CN113509431A - Mesenchymal stem cell extract and extraction method and application thereof - Google Patents
Mesenchymal stem cell extract and extraction method and application thereof Download PDFInfo
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- CN113509431A CN113509431A CN202110474597.3A CN202110474597A CN113509431A CN 113509431 A CN113509431 A CN 113509431A CN 202110474597 A CN202110474597 A CN 202110474597A CN 113509431 A CN113509431 A CN 113509431A
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Abstract
The invention discloses a mesenchymal stem cell extract and an extraction method and application thereof, wherein the extraction method comprises the steps of separating umbilical cord mesenchymal stem cells from umbilical cord blood, carrying out subculture, culturing the umbilical cord mesenchymal stem cells by using a cell growth medium until the cells are 80% fused, continuously culturing the umbilical cord mesenchymal stem cells in a serum-free culture medium for 70-75 hours by using a phosphate buffer solution, separating the serum-free culture medium from the cells, crushing the cells, centrifuging the cells, collecting supernatant, and mixing the supernatant with the separated serum-free culture medium to obtain the mesenchymal stem cell extract. The invention provides an efficient, quick and convenient way for the application of the mesenchymal stem cells by utilizing various cell factors secreted by the mesenchymal stem cells with high concentration.
Description
Technical Field
The invention belongs to the technical field of stem cell cosmetology, and particularly relates to a mesenchymal stem cell extract and an extraction method and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are adult stem cells with self-renewing and multipotent differentiation potential, and are present in bone marrow, adipose tissue, cord blood and various fetal tissues. It can secrete multiple cytokines and growth factors, and promote the proliferation and differentiation of Hematopoietic Stem Cells (HSC). MSCs also have immunomodulatory, anti-inflammatory, and tissue repair effects, and can reduce Graft Versus Host Disease (GVHD) and other transplant-related complications. Mesenchymal stem cells are currently extracted mainly from spinal cord and cord blood. Cord blood is the blood remaining in the placenta and umbilical cord after the fetus is delivered, the cord ligated and severed. The human umbilical cord blood mesenchymal stem cell is a multifunctional stem cell existing in umbilical cord tissues of newborns, can be differentiated into a plurality of tissue cells, and has wide clinical application prospect.
Clinical experiments at present prove that the yield and most of biological characteristics of human umbilical cord blood mesenchymal stem cells are similar to those of bone marrow-derived mesenchymal stem cells, and compared with the bone marrow mesenchymal stem cells, the umbilical cord blood mesenchymal stem cells have the following advantages:
(1) has no ethical problem, wide source, easy collection and easy amplification.
(2) Has stronger increment capability and is several times faster than bone marrow MSC.
(3) Is a more primitive MSC group.
(4) Lower expression of HLA-ABC (classical human leukocyte antigen class I antigen) and HLA-DR.
Therefore, the human umbilical cord blood mesenchymal stem cells are a more ideal cell group than the bone marrow mesenchymal stem cells. However, at present, the effective components can be obtained from the cells only by the human umbilical cord blood mesenchymal stem cells, or the secretory contents can be prepared by the human umbilical cord blood mesenchymal stem cells, and the research for simultaneously obtaining the effective components and the secretory contents is lacked.
People, especially women, pay more and more attention to skin care and beauty in daily life, and the demand for products capable of relieving skin cell aging and ultraviolet injury is continuously increased. And people pay more and more attention to the quality and safety of skin care products. It is necessary to study a cosmetic product by combining the effective components of umbilical cord blood mesenchymal stem cells with the secretory contents.
