CN110269833A - A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application - Google Patents
A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application Download PDFInfo
- Publication number
- CN110269833A CN110269833A CN201910489249.6A CN201910489249A CN110269833A CN 110269833 A CN110269833 A CN 110269833A CN 201910489249 A CN201910489249 A CN 201910489249A CN 110269833 A CN110269833 A CN 110269833A
- Authority
- CN
- China
- Prior art keywords
- umbilical cord
- stem cells
- mesenchymal stem
- cord mesenchymal
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- A61K8/33—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
-
- 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
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
-
- 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
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
- A61K8/442—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof substituted by amido group(s)
-
- 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
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
- A61K8/447—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof containing sulfur
-
- 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
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
- A61K8/4913—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid
-
- 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
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
- A61K8/4913—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid
- A61K8/492—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid having condensed rings, e.g. indol
-
- 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
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
- A61K8/4946—Imidazoles or their condensed derivatives, e.g. benzimidazoles
-
- 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
-
- 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
- A61K8/671—Vitamin A; Derivatives thereof, e.g. ester of vitamin A acid, ester of retinol, retinol, retinal
-
- 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
- A61K8/673—Vitamin B group
-
- 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
-
- 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
- A61K8/678—Tocopherol, i.e. vitamin E
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of preparations containing umbilical cord mesenchymal stem cells and its preparation method and application, pass through a large amount of experiment to grope and verify, the present invention selects the umbilical cord mesenchymal stem cells in 3-10 generation as raw material, and by first using Mechanical Method dispersion tissue, again with I containing Collagenase, Collagenase II, the mixed enzyme short time of trypsase, repeatedly several processing method, the vigor of the umbilical cord mesenchymal stem cells of separation ensure that.Using containing DMEM/F12 (without phenol red), 10%FBS, 20 kinds of amino acid and 8 kinds of vitamin umbilical cord mesenchymal stem cells preparation special culture media, by the mixed liquor for collecting multiple cell culture supernatant and cell lysate supernatant, obtain more umbilical cord mesenchymal stem cells activated proteins and active factors, preparation cost is saved, is met industrially to stem cell products formulation requirements.Umbilical cord mesenchymal stem cells preparation prepared by the present invention can promote epidermal cell and skin fibroblasts to be proliferated, safe without toxic side effect, not allergy, be applied to beauty skin care product significant effect.
Description
Technical field
The present invention relates to skin care product technical fields, and in particular to one kind preparation containing umbilical cord mesenchymal stem cells and its system
Preparation Method and application.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is derived from the mesoderm of mesoderm growing early stage and outer
Germinal layer, a kind of stem cell with self-renewing, proliferation, Multidirectional Differentiation ability, finds in marrow in nineteen sixty-eight, can induce
It is divided into the Various Tissues cells such as cardiac muscle, nerve, cartilage, fat, tendon.Mescenchymal stem cell can be from fat, marrow, navel
It is separated in the Various Tissues such as band, placenta, amnion, has clinically been widely applied to nervous system, angiocarpy, orthopaedics, glycosuria
The multiple fields such as disease, liver, skin, gastrointestinal tract.As the mesenchymal stem cell being found earliest, research is also the widest
It is general, but the quantity of mesenchymal stem cells in adult human with combined density gradient centrifugation, differentiation and proliferation ability by individual age, health states influenced compared with
Greatly, and sampling needs to puncture marrow, and larger to actual bodily harm, operating process is also vulnerable to pollution.With medulla mesenchyma
Stem cell is compared, and belongs to clinical waste from the mescenchymal stem cell of neonatal umbilical cord, materials are convenient, abundance, cost
Low, quantity is more and Proliferation, Differentiation ability is strong, and the efficiency factor of secretion is also more, in terms of medical treatment and beauty can be preferably applied for,
It is optimal mesenchymal cell source.
Umbilical cord mesenchymal stem cells can secrete mass efficient active constituent, including cell growth during culture
Factor S CF, bFGF, EGF, VEGF, KGF, IGF, HGF, immune correlation factor interleukins IL-6, IL-8, IL-10, I, II,
III, IV collagen type and other activated proteins, small-molecule substance etc., these ingredients are thin by directly or indirectly participating in
Born of the same parents' signal transduction, process of immune regulation can promote vascularization, promote cell metabolism, accelerating wound healing, repair thin
Cellular damage promotes collagen synthesis, alleviates cell ageing.Make umbilical cord mesenchymal stem cells in medical and beauty treatment in these features
Industry has great development potentiality.Above-mentioned active material is such as acted on into skin, epidermal cell and skin can be promoted at fibre
Cell Proliferation is tieed up, skin defence capability, repairing skin tissue are enhanced, it is raw to solve daily skin problem such as drying, water shortage, melanin
At, aging etc..
At present in beauty and skin care industry, umbilical cord mesenchymal stem cells can be used to prepare beauty injection or skin care item addition
The factor.Stem cell is mainly injected directly into skin nutritional deficiency and the aging for alleviating skin by beauty injection, still
Injection will form wound, have infection risk, and the requirement to injection environment and ejection preparation is relatively high, expensive, such as Shen
It please number patent of invention for being CN201010551722.And the additive as skin care item, stem cell can directly act on skin table
Layer, will not cause to damage to skin, be free from side effects being used for a long time.Cost of manufacture relative to ejection preparation want it is low very
It is more.But the effective component yield that the overwhelming majority method for preparing stem cell medicine obtains at present is all lower, such as application No. is
The patent of invention of CN201810621158 is however well-known using the supernatant culture solution of umbilical cord mesenchymal stem cells as essence
High generation stem cell can not only generate beneficial cell factor during the cultivation process, can also generate cell metabolism waste, and the hair
Bright cell factor is only detected containing IGF, bFGF and EGF, is obtained using the supernatant culture solution of umbilical cord mesenchymal stem cells
The type and quantity of cell factor are limited, and Shang Buneng makes full use of umbilical cord mesenchymal stem cells, are also unable to satisfy the reality of industry
Border demand.
