CN106282103A - Animal mescenchymal stem cell immunosuppressant can strengthen serum-free medium - Google Patents
Animal mescenchymal stem cell immunosuppressant can strengthen serum-free medium Download PDFInfo
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Abstract
The present invention provides a kind of cell culture fluid that can strengthen serum-free culture for animal mescenchymal stem cell immunosuppressant.In basic culture solution, add multiplicaiton factor and specific additive, for the serum-free culture of animal mescenchymal stem cell, promote animal mescenchymal stem cell healthy growth and propagation, and there is maintenance and strengthen the effect of animal mescenchymal stem cell immunosuppressant energy.And the immunosuppressant to mescenchymal stem cell can have potentiation.The experiment proved that animal mescenchymal stem cell immunosuppressant of the present invention can strengthen serum-free medium and can be used for the serum-free culture of animal mescenchymal stem cell, and apply for clinical research in terms of the immunosuppressant energy applied research of mescenchymal stem cell.
Description
Technical field
The present invention relates to the serum-free medium that a kind of animal mescenchymal stem cell immunosuppressant can strengthen, multiplicaiton factor and specific additive is added in basic culture solution, for the serum-free culture of animal mesenchymal cell, the immunosuppressant after animal mescenchymal stem cell propagation can be made to act on enhancing.
Background technology
Mescenchymal stem cell, in addition to having multiple differentiation potential, also has immunoloregulation function.Experiment in vitro shows, mescenchymal stem cell can suppress the propagation of T cell in vitro, makes T cell maintain state rather than the apoptosis of a dormancy.Mescenchymal stem cell, after inflammatory cytokine stimulates, shows the strongest inhibitory action to immune system.Owing to mescenchymal stem cell possesses the strongest immunoloregulation function and reduced immunogenicity, the therefore research emphasis of the disease of immune system that mescenchymal stem cell is that treatment is transplanted and autoimmune disease etc. is difficult to treat.Mescenchymal stem cell not only autoantigenic is the lowest, also has an impact the function of various immunocytes (T cell, B cell, NK cell, arborescent cell).Therefore, to the treatment with immunoreation relevant disease, using mescenchymal stem cell is desirable.
The clinical trial relevant with mescenchymal stem cell of U.S. FDA approval, mainly includes the following aspects: hematopoietic stem cell transplantation, the reparation of tissue injury, treatment of autoimmune diseases, carrier as gene therapy.China also has started to mescenchymal stem cell treatment some refractory diseases clinically, such as spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus (sle), systemic sclerosis, clone disease, apoplexy, diabetes, diabetic foot, liver cirrhosis etc., according to preliminary clinical report, mescenchymal stem cell has obvious curative effect to the treatment of these diseases.
The anti-host response of immunity after transplanting in regenerative medicine is a serious problem, for avoiding this immunological rejection, needs to use immunosuppressant.Owing to mescenchymal stem cell has immunosuppressive action, if it is possible to utilize this effect, perhaps can use immunosuppressant.Therefore, it is indispensable for manufacturing without animal component, the mesenchymal stem cell serum-free culture fluid that has an immunosuppressive action.
Culture fluid is supplied with the nutriment of the artificial preparation of animal and plant cells, tissue and microbial growth, generally individually contains carbohydrate, nitrogen substance, inorganic salt (including trace element) and auxin and water etc. in basic culture solution.The cultivating of traditional zooblast needs to add the serum in the heterogenous animal such as hyclone (FBS) source of 5～20% in basal medium, this serum is used as to promote the source of nutrition of cell growing multiplication in vitro, also serves as the supply source of the bioactive substance such as hormone, the factor simultaneously.It is to say, generally, the growth of zooblast all depends on the existence of serum, and in Nostoc commune Vanch liquid, such as not increase serum, overwhelming majority cells just can not be bred.
The shortcoming of above-mentioned application scheme is:
Using animal mescenchymal stem cell serum free culture system liquid, serum is expensive；
Because product batch number difference can cause difference between batch, cause the poor reproducibility of product and experimental result；
The protein ingredient that serum itself is brought into need to be removed, also need to if desired purify, make operation complicated；
Serum may be mixed into unknown pathogen (virus etc.), and then there is the possibility polluting cultivation cell.
