WO2021197459A1 - 从人经血中获得宫内膜间充质干细胞的方法 - Google Patents

从人经血中获得宫内膜间充质干细胞的方法 Download PDF

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WO2021197459A1
WO2021197459A1 PCT/CN2021/085202 CN2021085202W WO2021197459A1 WO 2021197459 A1 WO2021197459 A1 WO 2021197459A1 CN 2021085202 W CN2021085202 W CN 2021085202W WO 2021197459 A1 WO2021197459 A1 WO 2021197459A1
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cell
cell sieve
sieve
meshes
mesenchymal stem
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PCT/CN2021/085202
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金槿
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上海我武干细胞科技有限公司
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Priority to JP2022558395A priority Critical patent/JP7428823B2/ja
Priority to US17/907,318 priority patent/US20230116841A1/en
Priority to EP21778996.5A priority patent/EP4130249A4/en
Priority to CN202180025074.9A priority patent/CN115516083A/zh
Publication of WO2021197459A1 publication Critical patent/WO2021197459A1/zh

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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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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  • This application relates to the field of methods for preparing mesenchymal stem cells, in particular to a method for obtaining endometrial mesenchymal stem cells from human menstrual blood.
  • BMSC bone marrow-derived mesenchymal stem cells
  • the bone marrow needs to be obtained by puncture under anesthesia, and the number of autologous bone marrow stem cells varies with The aging of age is significantly reduced, all of which limit the clinical application value of BMSC.
  • the human endometrium is a highly dynamic tissue that periodically undergoes proliferation, secretion, and menstrual periods.
  • the female endometrium undergoes more than 400 times of self-renewal, proliferation, differentiation and shedding during its lifetime. It has a high degree of regeneration.
  • Mesenchymal stem cells derived from endometrial tissue not only exist in the basal layer, but also in the menstrual blood. It is convenient to obtain materials.
  • Human endometrial mesenchymal stem cells (human endometrial mesenchymal stem cells, hEMSC) have attracted more and more attention. Using endometrial mesenchymal stem cells as seed cells, they are used in cell therapy, tissue engineering, and regenerative medicine. Good application prospects.
  • the separation of mesenchymal stem cells mainly includes tissue digestion and gradient density centrifugation.
  • the tissue digestion method can obtain a large number of living cells from the living body in a short time, and the cell characteristics are similar to those in the body and can reflect the growth characteristics. It is very suitable for drug testing, cell differentiation and other experimental studies.
  • the digestion method is not only cumbersome and easy to contaminate. If the operator does not grasp the digestion time properly, it will directly affect the purity and biological characteristics of the primary mesenchymal stem cells, destroy the cell structure, and interfere with cell proliferation. And differentiation, and even get heterogeneous cell populations.
  • some digestive enzymes are expensive, which inevitably increases the cost of the experiment.
  • Gradient density centrifugation is based on the different density and diameter of different particles, the sedimentation speed in the solution is also different, under a certain centrifugal force, the particles each settle at a certain speed, using gravity and centrifugal force Function to sort cells, suitable for purifying mononuclear cells, but not suitable for separating and purifying cells from tissues. Therefore, finding another suitable for separating and purifying endometrial mesenchymal stem cells from human menstrual blood is a problem that must be solved in the current endometrial mesenchymal stem cell drug development.
  • the present invention provides a method for obtaining endometrial mesenchymal stem cells from human menstrual blood, the method comprising:
  • the menstrual blood sample is graded through a cell sieve to separate tissue fragments, and the pore size of the cell sieve is gradually reduced, including a filtering cell sieve for collecting filtrate and a retention cell sieve for collecting retentate, and retention of cells
  • the aperture of the sieve is smaller than the filtered cell sieve
  • Collecting adherent cells is endometrial mesenchymal stem cells.
  • the graded cell sieve includes at least a third-level cell sieve, preferably at least a fourth-level cell sieve.
  • the graded cell sieve includes a filtered cell sieve that has passed through one or two or more stages, and/or a retained cell sieve that has passed through one or two or more stages.
  • the graded cell sieve includes two or more stages of retained cell sieves, and the step 2) collecting the tissue fragments retained on the cell sieve includes combining the tissue fragments on the multi-stage retained cell sieve.
  • the step 2) collecting the tissue debris trapped on the cell sieve includes backwashing the tissue debris trapped on the trapped cell sieve with a cleaning solution.
  • the step 2) collecting the tissue debris trapped on the cell sieve also includes washing the tissue debris with a cleaning solution.
  • the present invention provides endometrial mesenchymal stem cells obtained by the method of the present invention.
  • Figure 1 shows the comparison of the expansion multiples of endometrial mesenchymal stem cells prepared by the mononuclear method and the method of the present invention.
  • Figure 2 shows the comparison of the doubling time of endometrial mesenchymal stem cells prepared by the mononuclear method and the method of the present invention.
  • Fig. 3A shows a staining diagram of the osteogenic differentiation ability of endometrial mesenchymal stem cells prepared by the method of the present invention
  • Fig. 3B shows a staining diagram of the chondrogenic differentiation ability of endometrial mesenchymal stem cells prepared by the method of the present invention.
