CN106754668A - A kind of stem cell medium and parenteral solution - Google Patents

A kind of stem cell medium and parenteral solution Download PDF

Info

Publication number
CN106754668A
CN106754668A CN201611007074.3A CN201611007074A CN106754668A CN 106754668 A CN106754668 A CN 106754668A CN 201611007074 A CN201611007074 A CN 201611007074A CN 106754668 A CN106754668 A CN 106754668A
Authority
CN
China
Prior art keywords
stem cell
injection
cell
culture
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
CN201611007074.3A
Other languages
Chinese (zh)
Other versions
CN106754668B (en
Inventor
杨柳松
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.)
Shenyang Cell Therapy Engineering Technology Research And Development Center Co Ltd
Original Assignee
Shenyang Cell Therapy Engineering Technology Research And Development Center 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 Shenyang Cell Therapy Engineering Technology Research And Development Center Co Ltd filed Critical Shenyang Cell Therapy Engineering Technology Research And Development Center Co Ltd
Priority to CN201611007074.3A priority Critical patent/CN106754668B/en
Publication of CN106754668A publication Critical patent/CN106754668A/en
Application granted granted Critical
Publication of CN106754668B publication Critical patent/CN106754668B/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
    • C12N5/0662—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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • 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
    • C12N2500/38—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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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

Abstract

A kind of stem cell medium, is made up of the following raw material：Amino Acid Compound Injection 10%, Vitamin H 1%, alanyl glutamine 0.5%, human serum albumin 10%, injection of fish 10%, micro-element injection 0.5%, Bomaili A liquid 68%；PH value is 6.8 7.0, and osmotic pressure is 310 mOsm/kg.Using the stem cell medium culture mescenchymal stem cell, and collect mescenchymal stem cell culture supernatant；The culture supernatant that will be collected collects filtrate, as stem cell injection liquid through membrane filtration.It is injected intravenously using the stem cell secretion factor of decellularization, effectively prevent dispute of the stem cell in terms of ethics；The stem cell medium, material composition belongs to clinical rank, it is safe and reliable, for stem cell living environment provides nutrition, the substantial amounts of cell factor of stem cell secretion can be effectively facilitated, keeps Stem Cell Activity, and nutritional supplement agent prescription to coordinate with stem cell secretion factor, body sub-health state can effectively be alleviated, body health is reappeared.

