WO2023050059A1 - Method for using magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes - Google Patents

Method for using magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes Download PDF

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WO2023050059A1
WO2023050059A1 PCT/CN2021/121304 CN2021121304W WO2023050059A1 WO 2023050059 A1 WO2023050059 A1 WO 2023050059A1 CN 2021121304 W CN2021121304 W CN 2021121304W WO 2023050059 A1 WO2023050059 A1 WO 2023050059A1
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cord blood
umbilical cord
stem cells
mesenchymal stem
magnetic
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PCT/CN2021/121304
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French (fr)
Chinese (zh)
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黄江鸿
赵玲
梁宇杰
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深圳市第二人民医院(深圳市转化医学研究院)
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Priority to CN202180102360.0A priority Critical patent/CN117980469A/en
Priority to PCT/CN2021/121304 priority patent/WO2023050059A1/en
Publication of WO2023050059A1 publication Critical patent/WO2023050059A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/16—Cyclodextrin; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention belongs to the technical field of chondrocytes, and in particular relates to a method for inducing umbilical cord blood mesenchymal stem cells to form chondrocytes by using a magnetic field.
  • PEMF therapy has been used to treat a variety of conditions, including knee osteoarthritis. Although its mechanism is not fully understood, it has been widely used clinically due to its safety and non-invasiveness.
  • MSCs injected through joints are considered as one of the future therapeutic approaches to regenerate damaged cartilage.
  • Extremely low frequency pulsed electromagnetic fields (PEMFs) can promote the migration and proliferation of MSCs, and enhance the paracrine activity of MSCs.
  • magnetic nanocomposite hydrogels have great potential for improving tissue engineering. We prepared magnetic nanocomposite hydrogels by ultrasonic dispersion and freeze-thaw crosslinking.
  • the magnetic nanocomposite hydrogel exhibited high biocompatibility for mesenchymal stem cells (MSCs), and the surface of the magnetic nanocomposite hydrogel showed uniform growth of MSCs and upregulated expression of chondrocyte-related genes.
  • MSCs mesenchymal stem cells
  • the differentiation ability of cartilage can be enhanced and cartilage repair can be promoted. This magnetic stimulation could help repair articular cartilage defects in rabbit knees in vivo.
  • miRNAs play an important role in the control of osteogenesis and chondrogenesis, and suggests that miRNA servers act as important epigenetic regulators mediated by external factors that induce cellular phenotypes.
  • PEMF pulsed electromagnetic fields can regulate a large number of miRNAs during the osteogenic differentiation of mesenchymal stem cells.
  • PEMF can promote the expression of miR-26a and miR-29b, which is beneficial to osteogenic differentiation, and inhibit the expression of miRNA miR-125b to counteract its negative effects.
  • miRNAs were differentially expressed under different magnetic field strengths of PEMFs. GO terms and KEGG pathways annotated the results of miRNA expression profiles, indicating that miRNAs may be involved in multiple signaling pathways.
  • the object of the present invention is to provide a method for inducing chondrocyte formation from umbilical cord blood mesenchymal stem cells by using a magnetic field, which realizes the problem of inducing umbilical cord blood mesenchymal stem cells to form chondrocytes through pulsed electromagnetic fields.
  • the technical solution adopted in the present invention is a method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes, which is realized through the following steps:
  • umbilical cord blood mesenchymal stem cells Place the umbilical cord blood mesenchymal stem cells on the magnetic hydrogel and place them in an incubator for incubation. After the umbilical cord blood mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, transfer them to the medium to continue culturing , to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells;
  • the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated by pulsed electromagnetic field to obtain chondrocytes.
  • the incubator is a CO2 incubator.
  • the incubation period is 3-6 hours.
  • the time to continue culturing in the medium is 5-10 days.
  • the induction and differentiation time is 20-24 days.
  • the magnetic hydrogel is a magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel
  • the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel is obtained by the following method:
  • the mass ratio of the gelatin to ⁇ -cyclodextrin is 10:(1 ⁇ 5).
  • the stirring temperature is 30-60° C.
  • the stirring time is 30-60 min
  • the stirring speed is 500-800 r/min.
  • the cross-linking agent is transglutaminase; the mass percentage of the CaCl 2 solution is 8-12%.
  • the stirring time is 5-10 min.
  • the umbilical cord blood mesenchymal stem cells are cultured by the following method:
  • the volume ratio of the umbilical cord blood to the PBS buffer solution is (2 ⁇ 4):(4 ⁇ 6).
  • the rotational speed of the centrifugal separation is 1000-1500 r/min, and the time of the centrifugal separation is 8-12 minutes.
  • the volume ratio of the PBS buffer solution added to the umbilical cord blood solution after the first centrifugation is (5-9):10.
  • the rotational speed of the centrifugal washing is 800 ⁇ 1200r/min.
  • the volume ratio of the high-glucose DMEM culture solution to the umbilical cord blood is (2 ⁇ 4):(4 ⁇ 6).
  • the high-sugar DMEM medium contains 8-12% FBS and 0.8-1.2% double antibody.
  • the specific method of S3' is: adding the precipitated cell suspension to high-sugar DMEM culture medium, culturing for 45-50 hours at a temperature of 36-38°C and a carbon dioxide mass fraction of 3-7%, And the high-glucose DMEM culture medium was replaced every 2-3 days to obtain umbilical cord blood mesenchymal stem cells.
  • Fig. 1 is the gelation process of magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel in the embodiment of the present invention
  • Figure 2 is an effect diagram of hUCB-MSCs planted on the surface of the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel material in the embodiment of the present invention
  • Figure 3 is a process diagram of the magnetic nano hydrogel stimulated by pulsed electromagnetic fields loaded with hUCB-MSCs in the embodiment of the present invention
  • Fig. 4 is a comparison diagram of the effect of a constant magnetic field on a magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel material in an embodiment of the present invention
  • Fig. 5 is a histogram of chondrogenic differentiation of hUCB-MSCs after 21 days of pulsed electromagnetic field stimulation in an embodiment of the present invention.
  • the embodiment of the present invention provides a method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes, the method is realized through the following steps:
  • umbilical cord blood mesenchymal stem cells Place the umbilical cord blood mesenchymal stem cells on the magnetic hydrogel, and place them in a CO 2 incubator to incubate for 3-6 hours. After the umbilical cord blood mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, transfer to Continue culturing in the culture medium for 5-10 days to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells;
  • the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated by pulsed electromagnetic field for 20-24 days to obtain chondrocytes.
  • the pulsed electromagnetic field was introduced using the pulsed magnetic therapy produced by Hebei Langfang Biomedical Equipment Co. Instrument, the magnetic therapy method used is time: 20 ⁇ 40min/time/day, magnetic field strength: 80 ⁇ 120T, frequency: 40-70Hz.
  • the above-mentioned magnetic hydrogel is a magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel
  • the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel is obtained by the following method:
  • umbilical cord blood mesenchymal stem cells are cultured by the following method:
  • the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel provided in Example 1 of the present invention is obtained by the following method:
  • the stability of the magnetic hydrogel can be effectively maintained by double cross-linking.
  • the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel provided in Example 2 of the present invention is obtained by the following method:
  • the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel provided in Example 3 of the present invention is obtained by the following method:
  • the umbilical cord blood mesenchymal stem cells provided in Example 4 of the present invention are obtained by culturing by the following method:
  • the umbilical cord blood mesenchymal stem cells provided in Example 5 of the present invention are obtained by culturing by the following method:
  • the umbilical cord blood mesenchymal stem cells provided in Example 6 of the present invention are obtained by culturing by the following method:
  • Example 7 of the present invention provides a magnetic field induced umbilical cord blood mesenchymal stem cells to form chondrocytes obtained by the following method:
  • Example 2 Take the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel prepared in Example 1, and concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4 to a concentration of 1 ⁇ 10 7 /ml concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were placed on the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel, and placed in a CO 2 incubator for incubation for 4 hours.
