CN113813289A - Preparation method of hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosome - Google Patents
Preparation method of hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosome Download PDFInfo
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- CN113813289A CN113813289A CN202111043984.8A CN202111043984A CN113813289A CN 113813289 A CN113813289 A CN 113813289A CN 202111043984 A CN202111043984 A CN 202111043984A CN 113813289 A CN113813289 A CN 113813289A
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- umbilical cord
- mesenchymal stem
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- stem cell
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0627—Hair cells
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- C12N5/0602—Vertebrate cells
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Abstract
The invention discloses a preparation method of a hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: s1: disinfecting and pretreating an umbilical cord tissue sample, cutting the umbilical cord tissue sample into tissue fragments, and storing the tissue fragments for later use; s2: adding modified DMEM culture solution, placing in a constant temperature incubator, and changing the solution once every 2d half amount and once every 3d full amount after three times; s3: carrying out subculture amplification culture; s4: extracting to obtain an umbilical cord mesenchymal stem cell exosome; s5: collecting umbilical cord mesenchymal stem cells of the fifth generation; s6: and (4) carrying out mixed culture on the umbilical cord mesenchymal stem cell exosome obtained in the step (S4), the fifth-generation umbilical cord mesenchymal stem cell obtained in the step (S5) and the hair papilla cell to obtain the hair follicle generation promoting fluid. The hair follicle generation promoting liquid can effectively improve hair follicle degradation, atrophy, alopecia areata and other symptoms, and has obvious and more lasting hair regeneration promoting effect.
Description
Technical Field
The invention relates to the technical field of extraction of umbilical cord mesenchymal stem cell exosomes, in particular to a preparation method of a hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosomes.
Background
The umbilical cord mesenchymal stem cells are a method for culturing and amplifying the umbilical cord mesenchymal stem cells of a culture system taking human umbilical cord blood serum as a main body, the human umbilical cord mesenchymal stem cells can be successfully amplified by applying an inactivated umbilical cord serum culture system, the cultured cells have the basic characteristics of the mesenchymal stem cells, theoretical basis is provided for establishing a mesenchymal stem cell bank and clinical application, and the method has wide clinical application prospect in tissue engineering aspects such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, cardiac muscle and the like.
The Exosome (Exosome) is a nano-scale lipid inclusion structure with the diameter of 30-100nm, and substances such as protein, mRNA (messenger ribonucleic acid), microRNA (ribonucleic acid) and the like are wrapped inside the Exosome. Exosomes are secreted and released by cells, spread in body fluids such as blood and finally phagocytosed by other cells, and are important mediators of intercellular communication. The method for separating and culturing the umbilical cord mesenchymal stem cell exosomes generally comprises the following steps: cleaning fresh healthy umbilical cord with PBS, removing blood vessel with scissors and forceps, removing Fahrenheit gelatin tissue, and sufficiently cutting the tissue to 1mm3Size, adding alpha-MEM culture solution, standing at 37 deg.C and 5% CO2Culturing in an incubator, wherein the culture solution contains 10% FBS, 100U/ml penicillin and 100U/ml streptomycin. After the umbilical cord tissue is cultured for 5-7 days, part of cells climb out from the periphery of the tissue block and are in a fine spindle shape, after one week, the cells begin to rapidly proliferate to form cell colonies with different sizes, and after the cells grow full, the cells are digested by 0.25% trypsin for passage.
Some patents have improved the preparation method, for example, CN111920828A discloses a lyophilized powder for hair regeneration based on umbilical cord mesenchymal stem cells, a preparation method, a hair growth nutrient solution and a hair regeneration method. The preparation method of the freeze-dried powder comprises the following steps: preparing a freeze-dried powder stock solution by adopting umbilical cord mesenchymal stem cells; uniformly mixing the freeze-drying protective agent and the freeze-drying powder stock solution to obtain an intermediate solution; and freeze-drying the intermediate solution under a preset freeze-drying condition to obtain the freeze-dried powder. The prepared freeze-dried powder and hair growth nutrient solution have obvious effect on at least one of promoting hair follicle restoration and promoting hair regeneration of a human body, and are beneficial to relieving the trend of alopecia and/or improving the hair state. But the preparation method is complex and may have negative effects on human skin.
Therefore, improvement of the mixed solution of umbilical cord mesenchymal stem cell exosomes and the preparation method thereof is needed, the generation speed of hair follicles is increased, and side effects are reduced.