Description

A kind of stem cell medium and parenteral solution
Technical field
The invention belongs to biological technical field, more particularly to a kind of Mesenchymal stem cell nutrient solution and parenteral solution.
Background technology
Mescenchymal stem cell（Mesenchymal stem cells）It is that a class has the thin into soma of multi-lineage potential Born of the same parents, come from the mesoderm of mesoderm growing early stage, are primarily present in connective tissue and organ interstitial, can be from marrow, peripheral blood, fat And obtained in the Various Tissues such as skin.Mescenchymal stem cell belongs to non-terminally differentiated cells, the feature of its existing interstitial cell, and There is the feature of endothelial cell and epithelial cell；Used as a class multipotential stem cell, it is in vitro under specific inductive condition, Ke Yixiang The direction such as bone, cartilage, muscle, tendon, liver, fat, nerve, endothelium and islet-like cells Proliferation, Differentiation.
In mescenchymal stem cell incubation, stem cell factor can be secreted（SCF）, EGF（EGF）, Ink vessel transfusing Skin growth factor（VEGF）, basic fibroblast growth factor（bFGF）, platelet derived growth factor A（PDGF-A）, turn Change growth factor（TGF-β）, HGF（HGF）, interleukins（IL）, IGF（IGF- Ⅱ）, macrophage colony stimulatory factor（GSFs）, TNF（TNF-α）, interferon（IFN）, nerve growth factor （NGF）And the excretion body of cell secretion（Exosome）, be loaded with functional protein, mRNA and microRNA（miRNA）Deng Material.Comprising this multiple cytokine of RNA molecule cell can be promoted to breed, break up in human body and regenerated, strengthen new old Metabolism, prevents cell ageing；Mediation body cell signal transduction, activation equipment somatic stem cell, promote new capillary vascularization, carry out Physiological is repaired；Regulation body inferior health, improves immunity of organism and removes damage, lesion, the cell of aging.There are some researches show making With using stem cell there is identical to act on stem cell secretion factor and Exosome.Therefore, do not used in cell therapy thin Born of the same parents and use cell-secretion factor decellularization concept, receive more and more attention and support, stem cell secretion factor It is with the obvious advantage compared with stem cell.
Cell growth in vitro depends on nutrient solution, and stem cell is trained directly by cytokine secretion to nutrient solution The composition of nutrient solution is directly connected to the Clinical practice of cell factor.The in vitro culture of zooblast depends on tire ox at this stage Serum, this like cell could preferably breed growth in vitro.But due to hyclone complicated component, use the culture containing serum There is potential cytotoxic effect, exogenous virus and virulence factor pollution problem in base cultured cells, make the standard of cell culture Change and finished product purifying difficulty is increased, the residual of foreign sera is also easily caused allergic reaction clinically.Therefore, free serum culture The development and application of base is imperative.So-called serum free medium, refers to not contain animal blood serum or other biological extract solution, but Cell long period growth, a kind of culture medium of breeding in vitro can still be maintained.
The World Health Organization by body without organic disease, but have some functions change state referred to as " the 3rd shape State ", China is referred to as " sub-health state ".Inferior health is a kind of low shape of physiological function of the finger between health and disease State.Clinical medicine substantially contributes to treat the medicine of disease, without the conditioning agent for inferior health colony.For sufferer, face Commonly use some nutritional supplements on bed to adjust organism metabolism and immune system, for example：Human serum albumin, circulates with increasing Blood volume, the effect for maintaining plasma colloid osmotic pressure, can be tissue with nutrient as nitrogen source, beneficial to cell metabolism, Different materials can be conveyed as carrier；Water soluble vitamin, with strengthen immunity, the work of anti-oxidant raising metabolism With the effect also with control and pre- anti-cancer；Amino acid, may participate in the synthesis and metabolism of protein, generates enzyme, swashs Element, antibody, structural proteins, promotion organization healing, recover normal physiological function；Glutamine, is a kind of nutritional supplement, is machine Body provides nitrogen source, promotes the synthesis of protein, improves body Nutrition and Metabolism, adjusts blood sugar, and can strengthen the function of immune system And it is anti-oxidant；Micro-element injection, belongs to parenteral nutrition additive, maintains the biochemical reaction in body to be normally carried out；Water Solution albumen, can enter histocyte, participate in protein anabolism, obtain positive nitrogen equilibrium, and generate enzyme, hormone, antibody, knot Structure albumen, promotion organization physiological function recover normal；Bomaili A liquid, is the replenishers and basifier of water, electrolyte, can be kept Osmotic pressure, protects cell.These clinical medicines in Clinical practice for many years, its security and validity obtain the accreditation of society.
Patent CN102920735A, patent CN1966080A, are each provided with a kind of mesenchymal stem cell injection, but its Injectable liquefied composition contains living cells, reduces the security and controllability of Clinical practice, also, make during culture mescenchymal stem cell With hyclone, foreign protein is introduced, the risk for using is bigger；Patent CN1618981A provides a kind of stem cell secretion Factor preparation method, its stem cell by being changed into immortality cell after genetic modification, because there is certain potential wind in genetic modification Danger, and stem cell secretion factor, by isolating and purifying, process is numerous and diverse, and stem cell secretion factor inevitably causes damage；Specially Sharp CN102008507A provides a kind of umbilical cord mesenchymal stem cells parenteral solution, but it only adds human serum albumin and heparin Calcium, formula nutritional structure is single, and certain influence is had on cytoactive and EF content, and contains work in its parenteral solution Stem cell, security there is also problem；Patent CN102703385A provides a kind of Mesenchymal stem cell nutrient solution, its application Cell culture fluid addition serum-free additive, bFGF, EGF, myosin and antibiotic are made stem cell medium, first carefully Born of the same parents' nutrient solution is not belonging to clinical grade reagent, it is impossible to directly apply to human body, secondly, antibiotic is added in nutrient solution, there is allergy Risk, finally, its trophic structure is single, it is impossible to meet cytotrophy, may have one to cytoactive and EF content It is fixing to ring.
Whether stem cell clinical research and treatment, or the application and development to stem cell secretion factor, are required to a large amount of Amplification of mesenchymal stem cells, therefore, how to obtain premise and key that substantial amounts of mescenchymal stem cell is research.
The content of the invention
Regarding to the issue above, the present invention provides a kind of stem cell medium and parenteral solution.The parenteral solution directly enters row vein Injection, without living cells.Operating process is simple and reliable, using safety, can effectively improve immunity of organisms, state of getting well.
To achieve the above object, the present invention provides a kind of stem cell medium, and its raw material is calculated by percent by volume, by such as It is lower into being grouped into：Amino Acid Compound Injection 10%, Vitamin H 1%, alanyl glutamine 0.5%, human blood are white Protein 10 %, injection of fish 10%, micro-element injection 0.5%, Bomaili A liquid 68%.
