WO2019196816A1 - Utilisation de butyrate de sodium et système de culture contenant du butyrate de sodium - Google Patents

Utilisation de butyrate de sodium et système de culture contenant du butyrate de sodium Download PDF

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WO2019196816A1
WO2019196816A1 PCT/CN2019/081854 CN2019081854W WO2019196816A1 WO 2019196816 A1 WO2019196816 A1 WO 2019196816A1 CN 2019081854 W CN2019081854 W CN 2019081854W WO 2019196816 A1 WO2019196816 A1 WO 2019196816A1
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hematopoietic stem
stem cells
cells
sodium butyrate
cell
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PCT/CN2019/081854
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English (en)
Chinese (zh)
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孙忠杰
刘德芳
肖雄
陈立功
郭潇
齐海龙
王晓芳
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诺未科技（北京）有限公司
诺未科技（银川）有限公司
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Priority claimed from CN201810330298.0A external-priority patent/CN109706119B/zh
Priority claimed from CN201811084025.9A external-priority patent/CN109207427B/zh
Application filed by 诺未科技（北京）有限公司, 诺未科技（银川）有限公司 filed Critical 诺未科技（北京）有限公司
Priority to CN201980001861.2A priority Critical patent/CN110799641B/zh
Publication of WO2019196816A1 publication Critical patent/WO2019196816A1/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
    • 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]

Definitions

  • the invention relates to the technical field of hematopoietic stem cells, in particular to the use of sodium butyrate and a culture system containing sodium butyrate.
  • Hematopoietic stem cells are an extremely important type of stem cells in the body. Although they account for less than one-tenth of the human blood cells, they have strong self-renewal ability and differentiation ability, and can rebuild the entire blood system and immune system of the body for a long time. The differentiation potential of blood cells and immune cells in various generations. Therefore, hematopoietic stem cells are widely used in the clinical treatment of malignant blood diseases such as leukemia and lymphoma. Not only that, hematopoietic stem cell transplantation can also help treat metabolic diseases, congenital immunodeficiency, diabetes and other diseases. According to statistics, there are more than 40,000 hematopoietic stem cell transplants every year in the world.
  • hematopoietic stem cells are mainly derived from the donor's bone marrow and mobilized peripheral blood hematopoietic stem cells that are self-contained or matched to the patient's HLA.
  • HLA matching about 70% of patients still cannot obtain suitable donors and cannot receive treatment; even if they receive treatment, most patients will experience degrees.
  • Different graft-versus-host disease (GVHD) is afflicted.
  • Umbilical cord blood hematopoietic stem cells have relatively low requirements for HLA matching, and their immunogenicity is low. Together with their convenient access and abundant sources, they have gradually become a major source of hematopoietic stem cell transplantation donors.
  • Hematopoietic progenitor cells are a type of cell that has a lower self-renewal ability and differentiation potential than hematopoietic stem cells. Although it expresses CD34 surface antigen, it does not express hematopoietic stem cell-specific CD90 surface molecules, but is CD45RA positive. Therefore, hematopoietic stem cells and hematopoietic progenitor cells can be distinguished by CD90 and CD45RA. Hematopoietic progenitor cells have short-term (less than one month) in vivo transplantation ability, and can be differentiated into red blood cells, lymphocytes, myeloid cells and other blood cells.
  • hematopoietic progenitor cells are much higher than that of hematopoietic stem cells (0.03% vs 0.0001%). If hematopoietic progenitor cells can be transformed into hematopoietic stem cells, the donor source of hematopoietic stem cells will be greatly expanded.
  • Apparent modification plays an important role in regulating cell fate. Methylation, acetylation of the locus, and various group modifications of histones directly affect the openness of genes in adjacent regions and the difficulty of binding transcription factors, thereby regulating the expression of genes and completing the regulation of cell status and fate. Therefore, apparent modification affects cell function at a higher level. Many apparent modifications have been developed to alter cell fate. Recent studies have found that apparent modifications have a significant effect on the in vitro expansion of human hematopoietic stem cells. Sodium butyrate is also highly valued in the field of cell reprogramming because it promotes cell fate changes and is used as a catalyst for IPS preparation.
