WO2023178465A1 - 一种制备经血源性间充质干细胞的方法 - Google Patents

一种制备经血源性间充质干细胞的方法 Download PDF

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WO2023178465A1
WO2023178465A1 PCT/CN2022/081910 CN2022081910W WO2023178465A1 WO 2023178465 A1 WO2023178465 A1 WO 2023178465A1 CN 2022081910 W CN2022081910 W CN 2022081910W WO 2023178465 A1 WO2023178465 A1 WO 2023178465A1
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menstrual blood
cells
blood
mesenchymal stem
stem cells
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French (fr)
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王新
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士泽生物医药(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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

Definitions

  • the invention belongs to the technical field of cell separation and culture, and specifically relates to a method for preparing menstrual blood-derived mesenchymal stem cells.
  • Cells used in stem cell therapy include embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, etc.
  • mesenchymal stem cells MSC
  • mesenchymal stem cells have attracted the attention of researchers due to their high proliferation ability, self-renewal ability, mesoderm differentiation ability, and low immunogenicity.
  • mesenchymal stem cells have also shown great development potential in aspects such as immune regulation and tissue regeneration. So far, researchers have discovered and isolated mesenchymal stem cells from different human tissues. The endometrium undergoes regular changes with the menstrual cycle. The high degree of regeneration of this tissue implies that there may be stem cells in the endometrium that promote its reconstruction. In 2004, Chan et al.
  • menstrual blood-derived mesenchymal stem cells can be obtained from menstrual blood through tissue enzymatic hydrolysis and density gradient centrifugation.
  • tissue enzymatic hydrolysis the damaging effect of the enzymatic hydrolyzate on cells is inevitable and the collagenase reagent used is expensive and the experimental cost is high.
  • Density gradient centrifugation method is based on the fact that the volume, shape and density of mononuclear cells in peripheral blood, including lymphocytes and monocytes, are different from other cells.
  • the density of red blood cells and white blood cells is relatively high, about 1.090g/mL, while The density of lymphocytes and mononuclear cells is 1.075 ⁇ 1.090g/mL, and the density of platelets is 1.030 ⁇ 1.035g/mL.
  • density gradient centrifugation in the lymphocyte separation solution cells of a certain density are distributed according to the corresponding density gradient, thereby dividing the specific density into of blood cells isolated.
  • menstrual blood-derived mesenchymal stem cells in uterine blood will exist in the form of single cells, finely divided tissues and tissue blocks after being shed from the endometrium, it is speculated that density gradient centrifugation cannot efficiently enrich menstrual blood-derived mesenchymal stem cells that exist in different forms.
  • Mesenchymal stem cells therefore, it is necessary to find a method to quickly and efficiently enrich primary menstrual blood-derived mesenchymal stem cells from menstrual blood to provide guarantee for its drug development and production.
  • the existing technology for preparing menstrual blood-derived mesenchymal stem cells has the following problems: due to the influence of sample blood clots and viscosity, the density gradient centrifugation separation effect is greatly different, and it is impossible to efficiently and simply enrich menstrual blood-derived mesenchymal stem cells in different forms. stem cells, and the cell recovery rate of simply collecting buffy coat cells is low.
  • the first object of the present invention is to provide a method for preparing menstrual blood-derived mesenchymal stem cells.
  • the second object of the present invention is to provide a method for constructing a bank of menstrual blood-derived mesenchymal stem cells.
  • the object of the third aspect of the present invention is to provide mesenchymal stem cells obtained according to the method of the first aspect of the present invention.
  • the object of the fourth aspect of the present invention is to provide the use of the mesenchymal stem cells of the third aspect in the preparation of medicines.
  • a first aspect of the present invention provides a method for preparing menstrual blood-derived mesenchymal stem cells, including the following steps:
  • (31) Collect the buffy coat layer and blood cell layer, resuspend them in culture medium respectively to obtain S1 liquid and S2 liquid, mix and adhere to the culture to obtain menstrual blood-derived mesenchymal stem cells;
  • the menstrual blood sample in step (1) is a mixture of menstrual blood and menstrual blood preservation solution.
  • the menstrual blood is the menstrual blood of at least one day from the first to the fourth day of the female menstrual cycle.
  • the menstrual blood is the menstrual blood of at least one day on the second or third day of the female menstrual cycle.
  • the menstrual blood preservation solution is a buffer commonly used in this field, such as PBS buffer.
  • Antibiotics and/or anticoagulants may be added as needed.
  • the antibiotics include but are not limited to gentamicin, penicillin, streptomycin, amphotericin B and other commonly used antibiotics or combinations thereof.
  • the anticoagulant includes but is not limited to other commonly used anticoagulants such as sodium heparin, sodium citrate, or combinations thereof.
  • the menstrual blood preservation solution is a PBS buffer containing heparin sodium, amphotericin B and gentamicin.
  • the gentamicin is gentamicin sulfate.
  • the menstrual blood preservation solution is a PBS buffer containing 0.1-0.3 mg/mL heparin sodium, 0.1-0.3ug/mL amphotericin B and 20-80ug/mL gentamicin.
  • the gentamicin is gentamicin sulfate.
  • the volume ratio of the menstrual blood to the menstrual blood preservation solution is 1: (0.5-1.5).
  • the menstrual blood sample in step (1) is passed through a 16-25 mesh sieve.
  • the menstrual blood sample in step (1) is passed through a sieve of 18 to 20 mesh.
  • step (2) before the filtrate in step (2) is subjected to density gradient centrifugation, the following steps are also included: pre-centrifugation, removing the supernatant, and mixing with the washing liquid.
  • the pre-centrifugation conditions are 300-500g and centrifugation at 15-25°C for 8-12 minutes.
  • the volume of the mixed liquid obtained after mixing with the cleaning liquid is 1 to 2 times the volume of the menstrual blood in the menstrual blood sample described in step (1).
  • the density gradient centrifugation method in step (2) is: add the filtrate after sieving in step (1) or the mixed solution prepared by pre-centrifugation in step (2) to the lymphocyte separation solution, and centrifuge.
  • the volume ratio of the filtrate or the mixed solution to the lymphocyte separation solution is 1: (0.5-1.5).
  • the centrifugation conditions are 200-800 g and centrifugation at 15-25°C for 20-40 min; further, the conditions are 400-600 g and centrifugation at 15-25°C for 25-35 min.
  • the rising and falling rates are adjusted to the lowest, that is, the rising speed is 1 and the falling speed is 0.
  • the method of collecting the buffy coat layer described in steps (31) and (32) is: removing most of the plasma layer, sucking out the remaining plasma layer and buffy coat layer, and adding cleaning solution.
  • the method for collecting the blood cell layer in steps (31) and (32) is a filtration method, which includes the following steps: mix the blood cell layer (bottom sedimentation) with the cleaning solution, filter it through a 40-70um filter device, and discard the filtrate. , rinse the trapped material with cleaning fluid, and collect the cleaning fluid containing the trapped material.
  • the step of rinsing the trapped substances with cleaning fluid also includes the following steps: cleaning the filter device with cleaning fluid, filtering the cleaning fluid through the filter membrane naturally, and discarding the filtrate.
  • the method of rinsing the trapped material with cleaning liquid is forward and backwash; further preferably, forward and backwash are used until there is no obvious tissue residue on the filter device; further preferably, forward and backwash are performed 1 to 4 times; and still further preferably, forward and backwash are performed. Backwash 2 to 3 times.
  • steps (31) and (32) the following steps are also included: centrifuge A, remove supernatant A, resuspend, centrifuge B, remove supernatant B.
  • the operations of centrifuging A and removing supernatant A are repeated 1 to 3 times.
  • a washing step is also included between each two times of centrifuging A and removing supernatant A.
  • the washing method is washing with a cleaning solution.
  • the operations of resuspension, centrifugation B, and removal of supernatant B are repeated 1 to 3 times.
  • the conditions for centrifugation A and centrifugation B are 300 to 500g and centrifugation at 15 to 25°C for 8 to 12 minutes.