Disclosure of Invention
Aiming at the problems, the invention provides a preparation method of a hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosomes.
The technical scheme of the invention is as follows:
the preparation method of the hair follicle generation promoting liquid based on the umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing and pretreating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical artery and vein, cutting into tissue segments, washing with sterile PBS solution for 3 times, and cutting into pieces of 2 + -0.5 mm3Placing the tissue fragments with the volume size in an ultra-clean culture dish for storage for later use;
s2: adding modified DMEM culture solution with 5-8 times of the volume of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant temperature incubator, introducing CO2When the volume fraction of the gas is 5-9%, the liquid is changed once every 2d half amount, and the liquid is changed once every 3d full amount after three times;
s3: when the fusion degree of the umbilical cord mesenchymal stem cells reaches 80-90%, digesting by using trypsin, and carrying out passage amplification culture;
s4: when the fusion degree of the third-generation umbilical cord mesenchymal stem cells reaches 70-80%, replacing serum-free basic culture solution for continuous culture, when the fusion degree of the umbilical cord mesenchymal stem cells reaches 85-95%, collecting culture solution supernatant to obtain culture solution containing the umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the third-generation umbilical cord mesenchymal stem cells and cell fragments, storing for later use, continuously ultracentrifuging and extracting the culture solution to obtain the umbilical cord mesenchymal stem cell exosomes, resuspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃;
s5: continuously culturing the third-generation umbilical cord mesenchymal stem cells until the fusion degree of the fifth-generation umbilical cord mesenchymal stem cells reaches 70-80%, and collecting the fifth-generation umbilical cord mesenchymal stem cells;
s6: mixing the umbilical cord mesenchymal stem cell exosome obtained in the step S4, the fifth generation umbilical cord mesenchymal stem cell obtained in the step S5 and hair papilla cells, culturing in a serum-free basic culture solution to obtain a mixed culture medium, mixing cholesterol and lecithin in a certain mass ratio, dissolving in a trichloromethane solution with the mass concentration of 50%, evaporating and drying to obtain liposome, adding the mixed culture medium, and performing ultrasonic dispersion to obtain a hair follicle generation promoting liquid.
Further, the specific steps of the sterilization and pretreatment in step S1 are as follows:
s1-1: soaking the umbilical cord tissue sample in 75% ethanol solution for disinfection for 1-2 min;
s1-2: and (4) washing the sterilized umbilical cord tissue sample for 1-2 times by using a sterile PBS solution to remove residual blood. The whole preparation process is ensured to be sterile and not to be interfered by impurities by a pretreatment mode.
Further, the length of the tissue segment in the step S1 is 2 + -0.5 cm. The operation of staff is convenient.
Further, the modified DMEM culture solution in step S2 contains 10% by volume of fetal bovine serum, wherein the mass concentration of streptomycin is 100g/L, the unit concentration of penicillin is 100U/mL, the mass concentration of hydroxyproline is 23-28mg/L, the mass concentration of chitosan is 55-60mg/L, and the pH is 6.8-7.2. The modified DMEM culture solution is rich in nutrient components, meets the growth requirements of umbilical cord mesenchymal stem cells with high growth speed and poor adhesiveness, and can realize rapid propagation culture of the umbilical cord mesenchymal stem cells.
Further, the temperature of the constant temperature incubator in the step S2 is 36.5 to 37.5 ℃, and the humidity is saturated humidity. By adjusting appropriate temperature and humidity and CO2The concentration can further promote the proliferation culture of the umbilical cord mesenchymal stem cells.
Further, in the step S3, the mass concentration of trypsin is 2.5g/L, and the trypsin is doped with 0.02% by mass concentration of EDTA, with the generation ratio of 1: 2-3. The winding ratio is controlled within a reasonable range, and the amplification is prevented from being too fast or too slow.
Further, the serum-free basal medium in the step S4 is phenol red-free alpha-MEM medium, wherein the mass concentration of D-glucose is 2250mg/L, the mass concentration of L-alanyl-L-glutamine is 380mg/L, the mass concentration of serine is 30-33mg/L, the mass concentration of tryptophan is 8-12mg/L, the mass concentration of biotin is 0.7-1mg/L, the mass concentration of vitamin B12 is 0.02-0.03mg/L, and the concentration of sodium pyruvate is 15-20 mmol/L. The serum-free basic culture solution removes phenol red and lysine, and has wide application range and good compatibility.