The present invention obtains the stem cell extract of high quality by improving stem cell acquisition and cultural method, selects first
Raw material obtain convenient and contained effective component more umbilical cord mesenchymal stem cells, in material materials, processing and transport
Keep low temperature, sterile.The umbilical cord mesenchymal stem cells for only selecting algebra less (3-10 generation), add 20 kinds of amino in culture solution
Acid and 8 kinds of vitamins, can make stem cell secrete more efficiency factors during the growth process, the production of metabolic waste can be greatly reduced
It is raw, optimize final product quality.Umbilical cord mesenchymal stem cells during growth, in addition to the effective active factor can be secreted
Outside, it can also consume nutriment, generate metabolite, therefore during we prepare extracting solution, be added to 20 toward culture medium
Kind amino acid and 8 kinds of vitamins, had both enriched the growing environment of cell in this way, in turn ensured that cell can divide in the state of preferably
Secrete more active factors.The generally organized adherent method of the separation of umbilical cord mesenchymal stem cells and two kinds of enzymic digestion in the prior art
Method, but adherent method cell is organized to climb out of that the time is longer, and simple enzyme digestion is since pre-processing cell stage is long, carefully
Born of the same parents' state can be weaker relative to tissue adherent method, and present invention improves over the separation methods of umbilical cord mesenchymal stem cells, first use machine
Xie Fa dispersion tissue, then the processing tissue with the mixed enzyme short time, repeatedly counted can not only shorten the growth time of cell, but also protect
The cultivation conditions of cell have been demonstrate,proved, and a large amount of effective cell factor component is obtained by low temperature ultrasonic lytic cell.This hair
Bright umbilical cord mesenchymal stem cells extract formulation can effectively promote skin epidermal cells and fibroblastic proliferation, carefully
Intracellular cytokine rich content, it is nontoxic and pollution-free it is without side-effects, to application on human skin not allergy, using safe, solve this field in umbilical cord
The many problems applied in mescenchymal stem cell application beauty skin care product, can satisfy the demand of industrial actual production, and existing
There is technology to compare and is more suitable for Skin Cell life, update, regeneration and the demand to nutrition.
Summary of the invention
The present invention provides one kind can promote skin repair and regenerated umbilical cord mesenchymal stem cells preparation, including between umbilical cord
Mesenchymal stem cells extract and umbilical cord mesenchymal stem cells preparation special culture media, the stem cell medicine special culture media include
Amino acid mixed liquor and vitamin mixture, the amino acid mixed liquor are selected from following any one or more: glycine, the third ammonia
Acid, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, egg ammonia
Acid, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, the vitamin are mixed
Close liquid be selected from it is following any one or more: vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin C,
Vitamin E, inositol；The umbilical cord mesenchymal stem cells are to be isolated from 3-10 generation after healthy, term birth fetal cord
Cell.
Preferably, the umbilical cord mesenchymal stem cells preparation special culture media is the free serum culture added with cell factor
Base, the cell factor include one or more in IGF, bFGF, EGF, PDGF, VEGF and HGF.
Preferably, the umbilical cord mesenchymal stem cells preparation special culture media further includes DMEM/F12 (without phenol red), 10%
FBS。
Preferably, the umbilical cord mesenchymal stem cells extract preparation method the following steps are included:
(1) separation of umbilical cord mesenchymal stem cells: the human fetal umbilical cord of acquisition health, term birth is added umbilical cord and saves
Liquid (DMEM/F12+1% mycillin), isolates magnificent Tong Shi glue, and Collagenase I, Collagenase II, pancreas are added after shredding
Protease mixed enzyme, 37 DEG C of digestion 10min will not digest tissue taking-up and add mixing enzymic digestion after soft concussion, repeatedly more
Secondary, the cell that digestion is obtained is collected kind and is entered in culture bottle, culture medium of the addition containing DMEM/F12+10%FBS+1%P/S, and 37
DEG C, 5%CO2Culture；
(2) passage and culture of umbilical cord mesenchymal stem cells: discarding culture medium, and PBS rinse is added, adds TrypLE and disappears
Change liquid, cell is resuspended with the culture medium containing DMEM/F12+10%FBS+1%P/S, after adherent, by culture medium in 37 DEG C of digestion 1min
It is changed to the stem cell medicine special culture media；
(3) preparation of umbilical cord mesenchymal stem cells extract: the umbilical cord mesenchyma in the 3-10 generation of selection step (2) separation
Stem cell is inoculated into culture bottle, and special culture media described in 10ml is added in culture bottle, collects culture medium afterwards for 24 hours, deposits in -4
DEG C, then the fresh special culture media of 10ml is replaced, it then collects again for 24 hours, until the cell confluency in culture bottle reaches
To 90%, by cell dissociation and collect.It is suspended with PBS, low temperature ultrasonic cracking is then centrifuged for removal cell fragment, and collection includes
The supernatant of cell lysate, it is all to be stored in -4 DEG C of supernatants and the supernatant mixing comprising cell lysate, as umbilical cord mesenchyma
Stem cell extract.
Preferably, the umbilical cord mesenchymal stem cells preparation further includes freeze drying protectant, and the freeze drying protectant is sweet dew
Alcohol, trehalose, dextran composition.
Preferably, the umbilical cord mesenchymal stem cells preparation is pulvis, liquid agent, emulsion, paste, creme, gelling agent, jelly
Dryed product.