Disclosure of Invention
Aiming at the prior art, the invention provides the mesenchymal stem cell extract, the extraction method and the application thereof, and provides an efficient, quick and convenient way for the application of the mesenchymal stem cells by utilizing various cytokines secreted by the high-concentration mesenchymal stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that: provided is an extraction method of a mesenchymal stem cell extract, comprising the following steps:
s1: isolating umbilical cord mesenchymal stem cells from the umbilical cord blood;
s2: subculturing the separated umbilical cord mesenchymal stem cells, taking subcultured P5-P25 generation human umbilical cord mesenchymal stem cells, culturing by using a cell growth solution until the cells are 80% fused, and discarding the culture solution;
s3: washing the cultured cells with a phosphate buffer solution, adding a serum-free culture solution, continuously culturing for 70-75 h, and then separating the serum-free culture solution from the cells;
s4: digesting the cells with digestive juice, then carrying out ultrasonic disruption, then centrifuging and collecting supernatant;
s5: and mixing the serum-free culture solution obtained in the step S3 with the supernatant in the step S4, filtering, collecting filtrate and concentrating to obtain the mesenchymal stem cell extract.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the isolation of umbilical cord mesenchymal stem cells comprises the following steps:
SS 1: mixing the cord blood with a PBS buffer solution according to the volume ratio of 1: 3-5, then dripping a lymphocyte separation solution with the same volume as the cord blood, and centrifuging for 10-20 min at 600-750 g;
SS 2: extracting the leucocyte layer cells, washing with a PBS buffer solution, and then resuspending with a basic culture solution;
SS 3: smearing the nonspecific adherence-promoting protein and the specific adherence-promoting protein on the bottom of a culture dish, then inoculating the resuspended white membranous layer cells into the culture dish, and culturing at 37 ℃ under the hypoxia condition;
SS 4: carrying out subculture when the fusion rate of the primary cells reaches 60%;
SS 5: continuously culturing the umbilical cord blood mesenchymal stem cells for passage at 37 ℃ under the hypoxia condition, and performing passage after the cell fusion rate reaches 80%;
SS 6: and collecting the umbilical cord blood mesenchymal stem cells to obtain the umbilical cord blood mesenchymal stem cells.
Further, the basal medium comprises the following components in volume concentration:
MCDB 13150-80 mL/L, human serum albumin 60-90 mul/L, lactoferrin 200-300 mul/L, dexamethasone 10-15 mul/L, natamycin 20-30U/L and penicillin 90-120U/L.
Further, the non-specific adherence-promoting protein is fibronectin; the specific adherence promoting protein is a CD90 monoclonal antibody.
Further, the hypoxic conditions were oxygen 15%, carbon dioxide 5% and nitrogen 80%.
Further, the cell growth solution comprises the following components in percentage by volume:
15% of compound amino acid solution, 5% of vitamin solution, 10% of human serum albumin solution, 10% of hydrolyzed protein solution, 2% of trace element solution and 58% of electrolyte solution.
Further, the serum-free culture solution consists of a basic culture solution and an additive; the basic culture solution is DMEM/F12, and the additives include L-glutamine, L-ascorbic acid, sodium selenite, fibronectin, hydrocortisone, human insulin, bFGF, coenzyme A and sodium pyruvate.
Furthermore, the concentration of L-glutamine in the basic culture solution is 1-3 mM, the concentration of L-ascorbic acid is 10-30 mg/L, the concentration of sodium selenite is 15-17 mu g/L, the concentration of fibronectin is 40-50 mg/L, the concentration of hydrocortisone is 8-12 mg/L, the concentration of human insulin is 10-15 mg/L, the concentration of bFGF is 15-20 mu g/L, the concentration of coenzyme A is 90-100 mg/L, and the concentration of sodium pyruvate is 1-5 mM.
In addition, the invention also provides application of the human umbilical cord blood mesenchymal stem cell extract or the human umbilical cord blood mesenchymal stem cell extract prepared by the preparation method in cosmetics.
