WO2022213704A1 - 一种高迁徙间充质干细胞及其制备方法和应用 - Google Patents

一种高迁徙间充质干细胞及其制备方法和应用 Download PDF

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WO2022213704A1
WO2022213704A1 PCT/CN2022/072878 CN2022072878W WO2022213704A1 WO 2022213704 A1 WO2022213704 A1 WO 2022213704A1 CN 2022072878 W CN2022072878 W CN 2022072878W WO 2022213704 A1 WO2022213704 A1 WO 2022213704A1
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mesenchymal stem
stem cells
culture
cell
migration
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吴耀炯
莫妙华
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清华大学深圳国际研究生院
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Definitions

  • the invention belongs to the technical field of stem cells, and in particular relates to a high migration mesenchymal stem cell and a preparation method and application thereof.
  • Mesenchymal stem cells are adult stem cells with multi-directional differentiation ability, which can differentiate into a variety of tissue cells, and participate in tissue regeneration by secreting a large number of growth and immune regulatory factors. microenvironment and promote tissue damage repair. Mesenchymal stem cells are widely present in various tissues of the body, but their numbers decrease significantly with age. A large number of studies have proved that supplementation of mesenchymal stem cells can promote the regeneration and repair of various tissues.
  • Mesenchymal stem cells come from a wide range of sources and can be isolated from placenta, umbilical cord, bone marrow and other tissues. Due to the special low immunogenicity of mesenchymal stem cells, allogeneic mesenchymal stem cell transplantation does not cause immune rejection and does not require tissue matching. It is especially suitable for tissue damage repair (such as the treatment of brain, myocardial infarction, etc.) For the treatment of diseases (such as diabetes, Parkinson's disease, etc.), intravenous injection is the main route of mesenchymal stem cell transplantation.
  • the number of mesenchymal stem cells obtained from the tissue is very small, and it needs to be expanded and cultured in vitro to meet the needs of clinical treatment.
  • the two-dimensional (2D) adherent culture method of mesenchymal stem cells is generally adopted.
  • microcarriers for co-culture such as: GE microcarriers (Cytopore, Cytodex, Cytoline), gelatin microcarriers, etc.
  • the cells are attached to the microcarriers, which are essentially 2D cultures.
  • WO2009134532A2 discloses a hanging drop culture method. Although this method can form cell aggregates, this method is a static culture method, and the obtained mesenchymal stem cells CXCR4 expression level. And the ability of targeted migration to ischemic tissue is still insufficient.
  • the first object of the present invention is to provide a method for preparing high-migration mesenchymal stem cells;
  • the second object of the present invention is to provide high-migratory mesenchymal stem cells prepared by the preparation method;
  • the third object of the invention is to provide the highly migratory mesenchymal stem cells in the preparation of medicines for the treatment of tissue damage and repair, degenerative diseases, immune diseases, immune rejection after organ transplantation, organ function decline, organ and tissue ischemia, and inflammation.
  • the present invention provides a kind of preparation method of high migration mesenchymal stem cell, it comprises the following steps:
  • the mesenchymal stem cells are inoculated and suspended in the mesenchymal stem cell culture medium, and the flow liquid culture is carried out in a cell culture spinner flask or agitated fermenter. There is no need to add microcarriers during the culture process. After digestion to form single cells, high migratory mesenchymal stem cells are obtained.
  • the inventor creatively adopts a three-dimensional (3D) dynamic cell-based culture method, in which the mesenchymal stem cells are seeded and suspended in the mesenchymal stem cell culture medium, and the cells are cultured in a spinner flask (with a stirring device) or a stirring type.
  • Flow liquid culture is carried out in the fermenter without adding any microcarriers during the culture process, which can reduce the volume of mesenchymal stem cells and increase their mobility in blood circulation and migration to damaged tissues; It is creatively found that cells flow with the liquid and gradually form cell aggregates (spheroids), and the shear force generated by the liquid flow caused by stirring can further significantly increase the migration ability of mesenchymal stem cells, and significantly increase the space between cells.
  • the seeding density of the mesenchymal stem cells is (0.5-10) ⁇ 10 5 cells/mL.
  • the mesenchymal stem cell culture medium contains 1%-20% (V/V) fetal bovine serum or 0.5%-10% (V/V) human platelet lysate or serum replacement.
  • low-glucose DMEM medium of substances such as PALL Ultroser G.
  • the low-glucose DMEM medium can be a commercial low-glucose DMEM medium (for example: purchased from Thermo Fisher Scientific).
  • the mesenchymal stem cell culture medium of the present invention can also adopt other conventional cell basal culture medium.
  • the mesenchymal stem cell culture solution is further added with 0.1%-5% (W/V, final concentration) of human serum albumin, 1-100 nmol/L (final concentration) of human serum albumin.
  • Trichostatin A 1 ⁇ 50 ⁇ g/mL (W/V, final concentration) of human fibronectin and 1 ⁇ 40ng/mL (W/V, final concentration) of bFGF (basic fibroblast growth factor, basic fibroblast) cell growth factor).
  • the conditions for carrying out the flow liquid culture in the cell culture rotary flask or the stirring fermentor are: under the conditions of 37° C. and 5% CO 2 , the stirring speed is 5-200 rpm, and the culturing time is 5- 200h.
  • the method for collecting the cell spheroids after culturing includes centrifugation, filtration or natural sedimentation.
  • the step of washing and digesting to form single cells includes: washing the collected cell spheres with phosphate buffer, and then digesting with trypsin to form single cells.
  • the phosphate buffer is a phosphate buffer without calcium and magnesium ions.
  • the trypsin is trypsin containing EDTA.
  • the present invention also provides a high-migration mesenchymal stem cell, which is prepared by the above-mentioned preparation method.
  • the average diameter of the highly migratory mesenchymal stem cells of the present invention is 11-14 ⁇ m, and the expression rate of CXCR4, a receptor for cell migration, is expressed on the surface thereof is more than 30%.
  • the present invention also provides that the above-mentioned highly migratory mesenchymal stem cells are used in the preparation and treatment of diseases such as tissue damage repair, degenerative diseases, immune diseases, immune rejection after organ transplantation, organ function decline, organ and tissue ischemia, inflammation and other diseases. application in medicines.
  • diseases such as tissue damage repair, degenerative diseases, immune diseases, immune rejection after organ transplantation, organ function decline, organ and tissue ischemia, inflammation and other diseases. application in medicines.
