CN113975221A - Umbilical cord mesenchymal stem cell factor freeze-dried powder and preparation method thereof - Google Patents
Umbilical cord mesenchymal stem cell factor freeze-dried powder and preparation method thereof Download PDFInfo
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- 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
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0216—Solid or semisolid forms
- A61K8/022—Powders; Compacted Powders
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- 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
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/345—Alcohols containing more than one hydroxy group
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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
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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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
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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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
- 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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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F26—DRYING
- F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
- F26B5/00—Drying solid materials or objects by processes not involving the application of heat
- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
- F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
Abstract
The invention discloses umbilical cord mesenchymal stem cell factor freeze-dried powder, which contains umbilical cord mesenchymal stem cell factor extracting solution and freeze-drying auxiliary agent, wherein the freeze-drying auxiliary agent comprises: mannitol, vitamin C derivatives, collagen and dipeptide diaminobutyrylbenzylamide diacetate. The umbilical cord mesenchymal stem cell factor freeze-dried powder prepared by the invention has the functions of quick response, long acting, long storage time and good beautifying and anti-aging effects.
Description
Technical Field
The invention relates to the technical field of freeze-dried powder preparation, and particularly relates to umbilical cord mesenchymal stem cell factor freeze-dried powder and a preparation method thereof.
Background
The factors of skin aging are divided into two categories, one is endogenous aging caused by aging, the other is exogenous aging caused by various external factors, and the skin aging is most directly reflected by dry and rough skin, relaxation, elasticity reduction, wrinkles and pigmentation; at the cellular level, skin aging is a problem of genetic variation, telomere abnormality, increased protein and cell damage, increased inflammation, increased cell aging, exhaustion of endogenous stem cells and the like.
The umbilical cord mesenchymal stem cells have self-renewal capacity and multi-directional differentiation potential, and can be differentiated into cells or organ tissues with different functions under specific physiological or in vitro induction conditions. The umbilical cord mesenchymal stem cells can secrete cell factors such as platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, acidic fibroblast growth factor, vascular endothelial growth factor, transforming growth factor beta, nerve growth factor and the like in the culture process, and the cell active substances exist in cell culture supernatant, and besides the cell factors contained in the cell culture supernatant, the stem cells also contain the cell factors such as vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, IL-6 and IL-7, megakaryocyte colony stimulating factor, deoxyribonucleic acid, ribonucleic acid, active polypeptide and the like. Various researches show that the cytokines can promote the proliferation of fibroblasts and the metabolism of cells, reduce inflammatory factors, repair aging and damage of cells and tissues, improve the internal environment of skin, play an important role in stable production and distribution of collagen and elastin and can be widely applied to stem cell beauty. At present, the cytokine cosmetic products on the market have the problems of short storage life, slow effect, low content of effective components and the like.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide umbilical cord mesenchymal stem cell factor freeze-dried powder and a preparation method thereof, so as to solve the problems of short storage life and slow effect of the existing cytokine cosmetic products.
The technical scheme for solving the technical problems is as follows: the umbilical cord mesenchymal stem cell factor freeze-dried powder comprises an umbilical cord mesenchymal stem cell factor extracting solution and a freeze-drying auxiliary agent, wherein the freeze-drying auxiliary agent comprises: mannitol, vitamin C derivatives, collagen and dipeptide diaminobutyrylbenzylamide diacetate.
The invention has the beneficial effects that: the freeze-drying auxiliary agent is added into the umbilical cord mesenchymal stem cell factor freeze-dried powder, and the vitamin C derivative is a low-irritation antioxidant, so that the active ingredients of the freeze-dried powder can be protected, the inactivation caused by oxidation can be prevented, and the storage life of the freeze-dried powder can be prolonged; after the cell factors are absorbed by the skin, the proliferation of fibroblasts is promoted, the synthesis of collagen is increased, and the problem of aging is relieved from the root of skin cells; meanwhile, the collagen is added to exogenously supplement the skin collagen and fill the skin; the dipeptide diaminobutyrylbenzylamide diacetate is a synthetic peptide simulating snake venom serum, and can prevent acetylcholine nerve conduction, achieve the effect of relaxing muscles and further improve wrinkles. The freeze-dried powder has excellent anti-wrinkle and anti-aging effects due to the cooperation of various components and cell factors.