  • hUCB-MSCs umbilical cord blood mesenchymal stem cells
  • the mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, they are transferred to the culture medium for 7 days to observe the growth state of the cells, and a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells is obtained; by observing under a microscope, As well as cell death and life staining, SEM scans showed that the cells proliferated well. This point of view can be clearly seen from Figure 2.
  • the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated for 21 days by using a pulsed electromagnetic field with a magnetic field strength of 100 mT and a frequency of 40-70 Hz for 30 min/time/day to obtain chondrocytes, specifically As shown in Figure 3.
  • the constant magnetic field has a superparamagnetic effect on the magnetic hydrogel material.
  • the strength of the constant magnetic field is 100mT. This strength has a good superparamagnetic effect on the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel. Specifically shown in Figure 4.
  • Example 8 of the present invention provides a magnetic field induced umbilical cord blood mesenchymal stem cells to form chondrocytes obtained by the following method:
  • Example 2 Take the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel prepared in Example 1, and concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4 to a concentration of 1 ⁇ 10 7 /ml Concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were placed on the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel, and placed in a CO 2 incubator for 3 h incubation.
  • hUCB-MSCs umbilical cord blood mesenchymal stem cells
  • the mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, they are transferred to the culture medium for 5 days to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; the magnetic therapy time is 30min/time/day, A pulsed electromagnetic field with a magnetic field strength of 100 mT and a frequency of 40-70 Hz induced and differentiated the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells for 20 days to obtain chondrocytes.
  • Example 9 of the present invention provides a magnetic field induced umbilical cord blood mesenchymal stem cells to form chondrocytes obtained by the following method:
  • Example 2 Take the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel prepared in Example 1, and concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4 to a concentration of 1 ⁇ 10 7 /ml Concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were placed on the magnetic Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel, and placed in a CO 2 incubator for 6 h incubation.
  • hUCB-MSCs umbilical cord blood mesenchymal stem cells
  • the mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, they are transferred to the culture medium for 10 days to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; the magnetic therapy time is 30min/time/day, A pulsed electromagnetic field with a magnetic field strength of 100 mT and a frequency of 40-70 Hz induced and differentiated the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells for 24 days to obtain chondrocytes.
  • Comparative Example 1 The difference between Comparative Example 1 and Example 7 of the present invention is that the hydrogel used in Comparative Example 1 is a common Gelatin/ ⁇ -CD/Fe 3 O 4 hydrogel, and other steps and parameters are the same.
  • the chondrocytes provided by Comparative Example 2 of the present invention are obtained by the following method:
  • hUCB-MSCs Concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4, and place the concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) at a concentration of 1 ⁇ 10 7 cells/ml in a CO 2 incubator After incubating and culturing in medium for 4 hours, after the umbilical cord blood mesenchymal stem cells grew tightly on the surface of the magnetic hydrogel, they were transferred to the culture medium and continued to culture for 7 days to obtain the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; The treatment time is 30min/time/day, the magnetic field strength is 100mT, and the frequency is 40-70 Hz pulsed electromagnetic field to induce and differentiate the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells for 21 days to obtain chondrocytes (i.e., hUCB -MSCs were cultured alone, and pulsed electromagnetic fields were applied to obtain chond
  • the chondrocytes provided by Comparative Example 3 of the present invention are obtained by the following method:
  • hUCB-MSCs umbilical cord blood mesenchymal stem cells obtained in Example 4, and place the concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) at a concentration of 1 ⁇ 10 7 cells/ml in a CO 2 incubator After incubating and culturing in medium for 4 hours, after the umbilical cord blood mesenchymal stem cells grew tightly on the surface of the magnetic hydrogel, they were transferred to the culture medium and continued to culture for 7 days to obtain the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells (i.e., using Composite of magnetic Gelatin/ ⁇ -CD/Fe3O4 hydrogel with hUCB-MSCs, no pulsed electromagnetic field) comparative example 4
  • Comparative Example 4 of the present invention is mainly an example where common Gelatin/ ⁇ -CD hydrogel is combined with hUCB-MSCs, and no pulsed electromagnetic field is applied.
  • Comparative Example 5 of the present invention is mainly an example in which hUCB-MSCs are cultured alone and a pulsed electromagnetic field is applied.

Abstract

Provided is a method for using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes. The method is implemented by means of the following steps: placing umbilical cord blood mesenchymal stem cells on a magnetic hydrogel, which is then placed in an incubator for incubation; after the umbilical cord blood mesenchymal stem cells grow tightly on a surface of the magnetic hydrogel, transferring to a culture medium to continue culturing, obtaining a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; using a pulsed magnetic field on the magnetic hydrogel loaded with the umbilical cord blood mesenchymal stem cells to perform induction and differentiation, obtaining chondrocytes.

Description

一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法A method for inducing chondrocytes from umbilical cord blood mesenchymal stem cells using a magnetic field 技术领域technical field
本发明属于软骨细胞技术领域，具体涉及一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法。The invention belongs to the technical field of chondrocytes, and in particular relates to a method for inducing umbilical cord blood mesenchymal stem cells to form chondrocytes by using a magnetic field.
背景技术Background technique
脉冲电磁场疗法已被用于治疗包括膝骨关节炎在内的多种疾病。尽管其机理尚不完全清楚，但由于其安全且无创性，它已广泛应用于临床。当前，通过关节注射的MSCs被认为是再生受损软骨的未来治疗方法之一。极低频脉冲电磁场（PEMFs）可以促进MSCs的迁移和增殖，增强MSCs的旁分泌活性。我们小组的早期研究发现，磁性纳米复合水凝胶在改善组织工程方面具有巨大潜力。我们采用超声分散和冻融交联成型法制备磁性纳米复合水凝胶。随后，磁性纳米复合水凝胶对间充质干细胞（MSCs）具有高生物相容性，磁性纳米复合水凝胶的表面显示MSCs均匀生长，并且软骨细胞相关基因的表达上调。利用磁场对磁性材料的响应原理，还发现在脉冲电磁场的作用下，软骨的分化能力可以增强，促进软骨修复。这种磁刺激可以帮助修复体内兔膝关节软骨缺损。越来越多的证据表明，miRNA在控制成骨和软骨形成中起着重要作用，并表明miRNA服务器作为重要的表观遗传调节因子介导的外部因素诱导了细胞表型。例如，脉冲电磁场可以在间充质干细胞的成骨分化过程中调节大量的miRNA。PEMF可以促进miR-26a和miR-29b的表达，这有利于成骨分化，并抑制miRNA的miR-125b的表达以抵消其负面作用。此外，研究还发现，miRNA在PEMFs的不同磁场强度下会差异表达。GO术语和KEGG通路注释了miRNA表达谱的结果，表明miRNA可能参与了多种信号通路。这些结果表明，miRNA作为调节PEMFs介导的信号通路的潜在调控元件，可能在PEMFs的磁性生物学效应中起重要作用。PEMF therapy has been used to treat a variety of conditions, including knee osteoarthritis. Although its mechanism is not fully understood, it has been widely used clinically due to its safety and non-invasiveness. Currently, MSCs injected through joints are considered as one of the future therapeutic approaches to regenerate damaged cartilage. Extremely low frequency pulsed electromagnetic fields (PEMFs) can promote the migration and proliferation of MSCs, and enhance the paracrine activity of MSCs. Earlier studies by our group found that magnetic nanocomposite hydrogels have great potential for improving tissue engineering. We prepared magnetic nanocomposite hydrogels by ultrasonic dispersion and freeze-thaw crosslinking. Subsequently, the magnetic nanocomposite hydrogel exhibited high biocompatibility for mesenchymal stem cells (MSCs), and the surface of the magnetic nanocomposite hydrogel showed uniform growth of MSCs and upregulated expression of chondrocyte-related genes. Using the principle of magnetic field response to magnetic materials, it is also found that under the action of pulsed electromagnetic field, the differentiation ability of cartilage can be enhanced and cartilage repair can be promoted. This magnetic stimulation could help repair articular cartilage defects in rabbit knees in vivo. Accumulating evidence indicates that miRNAs play an important role in the control of osteogenesis and chondrogenesis, and suggests that miRNA servers act as important epigenetic regulators mediated by external factors that induce cellular phenotypes. For example, pulsed electromagnetic fields can regulate a large number of miRNAs during the osteogenic differentiation of mesenchymal stem cells. PEMF can promote the expression of miR-26a and miR-29b, which is beneficial to osteogenic differentiation, and inhibit the expression of miRNA miR-125b to counteract its negative effects. In addition, the study also found that miRNAs were differentially expressed under different magnetic field strengths of PEMFs. GO terms and KEGG pathways annotated the results of miRNA expression profiles, indicating that miRNAs may be involved in multiple signaling pathways. These results suggest that miRNAs, as potential regulatory elements regulating PEMFs-mediated signaling pathways, may play an important role in the magnetobiological effects of PEMFs.
