CN110804587A - Method for modeling production of mesenchymal stem cells by adopting improved microcarrier cell culture method - Google Patents
Method for modeling production of mesenchymal stem cells by adopting improved microcarrier cell culture method Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C12N2531/00—Microcarriers
Abstract
The invention discloses a method for producing mesenchymal stem cells in a modeling manner by adopting an improved microcarrier cell culture method, which comprises the following steps: cutting umbilical cord with artery and vein removed, and washing thoroughly; adding low-sugar DMEM culture solution containing bovine serum and green chain double antibody, and placing in CO2Culturing in an incubator, and periodically replacing a culture medium; when the mesenchymal stem cells are fused to 70-80%, digesting with pancreatin solution; the mesenchymal stem cells are cultured at 2.6-2.9 × 105Inoculating the cells/ml in the microcarrier suspension for stirring suspension culture; after the cells are overgrown with the microcarriers, removing the upper culture medium, adding a trypsin solution for incubation, filtering by using a cell screen, collecting cell filtrate, centrifuging and collecting precipitates. The invention adopts the improved microcarrier cell culture method to culture the mesenchymal stem cells, thereby achieving the purpose of large-scale production of the mesenchymal stem cells and laying the experimental foundation for further expanding the range of the mesenchymal stem cells to be applied to clinic.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for producing mesenchymal stem cells in a modeling manner by adopting an improved microcarrier cell culture method.
Background
Microcarriers are microspheres or other shaped objects with a diameter of 60-250 μm that are suitable for adherent cell growth. Generally, the chitosan-chitosan composite material is composed of natural glucan or various synthetic polymers, and the basic types of the dextran-chitosan composite material mainly comprise liquid microcarriers, macroporous gelatin microcarriers, polystyrene microcarriers, PHEMA microcarriers, chitin microcarriers, polyurethane foam microcarriers, alginate gel microcarriers, magnetic microcarriers and the like. The commonly used commercial microcarriers are mainly Cytodex1, 2, 3, Cytopore and Cytoline, etc. The basic principle of microcarrier cell culture technology is to add microcarriers that are not harmful to cells to the culture broth of a culture vessel. As a carrier, cells are attached to the surface of the microcarriers and grown while the microcarriers are kept in suspension by continuous agitation. The microcarrier technology can be used for realizing the large-scale acquisition of stem cells, and provides a practical basis for the application of stem cell therapy to clinic.
Mesenchymal stem cells are widely present in connective tissues and organ mesenchyme of the whole body, are most abundant in bone marrow tissues, can be separated from umbilical cord blood of a fetus, and are also present in placenta, amniotic fluid, umbilical vein subendothelial layer, peripheral blood, liver, fat, muscle, skin and other tissues. The mesenchymal stem cells have the potential of high proliferation, self-renewal and multidirectional differentiation, can differentiate into various tissues and cells, are easy to separate, culture and amplify, are easy to introduce and express exogenous genes, always maintain the potential of multidirectional differentiation in the in vitro long-term culture process, and have quite stable genetic background.
In 1995, mesenchymal stem cells are first applied to clinical application, in 2012, the company Osiris declares that mesenchymal stem cells are approved by FDA in canada as a medicine to be marketed, and the clinical application of mesenchymal stem cells reaches a historical height. Mesenchymal stem cells have been the first choice in biomedical research by replacing placental stem cells, and are widely used in basic and clinical research of various diseases.
Therefore, it is very necessary to provide a method for producing mesenchymal stem cells in a large scale, and the method is a hot spot in the research of the mesenchymal stem cells at present.
Disclosure of Invention
The invention provides a method for modeling and producing mesenchymal stem cells by adopting an improved microcarrier cell culture method, aiming at more widely applying the mesenchymal stem cells to the basic and clinical research of various diseases.
The invention is realized according to the following technical scheme.