  • the present invention provides the use of sodium butyrate and a culture system containing sodium butyrate.
  • the present invention shows that sodium butyrate can promote the transformation of hematopoietic progenitor cells to hematopoietic stem cells, and can significantly increase the total amount of cells obtained by in vitro expansion of hematopoietic stem cells.
  • the present invention provides the use of sodium butyrate in promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells.
  • the present invention provides the use of sodium butyrate in the preparation of hematopoietic stem cells.
  • the present invention provides the use of sodium butyrate and hematopoietic progenitor cells for the preparation of hematopoietic stem cells.
  • the invention also provides a composition consisting of sodium butyrate, TPO, SCF and FLT3L.
  • a composition for promoting the transition of hematopoietic progenitor cells to hematopoietic stem cells which consists of sodium butyrate, TPO, SCF and FLT3L.
  • a composition for promoting in vitro expansion of hematopoietic stem cells which consists of sodium butyrate, TPO, SCF and FLT3L.
  • the composition provided by the present invention is used as an additive for a medium for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells and promoting hematopoietic stem cell expansion.
  • the solution of sodium butyrate was arranged in sterile ddH 2 O, and the mother liquor concentration was 200 mol/L.
  • the mass ratio of sodium butyrate, TPO, SCF and FLT3L is (5500 to 22000): (30 to 70): (80 to 120): (90 to 110) ).
  • the mass ratio of sodium butyrate, TPO, SCF, and FLT3L in the composition is 5500:30:80:90.
  • the mass ratio of sodium butyrate, TPO, SCF, and FLT3L in the composition is 11000:50:100:100.
  • the mass ratio of sodium butyrate, TPO, SCF, and FLT3L in the composition is 22000:70:120:110.
  • the mass ratio of the sodium butyrate, TPO, SCF and FLT3L is (10 to 200): (30 to 70): (80 to 120): (90 to 110).
  • the mass ratio of sodium butyrate, TPO, SCF, and FLT3L in the composition is 110:50:100:100.
  • composition of the invention for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells.
  • compositions of the invention in the preparation of hematopoietic stem cells.
  • composition of the invention and hematopoietic progenitor cells for the preparation of hematopoietic stem cells.
  • composition of the invention for promoting hematopoietic stem cell expansion.
  • the present invention provides a culture system for amplifying hematopoietic stem cells, comprising a cell culture medium containing sodium butyrate.
  • the sodium butyrate has a molecular formula of C 4 H 7 NaO 2 and a molecular weight of 110.0869.
  • the concentration of sodium butyrate is 50 to 200 ⁇ mol/L.
  • the concentration of sodium butyrate is 100 ⁇ mol/L.
  • the present invention provides a culture system comprising a basal medium and a composition of the invention.
  • the present invention also provides a culture system for promoting hematopoietic stem cell expansion comprising a basal medium and a composition of the present invention.
  • the invention also provides a culture system for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells, comprising a basal medium and a composition of the invention.
  • the concentration of the sodium butyrate is from 1 ⁇ mol/L to 200 ⁇ mol/L. In some embodiments, the concentration of the sodium butyrate is 1 ⁇ mol/L, 50 ⁇ mol/L, 100 ⁇ mol/L, and 200 ⁇ mol/L.
  • the concentration of the TPO is from 30 ng/mL to 70 ng/mL;
  • the concentration of the SCF is from 80 ng/mL to 120 ng/mL;
  • the concentration of the FLT3L is from 90 ng/mL to 110 ng/mL.