  • the tissue damage includes tissue damage caused by cerebral infarction or myocardial infarction;
  • the degenerative diseases include diabetes, Parkinson's disease, senile dementia or degenerative joint disease;
  • the immune Diseases include systemic lupus erythematosus, psoriasis or rheumatoid arthritis, etc.;
  • the immune rejection after organ transplantation includes the rejection of the host to the graft or the rejection of the graft to the host;
  • the organ dysfunction includes ovarian Or gonadal dysfunction;
  • the organ and tissue ischemia includes heart, brain or lower extremity ischemia, etc.;
  • the inflammation includes inflammatory reaction after mechanical injury or chemical injury, immune inflammation or chemical stimulation inflammation.
  • the phenomenon of pulmonary retention basically does not occur, and the pulmonary blood vessels are not blocked.
  • the high-migration mesenchymal stem cells prepared by the present invention are small in size, generally below 15 ⁇ m in diameter, and uniform in size, with an average diameter of 11-14 ⁇ m.
  • the cell surface of the high-migration mesenchymal stem cells prepared by the present invention can express a high level of cell migration-related receptor CXCR4, and the expression rate is as high as 30% or more.
  • the high migration mesenchymal stem cells prepared by the present invention have strong cell migration ability, and the high migration mesenchymal stem cells obtained by the three-dimensional carrier-free dynamic culture of the present invention are compared with ordinary two-dimensional adherent culture, ordinary three-dimensional hanging drop culture, etc. The migratory ability of the obtained mesenchymal stem cells was significantly enhanced.
  • the lung retention is greatly reduced, and the phenomenon of lung retention is basically not produced.
  • the high-migratory mesenchymal stem cells prepared by the present invention have strong targeted migration ability to ischemic tissue, and the number reaching ischemic myocardium is significantly increased after intravenous injection.
  • Figure 1 is a graph showing the comparison of the expression levels of CXCR4 on the surface of human placental mesenchymal stem cells in the adherent culture primary (P0) and the fifth passage (P5) by flow cytometry in Comparative Example 1 of the present invention.
  • Fig. 2 is Real-Time PCR to detect the 5th generation of the adherent culture (2D MSC) of Comparative Example 1 of the present invention, the hanging drop culture (3D MSC-H) of Comparative Example 2 and the spinner flask culture (3D MSC-S) of Example 1 Comparison of CXCR4 mRNA expression levels in human placental mesenchymal stem cells.
  • Fig. 3 is flow cytometry analysis of the fifth generation of the adherent culture (2D MSC) of Comparative Example 1 of the present invention, the hanging drop culture of Comparative Example 2 (3D MSC-H) and the spinner flask culture of Example 1 (3D MSC-S) Comparison of cell surface expression levels of CXCR4 in human placental mesenchymal stem cells.
  • Fig. 4 is a Transwell chamber measuring the adherent culture (2D) of Comparative Example 1 of the present invention, the hanging drop culture (3D-H) of Comparative Example 2, the spinner flask culture (3D-S) of Example 1 and the rotation after the blocking of CXCR4 function
  • Comparison chart of the migration ability of mesenchymal stem cells (3D-S-B) cultured in flasks (A in the figure is a comparison chart of the number of cells showing migration after DAPI staining; B in the figure is a comparison chart of the average number of cells counted in multiple fields).
  • Fig. 5 is the small animal imaging detection of mesenchymal stem cells in the adherent culture (2D MSC) of Comparative Example 1, the hanging drop culture (3D MSC-H) of Comparative Example 2 and the spinner flask culture (3D MSC-S) of Example 1 of the present invention Comparison of lung retention at 24 hours after intravenous injection of mice.
  • Figure 6 is the same number of mesenchymal stem cells in the adherent culture (2D MSC) of Comparative Example 1, the hanging drop culture (3D MSC-H) of Comparative Example 2 and the spinner flask culture (3D MSC-S) of Example 1 of the present invention. Comparison of the amount reaching the ischemic myocardium after intravenous injection.
  • the present embodiment provides a method for preparing high-migration mesenchymal stem cells, which specifically includes the following steps:
  • the human placenta-derived mesenchymal stem cells were suspended in the mesenchymal stem cell culture medium at a seeding density of 3 ⁇ 10 5 cells/mL, and the culture medium was placed in In the cell spinner flask, stir at 37°C and 5% CO 2 to realize flow liquid culture.
  • the stirring speed is 60rpm, so that the culture medium is in a flowing state in the incubator, and the culture medium is continuously cultured for 60h.
  • the mesenchymal stem cells The aggregates formed cell spheroids of uniform size, and the diameter of the cell spheroids was about 200 ⁇ m. After the culture, the cell pellets were collected by centrifugation;
  • the mesenchymal stem cell culture medium is low-glucose DMEM (Dulbecco's Modified Eagle Medium) containing 5% human platelet lysate (PLUS TM Human Platelet Lysate); the culture medium also includes the following additives: 1% human blood white Protein, 6.25nmol/L Trichostatin A (TSA), 5 ⁇ g/mL human fibronectin (fibronectin) and 5ng/mL bFGF (basic fibroblast growth factor, basic fibroblast growth factor) .
  • TSA Trichostatin A
  • fibronectin fibronectin
  • bFGF basic fibroblast growth factor, basic fibroblast growth factor
  • the human placenta-derived mesenchymal stem cells are a single cell suspension of mesenchymal stem cells harvested and digested by conventional 2D adherent culture for 5 generations.
  • RNA extraction and real-time quantitative PCR to measure CXCR4 gene expression level 1.
  • RNA is located in the upper aqueous phase, while the denatured protein is located in the lower organic phase or between the two layers. ;
  • RNA concentration was measured with nanodrop.
  • reverse transcription was performed. Take 1 ⁇ g of RNA as a template, and use the reverse transcription kit PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa) to further remove residual genomic DNA according to the reagent instructions to obtain high-purity RNA for reverse transcription, and the obtained cDNA is used for Real-time quantitative PCR reaction.
  • PrimeScript TM RT reagent Kit with gDNA Eraser TaKaRa
  • the primers used were:
  • CXCR4-F ATCCCTGCCCTCCTGCTGACTATTC (SEQ ID NO: 1);
  • CXCR4-R GAGGGCCTTGCGCTTCTGGTG (SEQ ID NO: 2);
  • Gapdh-F CGTGGAAGGACTCATGACCA (SEQ ID NO: 3);
  • Gapdh-R TCCAGGGGTCTTACTCCTTG (SEQ ID NO:4).
  • This comparative example provides 2D adherent culture of human placental mesenchymal stem cells of different passages. The specific steps are as follows:
  • Human placenta-derived mesenchymal stem cells were subcultured in 2D adherent subculture in low-glucose DMEM containing 10% fetal bovine serum or 5% human platelet lysate at 37°C and 5% CO 2 . When the confluence reaches 80%-90%, the cells are passaged at 1:3 or 1:4, and cultured continuously for 5 generations.
