CN110721199A - Preparation of exosome freeze-dried powder for promoting hair growth - Google Patents
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Abstract
The invention belongs to the technical field of regenerative medicine, and particularly relates to preparation of exosome freeze-dried powder for promoting hair growth. The exosome freeze-dried powder provided by the invention is bioactive freeze-dried powder, is derived from hair follicle stem cells and hair papilla cells, is rich in various bioactive substances required by hair follicle growth, is convenient to store and simple to use, greatly retains exosome activity after freeze-drying, can effectively promote hair growth, and can improve the hair loss problem.
Description
Technical Field
The invention belongs to the technical field of regenerative medicine and biology, and particularly relates to preparation of exosome freeze-dried powder for promoting hair growth.
Background
The hair follicle is a complex micro-organ, and the formation and development of the hair follicle is the result of the interaction between the epithelial and dermal components of the hair follicle, and the growth and regeneration of the hair follicle is regulated by its own stem cells. Among the stem cells present in hair follicles are mainly: hair Follicle Stem Cells (HFSCs), hair papilla cells (DPCs) and melanin stem cells (McSCs), among which hair papilla cells and hair follicle stem cells play an important role in hair follicle morphogenesis and hair cycle. The Hair papilla cells are a group of specially differentiated mesenchymal stem cells, are positioned at the Hair bulb part at the bottom end of a Hair follicle, are surrounded by Hair matrix at the periphery, play an important role in regulating and controlling the morphogenesis of the Hair follicle, the development of the Hair follicle and the cycle circulation process of the Hair follicle, are signal centers in the growth cycle of the Hair follicle, induce the activation of Hair follicle stroma cells (Hair matrix) and Hair follicle stem cells in a bulge area through various signal paths (such as Wnt and BMP) and cytokines at the early growth stage, and push the Hair follicle to enter the growth phase. The hair papilla cells cultured in the low generation have hair follicle induction characteristics, and a large number of researches prove that the hair papilla cells cultured in the low generation can be mixed with epithelial components according to a certain proportion to induce and generate complete hair follicles in a nude mouse body, so that the hair follicles in the tissue engineering open up a new research direction for treating alopecia.
The hair follicle stem cells are a group of adult stem cells which are planted in a hair follicle outer root sheath bulge area (bulbge area), belong to epithelial components, and have the characteristics of slow periodicity, self-renewal, undifferentiated property, strong in vitro proliferation capacity and the like. The hair follicle stem cells can be induced and differentiated into epithelial cells, vascular endothelial cells, melanocytes, smooth muscle cells and the like under certain conditions. In the growth phase, the division and proliferation of hair follicle stem cells are extremely active, and the hair follicle stem cells are differentiated to form outer root sheath cells, move downwards continuously and wrap the hair papilla a little bit. After the hair follicle enters the decline period, most of the epithelial cells of the outer root sheath die, only a small part of the epithelial cells are left to preserve partial stem cell characteristics, and the partial cells divide the inner root sheath, the hair shaft and the like in the next hair follicle cycle. It has been shown that inactivation of hair follicle stem cells and destruction of the hair follicle growth cycle are the root causes of hair loss.
Exosomes (exosomes) are vesicular substances secreted by cells, have a diameter of about 30-100nm, contain various bioactive substances, play an important role in intercellular signaling, and have been reported in a large number of fields. In the direction of hair follicle research, it has been demonstrated that exosomes promote entry of rat dorsal hair from telogen phase into anagen phase. But the exosome needs to be stored at low temperature and transported at low temperature, and the storage time cannot be too long, otherwise, the activity of the exosome is greatly reduced, and the function exertion of the exosome is influenced.
In view of the reasons, the exosome in the hair papilla cell culture supernatant is extracted, the exosome is prepared into the hair follicle stem cell conditioned medium to culture the hair follicle stem cells, the hair follicle stem cell culture supernatant is obtained, the hair follicle stem cell exosome is extracted to prepare the lyophilized powder, the bioactive substances of the lyophilized powder are greatly reserved, and the lyophilized powder only needs to be stored and transported at normal temperature, so that the practicability and the convenience are greatly improved.
Disclosure of Invention
The invention aims to provide a preparation method of exosome freeze-dried powder for promoting hair growth, which is characterized in that extracted hair papilla cell exosomes are added into a hair follicle stem cell culture solution, hair follicle stem cell culture supernatant is collected, and the hair follicle stem cell culture supernatant is freeze-dried after ultrafiltration concentration and is used for promoting hair follicle development and helping hair growth.
