EP0807168A1 - Anreicherung der hämiporetischen stammzellen aus blut oder knochenmark - Google Patents

Anreicherung der hämiporetischen stammzellen aus blut oder knochenmark

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Publication number
EP0807168A1
EP0807168A1 EP96902498A EP96902498A EP0807168A1 EP 0807168 A1 EP0807168 A1 EP 0807168A1 EP 96902498 A EP96902498 A EP 96902498A EP 96902498 A EP96902498 A EP 96902498A EP 0807168 A1 EP0807168 A1 EP 0807168A1
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EP
European Patent Office
Prior art keywords
cells
stem cells
stem cell
preparation
platelets
Prior art date
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Application number
EP96902498A
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English (en)
French (fr)
Inventor
Jacob het Nederlandse Rode Kruis KWEKKEBOOM
Daniel E. P. het Nederlandse Rode Kruis BUURMAN
Johannes A. het Nederlandse Rode Kruis LOOS
Clasina M. Slaper-Cortenbach
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Stichting Sanquin Bloedvoorziening
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Stichting Centraal Laboratorium Van de Bloedtransfusiedienst Van
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Priority to EP96902498A priority Critical patent/EP0807168A1/de
Publication of EP0807168A1 publication Critical patent/EP0807168A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B5/00—Other centrifuges
    • B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
    • B04B5/0442—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers with means for adding or withdrawing liquid substances during the centrifugation, e.g. continuous centrifugation
    • B04B2005/0471—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers with means for adding or withdrawing liquid substances during the centrifugation, e.g. continuous centrifugation with additional elutriation separation of different particles

Definitions

  • the invention is in the area of hematopoietic stem cell transplantation and is concerned with a novel process for the preparation of enriched suspensions of hematopoietic stem cells.
  • the invention relates to a method to deplete non-stem cells from hematopoietic stem cell suspensions used for transplantation, without the use of xenogeneic antibodies directed against cell surface molecules.
  • the process will be suitable for the processing of auto- logous transplants, as it will result in a reduction of the volumes of transplants to be cryopreserved, a reduction of the amount of (toxic) cryoprotectant to be infused into the patient, a diminished risk of transfusion problems related to lysis of granulocytes and erythrocytes upon freezing and thawing, and probably a reduction of the number of contaminating malignant cells.
  • GvHD Graft-versus- Host Disease
  • bone marrow transplants are bein used.
  • bone marrow is harvested in the remissio phase of the disease, cryopreserved, and reinfused into the patient after high-dose chemotherapy.
  • bone marrow infiltration with tumor cells is a common feature.
  • intensive pretreatment such as pelvic irradiation may lead to damage to bone marrow, thereby hampering the harves of sufficient bone marrow stem cells.
  • hematopoietic progenitor cells can be mobilized to the blood by treatment wit certain chemotherapeutic drugs. Moreover, it was observed that the mobilizing effect of chemotherapy can be enhanced by in viv administration of hematopoietic growth factors, such as G-CSF.
  • Peripheral blood stem cells PBSC are now increasingly used as an alternative for bone marrow in autologous transplantations.
  • PBSC are collected by leukocytapheresis. This is a centri- fugation technique in which low density cells (i.e. mononuclear cells, including the progenitor cells) are collected. The other blood components are reinfused (continuously or intermittently) into the donor.
  • low density cells i.e. mononuclear cells, including the progenitor cells
  • PBSC transplantation has several advantages over bone marrow transplantation: 1.
  • the chemotherapy applied before harvesting largely eradicates circulating tumor cells, thereby reducing the tumor load in peripheral blood stem cell transplants.
  • (low numbers of) malignant cells in the PBSC transplants can still b detected.
  • Umbilical cord blood stem cell transplantations have mainly been performed in children, in view of the limited numbers of stem cells harvested. Expansion in vitro may however facilitate transplantation of umbilical cord blood stem cells in adults.
  • purification of stem cells from the transplants would be desirable. Purification will provide major advantages: a. In autologous transplantations, purification will lead to a reduction in the number of malignant cells. b. In autologous and umbilical cord blood transplantations, purification will lead to a reduction in the volume of cells to be cryopreserved, to a reduction of the amount of (toxic) cryoprotectant to be infused into the patient, and to diminished risks of infusion which are related to the lysis of granulocytes and erythrocytes upon freezing and thawing (such as formation of aggregates, hemoglobinuria) . c. In allogeneic transplantations, the resulting depletion of T cells will lead to diminished GvHD. d. Growth-factor induced expansion and differentiation of stem cells in vitro is more effective when purified stem cells are cultured. e. For effective transfer of foreign genes into stem cells (for gene therapy) purified preparations of stem cells may be necessary.