Further, in the step S6, the mass ratio of the cholesterol to the lecithin is 7-9: 1, the volume ratio of the mixed culture medium, the trichloromethane and the liposome is 1.25-1.6: 1: 0.2, the mass concentration of the umbilical cord mesenchymal stem cell exosome in the mixed culture medium is 500-600ng/L, and the concentration of the fifth generation umbilical cord mesenchymal stem cell is 109Per L, concentration of dermal papilla cells 1010And L. The liposome can be degraded by the decomposition enzyme in the organism and discharged out of the body, has strong biocompatibility, is similar to the lipid bilayer structure of the skin stratum corneum, and can promote the transdermal absorption of the topical skin medicament.
Further, the method for acquiring papilla pili cells in step S6 includes: washing hair follicle tissue in sterile PBS solution for 2-3 times, and cutting into pieces of 2 + -0.5 mm3Dissolving the tissue fragments with the size of the volume in enough type I collagenase, stirring and reacting for 2-4h, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 2-4h, screening out hair papilla tissues under a microscope, placing the hair papilla tissues in an ultra-clean culture dish, adding modified DMEM culture solution for culturing, changing the solution once every half 2d, changing the solution once every 3d after three times, continuously culturing for two weeks, carrying out passage for 72-75h, and collecting primary hair papilla cells. The hair papilla is peeled off effectively, and two different collagens are usedThe enzyme can further reduce the adhesion part, and is beneficial to screening of the hair papilla.
Compared with the prior art, the invention has the beneficial effects that:
(1) the preparation method of the hair follicle generation promoting liquid provided by the invention has the advantages that umbilical cord mesenchymal stem cells are subjected to proliferation culture, umbilical cord mesenchymal stem cell exosomes are extracted and are mixed and cultured with fifth-generation umbilical cord mesenchymal stem cells and treated hair papilla cells, so that the hair follicle generation promoting liquid capable of effectively improving the symptoms of hair follicle degradation, atrophy, alopecia areata and the like is obtained, and the effect of promoting hair regeneration is remarkable and more lasting.
(2) The preparation method of the hair follicle generation promoting liquid can promote the transdermal absorption of the local skin externally applied medicine by taking the liposome as the main component, the liposome can be degraded by the decomposition enzyme in the organism and discharged out of the body, simultaneously has strong biocompatibility, is similar to the lipid bilayer structure of the skin cuticle, can be used as an excellent carrier for the hair follicle generation, and has important significance for the development of the liposome slow release preparation technology.
Detailed Description
Example 1
The preparation method of the hair follicle generation promoting liquid based on the umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing and pretreating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical artery and vein, cutting into 2cm tissue segments, washing with sterile PBS solution for 3 times, and cutting into 2mm pieces3Placing the tissue fragments with the volume size in an ultra-clean culture dish for storage for later use;
the specific steps of disinfection and pretreatment are as follows:
s1-1: soaking the umbilical cord tissue sample in 75% ethanol solution for disinfection for 1.5 min;
s1-2: washing the sterilized umbilical cord tissue sample for 2 times by using sterile PBS (phosphate buffer solution) to remove residual blood;
s2: adding modified DMEM culture solution with the volume 6 times that of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, and placing the mixture in a constant temperature cultureIn the incubator, the temperature of the constant temperature incubator is 37 ℃, the humidity is saturated humidity, and CO is introduced2When the volume fraction of the gas is 8%, the liquid is changed once every 2d half, and the liquid is changed once every 3d full after three times; the modified DMEM culture solution contains 10% volume fraction fetal calf serum, wherein the mass concentration of streptomycin is 100g/L, the unit concentration of penicillin is 100U/mL, the mass concentration of hydroxyproline is 25mg/L, the mass concentration of chitosan is 58mg/L, and the pH value is 7;
s3: when the fusion degree of umbilical cord mesenchymal stem cells reaches 85%, digesting with trypsin, carrying out passage amplification culture, wherein the mass concentration of the trypsin is 2.5g/L, the trypsin is doped with ethylenediamine tetraacetic acid (EDTA) with the mass concentration of 0.02%, and the passage ratio is 1: 3;