In order to solve the above problems, the present invention provides a method for preparing exosome freeze-dried powder for promoting hair growth, comprising the following steps:
1) culture of hair papilla cells and exosome extraction
A. Hair papilla cell culture and supernatant collection: selecting low generation human dermal papilla cells according to 3-5 × 105Inoculating at a density of 75cm2Collecting the culture supernatant of the hair papilla cells when the cells are cultured to the cell density of 85-90% in a cell culture bottle, centrifuging, removing cell debris, filtering and sterilizing the supernatant by a 0.22 mu m filter membrane, and storing at low temperature for later use;
B. extracting hair papilla cell exosomes: centrifuging the culture supernatant of the papilla pili cells obtained in the step 1) at 10000-.
Preferably, the low generation means that the generation number of hair papilla cells does not exceed P6 generation;
preferably, the dermal papilla cell culture medium is an alpha-MEM medium containing 8% -10% FBS;
preferably, the cell culture supernatant is centrifuged at 2000rpm for 5min to remove cell debris, leaving the supernatant.
2) Culture of hair follicle stem cells and extraction of exosomes
A. Preparing a hair follicle stem cell conditioned medium: adding the hair papilla cell exosome obtained in the step 1) into a hair follicle stem cell culture solution according to the adding proportion of 15% -25%, and preparing a hair follicle stem cell conditioned culture solution;
B. culturing hair follicle stem cells and collecting a culture solution: according to the conventional culture process, the human hair follicle stem cells within the P10 generation are cultured according to the ratio of 0.5-1 × 105Is inoculated at a density of 75cm2Adding a hair follicle stem cell conditioned medium into the cell culture bottle for culture, collecting cell culture supernatant when the cell density is 80-85%, centrifuging to remove cell debris, and filtering by 0.22 mu m for later use;
C. extraction of hair follicle stem cell exosomes: and D, transferring the culture supernatant of the hair follicle stem cells obtained in the step B to an ultrafiltration concentration tube with the density of 100KD, and centrifuging at a high speed at a low temperature to obtain a filtrate, namely the concentrated solution rich in the exosomes of the hair follicle stem cells.
Preferably, the hair follicle stem cell culture solution is a commercial K-SFM culture medium without serum;
preferably, the collected culture supernatant needs to be centrifuged to remove cell debris, and the centrifugation condition is 1000rpm centrifugation for 5 min;
preferably, the ultrafiltration concentration tube needs to be centrifuged at 3000-10000 Xg for 30-60min at 4 ℃, and the obtained filtrate is concentrated solution rich in exosome.
3) Freeze-drying: adding freeze-drying protective agent into the concentrated solution according to a proper proportion, mixing uniformly, balancing at 37 ℃ for 20-50min, filtering at 0.22 mu m, subpackaging penicillin bottles, adding 2mL of liquid into each 7mL penicillin bottle, half adding a rubber plug after subpackaging, pre-freezing at-80 ℃ for 12h, freeze-drying in a freeze dryer, and storing the obtained freeze-dried powder at normal temperature for later use
Preferably, the freeze-drying protective agent is trehalose, and is added in the same volume with the concentrated solution, and the final adding proportion is 10%;
preferably, consumables such as penicillin bottles and silicone tubes used for freeze-drying need to be sterilized by high-pressure steam in advance;
preferably, the freeze-dried powder obtained after freeze drying has the appearance ranging from white to light pink, and the product has the advantages of full appearance, smooth surface, no atrophy, uniform color and good porosity.
The exosome freeze-dried powder for promoting hair growth provided by the invention can be stored at room temperature, and can be used after being dissolved by adding a proper amount of water aqua.
The invention has the beneficial effects that:
1. the exosome freeze-dried powder for promoting hair growth is prepared by freeze-drying exosomes in human hair follicle stem cell culture solution, so that the activity of the exosomes is greatly preserved, and the exosome freeze-dried powder has the advantages of being convenient to store at room temperature and transport.
2. The exosome freeze-dried powder for promoting hair growth provided by the invention is rich in bioactive substances required by hair follicle growth, and can be used for preventing and treating alopecia and helping hair growth.
Drawings
FIG. 1 is a schematic diagram showing the growth of hair follicle stem cells in a conditioned medium of hair follicle stem cells;
FIG. 2 is a schematic diagram of the obtained lyophilized powder of exosomes of hair follicle stem cells;
Detailed Description
The present invention is further described in detail below with reference to specific examples and figures to enable one skilled in the art to practice the invention with the aid of the description text.