  • CD34 monoclonal antibodies which react specifically with hematopoietic stem cells, are being used for the isolation of progenitor cells.
  • mAbs monoclonal antibodies
  • One of the first reports was by Berenson et a (J. Clin. Invest. 81 (1988): 951) who isolated CD34 + cells from bone marrow, transplanted these cells into irradiated baboons and achieved hematological reconstitution. Because leukemic blasts from patients with acute leukemia also carry the CD34 antigen, this approach of stem cell purification cannot be used in acute leukemia. However, in solid tumors like breast carcinoma, neuroblastoma etc. and in other hematological disorders, the malignant cells are CD34-negative.
  • Soybean agglutinin (SBA) or CD34 mAb is covalently bound to polystyrene flasks.
  • SBA Soybean agglutinin
  • CD34 + cells are then selected from the SBA-negative population by binding to CD34 mAb. Finally, bound cells are released by physical agitation.
  • Baxter introduced the Isolex system, comprising immuno- magnetic beads coated with CD34 mAb for the selection of progenitor cells.
  • the positively selected stem cells are treated with chymopapain to strip the beads from the surface of the cells.
  • Dynal introduced a procedure similar to that of Baxter, but which differs in the method used to detach the beads from the cell surface. Cell release is performed using a polyclonal anti ⁇ body directed against murine Fab antibody fragments.
  • a major disadvantage of the available techniques is the large amount of CD34 monoclonal antibody needed, which makes these techniques very expensive.
  • a second disadvantage is the need of some technique to release the cells, which may result in damage to the progenitor cells.
  • the purity and yield achieved in isolations of stem cells from PBSC transplants is in general lower than purity and yield of stem cells isolated from bone marrow. Therefore, a process leading to substantial enrichment of stem cells by depletion of non-stem cells without the use of mAb would be a considerable advantage.
  • PBSC transplants are in general infused into the patients without any separation step.
  • PBSC leukocyta- pheresis products obtained after treatment of the patients with chemotherapy and G-CSF cannot be enriched by density separation because all cells collected by an optimal leukocytapheresis procedure have a low specific density.
  • the invention comprises a procedure for enrichment of hematopoietic progenitor cells based on selective adherence of certain non-progenitor cells to a fiber material coated with an opsonizing substance.
  • the adherence step is preceded by counterflow centrifugal elutriation.
  • the enrichment step with coated nylon fiber is preceded by elutriation, substantial depletion of all non-stem cell types from the transplant can be achieved.
  • the elutriatio technique separates platelets, erythrocytes and part of the lymphocytes from the progenitor cells.
  • the passage over coated nylon fiber results in depletion of monocytes and myeloid cells from the transplant.
  • substantial enrichment of stem cells is achieved without the us of monoclonal antibodies. In peripheral blood transplants approximately 10-fold reduction of the total number of non-stem cells is achieved.
  • the invention provides a process for the enrichment of hematopoietic stem cells in a stem cell preparation (usually umbilical cord blood, a peripheral blood stem cell suspension, or bone marrow) , the process comprising depleting non-stem cells (more particularly comprising monocytes and myeloid cells) from the preparation by adherence to an opsonized material and recovering a hematopoietic stem cell-enriched preparation.
  • a stem cell preparation usually umbilical cord blood, a peripheral blood stem cell suspension, or bone marrow
  • non-stem cells more particularly comprising monocytes and myeloid cells
  • said opsonized material comprises a fiber material, in particular a polyamide fiber material, treated with an opsonizing substance, wherein the opsonizing substance is preferably selected from the group consisting of IgG, IgA, C3b, C3bi, C3dg, Clq, iC3, conglutinin, mannan-binding protein (MBP), CL-43, SP-A and SP-D.
  • the opsonized material comprises a scrubbed nylon fiber material coated with human IgG.
  • the opsonized material is preferably treated with a blocking substance which reduces aspecific cell binding, wherein the blocking substance is preferably selected from the group consisting of human serum albumin, animal serum albumin, gelatin, Ficoll 70 and Ficoll 400.