The invention has the beneficial effects that:
separating human umbilical cord blood mesenchymal stem cells from human agent blood, and sequentially carrying out subculture and serum-free culture solution culture to obtain a stem cell culture, thereby improving the yield of secretory contents of the human umbilical cord blood mesenchymal stem cells in the stem cell culture and accelerating the propagation rate of the cells. The secretory type content of the human umbilical cord blood mesenchymal stem cells is obtained from the stem cell culture, the cultured human umbilical cord blood mesenchymal stem cells are crushed to obtain non-secretory type content of the human umbilical cord blood mesenchymal stem cells, then the secretory type content and the non-secretory type content are mixed to obtain human umbilical cord blood mesenchymal stem cell extract, and the human umbilical cord blood mesenchymal stem cells separated from the human agent umbilical cord blood are fully utilized to prepare the extract containing the secretory type content and the non-secretory type content.
Detailed Description
1. Cord blood collection
Selecting healthy caesarean section lying-in women under 30 years old, having no hereditary diseases, no infectious diseases and negative HIV and hepatitis B serum marker detection to collect cord blood. After collection, the cord blood is placed in an ice box and immediately transported to a laboratory for separation treatment. Wherein, the concerned parturient informed and agreed on the isolated umbilical cord blood protocol.
2. Isolation of umbilical cord mesenchymal Stem cells
The stem cell separation is carried out on the cord blood by adopting the following steps:
(1) mixing the cord blood with a PBS buffer solution according to the volume ratio of 1: 3-5, then dripping a lymphocyte separation solution with the same volume as the cord blood, and centrifuging for 10-20 min at 600-750 g;
(2) extracting the leucocyte layer cells, washing with a PBS buffer solution, and then resuspending with a basic culture solution; the basal medium comprises the following components in volume concentration:
MCDB 13150-80 mL/L, human serum albumin 60-90 mul/L, lactoferrin 200-300 mul/L, dexamethasone 10-15 mul/L, natamycin 20-30U/L and penicillin 90-120U/L;
(3) smearing nonspecific adherence-promoting protein (such as fibronectin) and specific adherence-promoting protein (such as CD90 monoclonal antibody) on the bottom of a culture dish, then inoculating the resuspended white membrane layer cells into the culture dish, and culturing at 37 ℃ under hypoxic condition; the hypoxia condition is 15 percent of oxygen, 5 percent of carbon dioxide and 80 percent of nitrogen;
(3) carrying out subculture when the fusion rate of the primary cells reaches 60%;
(5) continuously culturing the umbilical cord blood mesenchymal stem cells for passage at 37 ℃ under the hypoxia condition, and performing passage after the cell fusion rate reaches 80%;
(6) and collecting the umbilical cord blood mesenchymal stem cells to obtain the umbilical cord blood mesenchymal stem cells.
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1
An extraction method of a mesenchymal stem cell extract comprises the following steps:
s1: umbilical cord mesenchymal stem cells are separated from umbilical cord blood according to the method;
s2: subculturing the separated umbilical cord mesenchymal stem cells, taking subcultured P5-P25 generation human umbilical cord mesenchymal stem cells, culturing by using a cell growth solution until the cells are 80% fused, and discarding the culture solution; the cell growth liquid comprises the following components in percentage by volume:
15% of compound amino acid solution (with the concentration of about 5 wt%), 5% of vitamin solution (with the concentration of about 1 wt%), 10% of human serum albumin solution (with the concentration of about 1 wt%), 10% of hydrolyzed protein solution (with the concentration of about 1 wt%), 2% of trace element solution (containing iron, zinc and selenium, and the concentrations of iron, zinc and selenium are all about 1 wt%) and 58% of sodium chloride solution (with the concentration of about 5 wt%);
s3: washing the cultured cells by using a phosphate buffer solution, adding a serum-free culture solution, continuously culturing for 72 hours at 37 ℃, and then separating the serum-free culture solution from the cells; the serum-free culture solution consists of a basic culture solution and additives, wherein the basic culture solution is DMEM/F12, the additives comprise L-glutamine, L-ascorbic acid, sodium selenite, fibronectin, hydrocortisone, human insulin, bFGF, coenzyme A and sodium pyruvate, the concentration of the L-glutamine is 2mM, the concentration of the L-ascorbic acid is 20mg/L, the concentration of the sodium selenite is 16 mu g/L, the concentration of the fibronectin is 45mg/L, the concentration of the hydrocortisone is 10mg/L, the concentration of the human insulin is 12mg/L, the concentration of the bFGF is 18 mu g/L, the concentration of the coenzyme A is 100mg/L, and the concentration of the sodium pyruvate is 3 mM;
s4: digesting the separated cells with digestive juice, then carrying out ultrasonic disruption, then centrifuging and collecting supernatant; the digestive juice is 0.25% trypsin-0.02% EDTA mixed digestive juice which is filtered and sterilized by a filter membrane with the diameter of 0.22 mu m; ultrasonic crushing is carried out by adopting an ultrasonic crusher, the conditions of crushing cells are 4 ℃, 400W, 3s of ultrasonic treatment, 6s of interval and 99 cycles;
s5: mixing the serum-free culture solution separated from S3 with the supernatant in S4 according to the volume ratio of 1:1, filtering, collecting the filtrate, and concentrating to obtain the mesenchymal stem cell extract.
