CN112190533A - Stem cell striae gravidarum repair method - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
- A61K8/676—Ascorbic acid, i.e. vitamin C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
- A61K8/982—Reproductive organs; Embryos, Eggs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
Abstract
The invention discloses a stem cell striae gravidarum repairing method, which comprises the following steps: s1: preparing umbilical cord mesenchymal stem cell cytokine supernatant; s2: cutting part of the umbilical cord in the S1 into a plurality of small sections, and then cleaning the small sections with sterile PBS until the blood filaments cannot be seen by naked eyes; s3: shearing the small segment of umbilical cord after the completion of cleaning in the S2, transferring the small segment of umbilical cord into a cell culture dish, culturing by using an alpha-MEM complete culture medium containing fetal bovine serum, and adding trypsin for digestion and passage when the cells under a microscope are paved on 80% of the area of the culture dish; s4: when the cells in the S3 reach the 3 rd generation logarithmic growth phase, passing part of the cells to a stem cell serum-free culture medium for culture, obtaining a stem cell serum-free culture medium supernatant by a centrifuge through a low-speed centrifugation method, then adding connecting fibrin into the stem cell serum-free culture medium supernatant, and mixing to obtain an auxiliary repair solution for later use; s5: preparing adipose mesenchymal stem cell factor supernatant; s6: and (5) preparing a repair product.
Description
Technical Field
The invention relates to the technical field of striae gravidarum repair, in particular to a stem cell striae gravidarum repair method.
Background
The stretch marks are mainly formed because the elastic fibers and the collagen fibers of the skin are damaged or broken to different degrees due to the weight increase and the expansion of the abdomen of a woman during pregnancy, so that the skin becomes thinner and thinner, and the elasticity and the bearing force of the skin are lost, so that the stretch marks and the subsidence feeling are generated; and because the collagen and elastin in the dermis are not enough to load the skin and are supported too much at one stroke, the fibers are broken, so that the skin is sunken and the capillary vessels are squeezed to form scar-shaped stripes on the surface of the skin.
Although the striae gravidarum repair product on the market at present has a certain effect, the problems that the repair rate is low, the effect cannot meet the requirements of patients and the repair effect is not very ideal exist; meanwhile, the existing striae gravidarum repair cream, repair paste and the like contain chemical products, have certain toxic and side effects, and after long-term use, the skin is easy to generate allergic reaction or inflammation and has large damage to the skin.
Disclosure of Invention
The invention aims to provide a stem cell striae gravidarum repairing method to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a stem cell striae gravidarum repair method comprises the following steps:
s1: preparing umbilical cord mesenchymal stem cell cytokine supernatant;
s2: cutting part of the umbilical cord in the S1 into a plurality of small sections, and then cleaning the small sections with sterile PBS until the blood filaments cannot be seen by naked eyes;
s3: shearing the small segment of umbilical cord after the completion of cleaning in the S2, transferring the small segment of umbilical cord into a cell culture dish, culturing by using an alpha-MEM complete culture medium containing fetal bovine serum, and adding trypsin for digestion and passage when the cells under a microscope are paved on 80% of the area of the culture dish;
s4: when the cells in the S3 reach the 3 rd generation logarithmic growth phase, passing part of the cells to a stem cell serum-free culture medium for culture, obtaining a stem cell serum-free culture medium supernatant by a centrifuge through a low-speed centrifugation method, then adding connecting fibrin into the stem cell serum-free culture medium supernatant, and mixing to obtain an auxiliary repair solution for later use;
s5: preparing adipose mesenchymal stem cell factor supernatant;
s6: mixing an auxiliary repair solution, umbilical cord mesenchymal stem cell cytokine supernatant, adipose mesenchymal stem cell cytokine supernatant, arabinogalactan, zinc gluconate, copper gluconate and sodium ascorbate according to the proportion of 15-20: 20-30: 10-15: 1-3: 1-2: 1-2: 0.1-2 to obtain the repair product.