The present invention also provides a kind of preparation sides that can promote skin repair Yu regenerated umbilical cord mesenchymal stem cells preparation
Method includes the following steps:
(1) separation of umbilical cord mesenchymal stem cells: the human fetal umbilical cord of acquisition health, term birth is added umbilical cord and saves
Liquid (DMEM/F12+1% mycillin), isolates magnificent Tong Shi glue, and Collagenase I, Collagenase II, pancreas are added after shredding
Protease mixed enzyme, 37 DEG C of digestion 10min will not digest tissue taking-up and add mixing enzymic digestion after soft concussion, repeatedly more
Secondary, the cell that digestion is obtained is collected kind and is entered in culture bottle, culture medium of the addition containing DMEM/F12+10%FBS+1%P/S, and 37
DEG C, 5%CO2Culture；
(2) passage and culture of umbilical cord mesenchymal stem cells: discarding culture medium, and PBS rinse is added, adds TrypLE and disappears
Change liquid, cell is resuspended with the culture medium containing DMEM/F12+10%FBS+1%P/S, after adherent, by culture medium in 37 DEG C of digestion 1min
It is changed to stem cell medicine special culture media；
(3) preparation of umbilical cord mesenchymal stem cells extract: the umbilical cord mesenchyma in the 3-10 generation of selection step (2) separation
Stem cell is inoculated into culture bottle, and special culture media described in 10ml is added in culture bottle, collects culture medium afterwards for 24 hours, deposits in 4 DEG C,
The fresh special culture media of 10ml is replaced again, is then collected again for 24 hours, until the cell confluency in culture bottle reaches
90%, by cell dissociation and collect.It is suspended with PBS, low temperature ultrasonic cracking is then centrifuged for removal cell fragment, collects comprising thin
The supernatant of cellular lysate object, all to be stored in 4 DEG C of supernatants and the supernatant mixing comprising cell lysate, as umbilical cord mesenchyma is dry thin
Born of the same parents' extract；
(4) mannitol, trehalose, dextran will be added in the umbilical cord mesenchymal stem cells extract as freeze-drying to protect
Agent is protected, after 0.22 μm of filtration sterilization, is added in freeze-drying bottle, -80 DEG C of pre-freezes are placed in vacuum freeze drier for 24 hours and take out very
Sky is freeze-dried 24-36 hours and is lyophilized, freeze-dried powder is made, is stored in 4 DEG C；
The stem cell medicine special culture media includes DMEM/F12 (without phenol red), 10%FBS, amino acid mixed liquor and dimension
Raw element mixed liquor.
Preferably, the amino acid mixed liquor is selected from following any one or more: glycine, alanine, valine, bright ammonia
Acid, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, paddy
Glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine；The vitamin mixture is selected from following
It is one or more: vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin C, vitamin E, inositol.
The present invention also provides a kind of umbilical cord mesenchymal stem cells special culture media, the culture medium includes DMEM/F12
(without phenol red), 10%FBS, amino acid mixed liquor and vitamin mixture；Preferably, the amino acid mixed liquor is selected from following
It is one or more: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, silk ammonia
Acid, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, essence
Propylhomoserin, histidine；The vitamin mixture is selected from following any one or more: vitamin A, vitamin B1, vitamin B2, dimension
Raw element B5, vitamin B6, vitamin C, vitamin E, inositol.
The present invention also provides fill between a kind of umbilical cord prepared by above-mentioned umbilical cord mesenchymal stem cells preparation or the above method
The beauty of matter stem cell medicine preparation, skin-protection product.
The present invention also provides umbilical cord mesenchymas prepared by a kind of above-mentioned umbilical cord mesenchymal stem cells preparation or the above method
Application of the stem cell medicine in preparation beauty, skin-protection product.
The present invention also provides fill between a kind of umbilical cord prepared by above-mentioned umbilical cord mesenchymal stem cells preparation or the above method
Application of the beauty, skin-protection product of matter stem cell medicine preparation in reparation, the beautification unabroken skin of epidermis.
Compared with prior art, the invention has the following advantages:
Material of the present invention for separating mesenchymal stem cell comes from neonatal umbilical cord, belongs to Biohazard Waste, source is rich
Richness, is not related to ethics problem, and amplification, differentiation capability (are filled compared with other mescenchymal stem cells between such as mesenchymal stem cell, fat
Matter stem cell) it is higher, and the contaminated probability of stem cell separation operation process is lower.Umbilical cord mesenchymal stem cells can secrete generation
Various active albumen or cell factor, the composition identified at present have 200 various active albumen and cell factor.There is research
Report, these cell factor synergy have the effects that promote cell neogenesis, cell repair, generally use in the prior art dry
The type and quantity of activated protein or cell factor contained by cell culture supernatant are extremely limited, and in order to obtain a large amount of cell
Number, the prior art generally use repeatedly passage and obtain a large amount of high generation stem cells, these cells can generate more cell metabolisms
Waste, with the raising of cell passage number, cell state and cell activity are reduced, and the type and quantity of cell factor also drop
Low, these defects make the application of stem cell supernatant receive obstruction.The present invention verifies through a large number of experiments, overcomes existing
There is difficulty many in technology, and there is following technical advantage:
(1) stem cell for making umbilical cord mesenchymal stem cells preparation only selects 3-10 generation, has advanced optimized product quality,
It ensure that cell viability and cell state, efficiency factor is richer in acquired stem cell medicine, and metabolic waste is less.
(2) two methods of the generally organized adherent method of the separation of umbilical cord mesenchymal stem cells and enzymic digestion in the prior art,
But tissue adherent method cell climbs out of that the time is longer, and simple enzyme digestion is since pre-processing cell stage is long, it is cellular
State can be weaker relative to tissue adherent method, and the present invention gropes to improve separation method through a large number of experiments, first uses Mechanical Method
Dispersion tissue, then with I containing Collagenase, Collagenase II, the mixed enzyme short time of trypsase, repeatedly several processing group
It knits, can both shorten the growth time of cell, in turn ensure the state of cell, this method is mild, during the separation process by cell
The death rate be preferably minimized, be particularly suitable for the separation of umbilical cord mesenchymal stem cells.
(3) umbilical cord mesenchymal stem cells during growth, other than it can secrete the effective active factor, can also consume battalion
Substance is supported, metabolite is generated.Therefore, the present invention has carried out the umbilical cord mesenchymal stem cells special culture media in 3-10 generation excellent
Change, the culture medium of DMEM/F12 (without phenol red)+10%FBS is used by the verifying of a large amount of proportioning tests, and is added to 20 kinds of ammonia
Base acid and 8 kinds of vitamins, ensure that the vigor and state of the umbilical cord mesenchymal stem cells in 3-10 generation, can be in best shape
More active factors can be secreted under state, promote to maximizing skin epidermal cells and fibroblastic proliferation.
(4) cell culture supernatant is repeatedly collected, and the umbilical cord mesenchymal stem cells low temperature ultrasonic in 3-10 generation is cracked, is filled
Divide release cell lysate in supernatant, using the mixed liquor of a variety of supernatants as umbilical cord mesenchymal stem cells extract, no
Umbilical cord mesenchymal stem cells are only taken full advantage of, and can acquire and obtain activated protein that cell culture each stage generates and thin
Intracellular cytokine, the single generation cell culture supernatant used compared to the prior art, in the type for extracting the cell factor obtained
There is obvious technical advantage with quantitative aspects, be greatly saved preparation cost, meet industrially to stem cell products preparation need
It asks.