  • This comparative example provides a method for obtaining mesenchymal stem cells by 3D hanging drop culture. The specific steps are as follows:
  • the concentration of human placenta-derived mesenchymal stem cells was adjusted to 0.6 ⁇ 10 6 cells/mL with a mesenchymal stem cell culture medium (low glucose DMEM containing 5% human platelet lysate).
  • the human placenta-derived mesenchymal stem cells are a single cell suspension of mesenchymal stem cells harvested and digested by conventional 2D adherent culture for 5 generations.
  • the mesenchymal stem cells obtained by culturing the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 were subjected to Real-Time PCR and flow cytometry analysis experiments and showed that:
  • the single-cell suspension of human placental mesenchymal stem cells has an average diameter of 12.1 ⁇ m and a diameter range of 8-15 ⁇ m;
  • the single-cell suspension of human placental mesenchymal stem cells cultured in the spinning flask of Example 1 of the same passage has an average diameter of 12.1 ⁇ m and a diameter range of 8-15 ⁇ m.
  • the diameter is 12.3 ⁇ m, and the diameter ranges from 8 to 14 ⁇ m.
  • both the hanging drop culture of Comparative Example 2 and the spinner flask culture of Example 1 of the present invention significantly increased the gene expression level of CXCR4, but the spinner flask culture of Example 1 of the present invention increased the expression level of CXCR4. More obviously, its mRNA expression level was more than 2 times that of hanging drop culture (as shown in Figure 2).
  • This comparative example compares the effects of medium supplements on cell spheroidization and survival under the same culture conditions.
  • the comparative example is specifically designed as: the mesenchymal stem cell culture medium does not add human serum albumin and trichostatin in Example 1.
  • A. Human fibronectin and bFGF, other operations are the same as in Example 1.
  • the experimental results showed that after culturing for 10 hours, the spheroidization rate of the mesenchymal stem cells in Example 1 was above 90%, while the spheroidization rate of the mesenchymal stem cells in Comparative Example 3 was below 40%; The survival rate and recovery rate of mesenchymal stem cells were both above 90%, while the survival rate of mesenchymal stem cells in Comparative Example 3 was less than 50% and the recovery rate was less than 30%.
  • Example 2 Effects of different culture methods on the migration ability of mesenchymal stem cells and a comparative experiment of the role of CXCR4 in them
  • the mesenchymal stem cells are grouped as follows: 2D adherent cultured fifth-generation human placental mesenchymal stem cells, hanging drop cultured mesenchymal stem cells for 60 h, and spinner flask cultured for 60 h of mesenchymal stem cells, which were digested with trypsin to form single cells.
  • the cell suspension was washed with phosphate buffer, and resuspended in DMEM containing 1% BSA to form a single cell suspension with a density of 2 ⁇ 10 5 cells/mL; the mesenchymal stem cells were cultured in a spinner flask for 60 h.
  • Example 1 of the present invention increased the expression of CXCR4 on the surface of mesenchymal stem cells It is an important reason for the increase of its migratory capacity.
  • Example 3 Comparative experiment of pulmonary occlusion in mice injected with mesenchymal stem cells intravenously with different culture methods
  • the human placental mesenchymal stem cells of the fifth generation in the adherent culture of Comparative Example 1 were transfected with luciferase lentivirus (pLV-Luc), and some of the cells were further cultured in the spinner flask of Example 1 for 60 hours and the suspension of Comparative Example 2.
  • the cells were cultured in drops for 60 hours; the mesenchymal stem cells cultured by the three methods were digested with trypsin to form a single cell suspension, and the cell density was 1 ⁇ 10 6 cells/200 ⁇ L.
  • 1 ⁇ 10 6 mesenchymal stem cells cultured by different methods above were slowly injected into BALB/C mice (8 weeks, 18-22 g body weight) through the tail vein. 24 hours later, the mice were anesthetized, and fluorescein (D-luciferin, 150 mg/kg) was injected intraperitoneally, and the Bruker small animal imaging system collected the luminescent signal of the mouse body for 10 minutes.
  • Example 4 Comparative experiment of migrating ability of mesenchymal stem cells to ischemic myocardium in mice with acute myocardial infarction by tail vein injection of mesenchymal stem cells with different culture methods
  • the fifth generation of human placental mesenchymal stem cells cultured in Adherent Example 1, the mesenchymal stem cells cultured in the hanging drop of Comparative Example 2 for 60 hours, and the mesenchymal stem cells cultured in the spinner flask for 60 hours in Example 1 of the present invention were digested with trypsin respectively. Then, a single cell suspension was formed, washed with phosphate buffer, and resuspended with physiological saline to form a single cell suspension with a density of 1 ⁇ 10 6 cells/200 ⁇ L. 1 ⁇ 10 6 mesenchymal stem cells were slowly injected through the tail vein 24 hours after acute myocardial infarction in mice, and the mice were sacrificed 72 hours after the injection.
  • the primers were used to quantitatively detect the number of human mesenchymal stem cells in the myocardium by real-time quantitative PCR. The specific method is as follows:
  • Mouse myocardial infarction model 8-week-old BABL/c mice were injected intraperitoneally with 1% sodium pentobarbital (200 ⁇ L per 20 g mouse), the anesthetized mice were placed supine on the operation board, and the back chest was fixed. Depilation; blunt dissection of the subcutaneous tissue, exposure of the trachea, tracheotomy and intubation followed by a ventilator; thoracotomy at the level of the left fourth intercostal space, exposing the heart, and then ligating the left anterior descending coronary artery; repositioning the heart and suturing The incision is made until the tracheal cannula is removed when the small animal is awake.
  • Quantitative detection of human mesenchymal stem cells in mouse myocardium by real-time fluorescence quantitative PCR method The number of human mesenchymal stem cells migrated to the infarcted heart after intravenous injection was detected by fluorescence quantitative PCR method, and the primers were specific sequences Alu in human genomic DNA (F: CATGGTGAAAACCCCGTCTCTA (SEQ ID NO: 5); R: GCCTCAGCCTCCCGAGTAG (SEQ ID NO: 6)), this method has been widely used in previous studies.
  • PCR uses Taqman Universal PCR Master Mix probe method fluorescence quantitative PCR kit, each reaction is added with 900nmol/L primer, 200ng sample genomic DNA and 250nmol/L probe (TaqMan probe: 5'-FAM-ATTAGCCGGGCGTGGTGGCG-TAMRA- 3' (SEQ ID NO: 7)), the amplification procedure is: the first step: 50°C, 2min; the second step: 95°C, 10min; the third step: 95°C, 15s, 60°C, 60s, repeat 40 cycle. Three replicate wells were set for each sample. Using the logarithm of the amount of human genomic DNA in the sample and the corresponding CT value, a standard curve was prepared to obtain the amount of human genomic DNA corresponding to the CT value. According to previous studies, the amount of DNA contained in each diploid nucleus is about 5pg. By quantifying the amount of genomic DNA contained in each cell, the number of cells corresponding to the amount of genomic DNA in the sample can be obtained.