As a preferable technical scheme, the preparation method of the umbilical cord mesenchymal stem cell cytokine supernatant comprises the steps of cleaning umbilical cord tissue surface blood by using tissue protection solution, removing epidermis and vascular tissue, taking out and cleaning huatong glue, shearing into 5-20 blocks, and spreading in a culture dish.
As a preferred technical scheme of the invention, the special culture solution for the mesenchymal stem cells is slowly added along the edge of the culture dish until the tissue is submerged; placing the culture dish in CO2Culturing in a 5% culture box at 37 deg.C for 3-7 days; the culture medium is continuously added for 9-11 days, and then the culture medium is changed every 1-3 days.
As a preferred technical scheme of the invention, when the cell fusion degree in the culture dish reaches 50% -70%, digestive enzyme is heated in the culture dish to separate the tissue from the culture dish, the tissue is planted in a Corning T175 culture bottle, 30ml of culture solution is added for continuous culture for 3-4 days, and when the cell fusion degree reaches 90%, the supernatant is collected and centrifuged to remove cell debris; and adding the centrifuged supernatant into a 15ml ultrafiltration tube for concentration, removing redundant water to ensure that the volume is concentrated to be half of the original volume, and filtering the concentrated solution through a 0.22 mu m filter membrane for sterilization to obtain the umbilical cord mesenchymal stem cell cytokine supernatant.
As a preferable technical scheme of the invention, the tissue protection solution is prepared by adding 25ug/ml gentamicin sulfate and 5ug/ml amphotericin B into physiological saline.
As a preferred technical scheme of the invention, the centrifugal rate is 3000 r/min; the centrifugation time was 20 minutes.
As a preferred technical scheme of the invention, the fat particles are centrifuged for 4-8min under the conditions of 900-1200r/min, the upper layer fat tissue and the lower layer cells A are respectively reserved, collagenase with the mass concentration of 0.1% is added into the fat tissue, the fat tissue is digested for 25-35min in a water bath shaking table at 37 ℃, and then centrifuged for 4-8min at 1800-2200r/min, and the lower layer cells B are reserved; uniformly mixing the lower layer cell A and the lower layer cell B, resuspending, centrifuging for 4-8min at 1800-2200r/min, adopting serum-free component culture medium to suspend the cells, inoculating the cells to a six-hole plate for culturing for 40-50 h, changing the liquid every three days, and culturing for 20-24 days.
As a preferable technical scheme of the invention, the mixed cell population is identified after 20-24 days of culture, the cell population identified as the adipose-derived mesenchymal stem cells is subjected to mixed subculture, and the cell number reaches 3 x 107In the process, a serum-free culture system and a cell factory are adopted for carrying out enlarged culture for 3-4 days, and the supernatant of the adipose-derived mesenchymal stem cell factor is collected.
Compared with the prior art, the invention has the beneficial effects that:
the serum-free culture medium supernatant of the mesenchymal stem cells and the connective fibrin are used in a combined way, the collagen fibers and the elastic fibers in the dermis layer can be proliferated and rearranged by the serum-free culture medium supernatant of the mesenchymal stem cells, and the collagen is deposited orderly.
