Method for preparing mesenchymal stem cells by using differential settlement sorting cell spheres

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for preparing mesenchymal stem cells by utilizing differential settlement sorting cell balls. According to the method, the differential sedimentation method is adopted, the diameters of the embryoid cell balls are sorted according to the size, the diameter uniformity of the embryoid cell balls is remarkably improved, and then the uniformity and stability of subsequent differentiation products are improved. According to the method provided by the invention, reagent components are clear, facility requirements are simple, differentiation efficiency is high, the iMSC conforming to the identification standard can be obtained by rapidly differentiating for 20 days, the obtained iMSC is good in uniformity and less in undifferentiated cell residue, and the method has potential value in clinical transformation application.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method for preparing mesenchymal stem cells by sorting cell spheres using differential sedimentation. Background Technology

[0002] In stem cell differentiation, as well as organoid differentiation and culture, stem cell self-organization to form embryoid spheres is often required, enabling the determination of cell fate for specific germ layers through self-differentiation. Simultaneously, the formation and proliferation of three-dimensional embryoid spheres significantly increases cell yield, making it a crucial step in cell product preparation. However, due to differences in initial cell state (cell unit rate, cell stemness, and viability, etc.) and microenvironment (nutrient factors, mechanical stress), the volume of the formed cell spheres often varies. Cells within cell spheres of different volumes have varying levels of contact with external nutrients and oxygen, receive different signal concentrations, and exhibit differences in developmental speed and progress, ultimately affecting the uniformity of cell properties.

[0003] Mesenchymal stromal cells (MSCs) are pluripotent adult stromal cells with self-renewal and multi-lineage differentiation potential. Widely distributed in various tissues of the human body, MSCs exhibit specific responses to inflammatory responses and damage signals in tissues, enabling them to rapidly migrate to damaged tissues to exert anti-inflammatory, repair, and tissue regeneration functions. Primary MSCs are derived from adult bone marrow, adipose tissue, and peripheral blood, as well as neonatal placenta, umbilical cord, and cord blood. Due to the different tissue sources and preparation methods, the obtained MSCs exhibit variations in properties, significant heterogeneity in gene expression profiles, and limited proliferative capacity. The strict and limited availability of human tissues further increases the cost of cell preparation.

[0004] Induced pluripotent stem cells (iPSCs) possess unlimited proliferative capacity and multi-lineage differentiation potential. Recent induction techniques avoid integration of exogenous genes, significantly reducing the tumorigenic risk of iPSCs and the risk of gene mutations due to random insertion. Using iPSCs to induce differentiation into MSCs can increase cell homogeneity and batch-to-batch stability. Differentiating MSCs from patient-derived iPSCs can reduce the risk of immune rejection.

[0005] While various methods exist for differentiating iPSCs into MSCs, these methods still have some shortcomings. Current technologies for differentiating iPSCs into MSCs largely rely on animal serum (such as fetal bovine serum, animal-derived growth factors) and / or human serum substitutes (such as human platelet lysates), which carries the risk of heterologous proteins, unclear composition, and significant batch-to-batch variations, making standardization and large-scale production difficult.

[0006] For example, the methods in CN105754936A and CN106520687A use culture media containing FBS, which is of animal origin, with unknown composition and significant batch-to-batch variations, making it difficult to use for the production of clinical-grade MSCs. Another example is CN107574146A, which, after iPSC adhesion, requires changing the culture medium with different components at different stages, takes more than 30 days to induce expression and finally obtain iMSCs. A clinical study using iPSCs to differentiate into mesenchymal stem cells (Adrian JC Bloor et al., Nat Med. 2020) employed a method of forming embryoid spheroids using a semi-solid culture medium. While this reduces iPSC residue, the procedure is complex, the separation of embryoid spheroids is difficult, easily causing mechanical damage and contamination to cells, and the uniformity of spheroid diameter is uncontrollable. To reduce cell death, some methods (Adrian JC Bloor et al., Nat Med. 2020) use a hypoxic environment (5% O2) for mesodermal fate determination. However, this hypoxic environment requires a hypoxic incubator and a high concentration of nitrogen, which increases equipment requirements and preparation costs. Summary of the Invention

[0007] The purpose of this application is to overcome the shortcomings of the prior art and provide a method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres. This application provides a method for differentiating human iPSCs into mesenchymal stem cells (iMSCs) in a culture system that is entirely free of animal-derived components, serum, and has clearly defined components.

[0008] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres, comprising the following steps: S1. Human induced pluripotent stem cells are subjected to adherent and passage culture to obtain predifferentiated human induced pluripotent stem cells. S2. Differentiate and culture pre-differentiated human induced pluripotent stem cells in a mesodermal precursor cell differentiation medium to obtain mesodermal precursor cells. S3. Resuspend the mesodermal precursor cells obtained in step S2 in embryoid culture medium and culture them statically to form embryoid cell spheres. S4. Collect the embryonic somatic cell spheres obtained in step S3. Use the cell spheres to settle in the liquid at different rates to sort out the embryonic somatic cell spheres with uniform volume. Then place the embryonic somatic cell spheres in the mesenchymal stem cell maturation culture medium for homogenization and cell expansion. Then passage the cells to enrich mature mesenchymal stem cells.

[0009] In this application, embryoid somatic cell spheroids are a key component of cell differentiation and development. This application employs different differential sedimentation methods to sort the embryoid somatic cell spheroids by diameter, significantly improving the uniformity of spheroid diameter and thus enhancing the uniformity and stability of subsequent differentiation products. In some specific embodiments, human induced pluripotent stem cells (iPSCs) are adherently cultured and passaged in feeder-free medium.