A method for modeling production of mesenchymal stem cells by using an improved microcarrier cell culture method comprises the following steps:
s1, cutting the umbilical cord without the artery and vein into pieces, and fully washing;
s2, adding a low-sugar DMEM culture solution containing bovine serum and green chain double antibody, and placing in CO2Culturing in an incubator, and replacing the culture medium after 24-48 h;
s3, digesting the mesenchymal stem cells with a pancreatin solution when the fusion of the mesenchymal stem cells reaches 70 to 80 percent;
s4, filling the mesenchymal stem cells with the weight of 2.6-2.9 multiplied by 105Inoculating the cells/ml in the microcarrier suspension for stirring suspension culture;
s5, after the cells grow full of the microcarriers, removing the upper culture medium, adding a trypsin solution for incubation, filtering the cells by using a cell screen, collecting cell filtrate, centrifuging and collecting precipitates.
Further, in step S3, the pancreatin solution is 0.25% pancreatin +0.02% EDTA.
Further, the microcarrier suspension in step S4 comprises serum-free medium and microcarriers, and the density of the microcarriers is 2-3 mg/ml.
Further, the microcarrier is Cytodex1, Cytodex 2, Cytodex 3, Cytopore or Cytoline.
Further, the culture conditions of the agitation suspension culture in the step S4 are: intermittently stirring for 0-12 hr at a stirring speed of 20-30rpm for 5-10min, and stopping stirring for 15-25 min; stirring and culturing at 30rmp speed for 12-48 h; stirring and culturing at the speed of 50rmp for 49-96 h; the dissolved oxygen is 30-50% of air saturation.
Further, in step S5, the trypsin solution is EDTA-trypsin solution, and the volume ratio of EDTA-trypsin to mesenchymal stem cell microcarrier is 2-3: 1.
further, in step S5, the trypsin solution is incubated at 37 ℃ and 150rpm to 180rpm for 8 to 12 min.
Further, in step S5, the cell sieve is a 70 μm sieve.
Further, in step S5, the centrifugation condition is 150-200g centrifugation for 8-12 min.
The present invention obtains the following advantageous effects.
The invention adopts the improved microcarrier cell culture method to culture the mesenchymal stem cells, thereby achieving the purpose of large-scale production of the mesenchymal stem cells, laying an experimental foundation for further expanding the range of the mesenchymal stem cells to be applied to clinic, and laying a foundation for industrial transformation of the mesenchymal stem cells.
Drawings
Figure 1 is a map of mesenchymal stem cells produced by the method of the invention (40X);
fig. 2 is a diagram (100X) of mesenchymal stem cells produced by the method of the present invention.
Detailed Description
The invention is further explained below with reference to the drawings and the examples.
Example 1
A method for modeling production of mesenchymal stem cells by using an improved microcarrier cell culture method comprises the following steps:
s1, cutting the umbilical cord without the artery and vein into pieces, and fully washing;
s2, adding a low-sugar DMEM culture solution containing bovine serum and green chain double antibody, and placing in CO2Culturing in an incubator, and replacing the culture medium after 24-48 h;
s3, digesting with a 0.25% pancreatin and 0.02% EDTA solution when the mesenchymal stem cells are fused to 70%;
s4, filling stem cells are divided into 2.6 multiplied by 105Inoculating the cells/ml into a microcarrier suspension for stirring suspension culture, wherein the microcarrier suspension comprises a serum-free culture medium and a microcarrier, and the density of the microcarrier is 2 mg/ml; the microcarrier is Cytodex1, Cytodex 2, Cytodex 3, Cytopore or Cytoline. The culture conditions of the stirring suspension culture are as follows: intermittently stirring for 0-12 hours at a stirring speed of 20rpm for 5min,stopping stirring for 15 min; stirring and culturing at 30rmp speed for 12-48 h; stirring and culturing at the speed of 50rmp for 49-96 h; dissolved oxygen was 30% air saturation.