  • the concentration of the sodium butyrate is from 1 ⁇ mol / L to 200 ⁇ mol / L;
  • the concentration of the TPO is from 30 ng/mL to 70 ng/mL;
  • the concentration of the SCF is from 80 ng/mL to 120 ng/mL;
  • the concentration of the FLT3L is from 90 ng/mL to 110 ng/mL.
  • the culture system of the present invention is prepared by adding the composition provided by the present invention to StemSpan SFEM II medium until the concentration of each component is the concentration described in the present invention.
  • the composition of the present invention may be a dry powder which may be present as a mixture or a separate component of each component.
  • the composition may also be a solution, or a mother liquor.
  • the mother liquor includes all or part of the components of the composition.
  • the solvent of the sodium butyrate mother liquor is sterile ddH 2 O, and the solvent of the TPO, SCF and FTL 3L mother liquors is 0.1% BSA.
  • the concentration of the TPO is 30 ng/mL; the concentration of the SCF is 80 ng/mL; and the concentration of the FLT3L is 90 ng/mL.
  • the concentration of the TPO is 50 ng/mL; the concentration of the SCF is 100 ng/mL; and the concentration of the FLT3L is 100 ng/mL.
  • the concentration of the TPO is 70 ng/mL; the concentration of the SCF is 120 ng/mL; and the concentration of the FLT3L is 110 ng/mL.
  • the basal medium is StemPro, RPMI1640, IMDM, ⁇ -MEM or StemSpan SFEM II. In some embodiments, the basal medium is StemSpan SFEM II.
  • the culture systems provided by the present invention include StemSpan SFEM II medium, 1 ⁇ mol/L sodium butyrate, 50 ng/mL TPO, 100 ng/mL SCF, and 100 ng/mL FLT3L.
  • the culture systems provided by the present invention include StemSpan SFEM II medium, 50 ⁇ mol/L sodium butyrate, 30 ng/mL TPO, 80 ng/mL SCF, and 90 ng/mL FLT3L.
  • the culture systems provided by the present invention include StemSpan SFEM II medium, 100 ⁇ mol/L sodium butyrate, 50 ng/mL TPO, 100 ng/mL SCF, and 100 ng/mL FLT3L.
  • the culture systems provided by the present invention include StemSpan SFEM II medium, 200 ⁇ mol/L sodium butyrate, 70 ng/mL TPO, 120 ng/mL SCF, and 110 ng/mL FLT3L.
  • the culture system of the present invention is useful for promoting the transformation of hematopoietic progenitor cells into hematopoietic stem cells.
  • the present invention also provides a method for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells, which cultures hematopoietic progenitor cells by the culture system of the present invention.
  • the hematopoietic progenitor cells are CD34+CD90- and CD34+CD45RA+ cord blood hematopoietic progenitor cells.
  • the cultured CD34+CD90-cell density is 0.1 ⁇ 10 4 cells/mL ⁇ 10 ⁇ 10 4 cells/mL
  • the CD34+CD45RA+ cell density is 0.1 ⁇ 10 4 cells/mL ⁇ 10. ⁇ 10 4 cells/mL.
  • the cultured inoculated CD34+CD90-cell has a density of 0.55 ⁇ 10 4 cells/mL and the CD34+CD45RA+ cell density is 0.13 ⁇ 10 4 cells/mL.
  • the culture conditions were 37 ° C, 5% CO 2 , and the culture period was 5 to 10 days.
  • the present invention also provides a method of amplifying hematopoietic stem cells, which cultures hematopoietic stem cells with the culture system of the present invention.
  • the hematopoietic stem cells are cord blood hematopoietic stem cells; the density of the inoculation is 0.1 ⁇ 10 4 cells/mL to 10 ⁇ 10 4 cells/mL. In some embodiments, the inoculation has a density of 1 x 10 4 cells/mL.
  • the method for in vitro expansion of the hematopoietic stem cells specifically comprises: obtaining fresh blood-like hematopoietic stem cells as sample cells, and performing in vitro expansion culture by using the above-mentioned culture system for expanding hematopoietic stem cells to obtain hematopoietic stem cells.