Publication No. CN102703385A, date of publication is on October 3rd, 2012, and the Chinese invention patent application of invention entitled " a kind of Mesenchymal stem cell nutrient solution " discloses a kind of animal mesenchymal stem cell serum-free culture fluid；And Publication No. CN103805562A, date of publication is on May 21st, 2014, and the Chinese invention patent application of invention entitled " cultivating the serum-free medium of placenta mesenchyma stem cell " discloses a kind of placenta mesenchyma stem cell and cultivates the serum-free medium of amplification；But the serum-free medium composition of these inventions is relatively simple, limited to the growth of animal mescenchymal stem cell and the nutrition facilitation of amplification.
The patent relating to Mesenchymal stem cell nutrient solution such as aforementioned does not all account for the impact on cultivated mescenchymal stem cell immunosuppressive action, and cultivated mescenchymal stem cell immunosuppressive action may be made to reduce.
Summary of the invention
The present invention solves the problems referred to above, it is provided that a kind of animal mescenchymal stem cell immunosuppressant can strengthen serum-free medium, adds predetermined substance, strengthens mescenchymal stem cell immunosuppressant energy.This cell culture fluid can be used for the cultivation of animal mescenchymal stem cell, possesses the advantage of serum-free cell culture medium and maintenance and strengthens the immunosuppression capability of mescenchymal stem cell.
The technical solution adopted for the present invention to solve the technical problems is: the formula of the present invention adds various interpolation factor composition in basic culture solution；
Preferably, animal mescenchymal stem cell immunosuppressant of the present invention can strengthen in serum-free medium, and described basic culture solution is one or more the mixing compositions in Ham ' s F12, DMEM, RPMI-1640；
Above-mentioned basic culture solution is added with the conventional basic ingredient BSA of serum-free medium, insulin, transferrins, and is added with dexamethasone；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen three kinds of multiplicaiton factor of interpolation in serum-free medium；
Preferably, described multiplicaiton factor is FGF, PDGF-BB, TGF-β, HGF；
Preferably, the concrete kind of described multiplicaiton factor: FGF be FGF-2 (bFGF), PDGF be PDGF-BB or PDGF-AB, TGF-β be TGF-β 3；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen in serum-free medium adds one or more lipids；
Preferably, one or more during described lipid is phosphordithiic acid, lysophosphatidic acid, phosphatidylinositols, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, lecithin, phosphatidyl glycerol；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen in serum-free medium adds one or more fatty acids；
Preferably, described fatty acid is linoleic acid, oleic acid, alanine, arachidonic acid, myristic acid, palmityl acid, stearic acid；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen serum-free medium alternative and add cholesterol；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen in serum-free medium adds one or more lipid antioxidant；
Preferably, described lipid antioxidant is vitamin E, reduced glutathion, 2 mercapto ethanol；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen in the specific additive of serum-free medium containing IFN-γ and TNF-a；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen adds a kind of surfactant in serum-free medium；
Preferably, described surfactant is Pluronic-F (poloxamer) and Tween80；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen adds a kind of lithium salt compound in training serum-free nutrient solution；
Preferably, described lithium salt compound is lithium chloride；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen interpolation BSA in serum-free medium；
Animal mescenchymal stem cell immunosuppressant of the present invention can strengthen adds a kind of selenium-containing compound in serum-free medium；
Preferably, described selenium-containing compound is selenite.
