Patent Application
20070259330
In a first aspect, the present invention relates to a method of separating mononuclear cells from blood, which method comprises providing a human blood sample together with density gradient media (DGM) in a container; spinning the container comprising blood and DGM; and collecting the DGM fraction that comprises mononuclear cells; wherein the DGM has a density which is >1.080 and <1.090 g/cm3 as measured at 25° C.
In an advantageous embodiment, the density of the DGM is about 1.083 g/cm3, such as 1.083±0.003 g/cm3 as measured at 25° C. As known in this area, the density of a density gradient media will vary with temperature, and consequently needs to be specified by the temperature at which it was measured. For example, the above-mentioned advantageously used DGM presents a density of 1.0845 at 20° C.
In the general aspect, the blood from which mononuclear cells are separated may originate from any human source such as peripheral blood, embryonic blood, placental blood or umbilical cord blood. In a specific embodiment, the blood sample originates from cord or placenta. In one embodiment, the blood originates from human bone marrow, which has been processed according to well known methods into a form suitable for density gradient separation. In one embodiment, the mononuclear cells separated are characterized as CD34+ cells. In an advantageous embodiment, the present method is used to prepare a purified fraction of mononuclear cells for in vivo use, such as in cell transplantation or cell therapy.
As is well known, cord blood is frequently collected after birth, cryopreserved and stored for a period of time and then thawed to be used in the clinic or research lab. Thus, in one embodiment, the blood has been cryopreserved and thawed before the separation. In an advantageous embodiment, irrespective of whether it has been thawed or not, the blood is heated to a temperature close to room temperature, which is a suitable temperature to carry out the method of the invention.
In an advantageous embodiment of the present method, the DGM is comprised of neutral, highly branched, hydrophilic polymers of sucrose. In a more advantageous embodiment, the osmolality of the DGM is >300 Osm/kg H2O, such as >325 Osm/kg H2O. In an advantageous embodiment, the osmolality is in the range 325-350, such as 330-350 or 330-340 Osm/kg H2O. The DGM will be discussed in more detail below. In an alternative embodiment, the DGM used in the present method is comprised of iodixanol in water presenting the herein defined density and preferably the osmolality above.
In one embodiment of the present method, the blood sample has been diluted before the separation. Such dilution is advantageously carried out with a suitable buffer, such as a salt e.g. a phosphate buffer; a salt solution such a Hank's balanced; or cell culture medium. As the skilled person will appreciate, if and how the dilution is carried out will depend on the contents of mononuclear cells in the blood and the volume used. Further, if required, anti-coagulant may be added. Again, the skilled person in this field will be able to decide in which cases and which amount anti-coagulant can be added. In an advantageous embodiment, the total volume of the blood and DGM is 10-200 ml, such 20, 50 or 100 ml. In a specific embodiment, the total volume of the blood and DGM is above 200 ml.
In an advantageous embodiment, the blood sample is layered on top of the DGM in the container. In an advantageous embodiment, the container is a tube or a bag, as will be discussed in more detail below.
In the most advantageous embodiment, the spinning of blood sample with DGM is achieved by centrifugation. In a specific embodiment, the centrifugation is carried out at a speed of 400×G, and may last for about half an hour. Any commonly used centrifuge may be used, such as a temperature-controlled centrifuge.
As the skilled person will understand, the centrifugation of blood sample and DGM will result in an upper plasma fraction, and a DGM layer containing the mononuclear cells underneath. Red blood cells, which are regarded contaminants in the present method, will gather at the bottom of the container. Thus, in one embodiment, the desired monocyte-containing DGM fraction is recovered by aspiration after removal of the plasma fraction. The aspiration may be carried out with a commonly used syringe, or with an automated instrument. Preferably, the aspiration is carried out under sterile or aseptic conditions.
A specific aspect of the present method is a method of purifying lymphocytes from erythrocytes, thrombocytes and granulocytes in a blood sample, which method comprises a method according to the invention, as discussed above, and an additional step of isolating said lymphocytes from the DGM fraction. Lymphocytes have a number of roles in the immune system, including the production of antibodies and other substances that fight infection and diseases. Thus, lymphocytes isolated according to the present invention are useful e.g. for clinical and diagnostic use.
Another embodiment of this aspect is a method of purifying monocytes from erythrocytes, thrombocytes and granulocytes in a blood sample, which method comprises a method according to the invention and an additional step of isolating said monocytes from the DGM fraction. As is well known, a monocyte is a specific type of white blood cell, and like the isolated lymphocytes, monocytes isolated according to the invention are useful e.g. for clinical and diagnostic use.