但是，PEMFs刺激MSCs分化为软骨细胞的潜在分子机制仍不清楚。因此，在这项研究中，通过affymetrix芯片分析研究了PEMFs结合磁性纳米复合水凝胶暴露调节涉及间充质干细胞（hBMSCs）分化的miRNA的潜在能力。系统地分析了miRNA表达谱，以阻止miRNA在PEMFs结合磁性纳米复合水凝胶对MSCs软骨细胞分化的贡献中所起的作用和途径。However, the underlying molecular mechanisms by which PEMFs stimulate MSCs to differentiate into chondrocytes remain unclear. Therefore, in this study, the potential ability of PEMFs combined with magnetic nanocomposite hydrogel exposure to regulate miRNAs involved in the differentiation of mesenchymal stem cells (hBMSCs) was investigated by affymetrix chip analysis. The miRNA expression profile was systematically analyzed to block the roles and pathways of miRNAs in the contribution of PEMFs combined with magnetic nanocomposite hydrogels to the chondrocyte differentiation of MSCs.
技术问题technical problem
有鉴于此，本发明的目的在于提供一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，实现了通过脉冲电磁场诱导脐带血间质干细胞形成软骨细胞的问题。In view of this, the object of the present invention is to provide a method for inducing chondrocyte formation from umbilical cord blood mesenchymal stem cells by using a magnetic field, which realizes the problem of inducing umbilical cord blood mesenchymal stem cells to form chondrocytes through pulsed electromagnetic fields.
技术解决方案technical solution
为了解决上述问题，本发明所采用的技术方案是，一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，该方法通过以下步骤实现：In order to solve the above problems, the technical solution adopted in the present invention is a method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes, which is realized through the following steps:
将脐带血间充质干细胞放置于磁性水凝胶上，并放置于培养箱中孵育培养，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养，得到负载有脐带血间充质干细胞的磁性水凝胶；Place the umbilical cord blood mesenchymal stem cells on the magnetic hydrogel and place them in an incubator for incubation. After the umbilical cord blood mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, transfer them to the medium to continue culturing , to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells;
采用脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化，得到软骨细胞。The magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated by pulsed electromagnetic field to obtain chondrocytes.
优选地，所述培养箱为CO 2培养箱。 Preferably, the incubator is a CO2 incubator.
优选地，所述孵育培养的时间为3~6h。Preferably, the incubation period is 3-6 hours.
优选地，在所述培养基中继续培养的时间为5~10d。Preferably, the time to continue culturing in the medium is 5-10 days.
优选地，所述诱导和分化的时间为20~24d。Preferably, the induction and differentiation time is 20-24 days.
优选地，所述磁性水凝胶为磁性Gelatin/β-CD/Fe 3O 4水凝胶，所述磁性Gelatin/β-CD/Fe 3O 4水凝胶是通过以下方法得到： Preferably, the magnetic hydrogel is a magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel, and the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel is obtained by the following method:
S1、称取一定比例的明胶和β-环糊精混合均匀，并加入超纯水进行搅拌，再加入交联剂和CaCl 2溶液进行交联，得到水凝胶前体溶液； S1. Weigh a certain proportion of gelatin and β-cyclodextrin and mix evenly, add ultrapure water for stirring, then add a crosslinking agent and CaCl2 solution for crosslinking to obtain a hydrogel precursor solution;
S2、在搅拌的状态下，向所述水凝胶前体溶液中加入磁性纳米粒子并搅拌，得到磁性纳米水凝胶前体溶液；S2. In the state of stirring, add magnetic nanoparticles to the hydrogel precursor solution and stir to obtain a magnetic nano-hydrogel precursor solution;
S3、对所述磁性纳米水凝胶前体溶液进行冷冻干燥，并置于蒸馏水中浸泡1~3d，得到磁性Gelatin/β-CD/Fe 3O 4水凝胶。 S3. Freeze-drying the magnetic nano hydrogel precursor solution, and immersing in distilled water for 1-3 days to obtain a magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel.
优选地，所述S1中，所述明胶和β-环糊精的质量比为10:（1~5）。Preferably, in the S1, the mass ratio of the gelatin to β-cyclodextrin is 10:(1~5).
优选地，所述S1中，所述搅拌的温度为30~60℃，所述搅拌的时间为30~60min，所述搅拌的速度为500~800r/min。Preferably, in the S1, the stirring temperature is 30-60° C., the stirring time is 30-60 min, and the stirring speed is 500-800 r/min.
优选地，所述S1中，所述交联剂为转谷氨酰胺酶；所述CaCl 2溶液的质量百分数为8~12%。 Preferably, in the S1, the cross-linking agent is transglutaminase; the mass percentage of the CaCl 2 solution is 8-12%.
优选地，所述S2中，所述搅拌的时间为5~10min。Preferably, in the S2, the stirring time is 5-10 min.
优选地，所述脐带血间充质干细胞是通过以下方法培养得到：Preferably, the umbilical cord blood mesenchymal stem cells are cultured by the following method:
S1′、量取一定比例的PBS缓冲溶液和脐带血，并将其混合进行离心分离，弃上清液，获得一次离心后的脐带血溶液；S1′. Measure a certain proportion of PBS buffer solution and umbilical cord blood, mix them for centrifugation, discard the supernatant, and obtain the umbilical cord blood solution after one centrifugation;
S2′、向所述一次离心后的脐带血溶液中再加入PBS缓冲溶液并进行离心洗涤，弃上清液，获得沉淀的细胞悬液；S2', adding PBS buffer solution to the umbilical cord blood solution after the first centrifugation, performing centrifugation and washing, discarding the supernatant to obtain a precipitated cell suspension;
S3′、将沉淀的细胞悬液加入至高糖DMEM培养液中，并在二氧化碳的条件下进行培养，得到脐带血间充质干细胞。S3', adding the precipitated cell suspension into high-glucose DMEM culture medium, and culturing under the condition of carbon dioxide to obtain umbilical cord blood mesenchymal stem cells.
优选地，所述S1′中，所述脐带血与PBS缓冲溶液的体积比为（2~4）:（4~6）。Preferably, in the S1′, the volume ratio of the umbilical cord blood to the PBS buffer solution is (2~4):(4~6).