(5) umbilical cord mesenchymal stem cells preparation prepared by the present invention can be obviously promoted epidermal cell and skin into fiber finer
Born of the same parents' proliferation, and by the random test of 100 volunteers, product safety of the invention has no toxic side effect, and without allergic phenomena, tool
There is diminution skin pore, acne print desalination or small pox subside, and microgroove, the desalination of dry line, the remarkable efficacy that whiteness of skin brightens is suitable for answering
For beauty skin care product.
Detailed description of the invention
Fig. 1 is the influence that MTT detects that ordinary culture medium and special culture media are proliferated human umbilical cord mesenchymal stem cells.
Fig. 2 is expression of the Immunofluorescence test CD44 in human umbilical cord mesenchymal stem cells.
Fig. 3 is that MTT detects influence of the umbilical cord mesenchymal stem cells pulvis to epidermal cell proliferation.
Fig. 4 is the influence that MTT detects that umbilical cord mesenchymal stem cells pulvis is proliferated skin fibroblasts.
Fig. 5 is that MTT detects influence of the different algebra umbilical cord mesenchymal stem cells pulvis to epidermal cell proliferation.
Specific embodiment
Below by way of specific embodiment, invention is further described in detail, so that those skilled in the art can be more preferable
Ground understands the present invention and is practiced, but embodiment is not intended as restriction of the invention.
Experimental method used in following embodiment is conventional method unless otherwise specified.Material used, reagent
Deng being commercially available unless otherwise specified.
The separation of 1 umbilical cord mesenchymal stem cells of embodiment
Multipara's informed consent is voluntarily contributed, and mature healthy fetus fresh sterile umbilical cord tissue is acquired, and 4 DEG C of preservations are interior for 24 hours
Processing.Female blood is acquired in puerpera antenatal seven days carries out HIV detection, hepatitis B detection, hepatitis detection, Lues Assay, glutamic-pyruvic transaminase
Detection, cytomegalovirus detection and detection of mycoplasma can use its fetus fresh sterile umbilical cord group after all detection is qualified
It knits.
1) will added with umbilical cord preserving fluid (DMEM/F12+1% mycillin) umbilical cord tissue collecting bottle low-temperature transport to doctor
Institute, the fetal cord of acquisition health in aseptic operating room, term birth, removes surface contaminants, after 75% alcohol disinfecting, then uses
Normal saline flushing is clean, is put into collecting bottle, transports go back to laboratory.
2) it in constant temperature laboratory of the room temperature lower than 25 DEG C, takes out fresh umbilical cord tissue and is placed in the big ware of 10mm, with 4 DEG C of mistakes
The remained blood that the physiological saline (containing 1% mycillin) of night pre-cooling rinses umbilical cord surface well, with eye scissors and ophthalmic tweezers point
From and discard the artery on umbilical cord, vein and excess surface tissue, be soaked in serum-free DMEM/F12 culture medium.
3) scratch umbilical cord with scalpel, isolate magnificent Tong Shi glue, after shredding be added Collagenase I, Collagenase II,
Trypsase mixed enzyme, 37 DEG C of digestion 10min will not digest tissue taking-up and add mixing enzymic digestion, repeatedly after soft concussion
Repeatedly, cell digestion obtained is collected kind and is entered in culture bottle, addition DMEM/F12+10%FBS+1%P/S culture medium, 37 DEG C,
5%CO2Culture.
4) cell is observed, liquid was changed every 3 days, can be passed on when cells being waited to grow to 70~80% fusion rate.
The passage and culture of 2 umbilical cord mesenchymal stem cells of embodiment
In superclean bench, culture medium in culture bottle is discarded, PBS rinse is added, add TrypLE digestive juice, 37 DEG C
1min is digested, digestion is terminated, cell is resuspended with DMEM/F12+10%FBS+1%P/S culture medium, is carried out after blowing and beating uniformly by 1:3
Passage, after cell is adherent, is changed to stem cell medicine special culture media for culture medium.
Stem cell medicine special culture media are as follows: DMEM/F12 (without phenol red)+10%FBS+20 kind+8 kinds of vitamins of amino acid
20 kinds of amino acid include: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline,
Tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, paddy ammonia
Acid, lysine, arginine, histidine.
Vitamin includes: vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin C, dimension in 8
Raw element E, inositol.
The influence that 3 MTT of embodiment detection ordinary culture medium and special culture media are proliferated human umbilical cord mesenchymal stem cells
After human umbilical cord mesenchymal stem cells recovery culture, is spread in 96 orifice plates with 3000/hole, spread 2 groups, every group 5
Multiple holes spread 4 piece of 96 orifice plate altogether.2 groups of cells are cultivated with ordinary culture medium and special culture media respectively, are added every 24 hours
MTT, with the OD value in microplate reader detection 570nm wavelength lower opening.Ordinary culture medium is simultaneously not added with above-mentioned 20 kinds of amino acid and 8 kinds of dimensions
Raw element.For test result as shown in table 1 and Fig. 1, conversion culture medium of the present invention can be obviously promoted human umbilical cord mesenchymal
Stem cells hyperplasia and the vigor and preferable cell state for keeping stem cell, and detected after culture in 72 hours in microplate reader
OD value in 570nm wavelength lower opening is 0.627 ± 0.013.
The influence that 1 MTT of table detection ordinary culture medium and special culture media are proliferated human umbilical cord mesenchymal stem cells
| OD(570nm) | 0h | 24h | 48h | 72h |
| Ordinary culture medium | 0.454±0.016 | 0.481±0.008 | 0.529±0.008 | 0.546±0.018 |
| Special culture media | 0.452±0.008 | 0.508±0.010 | 0.573±0.011 | 0.627±0.013 |
Expression of the 4 Immunofluorescence test CD44 of embodiment in human umbilical cord mesenchymal stem cells
Respectively by P3, P10, P11 for human umbilical cord mesenchymal stem cells with 5 × 104The density in a/hole is spread in 24 orifice plates,
After cell is adherent, cell successively is fixed with paraformaldehyde, is closed with BSA, then applies CD44 antibody and fluorescence secondary antibody, is finally used
DAPI dyeing after fluorescence microscopy under the microscope, take pictures.Test result as shown in Fig. 2, in identical experiment and under the conditions of microscopy,
P3-P11 can express CD44 molecule for umbilical cord mesenchymal stem cells, but CD44 expression quantity is remarkably decreased for since P11, P3
For optimal, it is seen that the young generation of umbilical cord mesenchymal stem cells selects holding and cell for cell viability, cell state
The extraction of interior beneficial agents is necessary.