On the basis of the technical scheme, the invention can be further improved as follows:
further, the volume fraction of each component of the freeze-drying adjuvant in the cell factor extracting solution is as follows: 5-10% of mannitol, 0.5-1% of vitamin C derivative, 0.5-1% of collagen and 0.1-0.5% of dipeptide diaminobutyrylbenzylamide diacetate.
Further, the volume fraction of each component of the freeze-drying adjuvant in the cell factor extracting solution is as follows: 6% of mannitol, 0.5% of vitamin C derivative, 0.5% of collagen and 0.2% of dipeptide diaminobutyrylbenzylamide diacetate.
The beneficial effect of adopting the further technical scheme is as follows: the effective concentration of the cell factor can be seriously influenced by the freeze-drying auxiliary agent with higher content, and the addition of the freeze-drying auxiliary agent by adopting the proportion of the invention can effectively improve the concentration of the cell factor and ensure that the freeze-dried powder keeps good physical properties.
Further, the umbilical cord mesenchymal stem cell factor extracting solution is prepared by the following method: separating Wharton's jelly on an umbilical cord, performing isolated culture to obtain P0 generation umbilical cord mesenchymal stem cells, culturing with a serum-free culture medium until the fusion degree is 80-90%, digesting with pancreatin to obtain single cells, performing subculture until the 5 th generation, and collecting the supernatant of P5 generation cell culture and the supernatant of P5 generation cell lysate to obtain the umbilical cord mesenchymal stem cell factor extracting solution.
Further, the serum-free medium culture is Hyclone serum-free medium.
Further, the primary cells were subcultured in a 1: (2-4) performing seed distribution and subculture.
Further, before separation of Wharton's jelly on the umbilical cord, the umbilical cord is soaked in a physiological saline solution containing 0.8-1.2 vt% of double antibody, and then blood vessels in the umbilical cord are removed.
Further, the in vitro culture comprises cutting the isolated Wharton's jelly, and cutting the tissue fragments at 35-39 deg.C and 4-6% CO2Culturing for 4-6 days under the condition, performing liquid change culture for the first time, then performing liquid change culture every 1.5-2.5 days, and removing tissue blocks when adherently-grown cells appear around the tissue blocks to obtain the umbilical cord mesenchymal stem cells of P0 generation.
Further, the supernatant of the cell lysate from the P5 generation was prepared by the following method: digesting and cleaning P5 generation cells, then resuspending the cells with normal saline, freezing at-75-85 ℃ for 2-4h, then thawing at 3-5 ℃, repeating the freezing and thawing process for 2-4 times, centrifuging, and collecting cell lysate supernatant.
Further, the digestion of P5 generation cells was performed by digesting the cells into single cells with 0.25% pancreatin-EDTA solution.
Further, the cell washing of P5 generation was performed 2-3 times with PBS.
The beneficial effect of adopting the further technical scheme is as follows: the invention adopts a heat shock method to crack cells and collect cell factors, ice granules in the cells are formed and the salt concentration of the residual cell sap is increased to cause swelling through multiple times of freezing and thawing, so that the cell structures are cracked and broken to release the cell factors, and then supernatant containing the cell factors is collected through centrifugation.
The invention also provides a preparation method of the umbilical cord mesenchymal stem cell factor freeze-dried powder, which comprises the following steps:
(1) adding a freeze-drying auxiliary agent into the umbilical cord mesenchymal stem cell factor extracting solution, uniformly mixing, and pre-freezing at-75 to-85 ℃ for 10-15h to prepare a freeze-drying solution;
(2) and (4) freeze-drying the freeze-dried solution to prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder.
Further, the freeze-drying in the step (2) is as follows: putting the freeze-dried liquid at-35-45 ℃, and beginning to cool to-50-55 ℃ for 0.5-1 h; then a drying process was started: vacuumizing, controlling the vacuum degree to be 0.1-0.3mbar, heating to-25-35 ℃ at-50-55 ℃, and keeping for 4.5-5.5 h; heating to-25-35 ℃ to-10-20 ℃, and keeping for 5-6 h; heating to-3 to-8 ℃ at the temperature of-10 to-20 ℃, and keeping for 3.5 to 4.5 hours; heating to-1 ℃ at the temperature of-3 to-8 ℃, and keeping for 1.5-2.5 h; and (3) secondary drying process: heating to 25-35 ℃ at the temperature of-1 ℃, and keeping for 3.5-4.5 h.