Using the stem cell medium culture mescenchymal stem cell, and collect mescenchymal stem cell culture supernatant；To receive The culture supernatant of collection collects filtrate, as mesenchymal stem cell injection through 0.22 μm of membrane filtration.
The mescenchymal stem cell is umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, Cord blood mesenchyma are dry thin The adult stem cells such as born of the same parents, fat mesenchymal stem cell, mesenchymal stem cells MSCs or dental pulp mescenchymal stem cell.
To achieve these goals, the present invention also provides a kind of compound method of stem cell medium, comprises the following steps.
Step 1, the Bomaili A liquid for taking 1%, dissolve Vitamin H, obtain mixed liquor.
Step 2, respectively by the mixed liquor of step 1, Amino Acid Compound Injection, alanyl glutamine, human serum albumin, Injection of fish, micro-element injection, 67% Bomaili A liquid, mix, as stem cell medium in proportion.
The stem cell medium pH value is 6.8-7.0, and osmotic pressure is 310 mOsm/kg.
To achieve these goals, the present invention also provides a kind of preparation method of mesenchymal stem cell injection, including with Lower step.
Step 1, mescenchymal stem cell sample collection, treatment.
Step 2, the primary extraction and culture of stem cell：With the tissue of the method separating mesenchymal of physical shear, to organize Block stationary culture uses serum free medium culture mescenchymal stem cell；Centrifugation mononuclearcell, with adhere-wall culture Mode is separately cultured mescenchymal stem cell.
Step 3, the Secondary Culture of stem cell：When cell reaches more than 70% to be merged, cleaning adds cell dissociation afterwards twice Liquid, filtering removal tissue fritter, after supernatant is abandoned in centrifugation, obtains primary mescenchymal stem cell；Primary mescenchymal stem cell is with without blood After clear culture medium is resuspended, it is seeded in blake bottle or culture dish, when cell fusion degree reaches 80%-90%, between collecting the first generation Mesenchymal stem cells, as seed cell or continuation Secondary Culture.
The identification of step 4, the biology of mescenchymal stem cell and fluidic cell.
Step 5, preparation stem cell medium, change mescenchymal stem cell liquid, continue to cultivate mescenchymal stem cell, and collect Mescenchymal stem cell culture supernatant.
Step 6, the stem cell culture supernatant that will be collected in step 5 collect filtrate through 0.22 μm of membrane filtration, as dry thin Born of the same parents' parenteral solution.
Sample process uses alcohol-pickled time 1-4min in the step 1, and the alcohol is through 0.22 μm of aperture filter membrane mistake Filter bacterium.
The serum free medium is that 10% serum replacement is added in DMEM/F12 basal mediums, and uses preceding need Add Glu.
CO is used in step 2 adhere-wall culture2Incubator；The CO2The condition of incubator is：37 DEG C, 12% oxygen, The hypoxia condition of 5% carbon dioxide, 83% nitrogen.
Inoculum density is 5000-6000/cm in the step 32。
The stem cell injection liquid may be directly applied to human vein injection, dosage be 200m people/time.
Beneficial effects of the present invention.
Stem cell medium and parenteral solution that the present invention is provided, low-oxygen environment and nothing is used in simulation mescenchymal stem cell body Blood serum medium culture, when cell confluency reaches 70%, uses stem cell medium instead and continues to cultivate 2 days, collects stem cell training Supernatant is supported, stem cell secretion factor is contained in supernatant, stem cell injection liquid is obtained after filtering, may be directly applied to human body Intravenous injection.The present invention is injected intravenously using the stem cell secretion factor of decellularization, and decellularization contributes to neutralizing to lead Pipe portion door is to uncontrolled dangerous worry after implantation stem cell, and the supernatant of decellularization will not increase the micro- blood of tip The risk of pipe choking, also will not tumorigenesis；Stem cell medium of the invention, for stem cell living environment provides basic nutrition, energy The substantial amounts of cell factor of stem cell secretion and excretion body are effectively facilitated, nutritional supplement in Stem Cell Activity, and nutrient solution is kept Agent prescription coordinates with stem cell secretion factor, can effectively alleviate body sub-health state, reappears body health；What the present invention was provided Stem cell medium, material composition belongs to clinical rank, can be directly used for human body, safe and reliable.
Cell culture environment of the invention is culture under hypoxia condition, physiologically the ambient oxygen content of internal cells survival It is 1-13%, but most of cell injuring model of the prior art is completed under 21% oxygen concentration.Mescenchymal stem cell master To grow in the environment of hypoxemia in vivo, hypoxemia more meets its physiological status, oxygen-enriched to have damaging action to cell on the contrary.At present Research shows that hypoxemia culture can improve the multiplication capacity of mescenchymal stem cell, transfer ability and Adhering capacity, and reduces cell and decline Die rate.But more lactic acid can be produced under hypoxemia, used as metabolic by-product, excessive lactic acid can produce toxicity, limit to cell Cell further growth processed, therefore, the present invention is given and is suitable to the hypoxia condition 12% of interstital stem cell by substantial amounts of practice Oxygen.The present invention substitutes the hyclone used in traditional mescenchymal stem cell culture using serum replacement, in clinical practice In be avoided that the potential risk that the foreign protei of animal blood serum and animal origin brings.
Meanwhile, the nutrient solution and parenteral solution of the mescenchymal stem cell that the present invention is provided are adapted to large-scale industrial production, There is huge application prospect.
Brief description of the drawings
Fig. 1 is mescenchymal stem cell cell growth state figure.
Fig. 2 is mescenchymal stem cell FCM analysis figure；Wherein 2-1. mescenchymal stem cells CD90 expression rates oscillogram, 2-2. mescenchymal stem cell CD73 expression rates oscillogram, 2-3. mescenchymal stem cell CD105 expression rates oscillogram, 2-4. mesenchymas Stem cell CD45 expression rates oscillogram, 2-5. mescenchymal stem cell CD34 expression rate oscillograms.
Specific embodiment
With reference to specific embodiment, the present invention will be further described.
Embodiment 1.
The preparation method of a kind of umbilical cord mesenchymal stem cells nutrient solution and parenteral solution, comprises the following steps.
Step 1, sample collection.
Multipara's informed consent, gathers mature healthy fetus fresh sterile umbilical cord tissue, 4 DEG C of preservations, treatment in 24h.Produce Gathered in antenatal seven days of woman female blood carry out HIV detections, hepatitis B detection, hepatitis detection, Lues Assay, glutamic-pyruvic transaminase detect, it is huge Cell virus detection and detection of mycoplasma, after all detection is qualified, can use its fetus fresh sterile umbilical cord tissue.
Cleaning and sterilization：Umbilical cord is taken out in Biohazard Safety Equipment, first surface is carried out with 0.9% sodium chloride injection 10ml Cleaning, afterwards by umbilical cord immersion through 2min in 0.22 μm of 75% alcohol of filtration sterilization；Finally with 0.9% sodium chloride injection 10ml is cleaned again, removes residual alcohol and bloodstain.
Step 2, the primary extraction and culture of stem cell.