Example 2
An extraction method of a mesenchymal stem cell extract comprises the following steps:
s1: umbilical cord mesenchymal stem cells are separated from umbilical cord blood according to the method;
s2: subculturing the separated umbilical cord mesenchymal stem cells, taking subcultured P5-P25 generation human umbilical cord mesenchymal stem cells, culturing by using a cell growth solution until the cells are 80% fused, and discarding the culture solution; the cell growth liquid comprises the following components in percentage by volume:
15% of compound amino acid solution (with the concentration of about 5 wt%), 5% of vitamin solution (with the concentration of about 1 wt%), 10% of human serum albumin solution (with the concentration of about 1 wt%), 10% of hydrolyzed protein solution (with the concentration of about 1 wt%), 2% of trace element solution (containing iron and zinc, with the concentrations of both 1 wt%) and 58% of sodium chloride solution (with the concentration of about 5 wt%);
s3: washing the cultured cells by using a phosphate buffer solution, adding a serum-free culture solution, continuously culturing for 75 hours at 37 ℃, and then separating the serum-free culture solution from the cells; the serum-free culture solution consists of a basic culture solution and additives, wherein the basic culture solution is DMEM/F12, the additives comprise L-glutamine, L-ascorbic acid, sodium selenite, fibronectin, hydrocortisone, human insulin, bFGF, coenzyme A and sodium pyruvate, the concentration of the L-glutamine is 1mM, the concentration of the L-ascorbic acid is 30mg/L, the concentration of the sodium selenite is 15 mu g/L, the concentration of the fibronectin is 50mg/L, the concentration of the hydrocortisone is 8mg/L, the concentration of the human insulin is 15mg/L, the concentration of the bFGF is 15 mu g/L, the concentration of the coenzyme A is 100mg/L, and the concentration of the sodium pyruvate is 1 mM;
s4: digesting the separated cells with digestive juice, then carrying out ultrasonic disruption, then centrifuging and collecting supernatant; the digestive juice is 0.25% trypsin-0.02% EDTA mixed digestive juice which is filtered and sterilized by a filter membrane with the diameter of 0.22 mu m; ultrasonic crushing is carried out by adopting an ultrasonic crusher, the conditions of crushing cells are 4 ℃, 400W, 3s of ultrasonic treatment, 6s of interval and 99 cycles;
s5: mixing the serum-free culture solution separated from S3 with the supernatant in S4 according to the volume ratio of 2:1, filtering, collecting the filtrate, and concentrating to obtain the mesenchymal stem cell extract.
