WO2006069481A1 - Procede d'enrichissement de cibles en cellules souches et dispositif d'enrichissement magnetique pour localisation dans des cibles - Google Patents

Procede d'enrichissement de cibles en cellules souches et dispositif d'enrichissement magnetique pour localisation dans des cibles Download PDF

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Publication number
WO2006069481A1
WO2006069481A1 PCT/CN2004/001560 CN2004001560W WO2006069481A1 WO 2006069481 A1 WO2006069481 A1 WO 2006069481A1 CN 2004001560 W CN2004001560 W CN 2004001560W WO 2006069481 A1 WO2006069481 A1 WO 2006069481A1
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WIPO (PCT)
Prior art keywords
magnetic
stem cells
cells
enrichment
bracket
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PCT/CN2004/001560
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English (en)
Chinese (zh)
Inventor
Xuetao Pei
Yunfang Wang
Xue Nan
Yanhua Li
Wen Yue
Fang Yan
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Beijing Institute Of Transfusion Medicine
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Priority to PCT/CN2004/001560 priority Critical patent/WO2006069481A1/fr
Publication of WO2006069481A1 publication Critical patent/WO2006069481A1/fr

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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
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood

Definitions

  • the invention relates to a method for targeting and enriching stem cells and a magnetic targeting and enrichment instrument.
  • organ transplantation With the development of organ transplantation, transplantation immunology and the development and application of immunosuppressive agents, organ transplantation has become an effective means of treating end-stage diseases.
  • organ transplantation For the end-stage liver failure caused by various causes (including poisons, drug poisoning, genetic metabolic diseases, etc.), orthotopic liver transplantation is currently the only effective treatment.
  • the widespread use of liver transplantation in clinical settings is severely limited by the extreme shortage of donor liver, the high mortality associated with surgery and transplantation itself, and the lifetime use of immunosuppressants and the serious or even fatal complications they cause.
  • the treatment of refractory and end-stage diseases such as sexual diseases has increasingly shown good application prospects.
  • c-Met+p 2 M-cells can differentiate into mature hepatocytes
  • mesenchymal stem cells can differentiate into neurons, cardiomyocytes or islet ⁇ cells, and the like.
  • stem cell transplantation After stem cell transplantation, there is a process of chemotaxis, migration and colonization in the target tissue, which eventually colonizes the microenvironment and differentiates, matures, and even functions compensatory. In general, only a sufficient number of stem cells are enriched in the target tissue to perform the desired function.
  • common methods for stem cell transplantation include direct injection of target tissue and peripheral intravenous infusion. Although the former can ensure that stem cells can be relatively concentrated and concentrated in the target tissue, it needs to be realized through open surgery. The operation is complicated, the recipient is traumatic and has many complications. The latter method is simple and easy to operate, but the target stem cells follow the blood circulation.
  • the object of the present invention is to provide an efficient and simple method for targeting and enriching stem cells.
  • T N2004/001560 Magnetic Targeting and Enrichment Instrument.
  • the method for targeting and enriching stem cells comprises the following steps: 1) labeling stem cells with immunomagnetic beads having a particle diameter of 30-70 nm to obtain labeled cells; 2) using the labeled stem cells Injecting into the circulatory system of the target tissue; 3) placing the target tissue directly between the two opposite magnetic poles, and concentrating the stem cells in the target tissue during the circulation of the circulatory system;
  • the magnetic field strength between the two opposite magnetic poles is 9200-24500 Gs.
  • the stem cell is a stem cell having a multi-directional differentiation potential with a specific cell surface marker, and may be derived from human or other mammalian bone marrow, mobilized peripheral blood, cord blood or other tissues.
  • a specific cell surface marker for example, in the case of rat bone marrow c-Met M-cells, the process of labeling the stem cells with immunomagnetic beads is as follows: the specific antibody against the stem cell surface marker antigen is incubated with the stem cells, and after washing, The immunomagnetic beads conjugated to the specific antibody are incubated and washed to obtain the labeled cells.
  • the specific antibody is a rabbit anti-rat c-Met antibody and a ⁇ 2 ⁇ -antibody; the immunomagnetic beads are coupled with a goat anti-rabbit IgG.