  • Example 5 Comparative experiment of migrating ability of mesenchymal stem cells to ischemic brain tissue in rats with cerebral infarction by tail vein injection of different culture methods
  • Example 4 Using the same detection method as Example 4, the migration of highly migratory mesenchymal stem cells to the ischemic brain tissue was quantitatively analyzed.
  • the rat middle cerebral artery occlusion model was used for 1h-reperfusion cerebral infarction. Inject 1 ⁇ 10 6 cells/200 ⁇ L of mesenchymal stem cells, including 6 mesenchymal stem cells in the adherent cultured mesenchymal stem cell group of Comparative Example 1, and 6 mesenchymal stem cells in the hanging drop cultured mesenchymal stem cell group of Comparative Example 2, Example 1 spinner flask cultured 6 mesenchymal stem cells.
  • the animals were sacrificed 72 hours after the first injection of cells, and the brain tissue of the infarct edge was collected to determine the number of human mesenchymal stem cells in the tissue by real-time quantitative PCR.
  • the results show that the number of mesenchymal stem cells cultured in the spinner flask of Example 1 of the present invention reaching the ischemic brain tissue is 9.6 times that of the adherent cultured mesenchymal stem cells of Comparative Example 1, which is the same as that of the hanging drop culture of Comparative Example 2. 2.5 times of mesenchymal stem cells.
  • the high-migration mesenchymal stem cells prepared by the present invention are small in size, with an average diameter of 11-14 ⁇ m, the expression rate of CXCR4 on the cell surface is as high as 30%, and the cell migration ability is strong, and intravenous injection basically does not cause lung retention.
  • the number reaching the ischemic myocardium is significantly increased.

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Abstract

提供了一种高迁徙间充质干细胞及其制备方法和应用。该制备方法包括：将间充质干细胞接种混悬于间充质干细胞培养液中，在细胞培养旋转瓶或搅拌式发酵罐内进行流动液体培养，培养过程中无需添加微载体，培养结束后收集细胞球，冲洗并消化形成单细胞，即得到高迁徙间充质干细胞。制备的高迁徙间充质干细胞体积小，平均直径为11～14μm，其细胞表面CXCR4表达率高达30％以上，细胞迁徙能力强，静脉注射基本不产生肺滞留现象，到达缺血心肌的数量显著增加。

Description

一种高迁徙间充质干细胞及其制备方法和应用 技术领域
本发明属于干细胞技术领域，具体涉及一种高迁徙间充质干细胞及其制备方法和应用。
背景技术
间充质干细胞(mesenchymal stem cells,or mesenchymal stromal cells,MSC)是一种具有多向分化能力的成体干细胞，能够分化形成多种组织细胞，并通过分泌大量生长和免疫调节因子，参与构成组织再生微环境，并促进组织损伤修复。间充质干细胞广泛存在于身体的多种组织，但随着年龄的增加，其数量显著减少。大量研究证明，补充间充质干细胞能够促进多种组织的再生修复。
间充质干细胞来源广泛，可分离自胎盘、脐带、骨髓等组织。由于间充质干细胞具有特殊的低免疫原性，异体间充质干细胞移植不引起免疫排异，不需组织配型，特别适合组织损伤修复(如治疗脑、心梗死等)及多种退行性疾病(如糖尿病、帕金森氏病等)的治疗，静脉注射是主要间充质干细胞移植途径。