[0010] The feeder-free culture medium is E8 medium, which can also be a feeder-free complete culture medium for other induced pluripotent stem cells.

[0011] In a preferred embodiment of the method for preparing mesenchymal stem cells by differential sedimentation sorting of cell spheres as described in this application, in step S2, the mesodermal precursor cell differentiation culture medium includes IMDM culture medium and Ham's F12 culture medium. In some specific embodiments, the mixing ratio of IMDM culture medium and Ham's F12 culture medium is (40%~60%):(60%~40%). Preferably, the mixing ratio of IMDM culture medium and Ham's F12 culture medium is 50%:50%.

[0012] The mesodermal precursor cell differentiation medium also contains 5-15 μM Y-27632, 1-3 mg / mL sodium bicarbonate, 0.5-20 μg / mL human total transferrin, 0.5-20 μg / 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.

[0013] In some specific embodiments, the lipid concentrate is a chemically defined lipid concentrate, which is a commercially available concentrated fat emulsion used as a culture medium additive, and its components are: Arachidonic Acid, Cholesterol, DL-alpha-Tocopherol Acetate, Ethyl Alcohol 100%, Linoleic Acid, Linolenic Acid, Myristic Acid, Oleic Acid, Palmitic Acid, Palmitoleic Acid, Pluronic F-68, Stearic Acid, and Tween 80.

[0014] In some specific embodiments, 100×Non-essential amino acids (NEAA) (commercially available) contains seven non-essential amino acids: L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine.

[0015] In a preferred embodiment of the method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in this application, in step S3, the embryosome culture medium includes IMDM, Ham's F12, sodium pyruvate, L-alanyl-L-glutamine solution, non-essential 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.

[0016] In some specific embodiments, the culture time is 4 to 12 days, and the clonal diameter of the embryoid somatic cell spheres is >100 μM. Preferably, the culture time is 4 to 6 days, and the clonal diameter of the embryoid somatic cell spheres is 100 to 200 μM.

[0017] Preferably, the embryo-like culture medium also contains 1-6 mM glutamine, 1-100 μM 1-thioglycerol and 0.5-20 ng / ml fibroblast growth factor 2.

[0018] In some specific embodiments, glutamine was provided using Glutamax at a concentration of 2 mM, 1-thioglycerol at a concentration of 100 μM, and fibroblast growth factor 2 at a concentration of 20 ng / ml.

[0019] In a preferred embodiment of the method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in this application, in step S4, the mesenchymal stem cell maturation culture medium includes IMDM, Ham's F12, sodium pyruvate, L-alanyl-L-glutamine solution, non-essential amino acids, 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.

[0020] This application provides a method for differentiating human iPSCs into mesenchymal stem cells (iMSCs). By optimizing the starting cell state and culture medium composition (mesoderm precursor cell differentiation medium, embryoid culture medium, and mesenchymal stem cell maturation medium), this application achieves mesoderm fate determination under normoxic conditions, with cell viability comparable to that under hypoxic conditions, reducing equipment requirements and saving preparation costs. All consumables, coating solutions, and culture media used throughout the differentiation induction process are free of animal-derived components, with clearly defined compositions, which is beneficial for subsequent cell drug preparation and clinical translation applications.

[0021] In some specific embodiments, the working concentration of Y-27632 is preferably 10 μM. Y-27632 is an ATP-competitive rock inhibitor.

[0022] As a preferred embodiment of the method for preparing mesenchymal stem cells by sorting cell spheres using differential sedimentation as described in this application, step S4 further includes testing the sedimentation rate of embryoid cell spheres suspended in the lumen of a quartz glass tube, and determining the curve function relationship between the radius r of the embryoid cell spheres and the sedimentation time t. Taking a quartz glass tube perpendicular to the horizontal plane at a 90° angle as an example, the settling rate basically conforms to Stokes' law in terms of the sphere's volume (radius) and density: Stokes' Law: in: v Settlement velocity, ρ p : Density of a sphere, ρ f : Liquid density, g: gravitational acceleration, r: radius of the sphere, η: liquid viscosity.

[0023] In some specific embodiments, embryoid cell spheres are injected from the top opening of a quartz glass tube, and embryoid cell spheres of different volumes settle in the liquid pipe under the action of various mechanical forces such as gravity, buoyancy and friction.

[0024] In practical applications, ρ p MCA sphere density ρ f : Density of culture medium inside the tube g Gravitational acceleration η Liquid viscosity, the values ​​above are measurable or constants, and remain constant within the same system. Therefore, the settling rate of embryoid spheres depends only on their radius. r The velocity is directly proportional to the square of the velocity, meaning that the larger the radius (volume) of the embryoid sphere, the faster the settling rate and the more preferentially it reaches the outlet at the bottom of the tube; conversely, the smaller the radius of the embryoid sphere, the later it reaches the outlet.

[0025] The embryoid cell spheres obtained in this application are a mixture containing single cells ranging in diameter from a few micrometers to microspheres ranging from several hundred micrometers. Removing unformed single cells and particulate matter to obtain spheres of similar volume for further differentiation is of great significance for ensuring normal cell properties and improving the uniformity of differentiation products. To this end, this application designs a sphere homogenization collection scheme, utilizing the difference in sedimentation rate of cell spheres in a liquid environment to collect embryoid cell spheres of similar (or nearly similar) volume for further differentiation.

[0026] In a preferred embodiment of the method for preparing mesenchymal stem cells by differential sedimentation sorting of cell spheres as described in this application, the diameter of the embryonic somatic cell spheres is 10~250 μm, preferably 50~200 μm.

