CN113813289B - Preparation method of hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosomes - Google Patents
Preparation method of hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosomes Download PDFInfo
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- CN113813289B CN113813289B CN202111043984.8A CN202111043984A CN113813289B CN 113813289 B CN113813289 B CN 113813289B CN 202111043984 A CN202111043984 A CN 202111043984A CN 113813289 B CN113813289 B CN 113813289B
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- 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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Abstract
The invention discloses a preparation method of hair follicle generation promoting liquid based on umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: s1: disinfecting and preprocessing umbilical cord tissue samples, shearing into tissue fragments, and storing for later use; s2: adding the modified DMEM culture solution, placing the culture solution into a constant temperature incubator, and changing the solution once every 2d half, and changing the solution once every 3d full after three times; s3: carrying out passage amplification culture; s4: extracting to obtain umbilical cord mesenchymal stem cell exosomes; s5: collecting fifth generation umbilical cord mesenchymal stem cells; s6: and (3) culturing 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 cells in a mixed mode to obtain the hair follicle generation promoting liquid. The hair follicle growth promoting liquid can effectively improve the symptoms of hair follicle degeneration, atrophy, alopecia areata and the like, and has remarkable and more durable 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 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 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 mesenchymal stem cells, a theoretical basis is provided for establishing a mesenchymal stem cell bank and clinical application, and the umbilical cord mesenchymal stem cells have wide clinical application prospects in the aspects of tissue engineering such as bones, cartilages, muscles, tendons, ligaments, nerves, livers, endothelium and cardiac muscles.
Exosomes (exosomes) are nanoscale lipid-encapsulated structures with diameters of 30-100nm, and are internally encapsulated by proteins, mRNA, micrornas, and the like. Exosomes are secreted and released by cells and spread in body fluids such as blood, and finallyBut also can be phagocytized by other cells, and is an important medium for intercellular communication. The isolated culture method of umbilical cord mesenchymal stem cell exosomes generally comprises the following steps: collecting fresh healthy umbilical cord, washing with PBS, removing blood vessel with scissors forceps, removing Fahrenheit rubber tissue, and cutting the obtained tissue to 1mm 3 Adding alpha-MEM culture solution, standing at 37deg.C, and 5% CO 2 The culture medium is cultured in an incubator, and the culture medium contains 10% FBS,100U/ml penicillin and 100U/ml streptomycin. After 5-7 days of umbilical cord tissue culture, part of cells can climb out from the periphery of the tissue block, the form of the umbilical cord tissue culture is tiny, after one week, the cells start to proliferate rapidly to form cell colonies with different sizes, and after the umbilical cord tissue culture is full, the umbilical cord tissue culture is digested with 0.25% trypsin for passage.
Some patents have improved the preparation method, for example, patent CN111920828A discloses a freeze-dried powder based on umbilical cord mesenchymal stem cells for hair regeneration, 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 with the freeze-drying powder stock solution to obtain an intermediate solution; and freeze-drying the intermediate liquid under a preset freeze-drying condition to obtain the freeze-dried powder. The prepared freeze-dried powder and the hair growth nutrient solution have remarkable effects on at least one of promoting hair follicle repair and promoting hair regeneration of human bodies, and are beneficial to relieving the trend of alopecia and/or improving the hair state. However, the preparation method is complex and may have a negative effect on human skin.
Therefore, there is a need for an improved umbilical cord mesenchymal stem cell exosome mixture and method of preparation that increases the rate of hair follicle formation and reduces side effects.
Disclosure of Invention
Aiming at the problems, the invention provides a preparation method of 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-generating promoting liquid based on umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: navelSterilizing and pre-treating tissue sample, removing jelly, umbilical cord adventitia and umbilical artery and vein, cutting tissue section, washing with sterile PBS solution for 3 times, and cutting into pieces of 2+ -0.5 mm 3 Placing the tissue fragments with the size in an ultra-clean culture dish for storage;
s2: adding modified DMEM culture solution with volume 5-8 times of that of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant temperature incubator, and introducing CO 2 The gas is 5-9% in volume fraction, the liquid is exchanged once every 2d half, and the liquid is exchanged once every 3d full after three times;
s3: when the fusion degree of umbilical mesenchymal stem cells reaches 80-90%, carrying out digestion treatment by trypsin, and carrying out passage amplification culture;
s4: when the fusion degree of the 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 umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the umbilical cord mesenchymal stem cells and cell fragments of the third generation, preserving for later use, and continuously performing ultracentrifugation extraction on the culture solution to obtain umbilical cord mesenchymal stem cell exosomes, re-suspending 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 umbilical cord mesenchymal stem cell exosomes obtained in the step S4, the fifth-generation umbilical cord mesenchymal stem cells obtained in the step S5 and hair papilla cells, culturing in a serum-free basic culture solution to obtain a mixed culture medium, then mixing cholesterol and lecithin in a certain mass ratio, dissolving in a chloroform solution with the mass concentration of 50%, evaporating and drying to obtain liposome, adding the mixed culture medium, and performing ultrasonic dispersion to obtain the hair follicle generation promoting liquid.