（1）Umbilical cord is cut into the segment of 2-3cm, outside amnion is rejected and internal blood vessel is obtained magnificent Tong Shi glue, by gained China Tong Shi glue is cut into 2mm3Tissue fritter, after cleaning 3 times with 20ml physiological saline, obtain the magnificent Tong Shi glue tissue without bloodstain small Block.
（2）The magnificent Tong Shi glue tissue fritter without bloodstain that will be obtained, with 10ml NTx enzymes（0.1mg/mL）Digestion process 10min, extracellular matrix protein is discharged from tissue, that is, obtain the mixture of extracellular matrix protein and tissue fritter composition.
（3）2-3mL said mixtures are taken, is applied on the culture dish of a diameter of 10cm, with lucky confluent cultures ware bottom And tissue fritter is evenly distributed and is preferred, tissue tile position is moved with aseptic nipper, be uniformly distributed it；In Biohazard Safety Equipment Naturally 2min is dried, gluey coated layer is formed and is sticked to culture dish bottom, tissue fritter is fixed on culture dish bottom.
（4）To serum free medium 10ml is added in above-mentioned culture dish, CO is placed on237 DEG C, 12% oxygen in incubator Cultivated under gas, 5% carbon dioxide, the hypoxia condition of 83% nitrogen, a full dose is carried out after 24h and changes liquid；4-5 days are cultivated in microscope Lower observation has cell to be grown from tissue fritter, is further cultured for during to the 8th day, carrying out second full dose and changing liquid, while collection group Knit fritter.
The serum free medium is that 10% serum substitute is added in DMEM/F12 basal mediums（Serum replacement It is commercially available, PALL companies, 15950-017）, without animal derived material, and Glu need to be added before, make Its concentration is 15mg/L.Using serum substitute, the hyclone used in traditional human umbilical cord mesenchymal stem cells culture is substituted, The potential risk that the foreign protei of animal blood serum and animal origin brings is avoided that in clinical practice.
（5）Will be above-mentioned（4）The tissue fritter of middle collection is transferred in new culture dish, carries out the secondary adherent training of tissue fritter Support, primary mescenchymal stem cell is cultivated, according to cell growth status（General cell culture was to the 5-6 days）Carry out full dose and change liquid, With extra-nutrition material.
In the different cultural methods of tissue fritter, organize fritter adherent and cell growth status differences, such as the institute of table 1 Show.Wherein, fritter is organized to stick to the culture dish bottom used time in the present embodiment, during not adding culture medium, culture dish is inverted, tissue It is standard that fritter does not fall from culture dish bottom.
Table 1：Tissue fritter is adherent and cell growth status compare.
As shown in Table 1：Colloidal mixture is smeared in culture dish bottom, is conducive to tissue fritter quick wall attaching, and can be bright It is aobvious to promote cell to be grown from tissue block and in the growth of culture dish bottom；In same time, the tissue fritter of equal number By the way that after this method culture, the primary mescenchymal stem cell quantity of umbilical cord of acquisition increased one times than normal tissue block adherent method.
The coated layer that mixture of the invention is formed in culture dish bottom, on the one hand shorten tissue fritter it is adherent when Between；On the other hand cell is also made to be easier to dissociate from tissue fritter（Coated layer constructs the three-dimensional of solid for cell growth Growing environment, had both promoted adhesion of the cell to culture dish bottom, and the infiltration of nutrient solution is conducive to again, also helped cell in training Support propagation and the migration of ware bottom）, the primitive cell culture time is significantly shortened.And compared with traditional enzyme digestion, this Invention digests acquisition extracellular matrix protein in short-term using the gentle Collagenase I of property, and most cells are retained in tissue all the time In fritter, it is to avoid damage of the enzyme to cell membrane, it is ensured that cell viability.
When umbilical cord mesenchymal stem cells are cultivated be expanded to 70% fusion for the first time under different oxygen concentrations, required incubation time With flow cytometer detection result, as shown in table 2.
Table 2：Umbilical cord mesenchymal stem cells proliferation time and flow cytometer detection under different oxygen concentrations.
As shown in Table 2, the umbilical cord mesenchymal stem cells cultivated under the conditions of different oxygen concentrations, meet international cell therapy association Can the standard on mescenchymal stem cell：Positive expression rate >=95%, negative expression rate≤2%, and different oxygen concentrations CMC model Cell detection results it is consistent, show that growth of the hypoxia condition on umbilical cord mesenchymal stem cells does not influence；But in 12% oxygen Under hypoxia condition, umbilical cord mesenchymal stem cells incubation time is most short, but when oxygen concentration is more than 18%, incubation time is elongated, therefore The present invention uses the hypoxia condition of 12% oxygen.
Step 3, the Secondary Culture of stem cell.
（1）When microscope observation of cell reaches more than 70% and merges, passage amplification is carried out：Removal serum free medium, uses After 0.9% sodium chloride injection washs 2 times, 5ml cell dissociation buffers are added（Model：ACCUTASE GMP, producer Innovative Cell Technologies）, attached cell is departed from culture dish bottom, filtering removal tissue fritter, centrifugation 1000rpm processes 10min, after abandoning supernatant, obtains primary umbilical cord mesenchymal stem cells.
（2）The primary cell of acquisition with serum free medium it is resuspended after, be seeded in blake bottle or culture dish, inoculum density Control is in 5000-6000/cm2, after culture a period of time, when cell fusion degree reaches 80%-90%, collect first generation umbilical cord Mescenchymal stem cell, as seed cell or continuation Secondary Culture.
The umbilical cord mesenchymal stem cells of Secondary Culture, cultivate 3 days, according to ELISA under the conditions of 6%-21% different oxygen concentrations Kit（The U.S., R＆D companies）Specification is operated, and EF in its cells and supernatant is detected respectively, such as：Stem cell factor （SCF）, EGF（EGF）, VEGF（VEGF）, basic fibroblast growth factor（bFGF）、 Interleukin-6（IL6）, HGF（HGF）, tumor necrosis factor β 3（TNF-β3）Content, specific steps are such as Under.
（1）ELISA Plate is taken out, cytokine standards product conduct in blank well of 100 μ L is separately added into according to order correspondence Positive control；Sample number into spectrum being marked respectively, adding 100 μ L umbilical cord mesenchymal stem cells culture supernatants in blank well, 37 DEG C incubate Educate 90min.
（2）Board-washing machine is cleaned 4 times, and 10-20s is stood every time；The biotin labelled antibodies working solution of 100 μ L is added per hole, 37 DEG C of incubation 60min.
（3）Board-washing machine is cleaned 5 times, and 10-20s is stood every time；The enzyme conjugates working solution of 100 μ L is added per hole, 37 DEG C incubate Educate 30min.
（4）Board-washing machine is cleaned 4 times, and 10-20s is stood every time；The developer of 100 μ L is added per hole, 37 DEG C of lucifuges are incubated instead Answer 15 min.
（5）50 μ L terminate liquids, terminating reaction are added per hole.
Every part of sample sets 3 repeating holes, and absorbance (A) value at 450nm is surveyed with full-automatic ELIASA, takes its average value, root Cytokine content is calculated according to the standard curve drawn, as a result as shown in table 3.
Table 3：Umbilical cord mesenchymal stem cells EF assay under different oxygen concentrations（Unit：pg/mL）.