[0027] In a preferred embodiment of the method for preparing mesenchymal stem cells by differential sedimentation sorting of cell spheres as described in this application, the quartz glass tube is mounted on an adjustable slope scaffold, with the slope adjustment range being 0° to 90°.

[0028] Quartz glass tubes are the preferred material choice due to their ability to meet basic mechanical, cleanliness, optical performance, and biocompatibility requirements. Other materials, such as glass, resin, and acrylic, are not specifically limited depending on the application scenario. The material dimensions are determined within a preferred range after comprehensively considering factors such as product accessibility, available space within the aseptic workbench, and ease of handling, storage, disassembly, and sterilization. In some preferred solutions, the inner diameter of the quartz glass tube is 2-6 mm, and the length is 20-45 cm.

[0029] In some specific embodiments, sterile quartz glass tubes are placed on an adjustable-angle support (0-90°) inside a laminar flow hood. The quartz glass tubes are open at both ends, have smooth inner walls, and good optical properties. The bottom end of the quartz glass tube is immersed in a collection dish of liquid. The glass tube is rinsed with PBS and then filled with culture medium. Quartz glass is chosen primarily because of its advantages such as high light transmittance, high temperature resistance, and strong chemical stability.

[0030] In a preferred embodiment of the method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in this application, the radius r of the embryoid cell sphere and the sedimentation time t have the following curve function relationship: in, a The range is 5 to 150. b The range is 10 to 250; a , b It can also vary depending on the specific parameters of the device; More preferably: .

[0031] The above functional relationship is only obtained under the optimal parameters in the case study and is not the only functional relationship. The functional relationship followed by this differential sedimentation method is clear, namely the natural logarithmic relationship, but the specific coefficients vary with the parameters. In actual operation, embryoid spheres of different diameters (10-300 μm) will settle at different rates in the lumen and gradually separate. Through preliminary experiments to test the difference in sedimentation rate under different tube diameters and inclines, the optimal separation parameters are obtained. For example, at a 90° angle, with an inner diameter of 4 mm and a length of 25 cm, spheres with a diameter of 50-200 μm can settle differentially in the tube, reaching the bottom of the tube in stages with minimal adhesion loss to the tube wall. Sorting can be basically completed within 15 minutes, with minimal overall damage to the cell spheres (including environmental temperature changes, mechanical impact damage, etc.), and the time cost is feasible. The method used is simple to operate, has high facility accessibility, and the preparation cost is controllable. The cell properties are uniform, which is conducive to clinical translation applications.

[0032] In a preferred embodiment of the method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in this application, the culture conditions in step S2 include: Incubate at 37℃, 5% CO2, and 20% O2 for 24–72 hours; preferably for 48 hours. And / or, in step S3, the conditions for static incubation include: Incubate at 37°C for 24 hours with 5% CO2 and 20% O2. And / or, in step S4, the conditions for homogenizing cell expansion include: 5% CO2, 20% O2, stand at 37℃ for 24 hours.

[0033] In step S5, the following conditions apply to the transmission: During culture, passage should be performed every 48-96 hours, preferably every 72 hours. After passage 4-5, the cultured cells, iMSC cells, can be harvested for testing or cryopreservation.

[0034] As a preferred embodiment of the method for preparing mesenchymal stem cells by differential sedimentation sorting of cell spheres as described in this application, the method further includes identifying mesenchymal-like cells, wherein the positive rates of cell surface antigens CD105, CD90, and CD73 of the mesenchymal-like cells are all ≥95%, and the positive rates of CD45, CD34, CD14, and HLA-DR are all ≤2%.

[0035] This application provides a method for preparing mesenchymal stem cells (MSCs) using differential sedimentation sorting of cell spheres. In a culture system free of animal-derived components, serum, and with clearly defined components, human induced pluripotent stem cells (iPSCs) are induced to differentiate into mesenchymal stem cells (iMSCs) (iPSC-derived MSCs). The method first induces human iPSCs into a mesodermal fate-determining cell line, then forms embryoid cell spheres through liquid suspension culture. Uniform embryoid cell spheres are sorted using differential sedimentation, and then mesenchymal stem cells mature and proliferate through two-dimensional culture. Measurement and statistical analysis, as well as inverted microscope imaging, show improved cell homogeneity after sorting. The differentiated cells are identified as qualified based on morphology, surface marker detection, and trilineage differentiation capacity. Finally, a co-culture organelle transfer experiment preliminarily verifies the functional ability of differentiated cells to empower other cell types.

[0036] The differential sedimentation method provided in this application is not only applicable to the preparation of mesenchymal stem cells, but also applicable to other types of cell differentiation and organoid preparation. As long as the preparation of embryonic cell spheres is involved and sorting is required according to the volume (or diameter) of the spheres, the sorting device, parameters and functional relationships provided in this application can be used as a reference.

[0037] Compared with traditional centrifugation and filtration methods, the sorting method of this application has the advantages of less mechanical stimulation during the sorting process, which can effectively avoid mechanical damage to cells. The entire operation can be carried out in a sterile laminar flow hood, avoiding bacterial and other microbial contamination, and is suitable for live cell preparation.

[0038] Finally, this application provides a potential application method of the above-mentioned mesenchymal stem cells in regenerative medicine, wherein the mesenchymal stem cells, after being co-cultured with motor neurons, have their mitochondria transferred into the motor neurons, thereby achieving the repair of the mitochondrial function of the latter.