Further, the freeze-drying was: placing the freeze-dried solution at-40 deg.C, cooling to-50 deg.C, and maintaining for 1 h; then a drying process was started: vacuumizing, and controlling the vacuum degree to be 0.1-0.3 mbar; heating to-30 ℃ at the temperature of-50 ℃ and keeping for 5 hours; heating to-15 ℃ at the temperature of-30 ℃ below zero, and keeping for 5.5 hours; heating to-5 ℃ at the temperature of-15 ℃ and keeping for 4 h; heating to 0 ℃ below zero at the temperature of minus 5 ℃, and keeping for 2 hours; and (3) secondary drying process: the temperature is raised to 30 ℃ at 0 ℃ and kept for 4 h.
The beneficial effect of adopting the further technical scheme is as follows: the freeze-drying process comprises a primary drying step and a secondary drying step, wherein about 90% of water can be removed in the primary drying step, partial residual water can be removed in the secondary drying step, about 95% of water in the freeze-drying solution can be removed by the two drying steps, and the freeze-drying powder is ensured to be dried.
The invention also provides application of the umbilical cord mesenchymal stem cell factor freeze-dried powder in the aspects of skin aging resistance and beauty.
The invention has the following beneficial effects:
1. the cell factors are derived from culture supernatant and cell lysate supernatant of human umbilical cord mesenchymal stem cells, the types and the concentrations of the cell factors are effectively improved, the cell factors can promote proliferation of fibroblasts and metabolism of cells, reduce inflammatory factors, repair aging damage of cells and tissues, improve the environment in the skin, play an important role in stable production and distribution of collagen and elastin, and enable the freeze-dried powder to have the effects of beautifying and resisting aging.
2. The method adopts the serum-free culture medium to culture the umbilical cord mesenchymal stem cells, is safe and reliable, and does not cause immunological rejection.
3. The freeze-dried powder prepared by the method can effectively preserve the biological activity of the human umbilical cord mesenchymal stem cell factor, and the obtained freeze-dried powder has high cell factor yield, good activity, easy preservation and transportation, long preservation period, quick response and good market prospect.
Drawings
Fig. 1 is a picture of the face of a subject before using the umbilical cord mesenchymal stem cell factor lyophilized powder prepared in example 1;
fig. 2 is a picture of the face of a subject after using the umbilical cord mesenchymal stem cell factor lyophilized powder prepared in example 1.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1:
a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder comprises the following steps:
(1) soaking fresh umbilical cord in 1 vt% double-antibody-containing normal saline, removing residual bloodstain, removing blood vessel, separating Chinese TONG' ER gum from umbilical cord, cutting into pieces with diameter of 2mm, spreading the pieces in T-25 culture flask, culturing at 37 deg.C with 5% CO2After culturing for 5 days in the incubator, the liquid culture is changed for the first time,then half amount of liquid is changed every 2d, when cells growing adherently around the tissue block are observed under an inverted microscope, PBS is used for cleaning, the tissue block is removed, and umbilical cord mesenchymal stem cells of P0 generation are obtained;
(2) transferring the P0 generation umbilical cord mesenchymal stem cells into a culture flask, using Hyclone serum-free culture medium until 85% of fusion degree, digesting the cells into single cells by pancreatin, and mixing the primary cells according to the ratio of 1: 3, performing subculture in different varieties until the 5 th generation, collecting cell culture supernatant of the P5 generation, and storing at-80 ℃; digesting the cells of the P5 generation into single cells by using 0.25% pancreatin-EDTA solution, cleaning the single cells by using PBS for 2 times, removing residual culture medium, then re-suspending the cells by using normal saline, finally freezing the cells at-80 ℃ for 3 hours, taking the cells out, thawing the cells at 4 ℃, repeating the freezing and thawing process for 3 times, centrifuging the cells at 2000Xg for 10min, collecting cell lysate supernatant, and storing the cell lysate at-80 ℃;