从组织中获取的间充质干细胞数量很少，需要经过体外扩增培养后方能满足临床治疗需要。目前间充质干细胞普遍采用二维(2D)贴壁培养方法，基于生物反应器的培养过程中通常需要添加微载体共培养，例如：GE微载体(Cytopore、Cytodex、Cytoline)、明胶微载体等，细胞附着在微载体上，本质上也属于2D培养。2D培养过程中，细胞在增殖的同时发生表观遗传变化，导致诸多基因表达失常，表现为细胞体积增大，表面迁徙相关分子表达失常，如CXCR4(C-X-C Motif Chemokine Receptor 4)的表达显著下降，静脉注射后有高达高达90％以上的细胞阻塞在肺微血管，其中绝大部分在24小时内因局部缺血而死亡，难以到达肺外损伤组织。也有采用三维(3D)悬滴培养，例如：WO2009134532A2公开了一种悬滴培养方式，此种方法虽然能够形成细胞聚集体，但该方法为静态培养方式，其获得的间充质干细胞CXCR4表达水平及向缺血组织的靶向迁移能力仍然存在不足。
发明内容
基于现有技术存在的缺陷，本发明的第一目的在于提供一种高迁徙间充质干细胞的制备方法；本发明的第二目的在于提供该制备方法制备获得的高迁徙间充质干细胞；本发明的第三目的在于提供该高迁徙间充质干细胞在制备治疗组织损伤修复、退行性疾病、免疫性疾病、器官移植后免疫排异、器官机能衰退、器官和组织缺血、炎症的药物中的应用。
本发明的目的通过以下技术手段得以实现：
一方面，本发明提供一种高迁徙间充质干细胞的制备方法，其包括以下步骤：
将间充质干细胞接种混悬于间充质干细胞培养液中，在细胞培养旋转瓶或搅拌式发酵罐内进行流动液体培养，培养过程中无需添加微载体，培养结束后收集细胞球，冲洗并消化形成单细胞，即得到高迁徙间充质干细胞。
发明人创造性的采用了基于三维(3D)动态细胞的培养方法，该方法将间充质干细胞接种混悬于间充质干细胞培养液中，在细胞培养旋转瓶(带有搅拌装置)或搅拌式发酵罐内进行流动液体培养，培养过程中无需添加任何微载体，能够减小间充质干细胞体积，增加其在血液循环中的移动能力和向损伤组织的迁徙能力；此外，发明人在实验中创造性地发现，细胞随液体流动，并逐步形成细胞聚集体(细胞球，spheroids)，通过搅拌引起的液体流动所产生的剪切力能够进一步显著增加间充质干细胞的迁徙能力，并显著增加间充质干细胞表面迁徙相关受体CXCR4的表达水平；在细胞聚集和流动液体形成的剪切力的双重作用促进间充质干细胞CXCR4表达和向缺血组织的靶向迁移。
上述的制备方法中，优选地，所述间充质干细胞的接种密度为(0.5～10)×10 5个细胞/mL。
上述的制备方法中，优选地，所述间充质干细胞培养液包括含1％～20％(V/V)胎牛血清或0.5％～10％(V/V)人血小板裂解物或血清替代物(例如：PALL Ultroser G)的低葡萄糖DMEM培养液。所述低葡萄糖DMEM培养液可以为商品化的低葡萄糖DMEM培养液(例如：购自Thermo Fisher Scientific)。本发明的间充质干细胞培养液也可采用其他常规的细胞基础培养液。
上述的制备方法中，优选地，所述间充质干细胞培养液中还添加有0.1％～5％(W/V，终浓度)的人血白蛋白、1～100nmol/L(终浓度)的曲古菌素A、1～50μg/mL (W/V，终浓度)的人纤连蛋白和1～40ng/mL(W/V，终浓度)的bFGF(basic fibroblast growth factor,碱性成纤维细胞生长因子)。通过添加人血白蛋白、曲古菌素A、人纤连蛋白及bFGF的间充质干细胞培养液，能够保证细胞在悬浮流动的体系中成球和成活。
上述的制备方法中，优选地，在细胞培养旋转瓶或搅拌式发酵罐内进行流动液体培养的条件为：37℃、5％CO 2条件下，搅拌速度为5～200rpm，培养时间为5～200h。
上述的制备方法中，优选地，培养结束后收集细胞球的方法包括离心法、过滤法或自然沉降法。
上述的制备方法中，优选地，冲洗并消化形成单细胞的步骤包括：采用磷酸缓冲液冲洗收集的细胞球，然后用胰蛋白酶消化形成单细胞。
上述的制备方法中，优选地，所述磷酸缓冲液为无钙镁离子的磷酸缓冲液。
上述的制备方法中，优选地，所述胰蛋白酶为含有EDTA的胰蛋白酶。
另一方面，本发明还提供一种高迁徙间充质干细胞，其是采用上述的制备方法制备获得的。
本发明的高迁徙间充质干细胞的平均直径为11～14μm，其表面表达细胞迁移的受体CXCR4的表达率30％以上。
再一方面，本发明还提供上述高迁徙间充质干细胞在制备治疗组织损伤修复、退行性疾病、免疫性疾病、器官移植后免疫排异、器官机能衰退、器官和组织缺血、炎症等疾病的药物中的应用。
上述的应用中，优选地，所述组织损伤包括脑梗或心梗引起的组织损伤等；所述退行性疾病包括糖尿病、帕金森氏病、老年痴呆或退行性关节病等；所述免疫性疾病包括系统性红斑狼疮、银屑病或类风湿性关节炎等；所述器官移植后免疫排异包括宿主对移植物的排异或移植物对宿主的排异；所述器官机能衰退包括卵巢或性腺机能衰退；所述器官和组织缺血包括心、脑或下肢缺血等；所述炎症包括机械损伤或化学损伤后的炎症反应、免疫性炎症或化学刺激炎症等。
将本发明的高迁徙间充质干细胞通过静脉注射后基本不产生肺滞留现象，不阻塞肺血管。
本发明的有益效果：
(1)本发明制备的高迁徙间充质干细胞体积小，直径普遍在15μm以下，大小 均匀，平均直径为11～14μm。
(2)本发明制备的高迁徙间充质干细胞细胞的细胞表面能够表达高水平的细胞迁移相关受体CXCR4，表达率高达30％以上。
(3)本发明制备的高迁徙间充质干细胞的细胞迁徙能力强，本发明的三维无载体动态培养获得的高迁徙间充质干细胞相比普通二维贴壁培养、普通三维悬滴培养等获得的间充质干细胞迁徙能力显著增强。
(4)本发明制备的高迁徙间充质干细胞静脉注射后肺滞留大幅减少，基本不产生肺滞留现象。
(5)本发明制备的高迁徙间充质干细胞向缺血组织的靶向迁徙能力强，静脉注射后到达缺血心肌的数量显著增加。
附图说明
图1为本发明对比例1中采用流式细胞仪测定贴壁培养原代(P0)与第5代(P5)的人胎盘间充质干细胞表面CXCR4的表达水平对比图。
图2为Real-Time PCR检测本发明对比例1贴壁培养(2D MSC)、对比例2悬滴培养(3D MSC-H)和实施例1旋转瓶培养(3D MSC-S)的第5代人胎盘间充质干细胞的CXCR4的mRNA表达水平对比图。
图3为流式细胞仪分析本发明对比例1贴壁培养(2D MSC)、对比例2悬滴培养(3D MSC-H)和实施例1旋转瓶培养(3D MSC-S)的第5代人胎盘间充质干细胞的CXCR4的细胞表面表达水平对比图。
图4为Transwell小室测定本发明对比例1贴壁培养(2D)、对比例2悬滴培养(3D-H)、实施例1旋转瓶培养(3D-S)和经CXCR4功能阻断后的旋转瓶培养的间充质干细胞(3D-S-B)的迁徙能力对比图(图中的A为DAPI染色后显示迁徙的细胞数量对比图；图中的B为计数多视野平均细胞数对比图)。