  • the resuspension method is resuspension with cleaning solution.
  • the culture medium in steps (31) and (32) is DMEM/F-12, ⁇ -MEM, 7501 mesenchymal stem cell culture medium and other conventional culture media used for culturing mesenchymal stem cells in this field.
  • the culture medium (such as DMEM/F-12, ⁇ -MEM, 7501) contains FBS; further preferably, the culture medium contains 8 to 12% FBS.
  • the mixing method in step (31) is to use the "ten" figure or the "8" figure to mix.
  • the adherent culture in steps (31) and (32) includes primary culture.
  • the primary culture method is: culture at 35-40°C, 4-6% CO2 , change the medium after 20-28 hours, remove non-adherent cells, and change the medium every 1 to 3 days thereafter.
  • the adherent culture in steps (31) and (32) further includes subculture.
  • the subculture method is: when the primary cultured cells are 80% to 90% full, they are digested and subcultured.
  • the subculture conditions are 35-40°C and 4-6% CO 2 .
  • the passage density is 3000-7000/cm 2 .
  • the aforementioned cleaning solution in this application is a buffer commonly used in this field, such as PBS buffer.
  • Antibiotics can be added as needed.
  • the antibiotics include but are not limited to gentamicin, penicillin, streptomycin, amphotericin B and other commonly used antibiotics or combinations thereof.
  • the washing solution is PBS buffer containing amphotericin B and gentamicin.
  • the gentamicin is gentamicin sulfate.
  • the cleaning solution is a PBS buffer containing 0.1-0.3ug/mL amphotericin B and 20-80ug/mL gentamicin.
  • the gentamicin is gentamicin sulfate.
  • a second aspect of the present invention provides a method for establishing a bank of menstrual blood-derived mesenchymal stem cells, which includes the steps of the method for preparing menstrual blood-derived mesenchymal stem cells according to the first aspect of the present invention.
  • a third aspect of the present invention provides mesenchymal stem cells prepared according to the method described in the first aspect of the present invention.
  • the mesenchymal stem cell population prepared according to the first aspect of the present invention is provided.
  • a fourth aspect of the present invention provides the application of the mesenchymal stem cells and/or mesenchymal stem cell populations of the present invention and/or the method of the first aspect of the present invention in the preparation of medicines.
  • the medicine is used to treat at least one of the following diseases: hematological diseases, cardiovascular diseases, liver cirrhosis, tumors, neurological diseases, repair of partial meniscus resection of knee joints, and autoimmune diseases.
  • the present invention provides a method for preparing menstrual blood-derived mesenchymal stem cells, by passing the menstrual blood sample through a 10-30 mesh sieve to remove blood clots and large pieces of tissue to avoid affecting the centrifugation effect during subsequent centrifugation; the present invention is based on the traditional method (collection On the basis of the buffy coat layer), the step of collecting the blood cell layer is added.
  • the same sample can use the traditional method (collecting the buffy coat layer) and the method of collecting the blood cell layer at the same time to recover target cells from different separation layer samples of density gradient centrifugation.
  • This method The invented method of collecting the blood cell layer is to recover target cells from the waste separation layer of the traditional separation method.
  • the buffy coat layer can improve the recovery rate of the primary target cells based on the original traditional method (collecting the buffy coat layer), collect the buffy coat layer and collect blood cells.
  • the layers can be carried out at the same time, which is beneficial to later production and library construction; and compared with traditional separation methods, the cell proliferation ability of the cells prepared by the method of the present invention in the later culture process is stronger than that of the traditional separation method, which ensures that in the later stage Under the premise of ensuring cell quality, shortening the in vitro culture time and realizing early-generation cells as preparation products will play a positive role in improving the quality of cell drugs.
  • the present invention restricts the use of filtration method to collect the blood cell layer, so that the obtained target cells have obvious advantages in primary cell adhesion rate, primary cell acquisition amount, same-generation secondary cell acquisition amount, contamination rate, etc.; prepared Compared with existing technologies, mesenchymal stem cells have better proliferation ability, cell surface marker expression that is more in line with MSC standards, and better anti-inflammatory ability.
  • Figure 1 is a flow chart for the isolation of menstrual blood-derived mesenchymal stem cells in Example 1.
  • Figure 2 is a graph comparing the proliferation capabilities of menstrual blood-derived mesenchymal stem cells obtained in Example 1 (filtration method) and Comparative Example 1 (albuginea layer).
  • Figure 3 is a diagram of the cell state of primary blood-derived mesenchymal stem cells obtained by collecting blood cell layer cells through different methods from donors A to D in Comparative Example 2 after medium exchange.
  • Figure 4 is a diagram of the cell state of primary blood-derived mesenchymal stem cells obtained by collecting blood cell layer cells through different methods from donors E and F in Comparative Example 2 after medium exchange.
  • Figure 5 is a diagram of the cell state of primary blood-derived mesenchymal stem cells after medium replacement after 24 hours of cleaning culture medium supernatant obtained by different treatment methods in Comparative Example 2.
  • Figure 6 is a statistical graph comparing the number of cells collected by the cleaning method and the filtration method in Example 2.
  • Figure 7 is a diagram showing the cell cycle detection results of MSCs derived from human uterine blood isolated by different processing methods in the effect example.
  • Figure 8 is a flow chart showing the inhibition of Th1 by MSCs derived from human uterine blood isolated by different treatment methods in the effect example.
  • Figure 9 is a flow chart showing the inhibition of Th17 by MSCs derived from human uterine blood isolated by different processing methods in the effect example.
  • Figure 10 is a three-lineage differentiation diagram of MSCs obtained by different treatment methods in the effect example.
  • Example 1 A method of preparing menstrual blood-derived mesenchymal stem cells
  • Sample collection After signing the informed consent form, use menstrual cups to collect 10 to 20 mL of menstrual blood from 6 donors on the 2nd to 3rd day of their menstrual period, and store them in an equal volume of preservation solution to obtain a menstrual blood mixture.
  • the preservation solution contains heparin.
  • PBS buffer solution of sodium, amphotericin B, and gentamicin sulfate in which the final concentration of heparin sodium is 0.2mg/mL, the final concentration of amphotericin B is 0.2ug/mL, and the final concentration of gentamicin sulfate is 0.2mg/mL.
  • the concentration is 50ug/mL.
  • the menstrual blood mixture will be transported back to the laboratory within 48 hours at 4 to 8°C.
  • Isolation of menstrual blood-derived mesenchymal stem cells Take the menstrual blood mixture obtained in step 1 (based on 10 mL of pure blood) to separate menstrual blood-derived mesenchymal stem cells, including the following steps: sieve the menstrual blood mixture, coarsely separate, density Gradient centrifugation is used to collect buffy coat and blood cell layer cells, and the cells are cultured to obtain menstrual blood-derived mesenchymal stem cells.
  • the flow chart is shown in Figure 1. The specific steps are as follows:
  • Step (1) Pre-centrifuge the filtrate obtained in step (1) (400g, 10 minutes at 20°C);
  • the cleaning solution is PBS buffer containing amphotericin B and gentamicin sulfate, among which, amphotericin B
  • the final concentration of betaine B is 0.2ug/mL
  • the final concentration of gentamicin sulfate is 50ug/mL
  • the total volume of the sample after dilution is the initial pure blood volume (for more viscous samples, dilute to the initial pure blood volume. twice the volume), mix evenly;
  • the centrifuge tube includes four layers from top to bottom: plasma layer, buffy coat layer, lymphocyte separation liquid layer and blood cell layer;
  • Collect buffy coat cells Use a 3mL bus pipette to suck off most of the upper plasma layer (about 15mL). Use a 3mL Pasteur pipette to carefully suck out the remaining 2mL of plasma layer and buffy coat layer, add it to a new 50mL centrifuge tube, and then add The cleaning solution reaches 40mL/tube, which is recorded as liquid A;
  • Residue (allowing tissue fragments to be thoroughly washed off the filter membrane, minimizing the residue and improving the yield of the target cells), collect the cleaning solution after rinsing the filter membrane into a 50mL centrifuge tube, recorded as solution B, 40mL/tube;
  • Cell counting Use a 2 ⁇ 20uL pipette (single channel) to take 15 ⁇ L of the cell suspension in step 1) and 15 ⁇ L of the AO/PI staining solution, mix them in equal volumes, mix with a 20 ⁇ 100uL pipette, and aspirate Add 20uL of the mixed solution to the counting plate and perform cell counting. Use the AO/PI counting function of the countstar counter. Select MSC as the cell detection type. After the counting is completed, record the total cell volume, viable cell volume, viability rate, and average cell size. , cell clumping rate, etc.;
  • Inoculate cells Inoculate cells from solution A into T25-cellbind culture flask at a density of 3.00E+05 cells/ cm2 , and inoculate cells from solution B into T25- at a density of 3.00E+04 cells/ cm2 .