The preparation of 5 umbilical cord mesenchymal stem cells extracting solution pulvis of embodiment
The umbilical cord mesenchymal stem cells in 3-10 generation are selected, when passage is inoculated into the culture bottle of T75, and 10ml is added in culture bottle
Special culture media collects culture medium afterwards for 24 hours, deposits in 4 DEG C, then replaces the fresh special culture media of 10ml, then receives again for 24 hours
Collection by cell dissociation and is collected until the cell confluency in T75 culture bottle reaches 90%.
The cell gathered is suspended with PBS, and low temperature ultrasonic cracking is then centrifuged for removal cell fragment, collects supernatant, remaining
It include cell lysate in supernatant.
All cell culture medium supernatants and supernatant from cell lysate by Collection and conservation in 4 DEG C mix, addition mannitol,
Trehalose, dextran are as freeze drying protectant, after 0.22 μm of filtration sterilization, be added to freeze-drying bottle in, -80 DEG C of pre-freezes for 24 hours after
It is placed in vacuum freeze drier and vacuumizes, be freeze-dried 24-36 hours and be lyophilized, the agent of freeze-dried powder obtained freeze-drying is made, is stored in 4
℃。
Obtained freeze-drying powder includes: (1) umbilical cord mesenchymal stem cells extract and secretion factor；(2) 20 kinds of natural amino acids
(glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, half
Cystine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine)；
(3) 8 kinds of vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin C, vitamin E, fleshes
Alcohol) (4) frozen-dried protective ingredient (mannitol, trehalose, dextran).
6 MTT of embodiment detects influence of the umbilical cord mesenchymal stem cells pulvis extracting solution to epidermal cell
It after the epidermal cell recovery culture of people, is spread in 96 orifice plates with 3000/hole, spreads 2 groups, every group of 5 multiple holes are total
Spread 4 piece of 96 orifice plate.
Wherein 1 group of cell is as blank control group, not dosing, and stem cell liquid described in patent is added (by 1 part in another 1 group of cell
Freeze-dried powder is dissolved in 1ml physiological saline, and 100 μ l lysates are added in every hole).Every 24 hours addition MTT, detected with microplate reader
OD value in 570nm wavelength lower opening.As a result as shown in Table 2 and Fig. 3.
2 MTT of table detects influence of the umbilical cord mesenchymal stem cells pulvis to epidermal cell proliferation
| OD(570nm) | 0h | 24h | 48h | 72h |
| Control group | 0.328±0.010 | 0.349±0.018 | 0.409±0.018 | 0.430±0.024 |
| Experimental group | 0.337±0.019 | 0.443±0.017 | 0.492±0.018 | 0.521±0.027 |
7 MTT of embodiment detects influence of the umbilical cord mesenchymal stem cells pulvis to skin fibroblasts
After the skin fibroblasts recovery culture of people, is spread in 96 orifice plates with 3000/hole, spread 2 groups, every group 5
Multiple holes spread 4 piece of 96 orifice plate altogether.Wherein 1 group of cell is as blank control group, and not dosing, another 1 group of cell is added dry thin described in patent
Cytosol.(1 part of freeze-dried powder being dissolved in 1ml physiological saline, 100 μ l lysates are added in every hole) uses enzyme every 24 hours addition MTT
Mark the OD value in instrument detection 570nm wavelength lower opening.As a result as shown in table 3 and fig. 4.
The influence that 3 MTT of table detection umbilical cord mesenchymal stem cells pulvis is proliferated skin fibroblasts
| OD(570nm) | 0h | 24h | 48h | 72h |
| Control group | 0.402±0.018 | 0.434±0.010 | 0.465±0.009 | 0.510±0.014 |
| Experimental group | 0.415±0.007 | 0.471±0.013 | 0.527±0.011 | 0.545±0.013 |
8 MTT of embodiment detects influence of the umbilical cord mesenchymal stem cells pulvis of different algebra preparations to epidermal cell
It after the epidermal cell recovery culture of people, is spread in 96 orifice plates with 3000/hole, spreads 3 groups, every group of 5 multiple holes are total
Spread 4 piece of 96 orifice plate.3 groups of cells are separately added into the stem cell liquid that P3, P10, P11 are prepared into for umbilical cord mesenchymal stem cells pulvis,
(1 part of freeze-dried powder being dissolved in 1ml physiological saline, 100 μ l lysates are added in every hole) uses microplate reader every 24 hours addition MTT
Detect the OD value in 570nm wavelength lower opening.As a result as shown in table 4 and fig. 5, P3-P10 is for the dry thin manufactured pulvis of umbilical cord mesenchyma
The proliferation of epidermal cell is obviously relatively beneficial to for cell compared to P11, therefore the present invention selects the umbilical cord mesenchyma in P3-P10 generation dry
Pulvis made of thin.