优选地，所述S1′中，所述离心分离的转速为1000~1500r/min，所述离心分离的时间为8~12min。Preferably, in the S1′, the rotational speed of the centrifugal separation is 1000-1500 r/min, and the time of the centrifugal separation is 8-12 minutes.
优选地，所述S2′中，加入所述PBS缓冲溶液与所述一次离心后的脐带血溶液的体积比为（5~9）:10。Preferably, in the S2′, the volume ratio of the PBS buffer solution added to the umbilical cord blood solution after the first centrifugation is (5-9):10.
优选地，所述S2′中，所述离心洗涤的转速为800~1200r/min。Preferably, in the S2′, the rotational speed of the centrifugal washing is 800~1200r/min.
优选地，所述S3′中，所述高糖DMEM培养液与脐带血的体积比为（2~4）:（4~6）。Preferably, in the S3′, the volume ratio of the high-glucose DMEM culture solution to the umbilical cord blood is (2~4):(4~6).
优选地，所述S3′中，所述高糖DMEM培养液中含有8~12%的FBS和0.8~1.2%的双抗。Preferably, in the S3′, the high-sugar DMEM medium contains 8-12% FBS and 0.8-1.2% double antibody.
优选地，所述S3′的具体方法为：将沉淀的细胞悬液加入至高糖DMEM培养液中，在温度为36~38℃、二氧化碳质量分数为3~7%的条件下培养45~50h，且每2-3d更换一次高糖DMEM培养液，得到脐带血间充质干细胞。Preferably, the specific method of S3' is: adding the precipitated cell suspension to high-sugar DMEM culture medium, culturing for 45-50 hours at a temperature of 36-38°C and a carbon dioxide mass fraction of 3-7%, And the high-glucose DMEM culture medium was replaced every 2-3 days to obtain umbilical cord blood mesenchymal stem cells.
有益效果Beneficial effect
与现有技术相比，通过采用本发明采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，不仅有效的实现了获得了软骨细胞的目的，而且还解决了脉冲电磁场刺激脐带血间充质干细胞分化为软骨细胞的潜在分子机制仍不清楚的问题。Compared with the prior art, by adopting the method of the present invention to induce umbilical cord blood mesenchymal stem cells to form chondrocytes with a magnetic field, not only the purpose of obtaining chondrocytes is effectively achieved, but also the problem of pulsed electromagnetic field stimulation of umbilical cord blood mesenchymal stem cells is solved. The molecular mechanisms underlying the differentiation into chondrocytes remain a matter of unclear understanding.
附图说明Description of drawings
图1为本发明实施例中磁性Gelatin/β-CD/Fe 3O 4水凝胶成胶过程； Fig. 1 is the gelation process of magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel in the embodiment of the present invention;
图2为本发明实施例中hUCB-MSCs种植在磁性Gelatin/β-CD/Fe 3O 4水凝胶材料表面上的效果图； Figure 2 is an effect diagram of hUCB-MSCs planted on the surface of the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel material in the embodiment of the present invention;
图3为本发明实施例中脉冲电磁场刺激负载hUCB-MSCs的磁性纳米水凝胶过程图；Figure 3 is a process diagram of the magnetic nano hydrogel stimulated by pulsed electromagnetic fields loaded with hUCB-MSCs in the embodiment of the present invention;
图4为本发明实施例中恒定磁场对磁性Gelatin/β-CD/Fe 3O 4水凝胶材料的效果对比图； Fig. 4 is a comparison diagram of the effect of a constant magnetic field on a magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel material in an embodiment of the present invention;
图5为本发明实施例中脉冲电磁场刺激21天后hUCB-MSCs成软骨分化后的柱状图。Fig. 5 is a histogram of chondrogenic differentiation of hUCB-MSCs after 21 days of pulsed electromagnetic field stimulation in an embodiment of the present invention.
本发明的最佳实施方式BEST MODE FOR CARRYING OUT THE INVENTION
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
需要说明，以下实施例采用的所有组分均可以通过购买或自制得到。It should be noted that all components used in the following examples can be purchased or obtained by self-made.
本发明实施例提供的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，该方法通过以下步骤实现：The embodiment of the present invention provides a method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes, the method is realized through the following steps:
将脐带血间充质干细胞放置于磁性水凝胶上，并放置于CO 2培养箱中孵育培养3~6h，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养5~10d，得到负载有脐带血间充质干细胞的磁性水凝胶； Place the umbilical cord blood mesenchymal stem cells on the magnetic hydrogel, and place them in a CO 2 incubator to incubate for 3-6 hours. After the umbilical cord blood mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, transfer to Continue culturing in the culture medium for 5-10 days to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells;
采用脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化20~24d，得到软骨细胞，其中，脉冲电磁场的引入采用的是河北廊坊生物医疗器械有限公司生产的脉冲磁疗仪，采用的磁疗方式是时间：20~40min/次/天，磁场强度：80~120T，频率：40-70Hz。The magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated by pulsed electromagnetic field for 20-24 days to obtain chondrocytes. The pulsed electromagnetic field was introduced using the pulsed magnetic therapy produced by Hebei Langfang Biomedical Equipment Co. Instrument, the magnetic therapy method used is time: 20~40min/time/day, magnetic field strength: 80~120T, frequency: 40-70Hz.
进一步地，上述磁性水凝胶为磁性Gelatin/β-CD/Fe 3O 4水凝胶，磁性Gelatin/β-CD/Fe 3O 4水凝胶是通过以下方法得到： Further, the above-mentioned magnetic hydrogel is a magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel, and the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel is obtained by the following method:
S1、按照明胶和β-环糊精的质量比为10:（1~5）的比例称取明胶和β-环糊精并混合均匀，并加入超纯水，在温度为30~60℃、搅拌速度为500~800r/min的条件下搅拌30~60min，再加入交联剂转谷氨酰胺酶和质量百分数为8~12%的CaCl 2溶液进行交联，得到水凝胶前体溶液； S1. Weigh gelatin and β-cyclodextrin according to the mass ratio of gelatin and β-cyclodextrin as 10: (1~5), mix them evenly, add ultra-pure water, and put them under temperature of 30~60℃, Stirring at a stirring speed of 500-800 r/min for 30-60 min, then adding a cross-linking agent transglutaminase and a CaCl solution with a mass percentage of 8-12% for cross-linking to obtain a hydrogel precursor solution;
S2、在搅拌的状态下，向所述水凝胶前体溶液中加入磁性纳米粒子并搅拌5~10min，得到磁性纳米水凝胶前体溶液；S2. In the state of stirring, add magnetic nanoparticles to the hydrogel precursor solution and stir for 5-10min to obtain a magnetic nano-hydrogel precursor solution;
S3、对所述磁性纳米水凝胶前体溶液进行冷冻干燥，并置于蒸馏水中浸泡1~3d，得到磁性Gelatin/β-CD/Fe 3O 4水凝胶。 S3. Freeze-drying the magnetic nano hydrogel precursor solution, and immersing in distilled water for 1-3 days to obtain a magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel.