  • 7501 mesenchymal stem cell culture medium T25 10mL or T75 15mL
  • Comparative Example 1 Traditional method (collecting buffy coat cells) to prepare menstrual blood-derived mesenchymal stem cells
  • Sample collection After signing the informed consent form, use menstrual cups to collect 10 to 20 mL of menstrual blood from 6 donors on the 2nd to 3rd day of their menstrual period, and store them in an equal volume of preservation solution to obtain a menstrual blood mixture.
  • the preservation solution contains heparin.
  • PBS buffer solution of sodium, amphotericin B, and gentamicin sulfate wherein the final concentration of heparin sodium is 0.2mg/mL, the final concentration of amphotericin B is 0.2ug/mL, and the final concentration of gentamicin sulfate is 0.2mg/mL.
  • the final concentration is 50ug/mL.
  • the menstrual blood mixture will be transported back to the laboratory within 48 hours at 4 to 8°C.
  • the in vitro proliferation ability the results are shown in Table 1 and Figure 2: With the extension of in vitro culture time, the cell doubling time obtained by the traditional method (collecting buffy coat cells) is longer than that obtained by the filtration method.
  • the filtration method The obtained cell proliferation rate in vitro is relatively stable, mostly around 30 hours.
  • the same sample can use two different methods, the traditional method (collecting buffy coat layer cells) and the filtration method provided in Example 1, to analyze the target cells in different separation layer samples after density gradient centrifugation. Recycling and filtration recovery operation will not affect the normal recovery of buffy coat cells, because the filtration method recovers target cells from the waste separation layer of traditional separation methods, and can be improved on the basis of the original traditional method (collecting buffy coat cells)
  • the recovery rate of primary target cells is beneficial to later production and library construction.
  • the cell proliferation ability of the cells extracted by the filtration method provided in Example 1 during the subculture process is stronger than that of the traditional separation method.
  • the in vitro process can be shortened. The culture time plays a positive role in realizing early-generation cells as preparation products and improving the quality of cell drugs.
  • Sample collection After signing the informed consent form, use menstrual cups to collect 10 to 20 mL of menstrual blood from 10 donors on the 2nd to 3rd day of their menstrual period, and store it in an equal volume of preservation solution to obtain a menstrual blood mixture.
  • the preservation solution contains heparin.
  • PBS buffer solution of sodium, amphotericin B, and gentamicin sulfate in which the final concentration of heparin sodium is 0.2mg/mL, the final concentration of amphotericin B is 0.2ug/mL, and the final concentration of gentamicin sulfate is 0.2mg/mL.
  • the concentration is 50ug/mL.
  • the menstrual blood mixture will be transported back to the laboratory within 48 hours at 4 to 8°C.
  • Isolation of menstrual blood-derived mesenchymal stem cells Take the menstrual blood mixture obtained in step 1 to separate menstrual blood-derived mesenchymal stem cells, including the following steps: sieving the menstrual blood mixture for rough separation, density gradient centrifugation, and collecting the blood cell layer by different methods.
  • Cells, cell culture, to obtain menstrual blood-derived mesenchymal stem cells the specific steps are as follows:
  • Step (1) Pre-centrifuge the filtrate obtained in step (1) (400g, 10 minutes at 20°C);
  • the cleaning solution is PBS buffer containing amphotericin B and gentamicin sulfate, among which, amphotericin B
  • the final concentration of betaine B is 0.2ug/mL
  • the final concentration of gentamicin sulfate is 50ug/mL
  • the total volume of the sample after dilution is the initial pure blood volume (for more viscous samples, dilute to the initial pure blood volume. twice the volume), mix evenly;
  • the centrifuge tube includes four layers from top to bottom: plasma layer, buffy coat layer, lymphocyte separation liquid layer and blood cell layer.
  • Discard the plasma layer, buffy coat layer and lymphocyte separation liquid layer Dilute the remaining bottom sediment in the tube with 40mL of cleaning solution. Mix the blood cell layer mixture by inverting it upside down and divide the blood cell layer mixture into two parts. Pass one part through a 40um filter and discard it. filtrate, then clean the filter membrane once with 10mL cleaning solution, let it filter through the membrane naturally, and discard the filtrate; transfer the filter membrane to a 10cm vessel, add 40mL cleaning solution, and use a 3mL Pasteur pipette to remove the tissue fragments on the filtering membrane. Backwash 2 to 3 times until there is no obvious tissue residue on the filter membrane (allowing tissue fragments to be completely washed off the filter membrane to minimize residues and improve the yield of target cells).
  • the adherent cells in the washing method mostly exist in the form of clumps, and there are fewer clumps; the adherent cells in the filtration method include two forms: clumps and scattered cells.
  • the cell adhesion rate of the filtration method is much higher than that of the washing method;
  • the primary culture time of cells using the filtration method is much shorter than that of the washing method (under the same inoculation specifications, the cell density of the cells using the filtration method reaches more than 80% in 3 days, while the cell density of the cells in the washing method only reaches more than 80% in 8 days). ;
  • step (4) Take the D donor in step (4) and wash the culture medium supernatant of the group after 24 hours. Mix it upside down and divide it into two parts. Pass one part through a 40um filter membrane and discard the filtrate. Then wash the filter with 10mL cleaning solution. Filter the membrane once, let it filter through the membrane naturally, and discard the filtrate; transfer the filter membrane to a 10cm vessel, add 40mL of cleaning solution, and use a 3mL Pasteur pipette to wash the tissue fragments on the filter membrane forward and backwash 2 to 3 times until filtered There are no obvious tissue residues on the membrane (allowing tissue fragments to be thoroughly washed off the filter membrane, minimizing residues and improving the yield of target cells).
  • the cleaning solution after rinsing the filter membrane is collected into a 50mL centrifuge tube, 40mL/tube, It is recorded as the cleaning-filtration group; the other part is added directly into a 50 ml centrifuge tube, and the cleaning solution is added to 40 mL, which is recorded as the cleaning-cleaning group.
  • Centrifuge the washing-filtration group and the washing-washing group centrifuge conditions: 400g, 20°C for 10 minutes), discard the supernatant, resuspend the cells in 10 mL of culture medium and inoculate them into culture bottles of the same specification, 24 hours later Change the medium (co-culture for 48 hours), continue to culture, and observe the cell status. When the cell density reaches 80 to 90%, perform cell passaging.
  • the cell status of different treatment groups after 24 hours of medium replacement (48 hours of co-culture) is shown in Figure 5: It can be seen that 1) in step (4), there are target cells in the culture supernatant of the group washed after 24 hours, but due to limitations of the method, the cells The amount of adherent cells at 24 hours is less, that is, the washing method is not conducive to cell adhesion during medium change at 24 hours; 2) At the same time, the amount of cells adhered by the washing-filtration method is much greater than that by the washing-cleaning method, which further proves that the filtration method collects primary cells. The ability of the cells is stronger than that of the cleaning method.
  • the present invention uses a filtration method to collect blood cell layer cells, which makes the primary cell adhesion rate, primary cell acquisition amount, same-generation secondary cell acquisition amount, cell proliferation ability, contamination rate, etc. significantly better than the cleaning method to collect blood cell layer cells. .