As shown in Table 3, assay is carried out to 7 cell factors chosen, it is known that under the hypoxia condition of 12% oxygen, Umbilical cord mesenchymal stem cells secrete cytokines content highest, and 2-4 times is improve, therefore this experiment whole process uses 12% oxygen Hypoxia condition cultured cells, can greatly improve the yield for obtaining cell factor.
The identification of step 4, the biology of umbilical cord mesenchymal stem cells and fluidic cell.
1st, cell growth and its Morphological Characteristics：Using the digestion method of the present embodiment step 1, mixture is applied to training Supporting carries out adhere-wall culture on ware, culture is observed at the 3-5 days through inverted microscope, it is possible to find have more adherent cell growth； When culture was to 10 days, cell confluency reaches 70%, sees Fig. 1；Observe there is thin during the tissue secondary utilization of fritter, after culture 24h Born of the same parents carry out adherent growth, and cell confluency reaches 70% or so after 9 days.
2nd, fluidic cell identification mescenchymal stem cell surface marker.
Second generation cell is collected, PBS is washed 2 times（1000rpm centrifugal treatings 5min）, adjust cell dense Spend to 5 × 106Individual/ml；Be incubated 30min after adding the corresponding antibody of cell surface marker, wash again it is resuspended after, you can on Machine（Flow cytometer FACSCalibur, BD companies）Detection.Antibody used includes the antibody of following cell surface molecule：CD90、 CD105, CD73 are positive mark, and CD45, CD34 are negative marker.Concrete outcome is referring to table 4, umbilical cord mesenchymal stem cells streaming Detection, is as a result shown in Fig. 2.
Table 4：Umbilical cord mesenchymal stem cells surface marker testing result.
As shown in Table 4：The primary umbilical cord mesenchymal stem cells that fritter is harvested are organized in the present embodiment through passing on twice After culture, international standard of the cell therapy association on mescenchymal stem cell is still conformed to：Positive expression rate >=95%, radiolucent table reaches Rate≤2%；The cell for being obtained can be used as clinical research and the seed cell of application.
Step 5, preparation stem cell medium, then use stem cell medium instead and continue to cultivate stem cell, and collection is dry carefully Born of the same parents' culture supernatant.
1. stem cell medium is prepared.
（1）The Bomaili A liquid of 2ml is taken, Vitamin H is dissolved（Huarui Pharmaceutical Co. Ltd., lot number：Traditional Chinese medicines Quasi- word H32023002, specification 0.5g/ bottles）.
（2）By step（1）In mixed liquor, Amino Acid Compound Injection（Beijing Fresenius Kabi Pharmaceutical Co., Ltd, Lot number：Chinese medicines quasi-word H19993345）, alanyl glutamine（Huarui Pharmaceutical Co. Ltd., lot number：Chinese medicines quasi-word H20058281）, human serum albumin（Hualan Bio-Engineering Co Ltd., lot number：Chinese medicines quasi-word S19993024）, hydrolysis egg White parenteral solution（Liaoyuan City Hong Yuan pharmaceutical Co. Ltds, lot number：H22026405）, micro-element injection（The auspicious limited public affairs of pharmacy of China Department, lot number：Chinese medicines quasi-word H32023907）, Bomaili A liquid（Shanghai Baxter Healthcare Ltd., lot number： H20000475）, mixing in proportion, pH value is 6.8-7.0, and osmotic pressure is 310 mOsm/kg, as stem cell medium.It is dry thin The formula rate of born of the same parents' nutrient solution, as shown in table 5.
（3）When umbilical cord mesenchymal stem cells grow into 70% fusion, culture supernatant is abandoned in suction, and cell is cleaned with physiological saline Once, then step is used（2）The stem cell medium cleaning of preparation, takes stem cell medium and continues to cultivate 2 days afterwards.
（4）Continuous observation cell state, collects cells and supernatant, through cell dissociation buffer after 2 days（Model：ACCUTASE GMP, producer Innovative Cell Technologies）Cell is collected after digestion, cell analysis are carried out.
Table 5：Stem cell medium formula rate.
As shown in Table 5：Umbilical cord mesenchymal stem cells are cultivated with stem cell medium, Cell viability is determined after 2 days, from Two groups start, and stem cell motility rate is maintained at more than 96.4%, wherein second group of stem cell motility rate highest, is 97.5%, and comprehensively examine Consider value, the present invention selects second group of optimization formula as stem cell medium, i.e.,：Amino Acid Compound Injection 20ml, note Penetrate with water soluble vitamin 0.25g, alanyl glutamine 1ml, human serum albumin 20ml, injection of fish 20ml, micro- Secondary element parenteral solution 1ml, Bomaili A liquid 136ml.
（2）Using ELISA method, the concentration of umbilical cord mesenchymal stem cells parenteral solution EF is determined.
Selection determines stem cell factor（SCF）, EGF（EGF）, VEGF（VEGF）, alkalescence Fibroblast growth factor（bFGF）, platelet derived growth factor A（PDGF-A）, interleukin-6（IL6）, leucocyte Interleukin 1（IL1）, HGF（HGF）, tumor necrosis factor β 3（TNF-β3）, granulocyte colony stimulating factor（GM- CSF）Concentration, comprise the following steps that.
（1）ELISA Plate is taken out, cytokine standards product conduct in blank well of 50 μ L is separately added into according to order correspondence Positive control；Sample number into spectrum is marked respectively, and stem cell culture supernatant prepared by 50 μ L steps 5 of addition is in blank well；In sample The biotin labeling liquid of 10 μ L is added in hole；The enzyme label solution of 50 μ L is added in standard sample wells and sample well.
（2）37 DEG C of incubation 60min；Board-washing machine is cleaned 5 times, and 20s is stood every time；Substrate A, each 50 μ L of B liquid are added per hole；37 DEG C min of lucifuge incubation reaction 15；The H of 50 μ L is added per hole2SO4Terminate liquid, terminating reaction.
（3）Every part of sample sets 3 repeating holes, and absorbance (A) value at 450nm is surveyed with full-automatic ELIASA, takes its average value, According to the standard curve drawn, cytokine content is calculated, as a result as shown in table 6.
Albumin physiological saline and stem cell medium, each 10ml cultivate umbilical cord mesenchymal stem cells 2 days, then respectively Culture supernatant is collected, its cytokine content comparative result is as shown in table 6.
Table 6：Cytokine content compares（Unit：pg/mL）.
As shown in Table 6：To 10 selected measure of cytokine content, it is known that application stem cell medium culture Compared with albumin physiological saline culture, the cytokine content in its culture supernatant improves 2-4 times to stem cell.Therefore, profit With stem cell medium culture stem cell provided by the present invention, the yield for obtaining cell factor can be greatly improved.
Step 6, the stem cell supernatant using collection, prepare stem cell injection liquid.
（1）The cell conditioned medium that will be collected collects filtrate, as stem cell injection liquid through 0.22 μm of membrane filtration.
（2）Bacteriological surveillance, detects through Gram-negative and gram-positive bacteria, is as a result negative.
（3）Endotoxin detection is carried out using gel method, the 0.2EU of amount containing endotoxin in every 1ml meets NF to injection The requirement 0.5EU of liquid.
The human vein for directly applying to chain reaction in patients with premature ovarian using the stem cell injection liquid is injected, and is monthly injected 4 times, agent Measure for 200ml everyone/time, continuous injection 2 months, hectic fever hidrosis, vaginal dryness, sexual desire such as decline at the ovary morning of premenopausal rear symptom The symptom that declines be improved significantly, hectic fever hidrosis phenomenon disappears, and vagina point oozes, and thing is normal, and sexual desire recovers normal.