Example 1: dermal papilla cell culture and exosome extraction
1. Reviving and culturing hair papilla cells: taking out low-generation papilla cells from liquid nitrogen tank, quickly placing into 37 deg.C water bath, gently shaking for dissolving, and adding into 8ml PBS buffer solutionCentrifuging at 800rpm for 5min, discarding supernatant, suspending cells in hair papilla cell culture medium, and culturing at 5 × 105Inoculating at a density of 75cm2Placing in a cell culture flask at 37 deg.C and 5% CO2Culturing in an incubator, and changing the culture solution every other day;
2. collecting the hair papilla cell culture supernatant: collecting cell culture supernatant to 50mL centrifuge tube when the growth density of hair papilla cells reaches 85% -90%, centrifuging at 2000rpm for 5min, collecting supernatant, filtering the supernatant with 0.22 μm filter membrane for sterilization, collecting filtrate, and storing;
3. extraction of exosomes: transferring the filtrate obtained in the step 2 to a 50ml centrifuge tube, centrifuging at 12000 Xg for 1h at the temperature of 4 ℃, removing the supernatant, leaving a precipitate, adding PBS buffer solution into the precipitate, and suspending to obtain the hair papilla cell exosome.
Example 2: preparation of hair follicle stem cell exosome freeze-dried powder
1. Preparing a hair follicle stem cell conditioned medium: taking out hair papilla cell exosomes stored at low temperature, adding the hair papilla cell exosomes into a commercially available K-SFM culture medium according to the proportion of 15%, and uniformly mixing to prepare a hair follicle stem cell conditioned medium;
2. recovering and culturing hair follicle stem cells: taking out P4 generation hair follicle stem cells from liquid nitrogen tank, rapidly placing into 37 deg.C water bath, gently shaking for dissolving, adding into 8ml PBS buffer solution, centrifuging at 800rpm for 5min, removing supernatant, adding hair follicle stem cell conditioned medium suspension cells, and processing according to 1 × 106Inoculating at a density of 75cm2Placing in a cell culture flask at 37 deg.C and 5% CO2Culturing in an incubator, and changing the culture solution every other day;
3. collecting hair follicle stem cell culture supernatant: when the growth density of the hair follicle stem cells reaches 80% -85%, collecting cell culture supernatant to a 50mL centrifuge tube, centrifuging at 2000rpm for 5min, collecting supernatant, filtering the supernatant with a 0.22 μm filter membrane for sterilization, collecting filtrate, and storing;
4. extracting hair follicle stem cell exosomes: transferring the hair follicle stem cell filtrate obtained in the step 3 into a 100KD ultrafiltration concentration tube with the volume of 50mL, centrifuging at 4000 Xg for 30min at the temperature of 4 ℃, and collecting a permeate, namely a concentrated solution rich in hair follicle stem cell exosomes;
5. freeze-drying hair follicle stem cell exosome: weighing 50g of trehalose, stirring and dissolving in 5L of purified water, weighing an equal amount of hair follicle stem cell concentrated solution, uniformly mixing with the trehalose solution, placing the mixed solution at 37 ℃ for balancing 40min, filtering by 0.22 mu m, then subpackaging by penicillin bottles, adding 2mL of liquid into each 7mL of penicillin bottles, adding a rubber plug after subpackaging, pre-freezing at-80 ℃ for 12h, adjusting the temperature of a cold trap of a freeze dryer to-50 ℃, and carrying out freeze drying treatment for 24h to obtain the hair follicle stem cell exosome freeze-dried powder, wherein the vacuum pressure is less than 20 Pa.
The result shows that the obtained exosome freeze-dried powder is white, and the product has the advantages of full appearance, smooth surface, no atrophy, uniform color and good porosity (figure 2).