  • MSC are a type of stem cells derived from adults. They are used in the clinical treatment of articular cartilage damage, graft-versus-host disease, autoimmune diseases, and wound repair because of their capabilities of self-renewal and proliferation, multi-lineage differentiation, immune regulation, and promotion of angiogenesis. wait.
  • MSCs should express CD90, a marker related to stem cell proliferation and differentiation, CD73, a marker related to immune regulation, and CD105, a marker related to angiogenesis; they should not express the leukocyte marker CD45, hematopoietic stem cells Marker CD34, monocyte macrophage marker CD14 or CD11b, B cell marker CD19 or CD79 ⁇ , and MHC class II molecules such as HLA-DR, etc.
  • the positive rate of CD45 of menstrual blood-derived MSC obtained by the cleaning method in step (4) exceeds 2%, and the detection does not meet the standard requirements of MSC (suggesting that there are too many impure cells and insufficient purity); while step (4)
  • the positive rates of menstrual blood-derived MSCs obtained by the filtration method and the menstrual blood-derived MSCs obtained by the cleaning-filtration method in step (5) are all ⁇ 95%, and the positive rates of CD45, CD34, CD11b, CD19 and HLA-DR are all ⁇ 95%. 2%, which meets the testing standards. It can be seen that using the filtration method to collect blood cell layer cells can make the MSCs obtained meet the standard requirements of MSCs.
  • MSC has the function of regulating immunity.
  • the ability of MSC to inhibit Th1 and Th17 under inflammatory conditions can be evaluated by detecting the proportion of pro-inflammatory lymphocyte subpopulations Th1 and Th17 in PBMC after MSC is activated with inflammatory factors or co-cultured with PBMC. , thus reflecting the anti-inflammatory effect of MSC.
  • PBMC peripheral blood mononuclear cells
  • Comparative Example 2 Menstrual blood-derived MSC obtained by the cleaning method in step (4), menstrual blood-derived MSC obtained by the filtration method, and step (5) cleaning-filtration method.
  • the obtained menstrual blood-derived MSCs can inhibit the proliferation of activated Th1 and Th17 in PBMC, but the inhibition rate of menstrual blood-derived MSCs using the filtration method and the washing-filtration method (that is, including the filtration operation) on Th1 and Th17 is higher than that of washing.
  • the menstrual blood-derived MSC obtained by the method shows that the use of filtration method to collect blood cell layer cells can improve the anti-inflammatory effect of the obtained MSC.
  • MSC has multi-directional differentiation potential. After being induced by specific induction medium, MSC has the ability to form adipogenesis, osteogenesis and chondrogenesis.
  • Oil Red O staining was used to evaluate the induction differentiation of MSCs into adipocytes;
  • Alizarin Red S staining was used to evaluate the induction differentiation of MSCs into osteocytes;
  • Alcian blue staining was used to evaluate the induction differentiation of MSCs into chondrocytes.

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Abstract

提供了一种制备经血源性间充质干细胞的方法，包括取经血样品过筛得到滤液；将滤液进行密度梯度离心，得到四层分离液：血浆层、白膜层、分离液层和血细胞层；收集白膜层和血细胞层混合，贴壁培养得到经血源性间充质干细胞，其中收集血细胞层的方法为过滤法，包括如下步骤：将血细胞层与清洗液混合，经40～70um的过滤装置过滤，弃滤液，用清洗液冲洗被截留的物质，收集包含被截留的物质的清洗液。该方法可以在原有传统方法的基础上提高原代目的细胞的回收率，且在后期培养过程中细胞的增殖能力强于传统分离方法。

Description

一种制备经血源性间充质干细胞的方法 技术领域
本发明属于细胞分离、培养技术领域，具体涉及一种制备经血源性间充质干细胞的方法。
背景技术