  • fresh blood samples of hematopoietic stem cells can be derived from bone marrow, peripheral blood, and cord blood.
  • the culture conditions were 37 ° C, 5% CO 2 .
  • Fresh media and compositions provided by the present invention were added every 2 days.
  • the expansion ratio is 4 to 20 times in 5 to 10 days of culture.
  • the present invention provides the use of sodium butyrate for promoting hematopoietic stem cell expansion.
  • Studies have shown that in the process of expansion and culture of umbilical cord blood hematopoietic stem cells, the addition of sodium cyanoate while adding cytokines achieves the effect of increasing the number of hematopoietic stem cells and increasing the ability of hematopoietic stem cells to form CFU colonies, so that hematopoietic stem cells can be proliferated.
  • the state of differentiation meets clinical transplant needs.
  • the invention has the advantages of simple operation, low cost and more obtained hematopoietic stem cells, and solves the defects of low expansion rate and easy differentiation of hematopoietic stem cells in the prior art.
  • hematopoietic progenitor cells are expected to induce hematopoietic progenitor cells into hematopoietic stem cells through the regulation of epigenetic modification, and can realize hematopoietic stem cells in vitro, which can be applied to cord blood, placental blood, peripheral blood, bone marrow-derived hematopoietic stem cells; And hematopoietic progenitor cells have a large number of hematopoietic stem cells, and have the differentiation potential of each lineage, and can provide hematopoietic stem cell donors for clinical applications.
  • Figure 1a is a flow cytometric diagram of hematopoietic stem cells after 5 days of culture of CD34+CD90-progenitor cells (DMSO is the control group);
  • Figure 1b is a flow cytometric diagram of hematopoietic stem cells after 5 days of culture of CD34+CD45RA+ progenitor cells (DMSO is the control group);
  • Figure 2a is a statistical diagram showing the proportion and number of hematopoietic stem cells after 5 days of CD34+CD90-progenitor cell culture (DMSO is the control group);
  • Figure 2b is a statistical diagram of the proportion and number of hematopoietic stem cells after 5 days of culture of CD34+CD45RA+ progenitor cells (DMSO is the control group);
  • Figure 3 is a graph showing the results of statistical analysis of the total number of cells and the total number of CD34+ cells on day 7 of each sample of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 4 is a graph showing the results of the total number of CD34+CD45RA- and CD34+CD90+ cells on day 7 of umbilical cord blood hematopoietic stem cells in each concentration of sodium butyrate;
  • Figure 5 is a graph showing the expression of CD34 surface antigen on day 7 of a representative sample of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 6 is a graph showing the expression of CD34 and CD45RA surface antigens on the 7th day of representative samples of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 7 is a graph showing the expression of CD34 and CD90 surface antigens on the 7th day of representative samples of cord blood hematopoietic stem cells under the action of sodium butyrate;
  • Figure 8 is a representative map of colony formation of each lineage under an inverted microscope, wherein a picture is CFU-E; b picture is CFU-G; c picture is CFU-M; d picture is CFU-GM; e picture is CFU-GEMM;
  • Fig. 9 is a graph showing the number of colony forming units of each lineage of cord blood hematopoietic stem cell samples on the 7th day of each sodium butyrate culture.
  • the invention discloses a granular composition, a preparation method thereof and a preparation thereof, and those skilled in the art can learn from the contents of the present invention 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 method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • a method for preparing artificial blood stem cells using artificial blood progenitor cells comprising the following steps:
  • S1 obtains CD34+CD90- and CD34+CD45RA+ cord blood hematopoietic progenitor cells from human umbilical cord blood;
  • the CD34+CD90- and CD34+CD45RA+ cord blood hematopoietic progenitor cells are suspended and inoculated into a cord blood hematopoietic stem cell-specific transformation medium, and the cord blood hematopoietic stem cell-specific transformation medium is StemSpan SFEM II serum-free medium.