Claims (10)
1. The utility model provides a preparation of exosome freeze-dried powder of promotion hair growth, includes three steps of the cultivation of human hair papilla cell and the extraction of exosome, the cultivation of hair follicle stem cell and the extraction of exosome and the preparation of hair follicle stem cell exosome freeze-dried powder, its characterized in that:
the method comprises the following steps: the human hair papilla cell culture and exosome extraction method comprises the following steps:
1) culturing hair papilla cells and collecting supernatant: selecting low-generation human hair papilla cells, performing conventional culture, and aseptically collecting cell culture supernatant when the cells grow to the density of 85% -90%;
2) extracting hair papilla cell exosomes: extracting exosome in culture supernatant by adopting a low-temperature ultracentrifugation method, suspending exosome sediment in a culture medium, and storing at low temperature;
step two: the extraction of the human hair follicle stem cells and the exosomes comprises the following steps:
1) preparation of hair follicle stem cell conditioned medium: adding the hair papilla cell exosomes obtained in the step one into a hair follicle stem cell culture medium in a proper proportion to obtain a hair follicle stem cell conditioned medium;
2) culturing hair follicle stem cells and collecting supernatant: selecting human hair follicle stem cells within P10 generation, adding the human hair follicle stem cells into a hair follicle stem cell culture medium for culture, collecting cell culture supernatant when the cells grow to 80-85%, and filtering and sterilizing for later use;
3) extraction of hair follicle stem cell exosomes: centrifuging the filtered and sterilized cell culture supernatant at a low temperature by an ultrafiltration concentration centrifuge tube to obtain a hair follicle stem cell concentrated solution rich in exosomes;
step three: the preparation method of the hair follicle stem cell exosome freeze-dried powder comprises the following steps:
1) weighing a proper amount of freeze-drying protective agent, uniformly mixing the freeze-drying protective agent with the exosome-rich hair follicle stem cell concentrated solution obtained in the step two, balancing the mixture at 37 ℃ for 20-50min, filtering the mixture by 0.22 mu m, subpackaging penicillin bottles, adding 2mL of liquid into each 7mL of penicillin bottles, and adding a rubber plug into the subpackaged mixture;
2) pre-freezing, and freeze-drying in a freeze dryer to obtain freeze-dried powder.
2. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: and (3) performing conventional culture on the hair papilla cells in the step one, wherein the culture medium is alpha-MEM culture medium + 8% -10% FBS, and the cells can be subjected to continuous passage to collect supernatant.
3. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: the cell supernatant collected in the first step needs to be filtered and sterilized by a 0.22 mu m filter membrane, and then the subsequent exosome can be extracted.
4. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: the extraction of the hair papilla exosomes in the step one is specifically performed by centrifuging 10000-.
5. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: in the hair follicle stem cell conditioned medium in the second step, the adding proportion of the hair papilla cell exosome suspension is 15-25%.
6. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: and step two, extracting the hair follicle stem cell exosomes, wherein the specific operation is that the filtered and sterilized cell culture supernatant is transferred to an ultrafiltration concentration tube with the density of 100KD, and the cell culture supernatant is centrifuged for 30-60min at the temperature of 4 ℃ and at the temperature of 3000 plus 10000 Xg, so as to obtain the concentrated solution rich in the hair follicle stem cell exosomes.
7. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: the freeze-drying protective agent in the third step is trehalose solution, and the final concentration is 10-15%.
8. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: before use, the penicillin bottle in the third step needs to be autoclaved at the temperature of 121 ℃ and under the pressure of 0.1MPa for 30min, and then is dried for use.
9. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: the freeze-drying step in the third step is specifically pre-freezing at-80 ℃ for 12h, then setting the temperature of a cold trap of a freeze dryer to-50 ℃, and carrying out freeze-drying treatment for 24h under the vacuum pressure of less than 20 Pa.
10. Preparation of a lyophilized powder of exosomes for promoting hair growth according to claim 1, characterized by: the exosome freeze-dried powder is rich in various active factors for promoting hair follicle growth, and can effectively help hair follicle development and promote hair growth.
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| CN114392303A (en) * | 2022-02-17 | 2022-04-26 | 浙江卫未生物医药科技有限公司 | Exosome mixture for treating alopecia areata |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111411078A (en) * | 2020-04-21 | 2020-07-14 | 清远市人民医院 | Method for rapidly obtaining hair follicle cell source exosomes |
| CN111484969A (en) * | 2020-05-07 | 2020-08-04 | 内蒙古农业大学 | Application of hair follicle stem cell source exosome in promoting hair follicle stem cell proliferation and differentiation to hair follicle cells |
| CN111484969B (en) * | 2020-05-07 | 2023-04-07 | 内蒙古农业大学 | Application of hair follicle stem cell source exosome in promoting hair follicle stem cell proliferation and differentiation to hair follicle cells |
| CN113197918A (en) * | 2021-05-08 | 2021-08-03 | 奥镁(上海)医药科技有限公司 | Method and application of stem cell-based hair regeneration technology |
| CN114392303A (en) * | 2022-02-17 | 2022-04-26 | 浙江卫未生物医药科技有限公司 | Exosome mixture for treating alopecia areata |
| CN114392303B (en) * | 2022-02-17 | 2023-08-29 | 浙江卫未生物医药科技有限公司 | Exosome mixture for treating alopecia areata |
| CN114558031A (en) * | 2022-03-21 | 2022-05-31 | 南华大学 | Preparation method and application of exosome freeze-dried powder |
| CN114917250A (en) * | 2022-05-06 | 2022-08-19 | 广东发亿科技有限公司 | Application of fibroblast outer vesicles hFB-EVs in alopecia treatment product |
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