In addition, the invention also comprises adipose-derived mesenchymal stem cell secretion and umbilical cord mesenchymal stem cell secretion of various bioactive factors, and the adipose-derived mesenchymal stem cell secretion and umbilical cord mesenchymal stem cell secretion can effectively reduce or eliminate striae gravidarum, and simultaneously, cell growth factors with bioactive components can be led into skin deep activated cells, promote subcutaneous collagen fiber deposition, repair torn skin tissues, effectively prevent the formation of epilepsia marks and fundamentally repair the striae gravidarum; since these bioactive molecules are produced by normal cells, their concentrations and ratios to each other are more consistent with the physiological needs of the skin, which facilitates skin rejuvenation and repair without side effects.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Specifically, the method comprises the following steps: the invention provides a technical scheme that: a stem cell striae gravidarum repair method comprises the following steps:
s1: preparing umbilical cord mesenchymal stem cell cytokine supernatant;
s2: cutting part of the umbilical cord in the S1 into a plurality of small sections, and then cleaning the small sections with sterile PBS until the blood filaments cannot be seen by naked eyes;
s3: shearing the small segment of umbilical cord after the completion of cleaning in the S2, transferring the small segment of umbilical cord into a cell culture dish, culturing by using an alpha-MEM complete culture medium containing fetal bovine serum, and adding trypsin for digestion and passage when the cells under a microscope are paved on 80% of the area of the culture dish;
s4: when the cells in the S3 reach the 3 rd generation logarithmic growth phase, passing part of the cells to a stem cell serum-free culture medium for culture, obtaining a stem cell serum-free culture medium supernatant by a centrifuge through a low-speed centrifugation method, then adding connecting fibrin into the stem cell serum-free culture medium supernatant, and mixing to obtain an auxiliary repair solution for later use;
s5: preparing adipose mesenchymal stem cell factor supernatant;
s6: mixing an auxiliary repair solution, umbilical cord mesenchymal stem cell cytokine supernatant, adipose mesenchymal stem cell cytokine supernatant, arabinogalactan, zinc gluconate, copper gluconate and sodium ascorbate according to the proportion of 15-20: 20-30: 10-15: 1-3: 1-2: 1-2: 0.1-2 to obtain the repair product.
Further, the preparation method of the umbilical cord mesenchymal stem cell cytokine supernatant comprises the steps of cleaning umbilical cord tissue surface blood with tissue protection solution, removing epidermis and vascular tissue, taking out the Huatong glue, cleaning, shearing into 5-20 blocks, and spreading in a culture dish.
Further, slowly adding the special culture solution for the mesenchymal stem cells along the edge of the culture dish toSubmerging the tissue; placing the culture dish in CO2Culturing in a 5% culture box at 37 deg.C for 3-7 days; the culture medium is continuously added for 9-11 days, and then the culture medium is changed every 1-3 days.
Further, when the cell fusion degree in the culture dish reaches 50% -70%, heating digestive enzyme in the culture dish to separate the tissue from the culture dish, planting the tissue in a Corning T175 culture bottle, adding 30ml of culture solution to continue culturing for 3-4 days, collecting supernatant when the cell fusion degree reaches 90%, centrifuging the supernatant, and removing cell debris; and adding the centrifuged supernatant into a 15ml ultrafiltration tube for concentration, removing redundant water to ensure that the volume is concentrated to be half of the original volume, and filtering the concentrated solution through a 0.22 mu m filter membrane for sterilization to obtain the umbilical cord mesenchymal stem cell cytokine supernatant.
Further, the tissue protection solution is prepared by adding 25ug/ml gentamicin sulfate and 5ug/ml amphotericin B into physiological saline.
Further, the centrifugation rate is 3000 rpm; the centrifugation time was 20 minutes.
Further, the fat particles are centrifuged for 4-8min under the conditions of 900-; uniformly mixing the lower layer cell A and the lower layer cell B, resuspending, centrifuging for 4-8min at 1800-2200r/min, adopting serum-free component culture medium to suspend the cells, inoculating the cells to a six-hole plate for culturing for 40-50 h, changing the liquid every three days, and culturing for 20-24 days.
Further, identifying the mixed cell population after culturing for 20-24 days, and performing mixed subculture on the cell population identified as the adipose tissue-derived mesenchymal stem cells until the cell number reaches 3 multiplied by 107In the process, a serum-free culture system and a cell factory are adopted for carrying out enlarged culture for 3-4 days, and the supernatant of the adipose-derived mesenchymal stem cell factor is collected.