  • enrichment can be carried out in the magnetic targeting and enrichment apparatus of the present invention, and the distance between the magnetic poles can be adjusted to 5 - 25 mm. At this time, a high-intensity, high-gradient magnetic field is formed between the two magnetic poles.
  • the high intensity of the magnetic field enables the target cells labeled by the immunomagnetic beads to have a good enrichment effect, and the high gradient of the magnetic field enables the cells labeled with the immunomagnetic beads to Selectively retained in the target tissue rather than other tissues than the target tissue.
  • the magnetic targeted positioning enrichment apparatus comprises a bracket, wherein the bracket is vertically disposed opposite to the second magnetic pole, the lower magnetic pole is vertically disposed on the bottom surface of the bracket, and the upper magnetic pole corresponds to the lower magnetic pole Placed on the top surface of the bracket.
  • the upper magnetic pole is movably disposed on a top surface of the bracket.
  • a screw hole is disposed in a top surface of the bracket corresponding to the lower magnetic pole, a screw is disposed in the screw hole, and the lower end of the screw is connected to the upper magnetic pole.
  • a rotating handle is provided at the upper end of the screw.
  • the magnetic poles are cylindrical, and the diameters of the two magnetic poles near the opposite end are reduced.
  • the material of the bracket and the magnetic pole are selected from a permanent magnet material, such as neodymium iron boron.
  • DRAWINGS 0 is a schematic structural view of a magnetic targeting and enrichment analyzer of the present invention.
  • Figure 2 is a plan view of Figure 1;
  • Figure 3 is a schematic view showing the structure of immunomagnetic bead-labeled stem cells
  • Figure 4 is a schematic view of a magnetic field screening cell
  • Figure 5 is a schematic diagram showing the in vitro enrichment operation procedure of immunomagnetic bead labeled cells
  • Figure 6 is an enrichment photograph of immunomagnetic bead labeled cells in rat lung and liver.
  • the present invention mainly provides a method for stem cell targeted localization enrichment, and a magnetic targeting localization enrichment apparatus for use in the method, and the method and apparatus of the present invention are described below in conjunction with specific experiments.
  • the procedure of the present invention is specifically described by taking targeted localization enrichment of c-Met + p 2 MT cells as an example.
  • the liver of the rat model of liver injury is used as a target tissue, and other tissues may be used as a target tissue.
  • the immunomagnetic beads used were purchased from the School of Materials, Tianjin University. The magnetic beads were coupled with goat anti-rabbit IgG with a particle size of about 50 nm and good superparamagnetism.
  • the rats sacrificed by cervical dislocation were disinfected with 75% ethanol, and the femur was removed by laparotomy.
  • the DMEM medium containing 10% fetal bovine serum was used as the rinsing solution.
  • the medullary cavity was repeatedly washed with a 5 ml syringe and filtered through a 100-mesh sieve.
  • the cells were made into a single cell suspension of rat bone marrow. After centrifugation at 1000 rpm, the cells were resuspended in PBS, and the rat bone marrow mononuclear cells were collected by density gradient centrifugation at a relative density of 1.083 g/ml, washed and resuspended. In PBS.
  • the cells 21 obtained in the step 1 were first incubated with rabbit anti-rat ⁇ 2 ⁇ antibody 22 at 4 ° C for 30 min, washed with PBS buffer, and then coupled with goat anti-rabbit IgG 24 .
  • the immunomagnetic beads 23 were incubated at 4 ° C for 30 min and then washed with PBS, so that cells expressing ⁇ 2 ⁇ were labeled with immunomagnetic beads.
  • the cells are flowed through a plastic hose 27 between the two magnetic poles 29, wherein the cells 25 (ie, positive cells) expressing the ⁇ 2 ⁇ labeled by the immunomagnetic beads are retained in the magnetic field.
  • the unlabeled cells 26 i.e., negative cells
  • the cells 26 flow out of the magnetic field, and the cells 26 are collected by the Eppendorf tube 28; and the cells 25 retained in the magnetic field are collected by buffer washing after removing the magnetic field.