S5, after the microcarrier is full of cells, removing the upper culture medium, adding an EDTA-trypsin solution for incubation, wherein the volume ratio of the EDTA-trypsin to the mesenchymal stem cell microcarrier is 2: 1; the incubation condition of the trypsin solution is that the culture is carried out for 8min at the temperature of 37 ℃ and at the rpm of 150; filtering with 70 μm cell screen, collecting cell filtrate, centrifuging, collecting precipitate, and centrifuging at 150g for 8 min.
Example 2
A method for modeling production of mesenchymal stem cells by using an improved microcarrier cell culture method comprises the following steps:
s1, cutting the umbilical cord without the artery and vein into pieces, and fully washing;
s2, adding a low-sugar DMEM culture solution containing bovine serum and green chain double antibody, and placing in CO2Culturing in an incubator, and replacing the culture medium after 24-48 h;
s3, digesting with a 0.25% pancreatin and 0.02% EDTA solution when the mesenchymal stem cells are fused to 80%;
s4, filling stem cells are divided into 2.9 multiplied by 105Inoculating the cells/ml into a microcarrier suspension for stirring suspension culture, wherein the microcarrier suspension comprises a serum-free culture medium and microcarriers, and the density of the microcarriers is 3 mg/ml; the microcarrier is Cytodex1, Cytodex 2, Cytodex 3, Cytopore or Cytoline. The culture conditions of the stirring suspension culture are as follows: intermittently stirring for 0-12 hours, stirring for 10min at the stirring speed of 30rpm, and stopping stirring for 25 min; stirring and culturing at 30rmp speed for 12-48 h; stirring and culturing at the speed of 50rmp for 49-96 h; dissolved oxygen was 50% of air saturation.
S5, after the microcarrier is full of cells, removing the upper culture medium, adding an EDTA-trypsin solution for incubation, wherein the volume ratio of the EDTA-trypsin to the mesenchymal stem cell microcarrier is 3: 1; the trypsin solution is incubated at 37 ℃ and 180rpm for 12 min; filtering with 70 μm cell screen, collecting cell filtrate, centrifuging, collecting precipitate, and centrifuging at 200g for 12 min.
Example 3
A method for modeling production of mesenchymal stem cells by using an improved microcarrier cell culture method comprises the following steps:
s1, cutting the umbilical cord without the artery and vein into pieces, and fully washing;
s2, adding a low-sugar DMEM culture solution containing bovine serum and green chain double antibody, and placing in CO2Culturing in an incubator, and replacing the culture medium after 24-48 h;
s3, digesting with a 0.25% pancreatin and 0.02% EDTA solution when the mesenchymal stem cells are fused to 80%;
s4, filling stem cells are divided into 2.6 multiplied by 105Inoculating the cells/ml into a microcarrier suspension for stirring suspension culture, wherein the microcarrier suspension comprises a serum-free culture medium and microcarriers, and the density of the microcarriers is 3 mg/ml; the microcarrier is Cytodex1, Cytodex 2, Cytodex 3, Cytopore or Cytoline. The culture conditions of the stirring suspension culture are as follows: intermittently stirring for 0-12 h, stirring at a stirring speed of 30rpm for 8min, and stopping stirring for 20 min; stirring and culturing at 30rmp speed for 12-48 h; stirring and culturing at the speed of 50rmp for 49-96 h; dissolved oxygen was 40% air saturation.
S5, after the microcarrier is full of cells, removing the upper culture medium, adding an EDTA-trypsin solution for incubation, wherein the volume ratio of the EDTA-trypsin to the mesenchymal stem cell microcarrier is 3: 1; the trypsin solution is incubated at 37 deg.C and 180rpm for 10 min; filtering with 70 μm cell screen, collecting cell filtrate, centrifuging, collecting precipitate, and centrifuging at 200g for 10 min.
The obtained mesenchymal stem cells are shown in 1-2.