The invention has the beneficial effects as follows:
1. can strengthen the immunosuppression capability of cultivated mescenchymal stem cell；
2. can avoid the variation of quality between serum batch, improve cell and cultivate and the repeatability of experimental result；
3. avoid serum to the toxic action of cell and serum source contact scar, it is to avoid the serum component impact on experimentation；
4. effectively facilitate mitogen, shorten the cell population doublings time, cell proliferation, inhibited apoptosis can be promoted；
5. the condition making cell cultivate is more stable, maintains cell long period growth and breeding in vitro, the beneficially differentiation of cultured cell in vitro；
6. can improve the expression of product and make cell products be prone to purification；
7. when making mescenchymal stem cell expand, cell membrane is more stable, and can stablize the signal transduction path of cell；
8. additive introduces macromolecule nonionic surfactant, reduces the mechanical shear stress infringement to cell, effect protected to cell, albuminous use can be reduced simultaneously, cost-effective；
9. it is not only suitable for the cultivation amplification of any tissue-derived mescenchymal stem cell of human body, and is theoretically adapted to the cultivation amplification of the mescenchymal stem cell in almost all mammal source.
Accompanying drawing explanation
Fig. 1-a is that display utilizes 10%FBS-MSCGM culture fluid to co-culture human marrow mesenchymal stem cell and Mus source activating T cell, the T cell breeder reaction result figure stimulated for AntiCD3 McAb and CD28.Fig. 1-b is that display utilizes animal mescenchymal stem cell immunosuppressant of the present invention can strengthen serum-free medium to co-culture human marrow mesenchymal stem cell and Mus and originate activating T cell, the T cell breeder reaction result figure stimulated for AntiCD3 McAb and CD28.
Fig. 2-a is that display utilizes 10%FBS-MSCGM culture fluid to co-culture human marrow mesenchymal stem cell and Mus source activating T cell, the T cell breeder reaction result figure stimulated for anti-PMA/Ionomycin.Fig. 2-b is that display utilizes animal mescenchymal stem cell immunosuppressant of the present invention can strengthen serum-free medium to co-culture human marrow mesenchymal stem cell and Mus and originate activating T cell, the T cell breeder reaction result figure stimulated for anti-PMA/Ionomycin.
Fig. 3 is the immunosuppressive effect figure that the T cell under mouse lymphotactin hybrid reaction (MLR) is bred by bone marrow origin hMSC.The T cell breeder reaction stimulated for Mus lymph hybrid reaction, people MSC cultivates the immunosuppressive effect (Fig. 3-a) of the 3rd day；The T cell breeder reaction stimulated for Mus lymph hybrid reaction, people MSC cultivates the immunosuppressive effect (Fig. 3-b) of the 4th day.
Fig. 4-a display is applied mescenchymal stem cell immunosuppressant of the present invention can strengthen serum-free medium and is cultivated human adipose mesenchymal stem cells microscope inspection figure of cell growth state after the 8th day；Fig. 4-b display application 10%FBS-MEM culture fluid cultivation human adipose mesenchymal stem cells microscope inspection figure of cell growth state after the 8th day.
Fig. 5 (people synovial tissue mescenchymal stem cell difference culture fluid cultivates comparison result) can strengthen serum-free medium for two kinds of mescenchymal stem cell immunosuppressant of the present invention of application, multiplicaiton factor kind contained by two kinds of serum-free mediums is different, cultivate people's Synovial Mesenchymal Stem Cells, the cell proliferative conditions after cultivating the 8th day respectively.
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the range of application of the present invention.
Embodiment 1:
Take human bone marrow-derived mescenchymal stem cell to co-culture with Mus source activating T cell, culture fluid used is respectively 10%FBS-MSCGM culture fluid and animal mesenchymal stem cell serum-free culture fluid (SF-MSCGM) of the present invention is cultivated, and SF-MSCGM of the present invention composition is as follows: basic culture solution+multiplicaiton factor (bFGF, HGF, TGF-β 3, PDGF-BB)+CD+ cholesterol+VE+ lecithin+dexamethasone+transferrins+Monohydrated selenium dioxide+BSA+ insulin+INF-γ+TNF-α.The T cell breeder reaction stimulated for AntiCD3 McAb and CD28, result shows that the human mesenchymal stem cell cultivated with serum-free medium of the present invention has immunosuppressant energy potentiation (Fig. 1-a, Fig. 1-b).
