WO2018082316A1 - Application de cépharanthine et milieu de culture et procédé d'accroissement de cellules souches hématopoïétiques - Google Patents

Application de cépharanthine et milieu de culture et procédé d'accroissement de cellules souches hématopoïétiques Download PDF

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WO2018082316A1
WO2018082316A1 PCT/CN2017/089495 CN2017089495W WO2018082316A1 WO 2018082316 A1 WO2018082316 A1 WO 2018082316A1 CN 2017089495 W CN2017089495 W CN 2017089495W WO 2018082316 A1 WO2018082316 A1 WO 2018082316A1
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hematopoietic stem
stem cells
medium
cord blood
interleukin
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PCT/CN2017/089495
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Chinese (zh)
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王一飞
陈海佳
马岩岩
王小燕
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广州康琪莱生物科技有限公司
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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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2301—Interleukin-1 (IL-1)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the present invention relates to the field of biotechnology, and in particular to the use of alkaloids and a medium and method for amplifying hematopoietic stem cells.
  • Hematopoietic stem cells are a group of primitive hematopoietic cells present in hematopoietic tissues, adult stem cells in the blood system, capable of long-term self-renewal and the potential to differentiate into various mature blood cells. It is the longest and most in-depth study of adult stem cells, and has important guiding significance for the study of various stem cells, including cancer stem cells. Hematopoietic stem cells are effective methods for the treatment of malignant tumors such as leukemia, lymphoma, autoimmune diseases and innate immune defects, which can seriously alleviate human suffering and can effectively alleviate the suffering of patients.
  • cord blood transplantation Since Gluckman et al. first successfully performed HLA-matched cord blood transplantation for a 5-year-old Fanconi anemia boy in 1989, cord blood transplantation has become more and more prominent in the field of hematopoietic stem cell transplantation. However, the content of single cord blood hematopoietic stem cells is limited, which can not meet the needs of adults over 50kg. Therefore, the expansion of cord blood hematopoietic stem cells is a hot research topic at home and abroad.
  • the pathway for amplifying hematopoietic stem cells in vitro mainly adopts a liquid medium supplemented with serum and various cytokines, or co-culture with stromal cells or culture in a bioreactor, but the above method still cannot obtain sufficient transplantation activity.
  • Hematopoietic stem cells are used for clinical treatment and are prone to dry loss of hematopoietic stem cells and failure of transplantation.
  • Chinese patent CN105112374A discloses an in vitro expansion medium for umbilical cord blood hematopoietic stem cells and application thereof.
  • the in vitro expansion medium is based on IMEM basal medium, and serum, thrombopoietin, stem cell factor, FMS-like tyrosine are added.
  • the object of the present invention is to provide an application of the scutellaria alkaloids in culturing and expanding hematopoietic stem cells, thereby promoting the proliferative ability of hematopoietic stem cells, particularly umbilical cord blood hematopoietic stem cells, and significantly increasing the proliferation effect in terms of quantity and content. .
  • Another object of the present invention is to provide a medium comprising a thousand vines such that the medium can promote the proliferation of hematopoietic stem cells, particularly cord blood hematopoietic stem cells.
  • Another object of the present invention is to provide a method for in vitro expansion of cord blood hematopoietic stem cells using a medium containing ginsengine, such that the final result of the method significantly increases the proliferation effect of the number and content of umbilical cord blood hematopoietic stem cells.
  • the present invention provides the following technical solutions:
  • the hematopoietic stem cell is a cord blood hematopoietic stem cell.
  • kansine can increase peripheral blood leukopenia, and is used for leukopenia caused by tumor chemotherapy, radiotherapy, and other causes of leukopenia. There is no report on the in vitro value-added effect of scutellaria on umbilical cord blood hematopoietic stem cells.
  • the invention uses the ginsengine as a new component and is added to the umbilical cord blood hematopoietic stem cell expansion medium, which can significantly increase the proliferation effect of the cord blood hematopoietic stem cells in quantity and content compared with the common expansion medium, so that Hematopoietic stem cells are characterized by strong dryness and high activity. Based on this, the present invention provides an in vitro expansion medium for cord blood hematopoietic stem cells comprising scutellarin.
  • the saponin in the range of 50-100 ⁇ mol/mL, the saponin not only exhibits the advantages of low toxicity to cord blood hematopoietic stem cells, but also maximizes its excellent proliferation. Promote the effect.
  • the present invention is directed to the advantages of the genus sylvestine, and other suitable components are also selected as an in vitro expansion medium for constituting cord blood hematopoietic stem cells.
