CN110564675A - Separation and extraction method of hair follicle stem cells - Google Patents
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Abstract
The invention discloses a hair follicle stem cell separation and extraction method, which is characterized in that hair follicle stem cells are extracted by a secondary enzymolysis method, wherein pancreatin substitutes and collagenase IV are respectively adopted for secondary enzymolysis, the hair follicle cells are subjected to enzymolysis by the pancreatin substitutes for the first time, and the hair follicle cells are further subjected to enzymolysis by the collagenase IV for the second time. According to the invention, the hair follicle tissue is subjected to secondary enzymolysis by adopting a double-enzyme enzymolysis method, so that stem cells in the tissue are further released, the extraction quantity of the hair follicle stem cells is increased, and meanwhile, the damage to the hair follicle stem cells is reduced and the survival rate of the hair follicle stem cells is increased by adopting the enzyme with mild property.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a separation and extraction method of hair follicle stem cells.
Background
Hair Follicle Stem Cells (HFSCs) are adult stem cells that are present in the ectothecal root sheath and have the biological properties of self-renewal capacity and long cycle length. Under normal conditions, hair follicle stem cells do not participate in the self-renewal of the epidermal layer of the skin. However, when the skin is wounded, the hair follicle stem cells can be activated to migrate to the wound edge to help repair the wound and regenerate the skin. As an important stem cell for promoting wound healing, the hair follicle stem cell has strong proliferation capacity and multidirectional differentiation potential like other adult stem cells. The hair follicle stem cells can generate various cells required by organisms through the division and proliferation of the hair follicle stem cells so as to supplement the fallen and lost cells; the hair follicle stem cells can not only be transferred downwards to the root of the hair follicle to generate the hair follicle, but also can be transferred upwards from an outer root sheath of the hair follicle to generate epidermis and sebaceous gland, and can be differentiated to form bone and fat. In recent years, the research on hair follicle stem cells has attracted much attention and has become one of the hot spots.
At present, the separation and extraction methods of hair follicle stem cells respectively comprise: enzymolysis, tissue mass culture, micro-separation, differential adherence, immunomagnetic bead, and flow cytometry sorting; the above methods have different advantages and disadvantages, but none of them can overcome the characteristics of the hair follicle stem cells such as the appearance of the proliferation and easy differentiation of the cells during the in vitro proliferation. For example: the hair follicle stem cells are extracted by enzymolysis by using neutral protease, the enzymolysis effect is strong, the enzymolysis time is long, the damage to the cells is serious, and the cell in-vitro proliferation is not facilitated. The differential wall pasting method is adopted to avoid that a certain amount of mixed cells cannot be mixed, and the purity content of the hair follicle stem cells is not high in the subsequent culture process. The extraction process using the micro-separation technology requires the extraction part to be precise and accurate, has high requirements on the skills of technicians, and is not favorable for stable process establishment. The immunomagnetic bead method and the flow sorting method can obtain high-purity hair follicle stem cells, but the experiment cost is high, and the obtained cells are few.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for separating and extracting hair follicle stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
The hair follicle stem cell separation and extraction method provided by the invention is to extract the hair follicle stem cells by a secondary enzymolysis method, wherein the secondary enzymolysis method respectively adopts pancreatin substitute and collagenase IV, the first enzymolysis selects the pancreatin substitute to carry out enzymolysis on the hair follicle cells, and the second enzymolysis selects the collagenase IV to carry out enzymolysis on the hair follicle cellsCarrying out further enzymolysis; wherein the pancreatin substitute is Mpbio provided ACCUTASETMIs a replacement for pancreatin/EDTA, with protease and collagenase activity.
Preferably, the concentration of the pancreatin substitute is 0.125-0.3%, and the concentration of collagenase IV is 0.125-0.5%.
Preferably, the concentration of the pancreatin substitute is 0.125% and the concentration of collagenase iv is 0.25%.
Preferably, the conditions of the enzymatic hydrolysis reaction are as follows: the enzymolysis temperature is 37 ℃; the enzymolysis time is 10-30 min.