(3) thawing the collected supernatant at 4 deg.C, mixing, adding freeze-drying adjuvants such as mannitol, dipeptide diaminobutyrylbenzyl amide diacetate, vitamin C derivative and collagen, dissolving, mixing, and pre-freezing at-80 deg.C for 12 hr to obtain freeze-dried solution; wherein, the volume fraction of each component of the freeze-drying auxiliary agent in the cell factor extracting solution is as follows: 6% of mannitol, 0.2% of dipeptide diaminobutyrylbenzylamide diacetate, 0.5% of vitamin C derivative and 0.5% of collagen;
(4) starting a freeze dryer in advance, precooling to-40 ℃, putting the pre-frozen freeze-drying liquid into the freeze dryer, starting to cool to-50 ℃ for 1h, and then starting a drying process: vacuumizing, and controlling the vacuum degree to be 0.2 mbar; heating to-30 ℃ at the temperature of-50 ℃ and keeping for 5 hours; heating to-15 ℃ at the temperature of-30 ℃ below zero, and keeping for 5.5 hours; heating to-5 ℃ at the temperature of-15 ℃ and keeping for 4 h; heating to 0 ℃ below zero at the temperature of minus 5 ℃, and keeping for 2 hours; and (3) secondary drying process: heating to 30 ℃ at 0 ℃, and keeping for 4h to prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder.
Example 2:
a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder comprises the following steps:
(1) soaking fresh umbilical cord in 0.8 vt% double antibody-containing normal saline, removing residual bloodstain, and removing umbilical cordThe blood vessels of (4%) of the umbilical cord, then separating the Chinese Tong's jelly from the umbilical cord, cutting the jelly into pieces with a diameter of 1mm, spreading the pieces in a T-25 culture flask, and culturing at 35 deg.C with 4% CO2After culturing for 6 days in the incubator, firstly replacing the culture solution for culturing, then replacing the culture solution every 2.5 days by half, and when cells growing adherently around the tissue block are observed under an inverted microscope, cleaning the tissue block by PBS, and removing the tissue block to obtain the umbilical cord mesenchymal stem cells P0;
(2) transferring the P0 generation umbilical cord mesenchymal stem cells into a culture flask, using Hyclone serum-free culture medium until 80% of fusion degree, digesting the cells into single cells by pancreatin, and mixing the primary cells according to the ratio of 1: 2, performing subculture in a sub-species mode until the 5 th generation, collecting cell culture supernatant of the P5 generation, and storing at-75 ℃; digesting the cells of the P5 generation into single cells by using 0.25% pancreatin-EDTA solution, cleaning the single cells by using PBS for 2 times, removing residual culture medium, then re-suspending the cells by using normal saline, finally freezing the cells at the temperature of minus 75 ℃ for 4 hours, taking the cells out, thawing the cells at the temperature of 3 ℃, repeating the freezing and thawing process for 4 times, centrifuging the cells for 10min at 2000Xg, collecting cell lysate supernatant, and storing the cell lysate supernatant at the temperature of minus 75 ℃;
(3) thawing the collected supernatant at 3 deg.C, mixing, adding freeze-drying adjuvants such as mannitol, dipeptide diaminobutyrylbenzyl amide diacetate, vitamin C derivative and collagen, dissolving, mixing, and pre-freezing at-75 deg.C for 15 hr to obtain freeze-dried solution; wherein, the volume fraction of each component of the freeze-drying auxiliary agent in the cell factor extracting solution is as follows: 8% of mannitol, 0.1% of dipeptide diaminobutyrylbenzylamide diacetate, 0.5% of vitamin C derivative and 1% of collagen;
(4) starting a freeze dryer in advance to pre-cool to-35 ℃, putting the pre-frozen freeze-drying liquid into the freeze dryer, starting to cool to-52 ℃ for 0.75h, and then starting a drying process: vacuumizing, and controlling the vacuum degree to be 0.1 mbar; heating to-25 ℃ at the temperature of-52 ℃, and keeping for 5.5 h; heating to-10 ℃ at the temperature of-25 ℃ below zero, and keeping for 5 hours; heating to-3 ℃ at the temperature of-10 ℃ below zero, and keeping for 4.5 hours; heating to-1 ℃ at the temperature of-3 ℃ and keeping for 2.5 h; and (3) secondary drying process: heating to 25 ℃ at the temperature of minus 1 ℃, and keeping for 3.5 hours to prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder.
