in the source bone marrow or umbilical cord blood and that has a very small ratio of RBCs to CD34 + cells is an important advance for stem cell therapy and is an excess in stem cell transfusion. Safe cell products can be made that do not have the negative effects associated with red blood cells.

  The average ratio of RBC to CD34 + is initially 201,834: 1 and decreases to 5,007: 1 after processing. This dramatic reduction in the ratio of RBCs to stem cells reflects the success of this method that can selectively reduce RBCs without significant loss of CD34 + cells.

The unity of these cell populations is unique to the viable cells contained in them.
Table 13 contains an analysis of the results of CD45 + cell viability (viability) in cord blood as measured using Stem-Kit. There is no significant change in CD45 + cell viability, demonstrating the maintenance of stem cell viability in the cord blood products after treatment and is accepted as a surrogate.

Stem Cell Composition The method of the invention described above results in a composition of stem cells that is derived from bone marrow or umbilical cord blood containing stem cells, plasma, RBC, and red blood cells, and is free of xenobiotic additives. Stem cell composition derived from plasma is about 5 RBC for each TNC, about 10 RBC for each neutrophil, and RBC for each MNC as measured by CD34 + cells or ALDH Br + cells. About 18 and RBC has a ratio of about 400 to 500 for each stem cell (Table 6). Stem cell composition derived from umbilical cord blood is about 11 RBC for each TNC, about 24 RBC for each neutrophil, about 23 RBC for each MNC, as measured by CD34 + cells, And RBC has a ratio of about 5,000 for each stem cell (Table 12).

  Stem cell composition derived from plasma is recovered from about 79% to about 100% CD34 + cells and from about 74% to about 100% ALDH Br + cells, while about 97% to about 99% of RBCs are reduced. It has been. The stem cell composition derived from umbilical cord blood is more than about 77% and up to about 100% of CD34 + cells are recovered, while about 96% to about 99% of RBCs are reduced.

  Each of the above results indicate that stem cells in bone marrow or umbilical cord blood are relatively high in the WBC / stem cell and progenitor cell layers (from the red blood cell layer) through the process of separation and transfer of RBCs from the processing bag into the RBC enrichment bag. (Which is also close to the plasma phase), and neutrophils have demonstrated the unexpected result of maintaining a relatively low position in the WBC / stem and progenitor cell layers (closer to the red blood cell layer). RBCs flowing out of the processing bag are expected to cause significant mixing with other cell types along with stem cells, neutrophils, and red blood cells due to the effect of Coriolis and laminar flow as the RBC flows down toward the throttle valve. It was. By such mixing, a high rate of recovery of stem cells was expected for a low rate of recovery of neutrophils, and a high rate of recovery of stem cells was expected for a reduction of high efficiency of RBC.

  In the above, this invention was demonstrated based on the Example. Those skilled in the art will envision other embodiments and various embodiments of the invention within the scope of the claims.

100 Backset 102 Processing Bag 104 RBC Concentration Bag 106 Stem Cell Bag 138 Throttle Valve 200 Processing Device 218 Main Body 220 Stem Cell Bag Chamber 228 Centrifugal Bucket 230 Main Chamber 248 Servo Motor 254 Photosensor 272 Load Cell 286 Printed Circuit Board 288 Interface Printed Circuit Board 290 Micro Controller 296 Battery

Claims (8)

A method for preparing a stem cell composition from bone marrow or cord blood comprising:
(A) placing the collected bone marrow or umbilical cord blood in a treatment bag, wherein the treatment bag is part of a bag set, the bag set comprising an erythrocyte concentration bag, a stem cell bag, and a throttle valve; The throttle valve is connected to the processing bag, the red blood cell concentration bag, and the stem cell bag,
(B) including a step of installing the bag set in a processing device, wherein the processing device includes a microcontroller, a servo motor, an optical sensor, an LED, a load cell, and an accelerometer; Connected to the throttle valve to operate when stored in the processing device;
(C) The treatment bag containing the bag set is subjected to g force, the bone marrow or the cord blood cells in the treatment bag is a layer of red blood cells, a layer of white blood cells (WBC) / stem cells and progenitor cells, and plasma Centrifuging for a time sufficient to stratify into layers, the throttle valve being closed,
(D) holding the bag set for a time sufficient to separate most of the red blood cells from the processing bag into the red blood cell concentration bag with a lower g force than that used in the centrifuging step. Centrifuge the stored processing device, and the throttle valve opens from the processing bag to the red blood cell concentration bag,
(E) closing the throttle valve in response to the optical sensor detecting a predetermined volume of light transmittance from the LED;
(F) centrifuging the processing apparatus containing the bag set for a time sufficient to separate more of the red blood cells from the processing bag into the red blood cell concentration bag with g force. The throttle valve is repeatedly opened and closed many times,
(G) responsive to the optical sensor detecting a change in light transmittance at a preset rate, and closing the throttle valve;
(H) using the data from the load cell to tare the stem cell bag, wherein the tare is performed using the data from the load cell;
(I) The treatment device containing the bag set is moved from the treatment bag to the stem cell bag with g force, the remaining red blood cells, the WBC / stem cell and progenitor cell layer, and a part of the plasma. Having a step of centrifuging for a sufficient time to be separated, said throttle valve being opened and closed a number of times,
(J) closing the throttle valve in response to the load cell measuring a preset weight of the stem cell bag;
(K) removing the bag set from the processing device; and (l) removing the stem cell bag containing the stem cell composition from the bag set.   Between the steps (e) and (f), the processing apparatus containing the bag set is operated with g force so that the bone marrow or cord blood cells in the processing bag are in the processing bag with a red blood cell layer, WBC The method of claim 1, further comprising centrifuging for a time sufficient to re-stratify into a layer of / stem and progenitor cells and a layer of plasma.   The method according to claim 1, further comprising a step of selecting a volume of the stem cell composition in advance.   4. The method of claim 3, wherein the selected volume is determined by selecting a volume of the stem cell composition in the stem cell bag.   The method of claim 1, wherein the volume of the selected bone marrow is about 40 mL to about 240 mL. A system for preparing stem cell products from bone marrow and / or cord blood,
(A) a processing bag for holding collected bone marrow or umbilical cord blood, a red blood cell concentration bag, a stem cell bag, and a bag set including a throttle valve, wherein the throttle valve is the processing bag, the red blood cell concentration bag, And connected to the stem cell bag,
(B) The bag set is housed inside and includes a processing device including a microcontroller, a servo motor, an optical sensor, an LED, a load cell, and an accelerometer. The servo motor includes the bag set and the processing device. (C) the bag set and the processing device are adapted to extract stem cell products from the harvested bone marrow or umbilical cord blood with g force. A system that is capable of withstanding centrifugation for a time sufficient to separate and concentrate.   The system of claim 6, wherein the microcontroller receives and analyzes inputs from the accelerometer, the light sensor, and the load cell and opens or closes the throttle valve in response to the inputs.   The system of claim 6, wherein the light sensor is positioned in the processing apparatus such that the volume in the processing bag between the level of the light sensor and the level of the throttle valve is about 2 mL.

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