[0039] Compared with the prior art, this application has the following beneficial effects: This application provides a method for preparing mesenchymal stem cells (MSCs) using differential sedimentation sorting of cell spheres. In the iPSC maintenance stage, a well-defined coated matrix gel and culture medium are used, ensuring that the starting seed cells are free of animal-derived components and unidentified protein interference. In the first differentiation stage, Y-27632 is added to the well-defined coated matrix gel and culture medium, effectively improving cell activity and proliferation under normoxic conditions, ensuring a sufficient number of cells for the next differentiation stage. In the second differentiation stage, liquid suspension culture rapidly forms cell clones while avoiding iPSC residue. In the third differentiation stage, embryosome-like cell spheres of the same volume are sorted and enriched. In the fourth stage, differences in cell adhesion properties are utilized to further sort mature MSCs, removing contaminating cells and undifferentiated iPSCs, further improving cell homogeneity and batch stability of differentiation products. The method provided in this application has well-defined reagent components, simple facility requirements, and high differentiation efficiency. iMSCs meeting identification criteria can be obtained in as little as 20 days, with good homogeneity and few undifferentiated cell residues, demonstrating potential value for clinical translational applications. Attached Figure Description

[0040] Figure 1 A schematic diagram of the iPSC differentiation process into iMSCs and a flowchart of cell morphological changes; Figure 2 The figure shows the results of improving cell viability and spheroidization rate using the culture medium of this application and conventional culture (wherein, Figure 2 Image A shows the bright field results on day 2 of the mesodermal precursor cell differentiation medium. The traditional medium (a, b) and the medium used in this application (c, d) are shown. Images b and d are magnified views of parts of images a and c. Figure 2 Figure B shows the bright field results on days 4 and 8 of the embryoid culture medium. The traditional semi-solid culture medium (a, b) and the culture medium used in this application (c, d) are compared (scale bar: 50 µm). Figure 3 The results of adding Y-27632 during the mesenchymal embryonic spheroid formation stage significantly increased cell spheroid volume (where, Figure 3 a, Figure 3 b: Bright field results on days 2 and 4 of iMSC differentiation mesenchymal embryoid formation without the addition of Y-27632 (Y- for short). Figure 3 c, Figure 3 (d: Bright field results on days 2 and 4 of the iMSC differentiation mesenchymal embryoid formation period with added Y (Y+), scale bar 100 µm). Figure 4 This is a schematic diagram of a differential sedimentation sorting device. Figure 5 Flowchart for differential sedimentation sorting of cell spheres and sample loading / operation. Figure 6This is a schematic diagram of the overall process of differential sedimentation sorting of cell spheres. Figure 7 The image shows the results of cell spheroid sorting by differential sedimentation method (where, Figure 7 In the middle A field, a mixture of embryoid spheroids of varying sizes formed during iMSC differentiation is presented, representing a random field of view. Figure 7 In section B (al), embryoid spheres of different volumes were collected in different culture dishes from largest to smallest after preliminary sorting by suspension sedimentation over 1-12 minutes; the scale bar is 100 µm. Figure 8 A comparison of cell spheroid uniformity after sorting by differential sedimentation method; Figure 9 A graph showing the relationship between the radius of cell spheres and sedimentation time in differential sedimentation. Figure 10 The image shows the results of cell migration and cell community morphology differences after cell spheroids adhered to the wall after differential sedimentation sorting (where, Figure 10 In the middle section, A(ac) represents the differences in cell migration and morphology during subsequent adherence and expansion of embryo-like spheres of different volumes; Figure 10 Image B (ac) shows the migration and morphology of unsorted cell spheroids during cell adhesion and expansion on days 2-4. Figure 10 Image B (df) shows the cell spheroids adhered to the culture vessel and expanded during days 2-4, along with cell migration and morphology; scale bar 100 µm). Figure 11 The image shows the results of iMSC plastic adhesion and morphology identification (the right image is a partial enlargement of the left image). Figure 12 The growth status of induced differentiated mesenchymal stem cells (iMSCs) in the culture medium of this application and a certain commercial culture medium 1, 2, and 3 are shown in the figure (where, Figure 12 In the image, A represents the growth status of the induced differentiated mesenchymal stem cells (iMSCs) (7th generation) in this application in the culture medium (a) and commercial culture media 1, 2, and 3 (bd). Figure 12 In Figure B, the induced differentiated mesenchymal stem cells (iMSCs) of this application (generation 8) are shown in the growth state of the medium used in this application (a) and commercial mediums 1, 2, and 3 (bd), with a random field of view and local magnification in bright field; scale bar 100 µm. Figure 13 Figure I shows the results of iMSC surface antigen identification. Figure 14 Figure II shows the results of iMSC surface antigen identification. Figure 15 This is a diagram showing the results of trilineage differentiation identification in iMSC; Figure 16 This is a diagram showing the transport of iMSC mitochondria in microtubules. Figure 17 The image shows the results of iMSC mitochondrial transfer to co-cultured neurons. Detailed Implementation

[0041] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0042] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0043] In the following examples, the formulation of the mesodermal precursor cell differentiation medium (50 mL system) is shown in Table 1.

[0044] Table 1 The volume of factors and proteins smaller than 100 μL is negligible. *Add immediately before use.

[0045] The formulation of embryo-like culture medium (50 mL system) is shown in Table 2.

[0046] Table 2 The volume of factors and proteins smaller than 100 μL is negligible. *Add immediately before use.

[0047] The formulation of the mesenchymal stem cell maturation culture medium (50 mL system) is shown in Table 3.

[0048] Table 3 The volume of factors and proteins smaller than 100 μL is negligible. *Add immediately before use.

[0049] The following embodiments illustrate the process of iPSC differentiation into iMSCs and the flow of cell morphology changes as follows: Figure 1 As shown.

[0050] In the following embodiments: The formulation of commercial culture medium 1 is shown in Table 4.