Example 3:
a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder comprises the following steps:
(1) soaking fresh umbilical cord in 1.2 vt% double antibody-containing normal saline, removing residual bloodstain, removing blood vessel, separating Chinese TONGSHI gum from umbilical cord, cutting into pieces with diameter of 3mm, spreading the pieces in T-25 culture bottle, culturing at 39 deg.C with 6% CO2After culturing for 4 days in the incubator, firstly replacing the culture solution for culturing, then replacing the culture solution every 1.5 days by half, and when cells growing adherently around the tissue block are observed under an inverted microscope, cleaning the tissue block by PBS, and removing the tissue block to obtain the umbilical cord mesenchymal stem cells P0;
(2) transferring the P0 generation umbilical cord mesenchymal stem cells into a culture flask, using Hyclone serum-free culture medium until 90% of fusion degree, digesting the cells into single cells by pancreatin, and mixing the primary cells according to the ratio of 1: 4, performing subculture in a seed division manner until the 5 th generation, collecting cell culture supernatant of the P5 generation, and storing at-85 ℃; digesting the cells of the P5 generation into single cells by using 0.25% pancreatin-EDTA solution, cleaning the single cells by using PBS for 3 times, removing residual culture medium, then re-suspending the cells by using normal saline, finally freezing the cells at-85 ℃ for 2 hours, taking the cells out, thawing the cells at 5 ℃, repeating the freezing and thawing process for 2 times, centrifuging the cells at 2000Xg for 10min, collecting cell lysate supernatant, and storing the cell lysate at-85 ℃;
(3) thawing the collected supernatant at 5 deg.C, mixing, adding freeze-drying adjuvants such as mannitol, dipeptide diaminobutyrylbenzyl amide diacetate, vitamin C derivative and collagen, dissolving, mixing, and pre-freezing at-85 deg.C for 10 hr to obtain lyophilized solution; wherein, the volume fraction of each component of the freeze-drying auxiliary agent in the cell factor extracting solution is as follows: 10% of mannitol, 0.5% of dipeptide diaminobutyrylbenzylamide diacetate, 0.75% of vitamin C derivative and 0.75% of collagen.
(4) Starting a freeze dryer in advance to pre-cool to-45 ℃, putting the pre-frozen freeze-drying liquid into the freeze dryer, starting to cool to-55 ℃, keeping for 0.5h, and then starting a drying process: vacuumizing, and controlling the vacuum degree to be 0.3 mbar; heating to-35 ℃ at the temperature of-55 ℃, and keeping for 4.5 h; heating to-20 ℃ at the temperature of-35 ℃ below zero, and keeping for 6 hours; heating to-8 ℃ at the temperature of-20 ℃ below zero, and keeping for 3.5 hours; heating to 1 ℃ at the temperature of minus 8 ℃ and keeping for 1.5 h; and (3) secondary drying process: heating to 35 ℃ at 1 ℃, and keeping for 4.5h to prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder.