  • the “range” disclosed herein takes the form of the lower limit and the upper limit. There can be one or more lower limits, and one or more upper limits, respectively.
  • the given range is limited by selecting a lower limit and an upper limit.
  • the selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range.
  • the present invention first provides a method for obtaining endometrial mesenchymal stem cells from human menstrual blood.
  • the preparation method includes:
  • the menstrual blood sample is graded through a cell sieve to separate tissue fragments, and the pore size of the cell sieve is gradually reduced, including a filtering cell sieve for collecting filtrate and a retention cell sieve for collecting retentate, and retention of cells
  • the aperture of the sieve is smaller than the filtered cell sieve
  • Collecting adherent cells is endometrial mesenchymal stem cells.
  • the method includes
  • Tissue separation the menstrual blood sample is separated by grading through a cell sieve, and the aperture of the cell sieve is gradually reduced;
  • Tissue backwashing Backwash the tissue fragments trapped on the cell sieve with cleaning fluid
  • Tissue cleaning Resuspend the tissue fragments with cleaning solution, centrifuge to obtain tissue fragments precipitation;
  • Tissue culture Resuspend the tissue debris sediment with culture medium, inoculate it in a culture flask, add culture medium and place it in an incubator for culture;
  • Some embodiments further include expanding and culturing the collected endometrial mesenchymal stem cells.
  • the cell sieve can be any porous structure that can separate substances according to particle size without causing pollution to tissues, cells, etc.
  • Graded sieving refers to the use of cell sieves with different pores to pass through the sieves in order from large pores to small pores, thereby obtaining multi-stage retentates with different size distributions, or obtaining multi-stage retentates with different size distribution components.
  • Grade filtration material refers to the use of cell sieves with different pores to pass through the sieves in order from large pores to small pores, thereby obtaining multi-stage retentates with different size distributions, or obtaining multi-stage retentates with different size distribution components.
  • tissue fragments refer to fragment-like tissues and not specifically to products obtained through crushing operations.
  • the pore size of the retained cell sieve may be 70 meshes to 160 meshes, preferably 80 meshes to 160 meshes.
  • the present invention includes at least one level of retention cell sieves, but may and preferably includes two or more levels of retention cell sieves with decreasing pore size.
  • the tissue fragments trapped on the cell sieve of different levels can be combined for subsequent steps, such as backwashing with a cleaning solution and combined use, or scraping and combining before resuspension, cleaning, etc. .
  • the excessively large tissue fragments are removed by filtering the cell sieve and the filtrate is collected.
  • the filtered cell sieve includes a pore size not exceeding 30 meshes, not exceeding 35 meshes, not exceeding 40 meshes or smaller pores.
  • Cell sieve tissue fragments with a pore size of less than 36 mesh can be collected.
  • the filtered cell sieve includes at least one 36 mesh cell sieve. Removal of too large tissue fragments can be achieved by using one-stage sieving or multi-stage sieving.
  • the present invention includes at least one filter cell sieve, but may and preferably includes two or more filter cell sieves with decreasing pore size.
  • the pore size of the filter cell sieve can be 18 mesh to 60 mesh, preferably 36 mesh. -60 mesh, more preferably 36 mesh-50 mesh, still more preferably 36 mesh-40 mesh.
  • the step of stepping through a cell sieve of the present invention includes passing through at least a third-level cell sieve, preferably including at least a fourth-level cell sieve.
  • the step of grading through the cell sieve may include passing through at least two stages of filtering cell sieves and at least one stage of retaining cell sieves; or including passing through at least one stage of filtering cell sieves and at least two stages of retaining cell sieves; or including passing through at least two stages of filtering. Pass through a cell sieve and at least two levels of retention cell sieve.
  • tissue backflushing and “backflushing” both refer to the process of washing down the retentate with the cleaning fluid in the opposite flow direction of the molecular sieve. In order to recover as much of the retentate as possible, the end point of washing can be stopped based on the absence of visible tissue on the cell sieve.
  • the specific steps of backwashing may include: collecting a cell sieve with trapped tissue debris; placing a certain number of cell sieves with trapped tissue debris in a petri dish, the number of placement depends on the size of the petri dish, such as 10 cm 2 There are no more than 5 cell sieves in the petri dish; add the cleaning solution to the petri dish with the cell sieves that trap tissue debris, and add the amount of cleaning solution to just submerge the sieve in the petri dish; use a Pasteur pipette to suck The cleaning solution in the petri dish will backwash the tissue fragments trapped on the cell sieve; stop washing until there is no visible tissue on the cell sieve.
  • the method of the present invention further includes a tissue washing step, that is, washing tissue fragments harvested from the retained cell sieve.
  • the tissue fragments can be cleaned by resuspension in a cleaning solution.
  • the washed tissue debris can be precipitated by solid-liquid separation (for example, filtration or centrifugation).