  • suitable components include:
  • DMEM/F12 medium FBS, thrombopoietin, stem cell growth factor, human FMS-like tyrosine kinase 1 ligand, interleukin-1, interleukin-6.
  • each of the above components is based on DMEM/F12 medium, and the following concentrations are added:
  • FBS 5%-10% FBS, 15-30 ng/ml thrombopoietin, 100-200 ng/ml stem cell growth factor, 33-56 ng/ml human FMS-like tyrosine kinase 1 ligand, 0.2-0.5 ng/ml interleukin- 1. 0.2-0.5 ng/ml interleukin-6, 50-100 ⁇ mol/ml ginsengine.
  • each component has a concentration of 8% FBS, 20 ng/ml thrombopoietin, 150 ng/ml stem cell growth factor, 45 ng/ml human FMS-like tyrosine kinase 1 ligand, 0.4 ng/ml interleukin-1. , 0.4 ng / ml interleukin-6, 75 ⁇ mol / ml ginsengine.
  • the concentration of each component may also be selected as follows:
  • the present invention provides a method for expanding cord blood hematopoietic stem cells in vitro, and inoculation of cord blood hematopoietic stem cells into a medium mentioned in any one of the above technical solutions of the present invention.
  • cord blood hematopoietic stem cells can be obtained by extraction according to a conventional method in the art, and are obtained by an immunomagnetic bead cell sorting technique in the specific implementation of the present invention. Specific methods can be referred to as follows:
  • Step 1 Mix the cord blood with PBS in a volume ratio of 1:1, then add 6% hydroxyethyl starch solution according to the volume ratio of 4:1, and let it stand at room temperature for 20-30 minutes until the red blood cells naturally settle to When the boundary is clear, the upper solution is aspirated into a 50 ml centrifuge tube;
  • Step 2 centrifuge at 1500 rpm for 5 min. After centrifugation, remove the centrifuge tube, collect the lower mononuclear cells, wash with 3-5 ml of PBS, and resuspend the cells for use;
  • Step 3 the cells are counted with countstar, and the cell suspension having a density of 2 ⁇ 10 8 /ml is adjusted, and the mixture is added in a ratio of 100 ⁇ l of CD34 antibody per ml of the cell suspension, and incubated at room temperature for 15-20 minutes;
  • Step 4 According to the ratio of adding 50 ⁇ l of magnetic beads per ml of cell suspension, add CD34+ magnetic beads and mix thoroughly by pipetting, and incubate for 10-20 minutes at room temperature. CD34+ hematopoietic stem cell cells were sorted out in strict accordance with the procedure of the immunomagnetic bead cell sorting kit, and used.
  • the cord blood hematopoietic stem cells are seeded at a density of 1 ⁇ 10 5 /mL, and the culture is carried out at 37 ° C under 5% CO 2 , usually 3-7 days.
  • the present invention selects a plurality of control media including CN105112374A Example 3 amplification medium as a comparison object, cultures under the same cord blood stem cell source and culture environment, and collects cells on the 3rd and 7th day of culture. Cell number and CD34 cell content determination were performed. The results showed that the hematopoietic stem cells cultured in the in vitro expansion medium of the present invention had a magnification of about 60 times on the 7th day and a cell content of about 98%, which had an obvious amplification effect.
  • the present invention provides a related application of the aglycone in the expansion of hematopoietic stem cells, and the expansion effect of hematopoietic stem cells can be significantly improved by the action of a plurality of suitable components and a medium composed of aglycone. It has the characteristics of strong dryness and high activity.
  • Figure 1 shows the effect of different concentrations of sinomenine on the activity of cord blood hematopoietic stem cells; 1 is the result when cultured for 48 hours; 2 is the result when cultured for 72 hours.
  • the embodiment of the invention discloses the application of the scutellaria and a medium and a method for amplifying umbilical cord blood hematopoietic stem cells. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the application, the medium and the method of the present invention have been described by the preferred embodiments, and it is obvious that the application, the medium and the method described herein can be modified or appropriately changed without departing from the scope, spirit and scope of the present invention.
  • the techniques of the present invention are implemented and applied in combination.
  • the cord blood is mixed with PBS in a volume ratio of 1:1, and then mixed with 6% hydroxyethyl starch solution in a volume ratio of 4:1, and allowed to stand at room temperature for 20-30 minutes, until the red blood cells naturally settle to a clear boundary. , aspirate the upper layer solution into a 50 ml centrifuge tube;
  • Centrifuge at 1500 rpm for 5 min. After centrifugation, remove the centrifuge tube, collect the lower mononuclear cells, wash with 3-5 ml of PBS, and resuspend the cells for use;
  • the cells were counted with countstar, and the cell suspension having a density of 2 ⁇ 10 8 /ml was adjusted, and the mixture was added in a ratio of 100 ⁇ l of CD34 antibody per ml of the cell suspension, and incubated at room temperature for 15-20 minutes;
  • CD34+ magnetic beads Add 50 ⁇ l of magnetic beads per ml of cell suspension, add CD34+ magnetic beads, mix thoroughly by pipetting, and incubate for 10-20 minutes at room temperature.
  • CD34+ hematopoietic stem cell cells were sorted out in strict accordance with the procedure of the immunomagnetic bead cell sorting kit, and used.
