CN106497872A - Skeletal muscle stem Cells serum free medium and its preparation method and application - Google Patents

Skeletal muscle stem Cells serum free medium and its preparation method and application Download PDF

Info

Publication number
CN106497872A
CN106497872A CN201611087240.5A CN201611087240A CN106497872A CN 106497872 A CN106497872 A CN 106497872A CN 201611087240 A CN201611087240 A CN 201611087240A CN 106497872 A CN106497872 A CN 106497872A
Authority
CN
China
Prior art keywords
content
skeletal muscle
stem cells
muscle stem
free medium
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.)
Granted
Application number
CN201611087240.5A
Other languages
Chinese (zh)
Other versions
CN106497872B (en
Inventor
刘谋元
刘麟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Xin Feng Cmx Medical Technology Development Co Ltd
Original Assignee
Beijing Xin Feng Cmx Medical Technology Development Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Beijing Xin Feng Cmx Medical Technology Development Co Ltd filed Critical Beijing Xin Feng Cmx Medical Technology Development Co Ltd
Priority to CN201611087240.5A priority Critical patent/CN106497872B/en
Publication of CN106497872A publication Critical patent/CN106497872A/en
Application granted granted Critical
Publication of CN106497872B publication Critical patent/CN106497872B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/35—Polyols, e.g. glycerin, inositol
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/92—Medium free of human- or animal-derived components
    • 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
    • C12N2501/90—Polysaccharides
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses skeletal muscle stem Cells serum free medium and its preparation method and application.The culture medium includes following composition：DMEM：F12, serum substitute, cell factor, vitamin, amino acid, biosynthetic human insulin, help strong element and 6 chondroitin sulfates；The pH value of the culture medium is 7.2～7.4.The present invention further discloses the method for preparing the skeletal muscle stem Cells serum free medium.Serum substitute of the present invention is non-animal derived composition; composition determines; avoid the pathogenic risk that serum brings; the factor such as batch is unstable; culture medium of the present invention is applied to Human Skeletal Muscle stem cell primary and scale amplification cultivation, high with purity, and cell propagation is fast; amplification efficiency is high, the advantages of can stably pass on 20 more than generation.The present invention effectively increases skeletal muscle stem Cells amplification efficiency and purity, can be as scale, high-quality skeletal muscle stem Cells cultivating system meeting clinical research and application.

