CN112063582A - Culture method of mesenchymal stem cells of hair follicle - Google Patents
Culture method of mesenchymal stem cells of hair follicle Download PDFInfo
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- 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
- C12N5/0666—Mesenchymal stem cells from hair follicles
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
Abstract
The invention discloses a method for culturing mesenchymal stem cells of hair follicles, which belongs to the field of biology and comprises the following steps: step one, obtaining a primary hair follicle tissue block; step two, inoculating the obtained primary hair follicle tissue block into a cell culture medium for culture treatment; step three, performing biological identification on the amplified cells, and displaying the result: (1) cell adherence; (2) cell phenotype CD44+≥95％，CD105+≥95％，CD45+≤2％，CD34+Less than or equal to 2 percent; (3) can be made into fat, bone and cartilage. The invention can effectively improve the proliferation of the mesenchymal stem cells of the hair follicle, improve the amplification capacity of the mesenchymal stem cells of the hair follicle in the culture process, and has the advantages of convenient material taking, less pain in material taking, rich source and simple separation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method of mesenchymal stem cells of hair follicles.
Background
Mesenchymal Stem Cells (MSCs), which were first discovered in bone marrow, have been demonstrated to be present in various tissues and organs of the body, and are a group of pluripotent Stem Cells with multipotent differentiation potential.
Mesenchymal stem cells of dental pulp, fat and peripheral blood have the problems of small cell number, wound, and limited health conditions, and thus cannot be applied to autologous mesenchymal stem cells. Therefore, it is very necessary to find new mesenchymal stem cells and culture them successfully.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a culture method of mesenchymal stem cells of hair follicles, which aims to solve the technical problem of the limitation of the source of the existing autologous mesenchymal stem cells.
The invention discloses a method for culturing mesenchymal stem cells of hair follicles, which comprises the following steps:
step one, obtaining a primary hair follicle tissue block;
step two, inoculating the obtained primary hair follicle tissue block into a cell culture medium for culture treatment;
step three, performing biological identification on the amplified cells, and displaying the result: (1) cell adherence; (2) cell phenotype CD44+≥95％，CD105+≥95％，CD45+≤2％，CD34+Less than or equal to 2 percent; (3) can be made into fat, bone and cartilage.
As a preferred embodiment, the specific operation steps of the first step are as follows:
more than 9 intact hair follicles were extracted, washed and mechanically sheared into small pieces with a tissue cutter.
In a preferred embodiment, in the second step, the cell culture medium is a serum-free medium containing 5% serum replacement.
In a preferred embodiment, in the second step, the cell culture medium contains epidermal growth factor, fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor, and the content ratio of epidermal growth factor, fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor is 1:1:1: 1.
In a preferred embodiment, the content of the epidermal growth factor, the fibroblast growth factor, the platelet-derived growth factor and the vascular endothelial growth factor in the cell culture medium is 10-25 ng/ml.
In a preferred embodiment, in the second step, the culture conditions are: at a temperature of 37 ℃ and CO2The concentration was 5%.
In a preferred embodiment, in step two, the primary hair follicle tissue mass obtained is directly inoculated into a cell culture medium without using trypsin or collagenase in the culture treatment.
In a preferred embodiment, in step two, during the culture treatment, no treatment is performed on the primary tissue mass within 7 days, the fluid is changed by half on day 7, and the crawling out of at least 3 clonal cell masses is realized on day 10. Passage on day 10, followed by every third day.
The invention also provides the hair follicle mesenchymal stem cells obtained by the culture method of the hair follicle mesenchymal stem cells.
The invention has the beneficial effects that:
1. the invention provides a novel hair follicle mesenchymal stem cell and a culture method thereof, which can effectively improve the proliferation of the hair follicle mesenchymal stem cell.
2. According to the invention, the epidermal growth factor, the fibroblast growth factor, the platelet-derived growth factor and the vascular endothelial growth factor are added into the cell culture medium, and the addition proportion of the epidermal growth factor, the fibroblast growth factor, the platelet-derived growth factor and the vascular endothelial growth factor is controlled, so that each growth factor plays a synergistic effect in the amplification of the hair follicle mesenchymal stem cells, and the amplification capacity of the hair follicle mesenchymal stem cells in the culture process is improved.
3. The present invention does not use any pancreatin, collagenase, or the like in the culture process, and the cells are directly inoculated into a cell culture medium for culture. The method has the advantages of avoiding the damage of pancreatin or collagenase to the mesenchymal stem cells and improving the quality of the P0 generation mesenchymal stem cells.