  • Example 2 Safety test of scutellarin on cord blood hematopoietic stem cells
  • the drug concentration of 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 ⁇ mol/ml was diluted with DMEM/F12 basal medium.
  • Example 1 The cells isolated in Example 1 were seeded at a density of 1 ⁇ 10 4 /mL in a 96-well plate, and different concentrations of scutellarin were added at a concentration of 100 ul per well to culture the umbilical cord blood hematopoietic stem cells without the addition of scutellarin.
  • the base was a normal cell group, and cultured at 37 ° C, 5% CO 2 for 48 h and 72 h.
  • the OD value was measured by a microplate reader according to the instructions of the MTT kit, and the survival rate of the cells was calculated. The results are shown in Fig. 1.
  • the hematopoietic stem cell survival rate is the highest when the concentration of scutellarin is 50-100 ⁇ mol/ml, whether it is cultured for 48 h or 72 h, so 50-100 ⁇ mol/ml is used. It is the safe concentration of the sinomenine in the present invention.
  • Example 3 Comparative experiment on amplification effects of different amplification media
  • Control medium 1 DMEM/F12 + 10% FBS;
  • Control medium 2 CN105112374A Example 3 amplification medium
  • Positive control medium DMEM/F12 + 10% FBS + 15 ng / ml thrombopoietin + 100 ng / ml stem cell growth factor + 33 ng / ml human FMS-like tyrosine kinase 1 ligand + 0.2 ng / ml interleukin -1 + 0.2 Ng/ml interleukin-6+1uM SR-1 (Note: SR-1 is a puromycin derivative that has been obtained by screening technology and has been confirmed so far to promote large-scale expansion and self-renewal of human CD34+ hematopoietic stem cells. Small molecules, generally can increase the number of cells by 50 times, so it can be used as a positive control drug, but its cost is relatively high, and it is not easy to purchase);
  • Medium of the invention 1 DMEM/F12 medium + 8% FBS + 20 ng / ml thrombopoietin + 150 ng / ml stem cell growth factor + 45 ng / ml human FMS-like tyrosine kinase 1 ligand + 0.4 ng / ml interleukin - 1 + 0.4 ng / ml interleukin-6 + 75 ⁇ mol / ml ginsengine.
  • Medium of the invention 2 DMEM/F12 medium + 5% FBS + 15 ng / ml thrombopoietin + 100 ng / ml stem cell growth factor + 33 ng / ml human FMS-like tyrosine kinase 1 ligand + 0.2 ng / ml interleukin - 1 + 0.2 ng / ml interleukin-6 + 50 ⁇ mol / ml ginsengine.
  • Medium of the invention 3 DMEM/F12 medium + 10% FBS + 30 ng / ml thrombopoietin, 200 ng / ml stem cell growth factor + 56 ng / ml human FMS-like tyrosine kinase 1 ligand + 0.5 ng / ml interleukin - 1 + 0.5 ng / ml interleukin-6 + 100 ⁇ mol / ml of ginsengine.
  • the hematopoietic stem cells isolated in Example 1 were seeded at a density of 1 ⁇ 10 5 /ml in a 6-well plate, 2 ml/well, and added to each group of expansion medium, respectively, and cultured for 3 days and 7 days, respectively, and the cells were harvested. Quantity and determination of CD34 cell content. The number of cells was counted using a countstar automated cell counter. The results are shown in Table 1. The content of CD34+ cells was analyzed by flow cytometry. The results are shown in Table 2.
  • Control medium 1 Control medium 2 Positive control medium 0d 2*10 5 2*10 5 2*10 5 3d 2.3*10 5 4.5*10 6 7.2*10 6 7d 2.1*10 5 6.3*10 6 1.1*10 7 Training days Medium of the invention 1 Medium of the invention 2 Medium of the invention 3 0d 2*10 5 2*10 5 2*10 5 3d 7.12*10 6 6.42*10 7 7.05*10 7 7d 1.23*10 7 1.19*10 7 1.21*10 7
  • the number of hematopoietic stem cells of the control medium 1 did not change significantly; while the positive control medium and the medium of the present invention had significant amplification effects on hematopoietic stem cells.
  • the expansion ratio of the positive control medium was 55 times, and the expansion ratio of the culture medium of the present invention was 60 times; while the control medium 2 of the prior patent was significantly lower than the culture medium and the positive control culture of the present invention.
  • the amplification factor of the base indicates that there is no significant difference in the proliferation effect of hematopoietic stem cells between the ginsengine and the positive drug SR-1, and the amplification factor is even higher than that of the positive drug, and the umbilical cord blood hematopoietic stem cells can be rapidly expanded in vitro.
  • Control medium 1 Control medium 2 Positive control medium 0d 62.3% 62.3% 62.3% 3d 63.4% 71.2% 83.2% 7d 62.1% 80.1% 98.1% Training days Medium of the invention 1 Medium of the invention 2 Medium of the invention 3 0d 62.3% 62.3% 62.3% 3d 85.1% 84.2% 85.0% 7d 97.8% 98.2% 97.6%
  • the hematopoietic stem cell content of the control medium 1 did not change significantly; while the positive control medium and the medium of the present invention significantly increased the content of hematopoietic stem cells.
  • the CD34+ cell content of the positive control medium reached 98.1%, and the cell content of the culture medium of the present invention reached about 98%.
  • the control medium 2 of the prior patent was significantly lower than the present invention.
  • the cell content of the medium and the positive control medium indicates that there is no significant difference in the effect of the scutellaria and the positive drug SR-1 on the content of hematopoietic stem cells, and the dryness of the hematopoietic stem cells can be well maintained in vitro.