Further, 50 patients with striae gravidarum of the volunteers 3-6 months after birth are screened and divided into 5 groups (A, B, C, D and E), 10 patients in each group are tried, the striae gravidarum product is used, and the product is divided into the following test products according to the difference of the ratio S of the umbilical cord mesenchymal stem cell factor supernatant to the adipose mesenchymal stem cell factor supernatant, wherein the test product A (S is 20: 10), the test product B (S is 25: 10), the test product C (S is 30: 10), the test product D (S is 20: 12) and the test product E (S is 20: 15).
Experiment group one: applying test product A, B, C to volunteers of group A, group B and group C, respectively, cleaning abdomen of the volunteer, sterilizing with 75% medical alcohol, applying the product to striae gravidarum, and continuously applying for 4 weeks 2 times per week; 60% of striae gravidarum in the group A is completely recovered, 30% of striae gravidarum is gradually lightened, and 10% of striae gravidarum is not obviously changed; in group B, 70% striae gravidarum is completely recovered, and 30% striae gravidarum is gradually lightened; in the third group, 65% striae gravidarum is completely recovered, 20% striae gravidarum is gradually lightened, and 15% striae gravidarum is not obviously changed.
According to the first experimental group, under the condition that the ratio of the adipose-derived mesenchymal stem cell factor supernatant is not changed, the ratio of the umbilical cord mesenchymal stem cell factor to the adipose-derived mesenchymal stem cell factor supernatant is 25:10, which is the best.
Experiment group two: applying D, E test product to volunteers in group D and group E, respectively, cleaning abdomen of the volunteers, sterilizing with 75% medical ethanol, applying the product on striae gravidarum, and continuously applying for 4 weeks 2 times per week; it can be found that 65% of striae gravidarum in the group D is completely recovered, 25% of striae gravidarum is gradually lightened, and 10% of striae gravidarum is not obviously changed; in the group E, 80% of striae gravidarum were completely recovered, and 20% of striae gravidarum were gradually lightened.
According to the second experimental group, under the condition that the ratio of the umbilical cord mesenchymal stem cell cytokine is not changed, the ratio of the umbilical cord mesenchymal stem cell cytokine supernatant to the adipose mesenchymal stem cell cytokine supernatant is the best 20: 15.
In conclusion, the ratio of the umbilical cord mesenchymal stem cell cytokine supernatant to the adipose mesenchymal stem cell cytokine supernatant is 25:15, which is the best, according to the experimental group I and the experimental group II.
In the method for repairing the stem cell striae gravidarum, the steps are simple, clear and easy to operate, the product contains adipose mesenchymal stem cell secretion and umbilical cord mesenchymal stem cell secretion of various bioactive factors, the product can effectively reduce or eliminate the striae gravidarum, and simultaneously cell growth factors with bioactive components can be introduced into skin deep activated cells to promote subcutaneous collagen fiber deposition and repair torn skin tissues, so that the formation of the epilepsia can be effectively prevented, and the striae gravidarum is fundamentally repaired; since these bioactive molecules are produced by normal cells, their concentrations and ratios to each other are more consistent with the physiological needs of the skin, which facilitates skin rejuvenation and repair without side effects.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A stem cell striae gravidarum repair method is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing umbilical cord mesenchymal stem cell cytokine supernatant;
s2: cutting part of the umbilical cord in the S1 into a plurality of small sections, and then cleaning the small sections with sterile PBS until the blood filaments cannot be seen by naked eyes;
s3: shearing the small segment of umbilical cord after the completion of cleaning in the S2, transferring the small segment of umbilical cord into a cell culture dish, culturing by using an alpha-MEM complete culture medium containing fetal bovine serum, and adding trypsin for digestion and passage when the cells under a microscope are paved on 80% of the area of the culture dish;
s4: when the cells in the S3 reach the 3 rd generation logarithmic growth phase, passing part of the cells to a stem cell serum-free culture medium for culture, obtaining a stem cell serum-free culture medium supernatant by a centrifuge through a low-speed centrifugation method, then adding connecting fibrin into the stem cell serum-free culture medium supernatant, and mixing to obtain an auxiliary repair solution for later use;
s5: preparing adipose mesenchymal stem cell factor supernatant;
s6: mixing an auxiliary repair solution, umbilical cord mesenchymal stem cell cytokine supernatant, adipose mesenchymal stem cell cytokine supernatant, arabinogalactan, zinc gluconate, copper gluconate and sodium ascorbate according to the proportion of 15-20: 20-30: 10-15: 1-3: 1-2: 1-2: 0.1-2 to obtain the repair product.