  • the stem cell preparation is pretreated to remove platelets (thrombocytes) therefrom.
  • the pretreatment of the stem cell preparation preferably comprises a counterflow centrifugal elutriation in which smaller cells including platelets, erythrocytes and small lymphocytes are removed and larger cells including hematopoietic stem cells, monocytes and myeloid cells are recovered.
  • the platelets can be removed by sequential low-speed centrifugation steps.
  • a chelating substance e.g. EDTA or trisodium citrate
  • EDTA e.g. EDTA or trisodium citrate
  • a preferred process comprises providing a stem cell preparation from bone marrow or, preferably, from peripheral blood or umbilical cord blood, depleting platelets, erythro ⁇ cytes and small lymphocytes therefrom by a counterflow centri ⁇ fugal elutriation in which smaller cells including platelets, erythrocytes and small lymphocytes are removed and larger cells including hematopoietic stem cells, monocytes and myeloid cells are recovered, depleting monocytes and myeloid cells from said recovered larger cells by adherence to a material coated with an opsonizing substance and blocked against aspecific cell binding, and recovering a preparation enriched in hematopoietic stem cells.
  • a stem cell preparation from bone marrow, cord blood or peripheral blood is passaged directly (without prior removal of platelets) over a material coated with an opsonizing substance and blocked against aspecific cell binding, in a medium to which a chelating substance has been added.
  • monocytes and myeloid cells (and B cells) are depleted and a preparation enriched in hema ⁇ topoietic stem cells will be recovered.
  • erythrocytes, lymphocytes and platelets are not depleted, and the process will result in a more limited enrichment.
  • the invention covers the combination of both methods, i.e. prior depletion, e.g. by counterflow centrifugal elutriation, of platelets, erythrocytes and small lymphocytes, and passage of the resulting composition, which is enriched in the larger cells, over the non-stem cell depletion material in a medium containing a chelating substance.
  • the process may also comprise a step of immunoselection using an antibody which binds hematopoietic stem cells, or an antibody which binds non-stem cells or malignant cells, to effect a further enrichment of hematopoietic stem cells.
  • the invention also provides an assembly of means and instructions for use in a process for the enrichment of hematopoietic stem cells in a stem cell preparation, said means comprising (the constituents of) an opsonized material capable of binding non-stem cells including monocytes and myeloid cells present in a stem cell preparation.
  • the assembly may further comprise at least one member of the group consisting of elutriation media, cell suspending media, opsonization media, blocking media, washing media, and components of said media.
  • Figure 1 shows the principle of counterflow centrifugal elutriation.
  • CFU-GM a measure of the outgrowth of monocytic and myeloid precursors in semi-solid medium during a 12 day culture.
  • Figure 3 shows that yield of PBSC CD34 + cells upon passage over nylon wool in the presence of fetal bovine serum is enhanced after depletion of thrombocytes, erythrocytes and part of the lymphocytes by counterflow centrifugal elutriation. The figure depicts recovery of cells in the filtrate as percentage of the cells applied to the nylon wool.
  • Figure 4 shows the effect of an addition of paraformalde- hyde-fixed resting or thrombin-activated (non-autologous) platelets on the recovery of various cell types in elutriated PBSC upon nylon fiber filtration in the presence of fetal bovine serum.
  • the figure depicts the recovery of cells in the filtrate as percentage of the cells applied to the nylon wool.
  • Figure 5 shows the effect of elutriation on platelet binding to CD34 + cells in PBSC leukocytapheresis products.
  • Adhe- rence of platelets to CD34 + cells was measured by determination of CD41 mAb binding to CD34 + cells by FACS-analysis.
  • the figure depicts the percentage of CD34 + cells binding CD41 mAb (minus the percentage of CD34 + cells binding non-specific mAb) in unseparated leukocytapheresis product and in elutriated stem cell fraction.
  • Figure 6 shows that the yield of CD34 + cells after filtration of PBSC samples is enhanced when instead of removing the platelets by elutriation, a chelating agent is added to the medium prior to filtration.
  • the composition of the filtration medium is given.
  • the figure depicts the recovery of cells in the filtrate as percentage of the cells applied to the nylon wool.
  • another consequence of the addition of EDTA to the filtration medium is that myeloid cells are less well depleted from the cell preparation.
  • Figure 7 shows recovery of cells upon passage of elutriated PBSC cells over nylon fiber preincubated with: a. IMDM supplemented with 10% fetal calf serum and 9 iriM MgCl 2 ; b.