图5为小动物成像检测对比例1贴壁培养(2D MSC)、对比例2悬滴培养(3D MSC-H)和本发明实施例1旋转瓶培养(3D MSC-S)的间充质干细胞静脉注射小鼠后24h的肺滞留情况对比图。
图6为相同数量的对比例1贴壁培养(2D MSC)、对比例2悬滴培养(3D MSC-H)和本发明实施例1旋转瓶培养(3D MSC-S)的间充质干细胞在静脉注射后到达缺血 心肌的数量对比图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。以下实施例中所采用的试剂原料若无特殊说明，均为市售常规获得。
实施例1：
本实施例提供一种高迁徙间充质干细胞的制备方法，其具体包括如下步骤：
(1)采用细胞培养旋转瓶旋转培养方法，将人胎盘来源的间充质干细胞按3×10 5个细胞/mL的接种密度混悬于间充质干细胞培养液中，并将培养液置于细胞旋转瓶中，在37℃，5％CO 2条件下进行搅拌，实现流动液体培养，搅拌速度为60rpm，使培养液在培养器内呈流动状态，连续培养60h，培养期间，间充质干细胞聚集形成大小均一的细胞球，细胞球直径在200μm左右。培养结束后采用离心方式收集细胞球；
其中，间充质干细胞培养液为含5％人血小板裂解物(PLUS TM Human Platelet Lysate)的低葡萄糖DMEM(Dulbecco's Modified Eagle Medium)；该培养液中还包括以下添加物：1％的人血白蛋白、6.25nmol/L的曲古菌素A(Trichostatin A,TSA)、5μg/mL的人纤连蛋白(fibronectin)和5ng/mL的bFGF(basic fibroblast growth factor,碱性成纤维细胞生长因子)。
其中，人胎盘来源的间充质干细胞为常规2D贴壁培养5代收获并消化形成的间充质干细胞单细胞悬液。
(2)将收集的间充质干细胞球至15mL的离心管中，加入5mL的DPBS(无钙镁离子的磷酸缓冲液)洗一次；然后加入适量0.25％胰蛋白酶液(含0.02％的EDTA)进行消化约3min，至细胞球变得松散后，轻轻吹打使细胞分散成单细胞悬液；接着加入等体积含10％胎牛血清的DMEM培养基终止胰蛋白酶作用，1300r/min，离心5min，弃上清，最后加入含1％牛血清白蛋白(BSA)的磷酸缓冲液重新悬浮细胞形成单细胞悬液，获得高迁徙间充质干细胞悬液。
1、RNA提取和实时定量PCR测量CXCR4基因表达水平：
(1)直接采用上述培养的细胞球，将收集的细胞球去除细胞培养液，用磷酸缓冲液洗一次，加入1.0mL的
Figure PCTCN2022072878-appb-000001
吹打均匀，转移至1.5mL管中，将细胞进行裂解。
(2)用磷酸缓冲液再洗一遍，加入1.0mL的
Figure PCTCN2022072878-appb-000002
吹打均匀至细胞球完全消化，转移至1.5mL管中，将细胞进行彻底裂解。
(3)加入0.2mL的氯仿，混匀后室温离心15min，此时离心管中出现分层：RNA位于上层的水相，而被变性的蛋白则位于下层的有机相或介于两层之间；
(4)将上清转移至新的1.5mL管中，并加入等体积异丙醇沉淀核酸，混匀后于4℃放置30min后离心15min；弃上清，用75％的乙醇洗涤沉淀，待沉淀晾干后加入20μL的不含DNase/RNase的水溶解。
(5)用Nanodrop对RNA浓度进行测定后，进行反转录。取1μg的RNA为模板，按照试剂使用说明书，用反转录试剂盒PrimeScript TM RT reagent Kit with gDNA Eraser(TaKaRa)进一步去除残留基因组DNA，获得高纯度RNA用于反转录，得到的cDNA用于实时定量PCR反应。
(6)用TaKaRa SYBR premix Ex Taq试剂盒进行荧光定量PCR反应，实时荧光PCR扩增程序为：95℃、45秒；95℃、5秒；60℃、31秒；重复45个循环，每个实验样品需同时做三个重复。根据仪器反馈的Ct值，应用2-ΔΔCt法，并以Gapdh为内参，对样品中CXCR4基因的表达量进行定量。
所用引物为：
CXCR4-F：ATCCCTGCCCTCCTGCTGACTATTC(SEQ ID NO:1)；
CXCR4-R：GAGGGCCTTGCGCTTCTGGTG(SEQ ID NO:2)；
Gapdh-F：CGTGGAAGGACTCATGACCA(SEQ ID NO:3)；
Gapdh-R：TCCAGGGGTCTTACTCCTTG(SEQ ID NO:4)。
2、流式细胞仪测定细胞表面CXCR4表达水平：
(1)取高迁徙间充质干细胞悬液100μL，分别放到3个EP管中，其中一管为空白对照，第二管为同型对照(加PE标记的同型IgG)，第三管为抗体(加PE-标记的抗-CXCR4流式抗体)，在冰上孵育30min，避光。
(2)向每个EP管中加入1mL磷酸缓冲液，1300rpm离心5min，弃上清，用0.5mL含1％牛血清白蛋白的磷酸缓冲液重悬细胞，用BD流式细胞仪测定样本CXCR4的表达，用BD Csampler软件分析测定结果，设定同型对照细胞CXCR4的表达为0.5％以下。
对比例1：
本对比例提供2D贴壁培养不同代次的人胎盘间充质干细胞，具体步骤如下：
(1)人胎盘来源的间充质干细胞，在含10％胎牛血清或5％人血小板裂解物的低葡萄糖DMEM中2D贴壁传代培养，培养条件为37℃、5％CO 2，当细胞融合度(confluence)达到80％～90％时按1：3或1：4进行传代，连续培养5代。
(2)培养第5代的细胞去除细胞培养液后，一部分用磷酸缓冲液洗一次，在培养皿内裂解细胞，收集裂解液用于提取RNA，进行基因表达实时定量PCR分析；另一部分细胞采用胰蛋白酶(含EDTA)消化后，用含1％牛血清白蛋白的磷酸缓冲液重悬细胞形成间充质干细胞单细胞悬液(用于上述实施例1旋转瓶旋转培养和下述对比例2悬滴培养的起始人胎盘来源的间充质干细胞)。
采用间充质干细胞在2D贴壁培养传代后，采用流式细胞仪检测(方法同上述实施例1中的检测方法)，其细胞表面CXCR4的表达水平由原代(P0)的35％降低至第5代(P5)的不足1％(如图1所示)。
对比例2：
本对比例提供一种3D悬滴培养获得间充质干细胞的方法，具体步骤如下：
(1)用间充质干细胞培养液(含5％人血小板裂解物的低葡萄糖DMEM)调整人胎盘来源的间充质干细胞的浓度为0.6×10 6个细胞/mL。