Comparative example 1
A preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder comprises the following steps:
(2) soaking fresh umbilical cord in 1 vt% double-antibody-containing normal saline, removing residual bloodstain, removing blood vessel, separating Chinese TONG' ER gum from umbilical cord, cutting into pieces with diameter of 2mm, spreading the pieces in T-25 culture flask, culturing at 37 deg.C with 5% CO2After culturing for 5 days in the incubator, firstly replacing the culture solution for culturing, then replacing half amount of the culture solution every 2 days, and when cells growing adherently around the tissue block are observed under an inverted microscope, cleaning the tissue block by PBS, and removing the tissue block to obtain the umbilical cord mesenchymal stem cells P0 generation;
(2) transferring the P0 generation umbilical cord mesenchymal stem cells into a culture flask, using Hyclone serum-free culture medium until 85% of fusion degree, digesting the cells into single cells by pancreatin, and mixing the primary cells according to the ratio of 1: 3, performing subculture in different varieties until the 5 th generation, collecting cell culture supernatant of the P5 generation, and storing at-80 ℃; digesting the cells of the P5 generation into single cells by using 0.25% pancreatin-EDTA solution, cleaning the single cells by using PBS for 2 times, removing residual culture medium, then re-suspending the cells by using normal saline, finally freezing the cells at-80 ℃ for 3 hours, taking the cells out, thawing the cells at 4 ℃, repeating the freezing and thawing process for 3 times, centrifuging the cells at 2000Xg for 10min, collecting cell lysate supernatant, and storing the cell lysate at-80 ℃;
(3) thawing the collected supernatant at 4 deg.C, mixing, adding freeze-drying adjuvants such as mannitol, dipeptide diaminobutyrylbenzyl amide diacetate, vitamin C derivative and collagen, dissolving, mixing, and pre-freezing at-80 deg.C for 12 hr to obtain freeze-dried solution; wherein, the volume fraction of each component of the freeze-drying auxiliary agent in the cell factor extracting solution is as follows: 6% of mannitol, 0.2% of dipeptide diaminobutyrylbenzylamide diacetate, 0.5% of vitamin C derivative and 0.5% of collagen;
(4) starting a freeze dryer in advance, precooling to-40 ℃, putting the pre-frozen freeze-drying liquid into the freeze dryer, starting to cool to-50 ℃ for 1h, and then starting a drying process: vacuumizing, and controlling the vacuum degree to be 0.2 mbar; heating to-30 ℃ at the temperature of-50 ℃ and keeping for 5 hours; heating to-15 ℃ at the temperature of-30 ℃ below zero, and keeping for 5.5 hours; heating to-5 ℃ at the temperature of-15 ℃ and keeping for 4 h; heating to 0 ℃ at the temperature of minus 5 ℃, and keeping for 2 hours to prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder.
Effect verification:
the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared in the embodiments 1 to 3 has basically the same effect parameters, and the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared in the embodiment 1 is taken for effect verification.
First, detection of preservation time
The umbilical cord mesenchymal stem cell factor freeze-dried powders prepared in the example 1 and the comparative example 1 are subjected to moisture residual quantity detection, and the moisture residual quantity of the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared in the example 1 is 5%, and the moisture residual quantity of the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared in the comparative example 1 is 13%. The gradient drying process can remove about 95% of water on the premise of ensuring the activity of the freeze-dried components, can greatly improve the circulation and storage conditions of the cell factors, and can prolong the shelf life of the cell factors by one year.
Second, cosmetic effect verification
The umbilical cord mesenchymal stem cell factor freeze-dried powder prepared in the example 1 is subjected to cosmetic effect verification, and the specific method comprises the following steps: dissolving a proper amount of lyophilized powder with about 3ml of commercially available physiological saline, applying the solution on face, and detecting facial texture before and after application with VISIA respectively once a day in the morning and at night after application for 7 days, wherein the picture of facial wrinkles is shown in FIG. 1 and FIG. 2, and can be seen from FIG. 1 and FIG. 2: after the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared by the invention is used, the facial texture of a subject becomes good, which shows that the skin water locking property and the moisture retention are increased, the deep wrinkles of the facial wrinkles are reduced, and the fine wrinkles are reduced, and shows that the umbilical cord mesenchymal stem cell factor freeze-dried powder prepared by the invention has better effects of improving the skin, resisting aging and resisting wrinkles.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The umbilical cord mesenchymal stem cell factor freeze-dried powder is characterized by comprising an umbilical cord mesenchymal stem cell factor extracting solution and a freeze-drying auxiliary agent; wherein the lyophilization aid comprises: mannitol, vitamin C derivatives, collagen and dipeptide diaminobutyrylbenzylamide diacetate.
2. The umbilical cord mesenchymal stem cell factor freeze-dried powder of claim 1, wherein the volume fraction of each component of the freeze-drying adjuvant in the cell factor extracting solution is as follows: 5-10% of mannitol, 0.5-1% of vitamin C derivative, 0.5-1% of collagen and 0.1-0.5% of dipeptide diaminobutyrylbenzylamide diacetate.