Example 3
An extraction method of a mesenchymal stem cell extract comprises the following steps:
s1: umbilical cord mesenchymal stem cells are separated from umbilical cord blood according to the method;
s2: subculturing the separated umbilical cord mesenchymal stem cells, taking subcultured P5-P25 generation human umbilical cord mesenchymal stem cells, culturing by using a cell growth solution until the cells are 80% fused, and discarding the culture solution; the cell growth liquid comprises the following components in percentage by volume:
15% of compound amino acid solution (with the concentration of about 5 wt%), 5% of vitamin solution (with the concentration of about 1 wt%), 10% of human serum albumin solution (with the concentration of about 1 wt%), 10% of hydrolyzed protein solution (with the concentration of about 1 wt%), 2% of trace element solution (containing selenium and zinc, with the concentrations of both 1 wt%) and 58% of sodium chloride solution (with the concentration of about 5 wt%);
s3: washing the cultured cells by using a phosphate buffer solution, adding a serum-free culture solution, continuously culturing for 75 hours at 37 ℃, and then separating the serum-free culture solution from the cells; the serum-free culture solution consists of a basic culture solution and additives, wherein the basic culture solution is DMEM/F12, the additives comprise L-glutamine, L-ascorbic acid, sodium selenite, fibronectin, hydrocortisone, human insulin, bFGF, coenzyme A and sodium pyruvate, the concentration of the L-glutamine is 3mM, the concentration of the L-ascorbic acid is 10mg/L, the concentration of the sodium selenite is 17 mu g/L, the concentration of the fibronectin is 40mg/L, the concentration of the hydrocortisone is 12mg/L, the concentration of the human insulin is 10mg/L, the concentration of the bFGF is 20 mu g/L, the concentration of the coenzyme A is 90mg/L, and the concentration of the sodium pyruvate is 5 mM;
s4: digesting the separated cells with digestive juice, then carrying out ultrasonic disruption, then centrifuging and collecting supernatant; the digestive juice is 0.25% trypsin-0.02% EDTA mixed digestive juice which is filtered and sterilized by a filter membrane with the diameter of 0.22 mu m; ultrasonic crushing is carried out by adopting an ultrasonic crusher, the conditions of crushing cells are 4 ℃, 400W, 3s of ultrasonic treatment, 6s of interval and 99 cycles;
s5: mixing the serum-free culture solution separated from S3 with the supernatant in S4 according to the volume ratio of 1:2, filtering, collecting the filtrate, and concentrating to obtain the mesenchymal stem cell extract.
Analysis of results
The mesenchymal stem cell extract obtained in example 1 was used as an example to explain its effect.
The epidermal cell growth factor, the glutathione and the nicotinamide are mixed to prepare a mixed solution, the concentration of the epidermal cell growth factor in the mixed solution is 30 mug/mL, the concentration of the glutathione is 5 mug/mL, and the concentration of the nicotinamide is 2 mg/mL. Then, the mesenchymal stem cell extract obtained in example 1 was added to the mixture in a volume fraction of 0%, 0.5%, 1%, 2%, 5% to prepare a culture solution, each numbered A, B, C, D, E.
Using human skin fibroblasts, at a ratio of 1X 103The culture medium was added after inoculating to a 96-well plate, and cultured under optimal conditions. The proliferation rate of human skin cells was examined on day 3 (D3), day 5 (D5) and day 7 (D7) after inoculation, respectively. The results are shown in Table 1.
TABLE 1 proliferation rates of human skin cells treated with culture solutions containing mesenchymal stem cell extracts at different concentrations
| 0 | 0.5％ | 1％ | 2％ | 5％ | |
| D3 | 98.97％ | 108.34％ | 110.97％ | 112.72％ | 112.94％ |
| D5 | 100.88％ | 113.98％ | 125.62％ | 128.37％ | 129.31％ |
| D7 | 100.54％ | 111.88％ | 117.54％ | 118.47％ | 118.56％ |
The table shows that the proliferation rate of the human skin cells is obviously improved after the mesenchymal stem cell extract is added, which shows that the mesenchymal stem cell extract has a positive effect on promoting cell proliferation and can be used for preparing beauty products.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (10)
1. The extraction method of the mesenchymal stem cell extract is characterized by comprising the following steps:
s1: isolating umbilical cord mesenchymal stem cells from the umbilical cord blood;
s2: subculturing the separated umbilical cord mesenchymal stem cells, taking subcultured P5-P25 generation human umbilical cord mesenchymal stem cells, culturing by using a cell growth solution until the cells are 80% fused, and discarding the culture solution;
s3: washing the cultured cells with a phosphate buffer solution, adding a serum-free culture solution, continuously culturing for 70-75 h, and then separating the serum-free culture solution from the cells;
s4: digesting the cells with digestive juice, then carrying out ultrasonic disruption, then centrifuging and collecting supernatant;
s5: and mixing the serum-free culture solution obtained in the step S3 with the supernatant in the step S4, filtering, collecting filtrate and concentrating to obtain the mesenchymal stem cell extract.