进一步地，脐带血间充质干细胞是通过以下方法培养得到：Further, the umbilical cord blood mesenchymal stem cells are cultured by the following method:
S1′、按照脐带血与PBS缓冲溶液的体积比为（2~4）:（4~6）的比例量取PBS缓冲溶液和脐带血，将两者混合并在转速为1000~1500r/min的条件下离心分离8~12min，弃上清液，获得一次离心后的脐带血溶液；S1′, according to the volume ratio of cord blood to PBS buffer solution (2~4): (4~6), measure PBS buffer solution and cord blood, mix them and put them in a rotating speed of 1000~1500r/min Centrifuge for 8-12 minutes under the same conditions, discard the supernatant, and obtain the umbilical cord blood solution after one centrifugation;
S2′、按照PBS缓冲溶液与一次离心后的脐带血溶液的体积比为（5~9）:10的比例向一次离心后的脐带血溶液中再加入PBS缓冲溶液，并在转速为800~1200r/min的条件下离心洗涤，弃上清液，获得沉淀的细胞悬液；S2′. Add PBS buffer solution to the cord blood solution after the first centrifugation according to the ratio of the volume ratio of PBS buffer solution to the cord blood solution after the first centrifugation (5~9):10, and rotate at a speed of 800~1200r Under the condition of centrifugation/min, discard the supernatant to obtain the precipitated cell suspension;
S3′、将沉淀的细胞悬液加入至含有8~12%的FBS和0.8~1.2%的双抗的高糖DMEM培养液中，在温度为36~38℃、二氧化碳质量分数为3~7%的条件下培养45~50h，且每2-3d更换一次高糖DMEM培养液，得到脐带血间充质干细胞，其中，所述高糖DMEM培养液与脐带血的体积比为（2~4）:（4~6）。S3′. Add the precipitated cell suspension to the high-glucose DMEM culture medium containing 8-12% FBS and 0.8-1.2% double antibody, at a temperature of 36-38°C and a carbon dioxide mass fraction of 3-7%. Cultivate for 45-50 hours under certain conditions, and replace the high-glucose DMEM medium every 2-3 days to obtain umbilical cord blood mesenchymal stem cells, wherein the volume ratio of the high-glucose DMEM medium to cord blood is (2-4) : (4~6).
以下为具体实施例The following are specific examples
实施列1Implementation column 1
本发明实施例1提供的磁性Gelatin/β-CD/Fe 3O 4水凝胶是通过以下方法得到： The magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel provided in Example 1 of the present invention is obtained by the following method:
按照明胶和β-环糊精的质量比为10:5的比例称取明胶和β-环糊精并混合均匀，并加入超纯水，在温度为40℃、搅拌速度为600r/min的条件下搅拌30~60min，再加入交联剂转谷氨酰胺酶（mTG酶）和质量百分数为10%的CaCl 2溶液进行交联，得到水凝胶前体溶液；在搅拌的状态下，向所述水凝胶前体溶液中加入磁性纳米粒子并搅拌5~10min，得到磁性纳米水凝胶前体溶液；对所述磁性纳米水凝胶前体溶液进行冷冻干燥，并置于蒸馏水中浸泡1d，得到磁性Gelatin/β-CD/Fe 3O 4水凝胶。 Weigh gelatin and β-cyclodextrin according to the ratio of gelatin and β-cyclodextrin mass ratio of 10:5 and mix evenly, and add ultrapure water, under the conditions of temperature 40°C and stirring speed 600r/min Stir for 30-60min, then add cross-linking agent transglutaminase (mTG enzyme) and 10% CaCl solution for cross-linking to obtain hydrogel precursor solution; Adding magnetic nanoparticles to the hydrogel precursor solution and stirring for 5-10min to obtain a magnetic nano-hydrogel precursor solution; freeze-drying the magnetic nano-hydrogel precursor solution, and soaking in distilled water for 1d , to obtain magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel.
通过双交联作用可有效保持磁性水凝胶的稳定性。The stability of the magnetic hydrogel can be effectively maintained by double cross-linking.
此外，将制备好的磁性Gelatin/β-CD/Fe 3O 4水凝胶置于蒸馏水中浸泡1天，除去未交联的单体，通过超声分散、冷冻干燥成功制备实验用的磁性Gelatin/β-CD/Fe 3O 4水凝胶，关于这一观点，从图1中可以明确看出。 In addition, the prepared magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel was soaked in distilled water for 1 day to remove uncrosslinked monomers, and the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel for experiments was successfully prepared by ultrasonic dispersion and freeze-drying. β-CD/Fe 3 O 4 hydrogel, regarding this point, can be seen clearly from Fig. 1.
实施例2Example 2
本发明实施例2提供的磁性Gelatin/β-CD/Fe 3O 4水凝胶是通过以下方法得到： The magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel provided in Example 2 of the present invention is obtained by the following method:
按照明胶和β-环糊精的质量比为10:1的比例称取明胶和β-环糊精并混合均匀，并加入超纯水，在温度为30℃、搅拌速度为500r/min的条件下搅拌30~60min，再加入交联剂转谷氨酰胺酶（mTG酶）和质量百分数为8%的CaCl 2溶液进行交联，得到水凝胶前体溶液；在搅拌的状态下，向所述水凝胶前体溶液中加入磁性纳米粒子并搅拌5~10min，得到磁性纳米水凝胶前体溶液；对所述磁性纳米水凝胶前体溶液进行冷冻干燥，并置于蒸馏水中浸泡1d，得到磁性Gelatin/β-CD/Fe 3O 4水凝胶。 Weigh gelatin and β-cyclodextrin according to the ratio of gelatin and β-cyclodextrin mass ratio of 10:1 and mix evenly, and add ultrapure water, under the conditions of temperature 30°C and stirring speed 500r/min Stir for 30-60min, then add the cross-linking agent transglutaminase (mTG enzyme) and 8% CaCl solution for cross-linking to obtain the hydrogel precursor solution; Adding magnetic nanoparticles to the hydrogel precursor solution and stirring for 5-10min to obtain a magnetic nano-hydrogel precursor solution; freeze-drying the magnetic nano-hydrogel precursor solution, and soaking in distilled water for 1d , to obtain magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel.
实施例3Example 3
本发明实施例3提供的磁性Gelatin/β-CD/Fe 3O 4水凝胶是通过以下方法得到： The magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel provided in Example 3 of the present invention is obtained by the following method:
按照明胶和β-环糊精的质量比为10:3的比例称取明胶和β-环糊精并混合均匀，并加入超纯水，在温度为60℃、搅拌速度为800r/min的条件下搅拌30~60min，再加入交联剂转谷氨酰胺酶（mTG酶）和质量百分数为12%的CaCl 2溶液进行交联，得到水凝胶前体溶液；在搅拌的状态下，向所述水凝胶前体溶液中加入磁性纳米粒子并搅拌5~10min，得到磁性纳米水凝胶前体溶液；对所述磁性纳米水凝胶前体溶液进行冷冻干燥，并置于蒸馏水中浸泡3d，得到磁性Gelatin/β-CD/Fe 3O 4水凝胶。 Weigh gelatin and β-cyclodextrin according to the ratio of gelatin and β-cyclodextrin mass ratio of 10:3 and mix evenly, and add ultrapure water, under the conditions of temperature 60°C and stirring speed 800r/min Stir for 30-60min, then add cross-linking agent transglutaminase (mTG enzyme) and 12% CaCl solution for cross-linking to obtain hydrogel precursor solution; Add magnetic nanoparticles to the hydrogel precursor solution and stir for 5-10 minutes to obtain a magnetic nano-hydrogel precursor solution; freeze-dry the magnetic nano-hydrogel precursor solution, and soak in distilled water for 3 days , to obtain magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel.