  • the collected ⁇ 2 ⁇ -cell population 26 was incubated with rabbit anti-rat c-Met antibody and immunomagnetic bead-labeled goat anti-rabbit IgG, and washed with PBS. In this way, cells in ⁇ 2 ⁇ In the group, all c-Met M-cells expressing c-Met are labeled by immunomagnetic beads. When flowing through a magnetic field, the cells are retained in the magnetic field. After the plastic hose is removed from the magnetic field, the cells are washed out with PBS. , to obtain the target cell - immunomagnetic bead labeled c-Met + p 2 M - cells. In order to effectively detect the enrichment efficiency of transplanted cells in the recipient, the cells were labeled with fluorescent dye DAPI and fully washed.
  • F344 rats Male first inbred F344 rats, weighing 180 ⁇ 200g, were housed at room temperature 20 ⁇ 22°C, light and alternating for 12 hours, free to drink and eat.
  • the propylene alcohol was intraperitoneally injected into the F344 rat at a dose of 0.62 m O l / g body weight to prepare a drug-induced rat liver injury model, which was routinely reared for 3 days.
  • the rat is fixed in a magnetic targeting localization enrichment instrument, so that the body surface projection area corresponding to the liver faces the upper and lower magnetic poles, and then the target cells obtained in step 2 are obtained through the peripheral vein (tail vein or femoral vein) of the rat- - fluorescein-labeled ⁇ + ⁇ 2 ⁇ -cells carrying immunomagnetic beads are injected into the body, so that the target stem cells labeled with immunomagnetic beads can be distributed along the blood circulation to each other distributed in the liver under the guidance of magnetic force.
  • the target stem cells can penetrate into the liver tissue through the vascular endothelial cells and integrate into the hepatocyte plate, so that c-Met+p 2 M-cells can be targeted and enriched. In liver tissue.
  • the magnetic targeting positioning enrichment instrument used in the above operation includes a bracket 1 in which the upper and lower sides of the bracket 1 are oppositely disposed with two magnetic poles, and the lower magnetic pole 2 is vertically disposed on the bracket 1 On the bottom surface, the upper magnetic pole 3 is disposed on the top surface of the bracket 1 corresponding to the lower magnetic pole 2.
  • the upper magnetic pole 3 is movably disposed on the top surface of the bracket 1; the specific method is to provide a screw hole 11 in the top surface of the bracket 1 corresponding to the lower magnetic pole 2, and the screw hole 11 is A screw 12 is disposed, the lower end of the screw 12 is connected to the upper magnetic pole 3, and the upper end of the screw 12 is provided with a rotating handle 13. By rotating the rotating handle 13, the screw 12 can be rotated in the screw hole 11, so that the screw moves in the vertical direction with respect to the top surface of the bracket 1, so that the upper magnetic pole 3 can be moved in the vertical direction, thereby changing the upper and lower sides. The distance between the poles.
  • the two magnetic poles are formed in a cylindrical shape.
  • the diameter of the two magnetic poles near the opposite end is reduced, and the more the diameter is reduced, the higher the magnetic field gradient between the two magnetic poles.
  • the material of the bracket and the two magnetic poles are all made of neodymium iron boron.
  • the neodymium iron boron is a very good permanent magnet material, and the bracket is also made of the same material as the magnetic pole, which can effectively improve the magnetic field strength between the magnetic poles.
  • the rat is first fixed on the stent 1 in order to make it large.
  • 004 001560 The mouse can be better fixed on the bracket 1, and a spacer 4 can be placed inside the bracket, and the spacer 4 and the lower magnetic pole 2 are kept in the same plane; the body surface projection area corresponding to the target organ of the rat Directly facing the upper and lower magnetic poles, the distance between the magnetic poles is adjusted by rotating the rotating handle 13, so that it is adapted to the requirements of the body shape of the rat, and secondly, an ideal magnetic field strength can be obtained; and then the rat peripheral vein (tail) is passed.
  • Intravenous or femoral veins Inject cells labeled with immunomagnetic beads into the body, so that the cells labeled with the immunomagnetic beads can be enriched along the blood circulation to the blood vessels distributed in the target organs under the guidance of magnetic force.