s4: when the fusion degree of the third-generation umbilical cord mesenchymal stem cells reaches 75%, replacing serum-free basic culture solution for continuous culture, when the fusion degree of the umbilical cord mesenchymal stem cells reaches 88%, collecting supernatant of the culture solution to obtain culture solution containing the umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the third-generation umbilical cord mesenchymal stem cells and cell fragments, storing for later use, continuously ultracentrifuging and extracting the culture solution to obtain the umbilical cord mesenchymal stem cell exosomes, and resuspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃; the serum-free basic culture solution is phenol red-free alpha-MEM culture medium, wherein the mass concentration of D-glucose is 2250mg/L, the mass concentration of L-alanyl-L-glutamine is 380mg/L, the mass concentration of serine is 31mg/L, the mass concentration of tryptophan is 10mg/L, the mass concentration of biotin is 0.8mg/L, the mass concentration of vitamin B12 is 0.02mg/L, and the concentration of sodium pyruvate is 18 mmol/L;
s5: continuously culturing the third-generation umbilical cord mesenchymal stem cells until the fusion degree of the fifth-generation umbilical cord mesenchymal stem cells reaches 75%, and collecting the fifth-generation umbilical cord mesenchymal stem cells;
s6: mixing the umbilical cord mesenchymal stem cell exosome obtained in the step S4, the fifth generation umbilical cord mesenchymal stem cell obtained in the step S5 and the papilla pilaris cells, culturing in a serum-free basal culture solution to obtain a mixed culture medium, and then mixing the umbilical cord mesenchymal stem cell exosome, the fifth generation umbilical cord mesenchymal stem cell and the papilla pilaris cells in a mass ratio of 8: 1, mixing cholesterol and lecithin, and dissolving in mass concentrationEvaporating and drying 50% trichloromethane solution to obtain liposome, adding mixed culture medium, and ultrasonically dispersing to obtain hair follicle generation promoting liquid, wherein the volume ratio of the mixed culture medium, the trichloromethane and the liposome is 1.5: 1: 0.2, the mass concentration of the umbilical cord mesenchymal stem cell exosome in the mixed culture medium is 552ng/L, and the concentration of the umbilical cord mesenchymal stem cell in the fifth generation is 109Per L, concentration of dermal papilla cells 1010/L。
Example 2
This embodiment is substantially the same as embodiment 1, and mainly differs therefrom in that: the modified DMEM culture solution has different component content ratios.
The preparation method of the hair follicle generation promoting liquid based on the umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing and pretreating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical artery and vein, cutting into 1.5cm tissue segments, washing with sterile PBS solution for 3 times, and cutting into 1.5mm pieces3Placing the tissue fragments with the volume size in an ultra-clean culture dish for storage for later use;
the specific steps of disinfection and pretreatment are as follows:
s1-1: soaking the umbilical cord tissue sample in 75% ethanol solution for disinfection for 1 min;
s1-2: washing the sterilized umbilical cord tissue sample for 1 time by using sterile PBS (phosphate buffer solution) to remove residual blood;
s2: adding modified DMEM culture solution with the volume 5 times that of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant-temperature incubator at the temperature of 36.5 ℃ and the humidity of saturated humidity, introducing CO2When the volume fraction of the gas is 5%, the liquid is changed once every 2d half, and the liquid is changed once every 3d full after three times; the modified DMEM culture solution contains 10% volume fraction fetal calf serum, wherein the mass concentration of streptomycin is 100g/L, the unit concentration of penicillin is 100U/mL, the mass concentration of hydroxyproline is 23mg/L, the mass concentration of chitosan is 55mg/L, and the pH value is 6.8.
Example 3
This embodiment is substantially the same as embodiment 1, and mainly differs therefrom in that: the modified DMEM culture solution has different component content ratios.
The preparation method of the hair follicle generation promoting liquid based on the umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing and pretreating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical artery and vein, cutting into 2.5cm tissue segments, washing with sterile PBS solution for 3 times, and cutting into 2.5mm pieces3Placing the tissue fragments with the volume size in an ultra-clean culture dish for storage for later use;
the specific steps of disinfection and pretreatment are as follows:
s1-1: soaking the umbilical cord tissue sample in 75% ethanol solution for disinfection for 2 min;
s1-2: washing the sterilized umbilical cord tissue sample for 2 times by using sterile PBS (phosphate buffer solution) to remove residual blood;
s2: adding modified DMEM culture solution with the volume 8 times that of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant-temperature incubator at the temperature of 36.5 ℃ and the humidity of saturated humidity, introducing CO2When the volume fraction of the gas is 9%, the liquid is changed once every 2d half, and the liquid is changed once every 3d full after three times; the modified DMEM culture solution contains 10% volume fraction fetal calf serum, wherein the mass concentration of streptomycin is 100g/L, the unit concentration of penicillin is 100U/mL, the mass concentration of hydroxyproline is 28mg/L, the mass concentration of chitosan is 60mg/L, and the pH value is 7.2.