Claims (10)

1. a kind of stem cell medium, it is characterised in that including following raw material：Compound amido acid injection 10%, injection is water-soluble Property vitamin 1%, alanyl glutamine 0.5%, human serum albumin 10%, injection of fish 10%, micro-element injection 0.5%th, Bomaili A liquid 68%；PH value is 6.8-7.0, and osmotic pressure is 310.
2. the preparation method of stem cell medium as claimed in claim 1, it is characterised in that comprise the following steps：
Step 1, Bomaili A liquid is taken, be dissolved in Vitamin H, obtain mixed liquor；
Step 2, respectively by the mixed liquor of step 1, Amino Acid Compound Injection, alanyl glutamine, human serum albumin, hydrolysis Protein injection liquid, micro-element injection, Bomaili A liquid, mix, as stem cell medium in proportion.
3. a kind of stem cell injection liquid, it is characterised in that use the stem cell medium culture mescenchymal stem cell, and collect Mescenchymal stem cell culture supernatant；The culture supernatant that will be collected collects filtrate, as stem cell injection through 0.22 μm of membrane filtration Liquid.
4. the preparation method of stem cell medium as claimed in claim 3, it is characterised in that comprise the following steps：
Step 1, mescenchymal stem cell sample collection, treatment；
Step 2, the primary extraction and culture of stem cell：With the tissue of the method separating mesenchymal of physical shear, pasted with tissue block Wall cultivation culture mescenchymal stem cell；With lymphocyte separation medium centrifugation mononuclearcell, in the way of adhere-wall culture It is separately cultured mescenchymal stem cell；
Step 3, the Secondary Culture of stem cell：When cell reaches more than 60% to be merged, cleaning adds cell dissociation buffer afterwards twice, Filtering removal tissue fritter, after supernatant is abandoned in centrifugation, obtains primary mescenchymal stem cell；Primary mescenchymal stem cell is trained with serum-free After foster base is resuspended, it is seeded in blake bottle or culture dish, when cell fusion degree reaches 80%-90%, collects first generation mesenchyma Stem cell, as seed cell or continuation Secondary Culture；
The identification of step 4, the biology of mescenchymal stem cell and fluidic cell；
Step 5, the stem cell medium for taking preparation, change mescenchymal stem cell liquid, continue to cultivate mescenchymal stem cell, and collect Mescenchymal stem cell culture supernatant；
Step 6, the stem cell culture supernatant that will be collected in step 5 collect filtrate, as stem cell note through 0.22 μm of membrane filtration Penetrate liquid.
5. the preparation method of stem cell injection liquid as claimed in claim 1, it is characterised in that the mescenchymal stem cell is navel Filled with mescenchymal stem cell, between placenta mesenchyma stem cell, mesenchymal stem cells in umbilical cord blood, fat mesenchymal stem cell, marrow Matter stem cell and dental pulp mescenchymal stem cell.
6. the preparation method of stem cell injection liquid as claimed in claim 1, it is characterised in that sample process in the step 1 Using alcohol-pickled time 1-4min, the alcohol is degerming through 0.22 μm of aperture membrane filtration.
7. the preparation method of stem cell injection liquid as claimed in claim 1, it is characterised in that the serum free medium be 10% serum replacement is added in DMEM/F12 basal mediums.
8. the preparation method of stem cell medium as claimed in claim 1, it is characterised in that in step 2 adhere-wall culture Using CO2Incubator；The CO2The condition of incubator is：37 DEG C, 12% oxygen, 5% carbon dioxide, the hypoxemia bar of 83% nitrogen Part.
9. the preparation method of stem cell medium as claimed in claim 4, it is characterised in that what is be inoculated with the step 3 is close It is 5000-6000/cm to spend2。
10. the stem cell injection liquid as described in claim 4-9, it is characterised in that can apply to human vein injection, injection Dosage be 200ml/time.
CN201611007074.3A 2016-11-16 2016-11-16 Stem cell culture solution and injection Active CN106754668B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611007074.3A CN106754668B (en) 2016-11-16 2016-11-16 Stem cell culture solution and injection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611007074.3A CN106754668B (en) 2016-11-16 2016-11-16 Stem cell culture solution and injection