  • the prolongation of the residence time of the rat in the magnetic field the target cells can penetrate into the tissue into the target organ through the vascular endothelial cells, and proliferate, differentiate, mature, and finally play the specific cells in the appropriate microenvironment provided by the tissue. Functional compensation. It has been confirmed by in vitro enrichment and in vivo enrichment experiments that the method of the present invention can achieve a good enrichment effect.
  • the immunomagnetic bead labeled ⁇ 2 ⁇ + cells were obtained by the procedure of step 1 - 2 in the targeted localization enrichment experiment of c-Met + p 2 ]Vr cells, and washed with PBS. Then, physiological saline containing 20% dextran was prepared as a mobile phase, and the obtained cells were suspended therein. Adjust the peristaltic pump flow rate to 0.1 ⁇ 15cm/s, so that the magnetic bead-labeled cells flow through the magnetic field (adjust the distance between the two magnetic poles by 1.6 ⁇ 2.5cm, so that the field strength fluctuates
  • Fig. 5 The operation process is shown in Fig. 5.
  • 15 is a 20% dextran physiological saline solution with ⁇ 2 ⁇ + cells labeled with immunomagnetic beads; 16 is a peristaltic pump; 17 is a magnetic pole of two magnetic poles; Collect the resulting cells.
  • Inbred F344 rats were taken, male or female, and a liver injury model was prepared as in Example 1 step 4.
  • Animals were divided into 3 groups: Gl, peripheral vein (femoral vein) transplantation + magnetic field; G2, open abdominal portal vein transplantation; G3, peripheral vein transplantation (control group, no magnetic field).
  • Gl peripheral vein transplantation + magnetic field
  • G2 open abdominal portal vein transplantation
  • G3 peripheral vein transplantation (control group, no magnetic field).
  • the operation of the G1 group is as follows:
  • the rats are placed in the magnetic targeting locator and fixed in the limbs, so that the projection surface of the liver surface faces the upper and lower magnetic poles;
  • the rats were sacrificed on the 3rd and 10th day after operation, and the frozen sections were prepared from the liver and lung tissues. The distribution of fluorescently labeled cells in the liver and lung of the recipient rats was observed under a fluorescence microscope.
  • Fig. 6A and Fig. 6B after the cells were transplanted through the femoral vein, the rats were fixed in the magnetic field for 20 h and 2 h, and the cells were trapped in the lung; Fig. 6C is the transplantation of the cells through the portal vein. The cells were retained in the lungs; Figure 6D and Figure 6E show that after the transfemoral cells were transplanted, the rats were immobilized in a magnetic field for 20 h to remove the magnetic field, and after 3 d and 10 d, the rats were observed to be rich in the liver.
  • Figure 6F shows the enrichment of cells in the liver after portal vein transplantation
  • Figure 6G and Figure 6H show that after transfemoral transplantation, the rats were fixed in a magnetic field for 2 h and moved away from the magnetic field. After 3 d and 10 d, the cells were Enrichment of the liver.
  • an immunomagnetic bead having a diameter of about 50 nm is specifically labeled by an antigen-antibody affinity reaction. Due to the superparamagnetism of the immunomagnetic bead, the target cell is directionally enriched in a target in a magnetic field under the action of a magnetic field. Tissue, and when the magnetic field disappears, the immunomagnetic beads lose their magnetism, and the cells in the target tissue do not block the blood vessels due to mutual attraction and aggregation.
  • the main raw materials for the preparation of the immunomagnetic beads can be absorbed and excreted by the organism, and finally absorbed and metabolized by the body in the form of iron porphyrin, which has no toxic and side effects on the body.