  • CD34+CD90-cells are 0.1 ⁇ 10 4 cells/mL ⁇ 10 ⁇ 10 4 cells/mL
  • CD34+CD45RA+ cells are 0.1 ⁇ 10 4 cells/mL to 10 ⁇ 10 4 cells/mL
  • sodium butyrate 1 ⁇ M was added; and cultured at 37 ° C in a 5% CO 2 incubator.
  • the method for preparing artificial blood stem cells using artificial blood progenitor cells according to the present invention further includes:
  • the state of cell culture 500 ⁇ l of the umbilical cord blood stem cell-specific transformation medium is added every 2 days, and a large number of cells are obtained in 5 to 10 days, and the amplification factor is 4 to 20 times.
  • obtaining CD34+CD90- and CD34+CD45RA+ cord blood hematopoietic progenitor cells from human umbilical cord blood according to step S1 further comprises the following steps:
  • S11 obtains peripheral blood mononuclear cells PBMC
  • S12 obtains CD34 + cord blood hematopoietic stem and progenitor cells from the above PBMC by magnetic bead sorting MACS;
  • the obtaining the peripheral blood mononuclear cell PBMC described in step S11 further comprises the following steps:
  • S111 collects 80-120ml of cord blood with a disposable blood bag containing anticoagulant such as heparin sodium. Transfer the cord blood from the blood bag to a 500ml culture flask, dilute with physiological saline for 2 to 3 times, mix and add 0.4. Double volume lymphocyte separation solution;
  • the centrifuge tube was divided into four layers from top to bottom due to different densities: the first layer was the plasma layer, and the second layer was the ring-shaped milky white mononuclear cell layer PBMC.
  • the third layer is a transparent separation liquid layer, and the fourth layer is an red blood cell layer;
  • step S12 further includes:
  • Each umbilical cord blood PBMC of S121 was resuspended in a mixture of 50 ul of human CD34+ magnetic beads and 50 ul of FcR blocker and 150 ul of 0.5% BSA, and incubated at 4 ° C for 30 minutes;
  • step S125 resuspend the PBMC agglomerate in step S123 with 500ul 0.5% BSA, mix and transfer to the special adsorption column of MACS, until the liquid completely flows out;
  • the obtained liquid is frozen in liquid nitrogen with a cryoprotectant dimethyl sulfoxide DMSO.
  • the method for in vitro expansion of the hematopoietic stem cells of the present invention specifically includes the following steps:
  • step S2 adding the sodium butyrate concentrated solution obtained in step S1 to StemSpan SFEM II minimal medium to obtain a cell culture medium;
  • the purity of the sodium butyrate in the S1 is ⁇ 98.5%, and the concentration of the sodium butyrate concentrated solution is 200 mol/L.
  • the concentrated solution is directly used and diluted according to the sample volume. The concentrated solution must be stored at -20 ° C.
  • step S3 further includes the following steps:
  • S31 separating fresh blood samples into mononuclear cells by using a lymphocyte separation solution, and obtaining CD34+ cells, ie, hematopoietic stem cells, by MACS, and then storing the hematopoietic stem cells in liquid nitrogen;
  • the culture system for amplifying hematopoietic stem cells is for use in preparing hematopoietic stem cells using fresh blood-like hematopoietic stem cells as sample cells.
  • the culture conditions in the step S3 are: culture at 37 ° C under a condition of a CO 2 concentration of 5%.
  • the culture time in the step S3 is 5-10 days.
  • Sodium butyrate is expected to induce hematopoietic progenitor cells into hematopoietic stem cells by epithelial modification, thereby expanding hematopoietic stem cells in vitro, which can be applied to cord blood, placental blood, peripheral blood, bone marrow-derived hematopoietic stem cells;
  • the prepared hematopoietic stem cells are high in number and have the differentiation potential of each lineage, and can provide hematopoietic stem cell donors for clinical application.