4. In the culture treatment process, no treatment is carried out on the primary tissue blocks within 7 days, the liquid is changed by half on the 7 th day, and the climbing-out of at least 3 clone cell masses is realized on the 10 th day. Passage on day 10, followed by every third day. The beneficial effect of this step is to promote the climbing out of the mesenchymal stem cells from the tissue block and the adherence.
5. The invention also has the following advantages: (1) the materials are convenient to draw, and the pain of drawing the materials is small; (2) the source is rich; (3) the separation is simple.
6. The phenotype identification is carried out on the mesenchymal stem cells of the hair follicle, so that the conclusion is reached: low expression of hematopoietic cell marker CD45 on cell surface+2%) and CD34+Less than or equal to 2 percent, high expression mesenchymal stem cell surface antigen CD105+Not less than 95% and CD44+≥95％。
7. The invention determines the adipogenic differentiation potential of the mesenchymal stem cells of the hair follicle to obtain the conclusion that: the mesenchymal stem cells of the hair follicle obtained by the invention can be differentiated into fat cells; the determination of osteogenic differentiation potential of the mesenchymal stem cells of the hair follicle can be used to draw the conclusion that: the mesenchymal stem cells of hair follicles obtained by the invention can be differentiated into bone cells; the determination of chondrogenic differentiation potential of the mesenchymal stem cells of the hair follicle can be used to draw the conclusion that: the mesenchymal stem cells of the hair follicle obtained by the invention can be differentiated into chondrocytes.
Drawings
FIG. 1 is a diagram of adherent cells from a tissue block of mesenchymal stem cells of hair follicle cultured on day 10 in example 1.
FIG. 2 is a report chart of the flow detection result of IgG1-FITC as an isotype control of cells in test example 1.
FIG. 3 is a report chart of the flow detection results of the isotype control IgG1-PE of the cells in test example 1.
FIG. 4 is a report of the results of flow assay of CD44+ phenotype of cells in test example 1.
FIG. 5 is a report of the results of flow assay for CD34+ phenotype of cells in test example 1.
FIG. 6 is a report of the results of flow assay for CD45+ phenotype of cells in test example 1.
FIG. 7 is a report of the results of flow assay of CD105+ phenotype of cells in test example 1.
Fig. 8 is a view under the microscope for examining the adipogenic differentiation of mesenchymal stem cells of hair follicle in the experimental example.
Fig. 9 is a view under the scope of an osteogenic differentiation assay for mesenchymal stem cells of hair follicle in the experimental example.
Fig. 10 is an under-lens view of the assay for chondrogenic differentiation of mesenchymal stem cells of hair follicle in the experimental example.
Detailed Description
The culture method of the mesenchymal stem cells of the hair follicle can effectively improve the proliferation of the mesenchymal stem cells of the hair follicle, and mainly comprises the following steps:
step one, obtaining a primary hair follicle tissue block;
step two, inoculating the obtained primary hair follicle tissue block into a cell culture medium for culture treatment;
step three, performing biological identification on the amplified cells, and displaying the result: (1) cell adherence; (2) cell phenotype CD44+≥95％，CD105+≥95％，CD45+≤2％，CD34+Less than or equal to 2 percent; (3) can be made into fat, bone and cartilage.
Preferably, the specific operation steps of the first step are as follows:
more than 9 intact hair follicles were extracted, washed and mechanically sheared into small pieces with a tissue cutter.
Preferably, in step two, the cell culture medium is a serum-free medium containing 5% serum replacement.
Preferably, in the second step, the cell culture medium contains epidermal growth factor, fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor, wherein the content ratio of the epidermal growth factor to the fibroblast growth factor to the platelet-derived growth factor to the vascular endothelial growth factor is 1:1:1: 1; wherein the contents of the epidermal growth factor, the fibroblast growth factor, the platelet-derived growth factor and the vascular endothelial growth factor in the cell culture medium are all 10-25 ng/ml. Wherein, the epidermal growth factor is the growth factor discovered at the earliest and plays an important role in regulating the growth, proliferation and differentiation of cells; the fibroblast growth factor can promote the proliferation of mesenchymal stem cells and maintain the multidirectional differentiation capacity of the mesenchymal stem cells; the platelet-derived growth factor is an important mitogen and can stimulate the division and proliferation of various cells; the vascular endothelial growth factor can induce angiogenesis in vivo.
By adding epidermal growth factors, fibroblast growth factors, platelet-derived growth factors and vascular endothelial growth factors into a cell culture medium and controlling the adding proportion of the epidermal growth factors, the growth factors play a synergistic effect in the amplification of the mesenchymal stem cells of the hair follicle, so that the amplification capacity of the mesenchymal stem cells of the hair follicle in the culture process is improved.