[0051] Table 4 Commercial culture medium 2: MSC serum-free culture medium MH01, catalog number MH000-N011, Ekosei; Commercial culture medium 3: MSC serum-free medium MH01 + 5% platelet lysate.

[0052] Serum-free MSC medium MH01, catalog number MH000-N011, Ekosei; Platelet lysate, PLTGOLD100R, Biological Industries.

[0053] Example 1: A method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres. This embodiment provides a method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres, including the following steps: 1. iPSC culture and pre-differentiation preparation: (1) 6-well plates were coated with VTN-N (Gibco™, A14700) at a concentration of 0.5 μg / cm³. 2 Let it stand at 37℃ for 1 hour.

[0054] (2) Resuspend the resuscitated iPSCs in E8 (Essential 8™ Flex, Gibco™, A2858501), seed them in VTN-N coated 6-well plates, add culture medium to 2 mL, add 10 μM Y-27632 (Stemcell, 72302) to the culture medium (remove Y-27632 after 12~16 h), and incubate statically at 5% CO2 and 37℃.

[0055] (3) Change the E8 medium to fresh every day. After multiple passages, once the iPSCs are in good and stable condition, they begin to differentiate.

[0056] (4) Coating a 6-well plate with Collagen IV (0.5 μg / cm) 2 Let stand at 37°C for 1 hour. Add 1 mL of digestive enzyme Accutase (Sigma, A6964-500 mL) and digest at 37°C for 5 minutes.

[0057] (5) Gently disperse the cells into a single-cell suspension, transfer the cell suspension to a 15mL centrifuge tube, add 5mL of 1×DPBS, centrifuge to remove digestive enzymes, resuspend the cells in E8 medium, and count them. Add 2.0×10⁻⁶ cells to a centrifuge tube. 5 Cells were seeded into Collagen IV-coated 6-well plates, and the culture medium was added to a final volume of 2 mL. 10 μM Y-27632 was added (Y-27632 was removed after 12-16 hours). The plates were then incubated statically at 37°C and 5% CO2 for 24 h.

[0058] 2. Mesodermal precursor cell induction: Discard the old culture medium and use DPBS (Ca-free). 2+ and Mg 2+Carefully wash the cells and replace with mesodermal precursor cell differentiation medium, adding 10 μM Y-27632. Incubate at 37°C, 5% CO2, and normoxic (20% O2) conditions for 48 hours without changing the medium. This induces iPSC differentiation into mesodermal precursor cells. Using the above mesodermal precursor cell differentiation medium under normoxic conditions can yield differentiated cells and effectively ensure cell viability. Figure 2 (A)

[0059] 3. Formation of embryoid somatic cell spheroids: (1) Collect the mesodermal precursor cells obtained in step 2, add 0.5 mL of Accutase enzyme preheated to room temperature, incubate at 37°C for 5 min to digest the cells into single cells, and gently disperse them into a single-cell suspension. 300 × g Centrifuge for 5 minutes and discard the digestive enzymes.

[0060] (2) Resuspend the cells in 1 mL of embryoid culture medium, transfer the suspension to a low-adsorption 6-well plate (Corning, 3471), add embryoid culture medium to 3 mL, add 10 μM Y-27632, shake the cells well and place them in a static suspension culture at 37℃, 5% CO2, and normal oxygen (20% O2).

[0061] (3) Culture for 4 to 8 days. Replace half of the embryonic culture medium every 2 days.

[0062] Compared with traditional semi-solid culture media, the use of embryosome-like culture media is simple to operate, has a low risk of contamination, and significantly improves the efficiency of forming embryosome-like cell spheroids in the same time period. Figure 2 (B). After adding Y-27632, the cell spheroid volume significantly increased ( Figure 3 This increases cell yield and shortens the preparation cycle. Cell spheroids with a diameter >50 μm can proceed to the next step.

[0063] 4. iMSC formation and amplification: (1) Coating a 6-well plate with Fibronectin / Collagen I (0.67 µg / cm) 2 Fibronectin, 1.2 µg / cm 2 Collagen I was incubated at 37°C for 1 hour. The coating solution was removed, and 3 mL of iMSC maturation medium was added to each well of the 6-well plate for later use.

[0064] (2) Gently rotate and shake the petri dish to gather the embryosomal cell spheres in the center. Carefully collect the embryosomal cell spheres using a 1mL wide-mouth pipette tip (or a 1mL pipette tip with the tip cut off, or a Pasteur tube). Then sort the embryosomal cell spheres and hold the pipette vertically above the culture medium surface. Wait for the embryosomal cell spheres to settle and converge at the pipette tip. Make contact between the pipette tip and the culture medium surface to form a liquid channel. Under the action of gravity, the embryosomal cell spheres slowly settle into the culture medium along the channel (collect embryosomal cell spheres of similar volume and plant them in well plates coated with fibronectin (FN) and human type I collagen (hCo-I), and add iMSC maturation medium). Discard any excess liquid in the pipette tip.

[0065] As iPSCs differentiate into embryoid spheres, the degree to which cells within embryoid spheres of different sizes are exposed to oxygen and inducing factors varies, resulting in differences in cell stratification. This has a certain impact on subsequent cell migration and differentiation, ultimately leading to differences in the uniformity of cell products.

[0066] The specific steps for sorting embryoid somatic cell spheres include the following: ① Sorting device setup: The sorting device includes one quartz glass tube, two compatible sterile rubber stoppers, and an angle-adjustable support. Figure 4 , Figure 5 Inside the laminar flow hood, sterile quartz glass tubes (hereinafter referred to as glass tubes) are placed on an adjustable-angle support (0-90°). The glass tubes (inner diameter 2-6 mm, length 20-45 cm) are open at both ends, with smooth inner walls and good optical properties. The glass tubes are rinsed with 1×DPBS. Quartz glass is chosen primarily because of its advantages such as high light transmittance, high temperature resistance, strong chemical stability, and good biocompatibility. Currently, various specifications of quartz glass tubes are available on the market, and the supply is sufficient. Other materials with similar properties can also be used.