  • FIG. 8 shows recovery of cells upon passage of elutriat PBSC cells over nylon wool preincubated with either IMDM suppl mented with 10 % fetal calf serum or IMDM supplemented with immunoglobulin (1 mg/ml), without subsequent saturation with HSA.
  • the figure depicts the recovery of cells in the filtrate percentage of cells applied to the nylon wool.
  • Figure 9 shows recovery of cells upon filtration of elutr ated PBSC over nylon wool preincubated with either IMDM supple ⁇ mented with human serum albumin (10 mg/ml) or IMDM supplemente with human complement factor iC3 (0.1 mg/ml) followed by satu ⁇ ration of the fiber with HSA.
  • the iC3 was prepared by chemical cleavage of the intrachain thioester bond which is present in the ⁇ -chain of the molecule.
  • the figure depicts the recovery o cells in the filtrate as percentage of cells applied to the nylon wool.
  • Figure 10 shows the effect of addition of trisodium citrat to the filtration medium, instead of removal of platelets, on filtration of PBSC samples over IgG-coated nylon wool.
  • the figure depicts recovery of cells in the filtrate as percentage of the cells applied to the nylon wool.
  • On the x-axis the composition of the filtration medium is given.
  • Figure 11 shows the yields of various cell types present i PBSC leukocytapheresis products after counterflow centrifugal elutriation and passage over nylon wool coated with human immunoglobulin and saturated with HSA (three experiments with PBSC from different patients) .
  • the figure depicts recovery of cells after the whole procedure as percentage of the cells started with.
  • the invented procedure was developed in the first instance for enrichment of progenitor cells from peripheral blood stem cell leukocytapheresis preparations obtained after treatment with chemotherapy and G-CSF. These preparations contain leuko- cytes, erythrocytes (10 to 20 fold excess to leukocytes) and platelets (5 to 50 fold excess to leukocytes) . From about 1% to about 4% of the leukocyte fraction consists of progenitor cells (CD34 + cells), the remainder mainly consisting of lymphocytes, monocytes and myeloid cells. We looked for a non-immunological technique or a combination of techniques which would result in depletion of all these different non-stem cell types, with high recovery of progenitor cells.
  • leukocyte-depleted red cell concentrates are routinely prepared by passage over fibers ("filtration"). Such a procedure for removal of leukocytes is simple, fast, and does not require expensive equipment. A selective depletion of leukocytes occurs, while erythrocytes are recovered with high efficiency in these so-called “filtrations”. This is due to differences in size and adherence properties of the cells (cell sieving and cell adherence).
  • leukocyte removal filters are being used, which are composed of either polyester, cellulose acetate, cotton or nylon fibers. With nylon fiber not all leukocytes are depleted, but a selective removal of certain leukocyte types from blood can be achieved.
  • nylon fiber can be used for selective depletion of monocytes and myeloid cells from peri ⁇ pheral blood stem cell leukapheresis suspensions. The most important question was, whether progenitor cells would adhere to the nylon fiber or not. We observed that upon passage of PBSC leukocytapheresis products over nylon fiber in the presence of fetal calf serum, recovery of stem cells (enumerated as CD34 + cells) was strongly variable, ranging from 12 to 112% (Table 1) . The procedure resulted in considerable depletion of monocytes, but depletion of myeloid cells was only partial and variable.
  • myeloid cells The incomplete removal of myeloid cells is in strong contrast with the nearly complete removal of mature granulocyt achieved by passage of peripheral blood from normal donors ove nylon fiber.
  • Morphological examination of the cells showed tha in PBSC leukocytapheresis products obtained from patients pretreated with chemotherapy and G-CSF, in contrast to blood from normal donors, nearly exclusively immature myeloid cells (band forms, promyelocytes, myelocytes and metamyelocytes) are present. This is caused by the pretreatment of the patients wi chemotherapy and hematopoietic growth factors. In particular t more immature myeloid cells did not adhere to the fiber.
  • Platelets can be separated from leukocytes by sequential centrifugation washing steps, or by counterflow centrifugal elutriation.
  • Counterflow centrifugal elutriation is a techniqu in which particles are exposed to two opposite forces in a specially designed flow chamber within a centrifuge: the centrifugal force and a centripetal hydrodynamic force, caused by a fluid flow in the opposite direction of the centrifugal force.