Preferably, the secondary enzymolysis method comprises the following specific steps: firstly, adding the obtained hair follicle tissues into a cell culture solution containing 0.125% pancreatin substitute, placing the mixture in a constant-temperature shaking table at 37 ℃ for 200 revolutions, performing enzymolysis for 10-20 min, centrifuging for 5-10 min at 300-800g after enzymolysis, and removing supernatant; then adding the mixture into a cell culture solution containing collagenase IV with the concentration of 0.25%, placing the mixture in a constant-temperature shaking table at 37 ℃ and 200R for enzymolysis for 10-20 min, centrifuging for 5-10 min at 300-800g after enzymolysis, and removing supernatant to obtain hair follicle stem cells.
Preferably, the method for separating and extracting the hair follicle stem cells further comprises the following steps: inoculating the hair follicle stem cells treated by the enzyme, culturing, changing the liquid once every 2-3 days until the cell fusion rate is about 85% -95%, and carrying out cell passage.
Preferably, the culture medium selected in the culture process is an alpha-MEM as a basic culture medium, and the following components are added: PALL serum substitute, 1 Xglutamine, 10-20 ng/mL VEGF, 1 XQing-streptomycin; wherein the PALL serum is called completely Ultroser G serum substitete serum substitute, 20G/bottle, 2 bottles of alpha-MEM basal medium can be configured, the final concentration is 0.1G/mL, and the brand: pall, cat # s: 15950-017, purchased from Wayasi bio corporation, Shanghai; glutamine was purchased from shanghai huayasi bio-corporation, brand: lonza, cat # s: 17-605E L, original concentration 100 ×, final concentration 1 ×; qing-streptomycin was purchased from shanghai hua yasi bio-corporation, brand: lonza, original concentration 100X, final concentration 1X was used.
The invention has the beneficial effects that: according to the invention, the hair follicle tissue is subjected to secondary enzymolysis by adopting a double-enzyme enzymolysis method, so that stem cells in the tissue are further released, the extraction quantity of the hair follicle stem cells is increased, and meanwhile, the damage to the hair follicle stem cells is reduced and the survival rate of the hair follicle stem cells is increased by adopting the enzyme with mild property. Therefore, the composition and extraction conditions of the enzymatic hydrolysate in the method become important factors. In the invention, the inventor carries out screening optimization on the type and the proper concentration of the selected enzyme and the enzymolysis time to obtain the optimal technical scheme of the secondary enzymolysis method. The method has large extraction quantity, and the survival rate of target cells, namely hair follicle stem cells, is remarkably improved; in addition, the pollution rate of the cells extracted by the method is extremely low; the whole operation engineering is simple and effective, and the separation and extraction efficiency is obviously improved.
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FIG. 1 is a schematic diagram of flow-based result detection.
FIG. 2 is a schematic diagram of flow-based result detection.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1
1) Preservation of hair follicles: the hair with 10 hair follicles was pulled out, and the ends of the hair follicles were cut with scissors and then stored in a storage solution of about 1cm at 4 ℃ for 2 hours. Hair follicle preservation solution: 1640 basic medium contains 1x-2x double antibody, 3mg/mL vitamin C.
2) Hair follicle harvesting: primarily cleaning hair with hair follicle with 0-4 deg.C cleaning solution, clamping the hair with ophthalmologic forceps, cutting off hair follicle with ophthalmologic forceps close to hair follicle end, and discarding the redundant hair.
3) Enzymolysis: transferring the cleaned hair follicle to a 15mL centrifuge tube by using ophthalmic forceps, adding 5mL pancreatin substitute, and placing in a constant temperature shaking table at 37 ℃ for enzymolysis for 10min at 200R; centrifuging for 5-10 min at 300-800g, and discarding the supernatant; adding 5mL of 0.25% collagenase IV, placing in a constant temperature shaking table at 37 ℃, performing enzymolysis for 10min at 200R, centrifuging for 5-10 min at 300-800g, and discarding the supernatant. Enzymolysis liquid: the 1640 basic medium contains 0.125% of pancreatin substitutes and the 1640 basic medium contains 0.25% of collagenase IV.