干细胞治疗所用的细胞包括胚胎干细胞、诱导多能干细胞、间充质干细胞等。其中，间充质干细胞(Mesenchymal stem cell，MSC)以其高增殖能力、自我更新能力、向中胚层分化能力、低免疫原性等特点备受研究者瞩目。近年来，间充质干细胞也在免疫调节和组织再生等方面表现出巨大的发展潜能。到目前为止，研究者已从不同人体组织中发现并分离出间充质干细胞。其中子宫内膜会随月经周期发生规律性变化，这种组织高度再生的能力暗示着内膜可能存在促使其重建的干细胞。2004年Chan等从子宫组织中分离并培养出克隆能力强的干细胞，首次证实了子宫内膜干细胞的存在，到2007年Meng等从女性月经血中分离出具有间充质干细胞特性的细胞，称为经血源性间充质干细胞。经血源性间充质干细胞除具备间充质干细胞的特性外还具有取材方便、可定期从统一供体的经血中直接获得，提高细胞遗传背景的稳定性、不受伦理道德限制等优势，因此成为干细胞治疗的优势种子细胞。
目前经血源性间充质干细胞可以通过组织酶解法和密度梯度离心法从经血中获得。酶解过程中酶解液对细胞的损伤作用不可避免且所用胶原酶试剂价格不菲，实验成本较高。密度梯度离心法根据外周血中单个核细胞包括淋巴细胞和单核细胞等细胞的体积、形态和密度与其他细胞不同，如：红细胞和白细胞等细胞密度较大，为1.090g/mL左右，而淋巴细胞和单个核细胞密度为1.075～1.090g/mL，血小板为1.030～1.035g/mL等通过在淋巴细胞分离液中经过密度梯度离心使一定密度的细胞按相应密度梯度分布，从而将特定密度的血细胞分离出来。但由于宫血中经血源性间充质干细胞从子宫内膜脱落后会以单个细胞、细碎组织和组织块的形式存在，推测密度梯度离心法无法高效富集以不同形式存在的经血源性间充质干细胞，因此，需要寻找一种快速从月经血中高效富集原代经血源性间充质干细胞的方法为其药物研发及生产提供保障。
发明内容
现有技术制备经血源间充质干细胞存在如下问题：由于样本血块及粘稠程度的影响，密度梯度离心分离效果存在较大差异，无法高效简便地富集以不同形式存在的经血源性间充质干细胞，且单纯收集白膜层细胞的细胞回收率较低。
本发明第一方面的目的，在于提供一种制备经血源性间充质干细胞的方法。
本发明第二方面的目的，在于提供一种经血源性间充质干细胞的建库方法。
本发明第三方面的目的，在于提供根据本发明第一方面所述方法获得的间充质干细胞。
本发明第四方面的目的，在于提供第三方面的间充质干细胞在制备药物中的应用。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供一种制备经血源性间充质干细胞的方法，包括如下步骤：
(1)、(2)和(31)；或
(1)、(2)和(32)；
(1)取经血样品过10～30目筛，得到滤液；
(2)将滤液进行密度梯度离心，得到四层分离液：血浆层、白膜层、分离液层和血细胞层；
(31)收集白膜层和血细胞层，分别用培养基重悬，得到S1液和S2液，混合，贴壁培养，得到经血源性间充质干细胞；
(32)收集白膜层和血细胞层，混合，用培养基重悬，贴壁培养，得到经血源性间充质干细胞。
优选地，步骤(1)中所述经血样品为经血与经血保存液的混合液。
优选地，所述经血为女性月经周期第一天至第四天中至少一天的经血。
优选地，所述经血为女性月经周期第二天或第三天的中至少一天的经血。
优选地，所述经血保存液是本领域常用缓冲液，例如PBS缓冲液。可以根据需要添加抗生素和/或抗凝剂。
优选地，所述抗生素包括但不限于庆大霉素、青霉素、链霉素、两性霉素B等其他常用抗生素或其组合。
优选地，所述抗凝剂包括但不限于肝素钠、枸橼酸钠等其他常用抗凝剂或其组合。
优选地，所述经血保存液是包含肝素钠、两性霉素B和庆大霉素的PBS缓冲液。
优选地，所述庆大霉素为硫酸庆大霉素。
进一步优选地，所述经血保存液为包含0.1～0.3mg/mL肝素钠、0.1～0.3ug/mL两性霉素B和20～80ug/mL庆大霉素的PBS缓冲液。优选的，所述庆大霉素为硫酸庆大霉素。
优选地，所述经血与经血保存液的体积比为1:(0.5～1.5)。
优选地，步骤(1)中所述经血样品过16～25目筛。
优选地，步骤(1)中所述经血样品过18～20目筛。
优选地，步骤(2)中所述滤液进行密度梯度离心前还包括如下步骤：预离心，去上清，与清洗液混合。
优选地，所述预离心的条件为300～500g，15～25℃下离心8～12min。
优选地，与清洗液混合后得到的混合液的体积为步骤(1)中所述经血样品中经血的体积的1～2倍。
优选地，步骤(2)中所述密度梯度离心的方法为：将步骤(1)中过筛后滤液或步骤(2)中预离心所制备得到的混合液加入淋巴细胞分离液上，离心。
优选地，所述滤液或所述混合液与淋巴细胞分离液的体积比为1:(0.5～1.5)。
优选地，所述离心的条件为200～800g，15～25℃下离心20～40min；进一步为400～600g，15～25℃下离心25～35min。
优选地，离心过程中所述升降速率调至最低，即升速为1降速为0。
优选地，步骤(31)、(32)中所述收集白膜层的方法为：去除大部分血浆层，吸出剩余血浆层和白膜层，加入清洗液。
优选地，步骤(31)、(32)中所述收集血细胞层的方法为过滤法，包括如下步骤：将血细胞层(底层沉淀)与清洗液混合，经40～70um的过滤装置过滤，弃滤液，用清洗液冲洗被截留的物质，收集包含被截留的物质的清洗液。
优选地，所述用清洗液冲洗被截留的物质之前还包括如下步骤：用清洗液清洗过滤装置，清洗液自然滤过滤膜，弃滤液。
优选地，所述用清洗液冲洗被截留的物质为正反冲洗；进一步优选为正反冲洗至过滤装置上没有明显组织残留；更进一步优选为正反冲洗1～4次；再进一步优选为正反冲洗2～3次。
优选地，步骤(31)、(32)中培养基重悬前，还包括如下步骤：离心A、去上清A、重悬、离心B、去上清B。
优选地，所述离心A、去上清A的操作重复1～3次。
优选地，几次重复的离心A、去上清A的操作中，每两次离心A、去上清A之间还包含洗涤的步骤。
优选地，所述洗涤的方法为用清洗液洗涤。
优选地，所述重悬、离心B、去上清B的操作重复1～3次。
优选地，所述离心A、离心B的条件为300～500g，15～25℃下离心8～12min。
优选地，所述重悬的方法为用清洗液重悬。
优选地，步骤(31)、(32)中所述培养基为DMEM/F-12、α-MEM、7501间充质干细胞培养基以及本领域其他用于培养间充质干细胞的常规培养基。
优选地，所述培养基(例如DMEM/F-12、α-MEM、7501)含有FBS；进一步优选地，所述培养基含有8～12％FBS。
优选地，步骤(31)中所述混合的方法为采用“十”字或“8”字混匀。
优选地，步骤(31)、(32)所述贴壁培养包括原代培养。
优选地，所述原代培养的方法为：35～40℃，4～6％CO 2下培养，20～28h后，换液，去除未贴壁细胞，以后每1～3天换液一次。
优选地，步骤(31)、(32)所述贴壁培养还包括传代培养。
优选地，所述传代培养的方法为：待原代培养细胞长满80％～90％时，消化，进行传代。
优选地，所述传代培养的条件为35～40℃，4～6％CO 2。
优选地，传代密度为3000～7000/cm 2。
优选地，本申请前述清洗液是本领域常用缓冲液，例如PBS缓冲液。可以根据需要添加抗生素。
优选地，所述抗生素包括但不限于庆大霉素、青霉素、链霉素、两性霉素B等其他常用抗生素或其组合。
优选地，所述清洗液是包含两性霉素B和庆大霉素的PBS缓冲液。
优选地，所述庆大霉素为硫酸庆大霉素。
进一步优选地，所述清洗液为包含0.1～0.3ug/mL两性霉素B和20～80ug/mL庆大霉素的PBS缓冲液。优选的，所述庆大霉素为硫酸庆大霉素。
本发明的第二个方面，提供一种经血源性间充质干细胞的建库方法，包含本发明第一方面的制备经血源性间充质干细胞的方法的步骤。
本发明的第三个方面，提供一种间充质干细胞，根据本发明第一方面所述方法制备得到。
优选地，提供根据本发明第一方面所制备得到的间充质干细胞群体。
本发明的第四个方面，提供本发明所述间充质干细胞和/或间充质干细胞群体和/或本发明第一方面所述方法在制备药物中的应用。