Comparative example 1
Basically the same as example 1, the main differences are: a general DMEM medium was used.
Example 4
This embodiment is substantially the same as embodiment 1, and mainly differs therefrom in that: the serum-free basic culture solution has different component content ratios.
S3: when the fusion degree of umbilical cord mesenchymal stem cells reaches 80%, digesting with trypsin, carrying out passage amplification culture, wherein the mass concentration of the trypsin is 2.5g/L, the trypsin is doped with ethylenediamine tetraacetic acid (EDTA) with the mass concentration of 0.02%, and the passage ratio is 1: 2;
s4: when the fusion degree of the third-generation umbilical cord mesenchymal stem cells reaches 70%, replacing serum-free basic culture solution for continuous culture, when the fusion degree of the umbilical cord mesenchymal stem cells reaches 85%, collecting culture solution supernatant to obtain culture solution containing the umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the third-generation umbilical cord mesenchymal stem cells and cell fragments, storing for later use, continuously ultracentrifuging and extracting the culture solution to obtain the umbilical cord mesenchymal stem cell exosomes, and resuspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃; the serum-free basic culture solution is phenol red-free alpha-MEM culture medium, wherein the mass concentration of D-glucose is 2250mg/L, the mass concentration of L-alanyl-L-glutamine is 380mg/L, the mass concentration of serine is 30mg/L, the mass concentration of tryptophan is 8mg/L, the mass concentration of biotin is 0.7mg/L, the mass concentration of vitamin B12 is 0.02-mg/L, and the concentration of sodium pyruvate is 15 mmol/L.
Example 5
This embodiment is substantially the same as embodiment 1, and mainly differs therefrom in that: the serum-free basic culture solution has different component content ratios.
S3: when the fusion degree of umbilical cord mesenchymal stem cells reaches 90%, digesting with trypsin, carrying out passage amplification culture, wherein the mass concentration of the trypsin is 2.5g/L, the trypsin is doped with ethylenediamine tetraacetic acid (EDTA) with the mass concentration of 0.02%, and the passage ratio is 1: 3;
s4: when the fusion degree of the third-generation umbilical cord mesenchymal stem cells reaches 80%, replacing serum-free basic culture solution for continuous culture, when the fusion degree of the umbilical cord mesenchymal stem cells reaches 95%, collecting supernatant of the culture solution to obtain culture solution containing the umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the third-generation umbilical cord mesenchymal stem cells and cell fragments, storing for later use, continuously ultracentrifuging and extracting the culture solution to obtain the umbilical cord mesenchymal stem cell exosomes, and resuspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃; the serum-free basic culture solution is phenol red-free alpha-MEM culture medium, wherein the mass concentration of D-glucose is 2250mg/L, the mass concentration of L-alanyl-L-glutamine is 380mg/L, the mass concentration of serine is 33mg/L, the mass concentration of tryptophan is 12mg/L, the mass concentration of biotin is 1mg/L, the mass concentration of vitamin B12 is 0.03mg/L, and the concentration of sodium pyruvate is 20 mmol/L.
Comparative example 2
Basically the same as example 1, the main differences are: a general basal medium was used.
Example 6
This embodiment is substantially the same as embodiment 1, and mainly differs therefrom in that: the exosomes differ in mass concentration.
S5: continuously culturing the third-generation umbilical cord mesenchymal stem cells until the fusion degree of the fifth-generation umbilical cord mesenchymal stem cells reaches 70%, and collecting the fifth-generation umbilical cord mesenchymal stem cells;
s6: mixing the umbilical cord mesenchymal stem cell exosome obtained in the step S4, the fifth generation umbilical cord mesenchymal stem cell obtained in the step S5 and the papilla pilaris cells, culturing in a serum-free basal culture solution to obtain a mixed culture medium, and then mixing the umbilical cord mesenchymal stem cell exosome, the fifth generation umbilical cord mesenchymal stem cell and the papilla pilaris cells in a mass ratio of 7: 1, mixing cholesterol and lecithin, dissolving the mixture in a trichloromethane solution with the mass concentration of 50%, evaporating and drying to obtain liposome, then adding a mixed culture medium, and performing ultrasonic dispersion to obtain a hair follicle generation promoting liquid, wherein the volume ratio of the mixed culture medium to the trichloromethane to the liposome is 1.25: 1: 0.2, the mass concentration of the umbilical cord mesenchymal stem cell exosome in the mixed culture medium is 500ng/L, and the concentration of the umbilical cord mesenchymal stem cell in the fifth generation is 109Per L, concentration of dermal papilla cells 1010/L。
Example 7
This embodiment is substantially the same as embodiment 1, and mainly differs therefrom in that: the exosomes differ in mass concentration.