Further, the specific steps of the disinfection and pretreatment in the step S1 are as follows:
s1-1: immersing the umbilical cord tissue sample in an ethanol solution with the mass concentration of 75% for disinfection treatment for 1-2min;
s1-2: the sterilized umbilical cord tissue samples were rinsed 1-2 times with sterile PBS to remove residual blood. The whole preparation process is aseptic and can not 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 the staff is convenient.
Further, the modified DMEM culture solution in the step S2 contains fetal bovine serum with the 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 value is 6.8-7.2. The modified DMEM culture solution is rich in nutrient components, meets the growth requirement of umbilical cord mesenchymal stem cells with high growth speed and poor adhesiveness, and can realize rapid proliferation culture of umbilical cord mesenchymal stem cells.
Further, the temperature of the constant temperature incubator in the step S2 is 36.5-37.5 ℃, and the humidity is saturated humidity. By adjusting the proper temperature and humidity and CO 2 The concentration can further promote proliferation culture of umbilical cord mesenchymal stem cells.
Further, in the step S3, the mass concentration of trypsin is 2.5g/L, and EDTA (ethylene diamine tetraacetic acid) with the mass concentration of 0.02% is doped in the trypsin, and the passage ratio is 1:2-3. The proportion of the winding belt is controlled in a reasonable range, so that the amplification is avoided to be too fast or too slow.
Further, the serum-free basal culture medium in the step S4 is a 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 30-33mg/L, the 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 15-20mmol/L. The serum-free basal 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 chloroform and the liposome is 1.25-1.6:1:0.2, the mass concentration of umbilical cord mesenchymal stem cell exosomes in the mixed culture medium is 500-600ng/L, and the concentration of umbilical cord mesenchymal stem cells of the fifth generation is 10 9 Concentration of hair papilla cells of 10 10 and/L. The liposome can be degraded by the decomposition enzyme in the organism and discharged outside, has strong biocompatibility, has a similar lipid bilayer structure with the skin stratum corneum, and can promote the percutaneous absorption of the topical skin external medicine.
Further, the method for obtaining the papilla cells in the step S6 comprises the following steps: the hair follicle tissue is washed 2-3 times in sterile PBS solution and then sheared into 2+ -0.5 mm 3 The method comprises the steps of dissolving tissue fragments with the volume size in enough type I collagenase, stirring and reacting for 2-4 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 2-4 hours, sieving the hair papilla tissue under a microscope, placing the hair papilla tissue into an ultra-clean culture dish, adding modified DMEM culture solution for culturing, changing the solution once every 2d half amount, changing the solution once every 3d full amount after three times, continuing culturing for two weeks, and carrying out passage for 72-75 hours, and collecting and obtaining primary hair papilla cells. The hair papilla stripping effect is good, and the adhesion part can be further reduced by using two different collagenases, so that the screening of the hair papilla is facilitated.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the preparation method of the hair follicle growth promoting liquid, the umbilical cord mesenchymal stem cells are subjected to proliferation culture, umbilical cord mesenchymal stem cell exosomes are obtained by extraction, and are mixed with fifth-generation umbilical cord mesenchymal stem cells and treated hair papilla cells for culture, so that the hair follicle growth promoting liquid capable of effectively improving hair follicle degeneration, atrophy, alopecia areata and other symptoms is obtained, and the hair regeneration promoting effect is remarkable and more durable.
(2) The preparation method of the hair follicle stimulating liquid can stimulate the percutaneous absorption of local skin external medicine by taking the liposome as a main component, the liposome can be degraded by the decomposition enzyme in the organism and discharged outside the body, and has strong biocompatibility, is similar to the lipid bilayer structure of the skin stratum corneum, can be used as an excellent carrier for hair follicle generation, and has important significance for the development of liposome slow-release preparation technology.