优选地，所述药物用于治疗以下疾病中的至少一种：血液系统疾病、心血管疾病、肝硬化、肿瘤、神经系统疾病、膝关节半月板部分切除损伤修复、自身免疫性疾病。
本发明的有益效果是：
本发明提供了一种制备经血源性间充质干细胞的方法，通过将经血样品过10～30目筛， 除去血块、大块组织，避免后续离心时影响离心效果；本发明在传统方法(收集白膜层)的基础上，增加收集血细胞层的步骤，同一样本可同时用传统方法(收集白膜层)和收集血细胞层的方法对密度梯度离心的不同分离层样本进行目的细胞的回收，本发明收集血细胞层的方法是从传统分离方法的废弃分离层中回收目的细胞，可以在原有传统方法(收集白膜层)的基础上提高原代目的细胞的回收率，收集白膜层和收集血细胞层可同时进行，有利于后期进行生产和建库；且与传统的分离方法相比，通过本发明方法制备的细胞在后期培养过程中细胞的增殖能力强于传统分离方法的，对于后期在保证细胞质量的前提下，缩短体外培养时间，实现早期代次的细胞作为制剂产品，提高细胞药物的质量有着积极地作用。
进一步地，本发明限定采用过滤法收集血细胞层，使得得到的目的细胞在原代细胞贴壁率、原代细胞获得量、同代次细胞获得量、污染率等方面都有明显的优势；制备得到的间充质干细胞与现有技术相比，具有更好的增殖能力、更符合MSC标准的细胞表面标志物表达、更好的抑炎能力。
附图说明
图1是实施例1中经血源性间充质干细胞分离的流程图。
图2是实施例1(过滤法)和对比实施例1(白膜层)得到的经血源性间充质干细胞的增殖能力比较结果图。
图3是对比实施例2中A～D供体通过不同方法收集血细胞层细胞得到的原代经血源性间充质干细胞换液后细胞状态图。
图4是对比实施例2中E、F供体通过不同方法收集血细胞层细胞得到的原代经血源性间充质干细胞换液后细胞状态图。
图5是对比实施例2中24h后清洗组培养基上清通过不同处理方法得到的原代经血源性间充质干细胞换液后细胞状态图。
图6是对比实施例2中清洗法和过滤法收集的细胞数目对比的统计图。
图7是效果实施例中不同处理方法分离人宫血来源MSC的细胞周期检测结果图。
图8是效果实施例中不同处理方法分离人宫血来源MSC抑制Th1的流式图。
图9是效果实施例中不同处理方法分离人宫血来源MSC抑制Th17的流式图。
图10是效果实施例中不同处理方法所得到MSC的三系分化图。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
实施例1 一种制备经血源性间充质干细胞的方法
1、样本采集：在签署知情同意书后，用月经杯收集6个供体经期第2～3天的经血10～20mL，保存于等体积的保存液中，得到经血混合液，保存液是含有肝素钠、两性霉素B、硫酸庆大霉素的PBS缓冲液，其中，肝素钠的终浓度为0.2mg/mL，两性霉素B的终浓度为0.2ug/mL，硫酸庆大霉素的终浓度为50ug/mL。经血混合液在4～8℃的条件下48h内运送回实验室。
2、经血源性间充质干细胞分离：取步骤1得到的经血混合液(以10mL纯血量计)进行经血源性间充质干细胞分离，包括如下步骤：经血混合液过筛粗分离，密度梯度离心，收集白膜层、血细胞层细胞，细胞培养，得到经血源性间充质干细胞，流程图如图1所示，具体步骤如下：
(1)经血混合液过筛粗分离：取步骤1得到的经血混合液过20目不锈钢细胞筛，收集滤液；
(2)密度梯度离心
1)预离心：对步骤(1)得到的滤液进行预离心(400g，20℃下离心10min)；
2)用3mL巴士吸管轻轻吸去大部分上清，剩底部2mL上清和细胞沉淀，用清洗液(清洗液为含有两性霉素B、硫酸庆大霉素的PBS缓冲液，其中，两性霉素B的终浓度为0.2ug/mL，硫酸庆大霉素的终浓度为50ug/mL)稀释沉淀，稀释后样本总体积为初始纯血量(对于较粘稠的样本则稀释到初始纯血量的两倍体积)，混合均匀；
3)室温下将淋巴细胞分离液上下颠倒混合均匀，将步骤2)混合均匀后的溶液以1:1体积比例加入淋巴细胞分离液液面上，600g，20℃下离心30min(为保证分离效果，将离心机升降速率调至最低，即升速为1降速为0)，离心管从上到下包括四层：血浆层、白膜层、淋巴细胞分离液层和血细胞层；
(3)收集白膜层、血细胞层细胞
1)收集白膜层细胞：用3mL巴士吸管吸去大部分上层血浆层(15mL左右)，剩余2mL血浆层与白膜层用3mL巴氏吸管小心吸取，加入新的50mL离心管中，然后加入清洗液至40mL/管，记为A液；
2)过滤法收集血细胞层：弃去淋巴细胞分离液层后，管中剩余底层沉淀用40mL清洗液 稀释，混合均匀后过自然过滤40um滤膜，然后用10mL清洗液清洗滤膜1次，让其自然滤过滤膜，弃滤液；将滤膜转移到10cm器皿中，加入40mL清洗液，用3mL巴氏吸管将滤膜上的组织碎片正反冲洗2～3次，直到滤膜上没有明显组织残留(让组织碎片能彻底从滤膜上冲洗下来，最大程度减少残留，提高目的细胞的收率)，冲洗滤膜后的清洗液收集至50mL离心管中，记为B液，40mL/管；
3)将A液、B液分别离心(400g(1500rpm)、20℃下离心10min)，去上清，清洗液洗涤1次，再离心(400g(1500rpm)、20℃下离心10min)弃去上清；
4)用10mL移液枪往上述A和B液的离心管中分别加入5mL清洗液重悬沉淀(若有数管A液和B液则将其分别合并到一管中，记为A管和B管)，补足清洗液至40mL/管，离心(400g(1500rpm)，20℃下离心10min)，弃去上清；
5)用10mL移液枪往上述离心管中加入40mL清洗液重悬沉淀，离心(400g(1500rpm)，20℃下离心10min)，弃去上清。
(4)细胞培养
1)向步骤(3)中5)去上清后的离心管中加入沉淀量约5倍体积的7501间充质干细胞培养基(sciencell Mesenchyma Stem Cell Medium(cat:7501)+100X添加因子(cat:0503)+10％血清(cat:0025))，移液枪轻柔吹打混合均匀，重悬细胞沉淀；
2)细胞计数：用2～20uL移液枪(单通道)分别取15μL步骤1)的细胞悬液和15μL的AO/PI染色液等体积混合，用20～100uL移液枪混匀，并吸取20uL混合液加入到计数板中，进行细胞计数，使用countstar计数仪AO/PI计数功能，细胞检测类型选择MSC，计数完成后，记录细胞总量、活细胞量、活率、细胞平均粒径大小、细胞结团率等；
3)接种细胞：将A液所得细胞以3.00E+05个/cm 2的密度接种于T25-cellbind培养瓶中，将B液所得细胞以3.00E+04个/cm 2的密度接种于T25-cellbind培养瓶中，加入7501间充质干细胞培养基(T25 10mL或T75 15mL)；“十”字混匀细胞，置于37℃，5％CO 2培养箱中培养，细胞培养24h后，换液洗去未贴壁细胞，继续进行常规培养，得到经血源性间充质干细胞。之后每两天全换液一次，细胞长满约80％～90％时，消化，并按照5000/cm 2密度继续传代。
对比实施例1 传统方法(收集白膜层细胞)制备经血源性间充质干细胞
1、样本采集：在签署知情同意书后，用月经杯收集6个供体经期第2～3天的经血10～20mL，保存于等体积的保存液中，得到经血混合液，保存液是含有肝素钠、两性霉素B、硫酸庆大霉素的PBS缓冲液，其中，肝素钠的终浓度为0.2mg/mL，两性霉素B的终浓度为0. 2ug/mL，硫酸庆大霉素的终浓度为50ug/mL。经血混合液在4～8℃的条件下48h内运送回实验室。
2、采用传统方法(收集白膜层细胞，方法与实施例1中步骤(1)～(4)相同，区别仅在于未收集血细胞层)制备经血源性间充质干细胞。
分别取实施例1(过滤法)和对比实施例1(白膜层)得到的融合度90％以上的经血源性间充质干细胞(N＝6)置于7501培养基进行传代培养，比较细胞的体外增殖能力，结果如表1及图2所示：随着体外培养时间的延长，传统方法(收集白膜层细胞)获得的细胞倍增时间比过滤法所获得的细胞倍增时间长，过滤法获得的细胞体外增殖速度相对较稳定，大部分在30h左右。
表1 实施例1(过滤法)和对比实施例1(白膜层)得到的经血源性间充质干细胞的增殖能力比较
Figure PCTCN2022081910-appb-000001