  • the centrifugal force By increasing the fluid flow or decreasing the rotor speed, particles are separated ( Figure 1 and Lutz, M.P. et al, Analytical Biochemistry 200 (1992): 376). Separation occurs mainly according to size and to a lesser extent to density.
  • FIG. 2 shows that a nearly complete removal of thrombocytes, erythrocytes and a partial removal of lymphocytes from PBSC leukocytapheresis products was achieved by elutriation with a >85% yield of CD34 + cells and of myeloid progenitor cells (colony-forming units granulocyte-monocyte (CFU-GM) ) .
  • CFU-GM colony-forming units granulocyte-monocyte
  • Platelets or platelet fragments may adhere to the stem cells after which the stem cells adhere to the nylon wool.
  • Dercksen et al (Blood, in press) have observed that in PBSC leukocytapheresis products platelets and platelet fragments are bound to part of the CD34 + cells. This was reflected in binding of mAb directed against CD41 to the CD34 + cells.
  • CD41 is an antigen which is expressed nearly exclusively on platelets.
  • CD41 mAb binding to CD34 + cells was strongly diminished. It was proposed that the residual CD41 mAb binding after washing reflected cellular CD41 expression on megakaryocyte progenitors.
  • Figure 5 shows that elutriation not only results in removal of free platelets but also in removal of bound platelets or platelet fragments from the CD34 + cells, as indicated by the lowered percentage CD34 + cells which bind CD41 mAb.
  • platelets are removed from the stem cell graft by a counterflow centrifugal elutriation.
  • the same objective may be achieved by sequential differential (low-speed) centrifugation steps.
  • elutriation has in the invented procedure the followin advantages: 1. Non-bound platelets are more effectively removed by elutriation than by differential centrifugation.
  • Platelets which are bound to cells are more efficiently removed by elutriation than by low-speed centrifugation.
  • a chelating substance is included in the filtration medium to prevent the adherence of CD34 + cells to the nylon fiber, thereby circumventing the need of removal of platelets from the stem cell transplants.
  • opsonin in its most preferred form the opsonin being human IgG.
  • Myeloid cells are phagocytotic cells. Binding of phagocytes to foreign particles, such as bacteria, is strongly enhanced by opsonization, i.e. coating of the particle with specific proteins. Proteins with the ability to enhance the binding of phagocytotic cells to foreign particles are called opsonins.
  • the main opsonins are immunoglobulins (especially IgG and to a lesser extent IgA) and complement factors C3b, C3bi and C3dg. These complement factors are enzymatic cleavage products of complement factor C3, which are generated upon activation of the complement cascade. Moreover, complement factor Clq is thought to have opsonizing properties. We hypothesized that coating of the fiber with opsonins would result in enhanced adherence of the (immature) myeloid cells.
  • C3b, C3bi and C3dg cannot be purified from plasma.
  • the opsonizing capacity of C3 cleavage products can be mimicked by chemical cleavage of the thioester bond between a cysteine and glutamine residue in the ⁇ -chain of C3.
  • the resulting molecule is called iC3. Due to a conformational change of the molecule phagocytotic cells can bind to it via their complement receptors with high affinity.
  • FIG. 10 shows the effect of addition of trisodium citrate to the filtration medium on the yield of CD34 + cells fr PBSC leukocytapheresis preparations after filtration over IgG- coated nylon wool, without prior removal of platelets. Yield of CD34 + cells was enhanced in the presence of trisodium citrate, while depletion of monocytes or myeloid cells was similar to that in filtrations in which no chelating substance was include in the filtration medium. Similar results were obtained upon addition of EDTA to the filtration medium.
  • the invention shows that phagocytic cells can be removed from stem cell products by passage over a fiber coated with an opsonin. It can be used for the enrichment of stem cell from bone marrow, PBSC grafts, and umbilical cord blood grafts.
  • the passage over the opsonized fiber material is preceded by counterflow centrifugal elutriation, by which not only platelets, but also erythrocytes and a larger part of the lymphocytes can be removed from the grafts.
  • counterflow centrifugal elutriation similar depletions of non- stem cells can be achieved from bone marrow, from umbilical cor blood and from PBSC leukocytapheresis product. Therefore, the combination of these techniques may result in a significant enrichment of progenitor cells from all these types of grafts.
  • PBSC leukocytapheresis products This is illustrated for PBSC leukocytapheresis products in Figure 11.