4 MTT of table detects influence of the different algebra umbilical cord mesenchymal stem cells pulvis to epidermal cell proliferation
| OD(570nm) | 0h | 24h | 48h | 72h |
| P3 | 0.423±0.011 | 0.459±0.003 | 0.543±0.020 | 0.583±0.016 |
| P10 | 0.416±0.007 | 0.440±0.008 | 0.527±0.019 | 0.566±0.009 |
| P11 | 0.413±0.012 | 0.427±0.008 | 0.456±0.009 | 0.503±0.016 |
9 umbilical cord mesenchymal stem cells pulvis of embodiment uses example
100 people are randomly choosed, the age 22-50 years old, 2 weeks above-mentioned umbilical cord mesenchymas were used continuously in 46 people of male, 54 people of women
Using effect is fed back after stem cell pulvis.Umbilical cord mesenchymal stem cells pulvis application method: it is raw that 1 person-portion freeze-dried powder is dissolved in 10ml
It manages in salt water, face paper is impregnated wherein, face is then spread on, is taken off after 10-20 minutes, or freeze-dried powder is dissolved in 1ml physiology
In salt water, directly pat in face until absorbing.Now feedback result arrangement is as follows:
5 umbilical cord mesenchymal stem cells pulvis of table uses example fed back statistics table
| Without allergic phenomena | Hair pore shrinkage | Acne print desalination or small pox subside | Microgroove, the desalination of dry line | Whiteness of skin brightens |
| 100 | 49 | 56 | 68 | 72 |
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. one kind can promote skin repair and regenerated umbilical cord mesenchymal stem cells preparation, including umbilical cord mesenchymal stem cells extract
Object and umbilical cord mesenchymal stem cells preparation special culture media, the stem cell medicine special culture media include amino acid mixed liquor and
Vitamin mixture；The amino acid mixed liquor is selected from following any one or more: glycine, alanine, valine, bright ammonia
Acid, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, paddy
Glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine；The vitamin mixture is selected from following
It is one or more: vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin C, vitamin E, inositol；
The umbilical cord mesenchymal stem cells are the cell in 3-10 generation after being isolated from healthy, term birth fetal cord.
2. umbilical cord mesenchymal stem cells preparation according to claim 1, the dedicated training of umbilical cord mesenchymal stem cells preparation
Support the culture medium that base is following (1) or (2):
(1) the umbilical cord mesenchymal stem cells preparation special culture media is added with the serum free medium of cell factor, described thin
Intracellular cytokine includes one or more in IGF, bFGF, EGF, PDGF, VEGF and HGF；
(2) the umbilical cord mesenchymal stem cells preparation special culture media is to have serum containing DMEM/F12 (no phenol red), 10%FBS
Culture medium.
3. umbilical cord mesenchymal stem cells preparation according to claim 1, the system of the umbilical cord mesenchymal stem cells extract
Preparation Method the following steps are included:
(1) separation of umbilical cord mesenchymal stem cells: umbilical cord preserving fluid is added in the human fetal umbilical cord of acquisition health, term birth
(DMEM/F12+1% mycillin) isolates magnificent Tong Shi glue, and Collagenase I, Collagenase II, pancreas egg are added after shredding
White enzyme mixed enzyme, 37 DEG C of digestion 10min will not digest tissue taking-up and add mixing enzymic digestion after soft concussion, repeated multiple times,
The cell that digestion is obtained is collected kind and is entered in culture bottle, culture medium of the addition containing DMEM/F12+10%FBS+1%P/S, 37 DEG C,
5%CO2Culture；
(2) passage and culture of umbilical cord mesenchymal stem cells: discarding culture medium, and PBS rinse is added, and adds TrypLE digestion
Cell is resuspended with the culture medium containing DMEM/F12+10%FBS+1%P/S, after adherent, more by culture medium in liquid, 37 DEG C of digestion 1min
It is changed to the stem cell medicine special culture media；
(3) preparation of umbilical cord mesenchymal stem cells extract: the umbilical cord mesenchyma in the 3-10 generation of selection step (2) separation is dry thin
Born of the same parents are inoculated into culture bottle, and special culture media described in 10ml is added in culture bottle, collects culture medium afterwards for 24 hours, deposit in -4 DEG C, then
The fresh special culture media of 10ml is replaced, is then collected again for 24 hours, until the cell confluency in culture bottle reaches
90%, by cell dissociation and collect.It is suspended with PBS, low temperature ultrasonic cracking is then centrifuged for removal cell fragment, collects comprising thin
The supernatant of cellular lysate object, it is all to be stored in -4 DEG C of supernatants and the supernatant mixing comprising cell lysate, as umbilical cord mesenchyma are dry
Cell extract.
4. umbilical cord mesenchymal stem cells preparation according to claim 4, the umbilical cord mesenchymal stem cells preparation further include
Freeze drying protectant, the freeze drying protectant are the composition of mannitol, trehalose, dextran.
5. -5 any umbilical cord mesenchymal stem cells preparation according to claim 1, the umbilical cord mesenchymal stem cells preparation
For pulvis, liquid agent, emulsion, paste, creme, gelling agent or freeze-drying prods.
6. a kind of preparation method that can promote skin repair Yu regenerated umbilical cord mesenchymal stem cells preparation, includes the following steps:
(1) separation of umbilical cord mesenchymal stem cells: umbilical cord preserving fluid is added in the human fetal umbilical cord of acquisition health, term birth
(DMEM/F12+1% mycillin) isolates magnificent Tong Shi glue, and Collagenase I, Collagenase II, pancreas egg are added after shredding
White enzyme mixed enzyme, 37 DEG C of digestion 10min will not digest tissue taking-up and add mixing enzymic digestion after soft concussion, repeated multiple times,
The cell that digestion is obtained is collected kind and is entered in culture bottle, culture medium of the addition containing DMEM/F12+10%FBS+1%P/S, 37 DEG C,
5%CO2Culture；
(2) passage and culture of umbilical cord mesenchymal stem cells: discarding culture medium, and PBS rinse is added, and adds TrypLE digestion
Cell is resuspended with the culture medium containing DMEM/F12+10%FBS+1%P/S, after adherent, more by culture medium in liquid, 37 DEG C of digestion 1min
It is changed to stem cell medicine special culture media；
(3) preparation of umbilical cord mesenchymal stem cells extract: the umbilical cord mesenchyma in the 3-10 generation of selection step (2) separation is dry thin
Born of the same parents are inoculated into culture bottle, and special culture media described in 10ml is added in culture bottle, collect culture medium afterwards for 24 hours, deposit in 4 DEG C, then more
The fresh special culture media of 10ml is changed, is then collected again for 24 hours, until the cell confluency in culture bottle reaches 90%,
By cell dissociation and collect.It is suspended with PBS, low temperature ultrasonic cracking is then centrifuged for removal cell fragment, and collecting includes cell cracking
The supernatant of object, it is all to be stored in 4 DEG C of supernatants and the supernatant mixing comprising cell lysate, as umbilical cord mesenchymal stem cells extraction
Object；
(4) mannitol, trehalose, dextran will be added in the umbilical cord mesenchymal stem cells extract as frozen-dried protective
Agent after 0.22 μm of filtration sterilization, is added in freeze-drying bottle, and -80 DEG C of pre-freezes are placed in vacuum freeze drier for 24 hours to be vacuumized,
It is lyophilized within freeze-drying 24-36 hours, freeze-dried powder is made, is stored in 4 DEG C；
The stem cell medicine special culture media includes DMEM/F12 (without phenol red), 10%FBS, amino acid mixed liquor and vitamin
Mixed liquor.