Description

Skeletal muscle stem Cells serum free medium and its preparation method and application
Technical field
The present invention relates to a kind of cell culture medium, more particularly to a kind of Human Skeletal Muscle stem cell serum-free culture medium, this Bright preparation method further to Human Skeletal Muscle stem cell serum-free culture medium and in culture Human Skeletal Muscle stem cell Application, belongs to the culture medium field of Human Skeletal Muscle stem cell.
Background technology
Skeletal muscle stem Cells (also known as sarcoblast, myoblast, English name：Myoblast) can only directed differentiation be flesh Meat tissue, in the form of skeletal muscle satellite cell in skeletal muscle tissue, with natural cell fusion, is damaged in muscle After wound, skeletal muscle stem Cells are activated, and breed and produce contractile protein in a large number, repair and maintain skeletal muscle tissue.Skeletal muscle is done Cell transplant techniques treatment amyotrophia, heart disease, diabetes, tumour, pain, depression, in terms of bone and joint diseases in recent years By domestic and international follow-up story, skeletal muscle stem Cells implantation technique is increasingly subject to the whole world as a kind of New Type of Diseases solution Concern.Gusso ni etc. treat 8 DMD patients using double blind control ruling by law, prove that skeletal muscle stem Cells transplanting has to DMD patient Certain curative effect.The clinical research of the whole world 20 countries, 230 patients shows at present, injects Human Skeletal Muscle to myocardial infarction patient Infarct perilesional regional myocardial function can be improved after stem cell, increase Left Ventricular Ejection Fraction, perfusion, survival ability, power, wall Thickness, and the blood volume of diastasis and end-systole, without arrhythmia cordis.This treatment means will likely become the treatment heart Flesh infarct and the most important new way of heart failure.Skeletal muscle stem Cells are transplanted and have also been launched for treating type ii diabetes. All the above therapeutic effects rely heavily on cell transplantation amount, and according to relevant report, skeletal muscle stem Cells treat the heart The cell quantity that the cardiomyopathys such as popular name for, miocardial infarction are used is 2 × 109, the quantity that treatment DMD patient uses is 5 × 1010.Institute With, explore a kind of can high-purity, scale amplification skeletal muscle stem Cells and be applied to clinic cultivating system extremely urgent.
At present, most of research institutions are with DMEM：F12/1:The basal mediums such as 1, α-MEM add 10%-20%'s Hyclone is cultivating skeletal muscle stem Cells.But hyclone culture medium derived components containing animal, composition do not know, there is risk and The differences between batches of serum are big, be not easy to control, skeletal muscle stem Cells are difficult to purify, amplification efficiency low become difficult in clinical practice With the difficult point that goes beyond.
Content of the invention
The technical problem to be solved is to add hyclone for bone stem cell media, containing animal sources Composition, causes skeletal muscle stem Cells to be difficult to purify, the low problem of amplification efficiency, there is provided a kind of skeletal muscle stem Cells of optimization without Blood serum medium, for the culture of Human Skeletal Muscle stem cell primary and scale amplification cultivation, provides for clinical research and application excellent The skeletal muscle stem Cells of matter.
The technical problem to be solved is achieved through the following technical solutions：
A kind of skeletal muscle stem Cells serum free medium, comprising following composition：
DMEM and F12 is according to 1:The mixture of 1 mass ratio composition, serum substitute, cell factor, vitamin, amino Acid, biosynthetic human insulin, transferrins, Pidolidone acid amides, thiamine hydrochloride, inositol, niacinamide, cholesterol, P68 are told Temperature 80, ferric citrate and NaHCO3；PH value is 7.2～7.4.
The serum substitute is Ultroser G blood substitutes；
The cell factor is IGF, in epithelical cell growth factor or Basic Fibroblast Growth Factor Any one or more of；
Described vitamin is any one of vitamin C, vitamin B1 or vitamin B6 or combination.
The amino acid is 1 water altheine, L-Aspartic acid, L-Histidine, ILE, L-Leu, L- Lysine, L-Methionine, L- phenylalanines, Serine, L-Trp, Cys, L-Orn hydrochloric acid or to ammonia Any one of yl benzoic acid is multiple.
In order to reach more preferable effect, in every liter of culture medium, the content of each composition is：
DMEM：F12 contents are 15.6g/L, wherein, DMEM：The mass ratio of F12 is 1:1；
The serum substitute content is 20mL/L；
The vitamin B6 content is 0.95～1.05mg/L, preferably 0.975mg/L；The vitamin B1 content is 1.0～1.03mg/L, preferably 1.03mg/L；The Vitamin C content is 6～6.9mg/L, preferably 6.45mg/L；