  • the invention significantly increases the total amount of cells obtained by in vitro expansion of cord blood hematopoietic stem cells by adding sodium butyrate, and provides a preliminary technical basis for a wider range of clinical trials; the expanded hematopoietic stem cells contain a higher proportion of CD34+CD90+ cells. Group, and has better CFU colony forming ability, indicating that these hematopoietic stem cells are more primitive, have stronger differentiation potential of reconstituted blood system, can more effectively support clinical treatment needs; make hematopoietic stem cell expansion in vitro easier, safer and more efficient .
  • the hematopoietic stem cells are a kind of cells which have self-renewal ability and differentiation ability, can differentiate into various blood cells, and can reconstitute the entire blood system and the immune system of the receptor in the body for a long time, and express CD34 and CD90 surface antigens, CD45RA is not expressed, that is, CD34+CD90+CD45RA-, and in the present invention, both CD34+CD90+ and CD34+CD45RA- represent hematopoietic stem cells.
  • the hematopoietic progenitor cells are a type of cell having a self-renewal ability and a differentiation ability lower than that of hematopoietic stem cells, and can differentiate into a plurality of blood cells, but cannot completely reconstitute the entire blood system and the immune system in the body, although expression CD34 surface antigen, but does not express hematopoietic stem cell-specific CD90 surface molecule, and CD45RA positive, namely CD34+CD90-CD45RA+, in the present invention CD34+CD90- and CD34+CD45RA+ represent hematopoietic progenitor cells.
  • the transformation which may also be referred to as transformation, is the transformation of hematopoietic progenitor cells to hematopoietic stem cells; it may also be referred to as reprogramming, ie, hematopoietic progenitor cells are reprogrammed into hematopoietic stem cells.
  • the reprogramming refers to a state in which hematopoietic progenitor cells are reversed to return to hematopoietic stem cells.
  • the amplification refers to a process in which hematopoietic stem cells are cultured to increase the number thereof.
  • the number of cells can be amplified by 17 times or more.
  • the composition refers to a small molecule compound sodium butyrate, and a combination of growth factors TPO, SCF and FLT3L.
  • the components in the composition of the present invention may be present independently or in combination with each other, which is not limited in the present invention. Each component may be a solution or a powder. In the present invention, the components are present in the form of a solution, and the components are independent of each other.
  • the culture system refers to a nutrient for culturing cells under in vitro conditions, and may also be referred to as a medium.
  • the culture system of the present invention can be used immediately before use, or can be made into a long-term storage of the finished product.
  • the basal medium refers to a medium capable of realizing a desired essential nutrient for expansion or growth of hematopoietic stem cells.
  • the basal medium of the present invention may be in the form of a powder or a culture solution.
  • test materials, reagents or laboratory equipment used in the present invention are all commercially available products, and are commercially available. Specifically, regarding the raw material manufacturers involved in the following examples, the following table (Table 1):
  • StemSpan SFEM II is a serum-free medium supplied by StemCell Technologies under the trade number 09655;
  • RhSCF human stem cell factor
  • Recombinant human FMS-like tyrosine kinase 3 ligand the manufacturer is Stemimmune LLC, the product number is HHM-FT-1000;
  • PBMC Peripheral blood mononuclear cell
  • MACS magnetic bead sorting
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffer
  • MethoCult TM GF H4435 a semi-solid medium
  • CFU-E full name Conoly Forming Unit of Erythrocyte Chinese name is red blood cell colony forming unit
  • CFU-G full name Conoly Forming Unit of Granulocyte Chinese name is granulocyte colony forming unit
  • CFU-GM full name Conoly Forming Unit of Granulocyte-Macrophage Chinese name is granulocyte-macrophage colony forming unit
  • CFU-GEMM full name Conoly Forming Unit of granulocyte, erythrocyte, macrophage/monocyte, megakaryocyte, mixed colony, its Chinese name is granulocyte, red blood cell, macrophage/monocyte, megakaryocyte colony forming unit;
  • PBMC peripheral blood mononuclear cells
  • the centrifuge tube is divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, and the second layer is the ring-shaped milky white mononuclear cell layer (PBMC).