[0067] ②Measure the relationship between embryoid sphere volume and settling time: After collecting embryoid cell spheres, they were injected into a quartz glass tube through the top opening using a 1 mL wide-mouth pipette tip. Cell spheres of different volumes settled in the liquid tube under the influence of gravity, buoyancy, and friction. Taking a quartz glass tube perpendicular to the horizontal plane at a 90° angle as an example, the settling rate generally conformed to Stokes' law regarding the sphere's volume (radius) and density. Stokes' Law: in: v Settlement velocity, ρ p : Density of a sphere, ρ f: Liquid density, g: gravitational acceleration, r: radius of the sphere, η: liquid viscosity.

[0068] In practical applications, ρ p Cell spheroid density, ρ f : Density of the culture medium inside the tube, g; gravitational acceleration, η; viscosity of the liquid. In actual measurements, these values ​​are fixed or constants, and remain constant within the same system. Therefore, the sedimentation rate of cell spheroids depends only on their radius. r The sedimentation rate is directly proportional to the square of the volume of the cell sphere. That is, the larger the radius (volume) of the cell sphere, the faster the sedimentation rate and the more likely it is to reach the bottom outlet of the tube. Conversely, the smaller the radius of the cell sphere, the later it will reach the outlet.

[0069] ③ Use measurement time windows to sort and collect cell spheres of equal volume: Embryomorphic cell spheres of different diameters (10-250 μm) will settle at different rates within the lumen, gradually separating. Preliminary experiments were conducted to test the differences in sedimentation rates under different tube diameters and inclines, determining the optimal separation parameters. Preferably, a quartz glass tube with an inner diameter of 4 mm, a wall thickness of 1 mm, and a length of 25 cm was selected, with a support incline of 90°. The quartz glass tube sedimentation and sorting device was constructed at room temperature: Sterilized quartz glass tubes (hereinafter referred to as glass tubes), with smooth inner walls, good optical properties, and rinsed with 1×DPBS, were vertically fixed on the support in a clean bench. Both ends of the quartz glass tubes were open, and the tubes were filled with culture medium. Filling method: The bottom of the glass tube was sealed with a sterile rubber stopper. The quartz glass tube was vertically placed on the tube support, and liquid (iMSC amplification medium) was added from the top of the glass tube using a 1 mL pipette. Care was taken to avoid air bubbles and not to fill the glass tube completely (leaving a 1 mL empty volume at the top). Gently rotate the glass dish clockwise several times to gather the embryosomal cell spheres to the center. Carefully aspirate all the cell spheres using a 1mL pipette tip and transfer them (along with approximately 1mL of culture medium) to the empty volume at the top of the glass tube. Quickly seal the top of the glass tube with a sterile rubber stopper, then quickly remove the stopper from the bottom. Due to atmospheric pressure, the liquid inside the glass tube will not flow out, while the cell spheres will begin to settle at different rates under the influence of gravity and buoyancy. Start timing (...). Figure 5 The glass tube rack is moved above a culture dish pre-filled with iMSC amplification medium, with the glass tube suspended approximately 1 cm above the liquid surface (but not in contact), awaiting cell spheroid collection. Cell spheroids of different diameters at different time points sequentially settle and accumulate above the liquid surface at the bottom of the glass tube. By briefly (1 second) moving the culture dish upwards, the liquid surface at the bottom of the glass tube contacts the liquid surface inside the culture dish, forming a liquid channel. The cell spheroids accumulated at the bottom of the glass tube can then quickly fall into the culture dish, achieving cell spheroid collection. Subsequently, the bottom of the glass tube detaches from the liquid surface of the culture dish, initiating a new round of cell spheroid aggregation. By collecting cells in separate dishes at different time points, cell spheroids of different radii can be sorted. Figure 6).

[0070] ④ Determining the functional relationship between the radius of sorted cell spheres and sedimentation time: The average diameter of sedimented cell spheres collected at different time points was measured using microscopic imaging and specialized software (e.g., ImageJ). Statistical analysis of the results showed that the differences within the cell sphere group collected by differential sedimentation were significantly reduced, achieving uniformity in sphere diameter. Figure 7 , Figure 8 (Table 5) The unsorted spheres were in a mixed state, and the standard deviation was significantly greater than that of the sorted spheres.

[0071] Based on the measurement results, a distribution curve of cell spheroid radius versus sedimentation time can be plotted, establishing a functional relationship between cell spheroid radius *r* and sedimentation time *t*. In subsequent applications, this functional relationship can be used to estimate the radius range of cell spheroids collected at selected time points. For example, in a quartz tube with a 90° angle, 4mm inner diameter, and 25cm length, a 100μm diameter spheroid can settle to the bottom of the tube in 4 minutes; therefore, the time window for collecting spheroids with a diameter of approximately 100μm is 3 minutes 0.1 seconds to 4 minutes 0.2 seconds. (The curve showing the relationship between cell spheroid radius and sedimentation time in differential sedimentation sorting is also included.) Figure 9 The fitted curve function relationship is as follows: Table 5 (3) Place the well plate at 37°C, 5% CO2, and normal oxygen (20% O2) for 3 days without changing the medium. The embryonic somatic cell spheres adhere to the plate and spread outwards until they fill the bottom of the plate, thus achieving uniform cell expansion. This is recorded as generation p0.