  • the tissue cleaning step may include: collecting flushing fluid, solid-liquid separation to collect tissue fragments precipitation (for example, filtration or centrifugation, discarding the supernatant), adding cleaning fluid; repeat this many times The tissue fragments are cleaned, so as to obtain the cleaned tissue fragment sedimentation.
  • the conditions for centrifugal operation can be 400g centrifugal force, temperature 20-22°C, preferably 20°C, and centrifugation time 10 minutes.
  • the "washing solution” may be a buffer solution commonly used in cell operations, for example, a washing solution based on commonly used buffers such as phosphate buffer, in which other components may be added as needed, for example, antibiotics may be added in the present invention.
  • the cleaning solution is a phosphate buffer solution, and in some preferred embodiments, it is a phosphate buffer solution containing antibiotics.
  • the antibiotic is selected from gentamicin, penicillin, streptomycin, amphotericin B or a combination thereof.
  • the cleaning solution contains gentamicin, its concentration may be 50-100 U/mL, preferably 100 U/mL; if the cleaning solution contains penicillin, its concentration may be 180-200 U/mL, preferably 200 U/mL; If it contains streptomycin, its concentration may be 0.15-0.2 mg/mL, preferably 0.2 mg/mL; if the cleaning solution contains amphotericin B, its concentration may be 4-5 ⁇ g/mL, preferably 5 ⁇ g/mL.
  • Step 3 of the present invention culturing tissue fragments in mesenchymal stem cell culture medium, also known as "tissue culture”, includes inoculating tissue fragments in mesenchymal stem cell culture medium and culturing under mesenchymal stem cell culture conditions, thereby The tissue fragments grow mesenchymal stem cells with adherent properties.
  • the inoculation includes resuspending the tissue debris pellet with a mesenchymal stem cell culture medium for inoculation, and the culture can be carried out in a culture flask.
  • the non-adherent foreign cells can be removed to prevent the foreign cells from affecting the endometrial mesenchyme.
  • Stem cell culture causes contamination, and then continues to be cultured until the confluence reaches 90%-100% and then passage.
  • the mesenchymal stem cell culture medium used in the present invention can be any medium known to those skilled in the art for the cultivation of mesenchymal stem cells, and it can be obtained through self-preparation or through commercial channels.
  • the mesenchymal stem cell culture medium is selected from MSC primary cell serum-free culture medium (available from Beijing Youkang Technology Co., Ltd.) and amniotic fluid cell culture medium (available from Guangzhou Baiyunshan Baidi Biomedical Co., Ltd.).
  • the tissue culture conditions are conventional mesenchymal stem cell culture conditions, such as 5% CO 2 , 37°C.
  • the cultivation time is 4-6 days, preferably 5 days.
  • it further includes isolating the adherent endometrial mesenchymal stem cells after tissue culture for expansion culture.
  • a step can be added before step 1) to check the contamination of the collected menstrual blood sample, including combining the collected menstrual blood sample with a cleaning solution (preferably a phosphate buffer containing antibiotics, for example, a phosphate buffer containing an antibiotic) Amphotericin and amphotericin B (phosphate buffer) are mixed, and the mixed liquid is added to the culture medium and cultured in an incubator to observe the contamination.
  • a cleaning solution preferably a phosphate buffer containing antibiotics, for example, a phosphate buffer containing an antibiotic
  • Amphotericin and amphotericin B phosphate buffer
  • the mixing ratio of menstrual blood sample and cleaning solution is 1:1.
  • the invention also provides endometrial mesenchymal stem cells obtained by the method of the invention. Compared with the endometrial mesenchymal stem cells produced by the prior art such as mononuclear method, the endometrial mesenchymal stem cells obtained in the present invention have higher proliferation speed and passage expansion multiples, and have good differentiation potential.
  • the endometrial mesenchymal stem cells are prepared, and the specific experimental methods are as follows:
  • the menstrual blood sample in the 50 mL centrifuge tube was passed through an 18-mesh stainless steel cell sieve, a 36-mesh stainless steel cell sieve, an 80-mesh stainless steel cell sieve, and a 160-mesh stainless steel cell sieve in sequence.
  • step (1) 1) Put the 80-mesh and 160-mesh stainless steel cell sieves filtered from the menstrual blood sample in step (1) into a 10cm 2 petri dish, and no more than 5 cell sieves are placed in each petri dish, in each 10cm 2 petri dish Add 40mL of cleaning solution so that the cleaning solution just does not pass the sieve that retains tissue fragments; among them, the cleaning solution used is phosphate buffered saline, and contains gentamicin at a concentration of 100U/mL and amphoteric mold at a concentration of 5 ⁇ g/mL Vegetarian B;
  • the medium is changed to wash away non-adherent cells (such as red blood cells), and the culture is continued.
  • the subculture can be carried out when the degree of fusion reaches 90%.
  • Example 2 Detection of the growth of endometrial mesenchymal stem cells obtained by different preparation methods
  • the endometrial mesenchymal stem cells prepared in Example 1 and the endometrial mesenchymal stem cells prepared by the above mononuclear method were subcultured, and each generation was inoculated into T75 at a density of 5.0 ⁇ 10 3 cells/cm 2.