Publications (2)

Publication Number Publication Date
CN106754668A true CN106754668A (en) 2017-05-31
CN106754668B CN106754668B (en) 2020-08-25

Family

ID=58969877

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611007074.3A Active CN106754668B (en) 2016-11-16 2016-11-16 Stem cell culture solution and injection

Country Status (1)

Country Link
CN (1) CN106754668B (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107569516A (en) * 2017-08-16 2018-01-12 北京京蒙细胞生物科技股份有限公司 A kind of medicine for controlling cat distemper heat and its preparation method and application
CN107951904A (en) * 2017-11-06 2018-04-24 深圳市莱利赛生物科技有限公司 A kind of application of fat mesenchymal stem cell medicine in premature ovarian failure is treated
CN108096177A (en) * 2017-12-30 2018-06-01 广州润虹医药科技股份有限公司 A kind of compound stem cell factor cosmetic composition and its application
CN108815509A (en) * 2018-08-07 2018-11-16 天津昂赛细胞基因工程有限公司 Mescenchymal stem cell substitution product and its application comprising degradable biological medical slow-release material
CN109182259A (en) * 2018-08-29 2019-01-11 浙江大学 A method of improving mescenchymal stem cell excretion body yield
CN109337871A (en) * 2018-11-05 2019-02-15 潍坊市康华生物技术有限公司 A kind of placental hematopoietic stem cell preparation method
CN109453202A (en) * 2018-12-21 2019-03-12 广州益养生物科技有限公司 It is a kind of can direct injection application cell preparation and preparation method thereof
CN109943524A (en) * 2019-03-01 2019-06-28 宁波医诺生物技术有限公司 Human mesenchymal stem cell culture medium and its preparation method and application
CN110368404A (en) * 2019-07-16 2019-10-25 厦门艾赛生物科技有限公司 A kind of gynecological gel and preparation method thereof
CN113244160A (en) * 2021-05-31 2021-08-13 美卓(杭州)医疗科技有限公司 Multi-effect skin care injection and preparation method thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11951134B2 (en) 2019-09-30 2024-04-09 North Carolina State University Cationic platelet lysate compositions and related methods

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966080A (en) * 2005-11-17 2007-05-23 北京科宇联合干细胞生物技术有限公司 Neural stem cell injection for treating senile dementia and Parkinson's disease
CN102008507A (en) * 2010-11-21 2011-04-13 天津和泽干细胞科技有限公司 Human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and preparation method thereof
CN102920734A (en) * 2012-11-14 2013-02-13 青岛奥克生物开发有限公司 Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis
US20150190429A1 (en) * 2012-07-18 2015-07-09 Universitaet Duisburg-Essen Use of Preparations Comprising Exosomes Derived From Mesenchymal Stem Cells (MSCs) in the Prevention and Therapy of Inflammatory Conditions
CN104894064A (en) * 2015-07-08 2015-09-09 河南中科干细胞基因工程有限公司 Culture medium for culturing mesenchymal stem cells
CN105535022A (en) * 2016-01-12 2016-05-04 浙江生创精准医疗科技有限公司 Application of exosome to preparing of acute-hepatic-failure treating medicine and medicine composition
CN105920042A (en) * 2016-04-13 2016-09-07 上海华颜医药科技有限公司 Umbilical cord mesenchymal stem cell injection with anti-aging function and preparation method thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966080A (en) * 2005-11-17 2007-05-23 北京科宇联合干细胞生物技术有限公司 Neural stem cell injection for treating senile dementia and Parkinson's disease
CN102008507A (en) * 2010-11-21 2011-04-13 天津和泽干细胞科技有限公司 Human umbilical cord mesenchymal stem cell (HUMSC) anti-hepatic fibrosis injection and preparation method thereof
US20150190429A1 (en) * 2012-07-18 2015-07-09 Universitaet Duisburg-Essen Use of Preparations Comprising Exosomes Derived From Mesenchymal Stem Cells (MSCs) in the Prevention and Therapy of Inflammatory Conditions
CN102920734A (en) * 2012-11-14 2013-02-13 青岛奥克生物开发有限公司 Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis
CN104894064A (en) * 2015-07-08 2015-09-09 河南中科干细胞基因工程有限公司 Culture medium for culturing mesenchymal stem cells
CN105535022A (en) * 2016-01-12 2016-05-04 浙江生创精准医疗科技有限公司 Application of exosome to preparing of acute-hepatic-failure treating medicine and medicine composition
CN105920042A (en) * 2016-04-13 2016-09-07 上海华颜医药科技有限公司 Umbilical cord mesenchymal stem cell injection with anti-aging function and preparation method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
严冰浩: "间充质干细胞来源的外泌体的制备", 《解剖学杂志》 *
刘高米洋: "人脐带间充质干细胞外泌体的分离鉴定", 《干细胞研究专题》 *
杨向荣等: "人脐带间充质干细胞来源外泌体的生物学特性研究", 《华中科技大学学报》 *
肖漓等: "脐带间充质干细胞外泌体的分离和鉴定", 《中华细胞与肝细胞杂志》 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107569516A (en) * 2017-08-16 2018-01-12 北京京蒙细胞生物科技股份有限公司 A kind of medicine for controlling cat distemper heat and its preparation method and application
CN107951904A (en) * 2017-11-06 2018-04-24 深圳市莱利赛生物科技有限公司 A kind of application of fat mesenchymal stem cell medicine in premature ovarian failure is treated
CN107951904B (en) * 2017-11-06 2020-11-27 深圳市莱利赛生物科技有限公司 Adipose-derived mesenchymal stem cell medicine and preparation method and application thereof
CN108096177A (en) * 2017-12-30 2018-06-01 广州润虹医药科技股份有限公司 A kind of compound stem cell factor cosmetic composition and its application
CN108815509A (en) * 2018-08-07 2018-11-16 天津昂赛细胞基因工程有限公司 Mescenchymal stem cell substitution product and its application comprising degradable biological medical slow-release material
CN109182259A (en) * 2018-08-29 2019-01-11 浙江大学 A method of improving mescenchymal stem cell excretion body yield
CN109337871A (en) * 2018-11-05 2019-02-15 潍坊市康华生物技术有限公司 A kind of placental hematopoietic stem cell preparation method
CN109453202A (en) * 2018-12-21 2019-03-12 广州益养生物科技有限公司 It is a kind of can direct injection application cell preparation and preparation method thereof
CN109943524A (en) * 2019-03-01 2019-06-28 宁波医诺生物技术有限公司 Human mesenchymal stem cell culture medium and its preparation method and application
CN110368404A (en) * 2019-07-16 2019-10-25 厦门艾赛生物科技有限公司 A kind of gynecological gel and preparation method thereof
CN113244160A (en) * 2021-05-31 2021-08-13 美卓(杭州)医疗科技有限公司 Multi-effect skin care injection and preparation method thereof

Also Published As

Publication number Publication date
CN106754668B (en) 2020-08-25

Similar Documents

Publication Publication Date Title
CN106754668A (en) A kind of stem cell medium and parenteral solution
CN104726406B (en) It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell
CN104805054B (en) Stem cell serum-free culture medium
CN110564682B (en) Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes
CN105079859B (en) A kind of dressing and preparation method thereof
CN105238751A (en) Umbilical cord tissue mesenchymal stem cell isolated culture method
CN104762260A (en) Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof
CN104762257B (en) A kind of method preparing mescenchymal stem cell from umbilical cord
CN104651305A (en) Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells
CN109943526B (en) A kind of serum-free peptide composition promoting mescenchymal stem cell proliferation
CN113244272A (en) Composition for improving premature ovarian failure and preparation method and application thereof
CN102643784A (en) Expansion system in vitro for hematopoietic stem/progenitor cell
CN110623917A (en) Beautifying and skin repairing sodium hyaluronate gel coated with stem cell complex factor
CN104450613A (en) Cell culture medium beneficial to in-vitro culture of autologous bone marrow mesenchymal stem cells
CN102146359A (en) Method for extracting original mesenchymal stem cells from placenta and serum-free amplification
CN104726401A (en) Method for improving success rate of umbilical cord blood mesenchymal stem cell culture by using placental mesenchymal stem cells
CN108324993A (en) A kind of stem cell complex, preparation method and the application of induction hair regeneration
CN108588017A (en) A kind of amplification method of umbilical cord mesenchymal stem cells and its application in arthritis
CN110917217B (en) Application of muscle stem cells in preparation of anti-inflammatory drugs
CN104988111A (en) Inducing liquid for converting UC-MSC into islet cells and application thereof
CN102168063B (en) Method for separation and primary culture of intestinal mucosal cells of fishes
CN104789531A (en) Method for inducing umbilical cord mesenchymal stem cells to be differentiated into dopaminergic neurons
CN115820546B (en) Method for promoting chondrogenic differentiation of brown adipose-derived stem cells and application of method
CN108034634B (en) Method for separating endometrial mesenchymal stem cells from menstrual blood
CN105112367B (en) A kind of mescenchymal stem cell epidermal differentiation derivant and its application process

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
GR01 Patent grant
GR01 Patent grant