Detailed Description
Example 1
The preparation method of the hair follicle-generating promoting liquid based on umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing and pre-treating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical vein, cutting into 2cm tissue segments, washing with sterile PBS solution for 3 times, and cutting into 2mm pieces 3 Placing the tissue fragments with the size in an ultra-clean culture dish for storage;
the specific steps of disinfection and pretreatment are as follows:
s1-1: immersing the umbilical cord tissue sample in an ethanol solution with the mass concentration of 75% for disinfection treatment for 1.5min;
s1-2: washing the sterilized umbilical cord tissue sample for 2 times by using a sterile PBS (phosphate buffered saline) solution to remove residual blood;
s2: adding modified DMEM culture solution with volume of 6 times of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant temperature incubator with temperature of 37deg.C and humidity of saturated humidity, and introducing CO 2 The gas is changed into liquid at intervals of 2d for half a time until the volume fraction is 8%, and the liquid is changed into liquid at intervals of 3d for full time after three times; the modified DMEM culture solution contains 10% of fetal bovine serum by volume fraction, 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 mesenchymal stem cells reaches 85%, carrying out passaging amplification culture by using trypsin, wherein the mass concentration of trypsin is 2.5g/L, and the trypsin is doped with 0.02% of ethylenediamine tetraacetic acid EDTA (ethylene diamine tetraacetic acid) in mass concentration, and the passaging ratio is 1:3, a step of;
s4: when the fusion degree of the 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 culture solution supernatant to obtain culture solution containing umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the umbilical cord mesenchymal stem cells and cell fragments of the third generation, preserving for later use, and continuously performing ultracentrifugation extraction on the culture solution to obtain umbilical cord mesenchymal stem cell exosomes, re-suspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃; the serum-free basal culture solution is a 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 18mmol/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 hair papilla cells, culturing in a serum-free basic culture solution to obtain a mixed culture medium, and then mixing the culture medium with the mass ratio of 8:1 and lecithin, dissolving in 50% trichloromethane solution, evaporating and drying to obtain liposome, adding a mixed culture medium, and performing ultrasonic dispersion to obtain hair follicle generation promoting liquid, wherein the volume ratio of the mixed culture medium, trichloromethane and liposome is 1.5:1:0.2, the mass concentration of umbilical cord mesenchymal stem cell exosomes in the mixed culture medium is 552ng/L, and the concentration of the umbilical cord mesenchymal stem cells of the fifth generation is 10 9 Concentration of hair papilla cells of 10 10 /L。
Example 2
This embodiment is substantially the same as embodiment 1, with the main difference that: the component content ratios of the modified DMEM culture solution are different.
The preparation method of the hair follicle-generating promoting liquid based on umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing and pre-treating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical vein, cutting into 1.5cm tissue segments, and makingWashing with sterile PBS solution for 3 times, and shearing into 1.5mm pieces 3 Placing the tissue fragments with the size in an ultra-clean culture dish for storage;
the specific steps of disinfection and pretreatment are as follows:
s1-1: immersing the umbilical cord tissue sample in an ethanol solution with the mass concentration of 75% for disinfection treatment for 1min;
s1-2: washing the sterilized umbilical cord tissue sample for 1 time by using a sterile PBS solution to remove residual blood;
s2: adding modified DMEM culture solution with 5 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 with the temperature of 36.5 ℃ and the humidity of saturated humidity, and introducing CO 2 The gas is changed into liquid at intervals of 2d for half a time until the volume fraction is 5%, and the liquid is changed into liquid at intervals of 3d for full time after three times; the modified DMEM culture solution contains 10% of fetal bovine serum by volume fraction, 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 is 6.8.
Example 3
This embodiment is substantially the same as embodiment 1, with the main difference that: the component content ratios of the modified DMEM culture solution are different.
The preparation method of the hair follicle-generating promoting liquid based on umbilical cord mesenchymal stem cell exosomes comprises the following steps:
s1: sterilizing umbilical cord tissue sample, pretreating, removing jelly, umbilical cord adventitia and umbilical vein, cutting into 2.5cm tissue segments, washing with sterile PBS solution for 3 times, and cutting into 2.5mm pieces 3 Placing the tissue fragments with the size in an ultra-clean culture dish for storage;
the specific steps of disinfection and pretreatment are as follows:
s1-1: immersing the umbilical cord tissue sample in an ethanol solution with the mass concentration of 75% for disinfection treatment for 2min;
s1-2: washing the sterilized umbilical cord tissue sample for 2 times by using a sterile PBS (phosphate buffered saline) solution to remove residual blood;
S2：adding modified DMEM culture solution with volume 8 times of that of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant temperature incubator with temperature of 36.5deg.C and humidity of saturated humidity, and introducing CO 2 The gas is changed into liquid at intervals of 2d for half a time until the volume fraction of the gas is 9%, and the liquid is changed into liquid at intervals of 3d for full time after three times; the modified DMEM culture solution contains 10% of fetal bovine serum by volume fraction, 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 is 7.2.