  • the culture medium is amniotic fluid cell culture medium (YS for short, purchased from Guangzhou Baiyunshan Baidi Biomedical Co., Ltd.) for culture.
  • the cell fusion rate reaches about 90%, the culture medium is discarded, the cells are gently washed with phosphate buffered saline (PBS), the washing solution is discarded, and 3 ml of 0.25% trypsin is added to digest the cells for 4 minutes.
  • PBS phosphate buffered saline
  • P1 represents the first generation of cell subculture
  • FC refers to the abbreviation of the preparation method disclosed in the present invention
  • DH refers to the abbreviation of the mononuclear method.
  • the mesenchymal stem cells prepared by the preparation method of the present invention the expansion multiples of the cells of different generations in the culture medium are mostly higher than those of the endometrial mesenchymal stem cells prepared by the mononuclear method;
  • the doubling time is also shorter than that of the mononuclear method, and the doubling time of the mesenchymal stem cells prepared by the mononuclear method is affected by the generation and fluctuates greatly, while the doubling time required for the passage of mesenchymal stem cells prepared by the preparation method of the present invention The fluctuation is small. Therefore, from the two aspects of the doubling time and the expansion multiple of the obtained endometrial mesenchymal stem cells, the preparation method of the present invention has advantages compared with the mononuclear method.
  • Example 3 Differentiation ability test of endometrial mesenchymal stem cells prepared by the preparation method of the present invention
  • the endometrial mesenchymal stem cells 0031 prepared by the preparation method of Example 1 were resuspended in a cell concentration of 1.0 ⁇ 10 7 /ml amniotic fluid cell culture medium, and the suspension was evenly dropped onto the lid of a 10 cm 2 petri dish. Place them in a 5% CO 2 , 37°C incubator for 24 hours, pipette the balls into a 6-well plate in an ultra-clean table, and add 2 mL of chondrogenic differentiation medium (purchased from STEMCELL) to each well. The culture medium was changed every 3 days, and the chondrogenic differentiation section cells were stained with alcian blue when the chondrogenic differentiation was induced for 28 days. The results are shown in Figure 3B.