A further embodiment of this aspect is a method of purifying stem cells from erythrocytes, thrombocytes and granulocytes in a blood sample, which method comprises a method according to the invention and an additional step of isolating said stem cells from the DGM fraction. As is well known, stem cells are the cells from which other types of cells develop, and may be embryonic or human. Stem cells isolated according to the invention are useful for in vitro and/or in vivo purposes, such as for research, clinical and diagnostic use. In an advantageous embodiment, the present stem cells are used in vivo, and more specifically for transplantation purposes into patients. Such transplantation may e.g. be a method of replacing immature blood-forming cells that were destroyed, such as by cancer treatment. In this case, the stem cells are given to the person after treatment to help the bone marrow recover and continue producing healthy blood cells.
This embodiment is equally useful for the isolation of progenitor cells for in vitro and/or in vivo purposes. In this context, the term progenitor cell is used for immature or undifferentiated cells, typically found in post-natal animals. While progenitor cells share many common features with stem cells, the term is less restrictive.
The cells purified according to the present invention are useful in the context of cell therapy, such as in research related to cell therapy and/or for clinical or pre-clinical applications.
In a second aspect, the present invention relates to a density gradient media (DGM) as such. More specifically, the DGM according to the invention is comprised of neutral, highly branched, hydrophilic sucrose polymers, which DGM present a density as discussed above, such as in the range of 1.080-1.090 g/cm3, and an osmolality as discussed above, such as >325 Osm/kg H2O, preferably in the range of 325-350, such as 330-350 or 330-350 Osm/kg H2O. The DGM according to the invention may be prepared starting from a commercially available sucrose-based DGM, such as Ficoll-Hypaque™ (GE Healthcare, Uppsala, Sweden) or HistoPaque™ (Sigma-Aldrich), by careful modification of density and optionally also osmolality. In an advantageous embodiment, the DGM according to the invention is of GMP quality.
In a third aspect, the present invention relates to a container containing DGM and useful in the method according to the invention. Thus, in one embodiment, the container is a bag comprising the DGM according to the invention, which bag is made from synthetic polymers, preferably a plastic, e.g. a plastic laminate. In an advantageous embodiment, the bag is sterilizable. Thus, the bag may be sterilized separately and filled under aseptic conditions with sterile DGM, or more conveniently, the bag is filled with and sterilized with its DGM contents. In another embodiment, the container is a tube comprising the DGM according to the invention. The tube may be sterile and/or sterilizable, as the bag above. Further, the tube may contain some kind of physical partition means such as a horizontal wall to prevent mixing of the blood with the DGM. Such partition means have been described see e.g. U.S. Pat. No. 4,917,801.
In a fourth aspect, the invention relates to a kit for the purification of mononuclear cells from cord blood, which kit comprises a bag or a tube, which contains density gradient media according to the invention having a density as discussed above, such as in the range of 1.080-1.090 g/cm3, and an osmolality as discussed above, such as >325 Osm/kg H2O, preferably in the range of 325-350, such as 330-350 or 330-350 Osm/kg H2O. In one embodiment, the present kit is comprised of a container containing DGM comprised of neutral, highly branched, hydrophilic sucrose polymers, which DGM present a density in the range of 1.083±0.003 g/cm3 and an osmolality >325 Osm/kg H2O. In one embodiment, the kit is sterile and optionally adapted for use in an automated cell processing instrument. In one embodiment, the kit comprises instructions for use, preferably for use in a method of separating human mononuclear cells from blood such as cord blood, placenta blood or blood marrow.
The present examples are provided for illustrative purposes only, and should not be interpreted in any way as limiting the scope of the invention as defined by the appended claims. All references provided below and elsewhere in the present specification are hereby included herein via reference.
Cord blood (CB) Collection. Umbilical cord blood, which is a rich source of stem and progenitor cells, was obtained by direct drainage from the cord and/or by needle aspiration from the delivered placenta at the root and distended veins. Umbilical cord blood was collected from delivered placentas with syringes containing an anticoagulant, citrate phosphate dextrose (CPD) (CPD:blood 1:12).