The IGF content is 80～100 μ g/L, preferably 100 μ g/L；Epithelical cell growth factor Content is 18～22 μ g/L, preferably 20 μ g/L；Basic Fibroblast Growth Factor content is 18～22 μ g/L, preferably 20 μ g/ L；
Biosynthetic human insulin's content is 0.01～0.02mg/L, preferably 0.02mg/L；
The 1 water altheine content is 48.45～53.25mg/L, preferably 51.25mg/L；L-Aspartic acid contains Measure as 30.35～31.35mg/L, preferably 30.75mg/L；L-Histidine content is 85.12～89.12mg/L, preferably 87.12mg/L；ILE content is 38.55～42.13mg/L, preferably 41.13mg/L；L-Leu content is 33.55～36.55mg/L, preferably 34.55mg/L；1B content is 165.44～172.44mg/L, preferably 168.44mg/L；L-Methionine content is 4.9～5.1mg/L, preferably 5.0mg/L；L- phenyl alanine contents be 21～ 23mg/L, preferably 22mg/L；Serine content is 38.5～40.35mg/L, preferably 39.5mg/L；L-Trp content For 12.3～14.46mg/L, preferably 13.6mg/L；Cys content is 108.7～115.3mg/L, preferably 113.7mg/L；L-Orn content of hydrochloric acid is 27.3～30.3mg/L, and preferably 29.3mg/L, p-aminobenzoic acid content are 1.0～1.03mg/L, preferably 1.03mg/L；
The transferrin content is 11.5～12.5mg/L, preferably 12.5mg/L；Pidolidone amide content is 0.2mM；Thiamine hydrochloride cellulose content is 1.0～1.03mg/L, and preferably 1.03mg/L, inositol content are 34.5～37.01mg/L, Preferably 35.51mg/L；Niacinamide content is 1.0～1.03mg/L, preferably 1.03mg/L；Cholesterol level is 0.015 ～0.019mg/L, preferably 0.019mg/L；Pluronic F68 content is 97～100mg/L, preferably 98mg/L；Tween 80 Content is 5.1～5.4mg/L, preferably 5.25mg/L；Ammonium ferric citrate content is 179.1～186.6mg/L, preferably 181.6mg/L；NaHCO3Content is 1.2g/L.
It is a discovery of the invention that adding in above-mentioned culture medium equal to the proliferation activity of skeletal muscle stem Cells during 6- chondroitin sulfates There is certain facilitation；Preferably, the addition of 6- chondroitin sulfates is 50-500mg/L, wherein, 6- chondroitin sulfates Addition is that skeletal muscle stem Cells proliferation activity is optimal near 200 μ g/ml.
The present invention strengthens element it has furthermore been found that adding helping for certain consumption in above-mentioned skeletal muscle stem Cells serum free medium (N, N- dimethylpiperidine chloride) can effectively facilitate the growth of skeletal muscle stem Cells, and wherein, the addition for helping strong element is 8.7 The growth of skeletal muscle stem Cells can be obviously promoted during～10.7mg/L, and the addition for helping strong element can be notable when being 9.0mg/L Promote the growth of skeletal muscle stem Cells, skeletal muscle stem Cells proliferation activity reaches highest, during more than this concentration, skeletal muscle stem Cells Proliferation activity assumes moderate tone, shows that the concentration of 9.0mg/L is optimum addn amount.
μ g/L, mg/L and g/L in the present invention is the weight based on composition described in every liter of culture medium, and mL/L is based on every The volume of composition described in culture medium is risen, mM is the molal quantity based on composition described in every liter of culture medium.
Invention further provides a kind of method for preparing Human Skeletal Muscle stem cell serum-free culture medium, the culture medium by Following steps are obtained：
(1) by DMEM:F12(1:1) dry powder is dissolved in water for injection and obtains basal medium；
(2) remaining its composition each is added in basal medium, is stirred to being completely dissolved；
(3) pH value is adjusted to 7.2～7.4, Osmo detection ranges 290-320mOsm/kg；
(4) degerming with 0.22 μm of membrane filtration, obtain final product.
Serum substitute in skeletal muscle stem Cells serum free medium of the present invention be non-animal derived composition, composition determine, Avoid the pathogenic risk that serum brings, the factor such as batch is unstable；Skeletal muscle stem Cells serum free medium of the present invention is suitable for In Human Skeletal Muscle stem cell primary and scale amplification cultivation.Application culture medium of the present invention has purity high, and cell propagation is fast, expands Increasing Efficiency is high, and the advantages of can stably pass on 20 more than generation, its skeletal muscle stem Cells that turns out can be used for clinical research and should With.Skeletal muscle stem Cells serum free medium of the present invention effectively increases skeletal muscle stem Cells amplification efficiency and purity, is clinical Research and application provide the skeletal muscle stem Cells of high-quality, can be used as scale, high-quality skeletal muscle stem Cells cultivating system To meet clinical research and application.
Description of the drawings
Growth curve contrast and experiment of Fig. 1 skeletal muscle stem Cells in three kinds of culture mediums；
Fig. 2 P20For skeletal muscle stem Cells immunohistochemical staining result；
Fig. 3 P20Result is redyed for skeletal muscle stem Cells SABC；
Fig. 4 P20Figure (10 ×) is formed for skeletal muscle stem Cells induction differentiation myotube；
Fig. 5 P20Figure (40 ×) is formed for skeletal muscle stem Cells induction differentiation myotube；