其中，人胎盘来源的间充质干细胞为常规2D贴壁培养5代收获并消化形成的间充质干细胞单细胞悬液。
(2)以每滴35μL的体积把细胞悬液均匀的加到10cm的培养皿的上盖中，每板悬滴滴数控制在40滴左右，小心的把培养盖倒转，置于培养箱内，在37℃、5％CO 2条件下培养36h，收集细胞球，再置于不贴壁的培养皿中，在37℃、5％CO 2条件下继续培养至60h。
(3)收集细胞球，提取RNA，进行基因表达实时定量(Real-Time)PCR分析，或将细胞球胰蛋白酶消化形成单细胞悬液，纤维镜下拍照测定细胞大小，并进行流式细胞仪分析表面蛋白表达，方法参照上述实施例1。
上述实施例1、对比例1及对比例2培养获得的间充质干细胞进行Real-Time PCR及流式细胞仪分析实验表明：
(1)对比例1的2D贴壁培养第5代的人胎盘间充质干细胞单细胞悬液，其平 均直径为22.4μm，直径范围为15～41μm；相同代次的对比例2悬滴培养的人胎盘间充质干细胞单细胞悬液，其平均直径为12.1μm，直径范围为8～15μm；相同代次的实施例1旋转瓶培养的人胎盘间充质干细胞单细胞悬液，其平均直径为12.3μm，直径范围为8～14μm。
(2)关于间充质干细胞对CXCR4的表达，对比例2的悬滴培养和本发明实施例1的旋转瓶培养都显著增加CXCR4的基因表达水平，但本发明实施例1的旋转瓶培养增加更加明显，其mRNA表达水平是悬滴培养的2倍以上(如图2所示)。
(3)流式细胞仪分析显示，对比例1的2D贴壁培养5代的间充质干细胞CXCR4表达率为1％左右；对比例2的悬滴培养的间充质干细胞CXCR4表达率为14.7％，而本发明实施例1的旋转瓶培养的间充质干细胞CXCR4的表达率为42.8％(如图3所示)。
综上结果表明：间充质干细胞处在流动状态大幅提高了其表面的CXCR4表达水平。
对比例3：
该对比例对比相同培养条件下培养基添加物对细胞成球和成活的影响，该对比例具体设计为：间充质干细胞培养液不添加实施例1中的人血白蛋白、曲古菌素A、人纤连蛋白和bFGF，其它操作同实施例1。
实验结果显示，培养10h后，实施例1的间充质干细胞成球率在90％以上，而对比例3的间充质干细胞成球率在40％以下；培养60h后，实施例1的间充质干细胞成活率和回收率均在90％以上，而对比例3的间充质干细胞成活率小于50％，回收率小于30％。
实施例2：不同培养方法对间充质干细胞迁移能力的影响及CXCR4在其中的作用的对比实验
用孔径为8μm的24孔板的Transwell小室测定上述实施例1、对比例1和对比例2不同方法培养的间充质干细胞的迁徙能力。具体为：
孔板下室中分别加入600μL 0.5％的BSA-DMEM或50％小鼠心肌梗死组织提取液-DMEM(小鼠急性心肌梗死24h，梗死和缺血心肌水解物)，在小室内分别加入100μL经过不同处理的间充质干细胞悬液，细胞密度为2×10 5个细胞/mL。
间充质干细胞分组处理如下：2D贴壁培养的第5代人胎盘间充质干细胞、悬滴培养60h的间充质干细胞和旋转瓶培养60h的间充质干细胞，经胰蛋白酶消化后形成单细胞悬液，再经磷酸缓冲液洗涤，用含1％的BSA的DMEM重悬，形成密度为2×10 5个细胞/mL的单细胞悬液；在旋转瓶培养60h的间充质干细胞悬液中分别加入5μg/mL的CXCR4功能阻断抗体(R&D Systems，Clone#44717，货号MAB173)，或等量的非免疫同型对照IgG(对照)，37℃孵育30min。在上层小室加入间充质干细胞后，将Transwell在37℃、5％CO 2培养箱中孵育6h，取出小室，先将小室膜向上的面上附着的细用棉球擦除，然后将膜用4％多聚甲醛固定，用DAPI染色，荧光显微镜下观察迁徙到膜下面附着的细胞并拍照，每孔随机选取10个视野。每组设3个复孔，用Image J计算每个视野的细胞数。
结果显示，对比例1的2D贴壁培养的间充质干细胞、对比例2的悬滴培养的间充质干细胞和本发明实施例1旋转瓶培养的间充质干细胞向下室为DMEM的随机移动的细胞数量无差别，而向下室为梗死心肌提取物的膜移动并透过膜微孔粘附到膜下面的细胞数量有很大差别，其中旋转瓶培养的间充质干细胞较其它两组显著增加，而CXCR4阻断显著减少了其向梗死心肌提取物的迁移(如图4所示)，由此表面本发明实施例1的旋转瓶培养提高了CXCR4在间充质干细胞表面的表达是其迁徙能力增加的重要原因。
实施例3：不同培养方法的间充质干细胞静脉注射小鼠的肺部阻塞的对比实验
对比例1的贴壁培养第5代的人胎盘间充质干细胞进行荧光素酶慢病毒(pLV-Luc)转染，其中部分细胞再进行实施例1的旋转瓶培养60h和对比例2的悬滴培养60h；三种方法培养的间充质干细胞经胰蛋白酶消化后形成单细胞悬液，细胞密度为1×10 6个细胞/200μL。将1×10 6个上述不同方法培养的间充质干细胞经尾静脉缓慢注射到BALB/C小鼠体内(8周，18～22克体重)。24h后麻醉小鼠，腹腔注射荧光素(D-luciferin，150mg/kg)，Bruker小动物成像系统采集小鼠身体发光信号10min。
结果显示，对比例1的贴壁培养的间充质干细胞在静脉注射后大量滞留于肺，对比例2的悬滴培养也有相对滞留于肺，而本发明实施例1的旋转瓶培养的间充质干细胞在静脉注射后几乎未探测到肺内滞留(n＝3(每组3只小鼠)，如图5所示)；由此表明，采用本发明旋转瓶培养的间充质干细胞静脉注射后不发生肺阻塞。
实施例4：不同培养方法的间充质干细胞尾静脉注射急性心肌梗死小鼠的向缺血心肌迁徙能力的对比实验
对比例1贴壁培养的第5代人胎盘间充质干细胞、对比例2悬滴培养60h的间充质干细胞和本发明实施例1旋转瓶培养60h的间充质干细胞，分别经胰蛋白酶消化后形成单细胞悬液，再经磷酸缓冲液洗涤，用生理盐水重悬，形成密度为1×10 6个细胞/200μL的单细胞悬液。1×10 6个间充质干细胞在小鼠急性心肌梗死24h后经尾静脉缓慢注射，注射72h后处死小鼠，取心肌缺血部位(梗死边缘部位)提取DNA，用特异性针对人DNA序列的引物，用实时定量PCR法定量检测心肌内的人间充质干细胞数量。具体方法如下：
(1)小鼠心肌梗死模型：8周龄BABL/c小鼠，腹腔注射1％戊巴比妥钠(200μL每20g小鼠)，将麻醉后的小鼠仰卧在操作板上，固定后胸部脱毛；用钝性分离皮下组织，暴露气管，气管切开插管后接呼吸机；在左第四肋间水平开胸，暴露出心脏后结扎左冠状动脉前降支；将心脏复位，缝合手术切口，直至小动物苏醒时拔出气管插管。