Comparative example 1
Substantially the same as in example 1, the main difference is that: general DMEM broth was used.
Example 4
This embodiment is substantially the same as embodiment 1, with the main difference that: the serum-free basic culture solution has different component content ratios.
S3: when the fusion degree of umbilical mesenchymal stem cells reaches 80%, carrying out passaging amplification culture by using trypsin, wherein the mass concentration of trypsin is 2.5g/L, and the trypsin is doped with 0.02% of ethylenediamine tetraacetic acid EDTA, and the passaging ratio is 1:2;
s4: when the fusion degree of the 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 umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the umbilical cord mesenchymal stem cells and cell fragments of the third generation, preserving for later use, and continuously performing ultracentrifugation extraction on the culture solution to obtain umbilical cord mesenchymal stem cell exosomes, re-suspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃; the serum-free basal culture solution is a 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 15mmol/L.
Example 5
This embodiment is substantially the same as embodiment 1, with the main difference that: the serum-free basic culture solution has different component content ratios.
S3: when the fusion degree of umbilical mesenchymal stem cells reaches 90%, carrying out passaging amplification culture by using trypsin, wherein the mass concentration of trypsin is 2.5g/L, and the trypsin is doped with 0.02% of ethylenediamine tetraacetic acid EDTA, and the passaging ratio is 1:3, a step of;
s4: when the fusion degree of the 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 culture solution supernatant to obtain culture solution containing umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the umbilical cord mesenchymal stem cells and cell fragments of the third generation, preserving for later use, and continuously performing ultracentrifugation extraction on the culture solution to obtain umbilical cord mesenchymal stem cell exosomes, re-suspending the umbilical cord mesenchymal stem cell exosomes by using sterile PBS solution and freezing for later use at-80 ℃; the serum-free basal culture solution is a 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 20mmol/L.
Comparative example 2
Substantially the same as in example 1, the main difference is that: a general basal medium was used.
Example 6
This embodiment is substantially the same as embodiment 1, with the main difference that: the mass concentration of exosomes varies.
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 hair papilla cellCulturing in a serum-free basal culture solution to obtain a mixed culture medium, and then mixing the culture medium with the mass ratio of 7:1 and lecithin, dissolving in 50% trichloromethane solution, evaporating and drying to obtain liposome, adding a mixed culture medium, and performing ultrasonic dispersion to obtain hair follicle generation promoting liquid, wherein the volume ratio of the mixed culture medium, trichloromethane and liposome is 1.25:1:0.2, the mass concentration of umbilical cord mesenchymal stem cell exosomes in the mixed culture medium is 500ng/L, and the concentration of the umbilical cord mesenchymal stem cells of the fifth generation is 10 9 Concentration of hair papilla cells of 10 10 /L。
Example 7
This embodiment is substantially the same as embodiment 1, with the main difference that: the mass concentration of exosomes varies.
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 hair papilla cells, culturing in a serum-free basic culture solution to obtain a mixed culture medium, and then mixing the culture medium with the mass ratio of 9:1 and lecithin, dissolving in 50% trichloromethane solution, evaporating and drying to obtain liposome, adding a mixed culture medium, and performing ultrasonic dispersion to obtain hair follicle generation promoting liquid, wherein the volume ratio of the mixed culture medium, trichloromethane and liposome is 1.6:1:0.2, the mass concentration of umbilical cord mesenchymal stem cell exosomes in the mixed culture medium is 600ng/L, and the concentration of the umbilical cord mesenchymal stem cells of the fifth generation is 10 9 Concentration of hair papilla cells of 10 10 /L。
Comparative example 3
Substantially the same as in example 1, the main difference is that: the mass concentration of the exosomes is 300ng/L.
Comparative example 4
Substantially the same as in example 1, the main difference is that: the mass concentration of exosomes was 800ng/L.