针对月经血来源的样本，同一个样本可同时用传统方法(收集白膜层细胞)和实施例1提供的过滤法两种不同的方法对进行密度梯度离心后的不同分离层样本进行目的细胞的回收，过滤法回收操作不会影响正常的白膜层细胞回收，因为过滤法是从传统分离方法的废弃分离层中回收目的细胞，可以在原有传统方法(收集白膜层细胞)的基础上提高原代目的细胞的回收率，有利于后期进行生产和建库。且与传统的分离方法相比，实施例1提供的过滤法提取的细胞在传代培养过程中细胞的增殖能力强于传统分离方法的，对于传代培养过程中在保证细胞质量的前提下，缩短体外培养时间，以实现早期代次的细胞作为制剂产品，提高细胞药物的质量有着积极地作用。
对比实施例2 过滤法和清洗法的效果对比
1、样本采集：在签署知情同意书后，用月经杯收集10个供体经期第2～3天的经血10～20mL，保存于等体积的保存液中，得到经血混合液，保存液是含有肝素钠、两性霉素B、硫酸庆大霉素的PBS缓冲液，其中，肝素钠的终浓度为0.2mg/mL，两性霉素B的终浓度为0.2ug/mL，硫酸庆大霉素的终浓度为50ug/mL。经血混合液在4～8℃的条件下48h内运送回实验室。
2、经血源性间充质干细胞分离：取步骤1得到的经血混合液进行经血源性间充质干细胞 分离，包括如下步骤：经血混合液过筛粗分离，密度梯度离心，不同方法收集血细胞层细胞，细胞培养，得到经血源性间充质干细胞，具体步骤如下：
(1)经血混合液过筛粗分离：取步骤1得到的经血混合液过18目不锈钢细胞筛，收集滤液。
(2)密度梯度离心
1)预离心：对步骤(1)得到的滤液进行预离心(400g，20℃下离心10min)；
2)用3mL巴士吸管轻轻吸去大部分上清，剩底部2mL上清和细胞沉淀，用清洗液(清洗液为含有两性霉素B、硫酸庆大霉素的PBS缓冲液，其中，两性霉素B的终浓度为0.2ug/mL，硫酸庆大霉素的终浓度为50ug/mL)稀释沉淀，稀释后样本总体积为初始纯血量(对于较粘稠的样本则稀释到初始纯血量的两倍体积)，混合均匀；
3)室温下将淋巴细胞分离液上下颠倒混合均匀，将步骤2)混合均匀后的溶液以1:1体积比例加入淋巴细胞分离液液面上，600g，20℃下离心30min(为保证分离效果，将离心机升降速率调至最低，即升速为1降速为0)，离心管从上到下包括四层：血浆层、白膜层、淋巴细胞分离液层和血细胞层。
(3)不同方法收集血细胞层细胞
弃去血浆层、白膜层及淋巴细胞分离液层，管中剩余底层沉淀用40mL清洗液稀释，上下颠倒混匀后将血细胞层混合液一分为二，一份过40um滤膜后弃去滤液，然后用10mL清洗液清洗滤膜1次，让其自然滤过滤膜，弃滤液；将滤膜转移到10cm器皿中，加入40mL清洗液，用3mL巴氏吸管将滤膜上的组织碎片正反冲洗2～3次，直到滤膜上没有明显组织残留(让组织碎片能彻底从滤膜上冲洗下来，最大程度减少残留，提高目的细胞的收率)，冲洗滤膜后的清洗液收集至50mL离心管中，记为过滤组，40mL/管；一份直接补加清洗液至40mL，记为清洗组；将清洗组和过滤组样本离心，弃去上清，洗涤，离心，弃去上清，重悬，离心，弃去上清，重悬，离心，弃去上清(具体步骤与实施例1中步骤(3)中3)、4)、5)相同)。
(4)细胞培养
分别用15mL培养基重悬沉淀，接种于同一规格的培养瓶中，24h后分别收集上清至离心管中，用于后续实验。PBS清洗培养瓶2～3次，加入培养基后继续培养，并观察细胞状态，当细胞密度达到80～90％时，进行细胞传代处理。
分别使用过滤法和清洗法处理收集血细胞层细胞得到的原代经血源性间充质干细胞换液后细胞状态如图3～4所示(图3、4为10个供体中6个供体(A供体、B供体、C供体、D 供体、E供体、F供体的结果图)：
1)从原代24h换液后细胞贴壁情况来看：清洗法贴壁细胞多以团块形式存在，且其团块较少；过滤法贴壁细胞包括团块和散落细胞两种形式，24h换液时可看出过滤法细胞贴壁率远远高于清洗法；
2)同一培养规格下过滤法细胞原代培养时间远远短于清洗法(同一接种规格下，过滤法细胞3天细胞密度即达到80％以上，清洗法8天细胞密度才达到80％以上)；
3)从原代分离活细胞数及P0代收获细胞数如图6(6个供体的平均值)所示：过滤法原代分离的活细胞数是清洗法的7.3倍，同一培养时间内P0代收获细胞数过滤法是清洗法的86倍之多，可大大提高细胞的得率。
4)从培养的污染率来看：其中E、F供体清洗法样本24h换液后发现污染，而过滤法细胞未发现污染。清洗法所得细胞原代培养过程中污染率高于过滤法，10个供体中清洗法污染样本数是3个，过滤法为0个(图3、4仅为10个供体中6个供体的结果图)。
(5)取步骤(4)中D供体24h后清洗组培养基上清，上下颠倒混匀后一分为二，其中一份过40um滤膜后弃去滤液，然后用10mL清洗液清洗滤膜1次，让其自然滤过滤膜，弃滤液；将滤膜转移到10cm器皿中，加入40mL清洗液，用3mL巴氏吸管将滤膜上的组织碎片正反冲洗2～3次，直到滤膜上没有明显组织残留(让组织碎片能彻底从滤膜上冲洗下来，最大程度减少残留，提高目的细胞的收率)，冲洗滤膜后的清洗液收集至50mL离心管中，40mL/管，记为清洗-过滤组；另一份直接加入50毫升离心管中，补加清洗液至40mL，记为清洗-清洗组。将清洗-过滤组、清洗-清洗组进行离心处理(离心条件：400g，20℃离心10min)，弃去上清，分别用10mL培养基重悬细胞后接种于同一规格的培养瓶中，24h后换液(共培养48h)，继续培养，并观察细胞状态，当细胞密度达到80～90％时，进行细胞传代处理。
不同处理组的24h后换液(共培养48h)细胞状态如图5所示：可见，1)步骤(4)中24h后清洗组培养基上清还有目的细胞，但由于方法的限制导致细胞24h贴壁量较少，即清洗法不利于24h换液时细胞贴壁；2)同时，清洗-过滤法细胞贴壁量要远远多于清洗-清洗法的，进一步证明过滤法收集原代目的细胞的能力要强于清洗法。
可见，本发明采用过滤法收集血细胞层细胞，使得原代细胞贴壁率、原代细胞获得量、同代次细胞获得量、细胞增殖能力、污染率等方面显著优于清洗法收集血细胞层细胞。
效果实施例
1、不同处理方法分离经血源性MSC的细胞周期分布
(1)分别取不同处理方法分离经血源性MSC样品(对比实施例2中D供体步骤(4) 清洗法得到的经血源性MSC、过滤法得到的经血源性MSC、以及步骤(5)清洗-过滤法得到的经血源性MSC)至1.5mL离心管，1000g离心5min，弃上清液。
(2)加入1mL PBS，重悬细胞，1000g离心5min，弃去上清液。
(3)加入1mL 70％乙醇，重悬细胞，4℃固定30min以上，1000g离心5min，弃上清。
(4)加入1mL PBS，重悬细胞，1000g离心5min，弃去上清液。
(5)加入0.5mL碘化丙啶染色液，重悬细胞，37℃避光温浴30min。
(6)应用流式细胞仪检测红色荧光，采用适当分析软件进行细胞DNA含量分析，拟合统计处于不同细胞周期的比例(G0/G1期、G2/M期)。仪器型号：NovoCyte 3000(安捷伦科技)；基本参数：采用PI(碘化丙啶)染色，采集红色荧光(B615通道)。
结果如图7及表2所示：不同处理方法得到的经血源性MSC细胞周期分布相近，表明其增殖能力差异极小(S+G2/M期的比例越高，说明处在分裂过程中的细胞越多，提示细胞增殖能力越强)。
表2 不同处理方法分离人宫血来源MSC的细胞周期分布单位(％)
Figure PCTCN2022081910-appb-000002
2、不同处理方法分离经血源性MSC的细胞表面标志物表达情况
MSC是一类成体来源的干细胞，因其具有自我更新增殖、多向分化、免疫调节、促血管生成等能力而用于临床治疗关节软骨损伤、移植物抗宿主病、自身免疫性疾病、创口修复等。根据国际细胞治疗协会对MSC的最低标准要求MSC应表达干细胞增殖分化相关的标志物CD90、与免疫调节相关的标志物CD73、与血管生成相关的标志物CD105；不表达白细胞标志物CD45、造血干细胞标志物CD34、单核巨噬细胞标志物CD14或CD11b、B细胞标志物CD19或CD79α、以及MHC-II类分子如HLA-DR等。
(1)分别不同处理方法分离经血源性MSC(对比实施例2中D供体步骤(4)清洗法得到的经血源性MSC、过滤法得到的经血源性MSC、以及步骤(5)清洗-过滤法得到的经血源性MSC)至1.5mL离心管，离心弃去上清液。