实施例4Example 4
本发明实施例4提供的脐带血间充质干细胞是通过以下方法培养得到：The umbilical cord blood mesenchymal stem cells provided in Example 4 of the present invention are obtained by culturing by the following method:
量取3ml的脐带血和5ml的PBS缓冲溶液，将两者混合并在转速为1200r/min的条件下离心分离10min，弃上清液，获得一次离心后的脐带血溶液；按照PBS缓冲溶液与一次离心后的脐带血溶液的体积比为9:10的比例向一次离心后的脐带血溶液中再加入PBS缓冲溶液，并在转速为1000r/min的条件下离心洗涤2次，弃上清液，获得沉淀的细胞悬液；将沉淀的细胞悬液加入至5ml含有10%的FBS和1.0%的双抗的高糖DMEM培养液中，在温度为37℃、二氧化碳质量分数为5%的条件下培养48h，且每2-3d更换一次高糖DMEM培养液，得到脐带血间充质干细胞。Measure 3ml of umbilical cord blood and 5ml of PBS buffer solution, mix the two and centrifuge at a speed of 1200r/min for 10min, discard the supernatant, and obtain the umbilical cord blood solution after one centrifugation; The volume ratio of the umbilical cord blood solution after the first centrifugation is 9:10, then add PBS buffer solution to the umbilical cord blood solution after the first centrifugation, and centrifuge and wash twice at the speed of 1000r/min, discard the supernatant , to obtain a precipitated cell suspension; add the precipitated cell suspension to 5ml high-glucose DMEM culture medium containing 10% FBS and 1.0% double antibody, at a temperature of 37°C and a carbon dioxide mass fraction of 5%. The umbilical cord blood mesenchymal stem cells were cultured for 48 hours, and the high-glucose DMEM medium was replaced every 2-3 days.
实施例5Example 5
本发明实施例5提供的脐带血间充质干细胞是通过以下方法培养得到：The umbilical cord blood mesenchymal stem cells provided in Example 5 of the present invention are obtained by culturing by the following method:
量取2ml的脐带血和4ml的PBS缓冲溶液，将两者混合并在转速为1000r/min的条件下离心分离8min，弃上清液，获得一次离心后的脐带血溶液；按照PBS缓冲溶液与一次离心后的脐带血溶液的体积比为1:2的比例向一次离心后的脐带血溶液中再加入PBS缓冲溶液，并在转速为800r/min的条件下离心洗涤2次，弃上清液，获得沉淀的细胞悬液；将沉淀的细胞悬液加入至4ml含有8%的FBS和0.8%的双抗的高糖DMEM培养液中，在温度为36℃、二氧化碳质量分数为3%的条件下培养45h，且每2-3d更换一次高糖DMEM培养液，得到脐带血间充质干细胞。Measure 2ml of umbilical cord blood and 4ml of PBS buffer solution, mix the two and centrifuge at a speed of 1000r/min for 8min, discard the supernatant, and obtain the umbilical cord blood solution after one centrifugation; The volume ratio of the umbilical cord blood solution after the first centrifugation is 1:2, then add PBS buffer solution to the umbilical cord blood solution after the first centrifugation, and centrifuge and wash twice at the speed of 800r/min, discard the supernatant , to obtain a precipitated cell suspension; add the precipitated cell suspension to 4ml of high-glucose DMEM culture medium containing 8% FBS and 0.8% double antibody, at a temperature of 36°C and a carbon dioxide mass fraction of 3%. The umbilical cord blood mesenchymal stem cells were cultured for 45 hours, and the high-glucose DMEM medium was replaced every 2-3 days.
实施例6Example 6
本发明实施例6提供的脐带血间充质干细胞是通过以下方法培养得到：The umbilical cord blood mesenchymal stem cells provided in Example 6 of the present invention are obtained by culturing by the following method:
量取4ml的脐带血和6ml的PBS缓冲溶液，将两者混合并在转速为1500r/min的条件下离心分离12min，弃上清液，获得一次离心后的脐带血溶液；按照PBS缓冲溶液与一次离心后的脐带血溶液的体积比为7:10的比例向一次离心后的脐带血溶液中再加入PBS缓冲溶液，并在转速为1200r/min的条件下离心洗涤，弃上清液，获得沉淀的细胞悬液；将沉淀的细胞悬液加入至6ml含有12%的FBS和1.2%的双抗的高糖DMEM培养液中，在温度为38℃、二氧化碳质量分数为7%的条件下培养50h，且每2-3d更换一次高糖DMEM培养液，得到脐带血间充质干细胞。Measure 4ml of umbilical cord blood and 6ml of PBS buffer solution, mix the two and centrifuge at a speed of 1500r/min for 12min, discard the supernatant, and obtain the umbilical cord blood solution after one centrifugation; The volume ratio of the umbilical cord blood solution after the first centrifugation is 7:10. Add PBS buffer solution to the umbilical cord blood solution after the first centrifugation, and centrifuge and wash at a speed of 1200r/min, discard the supernatant, and obtain Precipitated cell suspension; add the precipitated cell suspension to 6ml of high-glucose DMEM culture medium containing 12% FBS and 1.2% double antibody, culture at a temperature of 38°C and a mass fraction of carbon dioxide of 7% 50h, and the high-glucose DMEM medium was replaced every 2-3d to obtain umbilical cord blood mesenchymal stem cells.
实施例7Example 7
本发明实施例7提供的一种采用磁场诱导脐带血间质干细胞形成软骨细胞是通过如下方法获得的：Example 7 of the present invention provides a magnetic field induced umbilical cord blood mesenchymal stem cells to form chondrocytes obtained by the following method:
取实施例1制备好的磁性Gelatin/β-CD/Fe 3O 4水凝胶，对实施例4获得的脐带血间充质干细胞（hUCB-MSCs）进行浓缩，将浓度为1×10 7个/ml浓缩后的脐带血间充质干细胞（hUCB-MSCs）放置于磁性Gelatin/β-CD/Fe 3O 4水凝胶上，并放置于CO 2培养箱中孵育培养4h，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养7d，观察细胞生长状态，得到负载有脐带血间充质干细胞的磁性水凝胶；通过在显微镜下观察，以及细胞死活染色，SEM扫描后，均显示细胞增殖良好，关于这一观点，从图2中可以明确看出。 Take the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel prepared in Example 1, and concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4 to a concentration of 1×10 7 /ml concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were placed on the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel, and placed in a CO 2 incubator for incubation for 4 hours. After the mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, they are transferred to the culture medium for 7 days to observe the growth state of the cells, and a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells is obtained; by observing under a microscope, As well as cell death and life staining, SEM scans showed that the cells proliferated well. This point of view can be clearly seen from Figure 2.
采用磁疗时间为30min/次/天，磁场强度为100mT，频率为40-70 Hz的脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化21d，得到软骨细胞，具体如图3所示。The magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated for 21 days by using a pulsed electromagnetic field with a magnetic field strength of 100 mT and a frequency of 40-70 Hz for 30 min/time/day to obtain chondrocytes, specifically As shown in Figure 3.
恒定磁场对磁性水凝胶材料具有一个超顺磁性的作用，恒定磁场作用的强度为100mT，这个强度对磁性Gelatin/β-CD/Fe 3O 4水凝胶具有较好的超顺磁性作用，具体如图4所示。 The constant magnetic field has a superparamagnetic effect on the magnetic hydrogel material. The strength of the constant magnetic field is 100mT. This strength has a good superparamagnetic effect on the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel. Specifically shown in Figure 4.
实施例8Example 8
本发明实施例8提供的一种采用磁场诱导脐带血间质干细胞形成软骨细胞是通过如下方法获得的：Example 8 of the present invention provides a magnetic field induced umbilical cord blood mesenchymal stem cells to form chondrocytes obtained by the following method:
取实施例1制备好的磁性Gelatin/β-CD/Fe 3O 4水凝胶，对实施例4获得的脐带血间充质干细胞（hUCB-MSCs）进行浓缩，将浓度为1×10 7个/ml浓缩后的脐带血间充质干细胞（hUCB-MSCs）放置于磁性Gelatin/β-CD/Fe 3O 4水凝胶上，并放置于CO 2培养箱中孵育培养3h，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养5d，得到负载有脐带血间充质干细胞的磁性水凝胶；采用磁疗时间为30min/次/天，磁场强度为100mT，频率为40-70 Hz的脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化20d，得到软骨细胞。 Take the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel prepared in Example 1, and concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4 to a concentration of 1×10 7 /ml Concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were placed on the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel, and placed in a CO 2 incubator for 3 h incubation. After the mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, they are transferred to the culture medium for 5 days to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; the magnetic therapy time is 30min/time/day, A pulsed electromagnetic field with a magnetic field strength of 100 mT and a frequency of 40-70 Hz induced and differentiated the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells for 20 days to obtain chondrocytes.