Preferably, in the second step, the conditions for culturing the primary mesenchymal stem cells in the cell culture medium are as follows: the culture temperature was 37 ℃ and CO2The concentration was 5%.
In the second step, cells are directly inoculated into a cell culture medium and cultured without using any trypsin, collagenase or the like in the culture treatment. The method has the advantages of avoiding the damage of pancreatin or collagenase to the mesenchymal stem cells and improving the quality of the P0 generation mesenchymal stem cells.
In the second step, in the culture treatment process, no treatment is carried out on the primary tissue blocks within 7 days, half the liquid is changed on the 7 th day, and the climbing-out of at least 3 clonal cell masses is realized on the 10 th day. Passage on day 10, followed by every third day. The beneficial effect of this step is to promote the climbing out of the mesenchymal stem cells from the tissue block and the adherence.
By the culture method of the mesenchymal stem cells of the hair follicle, the cultured mesenchymal stem cells of the hair follicle have higher amplification capacity.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 culture method of mesenchymal stem cells of hair follicle
(1) Isolation culture of primary mesenchymal stem cells
1) Preparation of complete culture medium: serum-free medium + 5% serum substitute +20ng/ml epidermal growth factor +20ng/ml fibroblast growth factor +20ng/ml platelet-derived growth factor +20ng/ml vascular endothelial growth factor.
2) 20 hairs with complete hair follicles were directly pulled out, the centrifuge tube was placed in an ice box, and the tube was put into a centrifuge tube containing 0.1% gentamicin in physiological saline and transported to a laboratory.
3) The hair was cut, washed three times with physiological saline containing 0.1% gentamicin, and then washed three times with physiological saline.
4) The tissue shears mechanically shear.
5) Resuspending the cut tissue with normal saline, centrifuging at 1500rpm for 5min, and removing the supernatant; inoculating into T75 culture flask containing complete culture medium, and standing at 37 deg.C under 5% CO2Culturing in an incubator.
6) No treatment was performed within 7 days.
7) As shown in fig. 1, adherent cell colonies, P0 passage, were observed on day 10.
8) On day 10, P0 passages were digested with 0.25% trypsin and digestion was stopped using complete medium.
9) Filtering the cell suspension by using a 100-micron cell screen, centrifuging at 1500rpm for 5min, and then removing the supernatant; after being resuspended in complete medium, the suspension was transferred to a T75 flask and placed at 37 ℃ with 5% CO2Culturing in an incubator.
Example 2-example 19A method for culturing mesenchymal Stem cells of Hair follicle
The same as in example 1.
Experimental example 1 relevant experimental tests and test results
Phenotypic identification of mesenchymal stem cells of hair follicle
1. The cells from P1 passage in examples 1-19 were digested and counted.
2. Respectively take 106The cells were placed in a flow tube and centrifuged to discard the supernatant.
3. After 100ul PBS was added for resuspension, CD45-FITC, CD44-FITC, CD105-PE and CD34-PE antibodies were added separately and incubated at room temperature in the dark for 20 min.
4. PBS was washed twice and the supernatant was centrifuged off.
5. 0.5ml PBS resuspended cells, the cell phenotype was detected by flow cytometry and at least 10000 cells were analyzed per sample.
6. As shown in FIGS. 2-7, the flow assay results showed that the hematopoietic cell marker CD45 was underexpressed on the cell surface in all the examples+2%) and CD34+Less than or equal to 2 percent, high expression mesenchymal stem cell surface antigen CD105+Not less than 95% and CD44+≥95％。
Second, determination of cell number obtained by culturing mesenchymal stem cells P0 generation and P1 generation
1. The P0 and P1 generation cells of examples 1-19 were digested.
2. Cells were mixed with equal volumes of trypan blue and analyzed by Counstar software.
3. The counting results showed that on average 1.06X 10 per hair follicle could be obtained5The cells of P0 generation can obtain 1.19 × 10 cells of P1 generation7And (4) generation of cells.
Measurement of adipogenic differentiation potential
1. Mesenchymal stem cells of hair follicle in a 2X 10 state4Individual cell/cm2In a 24-well plate.
2. After the cells grow to be completely fused, the culture medium is discarded; washing with PBS twice, removing the original culture medium containing growth factor, and adding adipogenic cell inducing liquid.