S5: continuously culturing the third-generation umbilical cord mesenchymal stem cells until the fusion degree of the fifth-generation umbilical cord mesenchymal stem cells reaches 80%, and collecting the fifth-generation umbilical cord mesenchymal stem cells;
s6: mixing the umbilical cord mesenchymal stem cell exosome obtained in the step S4, the fifth generation umbilical cord mesenchymal stem cell obtained in the step S5 and the papilla pilaris cells, and culturing in a serum-free basal culture solution to obtain the umbilical cord mesenchymal stem cell exosomeMixing the culture medium, and then mixing the culture medium in a mass ratio of 9: 1, mixing cholesterol and lecithin, dissolving the mixture in a trichloromethane solution with the mass concentration of 50%, evaporating and drying to obtain liposome, then adding a mixed culture medium, and performing ultrasonic dispersion to obtain a hair follicle generation promoting liquid, wherein the volume ratio of the mixed culture medium to the trichloromethane to the liposome is 1.6: 1: 0.2, the mass concentration of the umbilical cord mesenchymal stem cell exosome in the mixed culture medium is 600ng/L, and the concentration of the umbilical cord mesenchymal stem cell in the fifth generation is 109Per L, concentration of dermal papilla cells 1010/L。
Comparative example 3
Basically the same as example 1, the main differences are: the mass concentration of the exosome is 300 ng/L.
Comparative example 4
Basically the same as example 1, the main differences are: the mass concentration of the exosome is 800 ng/L.
Example 8
The present embodiment is different from embodiment 1 in that: the method for acquiring papilla pili cells in step S6 includes: the hair follicle tissue was washed 3 times in sterile PBS solution and then minced to 2mm3Dissolving the tissue fragments with the size of the volume in enough type I collagenase, stirring and reacting for 3 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 3 hours, screening out hair papilla tissues under a microscope, placing the hair papilla tissues in an ultra-clean culture dish, adding modified DMEM culture solution for culturing, changing the liquid once every 2d half, changing the liquid once every 3d full amount after three times, continuously culturing for two weeks, carrying out passage for 74 hours, and collecting primary hair papilla cells.
Example 9
This embodiment is substantially the same as embodiment 8 except that: the method for acquiring papilla pili cells in step S6 includes: the hair follicle tissue was washed 2 times in sterile PBS solution and then cut into 1.5mm pieces3Dissolving the tissue fragment with volume size in sufficient collagenase type I, stirring for 2 hr, filtering, oven drying, dissolving in sufficient collagenase type IV, stirring for 2 hr, sieving with microscope, placing into ultra-clean culture dish, adding modified DMEM culture solutionAnd (4) performing culture, wherein the liquid is changed once every 2d half amount, the liquid is changed once every 3d full amount after three times, continuously culturing for two weeks, performing passage for 72 hours, and collecting and obtaining primary dermal papilla cells.
Example 10
This embodiment is substantially the same as embodiment 8 except that: the method for acquiring papilla pili cells in step S6 includes: the hair follicle tissue was washed 3 times in sterile PBS solution and then minced to 2.5mm3Dissolving the tissue fragments with the size of the volume in enough type I collagenase, stirring for reaction for 4 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring for reaction for 4 hours, screening out hair papilla tissues under a microscope, placing the hair papilla tissues in an ultra-clean culture dish, adding modified DMEM culture solution for culture, changing the liquid once every 2d and half, changing the liquid once every 3d and full after three times, continuously culturing for two weeks, carrying out passage for 75 hours, and collecting primary hair papilla cells.