实施例9Example 9
本发明实施例9提供的一种采用磁场诱导脐带血间质干细胞形成软骨细胞是通过如下方法获得的：Example 9 of the present invention provides a magnetic field induced umbilical cord blood mesenchymal stem cells to form chondrocytes obtained by the following method:
取实施例1制备好的磁性Gelatin/β-CD/Fe 3O 4水凝胶，对实施例4获得的脐带血间充质干细胞（hUCB-MSCs）进行浓缩，将浓度为1×10 7个/ml浓缩后的脐带血间充质干细胞（hUCB-MSCs）放置于磁性Gelatin/β-CD/Fe 3O 4水凝胶上，并放置于CO 2培养箱中孵育培养6h，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养10d，得到负载有脐带血间充质干细胞的磁性水凝胶；采用磁疗时间为30min/次/天，磁场强度为100mT，频率为40-70 Hz的脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化24d，得到软骨细胞。 Take the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel prepared in Example 1, and concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4 to a concentration of 1×10 7 /ml Concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were placed on the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel, and placed in a CO 2 incubator for 6 h incubation. After the mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, they are transferred to the culture medium for 10 days to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; the magnetic therapy time is 30min/time/day, A pulsed electromagnetic field with a magnetic field strength of 100 mT and a frequency of 40-70 Hz induced and differentiated the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells for 24 days to obtain chondrocytes.
对比例1Comparative example 1
本发明对比例1与实施例7的区别在于：对比例1中所采用的水凝胶为普通Gelatin/β-CD/Fe 3O 4水凝胶，其他步骤和参数均相同。 The difference between Comparative Example 1 and Example 7 of the present invention is that the hydrogel used in Comparative Example 1 is a common Gelatin/β-CD/Fe 3 O 4 hydrogel, and other steps and parameters are the same.
对比例2Comparative example 2
本发明对比例2提供的软骨细胞是通过如下方法获得：The chondrocytes provided by Comparative Example 2 of the present invention are obtained by the following method:
对实施例4获得的脐带血间充质干细胞（hUCB-MSCs）进行浓缩，将浓度为1×10 7个/ml浓缩后的脐带血间充质干细胞（hUCB-MSCs）放置于CO 2培养箱中孵育培养4h，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养7d，得到负载有脐带血间充质干细胞的磁性水凝胶；采用磁疗时间为30min/次/天，磁场强度为100mT，频率为40-70 Hz的脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化21d，得到软骨细胞（即采用hUCB-MSCs单独培养，外加脉冲电磁场来获得软骨细胞）。 Concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4, and place the concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) at a concentration of 1×10 7 cells/ml in a CO 2 incubator After incubating and culturing in medium for 4 hours, after the umbilical cord blood mesenchymal stem cells grew tightly on the surface of the magnetic hydrogel, they were transferred to the culture medium and continued to culture for 7 days to obtain the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells; The treatment time is 30min/time/day, the magnetic field strength is 100mT, and the frequency is 40-70 Hz pulsed electromagnetic field to induce and differentiate the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells for 21 days to obtain chondrocytes (i.e., hUCB -MSCs were cultured alone, and pulsed electromagnetic fields were applied to obtain chondrocytes).
对比例3Comparative example 3
本发明对比例3提供的软骨细胞是通过如下方法获得：The chondrocytes provided by Comparative Example 3 of the present invention are obtained by the following method:
对实施例4获得的脐带血间充质干细胞（hUCB-MSCs）进行浓缩，将浓度为1×10 7个/ml浓缩后的脐带血间充质干细胞（hUCB-MSCs）放置于CO 2培养箱中孵育培养4h，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养7d，得到负载有脐带血间充质干细胞的磁性水凝胶（即采用磁性Gelatin/β-CD/Fe3O4水凝胶与hUCB-MSCs复合，未加脉冲电磁场）对比例4 Concentrate the umbilical cord blood mesenchymal stem cells (hUCB-MSCs) obtained in Example 4, and place the concentrated umbilical cord blood mesenchymal stem cells (hUCB-MSCs) at a concentration of 1×10 7 cells/ml in a CO 2 incubator After incubating and culturing in medium for 4 hours, after the umbilical cord blood mesenchymal stem cells grew tightly on the surface of the magnetic hydrogel, they were transferred to the culture medium and continued to culture for 7 days to obtain the magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells (i.e., using Composite of magnetic Gelatin/β-CD/Fe3O4 hydrogel with hUCB-MSCs, no pulsed electromagnetic field) comparative example 4
本发明对比例4主要是采用普通Gelatin/ β-CD水凝胶与hUCB-MSCs复合，且在未加脉冲电磁场的实例。Comparative Example 4 of the present invention is mainly an example where common Gelatin/β-CD hydrogel is combined with hUCB-MSCs, and no pulsed electromagnetic field is applied.
本对比例5This comparative example 5
本发明对比例5主要是采用hUCB-MSCs单独培养，外加脉冲电磁场的实例。Comparative Example 5 of the present invention is mainly an example in which hUCB-MSCs are cultured alone and a pulsed electromagnetic field is applied.
为了验证通过采用本发明方法获得的软骨细胞，现对本发明实施例7获得的软骨细胞以及对比例1-5获得的细胞的性能进行测试，具体测试结果如下图5所示：In order to verify the chondrocytes obtained by using the method of the present invention, the performance of the chondrocytes obtained in Example 7 of the present invention and the cells obtained in Comparative Examples 1-5 are now tested, and the specific test results are shown in Figure 5 below:
从图5中可知，经过脉冲电磁场刺激21天后hUCB-MSCs成软骨分化后的软骨标志物形成情况通过QPCR的检测结果显示A组：脉冲电磁场联合磁性水凝胶对hUCB-MSCs的成软骨分化作用最为显著。It can be seen from Figure 5 that after 21 days of pulsed electromagnetic field stimulation, the formation of cartilage markers in hUCB-MSCs after chondrogenic differentiation was detected by QPCR. Group A: the chondrogenic differentiation effect of pulsed electromagnetic field combined with magnetic hydrogel on hUCB-MSCs most notably.
综上所述，通过采用本发明采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，不仅有效的实现了获得了软骨细胞的目的，而且还解决了脉冲电磁场刺激脐带血间充质干细胞分化为软骨细胞的潜在分子机制仍不清楚的问题。In summary, by adopting the method of the present invention to induce umbilical cord blood mesenchymal stem cells to form chondrocytes with a magnetic field, not only the purpose of obtaining chondrocytes is effectively achieved, but also the problem of pulsed electromagnetic fields stimulating umbilical cord blood mesenchymal stem cells to differentiate into The underlying molecular mechanisms of chondrocytes remain unclear issues.
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (17)

  1. 一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，该方法通过以下步骤实现：A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes, characterized in that the method is realized through the following steps:
    将脐带血间充质干细胞放置于磁性水凝胶上，并放置于培养箱中孵育培养，待脐带血间充质干细胞在磁性水凝胶的表面贴紧生长后，转至培养基中继续培养，得到负载有脐带血间充质干细胞的磁性水凝胶；Place the umbilical cord blood mesenchymal stem cells on the magnetic hydrogel and place them in an incubator for incubation. After the umbilical cord blood mesenchymal stem cells grow tightly on the surface of the magnetic hydrogel, transfer them to the medium to continue culturing , to obtain a magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells;
    采用脉冲电磁场对负载有脐带血间充质干细胞的磁性水凝胶进行诱导和分化，得到软骨细胞。The magnetic hydrogel loaded with umbilical cord blood mesenchymal stem cells was induced and differentiated by pulsed electromagnetic field to obtain chondrocytes.