The 6- chondroitin sulfates of Fig. 6 variable concentrations promote the experimental result of skeletal muscle stem Cells growth.
The strong element that helps of Fig. 7 variable concentrations promotes the experimental result of skeletal muscle stem Cells growth.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and Apparent.But these embodiments are only exemplary, do not constitute any restriction to the scope of the present invention.People in the art Member is it should be understood that can enter to the details of technical solution of the present invention and form under without departing from the spirit and scope of the invention Row modification is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
The preparation of 1 Human Skeletal Muscle stem cell serum-free culture medium of embodiment
Cell Basal Medium (DMEM：F12(1:1) dry powder, article No.：SH30004, is purchased from Thermo companies), cell because Sub (being purchased from PEPROTECH companies), helps strong element (being purchased from Sigma companies, purity is more than 98%), and biosynthetic human insulin (is purchased from Sigma companies, purity are more than 98%), 6- chondroitin sulfates (are purchased from Hainan Gourd Doll Pharmaceutical Co., Ltd.), vitamin C (purchase In, Sigma companies, purity is more than 98%), (trade name is " Ultroser G " to serum substitute, and brand is Lonza；? Crescent Chemical Co., Inc. can be purchased from), NaHCO3(being purchased from Sigma companies, purity is more than 98%) and amino Acid (is purchased from Sigma companies, purity is more than 98%), and transferrins (is purchased from Sigma companies, purity is more than 98%), L- paddy Glutamine (is purchased from Sigma companies, purity is more than 98%), and vitamin B6 (is purchased from Sigma companies, purity is more than 98%), Thiamine hydrochloride (is purchased from Sigma companies, purity is more than 98%), and inositol (is purchased from Sigma companies, purity is more than 98%), Niacinamide (is purchased from Sigma companies, purity is more than 98%), and cholesterol (is purchased from Sigma companies, purity is more than 98%), P68 (Pluronic F68 is purchased from Sigma, the mainly addition polymers of polypropylene glycol and oxirane), Tween 80 (is purchased from Sigma Company, purity are more than 98%) and ferric citrate (being purchased from Sigma companies, purity is more than 98%).
Each composition of 1 skeletal muscle stem Cells serum free medium of the present invention of table and content
The preparation of Human Skeletal Muscle stem cell serum-free culture medium：
(1) 15.6g DMEM are accurately weighed:F12(1：1) dry powder, is dissolved in 800ml waters for injection.
(2) remaining each composition is weighed according to consumption in table 1, gradually added in above-mentioned solution, stir to being completely dissolved.
(3) 1.2g NaHCO are added3, continue stirring, be settled to 1L, adjust pH to 7.2-7.4, Osmo detection ranges 290- 320mOsm/kg.
(4) membrane filtration with 0.22 μm is degerming, obtains final product.
Growth curve contrast experiment of 1 skeletal muscle stem Cells of experimental example in three kinds of culture mediums
This experiment three kinds of culture mediums used are respectively the serum free medium of the preparation of the embodiment of the present invention 1 (hereinafter referred to as certainly Culture medium processed), the DMEM of 20% hyclone：F12 1:1 culture medium (hereinafter referred to as culture medium A), is purchased from the bone of Lonza companies Flesh stem cell special low blood serum medium SKGM-2 (hereinafter referred to as culture medium B).
1. skeletal muscle stem Cells are isolated and purified
(1) open surgical biopsy is aseptic takes young healthy people and voluntarily contributes rectus femoris or deltoid muscle 2-5g, proceeds to and contains equipped with 20ml In the 50ml centrifuge tubes of the PBS of 1% mycillin, 4 DEG C are transported to laboratory.
(2) musculature is taken out from 50ml centrifuge tubes, be put into sterilized petri dishes, remove fat and connective tissue, with aseptic Operating scissors are cut into about 1mm3Fritter, uses and flushes three times containing 1% dual anti-PBS.
(3) the II Collagenase Types that 10-15ml concentration is 0.1% are added, is mixed with pipette piping and druming, in 37 DEG C of constant temperature oscillations The digested 2h of device.
(4) 0.25% trypsase of 10-20ml is added, is mixed with pipette piping and druming, digested in 37 DEG C of constant temperature oscillators 20-30min.
(5) 10ml is added to terminate digestion containing 10% serum substitute culture medium.
(6) filter is sieved through with 400 mesh of advance moist heat sterilization, 200 mesh Stainless Steels successively, remove residue.
(7) filtrate is abandoned supernatant and is collected cell with 2000rpm, 5min centrifugation.
(8) PBS, 2000rpm, 5min is used to be centrifuged, in triplicate.
(9) with 4ml gentamicins containing 160IU/ml, the DMEM of 10% serum substitute:F12 1：1, it is inoculated into T25 cultures Bottle, density 104/ml.