3. The umbilical cord mesenchymal stem cell factor freeze-dried powder according to claim 2, wherein the volume fraction of each component of the freeze-drying auxiliary agent in the cell factor extracting solution is as follows: 6% of mannitol, 0.5% of vitamin C derivative, 0.5% of collagen and 0.2% of dipeptide diaminobutyrylbenzylamide diacetate.
4. The umbilical cord mesenchymal stem cell factor freeze-dried powder according to claim 1, wherein the umbilical cord mesenchymal stem cell factor extracting solution is prepared by the following method: separating Wharton's jelly on an umbilical cord, performing isolated culture to obtain P0 generation umbilical cord mesenchymal stem cells, culturing with a serum-free culture medium until the fusion degree is 80-90%, digesting with pancreatin to obtain single cells, performing subculture until the 5 th generation, and collecting the supernatant of P5 generation cell culture and the supernatant of P5 generation cell lysate to obtain the umbilical cord mesenchymal stem cell factor extracting solution.
5. The lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 4, wherein the in vitro culture comprises cutting the isolated Wharton's jelly, and cutting the tissue fragments at 35%-39 ℃ and 4-6% CO2Culturing for 4-6 days under the condition, performing liquid change culture for the first time, then performing liquid change culture every 1.5-2.5 days, and removing tissue blocks when adherently-grown cells appear around the tissue blocks to obtain the umbilical cord mesenchymal stem cells of P0 generation.
6. The umbilical cord mesenchymal stem cell factor lyophilized powder of claim 4, wherein the supernatant of the P5 generation cell lysate is prepared by the following method: digesting and cleaning P5 generation cells, then resuspending the cells with normal saline, freezing at-75-85 ℃ for 2-4h, then thawing at 3-5 ℃, repeating the freezing and thawing process for 2-4 times, centrifuging, and collecting cell lysate supernatant.
7. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor of any one of claims 1 to 6, comprising the steps of:
(1) adding a freeze-drying auxiliary agent into the umbilical cord mesenchymal stem cell factor extracting solution, uniformly mixing, and pre-freezing at-75 to-85 ℃ for 10-15h to prepare a freeze-drying solution;
(2) and (4) freeze-drying the freeze-dried solution to prepare the umbilical cord mesenchymal stem cell factor freeze-dried powder.
8. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 7, wherein the lyophilization in step (2) is: putting the freeze-dried liquid at-35-45 ℃, and beginning to cool to-50-55 ℃ for 0.5-1 h; then a drying process was started: vacuumizing, controlling the vacuum degree to be 0.1-0.3mbar, heating to-25-35 ℃ at-50-55 ℃, and keeping for 4.5-5.5 h; heating to-25-35 ℃ to-10-20 ℃, and keeping for 5-6 h; heating to-3 to-8 ℃ at the temperature of-10 to-20 ℃, and keeping for 3.5 to 4.5 hours; heating to-1 ℃ at the temperature of-3 to-8 ℃, and keeping for 1.5-2.5 h; and (3) secondary drying process: heating to 25-35 ℃ at the temperature of-1 ℃, and keeping for 3.5-4.5 h.
9. The method for preparing umbilical cord mesenchymal stem cell factor freeze-dried powder according to claim 8, wherein the freeze-drying is: placing the freeze-dried solution at-40 deg.C, cooling to-50 deg.C, and maintaining for 1 h; then a drying process was started: vacuumizing, and controlling the vacuum degree to be 0.2 mbar; heating to-30 ℃ at the temperature of-50 ℃ and keeping for 5 hours; heating to-15 ℃ at the temperature of-30 ℃ below zero, and keeping for 5.5 hours; heating to-5 ℃ at the temperature of-15 ℃ and keeping for 4 h; heating to 0 ℃ below zero at the temperature of minus 5 ℃, and keeping for 2 hours; and (3) secondary drying process: the temperature is raised to 30 ℃ at 0 ℃ and kept for 4 h.
10. The umbilical cord mesenchymal stem cell factor freeze-dried powder of any one of claims 1 to 6 for skin anti-aging and beauty.
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