Search

CN-122012390-A - Method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres

CN122012390ACN 122012390 ACN122012390 ACN 122012390ACN-122012390-A

Abstract

The application relates to the technical field of biology, and particularly discloses a method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres. The application adopts differential sedimentation method to sort the diameter of embryoid body cell spheres according to the size, which obviously improves the uniformity of the diameter of embryoid body cell spheres and further improves the uniformity and stability of the subsequent differentiation products. The method provided by the application has the advantages of definite reagent components, simple facility requirements, high differentiation efficiency, capability of obtaining the iMSC meeting the identification standard after the fastest differentiation for 20 days, good uniformity of the obtained iMSC, less undifferentiated cell residues and potential value of clinical transformation application.

Inventors

  • QIN DAJIANG
  • SHI JIANTAO
  • Ling Yixia
  • CHEN QING

Assignees

  • 广州医科大学附属第五医院(广州再生医学与健康广东省实验室附属医院)

Dates

Publication Date
20260512
Application Date
20260410

Claims (10)

  1. 1. A method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres, which is characterized by comprising the following steps: s1, adhering and subculturing the human induced pluripotent stem cells to obtain the human induced pluripotent stem cells before differentiation; s2, differentiating and culturing the human induced pluripotent stem cells before differentiation in a mesodermal precursor cell differentiation medium to obtain mesodermal precursor cells; S3, resuspending the mesoderm precursor cells obtained in the step S2 in embryoid body culture medium, and performing stationary culture to form embryoid body cell spheres; S4, collecting embryoid cell spheres obtained in the step S3, settling the embryoid cell spheres in liquid at different rates, sorting embryoid cell spheres with uniform volumes, placing the embryoid cell spheres in a mesenchymal stem cell maturation medium for uniform cell expansion, and then carrying out passage to enrich mature mesenchymal stem cells.
  2. 2. The method for preparing mesenchymal stem cells using differential sedimentation sorted cell pellet as claimed in claim 1, wherein in the step S2, the mesodermal precursor cell differentiation medium comprises IMDM medium and Ham' S F medium; The mesoderm precursor cell differentiation medium is further added with 5-15 mu M Y-27632, 1-3 mg/mL sodium bicarbonate, 0.5-20 mu g/mL human total transferrin, 0.5-20 mu g/mL insulin, 0.5-50 ng/mL fibroblast growth factor 2, 0.5-50 ng/mL bone morphogenetic protein 4, 0.5-20 ng/mL activin A, lipid concentrate and non-essential amino acids.
  3. 3. The method for preparing mesenchymal stem cells using differential sedimentation sorting cell spheres according to claim 1, wherein the embryoid body medium comprises IMDM, ham' S F, sodium pyruvate, L-alanyl-L-glutamine solution, nonessential amino acids, lipid concentrate, HEPES, human insulin, human transferrin, sodium selenite, ethanolamine, L-ascorbic acid, 1-thioglycerol, β -mercaptoethanol, lithium chloride, and basic fibroblast growth factor in step S3.
  4. 4. The method for preparing mesenchymal stem cells using differential sedimentation sorting cell pellet according to claim 1, wherein the mesenchymal stem cell maturation medium comprises IMDM, ham' S F, sodium pyruvate, L-alanyl-L-glutamine solution, non-essential amino acid, lipid concentrate, HEPES, human insulin, human transferrin, sodium selenite, ethanolamine, L-ascorbic acid, 1-thioglycerol, β -mercaptoethanol, lithium chloride, basic fibroblast growth factor, recombinant human epidermal growth factor and recombinant human insulin-like growth factor-1 in step S4.
  5. 5. The method for preparing mesenchymal stem cells by differential sedimentation sorting cell spheres according to claim 1, wherein the step S4 further comprises testing the suspension sedimentation rate of embryoid body cell spheres in the lumen of the quartz glass tube, and determining the curve function relationship of radius r of embryoid body cell spheres with sedimentation time t; The sedimentation rate and the volume and density of the sphere basically accord with Stokes law: stokes law: Wherein: v is sedimentation rate, ρ p is sphere density, ρ f is liquid density, g is gravitational acceleration, r is sphere radius, η is liquid viscosity.
  6. 6. The method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres according to claim 1, wherein the diameter of the embryoid body cell spheres is 10-250 μm.
  7. 7. The method for preparing mesenchymal stem cells by using differential sedimentation sorted cytoballs according to claim 5, wherein the quartz glass tube is arranged on a slope-adjustable bracket, and the slope adjustment range is 0-90 degrees.
  8. 8. The method for preparing mesenchymal stem cells by differential sedimentation sorted cytoballs according to claim 5, wherein the curve function relationship of radius r of embryoid body cytoballs and sedimentation time t is that ; Wherein a is 5-150, and b is 10-250.
  9. 9. The method for preparing mesenchymal stem cells using differential sedimentation sorting cell spheres according to claim 1, wherein the culturing conditions in step S2 include: culturing at 37deg.C under 5% CO 2 ,20% O 2 for 48 hr; And/or, in the step S3, the conditions of the stationary culture include: performing stationary culture at 37 ℃ for 24 hours with 5% CO 2 ,20% O 2 ; and/or, in the step S4, the conditions for homogenizing the cell expansion include: 5% CO 2 ,20% O 2 , at 37℃for 24 hours.
  10. 10. The method for preparing mesenchymal stem cells by differential sedimentation sorting cytoballs according to claim 1, further comprising identifying mesenchymal-like cells, wherein the positive rates of cell surface antigens CD105, CD90 and CD73 of the mesenchymal-like cells are all equal to or more than 95%, and the positive rates of CD45, CD34, CD14 and HLA-DR are all equal to or less than 2%.