2. The extraction method of mesenchymal stem cell extract according to claim 1, wherein the isolation of umbilical cord mesenchymal stem cells comprises the steps of:
SS 1: mixing the cord blood with a PBS buffer solution according to the volume ratio of 1: 3-5, then dripping a lymphocyte separation solution with the same volume as the cord blood, and centrifuging for 10-20 min at 600-750 g;
SS 2: extracting the leucocyte layer cells, washing with a PBS buffer solution, and then resuspending with a basic culture solution;
SS 3: smearing the nonspecific adherence-promoting protein and the specific adherence-promoting protein on the bottom of a culture dish, then inoculating the resuspended white membranous layer cells into the culture dish, and culturing at 37 ℃ under the hypoxia condition;
SS 4: carrying out subculture when the fusion rate of the primary cells reaches 60%;
SS 5: continuously culturing the umbilical cord blood mesenchymal stem cells for passage at 37 ℃ under the hypoxia condition, and performing passage after the cell fusion rate reaches 80%;
SS 6: and collecting the umbilical cord blood mesenchymal stem cells to obtain the umbilical cord blood mesenchymal stem cells.
3. Extraction method of mesenchymal stem cell extract according to claim 2, characterized in that the basic culture medium comprises the following components in volume concentration:
MCDB 13150-80 mL/L, human serum albumin 60-90 mul/L, lactoferrin 200-300 mul/L, dexamethasone 10-15 mul/L, natamycin 20-30U/L and penicillin 90-120U/L.
4. The extraction method of mesenchymal stem cell extract according to claim 2, wherein: the non-specific adherence-promoting protein is fibronectin; the specific anchorage-promoting protein is a CD90 monoclonal antibody.
5. The extraction method of mesenchymal stem cell extract according to claim 2, wherein: the hypoxic conditions are oxygen 15%, carbon dioxide 5% and nitrogen 80%.
6. The extraction method of mesenchymal stem cell extract according to claim 1, wherein the cell growth solution comprises the following components by volume percentage:
15% of compound amino acid solution, 5% of vitamin solution, 10% of human serum albumin solution, 10% of hydrolyzed protein solution, 2% of trace element solution and 58% of electrolyte solution.
7. The extraction method of mesenchymal stem cell extract according to claim 1, wherein: the serum-free culture solution consists of a basic culture solution and an additive; the basic culture solution is DMEM/F12, and the additives comprise L-glutamine, L-ascorbic acid, sodium selenite, fibronectin, hydrocortisone, human insulin, bFGF, coenzyme A and sodium pyruvate.
8. The extraction method of mesenchymal stem cell extract according to claim 7, wherein: the concentration of L-glutamine in the basic culture solution is 1-3 mM, the concentration of L-ascorbic acid is 10-30 mg/L, the concentration of sodium selenite is 15-17 mu g/L, the concentration of fibronectin is 40-50 mg/L, the concentration of hydrocortisone is 8-12 mg/L, the concentration of human insulin is 10-15 mg/L, the concentration of bFGF is 15-20 mu g/L, the concentration of coenzyme A is 90-100 mg/L, and the concentration of sodium pyruvate is 1-5 mM.
9. The mesenchymal stem cell extract obtained by adopting the extraction method of any one of claims 1 to 8.
10. Use of the mesenchymal stem cell extract of claim 9 for the preparation of a cosmetic preparation.
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