3. Half of the adipocyte-inducing solution was replaced every 3 days, and induction was carried out for 14 days.
4. Oil red staining was performed on day 14 and observed under an inverted microscope, as shown in fig. 8, showing: differentiation of mesenchymal stem cells of hair follicle into adipocytes
Determination of osteogenic differentiation potential
1. Mesenchymal stem cells of hair follicle in a 2X 10 state4Individual cell/cm2In a 24-well plate.
2. After the cells grow to 80% fusion, the culture medium is discarded; washing with PBS twice, removing the original culture medium containing growth factor, and adding adipogenic cell inducing liquid.
3. Half of the adipocyte inducing solution was replaced every 3 days, and induction was carried out for 28 days.
4. On day 28, formation of calcium salts was evident microscopically.
5. Alizarin red S staining was performed and observed under an inverted microscope, as shown in fig. 9, and the results showed: mesenchymal stem cells of hair follicle can be differentiated into bone cells.
Fifth, determining differentiation potential of chondroblast
1. 20 μ L of the extract containing 2X 105Dripping the hair follicle mesenchymal stem cells of each cell on the inner side of a plate cover of a 24-hole plate to prepare a cell drop; at intervals of 20. mu.L, the cell droplets did not fuse.
2. Gently snap the plate lid back onto the 24-well plate, allowing the cells to drip onto the plate lid; PBS was added to each well of the 24-well plate to increase the humidity in the wells and prevent evaporation of the cell suspension on the plate lid.
3. On day 2, the cell pellet was aspirated into the centrifuge tube, and chondrogenic induction solution was added, and half of every 3 days was replaced with chondrogenic induction solution, followed by induction for 21 days.
4. After 21 days, the medium was removed and the cell pellet was rinsed 2-3 times with PBS; the induced cell balls were wrapped with filter paper to prevent loss, fixed in 10% neutral formalin, dehydrated and paraffin-embedded. A5 μm paraffin section was prepared, stained with toluidine blue, and observed under a microscope, as shown in FIG. 10, showing: mesenchymal stem cells of hair follicle can be differentiated into chondrocytes.
The invention discloses a culture method of mesenchymal stem cells of hair follicles, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Claims (9)
1. A method for culturing mesenchymal stem cells of hair follicle is characterized by comprising the following steps:
step one, obtaining a primary hair follicle tissue block;
step two, inoculating the obtained primary hair follicle tissue block into a cell culture medium for culture treatment;
step three, performing biological identification on the amplified cells, and displaying the result: (1) cell adherence; (2) cell phenotype CD44+≥95％，CD105+≥95％，CD45+≤2％，CD34+Less than or equal to 2 percent; (3) can be made into fat, bone and cartilage.
2. The method for culturing mesenchymal stem cells of hair follicle according to claim 1, characterized in that the specific operation steps of the first step are as follows:
more than 9 intact hair follicles were extracted, washed and mechanically sheared into small pieces with a tissue cutter.
3. The method for culturing mesenchymal stem cells of hair follicle according to claim 1, wherein in the second step, the cell culture medium is a serum-free medium containing 5% serum substitute.
4. The method for culturing mesenchymal stem cells of hair follicle according to claim 1, wherein in the second step, the cell culture medium contains epidermal growth factor, fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor, and the content ratio of epidermal growth factor, fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor is 1:1:1: 1.
5. The method for culturing mesenchymal stem cells of hair follicle according to claim 4, wherein the content of the epidermal growth factor, the fibroblast growth factor, the platelet-derived growth factor and the vascular endothelial growth factor in the cell culture medium is 10-25 ng/ml.
6. The method for culturing mesenchymal stem cells of hair follicle according to claim 1, wherein in the second step, the culture conditions are as follows: at a temperature of 37 ℃ and CO2The concentration was 5%.
7. The method for culturing mesenchymal stem cells of hair follicle according to claim 1, wherein in the second step, the primary hair follicle tissue mass obtained is directly inoculated into the cell culture medium without using pancreatin or collagenase in the process of culture treatment.
8. The method for culturing mesenchymal stem cells of hair follicle according to claim 1, wherein in the second step, no treatment is performed on the primary tissue mass within 7 days during the culture treatment, the fluid is changed by half on the 7 th day, and at least 3 clonal cell masses are crawled out on the 10 th day. Passage on day 10, followed by every third day.
9. The mesenchymal stem cell of hair follicle obtained by the culture method of mesenchymal stem cell of hair follicle of any one of claims 1 to 8.
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| CN111849883A (en) * | 2020-07-28 | 2020-10-30 | 李鹏东 | Culture medium for in-vitro amplification of autologous human hair follicle mesenchymal stem cells and culture method thereof |
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| CN114958733A (en) * | 2021-07-29 | 2022-08-30 | 北京博百悦生物技术有限公司 | Preparation method of autologous hair follicle mesenchymal stem cells |
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