Examples of the experiments
The hair growth stimulating solutions prepared in examples 1 to 10 and comparative examples 1 to 4 were subjected to a hair growth test, and 140 general alopecia patients under alopecia, aged 30 to 50 years, were randomly selected and divided into 14 groups using the hair growth stimulating solutions of examples 1 to 10 and comparative examples 1 to 4, respectively. The using method comprises the following steps: before use, the hair and scalp were washed clean, 5mL of each product was evenly applied to the scalp while massaging the scalp for 15min, and then washed with clean water and used every 2 days for 8 weeks, and the growth of hair and the loss of hair of each patient were observed, and the results are shown in table 1.
TABLE 1 results of the folliculogenesis-promoting fluid development test of examples 1-10 and comparative examples 1-4
As can be seen from comparison of examples 1 to 3 and comparative example 1, the modified DMEM culture solution of the present invention has shortened initial hair growth time and increased total hair growth amount, so the modified DMEM culture solution of the present invention has obvious improvement on hair follicle growth promoting liquid, and the initial hair growth time in example 1 is the earliest, the total hair growth amount is almost the same, and the hair loss amount is low, so the content ratio of the components of the modified DMEM culture solution in example 1 is the most reasonable;
comparing examples 1, 4 and 5 with comparative example 2, it can be seen that the serum-free basal medium of the present invention is used, the hair growth time is shortened to some extent, the total hair growth amount is improved obviously, the hair loss amount is increased naturally after 8 weeks of hair washing, and the natural phenomenon is included, which indicates that the serum-free basal medium of the present invention has obvious improvement on the hair follicle growth promoting liquid, the hair growth time is shortest in example 5, but the total hair growth amount is less, so the serum-free basal medium of the present invention is selected and used in the most reasonable proportion of the components of the serum-free basal medium in example 1;
comparing examples 1, 6 and 7 with comparative examples 3 and 4, it can be seen that the initial hair growth time and the total hair growth amount of a patient are greatly influenced by changing the mass concentration of exosome, when the mass concentration of exosome is lower, the initial hair growth time is long, the hair growth is slow, the total hair growth amount is general, when the mass concentration of exosome is higher, the initial hair growth time is very short, the hair growth speed is high, but the total hair growth amount is reduced, meanwhile, the hair loss amount is greatly increased when hair is washed after 8 weeks, the higher the hair growth amount concentration is, the better the hair growth amount concentration is, the more reasonable the range of the mass concentration of exosome provided by the invention is selected, wherein the comprehensive hair growth effect of the patient under the mass concentration of exosome in example 1 is optimal;
it can be seen from comparison of examples 8-10 that the improved hair papilla cell harvesting method of the present invention has an effect on the performance of hair follicle growth promoting liquid, the hair growth starting time is generally short, the total hair growth amount is slightly increased, the hair loss amount is slightly increased after 8 weeks of hair washing, and the total hair growth effect is better than that of examples 1-7, wherein the comprehensive hair growth effect of the patient under the process parameters of the hair papilla cell harvesting method of example 8 is optimal.
Claims (9)
1. The preparation method of the hair follicle generation promoting liquid based on the umbilical cord mesenchymal stem cell exosomes is characterized by comprising the following steps:
s1: sterilizing and pretreating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical artery and vein, cutting into tissue segments, washing with sterile PBS solution for 3 times, and cutting into pieces of 2 + -0.5 mm3Placing the tissue fragments with the volume size in an ultra-clean culture dish for storage for later use;
s2: adding modified DMEM culture solution with 5-8 times of the volume of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant temperature incubator, introducing CO2When the volume fraction of the gas is 5-9%, the liquid is changed once every 2d half amount, and the liquid is changed once every 3d full amount after three times;
s3: when the fusion degree of the umbilical cord mesenchymal stem cells reaches 80-90%, digesting by using trypsin, and carrying out passage amplification culture;
s4: when the fusion degree of the third-generation umbilical cord mesenchymal stem cells reaches 70-80%, replacing serum-free basic culture solution for continuous culture, when the fusion degree of the umbilical cord mesenchymal stem cells reaches 85-95%, collecting culture solution supernatant to obtain culture solution containing the umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the third-generation umbilical cord mesenchymal stem cells and cell fragments, storing for later use, continuously ultracentrifuging and extracting the culture solution to obtain the umbilical cord mesenchymal stem cell exosomes, resuspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃;
s5: continuously culturing the third-generation umbilical cord mesenchymal stem cells until the fusion degree of the fifth-generation umbilical cord mesenchymal stem cells reaches 70-80%, and collecting the fifth-generation umbilical cord mesenchymal stem cells;
s6: mixing the umbilical cord mesenchymal stem cell exosome obtained in the step S4, the fifth generation umbilical cord mesenchymal stem cell obtained in the step S5 and hair papilla cells, culturing in a serum-free basic culture solution to obtain a mixed culture medium, mixing cholesterol and lecithin in a certain mass ratio, dissolving in a trichloromethane solution with the mass concentration of 50%, evaporating and drying to obtain liposome, adding the mixed culture medium, and performing ultrasonic dispersion to obtain a hair follicle generation promoting liquid.