4) Inoculation: suspending cell sediment by using an adherence promoting agent of mesenchymal stem cells and an alpha-MEM complete culture medium, and inoculating the cell sediment into a six-hole plate; 5 follicles/well; a total of 2 wells were inoculated. α -MEM complete Medium: alpha-MEM basal medium, PALL serum substitute, 1 Xglutamine, 10 ng-20/mL VEGF, 1 Xqing-streptomycin
5) Cell culture: after inoculation for 48 hours, liquid is changed; cell colonies were visible; and changing the liquid every 2-3 days until the cell fusion rate is 85-95%, and carrying out cell passage.
The invention also provides examples 2-5 and comparative examples 1-4, wherein the enzymolysis concentration and the enzymolysis time are different from those of the example 1, the other steps and parameters are the same as those of the example 1, and the parameters of the examples 1-5 and the comparative examples 1-4 are shown in the table 1.
Table 1: examples 1 to 5 and comparative examples 1 to 4 parameters
Note: the enzymolysis sequence in the embodiments 1 to 5 is to use pancreatin substitute for enzymolysis first, and then use collagenase IV for enzymolysis; in the table, "first" indicates that collagenase iv was used for the treatment in comparative examples 3 and 4, and "second" indicates that pancreatin substitute was used for the treatment in comparative examples 3 and 4, and the effect of the different enzyme sequences of the pancreatin substitute and collagenase iv on the extraction and separation of hair follicle stem cells in the secondary enzymatic hydrolysis method of the present invention was verified by the comparative examples 3 and 4.
Example 6 quality evaluation of Hair follicle Stem cells
The hair follicle stem cells obtained by the preparation methods of examples 1 to 5 and comparative examples 1 to 4 were subjected to quality evaluation, and the specific experimental method was as follows.
1. And (3) detecting the activity and the volume:
Taking freshly separated placental stem cells, adjusting cell density to 1 × 106cell/mL. According to the cell suspension: 0.4% trypan blue 3:1(v: v) was mixed well, and 20. mu.L of the cells were mixedThe homogenate was added to a cell counting plate, and the cell viability and volume were measured with a Countstar cell counter. Number extraction and viability assay of hair follicle stem cells P0, table 2 below:
Table 2: results of detecting vitality and volume of hair follicle stem cells obtained by preparation methods of examples 1-5 and comparative examples 1-4
From the above table, it can be seen that the hair follicle stem cell P0The number of generation cells obtained was 2.35X 106cells, number of viable cells 2.25X 106The cell viability is up to 95.88%; the cell activity rate and activity of the cell extracted by the double-enzyme method are high.
2. Wall adhesion rate detection
After the mesenchymal stem cells of the hair follicle are separated and cultured, the density is adjusted to be 2 multiplied by 105cells/mL, inoculated 8mL into a 9cm diameter dish (T ═ 1.6X 106cells) after the cells are naturally attached to the wall for 48 hours, after the nonattached cells are removed, enzymolysis is carried out by using 0.25% trypsin, the number of the attached cells is calculated, and the attachment rate is obtained, and the results are shown in table 3.
Table 3: hair follicle stem cell anchorage rate detection method obtained by preparation methods of examples 1-5 and comparative examples 1-4
| Grouping | Number of adherent cells (X10) at 48h6) | Wall sticking rate |
| Example 1 | 1.80±0.27 | 76.88％ |
| Example 2 | 1.65±0.18 | 68.46％ |
| Example 3 | 1.73±0.36 | 72.99％ |
| Example 4 | 1.77±0.29 | 72.84％ |
| Example 5 | 1.69±0.45 | 70.42％ |
| Comparative example 1 | 1.27±0.34 | 51.63％ |
| Comparative example 2 | 1.39±0.46 | 57.44％ |
| Comparative example 3 | 1.24±0.27 | 48.63％ |
| Comparative example 4 | 118±0.41 | 48.76％ |
3. Streaming result detection
Taking hair follicle stem cells in logarithmic growth phase, and regulating cellsDensity of 1X 106The cell suspension of cells is prepared by respectively taking 2.5 mu L of monoclonal antibodies of anti-human CD105, CD90, CD73, CD45, CD34 and HLA-DR, adding 500 mu L of cell suspension, incubating for 20min at room temperature in a dark place, simultaneously establishing a control in the same row, centrifuging for 5min at 1500r/min, discarding the supernatant, washing for 2 times with PBS containing 10% FBS, resuspending with 500 mu L of PBS, and detecting on a machine after 1640 resuspension, wherein the results are shown in figures 1 and 2.