  2. 根据权利要求1所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述培养箱为CO 2培养箱。 A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 1, wherein the incubator is a CO incubator .
  3. 根据权利要求2所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述孵育培养的时间为3~6h。A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 2, wherein the incubation time is 3 to 6 hours.
  4. 根据权利要求3所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，在所述培养基中继续培养的时间为5~10d。A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 3, wherein the time for continuing to culture in the medium is 5 to 10 days.
  5. 根据权利要求4所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述诱导和分化的时间为20~24d。A method for inducing chondrocyte formation from umbilical cord blood mesenchymal stem cells by using a magnetic field according to claim 4, wherein the induction and differentiation time is 20 to 24 days.
  6. 根据权利要求1-5任意一项所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述磁性水凝胶为磁性Gelatin/β-CD/Fe 3O 4水凝胶，所述磁性Gelatin/β-CD/Fe 3O 4水凝胶是通过以下方法得到： A method of using a magnetic field to induce chondrocytes from umbilical cord blood mesenchymal stem cells according to any one of claims 1-5, wherein the magnetic hydrogel is magnetic Gelatin/β-CD/Fe 3 O 4 Hydrogel, the magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel is obtained by the following method:
    S1、称取一定比例的明胶和β-环糊精混合均匀，并加入超纯水进行搅拌，再加入交联剂和CaCl 2溶液进行交联，得到水凝胶前体溶液； S1. Weigh a certain proportion of gelatin and β-cyclodextrin and mix evenly, add ultrapure water for stirring, then add a crosslinking agent and CaCl2 solution for crosslinking to obtain a hydrogel precursor solution;
    S2、在搅拌的状态下，向所述水凝胶前体溶液中加入磁性纳米粒子并搅拌，得到磁性纳米水凝胶前体溶液；S2. In the state of stirring, add magnetic nanoparticles to the hydrogel precursor solution and stir to obtain a magnetic nano-hydrogel precursor solution;
    S3、对所述磁性纳米水凝胶前体溶液进行冷冻干燥，并置于蒸馏水中浸泡1~3d，得到磁性Gelatin/β-CD/Fe 3O 4水凝胶。 S3. Freeze-drying the magnetic nano hydrogel precursor solution, and immersing in distilled water for 1-3 days to obtain a magnetic Gelatin/β-CD/Fe 3 O 4 hydrogel.
  7. 据权利要求6所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S1中，所述明胶和β-环糊精的质量比为10:（1~5）。According to claim 6, a method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes is characterized in that, in said S1, the mass ratio of said gelatin to β-cyclodextrin is 10:(1~ 5).
  8. 根据权利要求7所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S1中，所述搅拌的温度为30~60℃，所述搅拌的时间为30~60min，所述搅拌的速度为500~800r/min。A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 7, characterized in that, in said S1, the stirring temperature is 30-60°C, and the stirring time is 30 ~60min, the stirring speed is 500~800r/min.
  9. 根据权利要求8所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S1中，所述交联剂为转谷氨酰胺酶；所述CaCl 2溶液的质量百分数为8~12%。 A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 8, wherein, in the S1, the cross-linking agent is transglutaminase; the CaCl of the solution The mass percentage is 8~12%.
  10. 根据权利要求9所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S2中，所述搅拌的时间为5~10min。A method of using a magnetic field to induce chondrocytes from umbilical cord blood mesenchymal stem cells according to claim 9, characterized in that, in the S2, the stirring time is 5 to 10 minutes.
  11. 根据权利要求1-5任意一项所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述脐带血间充质干细胞是通过以下方法培养得到：A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to any one of claims 1-5, wherein said umbilical cord blood mesenchymal stem cells are obtained by culturing by the following method:
    S1′、量取一定比例的PBS缓冲溶液和脐带血，并将其混合进行离心分离，弃上清液，获得一次离心后的脐带血溶液；S1′. Measure a certain proportion of PBS buffer solution and umbilical cord blood, mix them for centrifugation, discard the supernatant, and obtain the umbilical cord blood solution after one centrifugation;
    S2′、向所述一次离心后的脐带血溶液中再加入PBS缓冲溶液并进行离心洗涤，弃上清液，获得沉淀的细胞悬液；S2', adding PBS buffer solution to the umbilical cord blood solution after the first centrifugation, performing centrifugation and washing, discarding the supernatant to obtain a precipitated cell suspension;
    S3′、将沉淀的细胞悬液加入至高糖DMEM培养液中，并在二氧化碳的条件下进行培养，得到脐带血间充质干细胞。S3', adding the precipitated cell suspension into high-glucose DMEM culture medium, and culturing under the condition of carbon dioxide to obtain umbilical cord blood mesenchymal stem cells.
  12. 根据权利要求11所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S1′中，所述脐带血与PBS缓冲溶液的体积比为（2~4）:（4~6）。A method of using a magnetic field to induce chondrocytes from umbilical cord blood mesenchymal stem cells according to claim 11, wherein in said S1′, the volume ratio of said umbilical cord blood to PBS buffer solution is (2~4) : (4~6).
  13. 根据权利要求12所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S1′中，所述离心分离的转速为1000~1500r/min，所述离心分离的时间为8~12min。A method for inducing chondrocytes from umbilical cord blood mesenchymal stem cells by using a magnetic field according to claim 12, characterized in that in said S1′, the rotational speed of said centrifugation is 1000-1500r/min, and said centrifugation The time is 8~12min.
  14. 根据权利要求13所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S2′中，加入所述PBS缓冲溶液与所述一次离心后的脐带血溶液的体积比为（5~9）:10。A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 13, characterized in that, in the S2′, the PBS buffer solution and the umbilical cord blood solution after the first centrifugation are added The volume ratio is (5~9):10.
  15. 根据权利要求14所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S2′中，所述离心洗涤的转速为800~1200r/min。A method for inducing chondrocyte formation from umbilical cord blood mesenchymal stem cells by using a magnetic field according to claim 14, characterized in that, in the S2′, the rotational speed of the centrifugal washing is 800-1200r/min.
  16. 根据权利要求15所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S3′中，所述高糖DMEM培养液与脐带血的体积比为（2~4）:（4~6）。A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 15, wherein in the S3′, the volume ratio of the high-sugar DMEM culture solution to the umbilical cord blood is (2~ 4): (4~6).
  17. 根据权利要求16所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S3′中，所述高糖DMEM培养液中含有8~12%的FBS和0.8~1.2%的双抗。A method of using a magnetic field to induce umbilical cord blood mesenchymal stem cells to form chondrocytes according to claim 16, wherein in said S3′, said high-sugar DMEM culture solution contains 8-12% FBS and 0.8 ~1.2% double antibody.
    18、根据权利要求17所述的一种采用磁场诱导脐带血间质干细胞形成软骨细胞的方法，其特征在于，所述S3′的具体方法为：将沉淀的细胞悬液加入至高糖DMEM培养液中，在温度为36~38℃、二氧化碳质量分数为3~7%的条件下培养45~50h，且每2-3d更换一次高糖DMEM培养液，得到脐带血间充质干细胞。18. A method of using a magnetic field to induce chondrocytes from umbilical cord blood mesenchymal stem cells according to claim 17, characterized in that the specific method of S3' is: adding the precipitated cell suspension to the high-glucose DMEM culture medium umbilical cord blood mesenchymal stem cells were obtained by culturing at a temperature of 36-38°C and a carbon dioxide mass fraction of 3-7% for 45-50 hours, and replacing the high-glucose DMEM medium every 2-3 days.
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