(10) 37 DEG C are put into, saturated humidity, are cultivated in 5% CO2gas incubator.
(11) by preculture 20min after supernatant liquid be transferred to a new blake bottle, remove adherent fibroblast, so Afterwards by 2h, the cell suspension of 8h, 24h, 48h, 72h updates and arrives new blake bottle.
(12) attached cell after 72h is collected, adds self-made medium formally to cultivate.
(13) pass on when bottom of bottle face 70%-80% is covered with to cell, pass on ratio 1：2, method of operating:Pour out culture in glassware Liquid, adds PBS to wash 2-3 time, adds 0.25% trypsase 2ml, 37 DEG C of constant temperature oscillators to digest 3min, add fresh culture 3ml terminates digestion, mixes, year 2000rpm, 5min, abandon supernatant, add fresh culture 8ml, assign in 2 blake bottles, in 37 DEG C, saturated humidity, 5%CO2Under the conditions of continue culture.
2. by the P5 for collecting as stated above, for skeletal muscle stem Cells, with culture medium of the present invention, (embodiment 1 is made respectively Standby), the DMEM of 20% hyclone (being purchased from Lanzhou Min Hai companies):F12 1：The skeletal muscle stem Cells of 1, Lonza company are special SKGM-2 culture medium equivalent is resuspended, by 105/ hole is seeded to 24 orifice plates, changes liquid per three days, totally 3 blocks of plates, counts three holes, note daily Record growth curve.
2 three kinds of medium culture results contrasts of table
Experimental result is shown in Table 2 and Fig. 1.As seen from Table 2, the skeletal muscle stem Cells culture medium amplification skeletal muscle that prepared by the present invention The efficiency of stem cell apparently higher than containing 20% hyclone DMEM:F121:1 culture medium and SKGM-2 culture mediums.
2 skeletal muscle stem Cells Purity of experimental example and differentiation capability evaluation
Desmin antibody and immunohistochemical kit and auxiliary reagent are purchased from Wuhan doctor's moral company；PBS is pH 7.2- 7.4 phosphate buffer；SABC reagents, DAB developers, DAB developers (being purchased from Wuhan doctor's moral company).
1. P20 skeletal muscle stem Cells are digested, collect and 2 × 10 are pressed with self-made medium5The density in individual/hole is added to 1:10 Coated 6 orifice plate of the poly-D-lysine of dilution is cultivated, and is used for SABC reality when cell covers with 6 orifice plate more than 90% Test.
2. cell 3 times, 2min/ time is cleaned with PBS solution.
3. 4% paraformaldehyde 1ml/ holes are added, under normal temperature, fix 30min.
4. cell 3 times, 2min/ time is cleaned with PBS solution.
5. add 0.5%triton x-100 1ml/ holes, 10min to strengthen the permeability of cell.
6. cell 3 times, 2min/ time is cleaned with PBS solution.
7. the H after methanol dilution is added2O2(1 part of H2O2+ 50 parts of methyl alcohol) 10 drops/hole, 10min.
8. cell 3 times, 2min/ time is cleaned with PBS solution.
10. lowlenthal serum (lowlenthal serum after dilution is added：PBS=1:10) 1ml/ holes, 20min.
After 11. addition dilutions, (one resists an anti-Desmin antibody：PBS=1:100) 1ml/ holes, lucifuge, 4 DEG C of overnight (16- 18h) or 37 DEG C, 1 hour.
12. clean cell 3 times, 2min/ time with PBS solution.
13. add dilution after two anti-(two resist：PBS=1:100) 1ml/ holes, 37 DEG C, 30min, or room temperature 1h.
14. clean cell 3 times, 2min/ time with PBS solution.
15. dropwise addition SABC reagents, 1ml/ holes, 37 DEG C, 20min.PBS 5min × 4 time.
16. addition DAB developers (1ml adds A, each drop of B, C), 1ml/ holes, lucifuge, 10min, basis of microscopic observation are thin , there is sepia and are reacted with distilled water color development stopping in born of the same parents.Distilled water cleans 5min × 3 time.
17. redye 0.5-1min with haematoxylin, then return indigo plant with PBS.Examine under a microscope.Take 5 visuals field at random, record Positive cell number and negative cells number, calculate positive rate.Experimental result is shown in Fig. 2, Fig. 3 and table 3.
3 skeletal muscle stem Cells culture purity result of table
Show from result in table 3：Three groups of parallel results are shown, are obtained using skeletal muscle stem Cells medium culture of the present invention Human Skeletal Muscle stem cell purity reach more than 97%.
The differentiation myotube identification experiment of 3 skeletal muscle stem Cells of experimental example
By skeletal muscle stem Cells to contain 2%FBS, containing IGF-1 (100ng/ml), the DMEM without BFGF:F12 1：1 culture Base presses 2 × 105/ hole is inoculated in 6 orifice plates, is changed liquid once within the 4th day, was observed that myotube was formed in the 7th day.Experimental result is shown in figure 4 and Fig. 5.
4 6- chondroitin sulfates of experimental example promote the experiment of skeletal muscle stem Cells growth
The skeletal muscle stem Cells culture medium that uses in this experiment, formula are shown in Table 4：
The skeletal muscle stem Cells culture medium that uses in 4 experiments of table