2. The method for repairing stem cell striae gravidarum according to claim 1, wherein: the preparation method of the umbilical cord mesenchymal stem cell cytokine supernatant comprises the steps of cleaning umbilical cord tissue surface blood by using a tissue protection solution, removing epidermis and vascular tissue, taking out the Huatong glue, cleaning, shearing into 5-20 blocks, and flatly spreading in a culture dish.
3. The method for repairing stem cell striae gravidarum according to claim 2, wherein: slowly adding the special culture solution for the mesenchymal stem cells along the edge of the culture dish until the tissue is submerged; placing the culture dish in CO2Culturing in a 5% culture box at 37 deg.C for 3-7 days; the culture medium is continuously added for 9-11 days, and then the culture medium is changed every 1-3 days.
4. The method for repairing stem cell striae gravidarum according to claim 3, wherein: when the cell fusion degree in the culture dish reaches 50% -70%, heating digestive enzyme in the culture dish to separate the tissue from the culture dish, planting the tissue in a Corning T175 culture bottle, adding 30ml of culture solution to continue culturing for 3-4 days, collecting supernatant when the cell fusion degree reaches 90%, centrifuging the supernatant, and removing cell debris; and adding the centrifuged supernatant into a 15ml ultrafiltration tube for concentration, removing redundant water to ensure that the volume is concentrated to be half of the original volume, and filtering the concentrated solution through a 0.22 mu m filter membrane for sterilization to obtain the umbilical cord mesenchymal stem cell cytokine supernatant.
5. The method for repairing stem cell striae gravidarum according to claim 2, wherein: the tissue protection solution is prepared by adding 25ug/ml gentamicin sulfate and 5ug/ml amphotericin B into physiological saline.
6. The method for repairing stem cell striae gravidarum according to claim 4, wherein: the centrifugation rate is 3000 rpm; the centrifugation time was 20 minutes.
7. The method for repairing stem cell striae gravidarum according to claim 1, wherein: the preparation method of the adipose-derived mesenchymal stem cell factor supernatant comprises the steps of centrifuging adipose particles for 4-8min under the conditions of 900 plus 1200r/min, respectively reserving an upper adipose tissue and a lower cell A, adding collagenase with the mass concentration of 0.1% into the adipose tissue, placing the adipose tissue into a water bath shaker at 37 ℃ for digestion for 25-35min, then centrifuging for 4-8min at 1800 plus 2200r/min, and reserving a lower cell B; uniformly mixing the lower layer cell A and the lower layer cell B, resuspending, centrifuging at 1800 plus 2200r/min for 4-8min, adopting serum-free component culture medium to resuspend the cells, inoculating to a six-hole plate, culturing for 40-50 h, changing the liquid every three days, and culturing for 20-24 days.
8. The method for repairing stem cell striae gravidarum according to claim 7, wherein: identifying the mixed cell population after culturing for 20-24 days, and performing mixed subculture on the cell population identified as the adipose-derived mesenchymal stem cells until the cell number reaches 3 × 107In the process, a serum-free culture system and a cell factory are adopted for carrying out enlarged culture for 3-4 days, and the supernatant of the adipose-derived mesenchymal stem cell factor is collected.
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CN111097041A (en) * | 2020-03-25 | 2020-05-05 | 白晋 | Preparation method of mixed solution for repairing striae gravidarum by using mesenchymal stem cell culture medium in combination with fibronectin |
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