  • Fig. 3A the endometrial mesenchymal stem cells prepared by the method described in Example 1 are obviously orange-red after staining with Alizarin Red S, which indicates that their osteogenic differentiation ability is good; it can be seen from Fig. 3B that the examples are adopted
  • the endometrial mesenchymal stem cells prepared by the method described in 1 were stained with alcian blue and the tissues appeared blue, indicating that they have the ability to form cartilage.
  • the endometrial mesenchymal stem cells prepared by the method of the present invention have good differentiation ability. .

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Abstract

提供了一种从人经血中获得宫内膜间充质干细胞的方法和由此制备的宫内膜间充质干细胞，所述方法包括：1）将经血样本分级过细胞筛以分离组织碎片，细胞筛的孔径逐级变小，包括用于收集滤过物的滤过细胞筛和用于收集截留物的截留细胞筛，截留细胞筛的孔径小于滤过细胞筛；2）收集截留细胞筛上的截留物即组织碎片；3）在间充质干细胞培养基中培养组织碎片；4）收集贴壁细胞即为子宫内膜间充质干细胞。

Description

从人经血中获得宫内膜间充质干细胞的方法 技术领域
本申请涉及间充质干细胞制备方法领域，具体涉及一种从人经血中获得宫内膜间充质干细胞的方法。
背景技术
近年来，间充质干细胞(Mesenchymal stem cells，MSC)因其具有增殖能力强、免疫原性低、多向分化潜能及定向迁徙至组织损伤部位等优势，受到研究者的广泛关注。骨髓来源间充质干细胞(bone marrow-derived stem cells，BMSC)是最早发现的间充质干细胞，也是目前临床研究最常用的干细胞，因骨髓需在麻醉下穿刺获取，且自体骨髓干细胞数目随着年龄的老化显著减少，这些均限制了BMSC的临床应用价值。人类子宫内膜是一种高度动态的组织，周期性地经历增殖期、分泌期、月经期，具有显著的再生能力，能从月经后最初的0.5-1毫米长到5-7毫米。女性子宫内膜一生中经过400次以上自我更新、增殖、分化和脱落，具有高度再生能力，来源于子宫内膜组织的间充质干细胞不仅存在于基底层，经血中也存在，取材方便，因此人子宫内膜间充质干细胞(human endometrial mesenchymal stem cell，hEMSC)越来越受到重视，以宫内膜间充质干细胞作为种子细胞，用于细胞治疗、组织工程和再生医学等方面，都具有良好的应用前景。
目前对于间充质干细胞的分离主要包括组织消化法和梯度密度离心法。组织消化法能短时间从活体上获得大量活细胞，且细胞特性与体内接近并能反应生长特性，很适合做药物测试，细胞分化等实验研究。不过其缺点也比较明显，消化法不但步骤繁琐，易污染，若操作者对消化时间把握不当，直接影响原代获得的间充质干细胞纯度及其生物学特性，并且破坏细胞结构，干扰细胞增殖及分化，甚至得到异种的细胞群。另外，一些消化酶价格昂贵，不可避免提高了实验成本。梯度密度离心法(单核法)，是根据不同颗粒之间密度和直径不同，在溶液中的沉降速度也不同，在一定的离心力作用下，颗粒各自以一定的速度沉降，利用重力及离心力的作用分选细胞，适合对于单个核细胞进行纯化，不适用于从组织中分离纯化细胞。因此，寻找到另外一种适用于从人经血中分离纯化宫内膜间充质干细胞是目前宫内膜间充质干细胞药物研发必须要解决的问题。
发明内容
本发明提供了一种从人经血中获得宫内膜间充质干细胞的方法，所述方法包括：
1)将经血样本分级过细胞筛以分离组织碎片，所述细胞筛的孔径逐级变小，包括用于收集滤过物的滤过细胞筛和用于收集截留物的截留细胞筛，截留细胞筛的孔径小于滤过细胞筛；
2)收集截留细胞筛上的截留物即组织碎片；
3)在间充质干细胞培养基中培养组织碎片；
4)收集贴壁细胞即为宫内膜间充质干细胞。
一些实施方式中，所述分级过细胞筛包括至少经过三级细胞筛，优选包括至少经过四级细胞筛。
一些实施方式中，所述分级过细胞筛包括经过一级或两级或更多级的滤过细胞筛，且/或包括经过一级或两级或更多级的截留细胞筛。优选地，所述分级过细胞筛包括两级或更多级的截留细胞筛，并且所述步骤2)收集细胞筛上截留的组织碎片包括将多级截留细胞筛上的组织碎片合并。
一些实施方式中，所述步骤2)收集细胞筛上截留的组织碎片包括将截留在截留细胞筛上的组织碎片用清洗液反冲洗下来。