Add the 6- chondroitin sulfates of variable concentrations in the culture medium of above-mentioned table 4, experiment is divided into six groups of 6- chondroitin sulfates Plain intervention group, i.e.,：1st, 2,3,4,5,6 groups, the interpolation concentration of the 6- chondroitin sulfates of each group is respectively：0μg/ml、50μg/ ml、100μg/ml、200μg/ml、300μg/ml、500μg/ml.4th day, the 5th day in being inverted into as basis of microscopic observation each group The myoblastic proliferative conditions of skeletal muscle stem Cells, prepare 6 parts of skeletal muscle stem Cells cells (1 × 10 according to packet on the 6th day8L-1) Suspension is inoculated in 12 orifice plates respectively, and per group of 3 holes are incubated at 37 DEG C per hole 1.5mL, the CO of volume fraction 5%2Incubator, the 6th day The incubation of 160 μ L lucifuges of MTT (tetrazolium bromide) solution is added, after 4h, terminates culture, remove supernatant, 1.6mL dimethyl is added dropwise per hole sub- Sulfone, vibrates and mixes 10min so that crystallization is fully dissolved, and using absorbance (OD value) of the ELIASA measure at 490nm, calculates Cell survival rate, this experiment repeat 3 plates.Impact of the comparative analysis 6- chondroitin sulfates to skeletal muscle stem Cells proliferation function.
Experimental result：MTT experiment result shows that increasing absorbance with 6- chondroitin sulfates concentration increases, and in concentration Proliferation activity highest during 200 μ g/ml.Draw variable concentrations cytoactive curve map and understand that skeletal muscle stem Cells number becomes in growth Gesture, is shown in Fig. 6.
Statistical analysis show that the 1st group and the 2nd, 3,4,5,6 groups has significant difference, P<0.05；Between 2nd, 3,5,6 groups No significant difference, P>0.05；4th group and the 2nd, 3,5,6 groups there is also significant difference, P<0.05；(table 5).
5 variable concentrations 6- chondroitin sulfates of table are intervened skeletal muscle stem Cells activity analysis result and skeletal muscle stem Cells are bred Activity impact (N=3)
Experimental result shows that 6- chondroitin sulfates have facilitation, and concentration 200 to the proliferation activity of skeletal muscle stem Cells Near μ g/ml, skeletal muscle stem Cells proliferation activity is optimal.
Experimental example 5 helps strong element (N, N- dimethylpiperidine chloride) to promote the experiment of skeletal muscle stem Cells growth
The skeletal muscle stem Cells culture medium prescription such as table 6 below that uses in this experiment：
Table 6
Add the strong element that helps of variable concentrations in the culture medium of above-mentioned table 6, experiment is divided into seven groups and helps strong element intervention group, i.e.,： 1st, 2,3,4,5,6,7 groups, the interpolation concentration for helping strong element of each group is respectively：0μg/ml、3μg/ml、6μg/ml、9μg/ml、12 μg/ml、15μg/ml、20μg/ml.4th day, the 5th day in being inverted into the propagation as basis of microscopic observation each group skeletal muscle stem Cells Situation, prepares 7 parts of skeletal muscle stem Cells cells (1 × 10 according to packet on the 6th day8L-1) suspension is inoculated in 12 orifice plates respectively, per group 3 Hole, is incubated at 37 DEG C per hole 1.5mL, the CO of volume fraction 5%2Incubator, adds 160 μ L of MTT (tetrazolium bromide) solution to keep away on the 6th day Light is incubated, and terminates culture after 4h, removes supernatant, and 1.6mL dimethyl sulfoxide (DMSO)s are added dropwise per hole, and vibration mixes 10min so that crystallization is filled Divide dissolving, using absorbance (OD value) of the ELIASA measure at 490nm, calculate cell survival rate, this experiment repeats 3 plates.Than Impact of the strong element to skeletal muscle stem Cells proliferation function is helped compared with analysis.
Experimental result：MTT experiment result shows, and concentration is in 9 μ g/ml Proliferation activity is higher, and during more than this concentration, proliferation activity compares presentation moderate tone.Draw variable concentrations cytoactive curve map Understand that skeletal muscle stem Cells number shows a rising trend, see Fig. 7.Statistical analysis draw the 1st group and the 2nd, 3,4,5,6,7 groups exist aobvious Write difference, P<0.05；No significant difference between 2nd, 3 groups, P>0.05；No significant difference between 4th, 5,6,7 groups, P>0.05； 4th group and the 1st, 2,3 groups has significant difference, P<0.05 (being shown in Table 8).
8 variable concentrations of table help strong element to intervene skeletal muscle stem Cells activity analysis result to skeletal muscle stem Cells proliferation activity Affect (N=3)
Experimental result shows and helps strong element to have facilitation to the proliferation activity of skeletal muscle stem Cells, and 9 μ g/ml groups of concentration are attached Closely, skeletal muscle stem Cells proliferation activity reaches highest, and assumes moderate tone more than this concentration skeletal muscle stem Cells proliferation activity, Show that this concentration is optimum addn amount.