7. a kind of preparation that can promote skin repair Yu regenerated umbilical cord mesenchymal stem cells preparation according to claim 7
Method, the amino acid mixed liquor are selected from following any one or more: glycine, alanine, valine, leucine, different bright ammonia
Acid, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, Soviet Union
Propylhomoserin, aspartic acid, glutamic acid, lysine, arginine, histidine；The vitamin mixture is selected from following any or more
Kind: vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin C, vitamin E, inositol.
8. a kind of umbilical cord mesenchymal stem cells special culture media, the culture medium include DMEM/F12 (without phenol red), 10%FBS,
Amino acid mixed liquor and vitamin mixture；Preferably, the amino acid mixed liquor is selected from following any one or more: sweet ammonia
Acid, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, half Guang ammonia
Acid, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine；It is described
Vitamin mixture be selected from it is following any one or more: vitamin A, vitamin B1, vitamin B2, vitamin B5, vitamin B6,
Vitamin C, vitamin E, inositol.
9. a kind of any described by claim 1-6 any the umbilical cord mesenchymal stem cells preparation or claim 7-8
The umbilical cord mesenchymal stem cells preparation of method preparation or umbilical cord mesenchymal stem cells special culture media as claimed in claim 9 preparation
Beauty, skin care item.
10. any any method system of the umbilical cord mesenchymal stem cells preparation or claim 7-8 of claim 1-6
Standby umbilical cord mesenchymal stem cells preparation or umbilical cord mesenchymal stem cells special culture media as claimed in claim 9 or claim
Beauty described in 10, skin care item, the application in reparation, the beautification unabroken skin of epidermis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910489249.6A CN110269833A (en) | 2019-06-05 | 2019-06-05 | A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910489249.6A CN110269833A (en) | 2019-06-05 | 2019-06-05 | A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110269833A true CN110269833A (en) | 2019-09-24 |
Family
ID=67960431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910489249.6A Pending CN110269833A (en) | 2019-06-05 | 2019-06-05 | A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110269833A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110934815A (en) * | 2019-12-24 | 2020-03-31 | 盛世荣恩生物科技有限公司 | Anti-saccharification mask containing mesenchymal stem cell culture supernatant and preparation method thereof |
| CN110974944A (en) * | 2019-12-31 | 2020-04-10 | 广州赛加生物科技有限公司 | Mesenchymal stem cell composite active factor freeze-dried powder and preparation method and application thereof |
| CN111690599A (en) * | 2020-07-17 | 2020-09-22 | 天晴干细胞股份有限公司 | Method for promoting mesenchymal stem cells to differentiate into chondroblasts |
| CN112089733A (en) * | 2020-11-05 | 2020-12-18 | 北京欣颂生物科技有限公司 | Application of modified umbilical cord stem cells in preparation of anti-aging pharmaceutical composition or health-care product |
| CN112852724A (en) * | 2021-01-05 | 2021-05-28 | 首玺(广州)医疗科技有限责任公司 | Beauty composition containing stem cell active factor and preparation method and application thereof |
| CN113975221A (en) * | 2021-11-23 | 2022-01-28 | 四川伍衍生物科技有限公司 | Umbilical cord mesenchymal stem cell factor freeze-dried powder and preparation method thereof |
| CN114432161A (en) * | 2020-10-20 | 2022-05-06 | 山东荆卫生物科技有限公司 | Preparation method and application of human umbilical cord mesenchymal stem cell active factor liquid with anti-aging and skin repair effects |
| CN115074321A (en) * | 2022-06-30 | 2022-09-20 | 清华珠三角研究院 | Preparation derived from Mesenchymal Stem Cells (MSC) and used for promoting angiogenesis and preparation method thereof |
| CN116410921A (en) * | 2023-02-09 | 2023-07-11 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
| WO2023143528A1 (en) * | 2022-01-28 | 2023-08-03 | 北京达尔文细胞生物科技有限公司 | Protein composition for repairing hair follicle, and preparation method therefor and use thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100110905A (en) * | 2007-07-20 | 2010-10-13 | 동국대학교 산학협력단 | Method for the preparation of dermal papilla tissue employing mesenchymal stem cells |
| CN104164405A (en) * | 2014-08-12 | 2014-11-26 | 赛业（苏州）生物科技有限公司 | Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro |
| CN107354131A (en) * | 2017-08-10 | 2017-11-17 | 河南省银丰生物工程技术有限公司 | A kind of culture medium for umbilical cord mesenchymal stem cells |
| CN108057014A (en) * | 2017-12-25 | 2018-05-22 | 北京汇智驰康生物科技有限公司 | A kind of preparation method of the stem cell medicine of beauty and skin care |
| CN108823156A (en) * | 2018-07-04 | 2018-11-16 | 陕西神州生物技术有限公司 | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder |
| CN109628394A (en) * | 2019-01-29 | 2019-04-16 | 劳敏翔 | A method of extraction umbilical cord mesenchymal stem cells of the grinding in conjunction with mixed enzyme |
| CN110564680A (en) * | 2019-08-27 | 2019-12-13 | 西安艾尔菲生物科技有限公司 | Human umbilical cord mesenchymal stem cell serum-free culture medium, preparation method thereof and method for obtaining human umbilical cord mesenchymal stem cell serum-free culture medium |
| CN110734893A (en) * | 2019-11-15 | 2020-01-31 | 华辰未来(北京)生物医学技术有限公司 | Culture medium containing vitamin C and used for promoting proliferation of umbilical cord mesenchymal stem cells |
-
2019
- 2019-06-05 CN CN201910489249.6A patent/CN110269833A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100110905A (en) * | 2007-07-20 | 2010-10-13 | 동국대학교 산학협력단 | Method for the preparation of dermal papilla tissue employing mesenchymal stem cells |