一些实施方式中，所述步骤2)收集细胞筛上截留的组织碎片还包括用清洗液清洗组织碎片。
另一方面，本发明提供了用本发明方法获得的宫内膜间充质干细胞。
附图说明
图1显示单核法与本发明方法制备所得宫内膜间充质干细胞的扩增倍数比较。
图2显示单核法与本发明方法制备所得宫内膜间充质干细胞的倍增时间比较。
图3A显示了本发明方法制备的宫内膜间充质干细胞的成骨分化能力染色图；图3B显示了本发明方法制备的宫内膜间充质干细胞的成软骨分化能力染色图。
具体实施方案
在本说明书中，如果没有特别的说明，所涉及的各组分或其优选组分可以相互组合形成新的技术方案。
在本说明书中，如果没有特别的说明，本文所提到的所有实施方案以及优选实施方案可以相互组合形成新的技术方案。
在本说明书中，如果没有特别的说明，本文所提到的所有技术特征以及优选特 征可以相互组合形成新的技术方案。
如果没有特别指出，本说明书所用的术语“一种”指“至少一种”。
本文所公开的“范围”以下限和上限的形式。可以分别为一个或多个下限，和一个或多个上限。给定范围是通过选定一个下限和一个上限进行限定的。选定的下限和上限限定了特别范围的边界。所有可以这种方式进行限定的范围是包含和可组合的，即任何下限可以与任何上限组合形成一个范围。
具体而言，本发明首先提供了一种从人经血中获得宫内膜间充质干细胞的方法，该制备方法包括：
1)将经血样本分级过细胞筛以分离组织碎片，所述细胞筛的孔径逐级变小，包括用于收集滤过物的滤过细胞筛和用于收集截留物的截留细胞筛，截留细胞筛的孔径小于滤过细胞筛；
2)收集截留细胞筛上的截留物即组织碎片；
3)在间充质干细胞培养基中培养组织碎片；
4)收集贴壁细胞即为宫内膜间充质干细胞。
实施方式之一中，所述方法包括
1)组织分离：将经血样本分级过细胞筛分离，所述细胞筛的孔径逐级变小；
2)组织反冲：将截留在细胞筛上的组织碎片用清洗液反冲洗下来；
3)组织清洗：用清洗液重悬清洗组织碎片，离心，获得组织碎片沉淀；
4)组织培养：用培养基重悬组织碎片沉淀，接种于培养瓶中，加入培养基置于培养箱中培养；
5)收集长出的贴壁细胞即为宫内膜间充质干细胞。
一些实施方式中还包括对收集的宫内膜间充质干细胞进行扩增培养。
其中，细胞筛可以是任何可将物质按照颗粒大小进行分离的一种多孔状结构且对组织、细胞等不造成污染。分级过筛是指采用不同孔径的细胞筛按照从大孔径到小孔径依次过筛的顺序通过细胞筛，由此可以获得不同大小分布的多级截留物，或者获得含有不同大小分布组分的多级滤过物。
本发明的一些实施方式中，首先用大孔径的滤过分子筛通过截留除去过大的组织碎片，接着用小孔径的截留分子筛截留收集用于培养的组织碎片。本发明中，“组织碎片”指碎片样的组织而非特指经过粉碎操作获得的产物。
一些实施方式中，截留细胞筛的孔径大小可为70目-160目，优选为80目-160目。本发明包括至少一级截留细胞筛，但可以并且优选包括孔径递减的两级或更多级截留细胞筛。包括多级截留细胞筛时，可以将截留在不同级细胞筛上的组织碎片 合并用于后续步骤，例如用清洗液反冲洗下来并合并使用，或者刮取后合并再进行重悬、清洗等处理。
本发明中，通过滤过细胞筛来去除过大的组织碎片并收集滤过物。优选地，收集小于30目、小于35目、小于40目孔径或更小的组织碎片，相应地，滤过细胞筛包括孔径不超过30目、不超过35目、不超过40目或孔径更小的细胞筛。例如，可以收集小于36目孔径的组织碎片，相应的，滤过细胞筛包括至少一级36目的细胞筛。去除过大的组织碎片可以采用一级过筛也可以采用多级过筛来实现。因此，本发明包括至少一级滤过细胞筛，但可以并且优选包括孔径递减的两级或更多级滤过细胞筛，滤过细胞筛的孔径大小可为18目-60目，优选36目-60目，更优选36目-50目，还要优选36目-40目。
一些实施方式中，本发明的分级过细胞筛步骤包括至少经过三级细胞筛，优选包括至少经过四级细胞筛。例如，分级过细胞筛步骤可以包括至少经过两级滤过细胞筛和至少一级截留细胞筛；或者包括至少经过一级滤过细胞筛和至少两级截留细胞筛；或者包括至少经过两级滤过细胞筛和至少两级截留细胞筛。
可采用任何合适的方法从截留细胞筛上收获组织碎片，例如刮取、挑取、反冲洗等，本领域技术人员能够视情况自行决定。一些实施方式中，本发明采用反冲洗的方法将截留在细胞筛上的组织碎片用清洗液冲洗下来。本文中，“组织反冲”和“反冲洗”都表示用清洗液按照与过分子筛时相反的流向将截留物洗下的过程。为了尽可能多地回收截留物，可以依据细胞筛上无肉眼可见的组织为终点停止冲洗。
举例来说，反冲洗具体的步骤可包括：收集有截留组织碎片的细胞筛；放置一定数量的有截留组织碎片的细胞筛于培养皿中，所述放置数量取决于培养皿的大小，例如10cm 2培养皿中放置的细胞筛不超过5个；加入清洗液到放置有截留组织碎片的细胞筛的培养皿中，加入清洗液的量为刚好淹没培养皿中的筛网；用巴氏吸管吸取培养皿中的清洗液将截留在细胞筛上的组织碎片反冲洗下来；直至细胞筛上无肉眼可见的组织时停止冲洗。
一些实施方式中，本发明的方法还包括组织清洗步骤，即对从截留细胞筛上收获的组织碎片进行清洗。例如通过用清洗液重悬来清洗组织碎片。然后，可以通过固液分离(例如过滤或离心)获得经清洗的组织碎片沉淀。举例来说，采用反冲洗来收集截留组织碎片时，组织清洗步骤可包括：收集冲洗液，固液分离收集组织碎片沉淀(例如过滤或离心，弃上清)，加入清洗液；如此反复多次清洗组织碎片，从而获得经清洗的组织碎片沉淀。如若采用离心，离心操作的条件可以是离心力400g，温度20-22℃、优选20℃，离心时间10分钟。