Description

Method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres Technical Field The application relates to the technical field of biology, in particular to a method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres. Background Stem cell differentiation, and organoid differentiation and culture, often require the formation of embryoid bodies from stem cells by self-organization, with self-differentiation to achieve specific germ layer cell fate decisions. Meanwhile, the formation and proliferation of the three-dimensional embryoid bodies greatly improve the cell yield, and are important links in the preparation of cell products. However, there are often differences in the volume of the formed cell spheres due to differences in the initial cell state (cell unit rate, cell stem property, activity, etc.), differences in microenvironment (nutrient factors, mechanical stress), etc. The cells in the cell spheres with different volumes are in different contact with external supply and oxygen supply, the received signal concentration is different, the development speed and the development progress are different, and finally the uniformity of the cell attribute is influenced. Mesenchymal stromal cells (MESENCHYMAL STROMAL CELLS, MSCs) are multipotent adult stromal cells with self-renewal and multipotent differentiation potential. The mesenchymal stromal cells are widely distributed in various tissues of a human body, have specific response properties on inflammatory response and injury signals in the tissues, and can rapidly migrate into the injured tissues to play the roles of anti-inflammatory, repairing and tissue regeneration. Primary MSCs are derived from adult bone marrow, fat and peripheral blood, neonatal placenta, umbilical cord and cord blood, and the like. Because of the different tissue source attributes and preparation methods, the obtained MSCs have attribute differences, the expression profile of the genes has large heterogeneity, and the proliferation capacity is limited. Human tissue acquisition channels are strict and the quantity is limited, so that the cell preparation cost is further increased. Induced pluripotent stem cells (induced Pluripotent STEM CELLS, IPSC) have unlimited proliferation capacity and multipotent differentiation potential. The latest induction technology avoids exogenous gene integration, and greatly reduces the risk of iPSC tumorigenesis and the risk of gene mutation caused by random insertion. The use of iPSCs to induce differentiated MSCs can increase cell uniformity and batch-to-batch stability. Patient self-derived iPSC differentiated MSCs can reduce the risk of immune rejection. Although there are a number of iPSC differentiation MSC protocols, these protocols still suffer from some drawbacks. In the prior art, the method for differentiating the iPSC into the MSC depends on animal serum (such as fetal calf serum and animal-derived growth factors) and/or human serum substitutes (such as human platelet lysate), has the problems of heterogeneous protein risk, undefined components, large batch difference and the like, and is difficult to realize standardized and large-scale production. For example, the media used in the methods of CN105754936a and CN106520687a contain FBS, which are animal-derived, of unknown composition, vary widely from batch to batch, and are difficult to use in the production of clinical grade MSCs. In another example, CN107574146A can induce expression after iPSC adhesion by changing culture mediums with different components at different stages for more than 30 days to finally obtain iMSC. The method for forming embryoid bodies by utilizing semi-solid culture medium to differentiate mesenchymal stem cells (Adrian J C Bloor et al., nat Med 2020) of iPSC for clinical study can reduce iPSC residues, but has the advantages of complex operation, difficult separation of embryoid bodies, easy mechanical damage and pollution to cells and uncontrollable sphere diameter uniformity. In order to reduce cell death rates, some methods (Adrian J C Bloor et al., nat med 2020) use a low oxygen environment (5% O 2) for mesodermal fate decisions, which requires a low oxygen incubator and high concentration nitrogen maintenance, increasing equipment requirements and manufacturing costs. Disclosure of Invention The application aims to overcome the defects of the prior art and provide a method for preparing mesenchymal stem cells by utilizing differential sedimentation sorting cell spheres. The application provides a preparation method for differentiating human iPSC Into Mesenchymal Stem Cells (iMSC) in a whole-course culture system without animal source components, serum and definite components. In order to achieve the above purpose, the technical scheme adopted by the application is as follows: the application provides a method for preparing mesenchymal stem cells by utilizing differen