  • the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer.
  • PBMC ring-shaped milky white mononuclear cell layer
  • CD34 + cord blood hematopoietic stem and progenitor cells were obtained from the above PBMC by magnetic bead sorting (MACS).
  • Each cord blood PBMC was resuspended in a mixture of 50 ul of human CD34+ magnetic beads and 50 ul of FcR blocker reagent and 150 ul of 0.5% BSA, and incubated at 4 ° C for 30 minutes.
  • CD34 + cells were stained with flow cytometry CD34, CD90, CD45RA for half an hour, and CD34+CD90- and CD34+CD45RA+ hematopoietic progenitor cells were sorted by flow sorter.
  • the CD34+CD90- and CD34+CD45RA+ cord blood hematopoietic progenitor cells are inoculated and cultured in a special transformation medium for cord blood hematopoietic stem cells.
  • a special transformation medium for cord blood hematopoietic stem cells 100 ng/ml SCF, 100 ng/ml FLT3, 50 ng/ml TPO; cell seeding density in 24-well plates: CD34+CD90-cell 0.55 ⁇ 10 4 /well, CD34+CD45RA+ The cells were 0.13 ⁇ 10 4 / well; Compound B (sodium butyrate) was added to 1 ⁇ M, and the control group was added with DMSO (0.1%); and cultured at 37 ° C in a 5% CO 2 incubator.
  • Compound B sodium butyrate
  • Transformation medium StemSpan SFEM II serum-free medium + 100 ng / ml SCF + 100 ng / ml FLT3 + 50 ng / ml TPO + 1 ⁇ M sodium butyrate;
  • Control medium StemSpan SFEM II serum-free medium + 100 ng / ml SCF + 100 ng / ml FLT3 + 50 ng / ml TPO + 0.1% DMSO;
  • the above-mentioned isolated human placental blood hematopoietic stem cells are subjected to phenotypic identification, viability and purity detection and functional identification, including the following steps:
  • the cultured CD34+CD90+ and CD34+CD45RA-hematopoietic stem cells were counted separately.
  • FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, and APC-labeled CD90 dissolved in 0.5% BSA were added. After vortexing, the tubes were incubated for 15 minutes at room temperature in the dark. Add appropriate amount of PBS, centrifuge at 1600 rpm for 5 minutes at room temperature, discard the supernatant, add 200 ⁇ l of PBS, and then analyze on the machine.
  • MethoCult TM GF H4534 semi-solid medium medium 1 ml/well was added to a six-well plate, CD34 + cells were seeded at a density of 1000 cells/well, and placed in a 37 ° C 5% CO 2 incubator for 14 days, and each lineage was calculated. Number of colonies and take photos.
  • a special transformation medium containing sodium butyrate can induce the production of hematopoietic stem cells of the CD34+CD90+ and CD34+CD45RA- phenotypes, whereas ordinary medium cannot.
  • StemSpan SFEM II medium is available from StemCell Technologies, Inc.
  • rhSCF, rhTPO, rhFLT3L is available from Stemimmune LLC; although the invention is preferably used in humans, it can also be used in laboratory animals such as mice; human hematopoietic stem cells can be sourced
  • umbilical cord blood hematopoietic stem cells are taken as an example, wherein umbilical cord blood is collected from healthy maternal pregnant infants, and hepatitis B, hepatitis C, syphilis, AIDS, and hepatitis B, hepatitis C, syphilis, AIDS, Cytomegalovirus, TORCH detection, mycoplasma, chlamydia, G-6PD and thalassemia are negative.
  • Human umbilical cord blood hematopoietic stem cells express several membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiation antigen CD34, leukocyte differentiation anti
  • the centrifuge tube is divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, and the second layer is the ring-shaped milky white mononuclear cell layer (PBMC).
  • the third layer is a transparent separation liquid layer, and the fourth layer is an red blood cell layer;
  • PBMC ring-shaped milky white mononuclear cell layer
  • Each umbilical cord blood PBMC was resuspended in a mixture of 50 ul of human CD34+ magnetic beads and 50 ul of FcR blocker reagent and 150 ul of 0.5% BSA, and incubated at 4 ° C for 30 min;
  • the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 80 ng/ml and the FLT3 to FLT3 is added by using StemSpan SFEM II serum-free medium.
  • the concentration was 90 ng/ml, the concentration of TPO to TPO was 30 ng/ml; the density of cells in the 24-well plate was 1 ⁇ 10 4 /well, sodium butyrate was added at 50 ⁇ M, and the cells were cultured at 37 ° C in a 5% CO 2 incubator.
  • Example 3 The difference from Example 3 is that the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 100 ng/s using StemSpan SFEM II serum-free medium.
  • Ml add FLT3 to FLT3 at a concentration of 100 ng/ml, add TPO to TPO at a concentration of 50 ng/ml; in a 24-well plate, the cell seeding density is 1 ⁇ 10 4 /well, sodium butyrate is added at 100 ⁇ M, and is placed at 37 ° C. Incubate in a 5% CO 2 incubator.
  • Example 3 The difference from Example 3 is that the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 120 ng/s using StemSpan SFEM II serum-free medium.
  • Ml the concentration of FLT3 to FLT3 was 110 ng/ml
  • the concentration of TPO to TPO was 70 ng/ml
  • the density of cells in a 24-well plate was 1 ⁇ 10 4 /well
  • sodium butyrate was added at 200 ⁇ M, and it was placed at 37 ° C. Incubate in a 5% CO 2 incubator.
  • This example is a blank control group, and the CD34+ cord blood hematopoietic stem cells are inoculated into the cell culture medium obtained in the step 2 for culture, and the concentration of SCF to SCF is 80 ng/ml by using StemSpan SFEM II serum-free medium.
  • Flow cytometry analysis was performed on different concentrations of sodium butyrate-cultured CD34+ cells on day 0 and day 7, respectively.
  • a FACS Verse flow Detector from BD
  • 20 ⁇ l of the cell suspension was taken, and FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, and APC-labeled CD90 dissolved in 0.5% BSA were added. After vortexing, the tubes were incubated for 15 min at room temperature, added with appropriate amount of PBS, centrifuged at 1600 rpm for 5 min at room temperature, the supernatant was discarded, 200 ⁇ l of PBS was added, and then analyzed by machine.
  • Colony forming unit analysis was performed on different concentrations of sodium butyrate-cultured CD34+ cells on day 0 and day 7, respectively.
  • MethoCult TM GF H4435 semi-solid medium the medium was added 1ml / hole in the 6-well plate, CD34 + cell seeding density of 1000 cells / well, placed in 37 °C 5% CO 2 incubator for 14 days, colonies were calculated for each lineage Number and take photos.

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Abstract

L'invention concerne une composition contenant du butyrate de sodium et son utilisation pour favoriser la transformation de cellules progénitrices hématopoïétiques humaines en cellules souches hématopoïétiques humaines. Pendant l'expansion et la culture de cellules souches hématopoïétiques du sang du cordon ombilical, l'ajout de butyrate de sodium tout en ajoutant des cytokines peut non seulement augmenter le nombre de cellules souches hématopoïétiques mais également augmenter la capacité des cellules souches hématopoïétiques à former des colonies CFU, ce qui permet de placer les cellules souches hématopoïétiques dans un état de prolifération et de non-différenciation et de répondre aux besoins de transplantation clinique.
PCT/CN2019/081854 2018-04-13 2019-04-09 Utilisation de butyrate de sodium et système de culture contenant du butyrate de sodium WO2019196816A1 (fr)

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