本发明中，“清洗液”可以是细胞操作中常用的缓冲液，例如基于磷酸盐缓冲液等常用缓冲液的清洗液，其中可以根据需要添加其他组分，例如在本发明中可添加抗生素。在一些实施方式中，清洗液为磷酸盐缓冲液，一些优选实施方式中为含有抗生素的磷酸盐缓冲液。优选地，抗生素选自庆大霉素、青霉素、链霉素、两性霉素B或其组合。优选地，清洗液如若包含庆大霉素则其浓度可为50-100U/mL，优选100U/mL；清洗液如若包含青霉素则其浓度可为180-200U/mL，优选200U/mL；清洗液如若包含链霉素则其浓度可为0.15-0.2mg/mL，优选0.2mg/mL；清洗液如若包含两性霉素B则其浓度可为4-5μg/mL，优选5μg/mL。
本发明步骤3)在间充质干细胞培养基中培养组织碎片，又称“组织培养”，包括将组织碎片接种在间充质干细胞培养基中在间充质干细胞培养条件下进行培养，从而由所述组织碎片长出具有贴壁特性的间充质干细胞。一些实施方式中，所述接种包括用间充质干细胞培养基重悬组织碎片沉淀用于接种，培养可以在培养瓶中进行。可选地，可以在确保宫内膜间充质干细胞贴壁贴牢后(例如组织培养48小时后)，去除不贴壁的杂细胞(例如红细胞)从而防止杂细胞对宫内膜间充质干细胞培养造成污染，然后继续培养至融合度达90％-100％后传代。
本发明所用的间充质干细胞培养基可以是本领域技术人员所知的任一种用于间充质干细胞培养的任何培养基，可以通过自己制备获得也可以通过商业渠道获得。所述间充质干细胞培养基选自MSC原代细胞无血清培养基(可购买自北京友康科技有限公司)和羊水细胞培养基(可购买自广州白云山拜迪生物医药有限公司)。
在一些实施方式中，组织培养条件即常规的间充质干细胞培养条件，例如5％CO 2，37℃。培养时间为4-6天，优选5天。
一些实施方式中，还包括在组织培养后分离出贴壁的宫内膜间充质干细胞进行扩增培养。
在一些实施方式中所述步骤1)之前还可以增加一步用于检查收集到的经血样本污染情况，包括将收集到的经血样本与清洗液(优选含有抗生素的磷酸盐缓冲液，例如含有庆大霉素和两性霉素B的磷酸盐缓冲液)混合，取混合后的液体加入培养基后于培养箱中培养，观察其污染情况。其中，经血样本与清洗液混合比例为1:1。
本发明还提供了用本发明方法获得的宫内膜间充质干细胞。本发明所得宫内膜间充质干细胞相比现有技术例如单核法所制得的宫内膜间充质干细胞具有更高的增殖速度和传代扩增倍数，并且具有良好的分化潜能。
实施例
下面将结合实施例进一步详细地描述本发明。然而应当理解，列举这些实施例只是为了起说明作用，而并不是用来限制本发明的范围。
实施例1：制备宫内膜间充质干细胞
按照本发明的方法制备宫内膜间充质干细胞，具体实验方法如下：
(一)组织分离：
将50mL离心管中的经血样本依次过18目不锈钢细胞筛、36目不锈钢细胞筛、80目不锈钢细胞筛和160目不锈钢细胞筛。
(二)组织反冲：
1)将步骤(一)中滤过经血样本的80目和160目不锈钢细胞筛放入10cm 2培养皿中，每个培养皿中放置的细胞筛不超过5个，每个10cm 2培养皿中加入40mL清洗液，使得清洗液刚好没过截留组织碎片的筛网；其中，所用清洗液为磷酸盐缓冲液，并且含有浓度为100U/mL的庆大霉素和浓度为5μg/mL的两性霉素B；
2)用3mL巴氏吸管吸取培养皿中的清洗液将截留在80目和160目细胞筛上的组织碎片反冲洗下来。
(三)组织清洗：
1)冲洗完成后，将上述步骤中含有组织碎片的冲洗液收集于50mL离心管中，每管40mL，在20℃，离心力为400g的条件下离心10分钟，弃上清；
2)用移液枪吸取清洗液加入离心管中，每管加入10mL清洗液，再次重悬沉淀后合并到一个50mL离心管中，在20℃，离心力为400g的条件下离心10分钟，弃上清；
3)用移液枪吸取30mL清洗液加入离心管中，再次重悬沉淀后，在20℃，离心力为400g的条件下离心10分钟，弃上清。
(四)组织培养：
1)用移液枪吸取1mL培养基(羊水细胞培养基，简称YS，购买自广州白云山拜迪生物医药有限公司)重悬沉淀，每500μL重悬液接种于1个T25培养瓶中，加入培养基10mL重悬；
2)“十”字或“8”字混匀细胞，置于37℃，5％CO 2培养箱中培养；
3)细胞培养48小时后，换液洗去未贴壁杂细胞(例如红细胞)，继续进行培养。融合度达到90％即可进行传代培养。
实施例2：不同制备方法获得的宫内膜间充质干细胞生长情况检测
按照周云帆、杨波、胡祥等的“经血源性子宫内膜间充质干细胞的分离、培养与鉴定”(中国组织工程研究,2010,14(32):5952-5956)所述单核法制备宫内膜间充质干细胞。
分别取实施例1制备的宫内膜间充质干细胞和以上单核法制备的宫内膜间充质干细胞进行传代培养，每代以5.0×10 3个细胞/cm 2的密度分别接种到T75培养瓶中，其中的培养基为羊水细胞培养基(简称YS，购买自广州白云山拜迪生物医药有限公司)培养。当细胞融合率达到90％左右，弃去培养基后，用磷酸盐缓冲液(PBS)轻柔清洗细胞后弃去清洗液，加入3ml 0.25％的胰酶消化细胞4分钟。加入6ml含有5％胎牛血清的PBS混合液终止细胞消化。将细胞悬液分别收集到两个离心管中，1500r/min，离心5min。弃上清，分别加入1mL羊水细胞培养基(简称YS，购买自广州白云山拜迪生物医药有限公司)重悬细胞。采用AO/PI(AO(Acridine Orange)吖啶橙，PI(Propidium Iodide)碘化丙啶)双染细胞凋亡检测试剂盒(DNA探针双染细胞核方法，购买自上海睿钰生物科技有限公司)检测每代收获的细胞数量，计算每代的扩增倍数和倍增时间，实验结果见表1，附图1-2。