Embodiment 2:
Take human bone marrow-derived mescenchymal stem cell to co-culture with Mus source activating T cell, culture fluid used is respectively 10%FBS-MSCGM culture fluid and animal mescenchymal stem cell immunosuppressant of the present invention can strengthen serum-free medium (SF-MSCGM), and SF-MSCGM constituent of the present invention is with embodiment 1.The T cell breeder reaction stimulated for anti-PMA/Ionomyein, result shows that the human mesenchymal stem cell cultivated with serum-free medium of the present invention has immunosuppressant energy potentiation (Fig. 2-a, Fig. 2-b).
Embodiment 3
The immunosuppressive effect experiment that T cell under mouse lymphotactin hybrid reaction (MLR) is bred by bone marrow origin hMSC.The culture fluid of experimental technique and use cell, use is the most same as in Example 1.The processing method of bone marrow origin hMSC is, sows 2 × 10 in 96 each holes, hole4The cell of individual density；The activation method of Mus spleen cell is, with 2 × 104The Mus spleen cell of density co-cultures with Os Mus marrow origin arborescent cell (BMDC), and the latter's thickness of sowing is 3.3 × 104Individual/hole.Embodiment result shows that bone marrow origin hMSC that bone marrow origin hMSC cultivated with SF-MSCGM of the present invention is cultivated with MSCGM culture fluid equally has immunosuppressant energy；And start to present immunosuppressive effect (Fig. 3-b) from the 4th day by bone marrow origin hMSC of MSCGM culture fluid cultivation, and bone marrow origin hMSC cultivated with SF-MSCGM of the present invention started to present immunosuppressive effect (Fig. 3-a) from the 3rd day. it is to say, present immunosuppressive effect earlier by bone marrow origin hMSC of SF-MSCGM of the present invention cultivation.
Embodiment 4
Fatty tissue origin hMSC separation and Culture in the following order, gathers human fat tissue, washes 2～3 times with serum-free DMEM culture fluid, the fine chopping of fatty tissue after cleaning with shears, (1mm3), with 0.1～0.2% collagen protein enzymatic solution process fat fragment, (37 DEG C, 30～60 points).The fatty tissue crossed with 100mm membrane filtration above-mentioned collagen protein ferment treatment, then 100 × g, the 10 separation hearts, isolated adipose tissue origin hMSC.Process the cell 5 after separating～10 points with Red lysis buffer (sigma R7757), then wash 2-3 time with serum-free DMEM culture fluid.Cell after Xi Jinging is seeded into plate (BECTON, DICKINSON (Falcon) 353047), cultivates with SF-MSCGM and 10%FBS-MEM of the present invention respectively.After cultivation the 8th day, every day was by microscope observation of cell form.Cultivate the vegetative state (Fig. 4-a) of the 8th day adipose-derived mescenchymal stem cell of descendant.
Embodiment 5:
Synovial membrane origin hMSC separation and Culture in the following order: gather people synovial tissue, after cleaning with PBS, add the collagen protein enzymatic solution 10ml of 0.4%, reaction in 1～4 hour at 37 DEG C, synovial membrane origin hMSC after being centrifuged after filtration is cultivated with following culture fluid, and 10%FBS-DMEM, culture fluid 1, culture fluid 2 are cultivated.Wherein, culture fluid 1 forms as follows: basic culture solution+multiplicaiton factor (bFGF, PDGF-BB)+CD+ cholesterol+VE+ lecithin+dexamethasone+transferrins+Monohydrated selenium dioxide+BSA+ insulin+INF-γ+TNF-a；Culture fluid 2 forms as follows: basic culture solution+multiplicaiton factor (bFGF, HGF, TGF-β 3, PDGF-BB)+CD+ cholesterol+VE+ lecithin+dexamethasone+transferrins+Monohydrated selenium dioxide+BSA+ insulin+INF-γ+TNF-α.That is, culture fluid 1 has lacked two kinds of multiplicaiton factor HGF and TGF-β 3 compared with culture fluid 2.From the beginning of cultivating the 12nd day (PO), microscope observes form, counting.Result shows that the facilitation effect of culture fluid 2 cell growth is about 8 times of 1, is 40 times of 10%FBS-DMEM.