  • PBSC grafts were subjected to counterflow centrifuga elutriation and subsequently subjected to passage over human immunoglobulin-coated nylon fiber. This resulted in nearly complete removal of erythrocytes and thrombocytes, in more than 95% removal of myeloid cells, monocytes, and B lymphocytes from PBSC, and in at least 80% removal of T and NK cells.
  • the mean enrichment of stem cells obtained by this procedure was 9-fold. No procedure is known which results in similar enrichment of stem cells from peripheral blood stem cell leukocytapheresis products, without the use of specific monoclonal antibodies.
  • stem cell containing preparations are directly passaged, without prior removal of platelets, over a fiber material coated with an opsonin in a medium containing a chelating substance.
  • This will result in removal of monocytes, myeloid cells and B lymphocytes, and therefore in a lower enrichment of stem cells than after processing stem cell grafts by consecutive elutriation and filtration.
  • the removal of these cell types may be of great advantage for further (immuno)selection steps, since especially monocytes are sticky cells, which have a tendency to adhere to many types of material, thereby interfering with purification.
  • myeloid cells are very susceptible to freezing/thawing damage, and removal of them will probably lead to better quality of stem cell grafts after thawing.
  • fiber materials may be used to remove phagocytotic cells from stem cell preparations.
  • Appropri ⁇ ate fibers should not bind progenitor cells, or, alternatively, should not bind progenitor cells after being coated with a non- opsonizing protein (i.e. blocked with a blocking substance).
  • Enhancement of the adherence of phagocytotic cells is achieved by coating with an opsonin.
  • the opsonizing substance is human IgG.
  • IgA may be used, or the complement proteins C3b, C3bi, C3dg or Clq.
  • collectins a new class of opsonizing proteins has been discovered, called collectins (Holmskov, U.
  • the fiber For most fiber materials, it may be necessary to cover the fiber with some non-opsonizing substance to prevent aspecific cell loss (i.e. as a blocking substance). This can be done with biocompatible substances, which do not have adherent properties for cells and bind easily to the fiber.
  • a specific cell loss i.e. as a blocking substance.
  • biocompatible substances which do not have adherent properties for cells and bind easily to the fiber.
  • human or animal proteins e.g. human serum albumin, bovine serum albumin
  • colloids e.g. gelatin
  • the invented procedure as such, or in combination with elutriation, may result in removal of malignant cells from the graft in several types of malignancies.
  • Malignant cells are different in adherence properties and/or size from normal blood cells.
  • malignant B cells may be depleted by passage over opsonized fiber, but also by counterflow centrifugal elutriation.
  • Carcinoma cells are generally more adhesive and larger than blood cells, and may also be separated from the progenitor cells during elutriation and/or passage over opsonized fiber.
  • the technique is simple and can be performed rapidly, is relatively inexpensive, and can be easily brought to a clinical scale on the basis of existing blood-filtration technology.
  • depletion of phagocytotic cells by the filtration technology described here may be more efficient than by using immunoglobulin-coated beads or cells.
  • the adherence in the filtration technique is not only based on affinity of the cells for immunoglobulin, but also on the natural affinity of phago ⁇ cytotic cells for polyamide fiber.
  • the invented procedure may be followed by an immunoselection step.
  • the immunoselection may be performed by a positive selection of progenitor cells (e.g. by using CD34 mAb) using an of the available immunoselection techniques described above, or by new immunoselection techniques.
  • residual non- progenitor cells may be removed by negative selection using mAb directed against the various types of non-progenitor cells and/or the malignant cells.
  • the immunoselection can be performed after applying the invented procedure.
  • an immunoselection may be integrated in the "filtration" procedure.
  • beads suited for capture of cells which are labeled with a specific antibody may be integrated in a column of opsonized fiber material.
  • beads to which cell-specific antibodie have been attached can be applied.
  • Counterflow centrifugal elutriation was performed in a Curame 3000 elutriation system (Dijkstra Vereenigde BV, Lelystad, The Netherlands) equipped with disposable polycarbo ⁇ nate separation chambers (International Medical BV, Zutphen, The Netherlands) .
  • a fluid counterflow within the elutriation chamber was achieved by a roller pump.
  • a pulse flattening air chamber was placed.
  • the cell suspension was introduced into the system at a fluid flow of 12.5 ml/min and a rotor speed of 3000 rpm.