Example 8
This embodiment differs from embodiment 1 in that: the method for obtaining the hair papilla cells in the step S6 comprises the following steps: the hair follicle tissue was washed 3 times in sterile PBS solution and then minced to 2mm 3 The method comprises the steps of dissolving tissue fragments with the volume size in enough type I collagenase, stirring for reaction for 3 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring for reaction for 3 hours, sieving 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 solution once every 2d for half, changing the solution once every 3d for full, continuing to culture for two weeks, and carrying out passage for 74 hours, and collecting and obtaining primary hair papilla cells.
Example 9
This embodiment is substantially the same as embodiment 8 except that: the method for obtaining the hair papilla cells in the step S6 comprises the following steps: the hair follicle tissue was washed 2 times in sterile PBS solution and then minced to 1.5mm 3 The method comprises the steps of dissolving tissue fragments with the volume size in enough type I collagenase, stirring and reacting for 2 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 2 hours, sieving 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 2d half, changing the solution once every 3d full amount after three times, continuing culturing for two weeks, and carrying out passage for 72 hours, and collecting and obtaining primary hair papilla cells.
Example 10
This embodiment is substantially the same as embodiment 8 except that: the method for obtaining the hair papilla cells in the step S6 comprises the following steps: the hair follicle tissue was washed 3 times in sterile PBS solution and then minced to 2.5mm 3 The method comprises the steps of dissolving tissue fragments with the volume size in enough type I collagenase, stirring and reacting for 4 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 4 hours, sieving 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 2d half, changing the solution once every 3d full amount after three times, continuing culturing for two weeks, and carrying out passage for 75 hours, and collecting and obtaining primary hair papilla cells.
Experimental example
The hair follicle stimulating liquids prepared in examples 1 to 10 and comparative examples 1 to 4 were subjected to an increasing test, 140 general hair loss patients who were losing hair were randomly selected, and aged 30 to 50, and 14 groups of hair follicle stimulating liquids were used in examples 1 to 10 and comparative examples 1 to 4, respectively. The using method comprises the following steps: before use, the hair and scalp are cleaned, 5mL of each group of products are uniformly rubbed on the scalp, the scalp is massaged for 15min, then the scalp is cleaned by clear water, the scalp is used once every 2 days, the test time is 8 weeks, and the hair growth condition and the hair loss condition of each group of patients are observed, and the results are shown in Table 1.
TABLE 1 results of hair follicle stimulating fluid enhancement test for examples 1-10 and comparative examples 1-4
As can be seen from comparative examples 1 to 3 and comparative example 1, the initial hair growth time is shortened and the total hair growth amount is increased by using the modified DMEM culture solution of the invention, so that the modified DMEM culture solution of the invention is obviously improved for the hair follicle growth promoting solution, the initial hair growth time in example 1 is earliest, the total hair growth amount is almost the same, the hair loss amount is lower, and the content ratio of the components of the modified DMEM culture solution in example 1 is most reasonable;
as can be seen from comparative examples 1, 4, 5 and comparative example 2, by using the serum-free basal culture solution of the invention, the initial hair growth time is shortened, the total hair growth increase is obviously improved, the hair loss amount naturally increases after 8 weeks of shampooing, and the serum-free basal culture solution belongs to a natural phenomenon, which shows that the serum-free basal culture solution of the invention has obvious improvement on the hair follicle growth promoting solution, and the initial hair growth time of example 5 is shortest, but the total hair growth increase is less, so that the content proportion of the components of the serum-free basal culture solution in the comprehensive consideration of the example 1 is most reasonable;
as can be seen from comparative examples 1, 6 and 7 and comparative examples 3 and 4, by changing the mass concentration of exosomes, the time for starting the hair growth and the total hair growth increase of patients are greatly influenced, when the mass concentration of exosomes is low, the time for starting the hair growth is long, the hair growth is slow, and the total hair growth is generally slow, when the mass concentration of exosomes is high, the time for starting the hair growth is short, the hair growth speed is fast, but the total hair growth is also reduced, and meanwhile, the hair loss is greatly increased after 8 weeks of shampooing, so that the higher the mass concentration of exosomes is not, the better, the most reasonable the range of the mass concentration of exosomes is selected, wherein the comprehensive hair growth effect of patients under the mass concentration of exosomes in example 1 is optimal;
as can be seen from comparative examples 8-10, the improved hair papilla cell harvesting method of the present invention has an effect on the performance of hair follicle growth promoting liquid, the time for starting hair growth is generally short, the total hair growth is slightly increased, the hair loss is slightly increased when the hair is washed after 8 weeks, 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 technological parameters of the hair papilla cell harvesting method of example 8 is optimal.