| CN104164405A (en) * | 2014-08-12 | 2014-11-26 | 赛业（苏州）生物科技有限公司 | Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro |
| CN107354131A (en) * | 2017-08-10 | 2017-11-17 | 河南省银丰生物工程技术有限公司 | A kind of culture medium for umbilical cord mesenchymal stem cells |
| CN108057014A (en) * | 2017-12-25 | 2018-05-22 | 北京汇智驰康生物科技有限公司 | A kind of preparation method of the stem cell medicine of beauty and skin care |
| CN108823156A (en) * | 2018-07-04 | 2018-11-16 | 陕西神州生物技术有限公司 | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder |
| CN109628394A (en) * | 2019-01-29 | 2019-04-16 | 劳敏翔 | A method of extraction umbilical cord mesenchymal stem cells of the grinding in conjunction with mixed enzyme |
| CN110564680A (en) * | 2019-08-27 | 2019-12-13 | 西安艾尔菲生物科技有限公司 | Human umbilical cord mesenchymal stem cell serum-free culture medium, preparation method thereof and method for obtaining human umbilical cord mesenchymal stem cell serum-free culture medium |
| CN110734893A (en) * | 2019-11-15 | 2020-01-31 | 华辰未来(北京)生物医学技术有限公司 | Culture medium containing vitamin C and used for promoting proliferation of umbilical cord mesenchymal stem cells |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110934815A (en) * | 2019-12-24 | 2020-03-31 | 盛世荣恩生物科技有限公司 | Anti-saccharification mask containing mesenchymal stem cell culture supernatant and preparation method thereof |
| CN110974944A (en) * | 2019-12-31 | 2020-04-10 | 广州赛加生物科技有限公司 | Mesenchymal stem cell composite active factor freeze-dried powder and preparation method and application thereof |
| CN111690599A (en) * | 2020-07-17 | 2020-09-22 | 天晴干细胞股份有限公司 | Method for promoting mesenchymal stem cells to differentiate into chondroblasts |
| CN114432161A (en) * | 2020-10-20 | 2022-05-06 | 山东荆卫生物科技有限公司 | Preparation method and application of human umbilical cord mesenchymal stem cell active factor liquid with anti-aging and skin repair effects |
| CN112089733A (en) * | 2020-11-05 | 2020-12-18 | 北京欣颂生物科技有限公司 | Application of modified umbilical cord stem cells in preparation of anti-aging pharmaceutical composition or health-care product |
| CN112089733B (en) * | 2020-11-05 | 2021-03-12 | 广东赛尔生物科技有限公司 | Application of modified umbilical cord stem cells in preparation of anti-aging pharmaceutical composition or health-care product |
| CN112852724B (en) * | 2021-01-05 | 2022-09-06 | 首玺(广州)医疗科技有限责任公司 | Beauty composition containing stem cell active factor and preparation method and application thereof |
| CN112852724A (en) * | 2021-01-05 | 2021-05-28 | 首玺(广州)医疗科技有限责任公司 | Beauty composition containing stem cell active factor and preparation method and application thereof |
| CN113975221A (en) * | 2021-11-23 | 2022-01-28 | 四川伍衍生物科技有限公司 | Umbilical cord mesenchymal stem cell factor freeze-dried powder and preparation method thereof |
| WO2023143528A1 (en) * | 2022-01-28 | 2023-08-03 | 北京达尔文细胞生物科技有限公司 | Protein composition for repairing hair follicle, and preparation method therefor and use thereof |
| CN115074321A (en) * | 2022-06-30 | 2022-09-20 | 清华珠三角研究院 | Preparation derived from Mesenchymal Stem Cells (MSC) and used for promoting angiogenesis and preparation method thereof |
| CN116410921A (en) * | 2023-02-09 | 2023-07-11 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
| CN116410921B (en) * | 2023-02-09 | 2024-01-23 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110269833A (en) | A kind of umbilical cord mesenchymal stem cells preparation and its preparation method and application | |
| US20110217385A1 (en) | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof | |
| US20140295555A1 (en) | Method of culturing cells | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN108992466B (en) | Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| JP2012504598A (en) | Method for promoting skin cell growth, composition for treating skin condition or hair condition | |
| KR101542847B1 (en) | Method for Differentiating Pluripotency Stem Cell Induced from Mesenchymal Stem Cell into Neuron | |
| CN108265023B (en) | Proliferation promoter and application thereof | |
| CN100508948C (en) | Production for skin beautification and care, and preparation method thereof | |
| CN112111451B (en) | Method for increasing yield of stem cell cytokines | |
| CN108057014A (en) | A kind of preparation method of the stem cell medicine of beauty and skin care | |
| CN105477626A (en) | Mixed stem cell-based medicinal product and preparation method thereof | |
| CN107779430A (en) | The collection method of umbilical cord mesenchymal stem cells supernatant | |
| CN113957040A (en) | Adipose-derived stem cell growth factor extract and preparation method and application thereof | |
| CN113171332A (en) | Exosome freeze-dried powder, preparation method thereof and skin care product | |
| CN112409456A (en) | Application of stem cell cytokine in preparation of cosmetics or medicines | |
| CN111821250A (en) | Anti-aging composition and preparation method and application thereof | |
| CN107338216A (en) | Artificial epidermal graft, its preparation method and application | |
| CN113116970A (en) | Stem cell skin repair liquid containing gardenia jasminoides extract and preparation method thereof | |
| KR101982835B1 (en) | Method for Differentiating Pluripotency Stem Cell Induced from Mesenchymal Stem Cell into Pancrease beta-cell | |
| CN115418341B (en) | Method for transdifferentiation of fibroblast to hair papilla cell and application thereof | |
| CN107441481A (en) | A kind of menses stem cell medicine for treating simple skin injury and preparation method thereof | |
| CN113018316A (en) | Application of mesenchymal stem cells derived from pluripotent stem cells in repairing skin injury | |
| CN115478077A (en) | Preparation and application of MANF gene modified umbilical cord mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190924 |