[0072] Cells of different diameters exhibited differences in migration pathways, cell community distribution, and cell morphology as they entered the next stage of adherent growth. Figure 10 (A); Unsorted cell spheres enter the adherent growth stage, and the above differences are significant ( Figure 10 In the middle B, ac), homogenized cell spheres enter the adherent growth stage, and the cell migration path, cell community distribution, and cell morphology show significant homogenization. Figure 10 (B, df), thereby ensuring the homogenization of the final cellular products.

[0073] 5. Cells that reached 90% coverage in step 4 were passaged. After 90% coverage, the cells were digested with Accutase, and the iMSCs were expanded and resuspended, then passaged 1:3 into Fibronectin / Collagen I-coated 6-well plates. The plates were then incubated statically at 37°C, 5% CO2, and normoxic (20% O2), designated as generation p1. For the first five passages, iMSCs with strong adhesion were screened and enriched using an adherent separation method, while weakly adherent cells, including undifferentiated iPSCs, were removed to improve the homogeneity of the obtained cell products. Characteristic markers of iMSCs were detected at p3-p5 (approximately day 20 of differentiation).

[0074] Example 2: Cell genus based on criteria such as cell morphology, plastic adhesion, surface antigen detection, and trilineage differentiation. Sexual identification When the cells were passaged to the 3rd to 5th generation, the cell morphology was observed and recorded. The results showed that the cells were spindle-shaped or triangular, consistent with the typical morphological characteristics of mesenchymal stem cells. Figure 1 , Figure 11 ).

[0075] 1) Identification of plastic adhesion: Differentiated iMSCs passaged to the 5th generation were digested with Accutase at 37°C for 5 min, then gently pipetted to obtain single cells, counted, and 1.0 × 10⁶ cells were collected. 5 Cells were resuspended in standard culture medium and seeded into 12-well plastic plates. The standard culture medium consisted of 89% DMEM, 10% FBS, and 1% PS antibiotics (penicillin-streptomycin). The plates were incubated in a 5% CO2, 20% O2, 37°C incubator for 24 hours. The adhesion of iMSCs was observed using an inverted microscope; the iMSCs adhered well. Figure 11 ).

[0076] Comparing the growth status of induced cellular mesenchymal stem cells (iMSCs) in this application with the mesenchymal stem cell maturation culture medium of this application (Table 3), and with three commercial culture media 1 (Table 4), 2, and 3, it can be seen that iMSCs still have good cell morphology at passages 7-9, while cells in commercial reagents 2 and 3 have shown obvious signs of flattening, fibrosis, and aging. Figure 12 Commercial reagent 1 also maintains cell morphology well in generations 7-9, but the cost of the culture medium in this application is significantly lower than that of commercial reagent 1. To prepare the same volume of culture medium, the cost of the culture medium in this application is about ⅓ to ¼ of that of commercial culture medium 1.

[0077] 2) Surface antigen identification: Differentiated iMSCs were passaged to the 5th generation (iMSC-p5), digested with the digestive enzyme Accutase at 37℃ for 5 min, gently pipetted to obtain single cells, counted, and 2.0 × 10⁶ cells were collected. 5Cells were resuspended in 400 μL of 1×DPBS and grouped. They were then incubated for 15 min with antibodies against CD73, CD90, CD105, CD34, CD45, CD14, CD19, and HLA-DR. After centrifugation to remove the antibodies, the cells were resuspended in 1×DPBS. Flow cytometry analysis was used to analyze iMSC surface antigen expression. The results showed that the positive rates of cell surface characteristic antigen marker expression were all ≥95%: CD105 (98.9%), CD90 (98.1%), and CD73 (99.7%). The positive rates of antigen marker expression in another group were ≤2%: CD45 (0.78%), CD34 (0.25%), CD14 (0.25%), and HLA-DR (0.019%). Figures 13-14 ).

[0078] 3) Identification of trilineage differentiation: ① Adipogenic differentiation: In this example, the StemCell MesenCult™ Adipogenic Differentiation Kit (Human), (Catalog #05412), hereinafter referred to as adipogenic differentiation medium, was used.

[0079] (1) iMSCs were seeded in gelatin-coated 6-well plates, and each well was seeded with 2 mL of mesenchymal stem cell maturation medium (Table 3).

[0080] (2) The cells were cultured in a 5% CO2, 20% O2, 37℃ incubator and reached about 90-100% confluence after 3-4 days.

[0081] (3) Remove the culture medium, replace 2 mL of lipid differentiation medium in each well, and incubate in a 5% CO2, 20% O2, 37℃ incubator. Record this as day 0 of differentiation.

[0082] (4) Replace the MesenCult™ adipogenic differentiation medium every 3-4 days and observe and record changes in cell morphology.

[0083] (5) After about 20 days, lipid vacuoles can be observed under low magnification. Oil Red O staining was used to label the lipid droplets, demonstrating that the cells can differentiate into adipocytes. Figure 15 (ab).

[0084] ② Osteoblast differentiation: In this example, the StemCell MesenCult™ Osteogenic Differentiation Kit (Human), (Catalog #05465), hereinafter referred to as osteoblast differentiation medium, was used.

[0085] (1) iMSCs were seeded in gelatin-coated 6-well plates, and each well was seeded with 2 ml of mesenchymal stem cell maturation medium (Table 3).

[0086] (2) The cells were cultured at 37°C and reached about 90-100% confluence after 3-4 days.

[0087] (3) Remove the culture medium, replace 2 mL of osteogenic differentiation culture medium in each well, and incubate in a 5% CO2, 20% O2, 37℃ incubator. Record this as day 0 of differentiation.