Isolation of Mononuclear Cells (MNC). The blood bag contents were mixed by gentle rotation for approximately 30 seconds to ensure that the contents were mixed well. The tubing was clamped approximately 2 inches from the opening of the bag with a sterile hemostat. The tubing was sterilized above the hemostat with alcohol wipes. The sterilized tubing was held with a hemostat, and the tubing was cut using sterile scissors approximately 2 inches above the hemostat. The blood was aliquoted into sterile, labelled, 50 mL conical tubes (25 mL of blood/tube) and the volume of the cord blood without anticoagulant was calculated by subtracting the amount of anticoagulant. DPBS (Dulbecco's Phosphate-Buffered Saline, pH 7.2-7.4, Cellgro) was added to each labelled tube of cord blood, to a volume of 35 mL and the tubes were inverted carefully 2 or 3 times. Each tube was underlaid, using a 10 mL sterile pipette, with diluted cord blood and 12.8 mL of Histopaque-1077 (Sigma-Aldrich, #10771., St. Louis, Mo.) and centrifuged at 400×g for 30 minutes at 22° C. The plasma was carefully removed approximately 1.5 cm above the MNC layer and stored in clean 50 mL tubes. The MNC layer was removed using a 10 mL sterile pipette. The cells were transferred into 50 mL tubes and RPMI was added to a volume of 45 mL. The tubes were centrifuged at 400×g for 15 minutes at 22° C. The supernatant was decanted and the cell pellets were resuspended by the addition of 5 mL of RPMI to each tube, and adjusting the total volume to 45 ml and centrifugation at 400×g for 10 minutes at 22° C. The supernatant was decanted and the cell pellet carefully resuspended in RPMI and then transferred to a sterile 15 mL conical tube. The tube was mixed gently by inversion. The tubes were centrifuged at 400×g for 10 minutes at 22° C. The remaining supernatant was discarded being careful not to disturb the cell pellet. 750 μL of plasma was added to resuspend the cell pellet. Once the cells were completely in suspension, the volume was aliquoted to 1 ml using a serological pipette and stored on ice. 20 μl of cell suspension was transferred to the Vi-Cell Viability Analyzer (Beckman Coulter) for cell counting and viability. Once the viable cell count had been determined by the Vi-Cell, the viable cell count number was typed into a cell solution program (MS Xcel), press enter, and this program calculated all volumes of reagents (90% autologous plasma, 10% DMSO) to be added to the final volume of the cell suspension prior to aliquoting into cyrovials. This program also determined the number of Research vials and 1 Quality Assurance (QA) vial based on a storage volume of 20 million cells per vial. In addition, 1 Archive (AR) vial was stored for each sample. The Archive (AR) vial contained the remainder of the cells after the Research and QA vials have been aliquoted. The program also determined the concentrations of reagents for the AR vial which contained a separate cell suspension since the total amount of cells present in this AR was much lower than the Research and QA vials. All contents within each vial were aliquoted properly and completely before the samples were frozen. The cryovials were placed in a rack, in a controlled rate freezer, and frozen using the assigned pre-set profile #1.
Human umbilical cord blood (HUCB) thawing. The cryovials were removed (Corning #430488) from the liquid nitrogen container and placed in a 37° C. water bath for 5 min. The thawed cell suspension was rapidly transferred from the cryovial to a 15-mL conical centrifuge tube (Falcon, #352057) containing 10 mL of DPBS and centrifuged at 400×g for 10 minutes at +21° C. The supernatant was removed without disturbing the cell pellet and 10 mL of DPBS was added to resuspend the cells again. After centrifugation, the pellet was resuspended in 1 ml of the DPBS, and 10 μl of HUCB suspension was removed for counting the cells using a hemacytometer or Vi-Cell Analyzer.
Blood smears for Giemsa. The blood samples were obtained from the fresh HUCB and were taken for morphological analysis. The smears were dried for 30 min, fixed in methanol for 7 min, then stained by Giemsa (Sigma-Aldrich, GS80, St. Louis, Mo.) as previously described (Brown and Febiger, 1993). After staining, blood smears were rinsed several times in distilled water and cover-slipped with Permount (Fisher Scientific, Fair Lawn, N.J.). The morphology of the peripheral blood cells was examined under an Olympus BX-60 microscope. The images were analyzed by Image-Pro Plus version 4.1 for Windows software (Silver Spring, Md.). Analyses for CBC (complete blood count) and white blood cell differential (WBC) were performed by Antech Diagnostics (NY, USA). The blood smears were treated as disclosed in Brown A, and L. Febiger, Hematology: Principles and Procedures (6th ed.), Lea and Febiger, Philadelphia (1993), p. 101.