  • the elutriation medium consisted of PBS supplemented with 4 mg/ml HSA and 5 mM EDTA. Alternatively, instead of EDTA, 0.38% (w/v) trisodium citrate was added. After introduction of the cells, the liquid flow was increased to
  • the syringe was placed in a 37°C water bath and approximately 20 ml of IMDM containing 1 mg/ml human immunoglobulin (Immuno- globuline I.M., CLB, Amsterdam; obtained from normal human plasma, and containing at least 90% IgG) was pumped into the syringe from below until the nylon wool was completely wetted.
  • the nylon fiber was incubated at 37°C during 30 min with the IgG, and subsequently 30 ml IMDM supplemented with 10 mg/ml HS was pumped through it in the opposite direction (from above). The nylon wool was incubated at 37°C during 20 min.
  • the cells (45xl0 6 - 150xl0 6 leukocytes), suspended in IMDM with 10 mg/ml HSA, were applied to the nylon wool and pumped through with a flow rate of 1.0 to 2.5 ml/min.
  • the non-adherent cells were washed from the fiber at the same flow rate with at least 30 ml IMDM supplemented with 10 mg/ml HSA.
  • Cells present in the filtrate were sedimented by centrifugation and suspended in PBS supplemented with 0.38% trisodium citrate and 2 mg/ml HSA. It was found that IMDM could be replaced by phosphate buffered saline (PBS), without any effect on the outcome of the filtration process.
  • PBS phosphate buffered saline
  • cells were suspended in PBS supple ⁇ mented with HSA plus EDTA (5 mM) or trisodium citrate (13 mM) .
  • PBS supple ⁇ mented with HSA plus EDTA or trisodium citrate was used as filtration medium.
  • the filtration medium was either or not supple ⁇ mented with 13 mM trisodium citrate or 5 mM EDTA.
  • Protocol using iC3 Similar to the protocol with human immunoglobulin, except that the nylon wool was preincubated with 0.1 mg/ml iC3 in IMDM instead of immunoglobulin; the iC3 was prepared from complement factor C3 purified from human plasma. C3 was incubated with 0.2 M methylamine in PBS (37°C, 60 min). Non-reacted methylamine was removed by dialysis.
  • Protocol using HSA Similar to the protocol using human immunoglobulin, except that the nylon wool was preincubated with IMDM supplemented with 10 mg/ml HSA instead of immunoglobulin.
  • the PBSC leukocytapheresis samples the fractions obtained after elutriation and the filtrate obtained after passage of cells over coated nylon wool were analyzed as follows: * Leukocytes and erythrocytes were counted using a Coulter Counter (model ZF) .
  • Thrombocytes were counted using a Cell-Dyn 100 thrombocyto- meter (Sequoia-Turner) .
  • mAb conjugated either with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) : Leu-4 (CD3) for T cells, Leu-M3 (CD14) for monocytes, Leu-16 (CD20) for B cells, HPCA-2 (CD34) for progenitor cells, Leu-19 (CD56) for NK cells (all obtained from Becton and Dickinson), and CLB-gran/2 (CD15) for myeloid cells. Platelet adhesion to progenitor cells was studied using non- conjugated CLB-thromb/7 (CD41) followed by incubation with FITC- conjugated goat anti-mouse immunoglobulin (CLB) .
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin

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EP96902498A 1995-02-02 1996-01-17 Anreicherung der hämiporetischen stammzellen aus blut oder knochenmark Withdrawn EP0807168A1 (de)

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EP96902498A EP0807168A1 (de) 1995-02-02 1996-01-17 Anreicherung der hämiporetischen stammzellen aus blut oder knochenmark

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EP95200254 1995-02-02
EP95200254 1995-02-02
EP96902498A EP0807168A1 (de) 1995-02-02 1996-01-17 Anreicherung der hämiporetischen stammzellen aus blut oder knochenmark
PCT/NL1996/000031 WO1996023872A1 (en) 1995-02-02 1996-01-17 Enrichment of hematopoietic stem cells from blood or bone marrow

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WO2021115912A1 (en) 2019-12-09 2021-06-17 Basf Se Formulations comprising a hydrophobically modified polyethyleneimine and one or more enzymes
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JPH10513054A (ja) 1998-12-15
AU704236B2 (en) 1999-04-15
CA2211380A1 (en) 1996-08-08
WO1996023872A1 (en) 1996-08-08

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