  • the magnetic pole positioning and concentrating instrument of the invention has two magnetic poles in a cylindrical shape, and the diameters of the two magnetic poles near the opposite end are reduced, and the materials of the magnetic pole and the bracket are all made of neodymium iron boron, which can provide a ⁇ gradient and a high intensity magnetic field. . And by rotating the handle to adjust the distance between the two magnetic poles, the adjustment of the distance between the magnetic poles can change the distance between the two magnetic poles, so that the magnetic field strength between the magnetic poles can be adjusted to
  • the invention skillfully according to the magnetic characteristics of the superparamagnetic magnetic beads, utilizes the high-strength and high-gradient magnetic field generated by the magnetic targeting and enrichment instrument to make the target cells locate and enrich in the target tissue, and has high enrichment efficiency and method. It has the advantages of simplicity and so on, and has broad application prospects.

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Abstract

La présente invention concerne un procédé d'enrichissement de cibles en cellules souches et un dispositif d'enrichissement magnétique destiné au positionnement dans des cibles. L'enrichissement de cibles en cellules souches décrit dans l'invention inclut les étapes suivantes : 1) marquage des cellules souches à l'aide de perles immunomagnétiques de 30 à 70 nm afin d'obtenir des cellules souches marquées ; 2) injection des cellules souches marquées dans le système circulatoire du tissu cible ; 3) positionnement de la région de la surface du corps qui correspond au tissu cible face à deux pôles magnétiques s'attirant mutuellement et enrichissement du tissu cible en lesdites cellules souches apportées par le système circulatoire. L'intensité magnétique desdits pôles magnétiques s'attirant mutuellement est de 9200 à 24500 Gs. Selon la présente invention, les cellules souches sont marquées à l'aide de perles immunomagnétiques et le dispositif d'enrichissement magnétique destiné au positionnement dans les cibles permet de créer un champ magnétique de gradient et d'intensité élevés. Une utilisation habile de la nature magnétique des perles super-paramagnétiques permet d'enrichir le tissu cible en des cellules données. L'invention présente plusieurs avantages tels que l'efficacité élevée de l'enrichissement, l'aspect pratique et d'autres et son champ d’application est vaste.
PCT/CN2004/001560 2004-12-29 2004-12-29 Procede d'enrichissement de cibles en cellules souches et dispositif d'enrichissement magnetique pour localisation dans des cibles WO2006069481A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045407A1 (fr) * 1997-04-09 1998-10-15 Childrens Hospital Los Angeles Selection magnetique de populations de cellules heterogenes pour le traitement d'affections malignes et hematologiques
US6148823A (en) * 1999-03-17 2000-11-21 Stereotaxis, Inc. Method of and system for controlling magnetic elements in the body using a gapped toroid magnet
US6296604B1 (en) * 1999-03-17 2001-10-02 Stereotaxis, Inc. Methods of and compositions for treating vascular defects
US20020164659A1 (en) * 2000-11-30 2002-11-07 Rao Galla Chandra Analytical methods and compositions
CN1470288A (zh) * 2002-07-24 2004-01-28 衡阳科晶微电子有限公司 顺磁材料在医药上的应用
CN1506120A (zh) * 2002-12-11 2004-06-23 中国科学院电工研究所 磁性药物靶向治疗的药物定位方法
CN1608677A (zh) * 2003-10-22 2005-04-27 中国人民解放军军事医学科学院野战输血研究所 一种干细胞靶向定位富集的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045407A1 (fr) * 1997-04-09 1998-10-15 Childrens Hospital Los Angeles Selection magnetique de populations de cellules heterogenes pour le traitement d'affections malignes et hematologiques
US6148823A (en) * 1999-03-17 2000-11-21 Stereotaxis, Inc. Method of and system for controlling magnetic elements in the body using a gapped toroid magnet
US6296604B1 (en) * 1999-03-17 2001-10-02 Stereotaxis, Inc. Methods of and compositions for treating vascular defects
US20020164659A1 (en) * 2000-11-30 2002-11-07 Rao Galla Chandra Analytical methods and compositions
CN1470288A (zh) * 2002-07-24 2004-01-28 衡阳科晶微电子有限公司 顺磁材料在医药上的应用
CN1506120A (zh) * 2002-12-11 2004-06-23 中国科学院电工研究所 磁性药物靶向治疗的药物定位方法
CN1608677A (zh) * 2003-10-22 2005-04-27 中国人民解放军军事医学科学院野战输血研究所 一种干细胞靶向定位富集的方法

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