表1宫内膜间充质干细胞生长规律结果
Figure PCTCN2021085202-appb-000001
注：1)P1代表细胞传代培养第一代；
2)FC是指本发明公开的制备方法的简称，DH是指单核法的简称。
实验结论：本发明制备方法制备获得的间充质干细胞，其不同代次细胞在培养基中的扩增倍数大部分高于单核法制备的宫内膜间充质干细胞；其所获细胞的倍增时间也比单核法短，且单核法制得的间充质干细胞的倍增时间受代次影响，波动较大，而本发明的制备方法制得的间充质干细胞传代所需的倍增时间波动较小。因此，从所获宫内膜间充质干细胞的倍增时间和扩增倍数两方面来看，本发明的制备方法较单核法相比，具有优势。
实施例3：本发明的制备方法制备的宫内膜间充质干细胞分化能力测试
实验方法：取实施例1的制备方法制备的宫内膜间充质干细胞0031，以细胞浓度为1.0×10 5个/孔，将其均匀铺到内含羊水细胞培养基(简称YS，购买自广州白云山拜迪生物医药有限公司)的十二孔板中。置于5％CO 2、37℃培养箱中培养3-4天至细胞融合率达到90％-100％。于超净台中吸弃孔内培养基，每孔加入1mL成骨诱导分化完全培养基(购买自赛业生物科技有限公司)。每3天更换一次培养基，成骨诱导分化21天时，用茜素红S对成骨分化细胞进行染色。结果见附图3A。
取实施例1的制备方法制备的宫内膜间充质干细胞0031，以细胞浓度为1.0×10 7/ml羊水细胞培养基重悬，取悬液将其均匀滴到10cm 2培养皿盖上。置于5％CO 2、37℃培养箱中培养24小时，于超净台中吸取球滴至6孔板中，每孔加入2mL成软骨诱导分化培养基(购买自STEMCELL)。每3天更换一次培养基，成软骨诱导分化28天时，用阿利新蓝对成软骨分化切片细胞染色。结果见附图3B。
由图3A可知：实施例1所述方法制得的宫内膜间充质干细胞用茜素红S染色后呈明显的橙红色，表明其成骨分化能力良好；由图3B可知：采用实施例1所述方法制得的宫内膜间充质干细胞用阿利新蓝染色后组织间呈现蓝色，表明其具有成软骨能力。综上，本发明所述方法制得的宫内膜间充质干细胞具有很好的分化能力。。
尽管本发明描述了具体的例子，但是有一点对于本领域技术人员来说是明显的，即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。因此，所附权利要求覆盖了所有这些在本发明范围内的变动。

Claims (10)

  1. 一种从人经血中获得宫内膜间充质干细胞的方法，所述方法包括：
    1)将经血样本分级过细胞筛以分离组织碎片，所述细胞筛的孔径逐级变小，包括用于收集滤过物的滤过细胞筛和用于收集截留物的截留细胞筛，截留细胞筛的孔径小于滤过细胞筛；
    2)收集截留细胞筛上的截留物即组织碎片；
    3)在间充质干细胞培养基中培养组织碎片；
    4)收集贴壁细胞即为宫内膜间充质干细胞。
  2. 根据权利要求1所述的方法，所述截留细胞筛的孔径大小为70目-160目，优选为80目-160目。
  3. 根据权利要求1所述的方法，滤过细胞筛设置为收集小于30目、小于35目或小于40目的组织碎片；
    或者，所述滤过细胞筛的孔径大小为18目-60目，优选36目-60目，更优选36目-50目，还要优选36目-40目。
  4. 根据权利要求1所述的方法，在所述步骤1)中，所述分级过细胞筛包括至少经过三级细胞筛，优选包括至少经过四级细胞筛；
    或者，所述分级过细胞筛包括经过一级或两级或更多级的滤过细胞筛，且/或包括经过一级或两级或更多级的截留细胞筛；
    优选地，所述分级过细胞筛包括两级或更多级的截留细胞筛，并且所述步骤2)收集细胞筛上截留的组织碎片包括将多级截留细胞筛上的组织碎片合并。
  5. 根据权利要求1所述的方法，所述步骤2)收集细胞筛上截留的组织碎片包括将截留在截留细胞筛上的组织碎片用清洗液反冲洗下来。
  6. 根据权利要求1所述的方法，所述步骤2)收集细胞筛上截留的组织碎片还包括用清洗液清洗组织碎片。
  7. 根据权利要求5或6所述的方法，所述清洗液为磷酸盐缓冲液；优选为含有抗生素的磷酸盐缓冲液；更优选地，所述抗生素选自庆大霉素、青霉素、链霉素、两性霉素B或其组合；
    或选地，所述庆大霉素浓度为50-100U/mL，优选100U/mL；所述青霉素浓度为180-200U/mL，优选200U/mL；所述链霉素浓度为0.15-0.2mg/mL，优选0.2mg/mL；和/或所述两性霉素B浓度为4-5μg/mL，优选5μg/mL。
  8. 根据权利要求1所述的方法，所述间充质干细胞培养基选自MSC原代细胞 无血清培养基和羊水细胞培养基。
  9. 根据权利要求1所述的方法，步骤3)组织培养的时间为4-6天，优选5天。
  10. 权利要求1-9中任一项所述方法获得的宫内膜间充质干细胞。
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