[0088] (4) Change the osteogenic differentiation medium every 3-4 days, observe and record changes in cell morphology.

[0089] (5) After about 20 days, bone matrix formation can be observed under low magnification. Alizarin Red S staining was used to mark osteoblasts with obvious calcium nodules, demonstrating that iMSCs can differentiate into osteoblasts. Figure 15 medium cd).

[0090] ③ Chondrogenic differentiation: In this example, the StemCell MesenCult™-ACFChondrogenic Differentiation Kit (Catalog #05455) was used, hereinafter referred to as chondrogenic differentiation medium.

[0091] (1) On day 0, add 2×10 to a 15 mL polypropylene tube (e.g., product number 38009). 6 Each iMSC was resuspended in 2 mL L PBS, the tube cap was tightened, and the solution was heated to 300 × 10⁻⁶ ppm. g Centrifuge using centrifugal force and discard the supernatant.

[0092] (2) Add 1 mL of chondrogenic differentiation culture medium to a 15 mL tube and carefully resuspend.

[0093] (3) Cover the centrifuge tubes (do not tighten the caps, keep the caps breathable).

[0094] (4) Place the centrifuge tubes vertically and incubate them at 37°C and 5% CO2.

[0095] (5) Change the medium every 3 days. Without disturbing the cells at the bottom, carefully aspirate half of the supernatant and replace it with fresh cartilage differentiation medium.

[0096] (6) By day 21, the chondrocyte clusters had fully differentiated and were ready for characterization analysis. The chondrocytes were fixed in 4% paraformaldehyde solution at room temperature, then embedded using OCT to prepare 6-micrometer sections for alixin blue staining. The alixin blue staining effect was observed under a microscope. The alixin blue stained portion showed the acidic mucopolysaccharides (AMPs) in the cartilage tissue. Figure 15 (e.g., eh).

[0097] Example 3: Mitochondrial-labeled iMSCs are transferred to other cells via organelles such as mitochondria to enable them to function. Fluorescently labeled iMSCs (GFP / mito-mcherry) were co-cultured with iPSCs to differentiate into motor neurons. After 3 days of co-culture, iMSCs formed microtubule channels containing mitochondria. Figure 16 (Red signal). Mitochondrial transfer signals were captured in motor neurons cultured for 3 days. Figure 17 (Yellow arrow) Preliminary evidence shows that iMSC mitochondria in the co-culture system can be transferred into motor neurons; indicating that iMSCs can empower other cells through organelles such as mitochondria.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres, characterized in that, Includes the following steps: S1. Human induced pluripotent stem cells are subjected to adherent and passage culture to obtain predifferentiated human induced pluripotent stem cells. S2. Differentiate and culture predifferentiated human induced pluripotent stem cells in a mesodermal precursor cell differentiation medium to obtain mesodermal precursor cells. S3. Resuspend the mesodermal precursor cells obtained in step S2 in embryoid culture medium and culture them statically to form embryoid cell spheres. S4. Collect the embryonic somatic cell spheres obtained in step S3. Use the cell spheres to settle in the liquid at different rates to sort out the embryonic somatic cell spheres with uniform volume. Then place the embryonic somatic cell spheres in the mesenchymal stem cell maturation culture medium for homogenization and cell expansion. Then passage the cells to enrich mature mesenchymal stem cells.

2. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, In step S2, the mesodermal precursor cell differentiation medium includes IMDM medium and Ham's F12 medium. The mesodermal precursor cell differentiation medium also contains 5-15 μM Y-27632, 1-3 mg / mL sodium bicarbonate, 0.5-20 μg / mL human total transferrin, 0.5-20 μg / 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. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, In step S3, the embryosome culture medium includes IMDM, Ham's F12, sodium pyruvate, L-alanyl-L-glutamine solution, non-essential 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.

4. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, In step S4, the mesenchymal stem cell maturation culture medium includes IMDM, Ham's F12, sodium pyruvate, L-alanyl-L-glutamine solution, non-essential amino acids, 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.

5. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, Step S4 also includes testing the settling rate of embryoid somatic cell spheres suspended in the lumen of a quartz glass tube, and determining the curve function relationship between the radius r of the embryoid somatic cell spheres and the settling time t. The settling rate and the volume and density of the sphere basically conform to Stokes' law: Stokes' Law: in: v Settlement velocity, ρ p : Density of a sphere, ρ f : Liquid density, g: gravitational acceleration, r: radius of the sphere, η: liquid viscosity.

6. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, The diameter of the embryoid somatic cell sphere is 10~250 μm.

7. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 5, characterized in that, The quartz glass tube is mounted on an adjustable support with an inclination range of 0° to 90°.

8. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 5, characterized in that, The curve function relationship between the radius r of the embryoid cell sphere and the sedimentation time t is as follows: ； in, a The range is 5 to 150. b The range is 10 to 250.

9. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, In step S2, the cultivation conditions include: The cells were incubated for 48 hours at 37°C with 5% CO2 and 20% O2. And / or, in step S3, the conditions for static incubation include: Incubate at 37°C for 24 hours with 5% CO2 and 20% O2. And / or, in step S4, the conditions for homogenizing cell expansion include: 5% CO2, 20% O2, stand at 37℃ for 24 hours.

10. The method for preparing mesenchymal stem cells using differential sedimentation sorting of cell spheres as described in claim 1, characterized in that, The method further includes identifying mesenchymal-like cells, wherein the positive rates of cell surface antigens CD105, CD90, and CD73 of the mesenchymal-like cells are all ≥95%, and the positive rates of CD45, CD34, CD14, and HLA-DR are all ≤2%.

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