Flow Cytometry. Surface antigens were detected by double-color immunofluorescence assay combining fluorescein isothyocyanate (FITC) or phycoerythrin (PE) conjugated monoclonal antibodies (Mab). These included: CD45-FITC/CD34-PE (#341070), Isotype Control (Mouse IgG1-FITC, #349041) (Becton Dickinson, BD), and CD133 (#130-090-422, MACS). All the Mab were used at the concentrations titrated for optimal staining. Treatment of leukocyte with Mab was done according to the manufacturer's recommendations. Irrelevant fluorochrome-conjugated murine isotype-matched Mab were always included in the staining protocols as a negative control. Detection of fluorescence of stained cells was performed with a FACScan flow cytometer (BD) equipped with Argon laser tuned to 488 nm. Calibration beads were used for monitoring and optimizing the instrument settings. Data were acquired with LYSIS II software (BD). Forward light scattering (FCS), orthogonal light scattering (SSC), and fluorescence signals (FL-1-FITC, FL-2-PE) were sorted in listmode data files. For data standardization gated acquisition of living lymphocyte population was performed routinely. A minimum of 50,000 cells were analyzed and at least 5,000 gated events were measured for each sample. All data were analyzed using PAINT-A-GATE software (Becton Dickinson).
Density gradient media (DGM) according to the invention were prepared in different densities and osmolalities by modification of Ficoll-Paque™ (GE Healthcare Bio-Sciences, Uppsala, Sweden), which is comprised of neutral, highly branched, hydrophilic polymers of sucrose. In the present experimental part and drawings, “Ficoll” refers to “Ficoll-Paque”™. In the present application, commercial products used for comparative purposes have been presented with the density provided by the supplier, while the density gradient media adapted by the present inventors presents a density of 1.083 as measured at 25° C.
Standard industrial protocol was used in all the experiments below, unless otherwise stated, to separate MNC fraction from human umbilical cord blood.
Density of FPP and number of CB samples used with each density:
All assessments were done prior to freezing.
MNC Yield—no significant differences were observed between densities 1.083 and 1.090 (
MNC Yield—densities 1.077 and 1.080 had a significantly lower number of cells as compared to 1.083 (
Cell Viability—no significant differences were observed between densities 1.083 and 1.077 (
Cell Viability—densities 1.080 and 1.090 exhibited significantly lower cell viability as compared to 1.083 (
Cell Diameter—all cell diameters were compared to the standard 1.077 density (
Density 1.083 had a significantly lower cell diameter as compared to 1.077 Note: this could be due to an increase of platelets or higher % of progenitor cells
Comparison of Unit Volumes—all unit volumes were averaged and compared for each density tested (
No significant differences were observed between any of the densities. This should rule out any chance of volume variability affecting the MNC outcome.
Density of FPP and number of CB samples used with each density:
All assessments in this example were done post-thaw.
In this section, the MNC yield was compared between Pre-Freeze and Post-thaw samples (see
(A) Pre-freeze MNC Yield—densities 1.077 and 1.080 had a significantly lower number of cells as compared to 1.083.
(B) Post-thaw MNC Yield—1.083 density produced a significantly higher cell number than densities 1.077 and 1.080.
In this section, cells were characterized by Flow Cytometry—cell number (see
(A) the number of CD45+ cells was significantly higher with 1.083 than 1.077, 63.17×106 and 23×106 cells respectively.
(B) the number of CD34+ cells was significantly higher with 1.083 than 1.077, 1.27×106 and 0.294×106 cells respectively.
(C) the number of CD133+ cells was significantly higher with 1.083 than 1.077, 0.2407×106 and 0.072×106 cells respectively.
The red blood cell (RBC) contamination was determined using Giemsa pre-Freeze and post-Thaw, N=5 for each density.
No significant differences were observed pre-freeze between densities 1.077 and 1.083, and no significant differences were observed post-thaw between densities 1.077 and 1.083.
The above examples illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed. Those skilled in the art having the benefit of the teachings of the present invention as set forth above, can effect numerous modifications thereto. These modifications are to be construed as being encompassed within the scope of the present invention as set forth in the appended claims.
This application claims priority to U.S. provisional patent application No. 60/797,645 filed May 4, 2006; the disclosure of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60797645 | May 2006 | US |