Claims (8)
1. The preparation method of the hair follicle growth promoting liquid based on umbilical cord mesenchymal stem cell exosomes is characterized by comprising the following steps:
s1: disinfecting and pre-treating umbilical cord tissue sample, removing jelly, umbilical cord adventitia and umbilical vein, cutting into small segments, washing with sterile PBS solution for 3 times, and cutting into pieces of 2+ -0.5 mm 3 Placing the tissue fragments with the size in an ultra-clean culture dish for storage;
s2: adding modified DMEM culture solution with volume 5-8 times of that of the tissue fragments, shaking to uniformly disperse the tissue fragments in the modified DMEM culture solution, placing in a constant temperature incubator, and introducing CO 2 The gas is 5-9% in volume fraction, the liquid is exchanged once every 2d half, and the liquid is exchanged once every 3d full after three times;
s3: when the fusion degree of umbilical mesenchymal stem cells reaches 80-90%, carrying out digestion treatment by trypsin, and carrying out passage amplification culture;
s4: when the fusion degree of the 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 umbilical cord mesenchymal stem cell exosomes, centrifuging the culture solution to remove the umbilical cord mesenchymal stem cells and cell fragments of the third generation, preserving for later use, and continuously performing ultracentrifugation extraction on the culture solution to obtain umbilical cord mesenchymal stem cell exosomes, re-suspending 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 umbilical cord mesenchymal stem cell exosomes obtained in the step S4, the fifth-generation umbilical cord mesenchymal stem cells obtained in the step S5 and hair papilla cells, culturing in a serum-free basic culture solution to obtain a mixed culture medium, then mixing cholesterol and lecithin with a certain mass ratio, dissolving in a chloroform solution with the mass concentration of 50%, evaporating and drying to obtain liposome, adding the mixed culture medium, and performing ultrasonic dispersion to obtain hair follicle generation promoting liquid;
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 chloroform and the liposome is 1.25-1.6:1:0.2, the mass concentration of umbilical cord mesenchymal stem cell exosomes in the mixed culture medium is 500-600ng/L, and the concentration of umbilical cord mesenchymal stem cells of the fifth generation is 10 9 Concentration of hair papilla cells of 10 10 /L。
2. The method for preparing the hair follicle stimulating fluid based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the specific steps of sterilization and pretreatment in step S1 are as follows:
s1-1: immersing the umbilical cord tissue sample in an ethanol solution with the mass concentration of 75% for disinfection treatment for 1-2min;
s1-2: the sterilized umbilical cord tissue samples were rinsed 1-2 times with sterile PBS to remove residual blood.
3. The method for preparing a hair follicle stimulating fluid based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the length of the tissue fragments in step S1 is 2±0.5cm.
4. The method for preparing hair follicle stimulating fluid based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the modified DMEM culture fluid 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 hair follicle stimulating fluid based on umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein the temperature of the incubator in step S2 is 36.5-37.5 ℃ and the humidity is saturated humidity.
6. The method for preparing the umbilical cord mesenchymal stem cell exosome-based hair follicle stimulating fluid according to claim 1, wherein in the step S3, the mass concentration of trypsin is 2.5g/L, and 0.02% mass concentration of ethylenediamine tetraacetic acid EDTA is mixed in trypsin, and the passage ratio is 1:2-3.
7. The method for preparing hair follicle stimulating fluid 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 15-20mmol/L.
8. According to the weightsThe method for preparing hair follicle stimulating fluid based on umbilical cord mesenchymal stem cell exosomes of claim 1, wherein the method for obtaining hair papilla cells in step S6 is as follows: the hair follicle tissue is washed 2-3 times in sterile PBS solution and then sheared into 2+ -0.5 mm 3 The method comprises the steps of dissolving tissue fragments with the volume size in enough type I collagenase, stirring and reacting for 2-4 hours, filtering and drying, dissolving the tissue fragments in enough type IV collagenase, stirring and reacting for 2-4 hours, sieving the hair papilla tissue under a microscope, placing the hair papilla tissue into an ultra-clean culture dish, adding modified DMEM culture solution for culturing, changing the solution once every 2d half amount, changing the solution once every 3d full amount after three times, continuing culturing for two weeks, and carrying out passage for 72-75 hours, and collecting and obtaining primary hair papilla cells.
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