From the flow results of fig. 1 and 2, the endometrial stem cells highly express CD73, CD90 and CD105, and the expression rates are 99.1%, 99.2% and 99.2%, respectively; low expression of HLA-DR, CD45 and CD34 at 0.2%, 0.2% and 0.2%, respectively. The surface antigen characteristics of the mesenchymal stem cells (the identification standard of the mesenchymal stem cells issued by the International Stem cell society ISCT, namely positive expression of CD73, CD90 and CD105 is more than 95.0 percent, negative expression of HLA-DR and CD45 is less than 2.0 percent) are met, the phenomenon that the endometrial stem cells are differentiated does not occur, and the characteristics of the stem cells are still maintained.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. The method is characterized in that the hair follicle stem cells are extracted by a secondary enzymolysis method, wherein pancreatin substitutes and collagenase IV are respectively adopted in the secondary enzymolysis, the hair follicle cells are subjected to enzymolysis by the pancreatin substitutes for the first time, and the hair follicle cells are further subjected to enzymolysis by the collagenase IV for the second time.
2. The method for separating and extracting hair follicle stem cells as claimed in claim 1, wherein the concentration of the pancreatin substitute is 0.125-0.3%, and the concentration of collagenase IV is 0.125-0.5%.
3. The method for separating and extracting hair follicle stem cells as claimed in claim 1, wherein the concentration of the pancreatin substitute is 0.125%, and the concentration of collagenase iv is 0.25%.
4. The method for separating and extracting the hair follicle stem cells as claimed in any one of claims 1 to 3, wherein the pancreatin substitute is Mpbio under the product number ACCUTASETMThe product of (1).
5. The method for separating and extracting hair follicle stem cells as claimed in claim 1, wherein the conditions of the enzymatic hydrolysis reaction are as follows: the enzymolysis temperature is 37 ℃; the enzymolysis time is 10-30 min.
6. The method for separating and extracting the hair follicle stem cells as claimed in claim 1, wherein the secondary enzymolysis method comprises the following specific steps: firstly, adding the obtained hair follicle tissues into a cell culture solution containing 0.125% pancreatin substitute, placing the mixture in a constant-temperature shaking table at 37 ℃ for 200 revolutions, performing enzymolysis for 10-30 min, centrifuging for 5-10 min at 300-800g after enzymolysis, and removing supernatant; then adding the mixture into a cell culture solution containing collagenase IV with the concentration of 0.25%, placing the mixture in a constant-temperature shaking table at 37 ℃ and 200R for enzymolysis for 10-30 min, centrifuging for 5-10 min at 300-800g after enzymolysis, and removing supernatant to obtain hair follicle stem cells.
7. the method for separating and extracting hair follicle stem cells as claimed in claim 1, wherein the method for separating and extracting hair follicle stem cells further comprises the following steps: inoculating the hair follicle stem cells treated by the enzyme, culturing, changing the liquid every 2-3 days until the cell fusion rate is 85% -95%, and carrying out cell passage.
8. The method for separating and extracting hair follicle stem cells as claimed in claim 1, wherein the culture medium selected in the culture process is an alpha-MEM (alpha-MEM) basal medium, and the following components are added: PALL serum substitute, 1 Xglutamine, 10-20 ng/mL VEGF, 1 XQing-streptomycin.
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Cited By (5)
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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 |
| CN112063582A (en) * | 2020-08-20 | 2020-12-11 | 吉林省银丰生物工程技术有限公司 | Culture method of mesenchymal stem cells of hair follicle |
| CN112280732A (en) * | 2020-10-21 | 2021-01-29 | 成都博润白癜风医院有限公司 | Suspension of external root sheath of hair follicle and preparation method thereof |
| CN112877276A (en) * | 2021-02-09 | 2021-06-01 | 北京生源充能生物科技有限公司 | Hair follicle cell extraction method |
| WO2022247848A1 (en) * | 2021-05-26 | 2022-12-01 | 上海我武干细胞科技有限公司 | Preparation method for and application of hair follicle mesenchymal stem cell |
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