Claims (10)

1. a kind of skeletal muscle stem Cells serum free medium, it is characterised in that comprising following composition：DMEM：F12, serum are replaced For thing, cell factor, vitamin, amino acid, biosynthetic human insulin, transferrins, Pidolidone acid amides, thiamine hydrochloride, flesh Alcohol, niacinamide, cholesterol, Pluronic F68, Tween 80, ferric citrate and NaHCO3；The pH value of the culture medium is 7.2 ～7.4.
2. according to the skeletal muscle stem Cells serum free medium described in claim 1, it is characterised in that the serum substitute is Ultroser G blood substitutes；
The cell factor is IGF, appointing in epithelical cell growth factor or Basic Fibroblast Growth Factor What one or more；
Described vitamin is any one of vitamin C, vitamin B1 or vitamin B6 or combination；
The amino acid is 1 water altheine, L-Aspartic acid, L-Histidine, ILE, L-Leu, the bad ammonia of L- Acid, L-Methionine, L- phenylalanines, Serine, L-Trp, Cys, L-Orn hydrochloric acid or p-aminophenyl Any or more than one any combination in formic acid；
The Pluronic F68 is mainly the addition polymers of polypropylene glycol and oxirane.
3. according to the skeletal muscle stem Cells serum free medium described in claim 1 or 2, it is characterised in that in every liter of culture medium, The content of each composition is：
DMEM：F12 contents are 15.6g/L, wherein, DMEM：The mass ratio of F12 is 1:1；
The serum substitute content is 20mL/L；
The vitamin B6 content is 0.95～1.05mg/L, preferably 0.975mg/L；The vitamin B1 content be 1.0～ 1.03mg/L, preferably 1.03mg/L；The Vitamin C content is 6～6.9mg/L, preferably 6.45mg/L；
The IGF content is 80～100 μ g/L, preferably 100 μ g/L；Epithelical cell growth factor content For 18～22 μ g/L, preferably 20 μ g/L；Basic Fibroblast Growth Factor content is 18～22 μ g/L, preferably 20 μ g/L；
Biosynthetic human insulin's content is 0.01～0.02mg/L, preferably 0.02mg/L；
The 1 water altheine content is 48.45～53.25mg/L, preferably 51.25mg/L；L-Aspartic acid content is 30.35～31.35mg/L, preferably 30.75mg/L；L-Histidine content is 85.12～89.12mg/L, preferably 87.12mg/L；ILE content is 38.55～42.13mg/L, preferably 41.13mg/L；L-Leu content is 33.55～36.55mg/L, preferably 34.55mg/L；1B content is 165.44～172.44mg/L, preferably 168.44mg/L；L-Methionine content is 4.9～5.1mg/L, preferably 5.0mg/L；L- phenyl alanine contents be 21～ 23mg/L, preferably 22mg/L；Serine content is 38.5～40.35mg/L, preferably 39.5mg/L；L-Trp content For 12.3～14.46mg/L, preferably 13.6mg/L；Cys content is 108.7～115.3mg/L, preferably 113.7mg/L；L-Orn content of hydrochloric acid is 27.3～30.3mg/L, and preferably 29.3mg/L, p-aminobenzoic acid content are 1.0～1.03mg/L, preferably 1.03mg/L；
The transferrin content is 11.5～12.5mg/L, preferably 12.5mg/L；Pidolidone amide content is 0.2mM； Thiamine hydrochloride cellulose content is 1.0～1.03mg/L, and preferably 1.03mg/L, inositol content are 34.5～37.01mg/L, preferably 35.51mg/L；Niacinamide content is 1.0～1.03mg/L, preferably 1.03mg/L；Cholesterol level be 0.015～ 0.019mg/L, preferably 0.019mg/L；Pluronic F68 content is 97～100mg/L, preferably 98mg/L；Tween 80 contains Measure as 5.1～5.4mg/L, preferably 5.25mg/L；Ammonium ferric citrate content is 179.1～186.6mg/L, preferably 181.6mg/L；NaHCO3Content is 1.2g/L.
4. according to the skeletal muscle stem Cells serum free medium described in claim 1, it is characterised in that containing 6- chondroitin sulfates Element.
5. according to the skeletal muscle stem Cells serum free medium described in claim 4, it is characterised in that the 6- chondroitin sulfates Content is 50～500mg/L；Preferably 180～200mg/L；Most preferably 200mg/L.
6. according to the skeletal muscle stem Cells serum free medium described in claim 1, it is characterised in that also contain N, N- dimethyl Piperidinium chloride.
7. according to the skeletal muscle stem Cells serum free medium described in claim 6, it is characterised in that the N, N- dimethyl piperazine The muriatic content of pyridine is 8.7～10.7mg/L, most preferably 9.0mg/L.
8. a kind of method for preparing skeletal muscle stem Cells serum free medium described in claim 1-7 any one, its feature exist In, including：
(1) by DMEM:The dry powder of F12 is dissolved in water for injection and obtains basal medium；
(2) remaining each composition is added in basal medium, is stirred to being completely dissolved；
(3) pH value is adjusted to 7.2～7.4, Osmo detection ranges 290-320mOsm/kg；
(4) 0.22 μm of filtration sterilizations, obtain final product.
9. in accordance with the method for claim 8, it is characterised in that：The DMEM:In the dry powder of F12, DMEM:The quality of F12 Ratio is 1:1.
10. skeletal muscle stem Cells serum free medium described in claim 1-7 any one is in culture skeletal muscle stem Cells Purposes；Preferably, the skeletal muscle stem Cells are Human Skeletal Muscle stem cells.
CN201611087240.5A 2016-12-01 2016-12-01 Skeletal muscle stem Cells serum free medium and its preparation method and application Active CN106497872B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611087240.5A CN106497872B (en) 2016-12-01 2016-12-01 Skeletal muscle stem Cells serum free medium and its preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611087240.5A CN106497872B (en) 2016-12-01 2016-12-01 Skeletal muscle stem Cells serum free medium and its preparation method and application