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Abstract

La présente invention concerne le domaine technique de la biologie. L'invention concerne une application de cépharanthine dans la préparation d'un milieu de culture d'accroissement ex vivo de cellules souches hématopoïétiques et/ou d'accroissement ex vivo des cellules souches hématopoïétiques et un milieu de culture contenant de la cépharanthine et un procédé d'accroissement de cellules souches hématopoïétiques du sang du cordon ombilical. Selon la présente invention, un milieu de culture d'accroissement ex vivo est composé de multiples constituants appropriés et de cépharanthine et l'effet d'accroissement peut être manifestement amélioré pendant l'accroissement ex vivo de cellules souches hématopoïétiques, de telle sorte que les cellules souches hématopoïétiques présentant les caractéristiques d'un "caractère souche" élevé et d'une activité élevée.
PCT/CN2017/089495 2016-11-04 2017-06-22 Application de cépharanthine et milieu de culture et procédé d'accroissement de cellules souches hématopoïétiques WO2018082316A1 (fr)

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CN113151172A (zh) * 2021-05-14 2021-07-23 郑州优倍得生物科技有限公司 一种用于扩增脐带血造血干细胞的培养基
CN114854678A (zh) * 2022-03-23 2022-08-05 郭昌春 一种用于培养生物干细胞的培养基及其制备方法

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CN107034189A (zh) * 2017-05-31 2017-08-11 东莞市保莱生物科技有限公司 一种造血干细胞培养方法
CN107998041A (zh) * 2017-12-04 2018-05-08 广州赛莱拉干细胞科技股份有限公司 一种含有千金藤提取物的抑菌护肤品
CN109735491A (zh) * 2019-01-16 2019-05-10 广东美赛尔细胞生物科技有限公司 一种可扩增造血干细胞的无血清培养基及其制备方法

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CN114854678A (zh) * 2022-03-23 2022-08-05 郭昌春 一种用于培养生物干细胞的培养基及其制备方法

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