Claims (10)
1. the serum-free medium that can strengthen for animal mescenchymal stem cell immunosuppressant.It is characterized in that: in basic culture solution, add multiplicaiton factor and specific additive, for the serum-free culture of animal mesenchymal cell, and the immunosuppressant after animal mescenchymal stem cell propagation can be made to act on enhancing.The experiment proved that the animal mescenchymal stem cell immunosuppressant of the present invention can strengthen the animal mesenchymal cell after culture fluid is cultivated, cell proliferation is fast, form good, stem cell immunosuppression capability is remarkably reinforced, and plays immunosuppressive treatment effect for clinical practice mescenchymal stem cell significant.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterized in that described basic culture solution consists of one or more mixing in Ham ' s F12, DMEM, RPMI-1640, and in basic culture solution, be added with the conventional basic ingredient BSA of serum-free medium, insulin, transferrins, and it is added with dexamethasone.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterized in that adding multiplicaiton factor in described basic culture solution, for FGF, PDGF, TGF-β, wherein FGF be FGF-2 (bFGF), PDGF be PDGF-BB or PDGF-AB, TGF-β be TGF-β 3.
Animal mesenchymal stem cell serum-free culture fluid the most according to claim 1, it is characterised in that containing lipid and fatty acid composition in described specific additive, and selectivity interpolation steroid composition；Lipid components can be one or more in phosphordithiic acid, lysophosphatidic acid, phosphatidylinositols, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, lecithin, phosphatidyl glycerol；Fatty acid composition can be one or more in linoleic acid, oleic acid, alanine, arachidonic acid, myristic acid, palmityl acid, stearic acid；The steroid composition that selectivity adds can be cholesterol.
Animal mesenchymal stem cell serum-free culture fluid the most according to claim 1, it is characterised in that also contain, in described specific additive, the antioxidant preventing lipid, for one or more in vitamin E, reduced glutathion, 2 mercapto ethanol.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterised in that possibly together with cell attachment factors in other specific additives described, for one or more in fibronectin, collagen protein, gelatin.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterised in that containing IFN-γ and TNF-α in specific additive.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterised in that containing surfactant in described specific additive, for the one in Pluronic-F (poloxamer) or Tween80.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterised in that containing lithium salt compound in described specific additive lithium salt compound, for lithium chloride.
Animal mescenchymal stem cell immunosuppressant the most according to claim 1 can strengthen serum-free medium, it is characterised in that containing selenium compound in described specific additive selenium-containing compound, for selenite.
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| CN110177872A (en) * | 2017-03-03 | 2019-08-27 | 日本乐敦制药株式会社 | Mescenchymal stem cell and pharmaceutical composition |
| CN110540678A (en) * | 2018-05-29 | 2019-12-06 | 怡诺博(北京)生物医学技术有限公司 | Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material |
| CN112522191A (en) * | 2020-12-18 | 2021-03-19 | 云南中科灵长类生物医学重点实验室 | Culture method of mesenchymal stem cells |
| CN112608891A (en) * | 2020-12-18 | 2021-04-06 | 云南中科灵长类生物医学重点实验室 | Mesenchymal stem cell serum-free medium and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110177872A (en) * | 2017-03-03 | 2019-08-27 | 日本乐敦制药株式会社 | Mescenchymal stem cell and pharmaceutical composition |
| CN110540678A (en) * | 2018-05-29 | 2019-12-06 | 怡诺博(北京)生物医学技术有限公司 | Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material |
| CN110540678B (en) * | 2018-05-29 | 2022-09-06 | 怡诺博(北京)生物医学技术有限公司 | Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material |
| CN112522191A (en) * | 2020-12-18 | 2021-03-19 | 云南中科灵长类生物医学重点实验室 | Culture method of mesenchymal stem cells |
| CN112608891A (en) * | 2020-12-18 | 2021-04-06 | 云南中科灵长类生物医学重点实验室 | Mesenchymal stem cell serum-free medium and application thereof |
| CN112522191B (en) * | 2020-12-18 | 2023-05-12 | 云南中科灵长类生物医学重点实验室 | Culture method of mesenchymal stem cells |
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Application publication date: 20170104 |