2. The method for preparing a solution for promoting hair follicle generation based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the steps of sterilizing and pretreating in step S1 are as follows:
s1-1: soaking the umbilical cord tissue sample in 75% ethanol solution for disinfection for 1-2 min;
s1-2: and (4) washing the sterilized umbilical cord tissue sample for 1-2 times by using a sterile PBS solution to remove residual blood.
3. The method for preparing a generation promoting solution for hair follicle according to claim 1, wherein the length of the tissue fragment in step S1 is 2 ± 0.5 cm.
4. The method for preparing a solution for promoting hair follicle generation based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the modified DMEM culture solution in step S2 contains fetal bovine serum with a volume fraction of 10%, wherein the mass concentration of streptomycin is 100g/L, the unit concentration of penicillin is 100U/mL, the mass concentration of hydroxyproline is 23-28mg/L, the mass concentration of chitosan is 55-60mg/L, and the pH is 6.8-7.2.
5. The method for preparing a solution for promoting hair follicle generation based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the temperature of the constant temperature incubator in the step S2 is 36.5-37.5 ℃, and the humidity is saturated humidity.
6. The method for preparing a solution for promoting hair follicle generation based on umbilical cord mesenchymal stem cell exosome according to claim 1, wherein the mass concentration of trypsin in step S3 is 2.5g/L, and ethylenediaminetetraacetic acid EDTA is added into trypsin at a mass concentration of 0.02%, and the generation ratio is 1: 2-3.
7. The method for preparing a solution for promoting hair follicle generation based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the serum-free basal medium in step S4 is phenol red-free α -MEM medium, wherein the mass concentration of D-glucose is 2250mg/L, the mass concentration of L-alanyl-L-glutamine is 380mg/L, the mass concentration of serine is 30-33mg/L, the mass concentration of tryptophan is 8-12mg/L, the mass concentration of biotin is 0.7-1mg/L, the mass concentration of vitamin B12 is 0.02-0.03mg/L, and the concentration of sodium pyruvate is 15-20 mmol/L.
8. The method for preparing a solution for promoting hair follicle generation based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the mass ratio of cholesterol to lecithin in step S6 is 7-9: 1, the volume ratio of the mixed culture medium, the trichloromethane and the liposome is 1.25-1.6: 1: 0.2, the mass concentration of the umbilical cord mesenchymal stem cell exosome in the mixed culture medium is 500-600ng/L, and the concentration of the fifth generation umbilical cord mesenchymal stem cell is 109Per L, concentration of dermal papilla cells 1010/L。
9. The method for preparing a hair follicle generation promoting solution based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the method for obtaining hair papilla cells in step S6 is: washing hair follicle tissue in sterile PBS solution for 2-3 times, and cutting into pieces of 2 + -0.5 mm3Dissolving the tissue fragments with the size of the volume in enough type I collagenase, stirring and reacting for 2-4h, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 2-4h, screening out hair papilla tissues under a microscope, placing the hair papilla tissues in an ultra-clean culture dish, adding modified DMEM culture solution for culturing, changing the solution once every half 2d, changing the solution once every 3d after three times, continuously culturing for two weeks, carrying out passage for 72-75h, and collecting primary hair papilla cells.
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| CN115814045A (en) * | 2022-11-03 | 2023-03-21 | 山东新创生物科技有限公司 | Exosome composite preparation for improving alopecia and preparation method and application thereof |
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| CN109432127A (en) * | 2018-11-21 | 2019-03-08 | 海门生原干细胞科技有限公司 | Application of the mescenchymal stem cell excretion body in the pharmaceutical preparation that preparation promotees hair regeneration |
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| CN115537393A (en) * | 2022-12-05 | 2022-12-30 | 广州优特佳生物科技发展有限公司 | Preparation method and application of umbilical cord mesenchymal stem cell directional secretion group for promoting hair growth |
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