Publications (2)

Publication Number Publication Date
CN106497872A true CN106497872A (en) 2017-03-15
CN106497872B CN106497872B (en) 2019-10-11

Family

ID=58329262

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611087240.5A Active CN106497872B (en) 2016-12-01 2016-12-01 Skeletal muscle stem Cells serum free medium and its preparation method and application

Country Status (1)

Country Link
CN (1) CN106497872B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108774630A (en) * 2018-06-13 2018-11-09 中国科学院成都生物研究所 A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell
CN111808801A (en) * 2020-07-20 2020-10-23 中国肉类食品综合研究中心 Method for extracting and culturing pigeon skeletal muscle satellite cells
CN111944752A (en) * 2020-08-28 2020-11-17 广州同康生物科技有限公司 Skeletal muscle stem cell serum-free medium and preparation method thereof
EP4144356A4 (en) * 2020-04-26 2024-04-17 Univ Soochow Stem cell drug for treating diabetes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104805054A (en) * 2015-04-30 2015-07-29 厦门中领精准医学产业发展有限公司 Serum-free medium of stem cell
CN106062205A (en) * 2013-12-20 2016-10-26 基本药品有限责任公司 Media for cell culture

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106062205A (en) * 2013-12-20 2016-10-26 基本药品有限责任公司 Media for cell culture
CN104805054A (en) * 2015-04-30 2015-07-29 厦门中领精准医学产业发展有限公司 Serum-free medium of stem cell

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MASSIMILIANO CERLETTI等: "Short-term calorie restriction enhances skeletal muscle stem cell function.", 《CELL STEM CELL》 *
P M GILLBERT等: "Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture.", 《SCIENCE (NEW YORK,N.Y.)》 *
王琳等: "适合人肌母细胞的无血清培养基", 《中国组织工程研究》 *
陈从波等: "成年大鼠骨骼肌干细胞纯化、培养及移植的初步实验研究", 《生物医学工程与临床》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108774630A (en) * 2018-06-13 2018-11-09 中国科学院成都生物研究所 A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell
EP4144356A4 (en) * 2020-04-26 2024-04-17 Univ Soochow Stem cell drug for treating diabetes
CN111808801A (en) * 2020-07-20 2020-10-23 中国肉类食品综合研究中心 Method for extracting and culturing pigeon skeletal muscle satellite cells
CN111944752A (en) * 2020-08-28 2020-11-17 广州同康生物科技有限公司 Skeletal muscle stem cell serum-free medium and preparation method thereof
CN111944752B (en) * 2020-08-28 2021-09-03 广州同康生物科技有限公司 Skeletal muscle stem cell serum-free medium and preparation method thereof

Also Published As

Publication number Publication date
CN106497872B (en) 2019-10-11

Similar Documents

Publication Publication Date Title
CN112322580B (en) Application of serum-free medium for mesenchymal stem cells
CN106497872A (en) Skeletal muscle stem Cells serum free medium and its preparation method and application
US20020039567A1 (en) Methods of treating bone or cartilage conditions by the administration of creatine
CN106591235B (en) A method of promoting endothelial cell function and characteristic
CN102634482B (en) Serum-free complete medium for mesenchymal stem cell
CN101374942A (en) Method for purifying cardiac myocyte and presumptive cardiac myocyte derived from stem cell and fetus
CN102787094B (en) Substratum, cell cultures test kit and cell culture processes
CN105112362B (en) A kind of serum free medium of placenta mesenchyma stem cell and preparation method thereof
CN103255103B (en) Serum-free adipose tissue-derived mesenchymal stem cell culture medium
CN102517251A (en) Mesenchymal stem cells, as well as preparation method and application thereof
CN110257328A (en) A kind of mesenchymal stem cell serum-free culture medium
CN109749997A (en) A kind of Limbus corneae stem cell serum-free culture medium and its cultural method
CN107502588B (en) Method for separating and preparing dental pulp stem cells
CN102505005B (en) Culture medium for keeping primary airway epithelial cells in physiological state in vivo
KR20180122288A (en) 3D bioprinting construct using human nasal inferior turbinate derived mesenchymal stem cell and uses thereof
CN108324993A (en) A kind of stem cell complex, preparation method and the application of induction hair regeneration
CN103184188A (en) Primary homologous three-cell four-dimensional model of pharmaceutical research on central nervous system and construction method
WO2019237812A1 (en) Adipose tissue digestive juice and method for rapidly obtaining stromal vascular fraction cells
RU2662172C2 (en) Method for producing regenerative veterinary preparation based on extract of mesenchimal stem cells and conditioned medium
CN108823158B (en) Application of ligustrazine and specnuezhenide in promoting proliferation of mesenchymal stem cells cultured in vitro and inhibiting replicative senescence
CN105087480A (en) Serum-free stem cell culture medium and application thereof
CN101392235A (en) Method for large-scale culture of immortalized porcine hepatocyte
CN101496815B (en) Medicament for treating hepatic failure and preparation method thereof
CN108220232A (en) A kind of cultural method of the culture medium based on fetal calf serum
CN109479873A (en) A kind of adipose tissue saves liquid and its application

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant