CN112680414A - Preparation method of aging-reversing mesenchymal stem cells - Google Patents
Preparation method of aging-reversing mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a preparation method of mesenchymal stem cells for reversing aging, which comprises the following steps: s1 donor selection and evaluation; s2 isolating human mesenchymal stem cells; carrying out in-vitro proliferation culture on the S3 cells; s4 cell assessment; s5, preparing a product; freezing and storing S6 cells; the invention has higher cell activity and less cell death number in a mouse body, has smaller preparation rejection, promotes preparation cells to replace regional aged cells in the mouse body by the characteristic, realizes reverse aging, and can be used for injection treatment of diseases such as diabetes, burn, osteoarthritis, graft-versus-host disease and the like; the serum-free culture medium is selected for the in vitro proliferation culture of the cells, and the human platelet lysate is added into the serum-free culture medium to increase the viscosity of the culture medium, protect the cells from mechanical damage, avoid cell modification in the proliferation process, and avoid the potential risks in the previous culture using fetal calf serum, such as human and livestock infectious sources, microbial pollutants and immune reaction.
Description
Technical Field
The invention relates to the field of cell processing, in particular to a preparation method of a mesenchymal stem cell for reversing senescence.
Background
Mesenchymal stem cells are derived from mesoderm in early development, belong to pluripotent stem cells, and are discovered in bone marrow at first, so that the mesenchymal stem cells are increasingly concerned by people due to the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. Under the specific induction condition in vivo or in vitro, the mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes.
The existing mesenchymal stem cells play an important role in modern treatment and regenerative medicine due to their regenerative and immunoregulatory properties and the ability to migrate to the exact site of injury. These characteristics make human mesenchymal stem cells (hMSCs) one of the active cells with a prospect in developing innovative cell therapy products, and have been applied to the treatment of specific diseases such as diabetes, burns, osteoarthritis, graft-versus-host disease, and the like.
Currently, over 500 worldwide hMSC-based cell therapy products are undergoing clinical trials to evaluate their safety and side effects, determine their dosage and route of administration, and examine their efficacy as specific diseases. However, only 8 hMSC-based products were marketed. Anti-aging properties in humans regarding hMCS have yet to be developed.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a preparation method of mesenchymal stem cells for reversing aging.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of mesenchymal stem cells for reversing aging comprises the following steps: s1 donor selection and evaluation; performing physical and serological studies on cell providers to exclude donors with signs and symptoms of infectious disease; s2 isolating human mesenchymal stem cells; separating the tissue mass from the human body and extracting mesenchymal stem cells therefrom; carrying out in-vitro proliferation culture on the S3 cells; preparing the cells separated from the S2 into cell suspension, and performing proliferation culture in a serum-free culture medium for 8-12 days; the serum-free culture medium comprises 55-70% of a mixture of fibronectin and laminin, 2-5% of growth promoting factors, 2-5% of enzyme inhibitors, 1-3% of trace elements and 17-40% of human platelet lysate in percentage by volume; the mass ratio of fibronectin to laminin is 3: 1; s4 cell assessment; extracting cells cultured in the proliferation of the part S3 to detect and identify the cell characteristics, activity, purity, potential, amplification capacity, gene stability, tumorigenicity and curative effect; s5, preparing a product; preparing the evaluated cells into an injection type preparation to obtain a finished product; freezing and storing S6 cells; the unused cells were frozen and stored.
Preferably, in the step of S1 donor selection and evaluation, the detection of viruses in serological studies includes detection of human immunodeficiency virus type 1 and 2, hepatitis virus type B and C and treponema pallidum, west nile virus, sepsis, vaccinia.
Preferably, in the step of isolating human mesenchymal stem cells at S2, the sources of mesenchymal stem cells include human bone marrow, umbilical cord and adipose tissue.
Preferably, in the step of isolating human mesenchymal stem cells at S2, methods including a tissue mass adherence method, a red blood cell lysis method and a gradient density method are used.
Preferably, in the step of separating the human mesenchymal stem cells in S2, the human mesenchymal stem cells are taken out of adipose tissues, the adipose tissues are firstly obtained by a minimally invasive method, and then the human mesenchymal stem cells are obtained by density gradient centrifugal separation by a gradient density method.
Preferably, in the step of freezing the S6 cells, the temperature reduction gradient in the process of freezing the cells is 0.05-0.1 ℃/min.
Preferably, in the step of freezing the S6 cells, the freezing solution of the cells is dimethyl sulfoxide, and the cells are frozen in a freezing tube with the concentration of 200-.
The invention has the beneficial effects that: the invention has higher cell activity and less cell death number in a mouse body, the human mesenchymal stem cell preparation has smaller rejection, and the characteristic promotes cells to replace regional aged cells in the mouse body, realizes reverse aging, and can be used for injection treatment of diseases such as diabetes, burn, osteoarthritis, graft-versus-host disease and the like; the method selects a serum-free culture medium for the in vitro proliferation culture of the cells, and human platelet lysate is added into the serum-free culture medium to increase the viscosity of the culture medium, protect the cells from mechanical damage, avoid cell modification in the proliferation process, and avoid potential risks such as zoonosis infection sources, microbial pollutants and immune reactions existing in the previous culture by using fetal calf serum.
Drawings
FIG. 1 is a table of experimental data for three examples of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example one
A preparation method of mesenchymal stem cells for reversing aging comprises the following steps:
s1 donor selection and evaluation; physical testing and serological studies were performed on cell providers to exclude donors with signs and symptoms of infectious disease.
S2 isolating human mesenchymal stem cells; the tissue mass is isolated from the human body and mesenchymal stem cells are extracted therefrom.
Carrying out in-vitro proliferation culture on the S3 cells; preparing the cells separated from the S2 into cell suspension, and performing proliferation culture in a serum-free culture medium for 10 days; the serum-free medium comprises 55% of a mixture of fibronectin and laminin, 2% of growth promoting factor, 2% of enzyme inhibitor, 1% of trace elements and 40% of human platelet lysate in percentage by volume; the mass ratio of fibronectin to laminin was 3: 1.
S4 cell assessment; extracting the cells cultured in the proliferation of the part S3 to detect and identify the cell characteristics, activity, purity, potential, amplification capacity, gene stability, tumorigenicity and curative effect.
S5, preparing a product; and preparing the evaluated cells into an injection type preparation to obtain a finished product.
Freezing and storing S6 cells; the unused cells were frozen and stored.
In this example, the step of S1 donor selection and evaluation, virus detection in serological studies includes detection of human immunodeficiency virus type 1 and 2, hepatitis virus type B and C and treponema pallidum, west nile virus, sepsis, vaccinia.
In this example, in the step of isolating human mesenchymal stem cells at S2, sources of mesenchymal stem cells include human bone marrow, umbilical cord and adipose tissue.
In the present embodiment, in the step of isolating human mesenchymal stem cells at S2, methods including a tissue mass adherence method, a red blood cell lysis method, and a gradient density method are used.
In this embodiment, in the step of S2, the human mesenchymal stem cells are taken out of adipose tissues, the adipose tissues are first obtained by a minimally invasive method, and then the human mesenchymal stem cells are obtained by density gradient centrifugation using a gradient density method.
In this example, in the step of freezing the cells at S6, the temperature gradient during the freezing process is 0.1 ℃/min.
In this example, in the step of freezing and storing S6 cells, the freezing solution of cells is DMSO, and is frozen in a freezing tube with a concentration of 200-.
Example two
A preparation method of mesenchymal stem cells for reversing aging comprises the following steps:
s1 donor selection and evaluation; physical testing and serological studies were performed on cell providers to exclude donors with signs and symptoms of infectious disease.
S2 isolating human mesenchymal stem cells; the tissue mass is isolated from the human body and mesenchymal stem cells are extracted therefrom.
Carrying out in-vitro proliferation culture on the S3 cells; preparing the cells separated from the S2 into cell suspension, and performing proliferation culture in a serum-free culture medium for 10 days; the serum-free medium comprises a mixture of 70% fibronectin and laminin, 5% growth promoting factor, 5% enzyme inhibitor, 3% microelement and 17% human platelet lysate by volume percent; the mass ratio of fibronectin to laminin was 3: 1.
S4 cell assessment; extracting the cells cultured in the proliferation of the part S3 to detect and identify the cell characteristics, activity, purity, potential, amplification capacity, gene stability, tumorigenicity and curative effect.
S5, preparing a product; and preparing the evaluated cells into an injection type preparation to obtain a finished product.
Freezing and storing S6 cells; the unused cells were frozen and stored.
In this example, the step of S1 donor selection and evaluation, virus detection in serological studies includes detection of human immunodeficiency virus type 1 and 2, hepatitis virus type B and C and treponema pallidum, west nile virus, sepsis, vaccinia.
In this example, in the step of isolating human mesenchymal stem cells at S2, sources of mesenchymal stem cells include human bone marrow, umbilical cord and adipose tissue.
In the present embodiment, in the step of isolating human mesenchymal stem cells at S2, methods including a tissue mass adherence method, a red blood cell lysis method, and a gradient density method are used.
In this embodiment, in the step of S2, the human mesenchymal stem cells are taken out of adipose tissues, the adipose tissues are first obtained by a minimally invasive method, and then the human mesenchymal stem cells are obtained by density gradient centrifugation using a gradient density method.
In this example, in the step of freezing the cells at S6, the temperature gradient during the freezing process is 0.1 ℃/min.
In this example, in the step of freezing and storing S6 cells, the freezing solution of cells is DMSO, and is frozen in a freezing tube with a concentration of 200-.
EXAMPLE III
A preparation method of mesenchymal stem cells for reversing aging comprises the following steps:
s1 donor selection and evaluation; physical testing and serological studies were performed on cell providers to exclude donors with signs and symptoms of infectious disease.
S2 isolating human mesenchymal stem cells; the tissue mass is isolated from the human body and mesenchymal stem cells are extracted therefrom.
Carrying out in-vitro proliferation culture on the S3 cells; preparing the cells separated from the S2 into cell suspension, and performing proliferation culture in a serum-free culture medium for 10 days; the serum-free medium comprises a mixture of 65% fibronectin and laminin, 3% growth promoting factor, 4% enzyme inhibitor, 2% microelement and 26% human platelet lysate by volume percent; the mass ratio of fibronectin to laminin was 3: 1.
S4 cell assessment; extracting the cells cultured in the proliferation of the part S3 to detect and identify the cell characteristics, activity, purity, potential, amplification capacity, gene stability, tumorigenicity and curative effect.
S5, preparing a product; and preparing the evaluated cells into an injection type preparation to obtain a finished product.
Freezing and storing S6 cells; the unused cells were frozen and stored.
In this example, the step of S1 donor selection and evaluation, virus detection in serological studies includes detection of human immunodeficiency virus type 1 and 2, hepatitis virus type B and C and treponema pallidum, west nile virus, sepsis, vaccinia.
In this example, in the step of isolating human mesenchymal stem cells at S2, sources of mesenchymal stem cells include human bone marrow, umbilical cord and adipose tissue.
In the present embodiment, in the step of isolating human mesenchymal stem cells at S2, methods including a tissue mass adherence method, a red blood cell lysis method, and a gradient density method are used.
In this embodiment, in the step of S2, the human mesenchymal stem cells are taken out of adipose tissues, the adipose tissues are first obtained by a minimally invasive method, and then the human mesenchymal stem cells are obtained by density gradient centrifugation using a gradient density method.
In this example, in the step of freezing the cells at S6, the temperature gradient during the freezing process is 0.1 ℃/min.
In this example, in the step of freezing and storing S6 cells, the freezing solution of cells is DMSO, and is frozen in a freezing tube with a concentration of 200-.
The product preparations obtained according to the three examples are product one, product two and product three, respectively, which are injected into mice, and the activity degree and the cell death number of the cells in the mice within one week are tracked by using marker observation, and compared with the product (control group) in the prior art, so as to obtain fig. 1. Obviously, the invention has higher cell activity and less cell death number in a mouse body, the human mesenchymal stem cell preparation has smaller rejection, and the characteristic promotes cells to replace regional aged cells in the mouse body, realizes reverse aging, and can be used for injection treatment of diseases such as diabetes, burn, osteoarthritis, graft-versus-host disease and the like.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A preparation method of mesenchymal stem cells for reversing aging is characterized by comprising the following steps:
s1 donor selection and evaluation; performing physical and serological studies on cell providers to exclude donors with signs and symptoms of infectious disease;
s2 isolating human mesenchymal stem cells; separating the tissue mass from the human body and extracting mesenchymal stem cells therefrom;
carrying out in-vitro proliferation culture on the S3 cells; preparing the cells separated from the S2 into cell suspension, and performing proliferation culture in a serum-free culture medium for 8-12 days; the serum-free culture medium comprises 55-70% of a mixture of fibronectin and laminin, 2-5% of growth promoting factors, 2-5% of enzyme inhibitors, 1-3% of trace elements and 17-40% of human platelet lysate in percentage by volume; the mass ratio of fibronectin to laminin is 3: 1;
s4 cell assessment; extracting cells cultured in the proliferation of the part S3 to detect and identify the cell characteristics, activity, purity, potential, amplification capacity, gene stability, tumorigenicity and curative effect;
s5, preparing a product; preparing the evaluated cells into an injection type preparation to obtain a finished product;
freezing and storing S6 cells; the unused cells were frozen and stored.
2. The method of claim 1, wherein the step of selecting and evaluating the donor of S1, the step of detecting the virus in serological studies comprises detecting hiv type 1 and 2, hbv type B and C, treponema pallidum, west nile virus, sepsis, vaccinia.
3. The method of claim 1, wherein in the step of isolating human mesenchymal stem cells at S2, the source of mesenchymal stem cells comprises human bone marrow, umbilical cord and adipose tissue.
4. The method of claim 1, wherein the step of isolating human mesenchymal stem cells at S2 comprises a tissue mass adherence method, a red blood cell lysis method and a gradient density method.
5. The method of claim 3, wherein in the step of S2, the step of separating the human mesenchymal stem cells comprises the steps of taking the human mesenchymal stem cells out of adipose tissues, obtaining adipose tissues by a minimally invasive method, and performing density gradient centrifugation by a gradient density method to obtain the human mesenchymal stem cells.
6. The method for preparing mesenchymal stem cells for reversing aging according to claim 1, wherein in the step of cryopreserving the S6 cells, the temperature gradient during the cryopreserving process is 0.05-0.1 ℃/min.
7. The method for preparing mesenchymal stem cells for reversing aging as claimed in claim 1, wherein in the step of cryopreserving the S6 cells, the freezing solution of the cells is DMSO, and the freezing solution is frozen in a freezing tube with a concentration of 200-.
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Cited By (2)
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| CN114540298A (en) * | 2022-03-25 | 2022-05-27 | 北京瑷格干细胞科技有限公司 | Stem cell serum-free medium and preparation method thereof |
| CN116426469A (en) * | 2023-06-07 | 2023-07-14 | 北京大学口腔医学院 | Application of LAP2 alpha in mesenchymal stem cell adipogenic differentiation |
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| CN116426469A (en) * | 2023-06-07 | 2023-07-14 | 北京大学口腔医学院 | Application of LAP2 alpha in mesenchymal stem cell adipogenic differentiation |
| CN116426469B (en) * | 2023-06-07 | 2023-09-08 | 北京大学口腔医学院 | Application of LAP2 alpha in mesenchymal stem cell adipogenic differentiation |
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Application publication date: 20210420 |