Example 4 examples 1-3 cell viability rates
| Rate of cell viability | |
| Example 1 | 96.21±1.97 |
| Example 2 | 96.87±2.01 |
| Example 3 | 97.35±1.84 |
Example 5 examples 1-3 culture for 96h cell density
| Density of inoculation | Cell density of 96h | |
| Example 1 | 2.6×105 | 3.9×106 |
| Example 2 | 2.9×105 | 4.1×106 |
| Example 3 | 2.6×105 | 4.8×106 |
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the embodiments of the present invention, and not for limiting the same; although embodiments of the present invention have been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for modeling production of mesenchymal stem cells by using an improved microcarrier cell culture method is characterized by comprising the following steps: the method comprises the following steps:
s1, cutting the umbilical cord without the artery and vein into pieces, and fully washing;
s2, adding a low-sugar DMEM culture solution containing bovine serum and green chain double antibody, and placing in CO2Culturing in an incubator, and replacing the culture medium after 24-48 h;
s3, digesting the mesenchymal stem cells with a pancreatin solution when the fusion of the mesenchymal stem cells reaches 70 to 80 percent;
s4, filling the mesenchymal stem cells with the weight of 2.6-2.9 multiplied by 105Inoculating the cells/ml in the microcarrier suspension for stirring suspension culture;
s5, after the cells grow full of the microcarriers, removing the upper culture medium, adding a trypsin solution for incubation, filtering the cells by using a cell screen, collecting cell filtrate, centrifuging and collecting precipitates.
2. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: in step S3, the pancreatin solution is 0.25% pancreatin +0.02% EDTA.
3. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: the microcarrier suspension in step S4 comprises serum-free medium and microcarriers, and the density of the microcarriers is 2-3 mg/ml.
4. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 3, wherein: the microcarrier is Cytodex1, Cytodex 2, Cytodex 3, Cytopore or Cytoline.
5. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: the culture conditions for the agitation suspension culture in step S4 were: intermittently stirring for 0-12 hr at a stirring speed of 20-30rpm for 5-10min, and stopping stirring for 15-25 min; stirring and culturing at 30rmp speed for 12-48 h; stirring and culturing at the speed of 50rmp for 49-96 h; the dissolved oxygen is 30-50% of air saturation.
6. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: in step S5, the trypsin solution is EDTA-trypsin solution, and the volume ratio of EDTA-trypsin to mesenchymal stem cell microcarrier is 2-3: 1.
7. the method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: in step S5, the trypsin solution is incubated at 37 ℃ and 150-180 rpm for 8-12 min.
8. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: in step S5, the cell mesh is a 70 μm mesh.
9. The method for modeling production of mesenchymal stem cells by using improved microcarrier cell culture method according to claim 1, wherein: in step S5, the centrifugation condition is 150-200g centrifugation for 8-12 min.
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Cited By (7)
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| CN111690604A (en) * | 2020-06-24 | 2020-09-22 | 杭州原生生物科技有限公司 | MSC in-vitro amplification method |
| CN113106059A (en) * | 2021-04-07 | 2021-07-13 | 清华大学深圳国际研究生院 | High-migration mesenchymal stem cell and preparation method and application thereof |
| CN114561348A (en) * | 2022-03-02 | 2022-05-31 | 北京圣美细胞生命科学工程研究院有限公司 | Soluble microcarrier for large-scale culture of stem cells |
| CN114703129A (en) * | 2022-03-09 | 2022-07-05 | 唐颐惠康干细胞产业平台(天津)有限公司 | Method for non-enzymatic 3D culture and amplification of therapeutic mesenchymal stem cells |
| CN115404208A (en) * | 2022-09-16 | 2022-11-29 | 深圳市儿童医院 | Method for obtaining mesenchymal stem cells by mixed culture of foreskin and application thereof |
| CN115466724A (en) * | 2022-10-21 | 2022-12-13 | 唐颐控股(深圳)有限公司 | Screening method and application of optimal factor value in human mesenchymal stem cell amplification |
| WO2023005119A1 (en) * | 2021-07-27 | 2023-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | System device for automated sustainable large-scale 3d cell production |
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