(2)组织及细胞DNA的提取：静脉注射间充质干细胞72h后处死小鼠，取心脏梗死边缘组织(或对应部位的正常心肌组织)，将每只鼠相同重量的心肌组织放入玻璃研磨器中，加入适量磷酸缓冲液进行研磨至匀浆，将其吸入到EP管中，加入抽提缓冲液(含10mmol/L的Tris-HCl，0.1mol/L的EDTA，0.5％的SDS)至1mL，加入5μL、20μg/mL的胰蛋白酶，37℃水浴1h。然后向每个EP管中加入10μL的蛋白酶K，在52℃水浴2h。向每管中加入1mL的酚氯仿异丙醇，剧烈震荡1min，12000rpm离心15min后，将EP管上方的无色透明液层转移到新的2-mL EP管中，再次加入等体积的酚氯仿异丙醇抽提，重复上述操作一次。离心后将上方无色透明液层转移到1.5-mL EP管中，加入等体积的异丙醇及十分之一体积的3mol/L醋酸钠溶液，混匀后10000rpm离心15min，丢弃上清，留白色沉淀(DNA)，用80％的乙醇洗DNA后加入TE溶液，4℃冰箱储存备用。
实时荧光定量PCR法定量检测小鼠心肌内的人间充质干细胞：人间充质干细胞在静脉注射后迁徙到梗死心脏的数量，采用荧光定量PCR方法检测，引物为针对人基因组DNA中的特别序列Alu(F：CATGGTGAAACCCCGTCTCTA(SEQ ID NO:5)；R：GCCTCAGCCTCCCGAGTAG(SEQ ID NO:6))，该方法在既往研究中被广为应 用。PCR使用Taqman Universal PCR Master Mix探针法荧光定量PCR试剂盒，每个反应分别加入900nmol/L的引物、200ng样本基因组DNA和250nmol/L探针(TaqMan probe：5’-FAM-ATTAGCCGGGCGTGGTGGCG-TAMRA-3’(SEQ ID NO:7))，扩增程序为：第一步：50℃、2min；第二步：95℃、10min；第三步：95℃、15s，60℃、60s，重复40个循环。每个样本设3个复孔。以样本中人类基因组DNA量的对数与相应的CT值为参数制作标准曲线，得到CT值对应的人类基因组DNA的量。根据既往研究，每个二倍体细胞核含有的DNA量约为5pg。通过定量每个细胞所含有的基因组DNA的量，可得到样品中的基因组DNA量所对应的细胞数。
结果显示，本发明实施例1旋转瓶培养的间充质干细胞在静脉注射后到达缺血心肌的数量显著多于对比例1贴壁培养的间充质干细胞和对比例2悬滴培养的间充质干细胞(n＝6(每组6只小鼠)，P<0.01，图6)；由此表明，采用本发明旋转瓶培养的间充质干细胞向缺血心肌迁徙能力显著提高。
实施例5：不同培养方法的间充质干细胞尾静脉注射脑梗死大鼠的向缺血大脑组织迁徙能力的对比实验
运用实施例4同样的检测方法，定量分析了高迁徙间充质干细胞向缺血大脑组织的迁徙，研究采用大鼠大脑中动脉阻塞1h再灌注脑梗死模型，分别梗死24h和48h后经尾静脉注射1×10 6个细胞/200μL的间充质干细胞，其中对比例1的贴壁培养的间充质干细胞组6只，对比例2的悬滴培养的间充质干细胞组6只，实施例1的旋转瓶培养的间充质干细胞组6只。第一次细胞注射72h后处死动物，取梗死边缘脑组织，用实时荧光定量PCR法测定组织中人间充质干细胞的数量。结果显示，本发明实施例1的旋转瓶培养的间充质干细胞到达缺血脑组织的数量是对比例1的贴壁培养的间充质干细胞的9.6倍，是对比例2的悬滴培养的间充质干细胞的2.5倍。
综上所述，本发明制备的高迁徙间充质干细胞体积小，平均直径为11～14μm，其细胞表面CXCR4表达率高达30％以上，细胞迁徙能力强，静脉注射基本不产生肺滞留现象，到达缺血心肌的数量显著增加。

Claims (10)

  1. 一种高迁徙间充质干细胞的制备方法，其包括以下步骤：
    将间充质干细胞接种混悬于间充质干细胞培养液中，在细胞培养旋转瓶或搅拌式发酵罐内进行流动液体培养，培养过程中无需添加微载体，培养结束后收集细胞球，冲洗并消化形成单细胞，即得到高迁徙间充质干细胞。
  2. 根据权利要求1所述的制备方法，其中，所述间充质干细胞的接种密度为(0.5～10)×10 5个细胞/mL。
  3. 根据权利要求1所述的制备方法，其中，所述间充质干细胞培养液包括含1％～20％胎牛血清或0.5％～10％人血小板裂解物的低葡萄糖DMEM培养液。
  4. 根据权利要求3所述的制备方法，其中，所述间充质干细胞培养液中还添加有0.1％～5％的人血白蛋白、1～100nmol/L的曲古菌素A、1～50μg/mL的人纤连蛋白和1～40ng/mL的bFGF。
  5. 根据权利要求1所述的制备方法，其中，在细胞培养旋转瓶或搅拌式发酵罐内进行流动液体培养的条件为：37℃、5％CO 2条件下，搅拌速度为5～200rpm，培养时间为5～200h。
  6. 根据权利要求1所述的制备方法，其中，培养结束后收集细胞球的方法包括离心法、过滤法或自然沉降法；
    优选地，冲洗并消化形成单细胞的步骤包括：
    采用磷酸缓冲液冲洗收集的细胞球，然后用胰蛋白酶消化形成单细胞；
    优选地，所述磷酸缓冲液为无钙镁离子的磷酸缓冲液；
    优选地，所述胰蛋白酶为含有EDTA的胰蛋白酶。
  7. 一种高迁徙间充质干细胞，其是采用权利要求1～6任一项所述的制备方法制备获得的。
  8. 根据权利要求7所述的高迁徙间充质干细胞，其中，所述高迁徙间充质干细胞的平均直径为11～14μm，其表面表达细胞迁移的受体CXCR4的表达率30％以上。
  9. 权利要求7或8所述的高迁徙间充质干细胞在制备用于治疗组织损伤修复、退行性疾病、免疫性疾病、器官移植后免疫排异、器官机能衰退、器官和组织缺血和/或炎症的药物中的应用。
  10. 根据权利要求9所述的应用，其中，所述组织损伤包括脑梗或心梗引起的组 织损伤；所述退行性疾病包括糖尿病、帕金森氏病、老年痴呆或退行性关节病；所述免疫性疾病包括系统性红斑狼疮、银屑病或类风湿性关节炎；所述器官移植后免疫排异包括宿主对移植物的排异或移植物对宿主的排异；所述器官机能衰退包括卵巢或性腺机能衰退；所述器官和组织缺血包括心、脑或下肢缺血；所述炎症包括机械损伤或化学损伤后的炎症反应、免疫性炎症或化学刺激炎症。
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