(2)染色液(含0.5％BSA的PBS缓冲液)重悬后平均分装至11个新的离心管中，每管加入Isotype、PE-IgG1、CD73、CD90、CD105、CD11b、CD19、CD34、CD45、HLA-DR、 Isotype PE-IgG2a抗体混匀后孵育20～30min。
(3)加入染色液洗涤一次，离心后弃去上清，用流式细胞仪检测，根据细胞大小和颗粒度设门圈出目标细胞群，排除死细胞和碎片，然后根据Isotype对照组荧光强度，在目标细胞群的基础上画出阳性细胞群，统计其阳性群比例。
结果如表3所示：步骤(4)清洗法得到的经血源性MSC CD45的阳性率超过2％，检测不符合MSC的标准要求(提示杂细胞过多，纯度不足)；而步骤(4)过滤法得到的经血源性MSC、以及步骤(5)清洗-过滤法得到的经血源性MSCCD105、CD73和CD90阳性率均≥95％，CD45、CD34、CD11b、CD19和HLA-DR阳性率均≤2％，符合检测标准，可见，采用过滤法收集血细胞层细胞可以使所得到MSC符合MSC的标准要求。
表3 不同处理方法分离人宫血来源MSC的细胞表面标志物表达情况单位(％)
Figure PCTCN2022081910-appb-000003
3、不同处理方法分离经血源性MSC抑制PBMC Th1和Th17的能力
MSC具有调节免疫的功能，在体外可以通过检测MSC与炎性因子激活或PBMC共培养后，PBMC中促炎性淋巴细胞亚群Th1和Th17的比例，评估炎症条件下MSC抑制Th1和Th17的能力，从而反映MSC的抑炎作用。
(1)分别取不同处理方法分离经血源性MSC样品(对比实施例2中D供体步骤(4)清洗法得到的经血源性MSC、过滤法得到的经血源性MSC、以及步骤(5)清洗-过滤法得到的经血源性MSC)接种至12孔板中，置于5％CO 2、37℃细胞培养箱中培养过夜，细胞贴壁。
(2)第二天，吸弃培养基，数量按MSC:PBMC为1：5的比例加入PBMC细胞悬液共同培养为实验组，同时单独培养的PBMC作为对照组，继续培养2天。
(3)在结束培养前4～6h，实验组和对照组每孔加入Leukocyte Activation Cocktail混匀后继续培养。
(4)结束培养后，收集上清中的PBMC，用染色液(含0.5％BSA的PBS缓冲液)洗涤一次。
(5)PBMC先加入表面流式抗体CD3和CD8标记，4℃孵育30分钟。
(5)直接加入染色液洗涤一次。
(6)加入250uL Fixation/Perm Buffer固定破膜孵育30分钟。
(7)直接加入1mL Perm/Wash Buffer洗涤一次。
(8)加入IFN-γ/IL-17A流式抗体进孵育30分钟。
(9)直接加入染色液洗涤一次后，用流式细胞仪检测，根据细胞大小和颗粒度设门圈出目标细胞群，排除死细胞和碎片，然后根据Isotype对照组荧光强度，在目标细胞群的基础上画出阳性细胞群，统计同时表达CD3阳性、CD8阴性、IFN-γ阳性的细胞比例，即为Th1细胞；统计同时表达CD3阳性、CD8阴性、IL-17A阳性的细胞比例，即为Th17细胞。
结果如表4、5、图8、9所示：对比实施例2中步骤(4)清洗法得到的经血源性MSC、过滤法得到的经血源性MSC、以及步骤(5)清洗-过滤法得到的经血源性MSC均能抑制PBMC中激活的Th1和Th17的增殖，但是采用过滤法和清洗-过滤法(即包含过滤操作)得到的经血源性MSC对Th1和Th17的抑制率高于清洗法得到的经血源性MSC，表明采用过滤法收集血细胞层细胞可以使所得到MSC的抑炎作用提高。
表4 不同处理方法分离人宫血来源MSC抑制Th1的能力比较单位(％)
组别 Th1表达1 Th1表达2 Th1表达3 平均数 抑制率
PBMC 11.30 11.51 11.61 11.47 -
过滤法 5.63 5.22 5.84 5.56 51.52
清洗法 5.69 6.94 6.28 6.30 45.07
清洗-过滤法 4.13 4.95 4.40 4.49 60.84
表5 不同处理方法分离人宫血来源MSC抑制Th17的能力比较单位(％)
组别 Th17表达1 Th17表达2 Th17表达3 平均数 抑制率
PBMC 1.37 1.68 1.40 1.48 -
过滤法 0.75 0.71 0.79 0.75 49.32
清洗法 1.05 0.88 1.03 0.99 33.1
清洗-过滤法 0.80 1.03 0.90 0.91 38.51
4、三系分化检测
MSC作为一种成体干细胞，具有多向分化潜能，经特定诱导培养基诱导，具有成脂、成骨和成软骨的能力。使用油红O染色评估MSC向脂肪细胞诱导分化结果；使用茜素红S染色评估MSC向骨细胞诱导分化结果；使用阿利新蓝染色评估MSC向软骨细胞诱导分化结果。
结果如图10所示：1)另采用一供体的经血，将经血一分为四，分别以四种不同分离方法所得细胞(对比实施例1的传统方法、对比实施例2中步骤(4)清洗法、过滤法、以及步 骤(5)清洗-过滤法)均具有成脂、成骨和成软骨的能力。2)四种方法来源的细胞成脂和成骨分化能力没有显著差异。3)从成软骨分化来看：清洗-过滤法和过滤法成软骨分化能力强。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种制备经血源性间充质干细胞的方法，包括如下步骤：
    (1)、(2)和(31)；或
    (1)、(2)和(32)；
    (1)取经血样品过10～30目筛，得到滤液；
    (2)将滤液进行密度梯度离心，得到四层分离液：血浆层、白膜层、分离液层和血细胞层；
    (31)收集白膜层和血细胞层，分别用培养基重悬，得到S1液和S2液，混合，贴壁培养，得到经血源性间充质干细胞；
    (32)收集白膜层和血细胞层，混合，用培养基重悬，贴壁培养，得到经血源性间充质干细胞。
  2. 根据权利要求1所述的方法，其特征在于：
    步骤(31)、(32)中所述收集血细胞层的方法为过滤法，包括如下步骤：将血细胞层与清洗液混合，经40～70um的过滤装置过滤，弃滤液，用清洗液冲洗被截留的物质，收集包含被截留的物质的清洗液；
    优选地，步骤(31)、(32)中所述收集白膜层的方法为：去除大部分血浆层，吸出剩余血浆层和白膜层，加入清洗液；
    优选地，所述清洗液为缓冲液；进一步为包含抗生素的缓冲液。
  3. 根据权利要求1所述的方法，其特征在于：
    步骤(1)中所述经血样品为经血与经血保存液的混合液。
  4. 根据权利要求1所述的方法，其特征在于：
    步骤(1)中所述经血为女性月经周期第一天至第四天中至少一天的经血。
  5. 根据权利要求1所述的方法，其特征在于：
    步骤(2)中所述密度梯度离心的方法为：将滤液加入淋巴细胞分离液上，离心；
    优选地，所述离心的条件为200～800g，15～25℃下离心20～40min。
  6. 根据权利要求1～5中任一项所述的方法，其特征在于：
    步骤(31)、(32)中所述贴壁培养包括原代培养；
    优选地，所述原代培养的方法为：35～40℃，4～6％CO 2下培养，20～28h后，换液，去除未贴壁细胞，以后每1～3天换液一次。
  7. 根据权利要求1～5中任一项所述的方法，其特征在于：
    步骤(31)、(32)中所述贴壁培养还包括传代培养；
    优选地，所述传代培养的方法为：待原代培养细胞长满80％～90％时，消化，进行传代；
    优选地，所述传代培养的条件为35～40℃，4～6％CO 2。
  8. 一种经血源性间充质干细胞的建库方法，包含权利要求1～7中任一项所述的制备经血源性间充质干细胞的方法的步骤。
  9. 一种间充质干细胞，根据权利要求1～7中任一项所述的方法制备得到。
  10. 权利要求9所述的间充质干细胞在制备药物中的应用，
    优选地，所述药物用于治疗以下疾病中的至少一种：血液系统疾病、心血管疾病、肿瘤、肝硬化、神经系统疾病、膝关节半月板部分切除损伤修复、自身免疫性疾病。
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